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·HEMATOLOGICẤL TECHNIQUE

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·HÆMATOLOGICAL TECHNIQUE
BY
L EVERARD NAPIER CIE
FRCP
LATE DIRECTOR AND PROFESSOR OF TROPICAL MEDICINE CALGUITA
CHOOL
OR
TROPICAL MEDICINE AYD SENIOR PHYSICIAN
CAR HFL HOSPITAL FOR TROPICAL DISEASES CALCUTTA
ASD
CR DAS GUPTA 1B
DIV (CAL )
IN CHARGE DEPARTMENT OF REHATOLOGY CALCUTTA SCHOOL OF
TROPICAL MEDICINE PHYSICIAS CARMICHEL HOSPITAL. FOR TROPICAL
DISEASES CALCUTTA
THIRD EDITION
REVISED BY
CR DAS GUPTA MB
DIN (CAL )
UN DHUR & SONS LIMITED
15 BANKLI CHATTERJEE STREET
CALCUTTA
1945

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·PREFACF TO THIRD EDITION Dr L E Napier left India early in 1943 As suggested by him I have to take up the responsibility of bringing out the third edition The second edition was exhausted as rapidly as the first edition and the book was out of print for over a y car as it had not been possible to bring out this edition earlier due to war time difficulties In the new edition the vection on the development of red cells has been re written following the Schemata outlined by Israels (1941) The chapters on blood grouping and blood transfusion have been enlarged in the table of contents sub headings have been given under each main heading I am grateful to Dr J B Chatterji MB Assistant research worker Hæmatological Unit IRFA for assistance with the proofs C R DG School of Tropical Vedicine Calcutta The 13th August 1945 PREFACE TO SECOND EDITION The rapid exhaustion of the first edition of this book indicates that there is a demand in India for a book of its kind In this second edition we have included blood grouping and transfusion Though neither is a diagnostic method we have felt that these two chapters would not be out of place in this book and with the closer approach of the war to India there is a special demand for knowledge on these subjects PREFACE TO FIRST EDITION In the past the attitude of the medical profession in India towards anæmia has been that it was an almost inevitable accompaniment of residence in a tropical country and that therefore per se it need not claim attention It i now beginning to be realized that when a patient is anæmic there is always some pathological process to account for the fact and that whilst other methods of ascertaining the nature of these pathological proces.es should not be neglected much information

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·CONTENTS PAGE 1- 3 3- 9 9-27 21-27 CHAPTER THE COLLECTION OF THE BLOOD SAMPLE Advantages in the use of venous blood Rules to be observed when venous blood is to be used Time for collection of blood Anti-coagulant Time lunits for using venous blood CI 4PTER 7 ESTIMATION OF HEMOGLOBLY Clinical methods of estimating hæmoglobin Vethods of expressing results Amount of hæmoglobin per 100 ccm of blood which are shown as 100 per cent by different instruments Technique of estimating hæmoglobin Hellige normal hæmometer Normal hæmoglobin levels of different populations CITAPTER 3 ENERATION OF ERYTIIROCYTES AVD LFUCOCITES Principle Counting chamber with improved Neubauer ruling Counting chamber with Thoma ruling Blood counting Pipette Diluting fluids Fnumeration of erythrocytes Rules for cells on boundary lines alculation erythrocyte coun Enumeration of erythrocytes in polycythæmia Normal red cell count of different populations Fnumeration of leucocytes with Meubauer ruling For high counts as in leukæmia Counting by fiend method for low count with Thoma ruling care of instruments Leucoryte counts in normal individual CHAPTER 4 ENUMERATION OF RETICULOCYTES Vethod of Osgond and Wilhelm Vodification of the above Method of choice Counting the reticulocytes Normal values Significance of retrculocytes Reticulocyte crises CHAITER 5 E\USERATION OF THRONROCYTES Direct method Indirect method Normal values CHAPTER 6 DETERMINATION OF CORPI SCULAR VOLUME Principle Cell volume tubes Method Calculation of corpuscular values Normal values CHAPTER 7 HYPLRBILIRUBIN EVIA AND VAN DEN BERGII S TEST The van den Bergh reaction Technique Different reactions Quantitative indirect reaction Cobalt standard solution prepara tion technique for its use Loubond comparator Normal bilirubin according to different observers CHAPTER 8 ICTERUS INDEX Pnnciple Technique CHAPTER 9 THE MAKING AND STAINING OF BLOOD FILMS Cleaning slides and coverslips Polishing the slides Spreading the film in the routine use of venous blood Spreading the film from capillary blood Staining of blood films Preparation of stains Staining with Leishmans Wrights or Jenner s stain Staining with (eimsa s stain Combined staining CHAPTER 10 WHITE CELL DIFFERENTIAL COUNT Number of cells to be counted Procedure Recording the 27-29 29- 34 34.- 40 40_42 42-47 47–54 ·[u] may be obtained from the blood picture Consequently far more attention is being paid to anæmia itself and the importance of accurate methods of blood examination for the proper study of a case of anæmia is igradually being appreciated Many books on laboratory technique give full details of all the various methods that can be adopted but such books are not always within the reach of practitioners further the methods described in these books are often embarrassing in their multiplicity We therefore felt that readers of the Indian Medical Ga ette particularly those working in India might find 2 short description of the methods followed at the Calcutta School of Tropical Medicine of some practical use and have prepared a series of short notes on hæmatological technique these were published in the Indian Medical Gazette during 1940 and early 1941 We are now issuing them as a collected series of papers In a few instances original modifications have been introduced but for the most part the methods we have described have been thoroughly tested by laboratory workers in many parts of the world and are generally accepted as sound and accurate in every instance they are methods that tve have used in our laboratory for some considerable time in most instances for many years and found not only accurate but suitable for the local conditions We have limited these articles to the description of procedures that are more-or less routine in the investigation of a case of anæmia in the Calcutta School of Tropical Medicine but we have not attempted to be comprehensive for in the majority of our cases in order to trace the ætiological factors many other examinations protozoological bacteriological helminthological and serological and in addition certain other biochemical examinations such as the estination of cholesterol blood protein blood calcium etc have been carried out usually in other departments of the School We also group the blood in every case on admission but as this is not in any sense a diagnostic procedure we have not included it in this series At the time we commenced this series all the apparatus we described was available in India we regret that now some of it is not but we hope that reliable British or American substitutes for the apparatus of German origin will soon be available Our thanks are due to Dr P C Sen Guptı MB (Cal) for allowing us to include the article on Red cell diameters Price Jones curve of which he was the joint author in this volume 1 Ε Ν School of Tropical Medicine Calcutta June 19th 1947 ·HÆMATOLOGICAL TECHNIQUE 1 THE COLLECTION OF THE BLOOD SAMPLE The first essential is that one constant practice should be decided upon and followed There is a difference between capillary which for all practical purposes is arterial and senous blood but the difference is only of practical importance if two samples from the same individuals at different times are to be compared or when normal standards are being worked out As there are very distinct advantages in the use of venous rather than capillary blood the former is preferred The advantages are that all the examinations eg the estimation of hæmo globin the determination of cell volume the total red and white cell count the reticulocyte count the differential white cell count the measuring of the red cell diameters the icterus under the van den Bergh reaction the fragility test the erythrocyte sedimentation test etc can be done from a single sample of blood obtamed by a single puncture Further the tests can be repeated by the same or by different workers from the same sample of blood For some of the tests the van den Bergh test for example blood will have to be taken from the vein so that sufficient might as well be taken at one time for all the tests and the patient saved a number of additional pricks In weak and anæmıc individuals it is sometimes difficult to draw enough blood from a finger or ear lobe without squeezing the part and thereby diluting the blood with the tis ue fluid The following rules should be observed when using venous blood (a) The syringe should be air tight and perfectly dry The syringe is conveniently dried by first washing it thoroughly in clean water removing the water with alcohol removing the alcohol with ether and finally drawing air through it If there is even a trace of water or alcohol there will be hæmolysis of the blood and this will make it unsuitable for examination (6) The tourniquet should not be kept tied for more than 2 minutes when drawing the blood from the vein as the red cell count begins to increase after 3 minutes stasis (c) The needle of the syninge should be removed before the blood is put into the flask as hæmolysis may result of the blood is forced through the needle ·[ 1111 ) 54-62 88-91 PAGE results Expressing the results Differential leucocyte counts in normal individuals CHAPTER I CELL IDENTIFICATION System of identification Development of blood cells Der elopment of granulocytes Development of monocytes CHAPTER I. TECHNIQUE OF STERNAL PUNCTURE 62— бо The sternal puncture needle Procedure Examination of material Normal standards CHAPTER 19 THE ARNETH AND SCHILLING COUNTS 69— 74 Principle Staining Arneth count Interpretation of Ameth count Schilling count CHAPTER 14 ERYTHROCY TE SEDIMENTATION RATE 74-77 Principle of the test Westergreen s method Wintrobe s method Correction for anæmnia Precautions CHAPTER 15 GASTRIC ANALYSIS 78_88 Introduction Methods Routine procedure Preparation of the patient Introducing the tube Drawing out the contents of the fasting stomach Test meals Withdrawal of post prandial specimens Histamine Examination of gastric contents Method of recording results Normal standards CHAPTER 16 THE Red CELI FRAGILITY TEST Introduction Qualitative method Quantitative method CH4PTER 17 PROCEDURES EMPLOYED IN INVESTIGATING HÆMOR RHAGIC DISEASES 92-98 Introduction Coagulation time Capillary tube (Wright s) method Method of Lee and White (modified) Bleeding time Duke s method Venous pressure method Quantitre determination of prothrombin in blood prothrombin time Hess capillary resistance test Clot retraction study CHAPTER 18 RED CFLL DIAMETERS PRICF JONES CURVE 98—105 Introduction Technique of measurement of cell diameters Recording the results Normals Significance of findings CHAPTER 19 BLOOD GROUPING AND BLOOD TRANSFUSION 106_113 Historical Hæmoagglutination and blood groups Crouping methods tube method slide method Pseudoagglutination Prozone phenomenon Direct crossmatching before transfusion CHAPTER 20 TRANSFUSION 113-122 Source of blood Transfusion of different elements of blood Indications for transfusion Technique of collection and transfusion of blood Selection of donors Rate of transfusion Cleaning of the apparatus Sterili ation Reactions after transfusion CHAPTER 21 THE WESTIGATION OF A CASE AND TIE REPORTING 122-128 OF RESULTS Investigation Different forms used in the School of Tropical Vedicine ·ESTIMATION OF ICVOGLOBIN 3 Icterus index and van den Bergh test--2 hours Ceil volume fragility test and sedimentation ratc-3 hours Hemoglobin estimation red cell count white cell count and reticulocy te count-24 hours 2 ESTIMATION OF HÆMOGLOBIN Hämoglobin is a conjugated protein consisting of a protein globin and an iron containing pigment Hæmoglobin readily combines with oxygen to form oxyhžemoglobin each molecule of hæmoglobin contains one atom of iron which is capable of com bining with two atoms of oxygen The important functions of hæmoglobin are to convey oxygen to the tissues to remove carbon dioxide from the tissues and in maintaining a constant reaction of the blood To estimate the amount of hæmoglobin in any sample of blood advantage is taken of certain properties of hæmoglobin Of these the most important is its Foxygen carrying capacity A fired amount of hæmoglobin will always combine with the same amount of oxygen actually I gramme of hæmoglobin with 134 ccm of oxygen the amount of oxygen that will combine with a sample of blood can be measured accurately and the hæmoglobin content of the sample thus estimated 1 Another property is its iron content Hæmoglobin has a known molecular formula in which there is one atom of iron so that by estimating the amount of iron in red cells from a gisen olume of blood it is possible to calculate the amount of hæmoglobin in the sample Hæmoglobin is a protein with a known refractometnc index By masuring this index it possible to calculate the amount of hæmoglobin in any sample 1 Yet another property of hæmoglobin is its colour and this can be measured in the various ways indicated below {-Whilst the property of hæmoglobin easiest to meastire its colour the most fundamentally important property is its oxygen carrying capacity There is evidence that these two properties are very closely correlated so that if an instru ment which measures the colour is first calibrated by the oxygen carrving method a very close approximation of the hæmoglobin content of a sample can be obtained hy the former method Most clinical hæmoglobinometers that are non sold are thus calibrated Clinical methods of estimating hæmoglobin Of the many methods of csti mating hæmoglobin that are in use none of the simpler methods is above criticism while the more accurate procedures eg van Slyke s orygen-carrying capacity method are hardly suitable for general clinical practice Most of the clinical pro cedures in common use are methods of measuring the colour of the hæmoglobin and are based on one or other of the following principles ·2 HÆMATOLOGICAL TECHNIQUE (d) The flask should always be kept closed with a rubber cork cotton wool should never be used The flash should be shaken at once vigorously to ensure complete solution of the oxalate powder (e) When not in use the flask must be kept corked and in a cool receptacle during the summer () The blood should always be mixed thoroughly for at least 3 minutes preferably in a shaking machine before any sample is withdrawn from it The blood should always be taken directly from the flask and not from blood poured out on a slide or watch glass Tume for collection of blood --When repeated examination is required blood should always be collected at the same hour under basal conditions in the morning Even for a single examination it is preferable to collect the blood in the morning under basal conditions this precaution will minimize the effect of the fluctuations in the total and differential count due to muscular and other physiologļcal activities Procedure -The blood is drawn from one of the prominent superficial veins at the bend of the elbow in a perfectly dry air tight syringe First of all without detaching the needle small drops of blood are put on clean glass slides and smears for the differential leucocy te count and red cell diameter measurements should be made* then the needle is detached and a measured amount of blood is transferred to a small 25 c cm Erlenmeyer flash containing the requisite amount of anticoagulant for the quantity of blood to be taken Anti coagulant —Two muligrammes of dry oxalate powder is required for each cubic centimetre of blood A mixture of potassium and ammonium oxalate powder in the proportion of 2 to 3 has been found to be ideal as in the above proportion the shrinkage caused by potassium oxalate is counteracted by ammonium oxalate (Wintrobe and Landsberg 1935) To measure the requisite amount of the oxalate powders make i per cent solution of the two oxalate powders ammonium and potassium accurately measure with a pipette 04 ccm of the potassium salt and 06 ccm of the ammonium salt and put them into the flask evaporate in a dry oven after which the oxalate will be found at the bottom of the flask in a powdered state this is the amount required for 5 c cm of blood for 3 c cm the proportions will be o 24 c an potassium and o 36 c cm ammonium salt The flasks are now hept corked and are ready for use It is a good practice to have two sets of flasks prepared containing enough antı coagulant for , c cm and 3 c cm respectively If potassium oxalate alone is used 2 milligrammes are required for each cubi. centimetre of blood that is 10 milligrammes or I com of 1 per cent solution for each 5 c cm of blood and in this case Wintrobe s correcting factor x 1 og mut be applied to compensate for shrinkage Time limils -Osgood and others (1931) gise the followin, time limits for different examinations - Vaking smears for differential count--I hour As stated above this can be done from the oxalated sample but it is better to inake *pantt smears ·FSTIMATION OF II ESIOGLOBIY 5 Sion unters vary from 138 to 179 gumes and in our personal experience we have found amongst groups of so called healthy coolics in Assam a figure lower than the former and amongst healthy Europeans in India onc higher than the latter If the expression 100 per cent hæmoglobin has any meaning at all it must imply that the par icular sample is what one would expect from that invidual if he were in perfect health This would mean that a different standard would have to be adopted for each class of individual 2 procedure which would lead to endless confu It is therefore better to abandon the second method namely that of going the hæmoglobin in terms of a percentage of an unknown and arbitrary standard and to adopt the first and express results in terms of grammes of dry hæmoglobin per Yoo c cm of blood Trastrument mahers who have hitherto made no attempt to adopt a uns ersal tanlard and hæmatologists who have not encouraged them to do so are both responsible for the pre ent unsatisfactory state of affaid's The confusion tha exists cannot be better exemphfied than by thc tible (1) Ielow which shows that not only arc there differences in the standırd hetueen Instruments of various mahes but instruments of the saithe name made at different times vary from one another and finally various authoritics give different figures for apparently the same instruments TABLE I Amounts of het moglobin per job c cm of blood which are shown as 100 per cent by different instruments Authority Tallqvist Sahli Dare ли com T 11allano Fleischi Mrch TetporanIarl y 1933 138 173 13 77 (old) 169 (ne) "58 cholson 193 1 158 138 if 9 1 1 171 (11) 45 (new) 173 (d) | i 138 hitby an Britton 1932 IS and Cayer 158 hock 1937 17 1 (011) 138 (newt 13 77 162 Lev non and Vacrat 1937 158 17 3 1б с 15 92 Orlwymam and I c 1937 158 13 8 to 172 137 160 161 138 158 - Beck 1938 138 138 to 173' 137 to 160 169 D fferent jostruments are supplied with different stan lards ·4 HÆVATOLOGICAL TECHNIQUE I Direct comparison of the colour of undiluted blood again t a graduated colour standard (a) On special blotting paper (Tailqvist) (6) Betueerglass plates (Dare) II Comparison of blood diluted to a fixed percentage against graduated colour standard (a) Diluted with sodium carbonate solution and compared with a coloured glass wedge (Fleischl Miescher) (b) Diluted with deci normal hy drochloric acid and compared with coloured glass wedge (Hellige Neoplan) III Comparison of blood diluted to a varying degree with a fixed colour standard (a) Diluted with water and compared with a permanent picrocarmine standard (Gow (6) Brought into contact with carbon monoxide diluted with water and compared with a permanent colour standard (Haldane) (c) Diluted with deci normal hydrochloric acid and compared with a perma nent standard of acid hæmatin (Sahlı) or a coloured glass block (Hellige) IV Comparison of blood diluted with deci normal hydrochloric acid to a fired percentage against a fixed colour standard in a colorimeter of the Duboscq type (a) Water placed in one chamber of the colorimeter and a standard coloured disc interposed (Newcomer) Other methods which require more elaborate instruments depend on the intensity rather than the colour of the light transmitted through hæmoglobin solutions this is measured by means of a photo electric cell In the writers laboratory the new Hellige normal hæmometer is used for all clinral purposes The great advantage in the Hellige instrument is that it is possible to match the brown coloured arıdulated blood solution with the colour standard exactly and as the standard 15 made of coloured glas it is unlikely to undergo any colour change one coloured prism which has been in use in our labora tory for more than six years has not undergone any colour change during this period Error excluding those of carelessness in technique using dirty pipettes etc may arise through variations in the diluting pipettes and in the calibres of the graduated tubes The method suffers the disadvantage that with each dilution only one reading can be made--a second observer can check the reading but not make an independent one--and that a time interval of 15 to 20 minutes must be allowed before reading the result Methods of expressing the results --The amount of hæmoglobin in an indu dual s blood may be expressed (0) As the number of grammes per too c cm of blood or (1.) as the pony centage of the amount present in the blood of the normal individual The disadvantage in the latter method is that there is no uniformity of opinton 15 to what is a normal individual Figures for the normal given by different ·ESTIMATION OF IS EMOGLOBIN 5 sion writers niry from 13 8 to 173 grammes and in our personal experience we have found amongst groups of so called healthy coolies in As am a figure lower than the former and amongst healthy Europeans in India one higher than the latter If the expression 100 per cent hemoglobin has any meaning at all it must imply that the par icular sample is what one sould expect from that lividual if he were in perfect health This would mean that 1 different standard would have to be adopted for each class of indivdual a procedure which would lead to endless confu It is therefore better to abandon the second method namely that of giving the hemoglobin in terms of a percentage of an unknown and arbitrary standard and to adopt the first and express results in terms of grammes of dry hæmoglobin per too c cm of blood Instrument makers who have hitherto made no attempt to adopt a universal standard and hæmatologists who hate not encouraged them to do so are both responsible for the present unsatisfactory state of affaits The confusion that exists cannot be better exemphfied than by the table (1) belos which shows that not only are there differences in the standard between instruments of various makes but instruments of the ame name made at different times vary from one another and finally various authorities gise different figures for apparently the same instruments TABLE I Amotents of hermoglobin per 100 ccm of blood u hich are shoren as 100 per cent by different instruments Authority Tallqvist Sahlı Dare Ital lane Fleisch Art shr comer 138 123 rint and Tarl y 1933 138 IS 8 1377 (old) 16 9 (new) 138 chol 1934 158 16) 172 (old) 145 (11ew) 17 3 (old) 138 159 158 177 (old) I 13 8 (new 13 77 169 W tby and Britton 1939 hrack and Crier 1937 Le in n and V rate 1937 Or Inay Corham and I sacs 1937 18 173 160 15 92 158 138 to 172 137 to 16 0 160 138 15 9 Beck 1938 138 138 to 173 137 to 160 169 D rent instruments are suppled with different standards ·4 HÆMATOLOGICAL TECHNIQUE I Direct comparison of the colour of undiluted blood against a graduated colour standard (a) On special blotting paper (Tallquist) (b) Betweer glass plates (Dare) II Comparison of blood diluted to a fixed percentage against graduated colour standard (a) Diluted with sodium carbonate solution and compared with a coloured glass wedge (Fleischl Viescher) (6) Diluted with deci normal hy drochloric acid and compared with coloured glass wedge (Hellige Neoplan) III Comparison of blood diluted to a varying degree with a fixed colour standard (a) Diluted with water and compared with a permanent picrocarmine standard (Gowers) (6) Brought into contact with carbon monoxide diluted with water and compared with a permanent colour standard (Haldane) (c) Diluted with deci normal hydrochloric acid and compared with a perma nent standard of acid hematın (Sahlı) or a coloured glaas block (Hellige) IV Comparison of blood diluted with deci normal hydrochloric acid to a fired percentage against a fixed colour standard in a colorimeter of the Duboscq tyre (a) Water placed in one chamber of the colonmeter and a standard coloured disc interposed (Newcomer) Other methods which require more elaborate instruments depend on the intensity rather than the colour of the light transmitted through hæmoglobin solutions this is measured by means of a photo-electric cell In the writers laboratory the new Hellige normal hæmometer is u ed for all clinical purposes The great advantage the Hellige instrument is that it is possible match the brown coloured acidulated blood solution with the colour standard exactly and as the standard is made of coloured glass it is unlikely to undergo any colour change one coloured prism which has been in use in our labora tory for more than six years has not undergone any colour change during this period Error excluding those of carelessness in technique using dirty pipettes etc may arise through variations in the diluting pipettes and in the calibres of the graduated tubes The method suffers the disadvantage that with each dilution only one reading can be made a second observer can check the reading but not make an independent one-and that a time interval of 15 to 20 minutes must be allowed before reading the result Methods of expressing the results --The amount of hæmoglobin in an india dual s blood may be expressed- () As the number of grammes per 100 ccm of blood or (m) as the per centage of the amount present in the blood of the normal individual The disadvantage in the latter method is that there is no uniformity of opinion as to what is a normal individual Figures for the normal given by different ·TABLE II V Normal hæmoglobin lezels of different populations Age Sex Locality Economic status Hæmoglobin in grammes per 100 ccm Standard deviation Number on which ba ed Authority Men 19-30 25+5 Bombay Calcutta 13 31 14 77 to 96 + 136 121 30 Students Mixed ervants Clerks and doctors Cool es Fohl et ul 1937 Napher and Das Gupta 19354 1936 25-45 15 70 to 91 30 4 Jults Assam Cachar Assam Shes rajpur US4 Britan 1263 12 60 11 83 13 74 17 5 36 00 15 60 14 50 12 99 12 63 + 1 41 + 1 S3 167 + 179 1 72 1936 Ner/and Majumdar 1938 Wapist and Das Gupta 19356 Cen (Napier 1939) ESTIVATION OF HEMOGLOBIN Women 18-30 1438 Borabay Calcutta Middle class HH 1 10 JOI jor 129 17-2 17-30 Students M Jdle class 13 73 13 17 15 81 093 081 2 54 62 TOO JUXO Child Madras Delhi Coonoor (6 000 feet) Cachar Assatn Bntain Ca tle and Minot 1936 Whitby and Bntton 1939 Pricy Jones 1931 Sokey ef al 1938 Napier Edwards and Das Gupta 1941 Sankaran and Raj gopal 1938 Benjamin 1939 Radhakn hna Rao 1938 Naper and Majumdar 1938 Naper and Bilimona 1937 Price Jones 1931 Ilh tby and Britton 1939 Bethcl 1936 Napier and Bilimona 1937 Napur and Das Gopta 19370 1937b HH IH baring Coolles 1 74 2 30 za 25 10 40 JO 80 1360 1370 13,6 100 18-22 Mich gan Pregnant women Awam Stud ot Coe t I 60 50 40 22 19 228 192 Obnou DPmics excluded) — M chigan Coonoor (6 000 feet) 1165 15 52 Ante natal cl nc (mixed) 1 252 Bethel 1936 Radhakrishna Rao 1938 8 ·6 21AMATOLOGICAL TECIINIQUE 1 may However many instruments that are now soll gate the readings in terms of frammes of hamoglobin per 100 c cm of blood others [11€ rcadings in duplicate so that it is easy to see what corresponds to 100 per cent or if they use the older method they state what their 100 per cent corresponds to in terms of dry hæmoglobin The excuse given for adhering to the older method (1) is that most practı tioners are familiar with this method of expressing the hæmoglobin It be true that they are familiar with the expression say hæmoglobin 80 per cent but they dont know what it means for, it may mean anything between 11 and 138 grammes of hæmoglobin per 100 ccm of blood a discrepancy which is far from negligible They must therefore learn something new so why should they not learn a new method which has a definite meaning accepted by all hæmatologists Most American medical books are now using the new momenclature and in Great Britain even the more conservative clinicians will follow the lea already given by British hæmatologists in course of time A tablc (II) showing the normal hæmoglobin leuch in different population in India with a few samples from other countries for comparison is given Apparatus required (1) Hellige normal hrmometer with extrı miting tubes and pipettes (11) Rack (ni) N/10 Hydrochloric ac! (11) Distilled water (1) Pipettes with rubber teats (vi) Hand leng Ilelltge normal hænomeler this cong ts of the following pirts A mxing tube with graduations from 10 to 170 ( pe figure 1 A) (1) A pipette with a mark at 20 c mm capacity (B) (1) A solid glass rod for stirnng the mixture in the tube to ensure thorough mixing (12) Coloured prism or posms against hich the blond has to be matched (C) (t) The housing of the hæmometer (D) vhich is made of steel vith a large bat to ensure safe standing Inside the housing are the coloured pri ms in th front and an opaque glass plate at the back Through a ho c in the housing the mixing tube is introduced and lies at the same distance from th eye as the prism Technique --Fill the graduated measuring tube up to mark 10 (using a large pipette) with N/10 hydrochloric acid After shaking the blood in the flask for 3 minutes with the pipette draw up the blood to the 20 c mm mark exactly wipe away any blood adhering to the outside of the pipette and transfer the blood into the measuring tube If the blood goes a little beyond the mark it is brought back to the mark by touching one s finger with the tip of the pipette a few times cotton wool or blottang paper hould never be used ·ENUMERATION OF ERYTHROCYTES AND LEUCOCYTES 9 It to by using the conversion table of which the skeleton is given (Table III) recommended that the full table be prepared and kept handy TABLE III Skeleton table of hæmoglobin values of the Hellige normal hæmometer converted into grammes of hemoglobin per 100 c cm of blood Per cent Heilige Gramines per 100 ccm Per cent Tieliige Grammes per 100 ccm 1 o 1375 10 I 375 2 0 2750 20 2 750 3 04125 30 4 125 4 5500 40 5 500 5 0 6875 50 6 875 6 o 8250 60 8 250 7 o 9625 70 9 625 8 I TOOO 80 11 000 9 I 7375 go 12 375 100 per cent Fellage = 13 75 grammes per 100 ccm 3 ENUMERATION OF ERYTHROCYTES AND LEUCOCYTES Erythrocyte and leucocy te counts are expressed as the number of cells per cubic millimetre (c mm ) of blood Principle-In enumerating the red or white cells oxalated venous blood from the flash (or capillary blood from the finger òr ear lobe) is taken into a red or white cell pipette up to a certain mark and the pipette is filled with the diluting fluid It is then shaken and a drop of the mixture is put into a special counting chamber where the cells within some specified ruled area are counted and finally the total number of cells per c m is calculated The dexterity necessary to carry out the various manipulations in the differ ent stages of making a blood count can only be acquired by practice Further 2 ·8 IfÆMATOLOGICAL TECIINIQUE By repeated filling and emptying the pipette should be completely freed from all vestiges of blood and the blood should be intimately mixed with the hydro chloric acid the red hæmoglobin now turns to acid hæmatın (brown) Wait for 15 to ho minutes the mixture which is now brown should be perfectly clear Now add water slowly drop by drop with a pipette mixing constantly with the solid glass rod until the mixture matches exactly the colour of the standard prism C in the housing During the process the solid rod should not be placed on the table as each time it is allowed to touch anything the small amount of mixture adhering to the rod will be lost and this source of error will be multiplied To match the solution and the standard the hemometer should be held up to 1 good source of indirect natural light direct sunlight and artificial light being av orded The level in the mixing tube opposite the lowest point of the meniscus of the diluted acid hæmatın is read this gives the percentage of hæmoglobin The end point is generally very sharp and there 15 seldom any difficulty in matching the solution and standards exactly except with leukæmic and jaundiced blood When a point is reached at which the solution appears to match the standards take a reading then add a drop of sater in the solution hold up to the light and take a second reading If at the second reading the solution is definitely too light the first reading should be taken On the other hand if the second reading still appears ICT-Hellig Dormal hemorheter to match the standards but after et another drop has been added the solution is definitely lighter than the standards the result should be the mean of the first two reidings The writers Napier and Das Gupta (1935) calculated that 100 per cent with the Hellige normal hæmometer represented 1367 grammes by the refractometer method but later by means of the san Slyke oxygen carrying method a slightly higher figure was obtained and they decided to consider 100 per cent as 13 75 grammes Other instruments of this pattern (Hellige normal hæmometer) that the writers have used have not differed appreciably from this standard but it is advisable to have the colour standard with the pipettes and mixing tubes to be used tested in some laboratory where the vin Slyke oxygen carrying method or the iron estimation method is also employed The reading in terms of percentage from the tube must be converted into this can be done conveniently grammes and the result expressed in grammes ·ENUNERATION OF ERYTHROCYTES AND LEL COCYTES 11 platforms Upon each ruled area there are dine large squares one square milli metre each separated by double lines Dz D 81 fir 2--Improved counting chamber Cover slip D A1 01 FIG 3-Section at x of figure 1 The four large comer squares A B C and D are the areas in which the leucory te count is made (figure 4) Each of these 4 square millimetres i subdivided into 16 squares to facilitate counting 8 N The se ning D C 1/8mm and oth Corc yos na FIC 4 - Vegbaper aliog ✓ The quare millimetre in the centre of the ruled area i used for the red cell count It is subdivided into 400 smaller squares each of which is therefore of a square millimetre I 400 ·IO NA YATOLOGICAL TECHNIQUE close attention must be paid to every detail of the technique of accurate results are to be obtained Apparatus required (1) Microscope (11) Hæmocytometer with counting chamber preferably with Neubauer ruling (171) Red and white cell pipettes (sv) Hæmocytometer coverslıp (u) Diluting funds (vi) Watch glass The ideal would be to use certified counting chambers and pipettes but where this is not possible on account of their high cost only tho e which are manufactured by reliable firms (viz Carl Zeiss Bausch and Lomb etc ) should be used and these should at least be checked against certified instruments Counting chambers - There are various types of counting chambers in use The new types of chamber have two main advantages over the old Thoma Chamber (I) The coverslıp can be placed in position upon its base before the blood is introduced and this may be done slowly and with the care that is essential to ensure the appearance and persistence of Neutons colour bands at the surface un contact (2) The uneven distribution of the corpuscles on the counting surface which is liable to arise in the original chamber devised by Thoma and is due to the rapidity with which the corpuscles especially the red ones tend to settle in the diluent is obviated the new chambers are so devised that the diluted blood that th rapidly enters the counting space by capillary attraction In the new types of chamber eg Neubauer Burker etc the counting space is oblong and divided into two compartments by a transverse groove both of which contain a ruled area for counting the red as well as the white corpu cles These two counting areas afford a means of making duplicate counts with a single application of the coverslip In our laboratory we have used chambers with Thoma Burker and Neubauer rulinys but for over eight years we have been using only chambers with the Neubauer ruling which 19 simpler to use than the Burker with a single application of the overslıp a larger area for the leucocyte count 4 square millinetres on each side of the chamber as opposed to 1 square millimetre in the Thoma type is obtained Counting chamber with improved Neubatier ruling The different parts of the counting chamber are shown in figures 2 and 3 The middle platform A A with the ruled areas BB are exactly or mm lower than the two side platforms The middle platforms are separated from each other by the transverse groove D and from the two side platforms by the trenches D and D When the coverslip is placed upon the platform there is a pace exactly or millimetre deep between it and the ruled areas on the middle ·ENUMERATION OF ERYTHROCYTES AND LEUCOCYTES 13 200 2 ccm Diluting fituds --For the red sell count we have found the following solution the most satisfactory Sodiam sulphate 12 5 grammes Glacial acetic acid 33 3 ccm Distilled water There is neither clumpin, nor hæmolysis with this fluid Other solution recommended are Hayem s fluid Mercuric chionde 05 gramm Sodian chlonde Io Sodium sulphate 50 gramines Distilled water 200 ccm and imple physiological salt solution We have not found either of these saty factory as clumping may occur with the former and hæmolysis with the latter solution For the white cell count we have found the following solution the best Clacial acetic acid Mercuric chloride Oi gramme Anuline gentian violet one drop Water to 100 cm The mercuric chloride will present growth of moulds when the solution I kept for a long time and the gentian violet will give a slight tinge to the leucocy tes and also make it easy to distinguish this solution from the red cell diluting fluid When the diluting fluids are used a small quantity should be put into a watch glass or other suitable receptacle into which the pipettes charged with blood should be plunged In no case should the pipettes be put into the original phials contain ing the diluting fluids as there is every possibility that in course of time cells will find their way into this fluid and accumulate at the bottom of the phial these may be taken up subsequently with the diluting fluid and vitiate the count Enumeration of Erythrocytes (1) Filling the pipette-Before u ing a pipette see that it is absolutely dry and clean and that the point is intact (the tips are easily damaged) With a dry red cell pipette suck blood up to the mark 0, (or 10 in the case of anamic patients) by holding the pipette almost horizontally and at right angles to the line of vision so that the exact height of the column of blood can be seen easily The blood should not go much beyond the mark but if it does go a little beyond it is brought back to the mark by applying the tip of the pipette to the top of the finger a few times cotton wool or blotting paper should never be used as they (will draw out the serum only and the cells in the pipette will become more con centrated The blood adhering to the outside of the pipette is wiped off and the Pipette us plunged into the red cell diluting fluid in a watch glass The diluting fluid is drawn up exactly to the mark 101 the pipette being held nearly vertical and gently rotated between the thumb and the forefinger The rubber tube is taken off and a tightly fitting flat rubber band is put round the pipette klosing both the ends to prevent any leakage It is now land flat on the table or in the box ·12 HAMATOLOGICAL TECHNIQUE To facılıtate counting these 400 small squares are cut up into 16 groups of 16 small squares each by extra lines which are drawn through every fifth square Red cells in the five small groups of squares one from each corner E F C and H and one from the centre J that is to say the contents of 80 small squares in all are counted is Counting chamber with Thoma ruling The disposition of the different part of the Thom1 counting chamber exact ly similar to that of the Neubauer count ing chamber with this difference however that there is only one big square on each of the Tuled areas B B in place of the nine big squares in the Neubauer type The area of each of the big squares is one square millimetre and each is subdivided into 400 small squares each of which therefore 1 of square millimetre To 400 facilitate counting these 400 small squares are cut up into 16 groups of 16 small squares each by extra lines drawn through every fifth square Thi square millimetre is thus an exact replica of the central square millimetre in the Neubauer ruling The covershp-Only the covership which is specially designed for blood-counting chambers must be used If any other coverslıp is used there may not be uniform depth in the counting chamber and the count will be inaccurate TIG 5 -Thoma ruling as seen with to third lens The blach circle hows the field seen with the one sixth icns is There 15 ES The blood counting pipettes (Thoma type) The capillary portion of both the red and white cell pipettes (figure 5) is divided into ten equal parts from the tip to the bottom of the bulb the fifth and the tenth marks being denoted by figures 05 and I respectively another mark just above the bulb where the figure 101 is given on the red cell pipette and the figure it on the white cell pipette To facilitate mixing there is a bead in the bulb which is ometimes coloured red in the red cell pipette to distinguish it from the white cell Pipette Leucocy te Red cells FIG 6-Counting pipettes ·ENUMERATION OF ERYTHROCYTES AND LEUCOCYTES 15 objective) may then be substituted by simply rotating the nose piece of the micro scope and readjusting the condenser and the diaphragm Counting the cells Focus the ruled area with the low power and also find out of the cells are evenly distributed—if not another preparation should be made The cells in the squares can now be counted with the high power one sixth objective The area from which the cells are to be counted will vary with the different types of ruling but the same rule for cells on boundary lines should hold good in all cases Rules for cells on boundary lines All the corpuscles touching the upper line and the left hand line of a square Are considered to be soside the square while those touching the lower line and the right hand line of a square are considered to be outside the square (figure 7) The cells contained on the five groups of small squares I F G H and J in the mddle of the ruled area of the Neubauer ruling (figure 4) are counted In all cases the cells on both sides FIG 7-One field with one sixth of the chamber should be counted and lens showing which cells should be counted and which neglected the mean of the two taken Should there be a difference of more than 5 per cent in the two counts the chamber should be recharged and the count done again Calculations for erythrocyte count Each small square -ado square millimetre Therefore the cubic dimensions each= 400 X01 (pace between the cover lip and the ruled area) = cubic millimetre And each group of 16 small squares = 16 4000250 zšo cubic millimetre Therefore cells from five groups of 16 squares each= 5 cubic 3050 millinetre of diluted blood Thus the total number of cells counted from five groups of small square multiplied by 50 will give the number of cells in Ic mm of diluted blood This wben multiplied by the diluting factor 200 (or 100) Will give the number of cells in one cubic millimetre of blood Crumpi - Blood i takes up to og mark Total number of cells counted in five groups of 16 small squares = 450 Therefore 450 x 50 = 22 500 cells per e mm of diluted blood and 22 500 x 200 - 4 500 oo cells per crom of un lulut d blood ·14 HLVATOLOGICAL TECHNIQUE When the pipette is filled it will be seen that the entire capillary end of the pipette from the tip to mark I is occupied by diluting fluid Hence the true dilution of cells is not 05 (or I) in 1or but 05 (or I) in rod ie 111 200 (or i in 100) Thus by taking blood up to the different marks from o I to n in the pipette dilutions ranging from I in 1 000 to 1 in 100 can be made Should there be any air bubbles at any stage during the filling of the tube with the blood or the diluting fluid the whole preparation must be rejected and another pipette filled up for it is never possible to get all the air out without loss of some fluid (11) Shaking the pipette-On every occasion prior to charging the counting chamber with the diluted blood the pipette should be shaken preferably in a shaking machine or by rotating the tube held horizontally between the palms of the hands first with the right hand on top and then the left for about 2 minutes The pipette should never be shaken in a horizontal direction 10 in the direction of its long axis as this tends to throw the cells into the capillary tube (111) Charging the counting chamber with Neubauer ruling -The counting chamber and the coverslıp must be perfectly clean and free from dust or threads of cotton Put the coverslip on the counting chamber so that it covers the ruled areas B and B (figure 2) and with the finger nail tap the coverslip to ensure gond contact Discard the first few drops of the dlıuted blood by blowing into the pipette Next holding the pipette in an inclined position of about 45 degrees apply a small drop of diluted blood to that part of the platform AA of the counting chamber which just projects from the coverslıp the drop will instantly pass under the coverslıp by capillary attraction The drop of blood should not be too large or it will overflow the chamber and pass into the overflow trenches D D and may even pass to the transverse trench D Neither should the drop be too small as in that case it will not completely fill the counting surface With a little practice a drop of the requisite volume can be put under the coverslıp any little excess on the platform should be removed by sucking with the pipette and never with blotting paper Small air bubbles are liable to be introduced into the counting chamber together trith the diluted blood – () if the counting surface and the coverslip are not previously cleaned with the requisite care or (11) of the point of the pipette 15 chipped If there be any overflow into the trenches or if there are any air bubbles remaining the chamber must be cleaned and charged again Place the counting chamber on the microscope stage which should be on a horizontal plane for about 2 to 3 minutes to allow the cells to settle properly minutos Focussing the ruled area Great care is necessary in locating the ruled area without injuring the objective or disturbing the distribution of cells This is most conveniently done by locating the ruled area first with the low power (two thirds objective and 10x eyepiece)—lowering the condenser and reducing the light by means of the arts diaphragm will show up the rulings distinctly) the higher power (one yixth ·TABLE V Shouing the normal red cell counts of different populations N Verred Age Eco C status cel per Local ty Number Standırd deviat on Authonty C31 11 mllions 5110 19-30 Males Bombay IZI Students etc $ 30 $ 633 25 45 Calcutta Mixed 50 5 362 20-45 30 5 533 to 490 19-30 Assam Coolies 24 5 353 to 620 19 30 O 3 270 +0,10 Cachar 25 5 057 #563 Adults USA 5 400 Britain 5 690 Sokhey et ? 1937 Nap r and Das Cupta 1935a 1936 1935) 1936 Vap er and Majumdar 1938 Castle and Minot 1936 Whitby and Britton 1939 Price JOIS 1931 Sokhey et al 1938 Naf et Edvards and Das Goptı 2941 Benjamın 1939 Napier and Majumdar 1938 Nap er and Blicona 1937 Whitby and Britton 1939 ENUMERATION OF ERYTHROCYTES AND LEUCOCITES 5 428 remales non pregnant Bombay 16e class 101 # 470 to 330 16 30 T4 38 Calcutta 125 +(15 to fog 17 30 Delhi V: 1 lle class 101 4560 o 250 Cachar Cool es 25 4 454 to 705 Assam 20 4 550 to 650 Bntain 4 800 5 012 Price Jones 1931 18 22 M chigan Students 50 4 750 Bethel 1936 Females pregnant A sin Coolies 4 650 ofzo Laper and Bilimona (1937) Bethel (1936) Vich gan 28 * 120 ·16 11ÆMATOLOGICAL TECIINIQUE When as in the above case blood is taken to the og mark and the dilution IS I In 200 the calculation can be simplified by adding four os to the total number of cells counted in the five small squares es in this instance 450 x 10 000= 4 500 000 For high counts as in polycythæmia I in 200 dilution is not enough as the cells are numerous and would be too crowded to allow an accurate count to be made so the blood is drawn up to the 0 2 mark and the diluting fluid as before up to the fol mark this produces a dilu tion of 1 in 500 and the total number of cells in the five squares must be multiplied by 25 000 instead of 10 000 as in the example above Conversely in an anæmic blood it is advisable to take blood up to the 1 o mark the dilution will be 1 in 100 and the total number of cells will be multiplied by 5 000 A table (IV) which will facilitate calculation when blood is taken up to the different marks of the red cell pipette and cells from the five groups of small squares of the Neubauer ruings are counted is given below TABLE IV Blood talen Dilution Multiplication factor to arrive at number of cells per cmim Oi mark I 1 000 X 50 000 O 2 1 500 x 25 000 04 1 250 x 12 500 05 I 200 X 10 000 1o Іоо X 5 000 i e Ilith Thoma Ruling Calculation for Erythrocytes Count the cells contained in the five groups of small squares a b c d and e (fig 5) on both sides of the counting chamber and take the mean of the two count As the area of each of the small squares is the same as in the Neubauer ruling 1 400 square millimetre the calculations are exactly the same as described for Neubauer ruling Normal standards The number of red cells per c mm is usually given in textbooks as 5 000 000 This figure is too low for men and too high for women The mean of a number of counts in different populations is given in table VI It will be seen that there is a striking uniformity in the counts in different populations compared for example with the hæmoglobin estimations in the same range of populations ·ENUMERATION OF ERYTHROCYTES AND LEUCOCYTES 19 practically impo sible In such a case blood is taken up to the OI or o 2 mark of a white cell pipette (or up to the 05 Or 10 mark of a red cell pipette) and 15 filled up with white cell diluting fluid The cells are counted from the four hig quares as in the ordinary leucocyte count and the number of cells per cubic millimetre is calculated A table (VI) for facilitating calculation when blood is taken up to the different marks of the pipette and the cells in four areas of 1 square millimetre each are counted is gren below TABI E VI Blad taken Vultiplication factor to Dilution 1 arrive at nuniber of cells per c mm 05 in red cell prette T 200 x 500 10 t 100 x 250 1 hit 1 100 x 250 02 1 50 X 125 04 1 25 1 5 1 20 X SO -- TO 1 TO x 25 WITH THOYA RULING CALCULATIONS FOR LEUCOCYTES tes in the whole of the cross ruled aren are counted at least s should be made and the average count taken square millimetre represents a cubic capacity of oi cubic hen multiplied by 10 gives the number of cells per cubic ced blood and when again multiplied by the diluting factor number of cells per cubic millimetre of whole blood e number oí cells multiplied by 100 (when the diluton is I 10) or on the dilution is I 20) gives the number of cells per cubic millimetre Aue blood Counting by field method for low counts This method is suitable in kala azar or other diseases in which there is usually a leucopænia Take blood up to og mark of the white cell pipette and dilute it I in 20 with 5x es epiece and one sixth objective focus the square in the centre of the field of the counting chamber with the Thoma tuling or the centre hlock of thc Neubauer and draw out the tube until the diameter of the field measures ·18 RÆYATOLOGICAL TECHNIQUE Enumeration of leucocy tes For enumeration of the leucocytes follow the technique given under enu meration of erythrocytes taking all the same precautions in the different steps With a dry white cell pipette suck blood up to the mark 05 (or I in cases of leucopænia) wipe off the blood adhering to the outside of the pipette plunge it into the white cell diluting fluid and draw the mixture up to the mark ii tale off the tube and put a tightly fitting flat rubber band round to close both the ends, and lay it flat on the table Here too the capillary end up to the i mark of the pipette is occupied entirely by the diluting fluid Hence the true dilution of the cells i a5 (or I) in 10 10 I in 20 (or I in 10) By taking blood up to the different marks from 0 1 to 1 in the Pipette dulu tions ranging from I in 100 to 1 in 10 can be made Counting the cells and calculation to find out the cells per c mm WITH NEUBAUER RULING Count the cells in the four corner areas A B C and D (fig 4) Divide by 4 to get the average number of cells in each area We know the area is I square millimetre and it is on millimetre deep Therefore the cubic capacity of each area 1 Of cubic millimetre (IXOT) Thus the cells counted are from or cubic millimetre of diluted blood This when multiplied by 10 gives the number of cells per cubic millimetre of diluted blood Again this figure multiplied by the diluting factor 20 (or 10) gives the number of cells per cubic millimetre of whole blood Example -B ood was taken up to og mark Number of cells counted from the four squares was 1o Therefore the average number of cells in 1 square ie o 1 cubic millimetre of diluted blood 18 30 Therefore 300 (30 x 10) is the number of cells in 1 cmm of diluted bood and 6 000 (300 x 20) is the number of cells per c mm of blood The number of cells can be calculated rapidly by multiplying the total 30 XI 50 20 of 25 when the dilution is I in to Thus in the above example 120 is the number of cells from the four squares and the dilution is I in 20 Therefore 120 x 50=6 000 is the number of cells per c mm of blood For high counts as on leukæmia In cases of leukæma greater dilution of the blood is necessary for a correct count of th cells as otherwise the cells are so crowded that an accurate count i ·ENUMERATION OF ERYTHROCITES AVD LEUCOCYTFS 19 practically impossible In such a case blood us taken up to the oi or oz mark of a white cell pipette (or up to the 5 or 10 mark of a red cell pipefte) and is filled up with white cell diluting fluid The cells are counted from the four hig squares as in the ordinary leucocite count and the number of cells per cubic millimetre is calcuhted A table (VI) for facilitating calculation when blond is taken up to the different marks of the pipette and the cells in four areas of 1 square millimetre cach are counted is given below - TABLE VI Binot taken Dilation Multipl cation factor to amive at number of cells pet c mm - 5 in the tell pipette 1 200 x 500 fo T 100 x 250 white 10) 250 1 50 125 04 25 623 5 t 20 SO IC 3 10 WITH TjomA RULING CAICELATIONS FOP LEUCOCITES ocytes in the whole of the cross ruled area are counted at leat unts should be made and the average count taken of 1 square millimetre represents a cubic capacity of or cubic when multiplied by 10 gives the number of cell per cubic duted blood and when again multiplied by the diluting factor < number of cells per cubic millimetre of whole blood the number of cells multiplied by 100 (when the dilution 18 1 10) or hen the dilution 13 I 20) gries the number of cells pas cubic millim tre d blood LIE Counting by field method for lou counts This method i suitable in kala azar or rther disea es in which there ! u ually a leucopænia Take blood op to og mark of the white cell pipette and dilute it I in 20 with 5x eyepiece and one sixth objectise focus the square in the centre of the field of the countiog chamber with the Thoma ruling or the centre block of the Neubauer and draw out the tube until the diameter of the field measures 3 ·20 HEMATOLOGICAL TECHNIQUE eight times the length of the side of a small squares The area of the whole field is now equal to the area of 50 small squares almost exactly ( 7+2=* *16=50 2857) Count the number of cells in 40 such different fields Calculation - The volume of each square is 1/4 000 cubic millimetre and each field contains 50 squares Thus if the total number of cells in 40 fields is 70, the number of cells per cubic millimetre of blood=4000 x 20 50 x 40 * 70=2 800 The same result may be obtained by multiplying the total number of cells in 40 fields by 40 if the dilution is I 20 or by 20 if the dilution is I in io With this method cells of a very large area are counted and the multiplving factor is only 40 or 20 and therefore greater accuracy is ensured Enumeration of the nucleated cells of the marrow The enumeration of the nucleated cells of the marrow from the material obtained by sternal biopsy is carried out in the same way as that of the leucocy le in cases of leukæmia Care of instruments After use the pipettes coverslip and the hæmocytometer chamber mu t be cleaned thoroughly 2 Cleaning the pipettes Thoroughly wash out all the diluted blood with water Remove water with absolute alcohol 3 Remove alcohol with ether 4 Pass air through to dry the pipette so that the bead rolls freely in the bulb It is very laborious process to clean the pipettes by sucking air through them and one cannot dry them properly by blowing as expired air is laden with mors ture The pipettes can be cleaned easily and dried by attaching them to a suction pump (figure 8) or when this is not avail able by attaching them to a syringe If the cells clog the tip or any part of the capillary tube loosen them by inserting FIG 8-Suction apparatus for a stuff horse hair cleaning pipettes If there is albuminous matter in the bullfill it up either with saturated solution of NaOH or potassium bichromate cleaning solution keep overnight in the 37 C incubator and clean next morning ·ENUMERATION OF RETICULOCYTES 21 Cleaning rounting chamber and coierslıp Wash the ruled side of the countin, chamber and the coverslip i rummo watet Thorou, hlv dry fint with a clean cotton handherchief and finally with a sık hindkerchief selyt cloth ou lens paper but avoid nulling the ruled zrca of the counting chamber From time to time the countin, chamber ind the corerslıp should be wind with alcohol or ncetont to remove iny klerse and then dried with the silk hand kerchief but on no account should the countin, Chamber be immersed or freely cleaned with alcohol If the rulings becoine fait after lon, use the lines may be made prommen hoy lightly rubbin, with a silh cloth on htch graphite ilead from a penal) hus liecul rubbed TABLE VII Leucocyte counts on normal individuals T Vumb Sex an! 1 es le 10 ) Total luoyte count Authority 0 1 lult Caleutta 18 Fetunle alults Lutta 16 +16 Napier en 1 Das Corta (unpubl led) Map r Elnardy and 149 Cupta 111 Olod et al 1137 199 300 (1 400 1 100) 19-39 veur 1 UBA Female 19-36 ) ar 14.0 (+100 to 11 00) 0.vl et al 19 4 ENUMERATION OF RETICULOCYTES Reticulocytes or retkulattd erythrocytes are youn, red cells thesipi sent the stime just after the culls have lost theit nuclei and before they are fully mature The reticulocyte count is expressed as a percentage of the rel cells their percentage incadence les information is to the netivity of the red blood cell formation at the moment The reticulation may appear as a net wol over the whole cell 25 a colledium tion of disciete rods or 15 granules These are not shown by ordin blood Romanowsky stains and a special suprasital stuinmg method is necessary, out 2 demonstrate the reticulation ·22 NIEMATOLOGICAL TECHNIQUE Staining methods - nuous dyes have been tried to stumn, reticulocytes but brilliant cresyl bluc has been found to be the best ind it is now used umersilly Althou.h brilliant crewvl blue is the dye of choice 1 number of differet methods of using it have been suomesteri Methods for reticulocytu staining Bury be classified in the following groups in all of which scrupulously clearl and polished slıdles and coverslips without sei itches should be usedlo A Vethods in luch 1 thin film of the dye is dried on the slide or core, slip and the blood added later B Methods in which the blood is mixed with a solution of the lye With either method (1) 1 dried sine ir ot (11) i wet coverslip preparation may be made Ipparatus ri juuel [1 r method choic L (11)] (1) I olished lide (i) (len coverslıp (preferably -*10 rum) (1) Cresvl bluu solution 1 il cent in 08) jer cunt solum chloride (W) Chpull try i'l tte u 1 ultur teit (10) Vaulino or rarthn (it) Gas or spirit Imp (int) Ehlt hscy plece (or oue nu as of buitin, thc in 10 copa fiull) (1x) Vacro Cose i klutional requirements for other methols (1) Surgical priching needle or blood gun (11) Al ohol ether an cotton vol (* ) Cresy) blue solution ) per cept Jolute ale uhol (12) 12e mouthci glus bottle (t) Spreimen tubu 1 (s) and (1) (11) Filter payer (11) Luhmany stain (t) und B (0) (2310) Di tilled water Mcthod A To prepare slices or (or aslips proceed is follows Mahe a 1 per cent solation of brilliant cr7 blue m solute alcohol al 1 cep it in a well stoppered bottle The slide may be prepared by one of the following methods - (a) Take a polished slide heat it a little over a fine and put it on a fut even surface With a capillars pipette tikt some brilliant cresyWuc * Slus_Ilese shoall be clearyd anl , lal in tl e follo ing way — (6) Only ner ships si oull be usei I ut the de in a glas jur conta ning pirit Keep them soaked in spirit for orer -í hours (11) 11th a pur of forceps take out one li le at a tine all the spirit to drain (fr the file and then flame it ovei prit lamı or gas burder (10) Polish well one ide of the slide x th jes eller rouge using a soft cloth Much the polt hel side with a glas pencil an 1 ke p it in a dust proof slide box Corerslips -heep the corerslijs 1 a wide mouthed glass jar soahol a pint Just bullre u e with pur of fine i topp the out one covers y at a tim lion thr fpirit to clear off flame them orer a spirit lamt or Iarner clein uith & roft cloth and kecp I covercu in a small Petri dish until ther dre wanteu ·ENUMERATION OF RETICULOCYTES 23 solution from the stoppered phral and put a drop on the middle of the slide If the slide is clean and it is on an even surface the stain will spread concentrically and will be distributed in a uniform manner The stain will dry in a minute or so the slide is then ready for use (b) Put some stain in a wide mouthed glass bottle Warm a polished slide by passing it through a Bunsen fiame and holding it in a pair of forceps dip it vertically into the jar of the dye up to three quarter of its length Take it out allow the excess of stain to drain back into the bottle and before it is dry put it in an upright position inside a specimen tube at the bottom of which a piece of blotting paper has already been placed Put in the cork and allow it to stay overnight take it out and wipe the stain from the unpolished de of the slide with a moist cloth it then ready for use Sudes prepared in either of these says may be kept for a long time in a dust proof slide box and used when required () To prepare a dried smear only prepared by the second methods are suitable Put a drop of blood oxalated or from the finger on one end of a prepared slide and draw a very thin smear across the stained surface of the slide in the usual way-the use of a hæmocytometer coverslıp as a spreader facilities the drawing of a thin smear Slides prepared in this way may be counterstained with any of the Romanowsky stains in the usual way (11) To make wet coverslip preparations slides prepared in either of the above methods may be used Put a drop of blood oxalated or from the finger on the stained surface of prepared slide gently place a coverslip on the drop of blood avoiding air bubble as far as possible If the drop of blood is of the proper size it will spread uniformis under the pressure of the coverslıp but in case it does not do so apply very gentle pressure with the top of the finger so that the blood spreads evenly but on no account should the coverslıp be pressed hard as some of the ceils may be ruptured Ring the coverslıp round with hard paraffin by dipping a match in melted paraffin and stroking it on to the slide at the junction of the slide and coverslıp Method B In this method small amount of blood 15 mixed with a quantity of die in some definite proportion Method of Osgood and Wilhelm (1934) Put o 5 c cm of 1 per cent brillant cresyl blue solution in o 85 per cent sodium chloride into a small test tube on a rack Add an equal quantity of oxalated blood from a well haken fiask Mix well and allow the mixture to stand for about 2 minutes Rotate the tube between the hand to get an eten mixture ·24 HÆMATOLOGICAL TECHNIQUE } Withdraw a little of the mixture with a capillary Pipette and put a drop at one end of a polished slide Make a thin smear and when it is quite dry counterstain with Leishman s stain in the usual way The slide is now ready for examination for reticulocy tes A differential count of the leucocy tes can also be done at the same time (11) Modification of the above method This is the method of choice Prepare a mixture of cresyl blue and oxalated blood as in the above method With a capillary pipette take out a little mixture and påt a small drop at about the centre of the polished side of a clean slide Apply a clean coverslip on the drop of the mixture when under the pressure of the coverslip the mixture will spread evenly but in case it does not do so very gentle pressure with the tip of the finger may be applied over the coverslıp The preparation is then sealed with vaseline or paraffin and is ready for examination for reticulocytes only one field Counting the reticulocytes Slides prepared by any of the methods described above are first examined Tapidly with a low power (two thirds objective) and a portion of the slide 1 selected where the cells appear discrete and there are not too many in This portion of the slide is now examined with the one twelfth oil immersion lens The total number of red cells in the field and also the number showing reticulation are counted The use of an Ehrlich s eyepiece or a piece of metal with a square hole in it placed inside the eyepiece to narrow down the field greatly facilities the counting Altogether one thousand red cells are counted in a more or less normal case but if the count is high 5 per cent or more -500 red cells are sufficient the number of cells showing reticulation is noted and from this the number of reticulocytes per 100 red cells is calculated A Veeder counter or some similar device is useful here one counts the red cells and each time a reticulocyte is encountered clichs the Veeder When a thousand red cells have been counted the number on the Veeder is read In the absence of an automatic counter a pencil and paper must be u ed for it is 1 mistake to try to keep two sets of figures in one s head Comment Osgood and others have shown that divergent results are obtained with the different methods in use They obtained the best results with the method described by them which has the additional advantage that a differential count of the leuco cytes can also be done in the same preparation We have however found that the modified Osgood method which is the routine procedure in our laboratory has given even better results in our hands than the original method With the modified method all the reticulocytes stain very well and there is no clumping nor overlapping of the cells thus the counting is very much facilitated With the original method a number of cells appear broken more so in the cast of næmic blood and the cells ·ENU FRATIOY OF RETICULOCYTES 25 are less discrete The percentage of reticulocytes appears to be slightly lower in the original than in the modified method In a small number of cases where the counts were done by both methods 5 16 per cent reticulocytes were found by the original method against 598 by the modified method On account of the disergent results obtained by the different methods in use for comparative studies a uniform technique should be adhered to in the enumera tion of the reticulocytes not only by ont group of workers but by all so that the results of different workers will be comparable The modified Osgood method described above is simple and as it appears to be superior to the other method, in many ways we recommend it as the method of choict for the enumeration of reticulocytes Normal values It is unnecessary to quote the figures of numerous obsen ers regarding the no mal percentage of reticulocytes When in a healthy individual whose red cells are at the normal physiological level the hemopoietic and bæmolytic tissues are functioning normally the number of reticulated red cells reaching the peripheral blood is less than per cent of the total red cells The normal range is usually given as 0 1 to 10 per cent The present writers found a mean percentage of 067 037 in a series of 50 city dwelling Indian males with a range from 2 to 24 and o 37 + 027 amongst 122 women On the other hand for Assam tea garden coolies the mean was 2 17 3 192 and the range 0 1 to 10 8 per cent It is probably true to say that reticulocyte counts un man of over 10 per cent are evidence of some unu ual stimulation to the hæmopoietic system though they may be encountered in an apparently healthy individual In our Asam cooli eries we accepted these findins of unusually high reticulocyte percentages & dciated as they were with a low hæmoglobin percentage as evidence of some abnormality of the erythorn in the individuals concerned Significance of reticulocytes Whenever the hemopoietic system meets an extraordinary demand for red blood cells the reticulocyte percentage will rise but the reticulocyte ne will not be maintained even thu h it will take some time for the deficiency of the red cells to be made good For example of a healthy individual loses a large quantity of blood there i an immediate steady rise in the reticulocyte curge which reaches its-maximum and falls to nomal again within a few days whereas it will probably be a few week Ixefore the lor's of blood cells is made up Similarly in deficiency anæmias when the deficient substances eg hæmo posetin in pernicious animia and iron in iron deficiency anemia are supplied the firopoietic tissues in the bone marrow are nos enabled to meet the demand for 4 ·26 HÆMATOLOGICAL TFCHNIQUE red cells and there is a rise in the reticulocyte percentage that starts within two or three days and reaches its maximum within five to ten days after which it falls as fast as it rose This reticulocyte rise gives a valuable indication that a cleficiency has been made good A third example is in toxic aplastic anæmia where a toxın is depre sing the function of the bone marrow In this case there may be no reticulocytes at all to be found in the peripheral blood but once the toxin is neutralized or otherwise ceases to exert its toxic effect the hemopoietic tissues will start to function properly Teticulocytes will appear in the blood their percentage will rise to a maximum within a few days and will fall again to the normal level but in the absence of any further intoxication the red cells will continue to increase until they reach their nomal level It should be emphasised that whilst a reticulocy te crisis as this sharp rise in called is evidence that the hæmopoietic tissues are functioning effectively a fall of reticulocytes to the normal level does not indicate any cessasion of this functioning but on the contrary it usually indicates that red blood cell formation is being carried on in an orderly manner In true hæmolytic anæmias there is a constant reticulocytosis as not only will the anoxemia stimulate erythrogenesis but there is a second source of stimulation in the debris of abnormal hæmolysis which has to be disposed of by the reticuloendothela! tissues A constant reticulocy te count of 5 to 10 per cent or higher is evidence of excessite hæmolysis and in a hæmolytic anaemia the return of the reticulocyte count to normal in usually evidence that the excessive blood des truction has ceased I N Roll Hecyf Art Treatment #Court M Ane Men Reticulocyte crises The extent of the reticulocyte response i governed mainly by the original les el of the red cells in the anæmic state For example in a case of pernicious anæmia the rise in the reticulocyte percentage after the same adequate dose of liver extract may be as high 15 55 per TIG 9-Riddles chart for calcu cent or practically negligible according to whe culating the maximum reticulo cyte response expected in perti: ther the red cell count was 500 000 per c mm d ຂຽa Yith dequate or 3 000 000 per cmm before treatment therapy Doubling the dose of lner extract would make no difference to the height of the reticulocy te response but reducing it to an inadequate dose would Therefore in pernicious anæma if one has a chart shoping the expected reticulocyte response one can CIOUS ·LNUVERATION Or THROMBOCYTES 27 judge whether or not the dose of livet extract given was an adequrte dose have given a chart here not so much for practical use as pernicious anemia is rare in India but to demonstrate this point In iron deficiency anæmia the response to adequatc iron therapy is also pro portionate to the degree of the anæmia but in this case the reticulocytc count does not respond with the same mathematical precision 5 ENUMERATION OF THROMBOCYTES Thrombocytes or blood platelets arc colourless or slightly bluish bodies spherical ovoid or pear shaped and are usually about one third of the diameter of a red blood cell but they may be larger especially in pathological conditions No method of enumeration of blood platelets is entirely satisfactory as plate Jets tend to form clumps and to stick to any foreign surface with which they may come into contact However with careful technique counts sufficiently accurate for practical purposes are not difficult to obtain The principal point to be re membered in the platelet count is that manipulation of the sample of blood should be avoided and if possible the blood should be taken directly on to the counting slide The platelet count is expressed as a number per cubic millimetre of blood There are several methods for estimating the number of platelets these fall under the two main heads the direct and the indirect methods 1ppar 1 is requ ed Efor method of clice B (0)] As in the reticulocyte couat Additonal rt įus ements for other n ethods Fox tet) od 1 (7--Red blool cell pipette (11) Sodium citrat 2 per cent solution (fresh and stenl zed) (1) Count ng chamber with Neubauer ruling an coverslip for method B-Magnesium sulphate 14 per cent solution A Direct method _In this method the number of platelets per cubic miltu metre is calculated without any reference to the red blood cell count Prick the finger with a sharp surgical needle and with a red cell pipette draw blood up to the 05 mark and dilute it with fresh sterile 2 per cent solution of sodium citrate up to the tor mark After gentle shaking put a drop in the counting chamber wait 3 to 4 minutes to allow the platelets to settle down focus the light carefully and count with the high power all the platelets in I square millimetre area The number of platelets per cubic millimetre of blood is then calculated in the usual way ·28 HÆMATOLOGICAL TECHNIQUE the many L Indirect method --As in the enumeration of the reticulocy tes the plate Iets are at first expressed as a percentage of the red cells calculated from the number of platelets observed Reticulocytes Platelets in counting one thousand red cells The number of platelcts per cubic millimetre can then be calculated from the total red cell count which must be done at the same time Of indirect methods advocated the following two methods have given consistent ly good result in our hands - Clean the tip of the finger first with soap and water and then with alcohol and ether and apply a thin layer of sterr lized vaseline over it Prick the finger with a blood gun or sharp pricking needle through As I GIO --Showing sıx reticulocytes Seven mature the thin layer of vaseline erythrocytes and nine platelets the blood comes out touch the uppermost portion of the blood drop with the centre of a 22 x 40 mm coverslıp Place the coverslip on a dne 1 cresyl blue stained slide prepared by method A (a) or (b) as described for the reticulocy te count With very gentle pressure try to obtain as uniformly spread a film as possible and finally seal the sides of the coverglass with vaseline or paraffin (11) Clean the tip of the finger as above Balance a drop of 14 per cent solution of magnesium sulphate on the back of the finger just above and to one side of the nail groove Mahe a small puncture through the drop The drop of blood should be such that the resultant blood mixture is about I in 20 Max rapidly with a paraffined* capillary pipette Transfer the blood mixture to clean slides make very thin smears and stain with Wr ghts or Leishman s stain The coverslip preparation ) or the slide (11) is examined with an oil immersion Jens A small rectangular field is more convenient for counting than a big circular one ( us) Note the number of platelets seen in counting 1 000 red cells Successive fields from the different areas of the smear should be examined so as to eliminate as far as possible the error from uneven distribution The red cell count is done as a separate procedure in the usuul way Latelets do not adhere to a paraffined surface so that if glass is covered with a thin layer of paraffin the platelets do not tend to adhere to it The pipette is prepared by drawing hot melted paraffin wax through it a thin layer of paraffin all acīhere to the glass when it cools ·DETERMINATION OF CORPUSCULAR VOLUE I 00 By a simple calculation the total number of platelets per com may now be ascertained Thus if the total red cell count 15 5 000 000 per c mm and if be the number of platrlets counted against 1 000 red cells then **5 com doo or **5000 is the total number of plateless per c mm Fra ple --Total red blood cells = 4 500 COO per c mm Number of platelets counted against 1000 red cells = 50 Therefore platelets per emm = 50% 4 500 DOO or 5)*4 500=2 5 000 1 000 The cnumeration of the platelets by the indirect method though more laborious ind time absorbing as a total red cell count has also to be made is nevertheless much superior to the direct method Results obtained by indirect methods especially by method ) are always higher than those of the direct method as thero is very little contact with forei n surfices Normal Lalues The figures gren by different writers sary considerably This variation is undoubtedly due to some extent to the differences in the methods adopted for counting them but there are considerable variations in th platelet counts donc at diffcrent times by the same method in the same individual Whitby and Britton (1939) in Creat Britain gise the range as 250 000 to 500 000 per cubic millimetre and Gram (1920) in America as 280 000 to 540 000 with the aserage at 350 000 The present writers found 36 000 + 248 000 in Calcutta and 423 000 + 343 000 in Assam in the latter series there were a number of so called normal individuals with platelet counts of over a million and a half 6 DETERMINATION OF CORPUSCULAR VOLUME Principle --A volume of uncoagulated blood is centrifuged at a high speed in 1 graduated tube until there is no further packin, of the corpu cles The level of racked red cells and of the serum are read off directly from the tube from this the proportion of the packed cells to the whole blood is calculated and cxpressed as a percentage Apparatus required () Graduated cell volumc tubes fitted wth rubber cerks (Cap Mary Pipettes th rubber teats (1) 11 gh speed centnfuge machine that will rotate at at leat 3 000 revolutions per minute (10) A bulance to ensur that the x ight of the totes and their contents are about equal (u) Iland lens ·30 I LVATOLOGICAL TECHNIQUE Cell volume tubes -Various types of tube are in use these vary in length bore and capacity Wintrobe uses small tubes II Centimetres long with 2 bore of 25 millimetres which are marked in millimetres and centimetres from o to to In our laboratory we use a tube 75 centimetres long with a bore of 7 millimetres and gradu ited from o to 110 These tubes were specially made for us by Mes rs Baird and Tatlock London to fit in our high speed centrifuge machine but comparatively short graduated tubes of this kind are now easily obtainable from instrument makers As the degree of packing of the cells depends to a certain extent on the calibre of the tube it is desirable that the same sct of tubes which were used in working out the normal standards should be u ed in all subsequent work Method - The blood is taken in the usual way into a 5 c cm or 3 c cm flask containing oxalate (see P 2) With a capillary pipette withdraw oxalated binod from the flask after it has been well shaken fill up the graduated cell volume tubo from the bottom upwards exactly up to the 100 mark put in a small rubber cork to prevent any evaporation dunng centrifugalızaion and place it in the centrifuge bucket If possible two specimens should be placed in the centrifuge at the simo time In any case the centrifuge buchet containing the graduated tube must be balanced against a similar bucket and tube so that their weights are exactly equal before these two buckets are placed in opposite sides of the centrifuge this elementary principle must be observed rigidly Loth for the sake of the centrifuge and for satisfactory packing Centrifuge at a high speed—2 500 to 3 000 revolu tions per minute—until there is no further packing of the red cells The maximum time required for complete packing of the red cells must be found out by a few preliminary experiments This will depend to a great extent on the centrifuge machine The tubes are first spun for 20 minutes at a high speed the actual speed being noted they are taken out and the readings taken they are again spun for another 5 minutes and the readings again taken The process is continued until there is no difference between two consecutive readings--the time at whuh the first of these two readings was taken is the time required for complete paching of the cells as there was no further paching The mean time in minutes for 10 to 20 such experiments is taken as the time required for complete packing of cells with that particular centrifuge machine at that particular speed However in all sub sequent examinations a little extra time say 5 minutes should be allowed over and above the mean time determined Generally the cells are fully packed if the tubes are spun for about 20 minutes in a centrifuge machine at a speed of 2 500 to 3 000 revolutions per minute but it is safer to allow 30 minutes The tubes are now taken out of the buckets and with a hand lerr the marking on the tube corresponding to the level of the packed red cells is noted ·DETERMINATION OF CORPUSCULAR VOLUSIE 31 also the top level of the serum > checked If this is at the mark 100 then the red cell reading can be translated directly into a percentage but if it is more or less than 100 the percentage figure must be calculated making an allowance for this fact Fra ple --If the top of the sprum 13 at the 102 level and the rel cells at 12 the 100 cell vo ume 19102*12=41 17 per cent With leukæmic blood it is almost imposible to read the top level of the red cell accurately and therefore it is difficult to ascertain the corrert cell volume This may be remedied at least partially by allowing the charged cell solume to stand for half an hour before centrifuging when the demarcation between the red and white cells levels will be more clearly seen Anh coagulant. This has been discussed above (p2) but perhaps we should again mention that if the isotonic mixture of potassium and ammonium which we recommended be used no shrinkage will occur On the other hand if potassium oxalate alone (o 2 per cent) be u ed then a factor * I og must be applied to the cell volume before the corpuscular value ar calculated Example – The uncorrected cell vo ame s 41 per cent the correttei cell olum will be 44*10)= 476 CALCULATIO OF CORPUSCULAR VALUES From the red cell count the tal moglobin expressed in grammes per cent and the corpus ular volume certain absoulte values can be calculated These values are - (1) the thean corpuscular volume (CV) (2) the mean corpuscular hamoglobin (VICH) and (3) the mean corpuscular ha moglobin concentration (HCHC) Mean corpuscular Loliume This is the mean or average volume of a single red ce!! It is expressed in cubic microns (cu ;) and obtained by the following method V lome of packed re 1 cells in c cm per 1 000 ccm of box! Rel cel in m ll un pes com Exa ple --Corpuscular yolume=40 c cm per 100 ccm of blood Red cely cout=500U 200 per com 40 10 Vi an corpuscular volume * 800 cubic Dxfony (cu > 5 VCV = ·32 HÆVATOJ DGICAL TECHNIQUE Mean corpuscular hæmoglobin This is the average hæmoglobin content of a single red cell expressed in micro microgrammes (YY) It is obtained by the following method MCH= Ilæmoglobin in grammes per 1 000 c cm of blood Red cells in millions per cmm Example --Hämoglobin= 14 grammes per roo c cm of blood Red cells=5 000 000 per com 14 X 10 Mean corpuscular hæmoglobin= = 28 Oy 5 Dean corpuscular hæmoglobin concentration roo 14 This is the mean or average of the hæmoglobin concentration in cach cell and is expressed as a percentage of the cell contents MCHC= Hæmoglobin in grammes per 100 c cm of blood Volume of packed cells in cem per 100 ccm of of blood Example -Hæmoglobin = 14 grammes per 100 c mm of blood Corpuscular volume - 40 cm Miean corpuscular hæmoglobin concentration = 40 x 100 = 350 per cent That is to say in this instance 35 per cent of the cell substance consists of hæmoglobin The great advantage of these mean values is that in calculating them it is not necessary to decide on any arbitrary normal as one does in calculating indices 18 the colour index for arriving at which one usually considers 5 000 000 red cells per cmm and 100 per cent hæmoglobin to be the normal values Unqualified a colour index is a meaningless expression as even if the above normal values lase been taken it is still uncertain what 100 per cent hæmoglobin means (vide p 5) If later one decides on the normal values for the particular population then it is a Very simple matter to calculate the colour index Example --MCH of the particular case = 3577 Mean MCH of the population= 309 35 Co our index (CI) = - = I 16=117 Similarly the volume index and the saturation index can be calculated from the MCV and MCHC respectively There is in our opinion little point in calculating these indices and we recom mend workers to get into the habit of thinking in terms of mean corpuscular values instead of indices just as they should get into the way of thinking in terms of grammes of hæmoglobin instead of percentages Normal values There are undoubtedly different normal values for different populations We will consider mean corpuscular volume first Whitby and Britton give 86 as the mean with the range from 80 to 94 but neither these writers nor the majority of others who give a normal range make any attempt to explain what they mean by the normal range They can scarcely mean that all normal observations fall within the range but they do not specify what percentage of observations may ·TABLE VIII Corpuscular values Mean Authority Mean Locality Number corpos cular volume Standard corpuscular Standard deviation hæmoglobin deviation Mean corpuscular Standard hæmoglobin deviation concea tration Sex 7.90 28 53 90 49 Calcutta 5 2 31 31 07 Male 30 30 01 Bombay 87 08 34 54 121 71 29 23 93 704 231 Assam 32 50 14 84 93 +10 78 25 14 + 370 Cachar 29 72 25 27 5 87 00 35 00 Un pecified USA Bntain 86 00 295 34 00 DETERMINATION OF CORPUSCULAR VOLUME # 776 Napier 2 89 31 57 Calcutta 36 82 +7 23 Females 128 27 42 + IO Napier and Das Gupta 1936 Sokhey et al 1937 + 3 10 Nap er and Das Gupta 196 2 94 Napier and Majumdar 1938 Castle and Minot 1936 Whitby and Bntton 1939 Edwards and Das Gupta 1941 Sokhey et al 1938 Benjamin 1939 +1 70 Napier and Bilimona 1937 + 337 Napier and Majumdar 1938 Bethel 1936 + 213 Napier and Edwards 1941 Napier and Bilimona 1937 Bethel 1936 906 32 86 Bombay 88 53 IOI Delhi 28,6 92 71 33 58 100 Assam 31 20 300 + 710 + 50 77 30 0 Cachar +1268 82 49 23 42 8 67 + 310 25 Michigan 50 86 30 Pregnant females Calcutta 6 62 86 83 64 10 08 + 334 30 57 Assam 32 60 23 80 72 10 40 Michigan 28 92 00 Calculated from data given after applying factor x 1 09 to cell volume to allow for shrinkage where this has not been done + Corrected figures recently supplied by worker 33 ·34 HÆMATOLOGICAL TECHNIQUE be expected to fall within it If one gives the mean (m) and the standard deviation (sd) of a number of observations that at least does mean something definite namely that two thirds of the observations will fall between metsd and m-sd and that 19 out of 20 will fall between m+2 x sd and m - 2 xsd The last figures seem reasonable ones for practical purposes and can certainly be applied amongst hæmopoietically more stable populations than we usually en counter in India but in most of our populations it provides too wide a range Esen for our Calcutta series in which the mean is 90 49 cu pi the range would be (90 49 +2x7 90=) 106 29 to (90 49-2x790) 74 69 or in round figures from 75 to 106 this is too wide to be considered a normal range though the mean is close to that given by the workers On the other hand some of the Assam so-called normal populations have a very low MCV and if the same rule were applied the range would be ridiculously low eg 57 21 to 85 37 cu ji in the first Assam series quoted but there is evidence in this case that the whole population is suffering from a degree of iron deficiency anæmia therefore as the figures are not based on a truly normal population this rule cannot be applied It is obvious that no hard and fast rule can be laid down Whenever possible it is best to examine normal individuals of the population concerned and where one can be certain of excluding most of the cases of sub clinical blood dyscrasia to calculate the range from the mean plus or minus twice the standard devitation Otherwise 80 to 100 cu xe may be taken as the normal range for general use in India That is to say any figure below 80 cu ye may be taken as indicating microcytosis and any above 100 cu pl as indicating macrocytosis Similarly for the mean corpuscular hæmoglobin on the Calcutta figures the normal range should be 28 53 + 2x2 31=23 91 and 33 14 or roughly 24 to 3377 This is in our opinion a good normal range for general use in India any figure below 24/7 indicates hypochromia and any above 33Yy hyperchromia Finally a good range for the mean corpuscular hæmoglobin concentration 1 30 to 35 this is not based on any of the data quoted for reasons into which we need not go now but on our general experience 7 HYPERBILIRUBINÆMIA AND VAN DEN BERGH S TEST Bilirubin the chief pigment of the bile is 4 by product of hæmolysis It is derived from hæmoglobin being the porphyrın (globin and iron free) fraction of the hæmoglobin molecule The bilirubin is formed by the disintegration of the red blood cells by specialised reticulo endothelial cells present in spleen liver bone marrow and elsewhere The result of the dısıntegration of the red celly is the formation of a simple pron compound which is preserıcd and re utilised for the formation of new red cells and an iron free pigment bilirubin which is excreted by the liver in the bile It was formerly believed that this disintegration of the red cells and elaboration of bil subin used to take place in the liver alone but recent researches have definitely ·Į HYPERBILIRUBIN ESIIA AND VAN DE DEPGII S TEST 35 shown that bilirubin is elaborated in any organ having these specialised reti culoendothelial cells but is mostly seen in spleen bone marrow and liver But where or it is formed the bilirubin enters the liver through thu hepatic vascular capillaries from where it passes through the polygonal celly into the bıle capıllaries lying in between the column of lyer cells and is finallv discharged into the bile ducts Normally, the bilirubin is concentrated in the gall blader and would pass into the small intestine In the large intestnie the bilirubin undergoes reduction by bacteria to forni urobilino, en (also called stercobilinogen) A part of the urobili no,en is excreted in the stools and on exposure is conserted into urobılın (stereo bilin) by oxidation I at the bulk of the urobilinogen kre absorbed in the portal circulation passes to the luser and is almost completely re excreted in the bila Any urololinomen which may escape into the general circulation is excreted Liv the kidney and converted to urobili by oxidation when the urine is sonded As a result of normal la molysis of the red blood cells bilirub 18 normally present in the plasma and serum usually in a dilution of 1 in 400 000 or 0 23 mgm per 100 cc of blood but may be present up to 7 dilution of 1 in 100 000 or 10 m, ni per 100 cc of blool The yellow colour of the plasma and serun is ir part due to the presence of bilirubin When bulirulin is present in the blood in excess of the normal amount the condition is known as hyperbilirubinemia Ihrlich observed that when ulphımlıc head and surium nitrite were adde 1 to a solution of bilirubin & reddish violet sulistance azo bilirubin was formed The reaction 14 specific and very delicate will show bilirubin in a dilution of 11 1 500 000 Van den Ber,h made use of these observations not only to test for the excess of bilirubin but to draw eertuin conclusions from the nature of the result HU original claims are now questioned but hi general conclusions hold good and form the basis of the test which we are describing The tan den Bergh reaction The reaction is classified into two main types the direct and the indirec The direct reaction may occur ether as immediate direct reaction or delayed direct reaction A tlure type of reaction the biphasic reaction may also oceur If the passage of the bile is obstructed due to obstruction in the hepatie or conimon bile duct the secretion of bile continues until the pressure in the ducts equalıses the secretors pressure of the liver cells When this occurs the bilirubin would enter the liser but instead of being secreted into the bile cupillaries usre absorbed by the liver cells when it combines in some loose way with bile salte and cholestrin and passes out of the liver through the hepatic vein and the lymphatics into the general circulation The association of the bilirubin with bile-salt ar cholestrin prevents its arlsorption by the proteins of the blood and therefore bilirubin in this state will give a prompt direct van den Bergh reaction When the destruction of the red cells is such that the baluce between the production and exerction of the bile pigment cannot be maintained even by a norint liver with its large functional reserve the concentration of the bilirubin ·36 HÆMATOLOGICAL TECHNIQUE rises above the normal level in the blood The excess bilirubin does not pass through the liver cells ind is in some loose combination with the proteins of the blood and therefore would be a positive reaction with the duzo reagent only when the proteins are precipitated with alcohol Thus indirect in den Bergh test is found to be positive in cases of excessive formation of bilirubin due to increased destruction of a large number of red blood cells In conditions of live dysfunction associated with eveessive hæmolysis the br phasıc ieaction will be positive indicating that both forms of bilirubin 910 present The tecbnical details of the test have been modified many times and it is done in a number of 1 lys in different laboratories Codfried (1935) recomiends the methoil described by Thannhauser und Anderson (1921) 94 tlıe most sutable for clinical purposes this is the method that we follow in our Inborntor Apparatus requireil () Graduated pipettes I com 2 em 5 cm and 2 е с (122) Small test tubes 4 inches hinch (in) Centrifuge tubes (12) Small glass leakers or flash (2) Capıllary pipettes with rubber teat (11) Centrifuge machine (in) Sealel cot alt ulphate standard tubes (1911) Lombond comparator with the coloured discs for bilirubin l'eagents Alcohol absolute anl 90 per cent Saturated solution of ammonium ulphate this 14 prejareu by di solving an exceye of ammonium sulphato in hot water (so that some un lie olvid salt 1e i ft at the bottom) Normal saline Tuzo reagent (A) Sulj hanılıc acid-i gramme Concentrated hydroellone 962 L...-13 e cm Distillet water 1 000 cm (B) Sodium nitrate-05 gn Installed water 1001 c em Stoch solutions of (A) and (P) are mide Sepırately they heep well for 11 long time The test can be clone either with the serum or plasmWhen the cell volume is also being estimated the supernatant plasmu in the cell volume tubes, after the paching of the red cells is quite sufficient for the test When the test is done by itself 3 cubic centimeties of blood is drawn and placed in a dry test tube or an oralated test tube The blood is collected in the usual way and even the slightest hænolysis muus! be avoided (vide supra) Preferably the blood should be collected in the fastin state as it is claimed that food intahe especially a big meal of carbohydrate and fat affects the level of serum bilirubin The specimen must always lx examined within 2 hours as otherwise piradoxical results may be obtained * The comparator can be obtured from British Dug House London lut stocks may be available at this Companys depots in India Imperiai Chemieal Hoi Ballur] Estate Bomber or c/o II Clurk Kent House 13 VI on R Cal utta ·HY PERBILIRUBIN EMIA AND VAN DEN BERGH S TEST 37 Technique - Prepare the diazo reagent by mixing 10 c cm of (A) and o 3 ccm of (B) in a small flask or beaker This mixture must be prepared just before use and must never be used later than one hour after it is made Pipette off the clear supernatant plasma from the cell volume tubes Take exactly I c cm of plasma un a centrifuge tube Add exactly 05 ccm of diazo reagent and mix well One of the following things may happen The mixture becomes reddish violet within 30 seconds Direct reachon (11) The mixture becomes sli, htly reddish at once and the colour gradually increases in intensity and becomes reddish violet-Biphasic reaction (111) A reddish colour appears 91 about a minute which gradually deepers into reddish violet --Delayed direct reaction No change of colour occurs within 10 minutes Indirect reaction - If no change occurs add to the mixture of plasma and diazo reagent 25 c em of alcohol (absolute or 96 per cent) and 10 c cm of saturated solution of ammonium sulphate Mix well by inverting the tube allow it to stand for about 2 minutes and centrifuge for 5 to 10 minutes A positive in direct reaction is indicated by the upernatant fluid becoming coloured a distinct bluish violet the intensity depending on the amount of bilirubin present As bilirubin is present in normal blood a faint violet colour nearly always appears such a reaction obviously has no pathological significance Quantitate indirect reaction -It will be clear that come form of quantitative cstimation is essential Many elaborate methods which require special apparatus have been devised but a fairly accurate estimation of the bilirubin content can be obtained by carrying out the test in the way described above and comparing the supernatant fluid with a prepared standard or with a permanent colour standard Comparing the supernatant coloured fluid with the cobalt sulphate standard (2 161 per cent) has given the most satisfactory colour matching in our hands Preparation of the cobalt standard solution -Dissolve 2 161 grammes of anhydrous cobaltous sulphate in 100 c cm distilled water The colour of this solution corresponds to that of 1 in 200 000 bilirubin or o 5 mg of bilirubin per 100 с cm Solutions equivalent to 04 03 02 01 and 005 mg per too c cm made by diluting the original solution About 2 c cm of each of these solutions is put into tubes made of hard glass The tubes are then sealed numbered and kept in the dark and are brought out only when required Properly stored the solutions retain the colour for a fairly long time In our laboratory we did not find any difference in colour in solutions which were in daily use for over six month when these were compared with freshly prepared solutions Technique for the use of cobalt tandard solutions - Take about 2 c cm of the coloured supernatant fluid in a tube having identically the same bore as the standard are ·HEMATOLOGICAL TECHNIQUE tubes and compare its colour in a comparator with the cobalt standard tubes The figure on the tube giving the correct matching with the supernatant fluid multiplied by four gives the bilirubin value of the undiluted serum for the dulation of the serum in the supernatant fluid containing azo bilirubin is x in 4 and not 1 in 5 because the saturated ammonium sulphate remains as a separate layer and does not contain azo bilirubin If the supernatant fluid is of a deeper colour than the cobalt standard tube marked os dilute the supernatant solution with normal salıne (dilution with 67 per cent alcohol which is recommended makes the solution hazy and difficult to match) until it matches one of the cobalt standard tubes Note the dilution required to match and calculate the amount of bilirubin in the specimen Exar ple One cubic centimetre of supernatant laid was taken to which war aided I cul centimetre of normal salıne to watch the tube marked 4 First dilution of serum in the supernatant flur is in 4 Second dilution to match the colour is I ID% Therefore total dilution is *** Then the original plasma contained 0 4x8= 32 mg of bilirubin git 100 cm (11) Lovidond comparator Quantitatis e estimation of the indirect van den Bergh reaction can be simplified by the use of a bilirubin disc in the Lossbond com parator The disc contains coloured glass standards marked 02 04 06 08 JO 125 150 75 and 20 mg which are calibrated in such a way that when the test is done in the way described above the watching with any of the discs gives directly the result for the undiluted serum (figure II) ME LOVIBOND COMPARATOR Szi Pro 11 Lovbond comparator with the bits subun disc and tubes 10 position Bilirubin dic for the Lovilond comparator ·HYPERBILIRUBINÆMLA AND VAN DEN BERGH S TEST 39 Technique for the use of the comparator Fit in the disc marked bilirubin in the Lovibond comparator Transfer the coloured supernatant fluid to the right hand tube of the comparator A tube of distilled water should be put behind the hole showing the coloured glass Hold the comparator against a source of light (day light is much better than any artificial light) Rotate the disc till a correct colour match is obtained the number that is visible indicates the bilirubin value in mg per 100 c cm of undiluted serum of plasma eg o 8 mg in the above figure When the colour of the supernatant fluid appears to lie between two conse cutite coloured discs and does not exactly match either of them an intermediate value is recorded If the bilirubin value exceeds 2 mg per 100 c cm dilute the supernatant fluid with normal salıne until a correct match is obtained with one of the coloured glasses Calculate the bilirubin value by multiplying the number on the disc by the dilution factor Discussion -.The significance of the positive indirect van den Bergh test in hæmatological investigation is that it gives a broad indication as to whether the arzemia is due to excessi e blood destruction or not In the true hæmolytic anemias the test always gives a high reading 20 milli grammes or more In pernicious anemia a reading well above the normal range is usual for though it is not a true hæmolytic anæmia the deficiency of hæmopoietin leads to the formation of abnormal cells which are very susceptible to destruction by the normal hamolytic tissues In tropical macrocytic anemias in a large number of cases especially in those with enlarged spleens the test is much above the normal range and in a small number of cases the test is well within the normal range This is probably due to destruction of an increased number of red blood cells on account of the proliferation of the reticula endothelial cells in the spleen The associated liver dysfunction » also a probable factor both in pernicious anæmia and tropical macrolytic anæmia in producing an increased indirect van den Bergh reaction In iron deficiency anæmias it is within normal limits and in aplastic anæmias at may be completely negative It is not however an absolute indication either of excessive blood destruction or of deficient blood formation as for example in an acute malarial attack when there may be enough red cells destroyed to lower the red cell count a million or more cells per c mm the van den Bergh often remains within normal limits for the liver cells are able to excrete this temporary excess of bilirubin in the blood bat of the red cell destruction is contidued the bilirubinæmia will inevitably rue above the normal range and a positive indirect van den Bergh reaction will result On the other hand in liver dysfunction not necessarily accompanied by anzemia a positive van den Bergh may be found for the liser cells are unable to utilize the products of normal hæmolysis ·40 HEMATOLOGICAL TECHNIQUE TABLE IX Showing the normal bilirubin according to different observers BILIRUBIN IN MILLIGRANTES PER 100 CCM OF PLASMA OR SERUM Author Range Mean SD van den Bergh (Vaughan and Haslewood 1938) O 08-02 Creene et al (19 5) 320 Perkın (1927) 0 05-035 Barron (1931) Oro 24 000 25 Exton (Vaughan and Haslewood 1938) Vaughan and Haslewood (1939) Mills and Mawson (1938) O217 054 +5 OIIO 031 + 016 Napier and Das Gupta (1941) OOIO O 20 8 ICTERUS INDEX Like the van den Bergh reaction the icterus index is a measure of the bilirubin content of the blood Though the test is simple to carry out it does not give all the information which is obtained by the van den Bergh test Principle of the test -The colour of the serum of plasma is compared with a standard solution of 1 in 10 000 potassium bichromate which is taken as the unit Apparatus required (1) Test tubes in a rack (11) Capıllary pipettes with rubber teats (tta) Potassium bichromate solution 1 on 10 000 (to be stored in the dark (tv) Colorimeter of the Duboscq or Klett type Technique ---Pipette off the supernatant plasma from the cell volume tube after complete packing of the cells Dilute the plasma with an equal part or with twice the amount of normal saline (more in cases of jaundice) di tion diminishes the cloudiness of the plasma and gives a better matching ·ICTERUS INDEX 41 Watching in the colorimeter —The following adjustments of the scales and of the illumination must alsays be made each time before the colorimeter is used 1) Adustment of the scales Gently raise the cups until the bottoms are in contact with the plungers The readings should be exactly at O on both sides If they are not they must be brought to O by adjustment (11) Adju tment for uniform illumination Half fill both the cups with the standard I IO 000 potassium bichromate solution Gently raise the cups until the bottoms touch the plungers—this will drive out any air bubbles that may be in the fluid Now slowly bring down the cups and set them at an equal depth say 15 millimetres Adjust the reflector in such a way that the same amount of light 1 reflected up through each cup Slight adjustment of the eyepiece may be necessary if both halves of the field do not match exactly Taking a readıng-heep the cup on the left side at a depth of 15 millimetres Take out the cup from the right side throw out the potassium bichromate solution wash the cup and plunger in water dry well with soft Imen Put the diluted plasma into the cup and replace it on the night side of the colorimeter Gently rase the cup until its bottom touches the plunger Now move this cup containing the plasma up and down until perfect match is obtained take a reading Vove the cup up or down and again adjust it to match take another reading Take the mean of three independent readings Calculation Icterus inders Reading of standard b cbromate olation x dilution factor Read ng of diluted plasma solution Frample --One part of plasma 13 diluted » th two parts of nortaal salae The cup containing the standard solut on is at 15 mm The cup containing the unkno ya plasma matches the standard at , mm Icterus Index - 1 *3=9 5 B Valching in tubes - In the absence of a colorimeter results sufficiently satisfactory for clinical purposes may be obtained by matching the plasma or serum diluted with normal saline against the standard 1 10 000 potassium bichromate solution Take two test tubes of equal height and calibre Into one put about 5 to IO c cm of standard potassium bichromate solution Into the other put I ccm of plasma and add measured amounts of normal saline from a graduated pipette until the colour matches the standard Calculation — The dilation of the plasma required to match the standard potassium bichromate solution giles the icterus index Example --To 1 cm of plasma was added 7 cm of normal salıne to match the standard Dilution of the plasma is I in 8 Ther foreacterus index=8 6 ·42 ILEMATOLOGICAL TECHNIQUE C Matching in a Lovibond comparator - If this comparator is available it is an inexpensive matter to obtain an icterus index disc The undiluted serum or plasma is placed in the right hand tube and matched directly against the colour standards in the Lovibond comparator When a correct matching is obtained the icterus index is read from the right hand corner of a comparator as in the case of the van den Bergh test (urde figure 11) The normal icterus Index is 4 to 7 9 THE MAKING AND STAINING OF BLOOD FILMS Properly made and well stained blood smears are essential for the white cell differential count the enumeration of the nuclear lobes of neutrophil polymorpho nuclears as in the Arneth and Schilling counts and also for the determination of the red cell size by the Price Jones method For ordinary work blood films are made on 3 by I inch glass slides and for special studies on 2 cm -square coverslips only new slides and coverslıps should be used if this is possible The slide and the coverslıp must be thin completely transparent scrupulously clean and free from grease and dust Cleaning glass slides and coverships Place the slides and coverslips in sulphuric acid bichromate mixture* and allow them to remain for 24 hours this mixture should be kept in a large jar for the slides and in a small one for the coverslips Decant off the cleaning mixture into another sınılar glass jar which is then ready for use again Transfer the slides or cover slips to a shallow enamelled tray or a big petri dish and place this under a tap for 3 to 4 hours stirring from time to time with a glass rod Finally wash with distilled water dry with a piece of soft linen and put them in a jar of absolute alcohol for 24 hours (methylated spirit may be used as a substitute) of forceps take out the slides or coverslips one by one allow the excess of spirit to drain off flame in a bunsen or spirit lamp clean again with soft linen and store in 2 dust proof container--in a slide box for the slides and in a small petrı dish for the coverslips These are now ready for use for all ordinary work Polishing the slides For special work polish one side of a slide cleaned as above with jewellers rouge for 2 to 3 minutes clean with soft linen ( elvyt cloth or silh is the best for this purpose) mark the polished side with a glass pencil and store for future use in a dust proof container (A) SPREADING THE FILM IN THE ROUTINE USE OF VENOUS BLOOD () On slides -Put a few polished slides on a flat surface With a syringe collect the blood from a vein (vide p 2) Before putting the blood into the With a pair Concentrated ulphuric acid Potassium bichromate (powdered) Water 100 ccm too gm to i tre ·TJE MAKING AND STAINING OF BLOOD FILMS 43 electric fan oxalated flask and while the needle is still attached to the syringe put a small drop of blood on to the middle of the slide a little away from the end Apply the thin cdge of another slide with the corners cut off so that the spreading edge is narrower than the slide or better still of a sprcader (hemocytometer cover slips mahe excellent spreaders on account of their smooth narrow edge) to the middle of the glass slide slide it along until it inales contact with the drop of blood which will now spread along the edge of the spreader (slight lateral movement will accelerate this) then with the spreader at an angle about 30 to 40 to the slide push the spreader with the blood following it along the slide and raise the spreader abruptly just before the whole drop of blood has been used up The film should be dried quickly by waving it in the air or under an An ideal film should FIG 12 occupy the middle third of the slide Ishould be of uniform thickness 3 should not have any tails and when viewed under the microscope the red cells should just touch one another and there should not be any rouleaux formation anywhere on the slide Figure iza shows a satisfactory film but with tails and figure 12b a good film with a stran, ht leucocytic edge They were drawn from above downwards Figure 13 shows the low power view of a satisfactory leucocytic edge the leucocytes are actually dis crete though the photograph scarcely g11es that impression The important points are a per fectly clean slide 2 a good spreader and a drop of blood of the right size this last can only bc gauged by trial and crror but compared with the size of the drop required from a normal individual 1 larger drop will be required from an anæmic patient and a smaller drop from one with polycythæma or leukæmia FIG 13 ·44 HEMATOLOGICAL TECHNIQUE (!) On coverships -Place a few clean coverslips on a flat even surface Proceeding as in put a very small drop of blood on the centre of a coterslip place another clean coverslıp of the same size on the top of the drop of blool in such a way that the sides of the two coverslips are not opposed (sce figure 14) It the two coverslips are clean the blood will spread uniformly between them Now draw apart the coverslips quickly but gently and dry them quickly by waving them in the air or under an electric fan (B) FROY CAPILLARY BLOOD FROM THE FINGER OR EAR LOBE FIO 14 Prick the finger or ear lobe fairly deeply with a sharp surgical needle or with a blood gun so that the blood flows freely from the wound Apply the surface of a clean slide or covership to a small drop of blood and proceed as in A) or (1) Comparing the tuo methods The method of choice is in our opinion the slide smear The advocates of the coverslıp method claim that it is the only way to obtain an even distribution of cells and that consequently reliable differential counts can only be made from coverslips On the other hand it is much easier to make a good film on a slide and if the whole drop of blood on the slide is utilized in making the smear and this drop is a small one examination of such a smear will give as reliable informa tion as examining a coverslıp smear the preparation of which demands considerable dexterity Staining blood films All blood smears should be stained within 24 hours If the smears cannot be stained immediately they must be fixed with methyl alcohol and stored in a dust proof slide box for staining at a later date The unstained slides must never be left uncovered on the working table as blood is readily eaten by flies during the day and by cockroaches at night Romanowsky stains are used for all blood work The most commonly used stains arc those originated by Leishman Wright Jenner and Gremsa All these stains depend for their action on the compounds formed by the interaction of methylene blue and eosin and the differences between the various stains are due to the proportion of the two stains Excepting Giemsa s stain the fluid stains are prepared by dissolving the dry powder in acetone free pure methyl alcohol so that a preliminary fixation with methyl alcohol is only required in the case of Gremsa s stain Leishman s and Wright s stains are used in the strength of 15 per cent and Jenner s stain in the strength of 05 per cent ·- whole of the film re minute to allow for proper fixing with a capillary THE BLAKING AND STAIVI GOH BLOOD FILMS 45 Preparing Leishmans Il rights and Jenner s stains Stains in powder or tablet forms and extra pure acetone free methyl alcohol for dissolving the stains should be obtained from some reliable firm He hile found Gurt se stains to be very satisfactory All the glassware used in preparing the stains and in storing them should be scrupulously clean and free from any trace of water they should be rinsed first with absolute alcohol and finally with a little methyl alcohol Take the requisite amount of stain in powder or tablet form in a mall glass mortar Measure out the requisite amount of methyl alcohol in a graduated glas cylinder Pour out about 2 c cm of methyl alcohol on the stain and grind well to make it into a thin paste Add in small quantities at a time about half the total amount of methyl alcohol grinding all the time Carefully decant the supernatant dissolved stain into a clean glass stoppered bottle Add more methyl alcohol to the undissolved stain grinding as before Again decant the supernatant stain into the bottle continue the process until all the methyl alcohol is used up If this is properly done all the stain will go into solution and no residue will be left at the end Inrubate the bottle with the stain at 37 C for 24 hours when it will be ready for use (A) STAINING WITH LEISIIBAN S WRIGHT S OR JENVER S STAIN Reagents a d appa atus required (1) Prepared stais preferably in a drop bottle (+1) Distilled water (PI 70) or buffer solution with pH of 6 4 and tra le with acıl monopnta sium phosphate and duolium phosphatet ( ) Fresh distilled water which is sightly acid in reaction (1) Staining rack 'Ilus can be made with plasticine and a pair of glas rout of equal size and thickness and may be placed at one end of a sink au ay from th water tap or on a rectangular esamell d tray placed near a tap Glass beakers cylinders a pair of forceps capillary Pipettes trats etc Technique -Put the slides on the staining rack takın, carc that the side with the blood film is upwards also see that the two ends of the slides are in the same plane From a drop bottle or with a pipette pour on sufficient stain to cover the wait pipette now add distilled water (pH 68 to 70) or the buffer solution—the same volume in the case of Leishman s and Wnghts stains but double the volumnc in the ca e of Jeoner s stains With a capillary pipette or gla s rod thoroughly mix the stain with the diluent to ensure a uniform mixture over the film Georg Te Gurr 136 New kings Road Lonn lon si | Vonopotass om phosphate Anhydrous disol um pho bat Distikdater Add 1 c cm of choloroform as preservat ve 6 England 6 63 in 2 56 gth up to 1 litre ·46 HEMATOLOGICAL TECHNIQUE When the mixture is allowed to settle a scum will form on the top if the proportion of the stain and diluent has been correct According to the depth of staining required allow the diluted stain to act for 5 to 10 minutes in the case of Leishman s and Wright s stains and 3 to 5 minutes in the case of Jenner s stain The diluted or undiluted stains on the slides must not be allowed to dry up at any stage of the staining Drying 1 prevented by covering the staining rack with a wide bell jar or other improvised device* When the staining is complete hold one end of the slide firmly with a pair of forceps and place the stain flooded slide under a running tap This will wash off all the stain from the upper surface while the bottom is cleaned by rubbing it well with the fingers of the left hand The slide is now transferred to the beaker containing fresh distilled water and gently shaken to and fro until the colour of the smear becomes faintly pink Now take it out of the beaker wash again under the tap and allow it to dry In order to dry it without allowing dust to adhere to the stained surface the slide should be sloped against a vertical surface eg wall or the side of a box with the film side inwards When it is dry the slide is ready to be examined a (B) STAINING WITH GIEZSA S STAIN Note-It is more difficult to prepare this stain and it is better to purchase it in solution Giemsa s stain as prepared by Gurr 1» very satisfactory In staining with Glemsa s stain preliminary fixing with methyl alcohol or some other fixing stain is absolutely necessary Preparing dilute solution Take about 20 c cm of prepared distilled water (pH 70) or buffer solution in a clean transparent glass cylinder add 20 drops of undiluted stain or in other Hordy as many drops of stain as there are cubic centunetres of water Njx well by inverting the cylinder and see that the depth of colour of the mixture is such that when held in front of the eyes it allows a distant object to be seen through it Place the slides to be stained on a staining rack flood the slide with methy1 alcohol and cover with a bell jar so that the methyl alcohol does not dry up on the slide Allow the methyl alcohol to act for about 2 minutes remove the bell jar and thoroughly wash the slide with distilled water Now flood the shde with the diluted stain cover with a bell jar and allow the stain to act overnight Next morning wash and dry the slide as in A The precaution will seldom be necessary i bumid climates eg of Ek igal and Assat except in the hottest months but will be imperative in drier provinces in India and in other dry countries such as Iraq Egypt etc ·WHITE CELL DIFFERENTJAL COUNT 47 (u) Combined staining with Lashmans Wright s 01 Jenner s and Gleisa s - This is done exactly in the same way as in A but substituting very dilute Gremsa s stain (1 drop in 2 ccm) as the diluent in place of distilled water or buffer solution (111) Combined staining with Ilay Grunwald* and Gremsa –Pour on undiluted May Grunwald s stain just sufficient to cover the slide place a bell jar over the staining rack allow the stain to act for 2 to 3 minutes remove the bell jar and add an equal amount of neutral distilled water (pH 7) Allow the diluted stain to act for 5 to 10 minutes according to the depth of staining desired wash thoroughly With neutral distilled water and then flood the slide with dilute Giemsa s stain to act for 15 to 30 minutes according to the depth of the staining desired and wash and dry as in A Methods used in our laboratory For all ordinary work Wright s staining method is used and for special work og examining smears from the marrow counting the nuclear lobes in Arneth and Schilling counts combined May Grunwald and Gremsa s staining are preferred For with the combined May Grunwald and Gremsa s staining all the component parts of both mature and immature red and white cells are very well shown 10 WHITE CELL DIFFERENTIAL COUNT When making a white cell differential count on slides one should attempt to fulfil the following requirements () The whole drop of blood should be utilized in making the smear as if only a portion of a large drop is used this portion may not contain a fair sample of the cells (eg the large cells tend to sink to the bottom of the drop) (vi) The film should be uniformly spread and should be neither too thin nor too thick-the ideal film is one where the margins of the red cells when seen under the microscope just touch one another without Overlapping (vide 43) (H1) The film should occupy the middle two thirds of the slide (iv) There should be no tails at the end of the film (v) The cells should be stained well and there should be no debris in between or on the cells A well stained film shows red cells—an orange buff colour lymphocytes with pale blue cytoplasm and neutrophil granules-a dull lilac This can be purchased in solution and is available from GT Gurr (us) ·48 KXMATOLOGICAL TECHNIQUE Number of cells to be counted This will vary directly with the total white cell count the higher the total count the larger is the number of the cells that should be counted to get a correct proportion of the different cells The following rule may be taken as a guide but need not be followed rigidly Count 100 cells when the total white count is below 5 000 per c mm Count 200 cells when the total white count is above 5 000 per c mm but below 10 000 per c mm Count 300 cells when the total white count is above 10 000 per c mm but below 20 000 per c mm Count 400 cells when the total white count is above 20 000 per c mm The differential count should be done with an oil immersion lens (one twelfth) and x5 eyepiece with a x Io ey epiece at hand This magnification will enable one to see the details of the cells distinctly but a higher magnification given by the a x 10 eyepiece may sometimes seem desirable eg in the case of doubt arışıng as to the true identity of a cell In actual practice it will be found that one seldom changes the eyepiece The microscope must be fitted with a mechanical stage that allows easy move ment in exther the horzontal or the vertical plane Northern daylight is preferable to artificial light as it shows the true colour of the different cells If artificial light has to be used a blue filter should be placed in front of the light iself or under the condenser of the microscope Procedure --Place the stained blood film on the mechanical stage of the microscope and fix it securely with the low power (two thirds objective) make a rapid survey of the different parts of the film to find out if the requirements mentioned above are fulfilled If not it is recommended that another properly made film should be stained for to attempt to make a differential count on an unsatisfactory blood film is laborious and irritating and the result obtained as never accurate If the blood film is satisfactory put a drop of cedar wood oil on the smear rotate the oil immersion lens into position open the diaphragm fully and raise the condenser to get the maximum amount of light With the coarse adjustment lower the oil immersion lens just to touch the stained film through the cedar wood oil then looking into the microscope gently raise the lens until the cells are seen and finally manipulate the fine adjustment until the focus is accurate The distribution of the white cells in the different portions of the blood films depends on their size the larger and heayter cells eg neutrophils cosinophils and large mononuclears are mostly found along the edges and at the tail end of a smear while the smaller and the lighter cells eg the lymphocytes tend to occupy the middle of the film Therefore in order to get an absolutely correct differential count every cell in the blood smear on the slide or on both the coverslips used in making the smear ·WHITE CELL DIFFERENTIAL COUNT 49 of the coverslıp method is used must be counted and classified and the percentage calculated This takes a long time particularly if the smear is a large one it is therefore advisable to make as small a smear as possible A smear 15 made from a small drop of blood and one of the methods suggested below is adopted from neither of these methods will the error be very great Counting the cells Method A (This is the method of choice) Start counting at a point A near the proximal end of the smear and along one edge moving the slide long tudinally counting and classi fying the cells all the tune up to B which is beyond the end of the film move the slide the breadth of one or two fields to B and again move back along the whole length of the film to C which is beyond the proxi mal end of the film move FIG 15 across to C and then back again up and down the film Continue to do this until about the middle of the film is reached or until a suffi cient number of cells has been counted but always end the counting at the end of the filin (tide figure 15) By this procedure you will have included one edge and one or two rows of fields from the rest of the film The majority of the heavy cells are drawn to the distal end and only a few to the sides so that the discrepancy will not be great of after counting one edge you count a little more or a little less of the rest of the film On the other hand it is important to finish at the end so that you will have covered an even proportion of body and tail of the film For this reason an exact ficure should not be aimed at but the point of stopping like the point of starting should be at one end of the film The percentages of the different cells are then calculated Example -Suppose that in makug a differential bite cell count of a blood film there are 141 neutrophils 63 lymphocytes 12 monocytes and 9 eosinoph.Is ie 225 cells altogether the percentage of the different cells are calculated as follows Neutrophus 141 X 100 -=626 or 627 per cent 225 Lymphocytes 63 x 100 = 28 o 225 Monocytes 12 x 100 -=5 3 or 53 225 Eosinophils .9 x 100 225 ·50 IIÆMATOLOGICAL TECIINIQUE Method B Four field meander technique - Though this method is recom mended in most textbooks we consider that it allows greater scope for individual variation in procedure and we prefer the previous method the method is as follows From a point at the edge of the film move towards the centre of the film for about four fields then using the other mechanical stage adjustment move about four fields towards the proximal end of the film then back towards and beyond the edge move few fields towards the proximal end and repeat the process until 50 ITCI cells have been counted and FIG 16 classified This process should be repeated in four different places at the edge of the film on either side and near each end of the film (vide figure 16) a no Recording the results Except in cases of leukaemia and some rare pathological conditions the types of white cell that are usually seen in the differential count of a peripheral blood film are neutrophils lymphocytes monocytes eosinophils and basophils in the order of frequency in which they are encountered As the slide is moved from one field of vision to another the exatniner must record both the total number and also the types of cell encountered This may be done in diferent ways The usual method is to write down on a piece of paper the names of the cells in the order of frequency in which they usually occur in the peripheral blood and to put vertical stroke against each name as that cell is seen in the field of 111on making the fifth stroke crosthe other four so that the strohes are in groups of five and easily counted (vide example 1) When all the cells in the film or in the case of a large film in the area decided upon have been counted thev are totalled and the percentage calculated Example I- N - NU MU I NU NU AU = 68 I L = 32 = 5 M 6 B II Another method is to memorize the numbers of the two main cell types until one of the other types is encountered then to enter the count on your prepared list and begin again (vide example 2) ·14 BITE CELL DIFFERENTIAL COUNT 51 The numbers against each type are added and the percentages calculated Example 2 - 10 12 4 10 14 1 20 JO 14 17 20 5 8 5 Total Percentage 13 = 680 51 = 263 7 = 36 4 3 1 8 4 1 IO Neutrophils Lymphocytes Monocytes Eosignphils Basophils 2 1 I 1 1 1 1 1 M T M 3 = 15 OS 1 I = 194 III A quicher method which can be learnt with a little practice is to remember the numbers of the different types of cell in the traditional sequence neutrophil lymphocytes monocy tes eosinophil and basophil untıl 2) cells have been counted the number of each type counted is entered against the respective name on a piece of paper and when the required number of cells have been counted they are totalled and the percentages calculated (vide example 3) Era ple 3 — Total Percentage IZO 60 0 18 17 10 14 16 14 20 II 6 5 12 8 7 9 4 9 Neutrophuls Lymphocytes Monocytes Eosinophils Basophil 3 2 1 2 I 1 3 60 = 300 13 65 30 05 I o 1 O I 0 2 0 O 0 I o 0 a 200 Method I is simple and fool proof but slow method II is also simple and saves the repeated interruptions of method I method III 15 very popular but tends to make one stop at a round number and thereby simplify the calculation which is a procedure not in the best interests of accurate countings In methods I and II theoretically one has to stop periodically and add up all the figures to see if sufficient cells have been counted but this can be obviated by the use of a mechanical counter eg the Veeder Root hand tally The counter fits nicely into the hollow of the palm and is conveniently kept in the left hand The knob is pressed with the thumb each time that a cell is seen and the numbers are at once recorded on the dial The total number of cells counted at any time during the process of the differential count can be seen by simply looking at the dial of the counter Expressing the results It is customary to express the result in the white cell differential count as a percentage of the various cells encountered without any reference to the total white cell count This does not always convey the full significance of the observation and both percentage and absolute figures should be given ·52 HÆMATOLOGICAL TECIINIQUE 68 0 50 Y o The absolute figures are readily obtained by striking out the last two zeros of the total leucocyte count or moving the decimal point back two spaces and multiplying each percentage by this figure as in the example given below Example –(A fairly bormal count) Total white cell count 500 per c mm Differential white cell count Total Per cent per c mm Neutrophils 5 TOO Lymphocytes 24 0 I 800 Monocytes 375 Eosinophile 25 187 Basophils 05 37 Now if the patient were to take some toxic drug which reduced the granulocytes to a low figure say to 540 per c mm but did not affect the other leucocytes the total count would now be 2 700 per c mm and the percentages Total Per cent per cmm Neutrophils 19 513 Lymphocytes 67 I 809 Monocytes 13 351 Eosinophils 27 Basophils In this count the attention is attracted by the high lymphocyte and monocy te percentages whereas actually these cell elements are normal and all that has happened is a decrease in neutrophils Again in some conditions eg asthma cosinophift lung idiopathic eosino philia etc the percentage of eosinophuls may be very high considerably reducing the percentage of other cell elements Example Total white cell count--25 000 per c mm Differential whste cell count- Total Per cent Per min Neutrophils 30 7 500 Lymphocytes 8 2 000 Monocytes 15 375 Eosinophits 60 75 000 Basophils 05 125 In this example though the high eosinophil count is apparent a wrong impression about the values of neutrophils lymphocytes and monocytes which are of the normal order is liable to be made from the percentage figures only Therefore a differential count should always be given in absolute figures as well as in percentages Normal u hite cell counts --The normal leucocyte count in an adult is usually given as 7 000 to 9 000 per c mm A table (IV) showing the normal white cell counts in different populations is given ·TABLE LX Differential Leucocy te counts 18 normal individuals Neutrophils Lyn phocytes Monocytes Losinophils Basophila Sex and Residence a. Authority Numben per cmm Pc per cmm pc pc per cmm pc ре per cin per cmm SO Male adults Calcutta Mean 7 00 590 Napier and Das Gupta (unpubli hed) SD$3911 #519 reme adults Calcutta Mean 62 6 4 507 69 I 937 55 300 346 02 14 Napier rward and Das Gupta 1944 WHITE CELL DIFFERENTIAL COUNT 199 USA Avge 538 390 388 2 800 310 2 152 05 30 Mules 1939 yerts Max 860 9850 630 4 900 120 1 360 30 380 40 340 Min 220 1 000 160 800 O good I al 1939 71 rem les USA 26 36 Avge 558 4 220 36 2 630 4 3 310 15 ITO OS 30 yeurs Mos 940 TI 000 GI 6 500 IIO 1 040 C90 3 720 Min 20 I 900 6 700 ·52 HÆMATOLOGICAL TECHNIQUE I 800 19 13 351 o o The absolute figures are readily obtained by striking out the last two zeros of the total leucocyte count or moving the decimal point back two spaces and multiplying each percentage by this figure as in the example given below - Example -(A fairly normal count) Total white cell count-7 500 per c mm Differential white cell count- Total Per cent per c mm Neutrophis 68 0 5 TOO Lymphocytes 24 0 Monocytes 50 375 Eosinophils 25 187 Basophils 05 37 Now if the patient were to take some toxic drug which reduced the granulocytes to a low figure say to 540 perc mm but did not affect the other leucocytes the total count would now be 2 700 perc mm and the percentages-- Total Per cent per cm Neutrophils 513 Lymphocytes 67 I 809 Monocytes Eosinophus 27 Basophils In this count the attention is attracted by the high lymphocyte and monocyte percentages whereas actually these cell elements are normal and all that has happened is a decrease in neutrophils Again in some conditions eg asthma cosinophilic lung diopathic eosino philia etc the percentage of eosinophils may be very high considerably reducing the percentage of other cell elements Example Total white cell count-25 000 per c mm Differential white cell count Total Per cent Neutrophus 30 7 500 Lymphocytes Monocytes 375 Eosinophits 15 000 Basophila 05 125 In this example though the high eosinophil count is apparent a wrong impression about the values of neutrophils lymphocytes and monocytes which are of the normal order is liable to be made from the percentage figures only Therefore a differential count should always be given in absolute figures as well as in percentages Norinal white cell counts -The normal leucocyte count in an adult is usually guen as 7 000 to 9 000 per c mm A table (IX) showing the normal white cell counts in different populations is given per c mm 8 2 000 15 бо ·TABLE LX Differential Leucocyte counts 5% normal sndividuals Neutrophils Lyrphocytes Monocytes Losilophus Basopbils Authonty Sex and Number Resideace ago per cmm per cm per cmo PC pc per cmm PC pc per cinn SO Male adalts Calcutta Mean 700 5 90 Napier and Das Gupta (unpublished) SD7.991 +5 19 114 Fema e adults Calcutta Mean 626 4 507 269 I 937 55 396 48 346 02 14 Nauer Edward and Die Gupta 1944 WHITE CELL DIFFERENTIAL COUNT 198 USA Asge 538 390 388 2 800 42 310 2 150 05 30 Males 19-38 years 11АТ 86 0 9 850 4 900 I2 O 1 360 10 980 40 340 Niin 270 I 000 160 800 Osgood tal 1939 71 Avge 55 S 4 zzo 36 2 630 43 310 15 110 05 30 Temales USA 26-36 years Mot 940 PI 000 61 6 500 II I 040 9 690 3 720 M11 290 3 900 6 700 53 ·54 HÆMATOLOGICAL TECHNIQUE It will be noted that in Indian the monocyte counts arc above the usual normal figure for Europeans and Americans the eosinophil figures are also high compared with European and American standards 10 CELL IDENTIFICATION The first essential for accurate identification of the cells of the blood or marrow is a satisfactory and properly stained blood film A well stained film should how no precipitate on or between the cells the red cells should stain an orange buff the neutrophil granules a dull shade of lilac and in the monocyte the so-called azurophil granules should just be visible with any of the Romanowsky stains The following system is recommended for the identification of different cells Granules ---First ascertain whether the cytoplasm of the cells contains any granules or not If there are granules decide whether the granules are neutro philic eosinophilic or basophilic Neutrophil granules are small uniform in size uncountably numerous but discrete and stain a dull shade of lilac Eosinophil granules are large round uniform in size and stain orange red with pale centres occasionally eosinophil granules take on a bluish stain even in a well stained specimen while in a badly stained one all may be blush The eosinophil granules are not so numerous nor so discrete as neutrophil granules Basophıl granules vary considerably in size in the same cell, they stain a dark blue violet colour which i entirely different from the colour of the nucleus they are always coarser and fewer in number than the neutrophil or eosinophil granules The granules are always found superimposed on the nucleus as well as in the cytoplasm Besides these specific granules the presence of which characterizes the cells of the granular series some cells of the non granular series eg monocy tes and lyrnphocytes may show some stained particles in the cytoplasm of the cell, which have been called azurophil granules These azurophil particles are of the same colour as the nucleus of the cell in which they occur but may be paler brighter or darker They vary in size in depth of staining and in number In the lympho cytes the particles are few in number they are large coarse and darkly stained and occur in small groups in the cytoplasm while the particles in the monocytes are more numerous paler and finer and are more evenly distributed in the cytoplasm ·CELL IDENTIFICATION 55 Having noted whether granules are present or not and if present the nature of the granules employ the following tables for the further identification of the cells Table gives the cells of the non granular series and Table \f those of the granular series The cells of the granular series all orginate from the primary differentiated cell of that series the myeloblast They are arranged in the table in order of maturation The mature neutrophul eosinophıl and basophil have their specific precursors in the earlier cells of the series and these can be distinguished by the size and colour of their granules (v s ) there are thus reutrophil eosinophil and basophil of each des elopmental stage pre myelocy tes meta myelocytes and taff cells Table XII is a supplementary table to aid in the identification of primary differentiated cells which are already shown in Table The points of difference in the morphology of the different primary cells are very fine and at times it is almost impossible to distinguish one from the other In cases of leukaemia or anzemri the identification of an individual cell has often to be made not on the cell alone but on the charactenstics displayed by other associated cells in other words the cell us judged by the company it keeps Table VIII is also supplementary It gives the nucleated cells of the red cell series The der elopment of blood cells We do not propose to discuss the ongin of the cells found in the blood stream for it is a controversial field in which unitarians dualists and trialists of the monophyletic and polyphyletic school argue sith one another to the confusion of the practical hematologist We are only concerned with the cells already differentiated the immediate precursors of the normal and abnormal cells of the peripheral circulation It 1 however impossible to asoid controversy altogether as this is intimately associated with nomenclature which is definitely our domain for at present much confusion in the literature is due to different writers adopting different names for the same cell and the same name for different cells We have followed Israel (1939) in using tbe term Pro-erythroblast for the earliest differentiated percursor of both normal and abnormal red cells and have used the expression megaloblast for the cell that are encountered in Addisons pernicious anæmia and in other macrocytic anemias With reference to the other cells we have taken the view that the granulocytes lynphocytes monocy tes plasma cell and platelets have each a separate differen tiated precursor cell and following the usual practice we hase called these myelo blast lymphoblast monoblast plasmablast and megakary ocyte respectively ·54 HÆMATOLOGICAL TECHNIQUE It will be noted that in Indian the monocyte counts are above the usual normal figure for Europeans and Americans the eosinophil figures are also high compared with European and American standards 10 CELL IDENTIFICATION The first essential for accurate identification of the cells of the blood or mirrow is a satisfactory and properly stained blood film A well stained film should how no precipitate on or between the cells the red cells should stain an orange buff the neutrophil granules a dull shade of lılac ant in the monocyte the so called azurophil granules should just be visible with any of the Romanowsky stains The following system is recommended for the identification of different cells Granules –First ascertain whether the cytoplasm of the cells contains any granules or not If there are granules decide whether the granules are neutro philic eosinophilic or basophilic Neutrophil granules are small uniform in size uncountably numerous but discrete and stain a dull shade of lilac Eosinophil granules are large round uniform in size and stain orange red with pale centres occasionally eosinophil granules tahe on a bluish stain even in a well stained specimen while in a badly stained one all may be blush The eosinophil granules are not so numerous nor so discrete as neutrophil granules Basophıl granules vary considerably in size in the same cell they stain a dark blue volet colour which is entirely different from the colour of the nucleus they are always coarser and fewer in number than the neutrophil or eosinophil granules The granules are always found superimposed on the nucleus as well as in the cytoplasm Besides these specific granules the presence of which characterizes the cells of the granular series some cells of the non granular series eg monocytes and lymphocytes may show some stained particles in the cytoplasm of the cells which have been called azurophil granules These azurophil particles are of the same colour as the nucleus of the cell in which they occur but may be paler brighter They vary in size in depth of staining and in number In the lympho cytes the particles are few in number they are large coarse and darkly stained and occur in small groups in the cytoplasm while the particles in the monocytes are more numerous paler and finer and are more evenly distributed in the cytoplasm or darker ·TABLE 1 Identificalon of cells containing granules ✓ 4 Nucleol Nuclens Granules Cytoplasm Cell Mask REMARKS Present fine Ia oval fine strac Round or relatively chromatin turc Very few scattered Pale blue Relative Pre myelocyte ly small amount This as well as the other gradu lar ces may be neutrophy) cosinophil or basophil Basc phu pre myelocytes are rarely dentified prominent Very coarse Myelocyte Ib Generally absent Round or oval coarsa chromatin structure may not be very well stailed Very pale blue var able in amount but usually more than a la not well seen as it us covered by gra aules In the cos nophul and tasophil myelocytes the cytoplasm is almost entirely covered by granules and is rarely visible In the basophil the nucleus also is blurred by granules Meta my encyte IC Bez ar shaped chromatin turt hidney coarse struc (ranules less promi Large 113 amount 1sent than corres Very pale blue ponding granules obscured by gra in Ib numerous nules CELL IDENTIFICATION The basophil meta myelocyto and stal cells are very difficult to identify Id le (0) Absent Curved rout coarse liner and mre Large amount Very Staff (or band) chromatı struc numerous that pale blue ture corresponding gta nules in IC Lobed or segment Very fine and nume Larg amount Very Neutrophil rd z to 5 or Tous fight lilac light greyish bitte polymorphoriore lobes colour naclear granu locyte Usually bilobed Finer than eosia Very pale blue Losinophil rarely may have ophilic precursor scarcely seen granulocy e moro lobes id but coarser than Io (1) Segmentation of the very coarse dark ry pale blue B Sophil lobes 1 lifficult violet blue almost scarcely seen &ya Dulocyte to make out but back o'ten obs never more this cùns nucleus to lobes Je (18) Te (11) - 57 ·TABLE Y Identification of cells without specific granules Chromatin Size of nucleus in Structure Cytoplasm relation to cell Nucleo) Nucleus Azurophil particles Name of cell Identi fication mark - 111 (a) very fine Round oval or litegular Tine Light blue cear More than three quarters Myelobiast I Round or oval Finer than I Deeper blue than I clear Lymphoblast II Present Round oval or irregular Relatively coarse Light blue opaque More than two third's None Monoblust III Round or oval Fine stippled Light grey blue Opaque Akore than three quarters Proery throblast IV Round or oval Fone but not so Deep blue opaque More than half Pla mablast V fine as I and III Present or absent Kidney haped or are Coarser than III Light blue opaque May be p esent Premonocyte gular Round or oval Coarse Light blue clear May be present Large II (a) Lymphocyte Generally round or Coarse in clumps Clear transparent bluc may More than half in larger May be present Lymphocyte JIL (5) oval may be clover appear az thin run in cells but almost fills the few leaf unevenly smaller cells cells in smaller ones distributed strands Horse shoe or regular Coarse clumps or Ligat faded blue Opaque More than halk Usually present Monocyte III (b) Dume rous scattered Round or oval Coarse Basophils More than two thirds Nomoblast A IV (a:) Round or oval Coarser than Basophilic Polychromatic Not more than halt (IV(ai) Normoblast B IV (asi) or slightly eosinophilic Ronad or regular Pycnotic Polychromatic or Vanable Normoblast C IV (arsi) eosinophilic Round or oval Tine charactens Dark Grey blue opaque More than two third Megaloblast A IV (br) tic open net periouclear pale area inay work be present Round or oval Network is stall Basophilic polychromatic Less than two thirds open than None Megaloblast B IV (hu) or slightly eosinophilic Round oval or irregular Indefinite Polychromatic or Less than half Round or oval Eosinophilic Megaloblast C IV (611) Coarse Deep blue opaque with a Less than half Preplasmacyte V (u) Round or oval perinuclear pale area Very coars cart Deep Greyish blue ground Less than half ecentric Plasına cell glass V (6) Irregular Vulti Coarse lobed Light bue Variable Present fine Mega karyo VI HÆMATOLOGICAL TECHNIQUE more IV(61) wheel appear ince and Numeibus cyte* Mature platelets (Vla) which develop from megakaryocyte are small (2 34) ill defined structures not clearly differertiated into oucleus and cytoplasm ·TABLE XIII Identification of the diffrent nucleated cells of red cellsertes V Cell outline Cell Chromato Size Cytoplasm Nucleus Yucleoli Pr erythroblast 16 20: Irregular Light grey blue Large occup es must Fine stapple 1 stains lightly paque (basoph !!c) of the celi re embles a mass of unravelled Present fe y ap rear 2$ irre gular gaps yarn Absent Normobla t 4 Ir 171 Regular Blush grey opaque Large nccupies over two thirds of the cell Coarse arrangel rather half hazard in lumpy, dark staining masses with light areas be t een Normoblast B Rgular 3 141 Absent Basophilic polychro Occup es about hals Coarser than n normnhlat matc or eo TO of the cell sten philic n lumps o ca fonally has a cart wheel appearance CELL IDENTIFICATION Normoblast C 6 τομ Regular P lychromatic or Sometimes eccentric No definite structure Pycnotic 1bsent eosinophilic relativa ize variable appears as a drp of unk Dark grey blue Larve occupies more Tine has a charactenst c ofen Absent opique generally than two thurds of net ork yith 3 pale area the cell aroun i the nucleu Megalobin tA 14 21 Irre ular Megaloblat B 2 og Sightly. rregular than 10 Basophilc or poly Large but smaller | The characteristic open net york Threat cb omatic in megalo 15 still more open than blast A megaloblast A Plychromatic or Small stalas darkly No very definite pattern but still Ab ent co inophilic and generally may have some re emblance to eccentric the open network arrangement of the nucleus of the megalo 1 1?t er es Megaloblast C 9 15 Regular 59 ·58 TABLE XII Identification of the earhest differentiated cells Name of cell Nucleol Nucleus Chromatin Nuclear membrane Cytoplasm Auer s bodies I Myeloblast Fey not very Round or oval distinct usually central Fine reticulation seen Not distinct and no CD densation of chromatın at edges of nucl us Cleur light transparent blue May be occasionally II Lymphoblat Many distinct very Round or and central oval Fiber reticulation Distinct with condensa tion of chromatin at Clear blue deeper Not seen HAMATOLOGICAL TECHNIQUE the edges III Manoblast Of One often two Round or oval cl an often folde] Relatively coars Frequently cut Not distinct and no Light blue condensation of chc Dot SO matin at edge transparent Seen IV Pro-erythroblat Few appear Round or oval as irregular aps Fige stippled grey None Not distinct and no Light condensation of chro matin at edges glass blue ground Plasmablat res not 415 tinct Round or oval I ne Not di tinct Deep blue opique None

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·60 SCHEMA SHOWING ORIGIN OF BLOOD CELLS Frons retici lo endol selial system in bore arroz spleen lymph glands etc Extrar a cular development Intravascular dei elopment from bone baryo Probably ex (raiascular from bone maryoti Irom bor maryota From lyinphala tissue all over the body 175 cluding bone marrow Fronx reticulus mostly of spleen Pro erythroblast 1 Lymphoblast Monoblast Plasınablast Normoblast A Me aloblast A aloblast Megakaryocyte Pre monocyte Norinoblast B Ve aloblast B Mgeloblas Pre myelny te Myelocvie Veutrophil Eosinophil Basophal Bone in arrow HEMATOLOGICAL TECHNIQUE Normoblast C Vegaloblast Melangelocyte Ventrophil Eosinophil Basophil Pro plasmacyte (Turk celi) Reticulocyte Reticulocyte Large lymphocyta Large lymphocyte children) Reticulocite Pro plasmacyte (Turk ce 1) Rettculocyte Staff Neutrophil Cost of ke Basoph! Peripheral blood Segmented Veutrophil Eosinophu Basophi Small Ironphocyte }{onocyte Plasma cell Erytrocyts Mego ocylo Platelet

·Explanation of Plate No I
I
Vyeloblasts (\ucleou have not been reproduc d)
la Premyelocytes showing fine granules the fou th cell an eo inophil precurso
18 slightly more mature than the rest
Ib Nyelocytes the cells in the upper are less mature than those in the lower row
the first three in eich row are neutrophils and the lat two eos noph Is
Ic Jlett mycincytes the lat two (Ie 1) are eo inophils
Id Stall or band forms the last two are eosinophils (11) (Tle granule a ti
list cell have not been well reproduced)
le Jature granulocytes the first three are neutroph Is and the last t o eo inophil
Ib(11) I(1) and le(1) Lasop) tls myelneyte me ta myel vte and matur
The shape of the nu leus 19 the only feature on Zich the immature form
are identified in the original Irasing the shapes of the nucles were just
dis ernible but in the reproduction they are oba ured
III
111a
Ilib
Monoblasts the lower two sho ving Auers bod es
I remonocytes
Monocytes the se onl cell in the top rou might be ela ed sa premonocyt
but tie azure particles are well develoj el (There lould be more blue
colour n the cytoplam of these cells )
VI
Megaharyocyte these cells are often proportionately mua bgger than the ob
show
VIR
Platelets
Magnification about X 1 DUO
Sta nung-Vay Gun ald and Gems.

·PLATE No 1
la
Ta
Ia(1)
12 (10)"
Ib
IIb
1b (11)
Ic
Id(1)
n (LT)
Id
Id(1)
Id(11)
le
Tell'
Teh
Va
Ib (III)
IC(I) le (III)
4 Roy to

·Plate No 111 tylenat, mof 11 te in 11 Lymposiasis Ila Targe lume cytes with ature surtuk 111 Alature (mall) tu il cute the firt Sit Zut ir le (above) Irelanneute (Turk tell ) el ) I' ma cells (The elartt ritu tru tu of the ulu cell of the pl my cell serve not rert lens in ROS cell Tu ere hrull! n I lve colou in tleet sle of the Ia Lelt la et corner Creuslllue Aina Frythrocytes reticulo y es platelets Magnific ti n about x 1109 O Stain gay Goal Anima es et t? Lun in the left lus e corner in wait te ell strud with cre 1 blu (Ile Tethol Bs ) & n16 Cresyl blue starring Red cells Reticulocybes Platelets H Row ·PLATE NO II IV Tai Ib Vali Ib th Iam Mb IC Na a M Fyri ation of 1 lite lo II IT 110erythr blast lugornya! F71) rocyte Iar Tormoblast ! (Ar three tell ) () tel cell th wiog Hortelt 11111 Tormodlit B (first three celt) Jolly bol Ilain Tormodlast (fitt four cell (1) Kid cell showing Calots 11! Jegaloblast (Ia tino ell } tings Veganvlast B (m) Poisloste 11 jatoblait C (19) Het cell titi at pilog It 2. treulocytes Anpined runs all and Cirant evett figure ile in are starter fir thith cre il llur axl the systh right thin ile 23 wethol Br] FOS ·CELL IDENTIFICATION бI Red cells-Normally the red cells originate intravascularly from the endothe 1:al cells of the sinusoidal spaces and capillaries of the bone marrow The first differentiated cell is the pro erythroblast As da elopment proceeds the proery throblast passes through the different stages of normoblast and reticulocyte to dsvelop into the mature erythrocyte (1 :de schema p 60) in the process of matu ration the nucleus loses its nucleoli very early so that these are pot seen in the slommoblast A the nucleus also loses its reticular structure and becomes gradually coarser and coarser as development proceeds until in the normoblast C it becomes completely pycnotic after this the nucleus 1y lost (by fragmentation or extrusion) and the erythrocyte becomes fully mature The reticulocy te is a stage between the rormoblast C and the fully mature red cell but the reticulations are not seen itu a Romanowsky stained film In pathological condition as in cases of macrocytic anæmias some of the pro erythroblasts instead of developing into normoblasts develop into megalo blasts which passing through the different stages mature into megalocytes Nucleols are not seen open in the earliest megaloblasts megaloblast A In the earlier cells megaloblast A and megaloblast B the nucleus has a characteristic open network of the chromatin which is not een in the later stages megaloblast C Granulocytes The granular cells develop extras ascularly from the reti alo endothelial cell of the bone marrow The earliest differentiated cell is the myeloblast The first evidence of maturation of the myeloblast is the appearance of granules in the cytoplasın At first the granules are very fine and few as in the pre myelocytes bat gradually they become more numerous and coarser ind are coarsest at the myelocytic stage after which the granules again become finer and less numerous as development proceeds the fully developed neutrophil polymorphonuclear cells the granules are very fine and numerous The cells can be differentiated as neutrophil eosinophil and basophil according to the colour and size of their respective granules with difficulty the pre myelocy te stage but qaite easily from the myelocyte stage onward In the process of desclopment the rucleus loses its nucieoit these are ult defined in the pre myelocyte and are almost always absent from the myeloci te stage onwards The chromatin structu e of the nucleus which is finely reticular 11 the myeloblast becomes courser and more condensed as development proceeds and is iery dense at the later stages this condensation makes the nucleus smaller The shape of the nucleus too changes from round or oval at the earlier myeloblast pre myeloryte and myelocyte stages to kidney or bean shape at the meta myelo cyte stage and then it becomes elongated in the staff and finally lobulated in the Segmented forms The cytopla m shick blue and ranty in the myeloblast loses its colour gradually and becomes more abundant as dieselopment proceeds—in the later tages the cytoplasm is hardly visible as it is covered by the granales

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·CEIL IDFVTIFICATION 61 Red cells._ormally the red cells originate intravascularly from the endo he 1111 cells of the sinusoidal spaces and capillaries of the bone marrow The first differentiated cell is the pro erythroblast As des elopment proceeds the pro ery throblast passes through the different stages of normoblast and reticulocy te to dsvelop into the mature erythrocyte (lide schema p 60) In the process of matur ration the nucleus loses its nucleoli very early that these are not seen in the normoblast A the nucleu alo lo-es its reticular structure and becomes gradually coarser and coarder as development proceeds until in the normoblast C it becomes completely pycnotic after this the nucleus i lost (by fragmentation or extrusion) and the erythrocyte becomes fully mature The reticulocyte 1 a stage between the bormoblast C and the fully mature red cell but the reticulations art not seen in a Romanowsky stained film In pathological conditions as in ca es of macrocytic anæmias some of the pro-ers throblasts instead of developing into normoblaşts develop into megalo blasts which passing through the different stages mature into megilocytes Vucleoli are not seen even in the earliet megaloblasts megaloblast A the earlier cells megaloblast A and megaloblast B the nucleus has a characteristic open network of the chromatin which is not seen in the later stages megaloblast C Granulocytes --The granular cells develop extra ascularly from the retirulo endothelial cells of the bone marrow The earliest differentiated cell is the my eloblast The first evidence of maturanon of the my elablast as the appearance of granules in the cytoplasm At first the granules are very fine and few as in the pre myelocytes but gradually they become more numerous and coarser and are coarsest at the myelocytic stage after which the granules again become finer and less numerous as development proceeds In the fully developed neutrophul polymorphonuclear cells the granules are very fine and numerous The cells can be differentiated as neutropbil eo inophil and basophil according to the colour and size of their re pective granules with difficulty at the pre myelocy te stage but quite easily from the myelocyte stage onwards In the process of des elopment the rucleu loses its nucleolithec are all defined in the pre myelocyte and are almost always ab ent from the myelocy te stage onwards The chromatin structu e of the nucleus which is finely reticular in the myeloblast becomes coarser and more condensed as deselopment proceed and is very dense at the later sta.es this condensation makes the bucleus smaller The shape of the nucleus too chan,es from round or oval at the earlier myeloblast pre myelocyte and myelocy te stages to kidney or bean shape at the meta myelo cyte stage and then it becomes elongated in the staff and finally lobulated in the segmented forms The cytoplam which is blue and canty in the myeloblast lo es its colour gradually and becomes more abundant as development proceed in the later tages the cytoplasm is hardly visible as it is covered by the granules ·62 HÆMATOLOGICAL TECHNIQUE lonocytes -The monocy tes originate from the reticulum all over the body but particularly from that in the spleen The first differentiated cell is the monoblast it proceeds through the stag of the pre monocyte to the stage of fully developed monocyte As development proceeds the nucleus loses its nucleoli and the fine reticular structure becomes coarser and very fine and numerous azure particles appear in the cytoplasm of a fully developed monocyte The nucleus which is round or oval in the monobla + becomes slightly indented in the pre monocyte and may take on rarious shapes in the mature monocy tes (We have made no attempt to differentiate betwen monocytes and histiocytes this can only be done by a supra vital staining process which we are not describing here Some of the large mononuclear cells in the peripheral blood are undoubtedly histiocy tes who e origin is probably different from that of the monocytes ) marrow Lamphocytes The lymphocytes originate mainly from the lymphatic tissue of the lymph glands all over the body and to a small extent from the lymphoid tissue in the The first differentiated cell is the lymphoblast which proceeds througn the stage of large lymphocyte to the fully developed (small) lymphocyte In the process of development the nucleus loses the nucleolt the fine chromatın structure becomes coarser and finally in the mature cells it is Pycnotic The cytoplasm which is definitely blue in the lymphoblast becomes lighter in colour as development proceeds but is always trursparent In some of the cells a few coarse irregularly scattered azure particles may be seen 12 TECHNIQUE OF STERNAL PUNCTURE Bone puncture as an aid to accurate diagnosts of blood diseases and to the study of their ætiology has only come into general use during the last few years the main reason for this was that the methods used for obtaining material from the bones were comparatively difficult and usually painful unless anästhetic was given before the Salah sternal puncture needle was introduced This handy and inexpensive instrument is now used widely not only in hæmatological work bat in the diagnosis of kala azar and other protozoal and bacterial disea es We have used this needle for about four years and have adopted sternal puncture as a routine procedure in all cases of aflæmnia The senior writer described this technique in a paper written in co operation with Dr P C Sen Gupta* (Napier and Sen Gupta 1938) since then as a result of further experience certain modifications in technique have been introduced and these are incorporated in this description but the technique described materially the same as that given in the above mentioned paper lith wbose kund permis, on the figures from this paper have been reproduced ·TECHNIQUE OF STERVAL PUNCTURE 63 Apparatus required The sternal puncture needle (0) Two Record syringes 2 c cm ( A pair of scissors and a shaving set (12) Two per cent solut on of novocaine or any of its substitutes cent solution podues gnod an, sth w) (v) Absolute alcohol and ether (vi) Collodion or tincture of benzoin (11) Cotton wool etc (Pitumenit + per The syninge The needle 1 dry sterilized in a hot air sterilizer in a test tube is sterilized by boiling and then dried thoroughly by first driving out water with alcohol and later alcohol with ether and finally by drawing in and expelling hot air (through the flame of a gas burner or Pirit lamp) as B The sternal puncture needle The Salah needle used for sternal puncture is shown (figurē 17) It i made of rustless steel and the bore is about the sime as that of a lumbar puncture needle The guard Con the needle can be moved SO to A adjust the depth of the puncture Usually the guard has to be fixed at a distance of 1 to I) cm from the tip in order that the mar To 17 -Needle B with stylet A remo guardi C (actual size) row may be reach ed This distance will vary with the thickness of the skin and subcutaneou ti te of the thoracic wall in fat individuals as much as 2 cm may be required and in very emaciated ones less than I cm It may be found advisable to readju t the guard after the needle has reached the periosteum before it is pushed through the puter plate into the marrow cavity The stylet A is kept in while the puncture 1» being made and is withdrawn after the cavity is reached muv bi Procedure The hair over the sternum f there is any first clipped with a pair of scissors shaved with a razor and the skın finally cleaned thoroughly with alcohol The best site for the puncture i just to on side of the middle line at the level of the second intercostal space This area is first anesthetized by infiltration with a 2 per cent solution of norocaine or its ub titute Some solution is first injected into the skin with a fine needle attached to a 2 c cm syringe then the needle is pushed down to the periosteum and the rest of ·64 HÆMATOLOGICAL TECHNIQUE FIC JB -Needle s position after the marrow cavity has been penetrated the solution injected About 1 cem is usually sufficient in a thin individual but more is required where the subcutaneous tissue as deeper After an sterial of a to wo minutes the actual puncture is made The apparatus is held with the knob of the stylet in the palm of the hand and the needle itself between the thumb and index finger the latter on the guard C of the needle Pressure is applied and the shin and subcutaneous tissues are prerced a rotatory movement will then facilitate puncture of the outer plate of the sternum (figure 18) As the external plate of the sternum B pierced and the marrow cavity u Entered there is a sensation of loss of resistance just as felt on entering the spisal canal during lumbar puncture The stylet is now taken out Record syringe is attached to the end of the needle and the marrow blood is aspirated When the fuld s aspirated the patient feels a dragging pais (figure 19) which is a guide as to whether the needle ss in the marrow cavity or not About o scom of marrow (sinusoidal) blood is removed and the syringe and the sternal puncture needle are withdrann digital pre sure is applied over the puncture for a minute or tud and the passicture is sealed ss3th collodion The needle is detached from the syringe and the latter 13 interted several times so as to mix the contents thoroughly thes small drops are placed on clean shdes and smears are made 126 cm ·TECHNIQUE OF STERNAL PUNCTURE 65 (vide p 43) The rest of the fluid is put into in orlate tube for total nuclcatel cell count Only very rarels will onc fail to obtain blood The commonest error is to ful to allow a sufficient length of needle In this case the guard must be adjusted slightly the stylet replaced and the needle pushed in 1 little deeper Occa sionally the needle goes too deeply and has to be withdrawn slightly before blood will come Our only complete failure was in 1 case of leukæmia and in none of our cases of leukæmin has the blood come freely as the marrow N piched with cells in such cases Fro 19._The stylet tas hen removed and nu o lal bloo1 being drawn into a Record svringi usually the only painful part of the operation Not more than 0 cm of lilond is aspirated because if more is drawn there 19 a probability that the negatne pressure in the marrow cauty will draw blood from the vessels in the locality and thus dilate the sinusoidal blood It is prob ably impossible entirel to present this occurrin, therefore a constant amour t of blood is drawn to obviate gross differences in the degree of di ution in the samples taken from different persons The syrin e is inverted several times in order that the contents may be mixed thoroughly This is necessary because the fluid that comes out at the beginnin, is not the same as regards cellular content as the fluid that comes out towards the end of the aspiration Ile Aar Pep Calcutta $ horl of Trop ved for 1938 ·66 II TBIATOLOGICAL TECHNIQUE Examination of material (1) Fxamination of the stoned smerrs -- The sternal puncture smears are best stamed with combined Muy Grunwall and Gremsz stamng this shows the nuclear details very clearly railing this satisfactory results for ordinary work may be obtained by stuning witu Leishmans or Wright s stain (ude p 45) An accurate diflerential count of the different nucleated cells is made by counting 500 or more nucleated cells from different parts of a well stuned smear Tne criterin on which the different cells are identified are given above (2) The ovalated specimen is examined for enumeration of total nucleated cells (vde p 20) (3) The oxalated specimen may also be examined for estimation of humo globm (vrde p 3), enumeration of red cell (ride p 13) and estimation o reticulocyte percentnge (inde p 21) if the informations are required for any special purpose Discussion The material which is obtained by this procedure is neither peripheral blood (obviously) nor bone marrow but is blood from both the patent and the closed sinusoidal spaces in the hamopoietic tissue of the bone marrow, in which are me ed a few cells detached from the walls of these sinuses by the intruding needle or by the act of aspiration The extent to which the detached tissue cells are add ed tothe sinusoidal blood probably varies with each puncture and constitutes tlı weahness of the procedure from the point of view of obtaining a true and unvary ing picture of bone marrow hæmopoietic actuty However the extreme vara tions in a single subject that have been reported by some workers and the differ ences in the normals given by diflerent workers can probably to some extent be accounted for hy variations or defects in the technique employed eg by the fail ure to remove small and constant amount of blood by failure to mix the blood before making smears or by mal ing counts from one part of the blood film only Zanaty (1937) his pointed out that wide variations may exist in the total nucleated cell counts and that from these counts little idea of the actity of the bone marrow can be obtained but from our esperience of sternal puncture we believe that there is generally a distmet correlation between hainopoietic activity and the total nucleated cell count though this may on occasions le misleading Both the hæmoglobi and red cell count are s),htly lower and reticulocyte count slightly higher in the sinusoidal blood than in the senous blood but these estimations are of little practical alue Normal standards It is useful to know what proportions of the various cc]ls one may expect o find in a sternal puncture The data given by trous writers are difficult to correlate beciuse they hase adopted different methods of classification and different nomenclatures and we prefer to quote only our own findings ·TECHNIQUE OF STERVAL PLUCTURE 67 The data gien in Tables VIV and \l are from two sources (a) First senes to normal individuals males 2 c cm of fluid taken (6) Second series 53 subjects infected with filanasis 05 ccm of Aud taken We have quoted the latter series because we beliese that they represent the findings in normal individuals very clea and because it is not easy to persuade 50 perfectly healthy individual to undergo a sternal puncture One might expect them to differ from the normal in the matter of the eosinophil cells but in actual fact the percentage is lower than in the first normal series The material differences that will be noted between the two series are almost certainly due mainly to the fact that in the second series we took a smaller amount of fluid and that there was consequently less dilution by systemic blood The main discrepancies are the higher total nucleated cell count in the second series_higher than the normal figure u.ually quoted a lower myeloid nucleated red-cell ratio-also lower than the usual normal figure a hi, her lymphocyte per centage and a higher maximum figure for most of the cells_which may be due to the larger number of subjects insolied in the second series For the findings of other workers reference hould be made to Zanaty (local) and Scott (1939) TABLE VIV Sternal puncture nomal data FIRST SERIES SECOND SERIE Vean Standard deviation Vean Standard deviation 13 43 to 93 Hemoglobin in grmmes per 100 ccm Ked c Ils per c mm in millions 499 t 40 Rticu ocytes-percentage of red cells o 75 # 039 Total nucl atel cells per c mri 53 500 + 26 500 UT 678 832 Yucleated red cells-percentage of total pucleated cells 25 75 # 407 27 606 + II 745 Leucocytes—percentag of tota nacealed celis 74 25 + 407 724 Cranul cyt su-percentage of total nu leat ed cells 64 40 5 10 51 529 II 431 Non granular 1 cocytes-percentage of total ) ucocytes My lou1/nucleated red-cell rati) 9.85 + 213 17 839 16 796 250 1975 We seldom identify cell as lymphoblasts or monoblasts except in cases of leukæmia but myeloblasts are occasionally found and appear in most of our ·68 TABLE YV Sternal puncture differential nucleated cell count SERIES I Range Blean SERIES II Range Mean Standard deviation o o to 15 07 oo tu 325 092 to 7366 Megaloblasts Erythroblasts oo to 33 049 + o 6619 Nucleated red cells 0 4 to 1o O 34 Macroblasts voto 875 2 075 # 1 7675 Normoblast Ibo to 2,5 21 6 75 to 63 2 24 17 + IO 450) White cell series Myeloblasts 04 to 15 I 2 oo to o 75 015 to 2670 Pre myelocytes oo to 15 07 bo to 20 04 + 0 4409 Myelocytes Neutrophil Io to 95 4 ro to 18 75 8 30 3 2061 Eosinophil 0 4 to 26 13 oo to 40 1 445 + 0 9198 Basophil None found None found A Granular senes Meta myelocyte neutrophil 6 4 to 150 97 0 4 to 8 5 2 76 1 7257 Stafi or band neutrophil 14 6 to 38 5 53 12 4 to 600 33 91 + 9 1933 Segmented neutrophul 65 to 256 16 8 o o to 236 5 35 + 5 0232 Mature eosinophils oo to 76 47 ooto 7+ 2 77 + 1 6539 Basophils oo to 10 02 oto 1 2 to 090 Lymphocytes 30 to 120 6 25 29 to 34 5 15 68 + 7 2623 Non gripular series Large thononuclear3 I 2 to 70 30 oo to 35 I 585 to 9386 Plasma cells oo to 1 6 06 oo to I 5 0 31 After Sabia School and not according to Isræls nomenclature which has been given in the tert The term megalobla t is us 1 here to denote the earliest differentiated percursor of both normal an abnormal red cells HEMATOLOGICAL TECHNIQUE o os ·THE ARSETH AND SCHILLING COUNTS 69 counts forming on to o2 per cent of the nucleated cells In a properly drawn and well stamed smear there are very few disintegrating cells and practically all the nucleated cells seun can be identified in a highly actre martou however there are some cells which show mitotic dr ision and are difficult to identify properly 13 THE ARNETH AND SCHILLING COUNTS Amcth (1904) divided the neutrophul polymorphonuclears into five main classes according to the number of segments in the nuclei which in their turn were classi fied into various groups according to the shape of the lobes Under the original classification the count was very complicated and had only limited application in general practice Cooke and Ponder (1927) modified and simplified the Arcth count taking into account only the five original basic divisions of the Arneth count without going into the intricate ubdivi ions of Ameth Schilling deui ed a method in which the differential leucocyte count and a simple nuclear lobe count of the neutrophils were incorporated and considered in the form of a hemogram Apparatus required (vide 4 et seq) Smears -Smears for the Arneth and Schilling counts should be very thin and uniformly spread they should be dn d quichly by being waved to and fro or held under a fan (uide P 43) Slatning -Combined staining with Vay Grunwald and Gremsa stains gives the most satisfactory result--the nuclei stain deeply and the connecting chromatin filaments are well shown while the granules also show characteristic colour and size If Vay Grunwald and Giemsa stains art not available proper staining with Wright s or Leishman s stains though not as satisfactory grues quite good results Iron hæmatorylın or hæmaton lin staring for these counts in preference to any of the Romanowsky stains is sometimes advocated while these stains bring out the nuclei and the chromatin filaments well the colour of the granules is not distinc tre so that differentiation between neutrophils and eosinophils is very difficult (A) ARNETH COUNT The nucleus of the neutrophil prlymorphonuclear cells is divided into lobes which may be separate or joined together by fine filaments of chromatin Cooke s criterion of separate lobulation is that either the lobes should be distinctly separate or should be joined together by a fine chromatın filament but not by broad bands of nuclear materal Lobulation of the nucleus it is assumed gives usually an indication of the age of the cell—the fewer the lobes the younger is the cell youngest cell in the normal peripheral circulation is one which has a nucleus shaped Jike the letter C while the cells with five or r orè distinct nuclei are the oldest The ·70 HEMATOLOGICAL TECHNIQUE The principle of the method is to make a differential count of the neutrophil cell to group the cells according to the lobulation of the nucle and to calculate the percentage of cells in each group The cells of the various types can be described as follows Class I Neutrophil granulocy tes in which the nucleus has only one defi nite lobe or two or more lobes joined by a definite band of chromatin and not by thin filaments Class II When there are two separate lobes joined by a thin filament or when there are two completely separate lobes Class III When there are three separate lobes connected by thin filaments or there are three completely separate lobes Class IV When there are four separate lobes connected by thin filaments or there are four completely separate lobes Class V When there are five or more separate lobes connected by thin fila ments or there are five or more completely separate lobes When in doubt about the number of lobes in cells with three or more lobes they must always be placed into the next lower class for example if there is any doubt whether a cell has three or four lobes it should always be placed in class III The count is made by identifying and classifying one hundred consccntia neutrophil granulocytes from different parts of the slide and the result is usually reported in the following form – Example 1 a normal count (European standard) Class I II III Number 25 47 V IV 16 Total 100 ΤΟ 2 When there is an increase in the number of cells with one and two separate nuclear lobes ie of class I or class II at the expense of those with three or more lobes there is said to be a left shift in the Arneth count as in the example gren below Erampie z a lejt shift count Class 1 Number 41 II IV V Total 100 14 1 0 Conversely when there is an increase in the 4-- and multi lobed nuclei it is known as a right shift V Example 3 a night shift count Class I Number 9 II 22 III 36 IV 23 Total 100 10 Various indices and methods of expressing more concisely the results of the Ameth count (Cooke s modification) have been proposed Probably the most suitable of these methods is the calculation of the weighted mean of the nuclear ·THE ARNETH AND SCHILLING COUNTS 71 lobes this is obtained by multiplying the number of cells in class I by I thu number in class II by 2 the number in class III by the number in class IV by 4 the number in class V by, and then adding together and dividing the sum total by the number of cells counted The weighted means of the nuclear lobes in the three examples are calculat ed in the following way - Example Example 2 Example 3 1 x 10 = 10 IX 4 = 41 9 2 x 25 = 50 2 X 44 = 88 2 X 22 S ++ 3 x 47 141 3 x 14 = 42 3 X 36 = 4 x 16 64 4 X 4 4 X 23 9 5 X TO 0 O 5 ΙΟ 50 IOO 275 100 175 TOU 303 Weighted mean 275 I 75 303 Interpretation - Immature granulocytes and cells of Argeth class I and some of class II but none of cla s III IV or are found in the bone marrow If there be a sudden stimulus to the bone marrow to produce granulocytes a in an infeu tion the leucocytosis is brought about by young cells appearing in the blood the number and the proportion of these depending upon the nature of the stimulu and the reacting power of the marrow of the individual and there is a hift to the left in the Arneth count But leucocyto is as a result of muscular exercise i only due to redistribution of cell to not accompanied by any increase of young leuco cytes in the peripheral circulation and therefore not associated with any left shift Again in chronic infection or toxæmia there is an increased leucocyte destruc tion which may or may not be balanced by next leucocyte formation in the for mer case there will be leucocytosis or a normal count and in the latter leucopenia In either case there is an increase in cells of class I and II actual or relative wbich results in a shift to the left and a decrease in the weighted mean It wat be apparent that this shit is quite independent of the total lencocyte or total granu locyte count and provides another indication of the degree of the tovæma In pernicious anæmia and certain other conditions of bone marrow dysfunc tion there is a shift to the right in the Araeth count and therefore an increase in the weighted mean in such conditioes there are many multi lobulated cells and only very few young cells coming into the circulation ·72 HÆMATOLOGICAL TECHNIQUE Normals I II IV Weighte! men Cook and Ponder (1927) I 25 47 ut 275 13 39 10 4 262 hennedy (193) (fraq) Das rupta (Calcutta) 37 44 16 3 0 TRS I (B) SCHILLING COUNT In this count the neutrophil granulocytes are divided into fosir groups and classified as follows Myelocytes (M) Juveniles (J) these have a distinct indentation in the nucleus they correspond to our meta myelocytes (vide p 56 table XI) 3 Stab cells (St) these have a C- or horse shoe shaped nucleus but no true lobulation they correspond to class I of the Arneth count and to our staff cells 2 Arreth 1 11 111 2 (Dig) M st seg Schilling Stab cells may be of the regeneratie of degenerative tvpe and are differen tiated by the shape and staining reaction of the nucleus in the latter the nucleus bnbbon like and pycnotic with clumps of chromatin and usually irregular shaped and the cytoplasm may be sacuolated ·THE ARNETII AND SCHILLING COUNTS Segmented (Seg) polymorphonuclears showing two or more distinct lobes According to Schilling the following are the constituents of a normal hæmogram in a German population - Total leucocytes Percentages St Seg Mon 5 000 to 8 000 35 57 67 21 35 48 60 70 B = basophil L = lymphocyte E = eosinoph? Mon large mononuclear Discussion --The Schilling hemogram is much more than a differential granu locyte count as is the Arneth count and Schilling claws that it gives far more information The interpretation depends on a highly imaginatie series of res ponses to infection and toræmia that are supposed to and possibly do occur in the hæmopoietic system in which there are three phases neutrophilic struggle monocytic defence and lymphocytic cure but it does not seem to take into account the nature of the infection and the consequent variations in the response Further there are so many directions in which changes may take place that the hæmoram seerns peculiarly susceptible to facile interpretation to suit the consci ous or unconscious wishes of the uterpreter We do not propose to give a full discussion on the interpretation of the hæmo gram as it has only an indirect bearing on the subject of anæmra but as far as the neutrophil granulocyte elements are concerned Schilling s contention is that infec tron may cause two types of reaction the regenerative reaction and degenerative reaction in the regenerative reaction new leucocytes are formed in the bone mar row and this is reflected in the peripheral circulation by a rise in the total white cell count an increase in the juveniles and regenerative forms of stab cells while m the degeneratve reaction whichi 13 ivand in severe toxærma on accurant of the degenerative influence of the toxio on the marrow the formation of new cells u retarded this may be shown by a leucopenia In the differential count of such a case there are few juvenile cells and the stab cells that reach the circulation fail to segment at the usual rate and may degenerate with the result that there is a preponderance of stab forms including many degenerative forta in the blood The reaction however may not be purely regenerative or degenerate in character but may be a mixture of the two and the hæmogram may be intermediate with slight leucoperta and a greater preponderance of the younger cells than in a purely degenerative process 10 ·1 FVATOLOGICAL TICINIQUE Normals 1 1 j1 It 1 Weighted mean Cooke and Pander (1927) 10 25 Z 275 *3 37 13 10 4 1 G2 henne ly (1939) (Iraq) Das (upta (Calcutta) 37 3 0 + 85 (B) SCHILLIAT COUNT In this count the neutrophil granulocy tes are desided into four groups and classified as follows Myelocytes (1) Juveniles (J) these have a distinct indentation in the nucleus they correspond to our met my elocs tes (wide r su table XI) 3 Stab cells (St) these have a C- or horse shoe shaped nucleus but no true lobulation they corre pond to class I of the Arcth count and to our staff cells 2 Arneth 1 11 it IV V (Det? St seg Schilling Stab cells may be of the regenerative or degeneratre type and are different tiated by the shape and staining reaction of the nucleus in the latter the nucleus bnbbon like and py cnotic with clamp of chromatin and usually irregular shaped and the cytoplasto may be vacuoated ·THE ERYTHROCYTE SEDIMENTATION RATE 75 WESTERGRENS METHOD Apparatus required () Sumantatın tubes Mestergren tube This is about 300 m long and is graduated from o to 200 mm in i midterv 1 it has a uniform bore of about 3 mm and is open at both ends (cial Il est rgren rack or an improved rack to keep the tubes in a vertical position anl to prevent the blood escaping ( ) Inti-coagulant-38 per cent sodium citrate solution (10) Syringe etc for collection of blood (v) Stop watch Technique - In this method 38 per cent solution of sodium citrate is used as anticoagulant in the proportion of one part of citrate solution to four parts of the blood Put exactly 2 c cm of blood into a flask to which o 5 c cm of 38 per cent solution of sodium citrate has already been places! Mie immediately by rotating the flash and subsequently if possible in a shaker then draw up the mixture into a Westergren s standard tube to the zero mark which is exactly 200 mm from the tip The tube is now set upright in a stand in which a spring clip holds the points of the tube firmly against a rubber cork at the bottom end The tube is now I ft to stand and the upper level of the red cell column which is generally sharp rcad at the end of one and again after two hours* The length of the column of plasma from the zero mark to the top of the red cell column is the amount of edimentation and this is read off in millimetres this us reported as the sedimenta tion rate in millimetres for one hour and for two hours Vom al lumits (afte ono hour)- Ven 3 to 5 mm Women 4 to 7 min Textbooks Our own experience in Indians a Calcutta 3 to 15 mm 5 to 40 mm The high figures that we obtained in apparently normal subjects are partly explained by the higher temperatures that preva:led compared with those of the places where the tests on which other normal figures are ba ed were carried out WINTROBE S METHOD Apparatus requ red (Introbes tubeta At bottomed glass tube 115 mm long graduated from o to 100 in i mm intervals at bas a uniform bore of 25 mm (1) Anticoagulant-solium and potass um oxalate mixture (vide P32) (1) Electric centrfuge Together with a syringe etc and stop watch as in Westergren s method Technique-Wintrobe used his cell volume tube for the determination of the sedunentation rate Only one hour readings are usually recorded ·74 HEMATOLOGICAL TECHNIQUE (It is surprising to the writers that no provision is made in the classical Schil ling hæmogram for the separate counting of the regenerative and degenerative forms of stab cell when obviously the presence or absence of the latter is an important factor in the interpretation of the hæmogram) Conclusion - The interpretation of the Arneth count is simpler though the implications are more limited but it presents a minor practical objection in that more skilful staining is required to ensure accurate counting of the separate lobes The value of these counts is mainly in establishing the presence of infectne or tovæmic factors which may be important contributory causes of the anæmia and/or muy prevent response to appropnate hæmatınıcs The presence of multi lobular cells shown only in the Arneth count ?important in the diagnosis of pernicious anæmi? 14 THE ERYTHROCYTE SEDIMENTATION RATE The determination of the erythrocyte sedimentation rate (ESR) is not a test that gives much direct information regarding the nature of the anæmia but it is of value in differentiating functional" disorders such as pernicious anæmia in which the true ESR is within normal limits from anæmia due to such causes as sep»rs and malignant disease in which the ESR i murhedly increased Further as from the single specimen taken in the normal course of a routine blood examination the ESR can be estimated without additional labour it should always be included The test suffers from the multiplicity of the methods by which it is performed and by the variety of the forms in which the results are expressed The most commonly used technique is that of Westergren (1926) but as it is important that the correction for anæmia should be made and as no correction chart has been worked out for this technique this method has its limitation Wintrobe s method (1933) is really a modification of this method wt has the advantage that the cell volume can be estimated on the same sample of blood and also that a correction for anæmia can be made Principle of the test -A known volume of blood is intimately mixed with a definite amount of antı coagulant and the mixture is drawn into a tube of known calibre up to a certain point the tube i then allowed to stand in an upright posi tion at room temperature and the level of the red blood cells in the tube is noted at the end of one hour and again after two hours The fall in the level of the red cells is expressed in millimetres ·THE ERYTHROCYTE SEDIMENTATION RATE 77 To make the correct allowance for this fact Ily nes and Whitby (1938) norked out a chart (fig 20) showing the relationship of cell volume and sedimentation rate in normal blood and in blood showing various degrees of anæmia lonial Is its (after one kous)- len Women oto 9 mm oto O mot Textbooks Our own experi nce in Indians 1a Calcutta 2 to 20 min 2 to 30 mm Precautions All apparatus must be scrupulously clean and dry (11) The dilution with sodium citrate solution in Westergren s method should be absolutely correct (:) The specimen of blood should be collected during the fasting state and examined within one hour of collection (10) Inuomen its advisable to avoid the men trual period and the few days before and after it (v) The specimen should be rejected if there is a slight trace of coagulation or any marked hæmolysis (ur) The tubes should be set up in an absolutely vertical position in a quict place well away from any apparatus that ubrates The temperature of the room should be between 72 and 80 T whenever possible (uni) In recording the results correction for anemia must always be applied Discussion -There are a number of factors insolied in the phenomenon of erythrocyte sedimentation but the mot unportant is the size of the rouleaux for mation and this is mainly determined by the composition of the plasma and to a certain extent by changes in the hape and size of the red cells Increase in plasma fibrinogen will cause an increa e in the size of the rouleaux formation and therefore an increase in the sedimentation rate It is probably on this fact that the value of thi test depends Erythrocyte sedimentation rate is also greatly influenced by changes in the concentration of the red cell in the plasma that is any associated arma Thy may overshadox other changes but being a mea urable factor must be allowed for in estimating the true sedimentation rate The sedimentation rate is increased in so many conditions that it is very danger ous to attach any specific diagnostic significance to it This must be quite obvious from the wide range of the ESR rates that has been recorded in apparently healthy individuals However the ESR is a test of great value in estimating the progress in chronic infections such as tuberculosis and leprosy A standard technique hould be adopted and in reporting the results the method employed must be stated or the information losps much of its value ·70 HEMATOLOGICAL TECHNIQUE The blood is collected in an Oxalated flask and is thoroughly mixed the cell volume tube is filled with the oxalated blood by means of a capillary pupette up to the zero mark which is exactly 100 mm from the bottom The tube is now allowed to stand upright in a vertical position on a special stand or on a tray of plasticine and the reading is taken at the end of one and again after two hours in the same way as in Westergren s method the cell volume tube is then put into a centrifuge and centrifuged at 3 000 revolutions per minute for half an hour to obtain the pached cell volume percentage thus both procedures are done in the same tube with the same blood sample Correction for anæmia -One important factor in the rate of sedimentation of bodies in fluid is the concentration of these bodies The sedimentation standards for red cells in plasma are based on there being a normal number of red cells in a given volume of blood and in anæmia when these are reduced the sedimentation rate is increased above the normal irrespective of the other changes in the blood 100 90 80 70 80 Sed mentation rate mn in Aour 594 42 30 10 60 10 20 20 40 50 Volume of packed & cefis com De 100 CM of Blood JIG 20 Chart for correction of sedimentation rate for anæmia by means of the corpuscular volume To correct for anæmia find the junction of the lines of the observed sedimentatınn rat and the observed corpuscular volume in the chart this point will fall in on of the fire zones (normal doubtful ets) which indicate the approximate degree of increase in the fate If a compensated figure is required follow the appropriate curve down to the point where it cuts the 45 c cm vertical lines for men and the 40 c cline for women Example -Observed rate=50 mm obsen ed corpuscular volume 30 per cent point of Junction lies ia area of slight increase compensated rate for men=18 and for women = 28 ·GASTRIC ANALYSIS 79 Fractional gastnc analysis gives valuable information as to the motility and secretory functions of the stomach This method is followed by us in the investi gation of all cases of anæmia in the hospital and in many of the cases attending the anamia outdoor clinic In the course of the fractional gastric analysis an injection of o5 mg of histamine is given one hour after the test meal to the patients who fail to show any free acid in any of the previous specimens Apparatus requ red () Rehfuss or Ryles tube or any suitabe modifi cation (ride figure 21) These are tubes made of good rubler They are about 3 inches long with and inter FIG 21 nal duameter of about th inch One end of the tube 19 open while the other the tip ends in an Olive haped bullous protuberance filled with metal to weight it and keep it ngid There are h es a the rubber at ornar the tip for the passage of the gastric juice through the tube The lastance up to which the tube has to be passed is shown by a mark near the open end al out 20 to 22 inches from the tp and is kept ith teats Before use the tube is sterilized in boiling water for a minute or two coile ! up in a stenlız d petr dish until required (11) Record syringes 10 C CITI andt (v) Cap llary Pipettes I ccm (vi) Smal glass funnel (1) Twenty five ccm burette gra (zu) Porce ain di hes-3 duated in 1/10th of a c cm (011) Glass rous -3 with a stand (1x) Centrifuge machine (iv) Twelve labelled te t tube а а (*) Slides rack plus a few extra tubes (xi) Microscope I 10 Chem cals required () One per cent solution of cocaine (11) Adrenaline chloride olation or a suitable substitute 1 000 fo injection ( ) Seven per cent alcoho! 100 ccm (Un) Sulphur po der (1) N/10 2011 um hydroxide fur) Benzijine Dimethyl am do a obenz ne 05 (1) Glacial acetic ac d per cent in 95 per cent alcohol (xt) Hydrogen perox (Topfer 3 re-gent) (*11) Pepsin (v) Ph nolphthalein I per cent on (**) Mett s albumin tutes (ur) alcohol (XIV) D lute lyd olonc acid (21) Histamine 5 mg in 5 ccm (ru) Fresh mlk st nlized dist lled water (rt ) Lugols dine solut on ROUTINE PROCEDURE The following is our usual procedure in carrying out a fractional gastric analy1 A Preparation of the patient On the previous night the patient has hi usual meal at 8 pm and at 10 pm he is given four charcoal tablet with a glass of milk The next morning he is not ·78 HEMATOLOGICAL TECHNIQUE 15 GASTRIC ANALYSIS Introduction - The gastric juice in the normal individual contains hydrochloric acid free and in the combined state the enzymes pepsin and rennin and the intrinsic factor of Castle Examination of the aspirated gastric juice primarily for acidity but also for the presence of the enzymes and for other normal and abnormal characters is known as gastric analysis We have included gastric analysis in this series on hæmatological technique because normal hæmopoiesis is to a large extent dependent on normal gastric function and consequently the knosledge that we obtain from this test is important in both the diagnosis and the treatment of the anæmias Achlorhydria or hypochlorhydria is associated with deficient digestion and absorption of a number of food substances In some cases of microcytic anamia achlorhydria is considered to be the main ætiological factor iron is more easily absorbed from an acıd than from an alkaline medium Free acid is necessary also for ensuring proper peptic digestion (vi) Further in the absence of hydrochloric acid fermenting organisms flourish causing fatulence meteorism and darfhcea This mucosal dysfunction leads to a macrocytic anæmia the anæmia of sprue is often of this type and due to this cause but neither in sprue nor in nutritional macrocytic anæmia is achlorhydria constant nor is there any evidence that the intrinsic factor is also absent Finally in pernicious anæmia there is complete and constant achylia * which is associated with the absence of the intrinsic factor though this latter deficiency cannot be demonstrated directly in the laboratory In practically all cases of anæmia associated with achlorhy dra or hy pochin rhydria the giving of dilute hydrochloric acid alone or with pepsin will be bene ficial and may in fact constitute an important part of the treatment 1/ethods Gastric analysis can be done by either of the following methods (1) Single examination This 1 an old method and is obsolete non In this method after complete evacuation of the fasting juice an Ewald meal consisting of bread and water is gren to the patient and the stomach content withdrawn again after one hour (11) Fractional analysis of Rehfuss In this method after complete evacua tion of the fasting juice a suitable test meal as given to the patient small samples about 10 c cm of the gastric ffud are drawn off every 15 minutes up to 21 to 3 hours The following terms are in common us for the different stages of gastric d ficiency Achlorhydra (or false = absence of free hydrochloric acid dunag an ordinary ach orhydna) test meal in which histamine was not given Complete achlorhydna = absence of free acid even after histamine injection Achylia = absence of free acıd and the gastric enzymes even after bistamine ·GASTRIC ANALISIS 81 After complete evacuation of the stomach contents the test meal 1 gen to the patient with the tube in situ D Test meals An ideal test meal is obsou ly one that bears a close similarity to the ordinary diet of the patient but for many reason it is not po sible to give such a te t meal Iarious test meal, have been adı ocated but here we shall describe only two with which we have had personal experience Cruel fest meal In making this we have always u ed Quaker oats but any form of prepared oats can be used and in this country some worker prefer to use a nice gruel Take a tablespoonful of Quaker oats in two pints of water add a pinch of salt boil down to a pint and strain through fine mu lin This meal does not contain any lactic acid and is thus almo t an ideal test meal but with the tube in situ it becomes very difficult and sometimes impossible to swalloss such a large quantity of thick gruel (1) Alcohol test meal One hundred ccm of 7 per cent alcohol is used for thi meal The measured quantity of alcohol is placed in a beaker from which it 13 drawn up into a syringe and introduced into the stomach through the tube the process is repeated until the whole amount has been introduced Thi meal is very ea y to administer while the fluid that is sub equently with drawn is almost clear this allows of easy titration for acıdımetry S 11 ithdrau al of post prandial specimens Note the time when the test meil is given a pirate with a syringe about to c em of gastric contents esery 15 minutes up to 2l or 3 hours The specimen are hept in labelled te t tubes until the time of examination Histamine - As the fasting and post prandial junces are withdrawn they are examined for the presence of free hy drochloric acid by the bedside Take one c cm of the ga tric juice in a small test tube add a mall drop of Topfer s rea,ent Note the coloursed or orange colour indicates the presence of free hy drochloric acid and further bed ide examination of sub-equent specimens 1> not necessary If free hy drochlonc acid is not present in the fasting juice and in the first four post prandial specimens an injection of o, mg of hi tamine is gren and the pro cedure of withdrawing samples is continued a before A little flushing of the face is seen after injection of hi tamine and occasionally the patient may complain of palpitation which generally pass off quickly In the event of the patient be coming distressed by these symptoms an injection of occm adrenalın chloride should be gren this will give in tantaneous relief II ·80 HÆMATOLOGICAL TECIINIQUE allowed any food or drink before the test is finished Indian patients must be warned not to chew any pan as it may impart a red colour which might be mistaken for blood to the gastric juice Before introducing the tube in very sensitive patients the nasal mucous membrane and the posterior part of the pharynx may be sprayed or swabbed with I per cent solution of cocaine or with a suitable substitute B Introducing the tube (1) Through the mouth With a patient in a comfortable position sitting of possible, but the test is best done with the patient lying down the tip of the tube is placed on the posterior portion of the dorsum of the tongue and allowed to drop slowly backwards and downward When the tip strike the posterior pharynx the patient is instructed to make swallowing movements and the tip will then pass into the cesophagus The patient should be told to continue to swallow slowly when the tube will gradually descend until the end has reached the fundus of the stomach the mark on the tube gives an approximate indication of when this point is reached (11) Through the nose With a little practice the introduction of the tube through the nose is much easier than through the mouth and can be carried out even in the most sensitive patient The tip of the tube is introduced into one of the nasal orifices and gently pushed through the nose until it reaches the posterior wall of the pharyne The patient is now told to make swallowing movements while the tube is pushed gently down until it reaches the fundus of the stomach Some obstruction may be felt in passing the tube through the nose this 13 casily overcome by a little manipulation but if the resistance is great the tube must be taken out and introduced through the other na al orifice as not infre quently the septum is deviated to one side с Drau ing out the contents of the fasting stomach When the tube has been introduced up to the required distance introduce the nozzle of a 10 c cm syringe into the tube with the piston drawn out push down the piston so that the air in the syringe is forced through the tube to dislodge any mucus or food debris that may be blocking it at its distal end then aspirite the contents of the fasting stomach If there is any difficulty in getting the juice vary the position of the tube in the stomach by drawing it out or pushing it in and/or by forcing more air through the tube Rarely difficulty in obtaining juice may be due to contraction of the gastric muscles which may be difficult to overcome An attempt should be made to draw out all the fluid of the fasting stomach-by altering the position of the tube in the stomach by putting the patient in different postures and by applying a little pressure to the stomach from outside The contents of the stomach are placed in the test tubes previously labelled ·GASTRIC WALISIS 83 (a) Estimation of free hydrochloric acid Titrate with V/10 sodium hydroxide until the red or orange colour is dis charged thi is done as follows Fill a graduated 25 ccm burette with V/10 \aOH up to the zero mark With one hand regulate the flow of NaOH and allow it to fall drop by drop from the burette into the porcelain di h stir all the time with a clean glass rod with the other hand The end point will be shown by a brownish and not a yellow colour Take the reading of the burette the difference between the two readings (the first should be zero) gnes the amount of VaOH that was required to neutralize ) or 10 c cm of gastric juice In clinical work the result is always expres ed as the number of cubic centi metres of M/10 NaOH which would be requhred to neutralize 100 c cm of the gastric juice each cubic centurnetre repte enting one degree of acidity So the number of degrees of acidity is calculated by multiplying the number of cubic centi metres of V/10 NaOH by 10 of 10 cubic centimetres of gastric juice was used or by no f 5.6 cm of gastric juice was ed The re ult may be expressed in grammes of hydrochloric acid by multiplying the number of degrees of acidity by 0 0036, (an easy way to remember this is that 365 is the number of dats in the car the lat figure-5-indicating the number of decimal places) Franple - o ccm of pastric juce was taken an 1 of the end print a rache] wlen 23 ccm of M/10 NaOH hal been ad led the degree i acıtty is IS 3* TO 23 Alin term of hs drochloric acil 23 000357 = 0 0839) 8 of hydrochloro act 1 (6) Estimation of total acid After the estzination of free hydrochloric aud or if there is no free HCI at all I or 2 drops of phenolphthalein (I per cent solution in alcohol) is added as an indicator and the titration with V/10 VaOH >> continued until a permanent fairt red colour develop The third reading manus the first reading (zero in this case) multiplied by to of 10 c cm was taken or 20 of 5 < cm was taken gives the degree of total acid The total acidity is made up of free HCl together with the HCI which is com bined with protein and mucus plus the organic acids such as lactic and butyric which result from fernentation The estimation of total acidity is of little practical importance It is usually about to degrees higher than the free HCI except in cases of marked lactic or butyric acid fermentation when it may be ser much higher than the free Hul In these cases the typical rancid butter odour enables one to detect fermentation (c) Lactic acid Test for lactic acid must be applied in all es where there is no free HCl in the fa ting juiCL ·82 11 CMATOLOCICAL TECHNIQUE TYAVINATION OF GASTRIC CONTENTS A Macroscopic examinations (a) In the fasting juice (1) Amount measure and note the amount Normally 20 to 2, ccm arc found Marked increase over 50 ccm suggests hy po motility obstruction or hypersecretion (11) Odour normally it has no striking odour An offensive odour suggest cancer and a sour odour fermentation (111) Remnants of food or charcoal normally no food remnants or charcoal are found after 10 hours interval The presence of food remnants or charcoal particles suggests hypo motility pyloric obstruction or ptosis (6) In the fastıng price and in the post prandial specimens (sv) Mucus in large quantity in the fasting juice and in many of the later post prandial specimens indicates catarrhal gastritis (v) Bile traces of recently regurgitated lemon yellow bile may be seen in a few specimens and are usually due to retching caused by the Introduction of the tube A large quantity of turbid green bile in the fasting and in any of the carly post prandial samples is almost always pathological (v1) Blood macroscopic examination for blood gives more valuable in formation than the chemical examination Flechs of fresh blood are usually the result of trauma in passing the tube while large quantı ties of fresh blood in any specimen would indicate varices erosions or even malignant ulceration of the cesophagus Blood from a gastrıc ulcer or carcinom3 of the stomach is changed to brown acid hæmatın by the acid in the stomach juices it is found most frequently in the fasting juice but it may be found also in any post prandial specimen B Chemical examinations Acidimetry Place 5 or to c cm of clear gastric contents in a shallow porcelain dish If there is an excess of mucus in any specimen filter the gastric juice through a plug of cotton wool to remove the mucus this will then allow of easy titration Add a drop of Topfer s reagent to the gastric juice in the porcelain dish the presence of free hydrochloric acid will be indicated by the red or orange colour of the juice ·GASTRIC LILYSIS 83 (a) Estimation of free hydrochloric acid Titrate with 1/10 sodium hydroxide until the red or orange colour i dis charged this is donc as follows - Fill a graduated 25 ccm burette sith M/10 NaOH up to the zero mark With one hand regulate the flow of \aOH and allow it to fall drop by drop from the burcite into the porcelain di h stur all the time sith a clean glass rod with the other hand The end point will be hown by a brown.h and not a vcllou colour Take the reading of the burette the difference between the two reading the first hould be zero) ges the amount of NaOH that was required to neutralize 3 or 10 c cm of gastric juice În clinical work the result i always expres ed as the number of cubic centi metres of \/10 VaOH which would be required to neutralize yoo c m of the gastric juice each cubic centimetre representing one degree of acidity So the number of deftes of acidity i calculated by multiplying the number of cubic centi metres of 1/10 \aOH by 10 il ro cubic antimetres of gastric juice was used or by 20 of Soccm of gastric juice was u ed The result may be expressed in grammes of hydrochloric acid by multiplying the numbur of dosees of acidity by 00036, (an easy way to remember this is that 365 is the numbur ol days in the year the last figure--5--indicating the nembur of decimal places) Tranne --If to cart of gi tre juice a taken and if the end print v rache wlen 23 com o N to Maol hal been alled the degree i acility is 1 2 3X TO : 23 11 in terms of hydrochl nc acıl 23 00035 +0 03395 6 of hufrochloric 2011 (b) Estimation of total açıd After the estimation of free hy drochloric acid or if there is no frec HC1 at all I or drop of phenolphthalein (i per cent wlution in alcohol) is added as an indicator and the titration with V/10 \aOH I continued until a permanent faint red colour des clops The third reading minus the first reading (zero in this case) multiplied by 10 of 10 ccm was talen or do if 5 ccm was taken gives the degree of total acid The total acidity is made up of free HCI together with the HCI which is com bined with protein and mucus plus the organic acids such as lactic and butyric which result from fermentation The estimation of total açıdity is of little practical importance It is u ually about to deorces higher than the free HCI except in cases of marked lactic or butyric acid fermentation when it may be very much higher than the free HUI In these ca es the typical rancid butter odour enables one to detect fermentation (c) Lactic acid Test for lactic acid must bı applied in all ca is where there is no free HCI in the fasting juice ·82 HEMATOLOGICAL TECHNIQUE EXAMINATION OF GASTRIC CONTENTS Macroscopic eramınahons (a) In the fasting juice Amount measure and note the amount Normally 20 to 2, ccm arc found Marked increase over 50 ccm suy gests hy po motility obstruction or hypersecretion (15) Odour normally it has no striking odour An offensne odour suggest cancer and a sour odour fermentation (11) Remnants of food or charcoal normally no food remnants or charcoal are found after 10 hours interval The presence of food remnants or charcoal particles suggests hypo motility pyloric obstruction or ptosis (6) In the fastıng juice and in the post prundial specimens (10) Mucus in large quantity in the fasting juice and in many of the later post prandial specimens indicates catarrhal gastritis (v) Bile traces of recently regurgitated lemon yellos bile may be seen in a few specimens and are usually due to retching caused by the introduction of the tube A large quantity of turbid green bile in the fasting and in any of the early post prandial samples is almost always pathological (11) Blood macroscopic examination for blood gives more valuable in formation than the chemical examination Flechs of fresh blood arc usually the result of trauma in passing the tube while large quantı ties of fresh blood in any specimen would indicate varices erostons or even malignant ulceration of the csophagus Blood from a gastric ulcer or carcinoma of the stomach is changed to brown acid hæmatın by the acid in the stomach juices it is found most frequently in the fastıng juice but it may be found also in any post prandial specimen B Chemical eranınations (1) Acıdımetry Place 5 or lo c cm of clear gastric contents in a shallow porcelain dish If there is an excess of mucus in any specimen filter the gastric juice through a plug of cotton wool to remove the mucus this will then allow of easy titration Add a drop of Topfer s reagent to the gastric juice in the porcelain dish the presence of free hy drochloric acid will be indicated by the red or orange colour of the juice ·(ASTRIC AVALYSIS 85 Interpretation --Digestion in tube 2 indicates the presena of both pxepin 25/1 free hydroxchionc acid in the gastric juku If digestion fail in tube 2 but occurs in tutx 3 pepsinogen is present but it required the free HCI to In added to the tube to convert it into pepsin If there is no di,estion even in tulu 3 then both papan and pepsinngen are absent (m) Delection of rennir --The enzyme rennın angulates the protein of milk 1 resh milk I used as the rea,ent Neutralize o c cm of clear gastric flud with in Jilute wodum hidroude Add an equal amount of fresh milk and place in 1 water bath at 40 C for 1, minutes Interfrelation Coagulation of the milk in 10 to 15 minutes de notes the presence of a normal amount of renain delayed congulation denotes a decreased amount D Microscopic cramination of the gastric residue This (lamination ha very limited application and need only be done if carcinoma is susprcted The fasting contents when no frec HCI 1» present are mot suitable for the test Centrifugalize the fasting contents take a small loopful from the bottom and mcar it on a glass slide Fix by heat and stain by Lugol s rodine and camine with the oil immer ion lens for Boas Oppler bacıllı Boas Oppler bacill are large (5 to 10. long) non motile and usually arranged in clumps or end to end in zig zag chains They stain yellow to brown with rodine solution which distingui hes them from leptoincha buccalis which is not infrequently swallowed and hence found in stomach fluid (Todd and Sanford 1939) Vethod of recording results The rcsults are best recorded on a pecial chart which may be printed or cyclosty led Figure 2 shows the form of chart that we use cocept that in tins figure 7 shaded area has been added to show the range within which 80 per cent of gastric analyses in normal individuals fall Both free acid and total acid ate recorded in the chart preferably in different colours The presence of mucus bile and blood is indicated by a piros or a double plus sign in the appropriate place ·84 HLSTATOLOGICAL TECHNIQUE Add 2 drops of 5 per cent aqueous solution of ferric chloride to half an inch of clear gastric fluid in one tube and to half an inch of water in another tube of the same size which will serve as a control To each add 6 drops of saturated solution of mercuric chloride If lactic acid is present in the gastric contents a dccp vellow colour will be produced in that tabu but not in the other (11) Bile The presence of bile will be denoted by the colour in the gastric fluid If in doubt Hays sulphur test can be done by sprinkling sulphur dust on the surface of a specimen of clear juict If bile salts are present the sulphur will fall to the bottom (111) Blood Dissolve a little benzidine in 2 c cm of glacial acctic acid warm if necessary add 2 c cm of 3 per cent hydrogen peroxide Finally add 1 c cm of the gastriu Juick and mix well by shaking A blue or bluish green colour indicates the presency of blood The test is very sensitive and will detect blood in dilution of 1 3 000 000 C Examination for en pics (1) Detection of pepsin - Normally pepsinogen is secreted by the stomach and is transformed into pepsin by the free HCl in the stomach Its presence is detected by the digestion of egg albumin Tethod -Take threc small test tubes In tube I put i g of pepsin 2 c cm of the HCl solution and one inch lenkth of capillary tube of albumin * In tube 2 put 2 c cm of clear gastric fluid and a capilları tube of albumin In tube 3 put 2 c cm of clear gastric fluid 2 c cm of the HCl solution and a capillary tube of albumin Place all the three test tubes in a warm incubator overnight cximine them for digestion of the albumin next morning Tube I in which digestion should be compk te serves as a control Vett s capillary tubes of albumin are prepared 29 the following Mis the white of 3 or 4 egs beating them gently Vake some capillary tubes of uniform bore Fl the tut *ith the egg alumiin) cut them into pieces of about ro to I inches long Seal the ends ith caling vax B1 a larg quantity of water in a big vesse! Just as the later I giu to bol put tle capil lary tube containing albuma into the bulling ater Remove the esset from the sourc of the heat After 5 minutes take out the cap llary tubes-the egg bum n ille lightly coagulated Store the tubes in a refrigerator Before de cut the egg albumin tubes into length of of unch taking care that the en Is are clean cut ao circular ·GASTRIC ANALYSIS 87 Vs These criterii are based on our personal experience in India (apier and Da Gupta 1933 lapier Chaudhur and Rar Chaudhur 1939) but they do not differ materially from tho e adopted by workers in other countries About Ro per cunt of normal individual full within the sochlorhydril range within the range women will u calls Le lower than men (figurt 23) There no evidence that the ga tric acidity of Indians in India is lower than that of Europeans in their own countries 1 em The data available from our Own experience suggest that in South Indian and Bengali (figure 24) the acid range I a little higher than the textbook figures which are based on Luropean 11 23 Asam cron TIL! 171 and North American subjects (n. $ igru 1 tm 1) 1 From 2 to 4 per cent of apparently normal Indian are completely achlorhydric ssen with h: timin with experience in other countries this 1 in heeping 40 TIG 24-ormal Indians in Calcutta (gruel test meal) With the alcohol test meal the and cure on the whole tends to be higher this is particularly noticeable in the first post prandial sample in which the initial drop in acidits due mainly to the neutralization of the acıd by the meal may be absent Another difference is that the maumum acidity is usually reached earlier when alcohol + giien as a test meal ·86 HLVATOLOGICAL TECHNIQUE Normal standards Acid curves will vary not only in height but in shape and one cannot jude them solely on one feature is on the highest point reached during the test SIN ANEM DONT GASTRIC ANALYSIS REPORT Nm -se Runthy Chere! Fondem Mikroscopk MC B!! E000 120 110 100 90 BO 70 60 50 40 30 20 30 o } 1 1 1 1 1 2 212 213 bu F -Tilty Ppt dg to - Oposs Dte FIG 22 Nevertheless if a curve is to be classified on a single feature this i probably the best one as it usually reflects fairly accurately the rest of the curve We haie adopted the following criteria for classifying acid curves according to the highest free acid reading ccm of N, 10 VaOH rt qoires to ne truie TOK com gastric ju! 0 <IO 10 D< 25 Achl rhydrit Hypochlorhydri Isach orliyana los medium high Hyperchlorhy dnia 25 to 4 <45 to 65 <65 ·TIIE RED CELL TRAGILITI TEST 89 the numbers of the two tubes that show the beginning of hemolysis and complete hemolysis are noted and from these the strength of the corresponding saline solutions are calculated Aparatus and reagents required ) Accurate solutions of sodium chloride os per cent and 10 per cent standardizeit against slver nitrate solution (1) Treshly boiled distille ! wafer (plt 7 ) (0) Small test tubes (vv) Aletal rack for holding the tubes (u) 5 or 10 c cm cyninge with needle of fairly wido bore (vi) Capillary pipette with teat A convenient form of rack is that used for the Wassermann fraction The holes are in two rows with single tubes at either end of the rack The holes are numbered 12 to 24 and those at each en] S and W (figure 25) U U 1974 Fio 25 With a syringe fitted with a needle of fauly large bore or with a capillary pipette put into each tube the number of drops of o, per cent sodium chloride solution indicated by the number on the rack opposite that tube apd 25 drops in the tube marked S Wash out the syringe or the pipette thoroughly with distilled water With the same needle or pipette add a sufficient number of drops of distilled water to bring the total number of drops of fluid in each tube up to 25 and put 25 drops of distilled water in the tube at the end of the rack mirked W I2 13 14 15 16 17 24 S O 12 13 14 15 16 17 24 75 Framples Numb rs on racks oppo site test tubes Number of drops of o5 per cent NaCl to be put to each test tube Number of drops of dys tillel witer to be added to each test tobe Concentration of NaCl (per cent) 12 25 13 12 11 10 9 8 1 14 26 28 30 32 34 48 50 ·88 HI EVATOLOGICAL TECHNIQUE tion Other indications given by gastric analysis In chronic gastritis in the early stages there is hyperacidity and hypersecr this latter is shown by the large quantity of fastig juice but in the later stages there is gastric atrophy with accompany ing hypoacidity or achlorhy drza In gastric ulcer there is usually increased acidity and blood may be present In gastric carcinoma the findings are sometimes very characteristic but in the early stages there may be no indications so that it is dangerous to exclude carcinoma on the strength of a normal gastric analysis There is achlorhydria excess of lactic acid and usually blood and the Boas Oppler bacillus is often present If the cancer is at the pylorus there may be delayed emptying and food or charcoal wall be found in the fasting juice which will usually be very sour and offensive In gastric neuroses there is usually hypersecretion and in dilatation of the stomach food retention and fermentation 1 16 THE RED CELL FRAGILITY TEST Introduction ---When normal red cells are suspended in 08, per cent salını they remain unaltered but they become crenated when the concentration of the salıne as raised above o 9 per cent whereas they are hæmoly sed or laked when the concentration is lowered below 0 45 per cent The study of the reaction of the red cells in hypotonic salıne solution is known as the fragility test of the red cells The fragility of the red cells is influenced by the CO content of the blood arterial blood is less fragile than venous blood while the fragility of the capillary blood is intermediate Further the red cell us to some extent protected by its own plasma from salıne hæmolysis which is more marked in case of anemic patients (Creed 1939) The test is conveniently done with fresh oxalated venous blood collected for routine examination of the blood (p I) but for reasons given above the te t should be done with washed red blood cells which have been thoroughly orvgenated by rotating the blood around in a wide mouthed bottle for over 5 minutes The fragility of the red cells is expressed in terms of percentages of sodium chloride solution namely the points at which hæmolysis commences and at which it is complete The test can be done either by a qualitative or quantitative method Qualitative method To each of a series of numbered test tubes containing salıne solution of different graded concentrations one drop or two drops of blood are added the contents of the tubes are mixed well and allowed to stand for about 2 hours after which ·THE RED CELL FRAGILITY TEST 97 1p paralis rewired 1 in telcell count (10) \!accurate slut ol solium chloride (stan Jardined against silvr hitrate solution) from 3 to 07 per cent with intervals of 205 part ceat Technique -Till red cell pipettes as in ad-cell count using different strengths of salt solution ranging from o 3 to 07 per cent with interval of 005 per cent Allow the mpette to stand on an esen surface for about 15 minutes shake it well and then count the red cells in the usual way (see cnumeration of red cell P 13 et «eq) Repeat this count with each solution and include a control with the usual diluent t M 3 ts 40 } 1 w $ From the above counts calculate the number of cells becomc hemolyser in different strengths of the salt solution and plot a cune The normal limit of fragility of the red blood cells as deter mined by the quantitative method of Whitby and Hynes (1935) Is shown in figure ng Discussion The fagility tost is based on the assump tion that there is some parallelism between the fragility of the erythrocytes in extro and thir susceptibility to haemolysis 1% tilo Though it is very unlikely that the mecha 111 ms of these two processes neither of which 18 fully understood are identical there is the puidence that in certain conditions & acholuric jaundice where the red cells are particularly liable to hæmolysis :* VILO they 1626 -Tragility of mod corpu«cle as determ ned by qua titative method are hæmoly ed by hypotonic saliac solutions (12itby and flynes 1935) that would not affect normal erythrocytes In the physiological process of red cell demolition there is ome evidence that the red cell normally a biconrase dixc becomes more spherical in hape before it disintegrates and in many hæmolytic anæmias it has been shown that the red cells are more spherical than tha normal red cells These spherical cell, appear to be more susceptible to hæmolysi by hypotonic salt solutions but it is certain that this is not the only change that brings about this increased fragility In all cases of obscure anæmia it is certainly worth carrying out the fragility test by the quanutatic method if this is feasible not only in the interests of accurate diagnosis but in order to add to our knowledge of the ætiology of the many types of anæmia about which our knowledge is incomplete ·go REMATOLOGICAL TECHNIQUE Shake each tube thoroughly to get a uniform mixture To each tube add one drop or tuo drops of the patient is very anæmic of oxygenated blood (vs) Shake all the tubes again Place the rack with the tubes on a fat even surface and allow it to stand for about 2 hours at room temperature If there is any special need for hurry centrifuge all the tubes for about 10 minutes and take the readings at once If possible put up a parallel test with normal blood as a check to the accuracy of the sodium chloride solution Reading the results –Hold the rack with the tubes level with your eyes against daylight Note the number of the tube in which hæmolysis is first shown and also the number of the tube in which it is quite complete -Examine the tube commencing from tube No 25 and proreed towards the other end the first tube showing a tinge of reddish colour in the supernatant fluid indicates the beginning of hæmolysis and the first tube in which all the corpuscles are laked and no cor puscular residue is visible even after centrifugalgation indicates the point of complete hæmolysis The result is expressed as a percentage of sodium chloride solution and 13 calculated by multiplying the figures on the tubes by o 02 Example --The first tube in which the supernatant fluid sbows any pink colour 19 numbered 20 The first tube in which there is no corpuscular resi lue is numbered 15 Therefore hæmolysis begins at 20 x 0 02=0 40 per cent saline hæmolysis 19 complete at 15 * 0 D2=0 30 per cent saline When the test has to be carried out with very fragile cells eg in acholuric jaundice concentration of sodium chloride of over o 5 per cent may be required in such a case use I per cent solution instead of o5 per cent and proceed with the test in the same way as before Take the readings and multiply the numbers on the tubes by 004 to get the percentage of the saline Normally partial hämolysis begins at about o 44 per cent and complete at about o 30 per cent saline This quanti Quantitat e method Thc qualitative method would be quite accurate if all the cells in any particular sample were of the same fragility but the individual cells vary in their fragility just as they vary in their size It is therefore important to knox what percentage of cells have been hæmolysed by each solution tative method is more laborious but is worth doing in special cases Principle A number of accurate red cell counts are done in the usual way except that instead of the standard diluting fluid different strengths of salt solutions are used from the results an accurate quantitative graph of the fragility is plotted Simmel (1923) used mixtures of salts identical with those in the blood but Ilhitby and llynes (1935) obtained identical results by using solum chloride soluti ng we recom mend the latter method on account of its simplicity ·PROCEDURES FUPLOYED IY IV ESTIGATING HI ETORRILAGIC DISEASES 93 known causes of the hæmorrhagic state are — (7) Duficiercy of platelets (and con equent deficiency of thrombokinase) as in thrombocytopenic purpura (1) A defect of the platelets so that they do not disintegrate at the usual rate as in hæmophilia (1) A deficuncs of prothrombin in the blood as in vitamin h deficiency (11) Fibrinogen deficiency as in prolonged chronic hæmorrhage (U) A defect in the capillary wall as in vitamin C deficiency In there is a decrease in the platelet count a normal coagulation but a prolonged bleeding time and poor clot retractoon the prothrombin is normal In (11) the bleeding time is normal and the coagulation time is prolonged In (H) the bleeding time is normal and coagulation time prolonged prothrombin is decrea.ed frut the clot retracts normally in (it) the bleeding time is normal and the coagulation time prolonged and the clot retraction is delectus but prothrombin is normal In (t) the bleeding time s prolorged and the capillary resistance decreased but the coagulation time prothrombin and clot retraction are normal A COAGULATION TIME Various methods have been described for the determination of the coagulation time but it is essential to adopt one method and to follow the technique as uniform ly as possible for the normal coagulation time vanes very considerably with the technique with the temperature and rith the diameter of the tube used it is also influenced by admixture of the blood with tissue juice or any other foreign substances It i advisable first to determine the coagulation time of a few normal persons in each laboratory all subsequent examinations must be done exactly in the same way and the results interpreted in relation to the normal finding Two methods are described as we have had considerable experience with each I CAPILLARI TUBE (1) RIGHT S) VETHOD Appu olms and reagents required (Fine capillary tube with llameters from o8 to i 2 mm ani x incbac long ( ) Frxking need e or blood gun () Kater bath or warm water in a beaker at 37 with a therm mtr (1v) Spint lamp (u) Stop watch (t) Alcohol (1) Ether (v) Coton wool Thus is udgecessary in the hot weather months n India ·92 HEMATOLOGICAL IFCHNIQUE 17 PROCEDURES EMPLOYED IN INVESTIGATING HÆMORRHAGIC DISEASES Introduction - In the study of hæmorrhagic diseases and hæmorrhagic diatheses a number of procedures are commonly carried out that should be in cluded in this series on hæmatological technique These are Estimation of the coagulation time B Estimation of the bleeding time C Quantitative estimation of prothrombin in the blood or determination of prothrombin time D Capillary resistance test E Study of clot retraction F Platelet count (vide p 27 et seq) The study of clot retraction gives little additional information of value as it appears to be dependent entirely on the platelet count but as it provides confirm atory evidence we have indluded it The estimation of blood calcium also has some bearing on this subject but is essentially a biochemical test and will not be described here further though calcium deficiency could theoretically lead to a hæmorrhagic state in actual practide it does not appear to do so unless associated with other deficiencies Discussion --Briefly stated and essentially coagulation of the blood is depen dent on filbrinogen ionized calcium prothrombin and thrombokinase (thromboplastin) The first three are present in the plasma while thrombokinase is derived from the disintegration of the platelets and damaged tissue the chain of events ty as follows - Prothrombin + ionized calcium + thrombokinase (thromboplastın) → thrombin Thrombin+fibrinogen → fibrin Fibrın + cellular elements of blood → blood clot Clot + platelets > the retracted clot (This leaves out of account the interaction between antı prothrombin pro thrombin and thromboplastin substances Heparin-anti prothrombin—is a very real substance but whether it exists in the circulating blood in health does not seem definitely established so for the sake of brevity and we hope clarity we have not considered the part it may possibly play in preventing-clotting in the circulating blood ) All the above substances are normally present in the blood a defect in any of them will cause a break in the continuity of the chain and will lead to hæmorrhage ·I ROCEDLRFS EBITLOYED IN INVESTIGATIVG I CHORRILAGIC DISEASES 93 Comment -The second method is more accurate as certuin disturbing factor % tissit juke te eliminated BLEEDING TIDIE paralus required O Pakng ntel bind 1 gun (ui) Stor watch () Istruper (1L) Slygi to manomets I DUKF S METHOD Clean the ear lobe or the dorsal surface of a finger firt with alcohol and then with ether (The tip of the finger is not suitable for this test as it is horny in many cases ) Prick the selected site fairly decply with a blood gun or a pricking netalle o that blood flow's freely without squecuing Soak up the blood which comes out with a clean piece of filter paper without touching the skin The flow of the bloou hould be such that the first impression on the filter paper has a diameter of about half an inch Without squeezing or pressing the finger continue to soak ur the blood with the filter paper every 30 seconds until the bleeding stops The time from the first appearance of the blood until the cessation of bleeding is the bleeding time Mornal limit:=1 to 3 minutes II VELOCS PRESSL RE METHOD (Ivy et al 1935) Put the cuff of a sphygmomanometr around the arm above the elbos as 13 done in taking blood pressure Select a portion of the forearm below the elbow and clean it with alcohol and ether Rai e the pressure and maintain it at210 mm of mercury which sufficient to check any enous return Prick sharply up to a depth of about 1/10th inch at a point frec from any superficial veins so that blood will flow freely Soak up the drop of blood estry 30 seconds as in method I until the bleeding stops This method is superior to method I a the capillarses are kept wide open and their tone eliminated also the area selected for punctare is uniformly thun For these reasons more consistent results are obtained with thirs method than with method i Normal limits=2 to 4 minutes с QUANTITATIVE DETERMINATION OF PROTHROWBIN IN BLOOD PROTHROUBIN TIME ( Ten com record syringe O) Centnfuge tubes (11) Small te t tubes in a rack Appa als qu (v) Water bath with a thermometar (v) Capillary P pettes with teita (v) Stop waten ·HIAMATOLOGICAL TLCHNIQUE Technique -Clean a finger or an ear lobe first with alcohol and then with ether Puncture sharply and fairly deeply with a blood gun or a picking needle so that blood will flow freely without the necessity of squeezing Discard the fir t two drops of blood and then fill a capillary tube up to three fourths of its total len, th with blood (the capillary tube will fill by capillary attraction when þeld against the drop of blood) Note the time by the stop watch when the blood first enters the sapıllary tube Seal the free ends of the tube in a flame and put it in warm water (37 C) in a beaker Repeat the process with two other tubes notine the time of the commencernent in each case After one minute take out the first tube gently break off a portion of the fubu from one end and repeat this every 1, seconds until a thin line of unbroken coagulum is seen stretched between the two broken ends note the time The difference between the two times by the stop watch is the coagulation timc Repeat the procedure with the other two tubes breaking them at intervals of 15 seconds and find out the coagulation time for each tube Take the mean of the three readings as the coagulation time Normal time limits=1 to 3 minutes 1 II METHOD OF LEE AND WHITE (MODIFIED) WITH VENOUS BLOOD Apparatus and reagents required (additional) (3) An all glass 5 ccm syringe with a needle of fairly large bore (*) Small parafined test tubes inch in diameter with rubber corks in tack (111) Nortnal saline (1x) Hard paraffin in a metal or porc, ain bowl Prepare a water bath at 37 C plane the paraffined tubes ready in the bath in an upright position Collect 5 ccm of blood from a vein ir an all glass syringe immediately after washing it out sith normal suline Note the time by the stop watch when the blood enters the syringe Take out the needle from the syringe and put exactly I c cm of blood into each of the three tubes Cork the tubes with rubber corks and allow them to stand in the water bath Wait for 3 minutes and then every 15 seconds in rotation take a tube and tilt the contents to see if the blood has et Conținue doing this until the blood is found to have set so that the tube can be inverted without spilling note the time any blood Normal time limuts=3 to 6 minutes Prepanng paraffin tubes --Delt some paraffin in a clean vessel Place a test tube with a glass funnel which has been previously warmed in a sack Poss the melte i paraffin through the funnel into the tube up to about two thirds of its length Remove the furt 1 and pour back the excess paraffin from the ube into the vessel Immerse the tube in cold mater taking care that no water enters the tube when the test tube is cold there will be a thin coating of paraffin inside the tube ·PROCEDURES EMPLOYED IN INVESTICATING HCVORRIAGIC DISEASES 97 Repeat the experiments with the other two tubes Take the mean of the three reading this gives the prothrombin time Norma)=15 to 25 seconds [More recently we have determined the prothrombin time in a few cases bv using Russell siper venom alone and with ovolecithin added to it in the proportion recommended by Witts and Hobson (1940) With other workers we have found that the addition of ovoletithin shortens the coagulation time and the results tre more uniform Normal prothrombin time with osolecithin = 10 to 15 seconds ] D HESS CAPILLARY RESISTANCE TEST (1) Sphygmomanometer (21) Stethoscope Apparatus required (1) Hand lens (iv) Skin marking pen 1 Place the cuff of a sphygmomanometer on the arm above the elbow as in taking the blood pressure Determine the systolic and diastolic pressures release the pressure Wita a skin (or glass) pencil mark any purpuric spots that may be present on the forearm - Raise the pressure to a point midway between the systolic and the diastolic and maintain it at this point for 5 minutes Remove the armlet and examine the forearm in a strong light and if necesity with a hand lens for purpuric spots (other than those already marked) A positive re ult indicating decreased capillary resistance is shown by the presence of a number of small fresh purpuric spots dotributed over the forearm below the point at which the armlet was applied The process should be repeated on the other arm E CLOT RETRACTION STUDY When blood coagulates a clot is formed which after a time contracts and expresses the serum the clot itself then shrinks and becomes hard Retraction of the clot as it is called varies directly with the number of platelets in the circu lation but is independent of the coagulation time with a low platelet count 60 000 per c mm or below it takes over 24 hours for the clot to separate and the clot is soft and friable Apparatus required ( Same as for collecting blood (1) Craduate 1 10 cm centnfuge tubes each fitted with a cork baving a hole in the centre (1) Class rods with flanges about the size of a silver 4 apna piece half an uch from the end (tu) Test tube rack Method of Macfarlane - Collect blood from the patient in dry syringe (tide PI) 13 ·96 HÆMATOLOGICAL TECHNIQUE Solutions required (1) Sodium oxalate solution dissolye 134 gm of chemically pure anhydrous sodium oralate in 100 ccm of distilled water (1) Calcium chlonde solution des ole o 275 gm of chemically pure anhydrous calcium chloride in 100 ccm of distilled water Check the strength of the soluloh by titration against silver nitrate solution (111) Thromboplastın solution Preparation of thromboplastın solution Satisfactory results will be obtained with either of the following substancts (a) From rabbit s brain Dissolve 03 c cm of dehydrated* rabbit s brain in 5 c cm of normal saline containing on c cm of sodium oxalate solution incubate at 45 C for 10 minutes and finally centrifuge at a low speed for 5 minutes Pipette off the milky supernatant fluid and keep the solution in a cold incubator floating a little toluene on the top of the olution The solution will keep for more than a month () From Russell s riper venom This is the method of choice A I in 20 000 solution of Russell s viper venom gives a potent thromboplastic solution In the dried state the venom maintains its full potency indefinitely kept in a refrigerator with toluene the solution also retains full potency for more than a month (Iyengar et al 1941) Technique --A slightly modified Quick s method is followed Put o 5 ccm of sodium oxalate solution into a centrifuge tube Collect blood in a dry syringe (vide p 2) and put 45 c cm into the centrifuge tube containing oxalate solution Centrifuge until the plasma completely separates carefully pipette of the supernatant plasma and put it into another dry sterilized tube Put exactly 02 c cm of plasma into each of the three tubes marked A B and C and place them in the water bath at 37 C Take out one tube at a time and to each add exactly 02 c cm of thrombo plastın solution and o2 c cm of calcium chloride solution Note the time when the calcium chloride solution is added shake gently keeping the tubes in the water bath and again note the time when the clot firt appears mix well Dehydrating the brain Remove all the blood vessels and then macerate u ell in a glass mortar Add acetone and macerate again Decant off the acetone add ome fresh acetone macerate and again decant of the excess of acetone Continue doing this unti! the whole thing is converted intd gragular powder keep is a cold incubator In the dried state its activity is retained for a long time ·RED CELL DIAMETERS PRICE JOVES CURVE 99 In different forms of anemia this curte still vary in shape its median will mose to the n,ht or to the left and it may be of the normal het,ht or flatter than normal Thest variations in the curse can be expressed in figures The median can be given in terms of micron but it is more usual to give the mean corpuscular dia meter (VCD) also in micron the flatness of the curve is shown by the standard deration (c) of the drameters of the cells from the mean diameter or more accu rately by the co-ethccnt of variation (u) and also by the percentage microcytosis and macrocytosis These data can all be calculated from the Price Jones curve The measurement of cell volume percentage and calculation of the mein corpuscular volume (MCV) have been described alreadus (p 29 et scq) this VCV ghes the volume of the cells but not their diameter Further it is a mean measurement and from it one 5C no idea of the range of size of the individual cells it is therefore more limited in its application than the Price Jones curve Neither measurement however alone will give informatiot on the thickness of the cells but this can be calculated from VCV and VCD The method of makin these calculations will be described later The technique of the measurement of cell diam ters - The technique describd below is a modification of Hynes and Martin s method of measurement of red cell diameters The images of the cell are projected on the vertical ground glass screen of a Bausch and Lomb euscope at a magnification of 2000 and the measurements are made with the help of celluloid protractor on which a enes of circles with diameters increasing by the equivalent of 0 254 have been drawn Apparatus equ red (A Bausch and Lomb euscope with projection screen (A mechankal feed arc lamp with cond ases working 45 amperes (1) A glass contain r filled with distilled water for cooling the bam of light from th arc lamp (filter) (10) A microscope with oil mersion objective and eye pec to giv a magnification of 2 000 on the projection streen of the euscope (U) A stage micrometer scale (Zeiss) with divisions 10 apart (11) A cellulo.1 cm /MI scale (w1i) Celluloid protractor for measuring the cells (v 1) Prepanng the celluloid protractor --At a magnification of 2 000 If corresponds to 2 mm and o 2544 to 05 mm The circles on the protractor are drawn with diameters increasing by the equivalent of o 25 (05 mm) ie the radi of the circles increa e by o 25 mm The measurements of the radu of the scries of circles are best obtained from a diagonal scale drawn to give measurements in multiples of o 25 mm ·98 HIAMATOLOGICAL TI CHNIQUE Put 5 c cm of blood into the centrifuge tube Put in the cork with the glass rod passed through the hole in the centre of the cork See that the flanged end of the rod is near the bottom of the centrifuge tube (figure 27) Place the tube in an upright TU position in the rack and put the rack into an incubator at 37 C From one hour onwards Frc 27 --Showing centrifuge tube with glass rod in position examine the tube from time to time for clotting After one hour of clotting remove the clot by drawing out the glass rod (the clot will adhere to the flanged end of the rod) Note the amount of flaid in the tube The amount of fluid in the tube divided by the amount of blood put in the tube and multiplied by 100 gives the percentage of retraction Example --Suppose 5 ccm of blood was taken and 3 c cm of Aaid was left after the removal of the clot Retraction = x 100 = 60 per cent Norinal retraction = 43 to 64 per cent The clot is also examined for firmness and inability Clot retraction is low in thrombocytopenic purpura but is normal in hæmophilia 18 RED CELL DIAMETERS PRICE-JONES CURVE Introduction –In the normal blood the red cells are not all exactly of the same size but in no instance does the cell size deviate very much from the mean However in some forms of anæmia the inean size of the red cells is much below the normal mean in others it is far above it But in the latter case there are nearly always some exceptionally small cells as well so that the picture presented is that of cells of a great range of differe it sizes This blood picture is best pre sented in the form of a curve the absui sa being the size and the ordinates the number of times that cells of the particular size have been encountered (1 e the frequency) In the normal count the curve rises sharply from the base line at about 6 on to a peak at 7 25/4 and then falls almost to the base line again at This graph is usually known as a Price Jones curve In the normal individual the shape of the curse is more constant than the mean diameter of the cells which in a group of individuals will vary within but rarely fall outside the range 674 to 7 8u Price Jones calculated what he called ideal maximum and minimum normal curves (Price Jones 1933) 8 504 ·RED CELL DIA VETERS TRICE JOVES CLKTE JO1 2003 The magnification of the apparatus is adju ted to be 2000 by 110g a suitable oil immersion objective and eye piece and varying the length of the drax tube of the microscope (& Zeiss microscope with objective go and a Leitz ese piece 15* with a draw tube length of 154 mm gives a magtulication of 2 000 on the projection screen of the particular euscose u.ed in our laboratory ) To find the magnification the distance between the images of tro lines of the scale is ineasured with the cellulosd cm mm scale and at a magnification of 2 000 the images of the lines of the scale are 2 cm apart (2 cm = 20 000u) different components of the apparatus are kept fired after the above adjustments the measurements cus be carried out sssthout repeating these adjustments every time) (34 the O O Jeasuring the cells ---The miscrometer scale is next remosed the blood film stained with a Romanowsky stain and counter stained with r per cent aqueous Costa soutuon is placed under the oil immersion objective and the image of the cells as focused on the ground glass screen of the euscope Osly those images of thin cells that fall withun the reduced field are measured The celluloid protractor with the series of graduated circles is superimposed on the image of the corpuscles to find the circle that fits the image of each cell Each cel is measured to the nearest 0 23 The measurement of circular cor 910 30 puscles is quite easy In the case of the trregular shaped corpu cles a cstcle is found such that the area of the corpuscle falling outside the circle us about the same as the area of the circle unfilled by the uage of the corpu cle (see figure 301 Fre hundred cells are measured from different parts of the blood filma It is better to avoid the tar! end and the thick parts of the smear as ss the former the cells are excessively spread out and in the latter the cells overlap and are not sell spread Recording the results By thus method 500 cells can be measured in about 40 to 90 minutes the time depending on the size of the cells and the degree of poikilocytosus the larger and more irregular the cells the longer is the time required An 25525 tant is needed to note down the measurements and record the kumber of cells counted sth a counting machine of the type of a Veeder counter From the figures obtained the mein diameter standard deviation and co eficient of variation are calculated A Price Jantes curve is drawn by plotting the figures on graph paper The degree of microcytosis and macrocytosis can also be determined from this curse by comparison with the maximum and mint ideal curves the Overlapping of a cure beyond the minimum deal cune on the low side and the maxsmum curve on the hi, h side indicates the degree of microcytosis and macrocytosis respectului ·100 ILÆMATOLOGICAL TECHNIQUE 00000000 ୦୦୦୧ OOO QO OO . O Size The circles are drawn in Indian ink with very fine bow pen compasses on a celluloid sheet with radii increasing from 4 mm to 12 mm by steps of 25 mm so as to give measurements corresponding to 44 to 121 diameter Circles smaller and larger than the above are drawn on another sheet The first sheet is generally suffi cient for routine work the second is needed rery exceptionally (se figure 28) Assembling and adjusting the apparatus Figure 29 shows the euscope with micro Reduced to scope and arc lamp assembled Origiral The concentrated bean of light from the arc lamp cooled FIO 28 by passage through water in the filter is focussed on to the concave mirror of the micro scope which reflects it through the condenser the objective and the eje prece of the microscope The total reflexion prism of the euscope placed over the ocular reflects the light on to the opaque screen of the euscope The condenser la fully raped up and with the high power objective turned on a bright beam of light is focussed on the opaque screen After the illumination has been adjusted the micrometer scale is placed under the obective of the microscope and the image of the scale us focussed on the opaque screen first with the low power and finally the oil immersion objective A final adjustment of light is made to give the maximum illumina tion FIG 29 The opaque screen is now moved off and the image is focussed on to the ground glass pro ectron screen of the euscope By moving the microscope and/or the prism the image of the scale is so projected that the lines of the scale are vertical parallel and entirely free from spherical aberration at least within an area of about 4 inches Dy 3 inches marked out around the centre of the viewing Screen That there is no spherical abberation within this area can be shown by noting that the distances between the images of the lines are the same in all parts of this ·RED CELL DIAMETERS PRICE JOYFS CURVF 103 Trom the mean corpuscular diimeter (VCD) and the mean corpuscular Volume (lide p 31) the mean corpuscular average thickness (NICAT) can be calculated by means of the following formula - VICH VCAT VICV VICDIER Frumple --Let go cur le the mean corpuscular volume (cy) ani! (52p the mean con uscular diameter (CD) Vean corpuscutar average thickness (CAT)= 90 90 ** 263 311 3 26" The normal range of VCAT is from 17 to 256 (Price Jones Vaughan and Goddard quoted by Whitby and Britton 1939) In order to calculate the percentages of microcytosis and macrocytosis it 15 necessary to have ideal minimum and maximum curses for the population in which the investigations are being carried out Price-Jones calculated the ideal curves for subjects in Great Britain Working on similar lines we (Napier Sen Gupta and Chandra Schar 1941) hase worked out two similar cun es Two smoothed curses are shown in figure 31 and the expıcted frequency distributions in the ideal minimum and maximum curies are given in table VII To arrive at the percentage microcytosis and macrocytosis the number of cells of each size (below 694) in excess of the number of that particular size that appear in the minimum deal curve are summed the number divided by 5 to reduce it to a percentage is the percentage microcytosis Similarly the number of cells of cach size (above 781) in excess of the number of that particular size that appear in the maximum ideal curve are summed and divided by 5 to obtain the percentage macrocytosis (vide table \VII) Normals OF VEAN CORPUSCULAR DIAMETER (MCD) STANDARD DEVIA TION (1) CO EFFICIENT VARIATION (v) Number of obser vations Max Win Vean Mar Vin Mlean Blat Min Mean 100 Lont (Price Jones 1933) 7 718 6 686 7 202 500 0400 487 7 30 5 30 6 326 25 7 750 6937 7314 0613 0 341 0 492 & 74 4 66 6 697 Cal utta (Nap er Sen Gupta and Chandra Sekar 1941) ·102 HEMATOLOGICAL TECIINIQUE Vethod of calculating The following figures show the method of calculating the mean cell diameter (MCD) the standard deviation (o) and the co efficient of variation (v) 65g the median has bcen taken as the arbitrary mean TABLE IVI Diameters in microns fxd fx d Deviation (d) Number of of each class corpuscles in from arb mean each class in 0.54 (f) 1c the class interval 10 72 10 131 74 5 25 5 50 5 75 6 00 65 6 50 6 75 7 00 7 25 750 7 75 - 402 18 34 67 74 107 70 50 46 18 5 4 2 I TOO 288 306 268 74 0 70 200 414 289 I 5 + 3 + 4 + 5 + 6 + 7 + 8 70 + 100 + 138 + 72 + 25 24 + 14 + 80 8 25 98 64 8 50 + 457 goo + 49 2437 (x class interval 1) n=total number of observations=sum of frequencies ()=500 Sfd=sum () of the product of the frequencies () and the degrees of deviation (d) from the mean= +49 Sf d=sum of the product of the frequencies and the squares of the deviations (da) Class interval=0 254 sid Mean corpuscular diameter (MCD)=arbitrary mean + *025 )u=65 1=6524544 500 sid Standard deviation ()= x class intervalx0 25 = 4878- (0 098)2 x 0 25u= 4878--00096 x 0 25 1= 4 868 x 0 2544 = 2 206 x 0 25u=0 5514 0 551 X 100 =84 per cent Co efficient of variation (U)= MCD 6 525 --65+ ( 49 F 439 500 5070 ox100 ·RED CELL DIAMETERS PRICE JOVES CURVE 105 Significance of the findings --It is not possible here to enter into any cxtensnc discussion on the significance of the different findings but as a general rule it may be taken that in cases that have a high microcytosis percentage and a low MCV thc anemia ts due to uron deficieny When such microcytosis is assocuted with a normal or even a high MCV it means that the cells arc thicker than 7 144 to thanders 110 31 Ideal maxunum and minimunt curves based on Indun data (Naples Ses Gupta and Chandra Sekar 1941) normal and there is a tendency to spherocytosis such a condition is usually found in hemolytic anemias Pernicious anaemia shows a flat curve with a wide base usually a high percentage of macrocytes and a few mxcrocytes whereas tropical macrocytic arema is usually a socated with a moderately tall cune with an MCD slightly on the macrocytic side a few macrocytes and probably no microcytes 14 ·10.4 HÆMATOLOGICAL TECHNIQUE TABLE XVII Frequency distributions for Case X CASE Y lo Measurement to nearest Q 2514 ideal minimum curse MCD=69374 = 49484 ideal maximuni Curve MCD=7 7504 = 49484 microcytes Excess macrocytes Ercess Dicrocytes Excess Exces macrocytes Count Count кн 30 30 6 38 4 00 4 25 4 507 4 75 5 00 525 50 5 75 600 6 25 6 50 6 75 7 00 / 5 7 50 7 75 8 00 39 IT 14 32 39 68 93 99 82 53 27 10 3 1 8.25 88 100 88 61 32 14 + 850 8 75 goo 925 950 975 10 00 20 25 TO 50 10 75 11 00 II 25 II 50 Yr 75 12 00 I 500 73 500 196 7 500 Case X Percentage of microcytosis = 3 x 100 - 06 per cent 500 macrocytosis = 73 X 100 =146 per cent MCD= 779556 = O GO3M v=I1 5 per cent Case Y Percentage of microcytosis = 194 100 = 38 8 per cent = 14 per cent MCD= 6 434 r= |o7 v=16 6 per cent 500 macrocytos sa 7x100 500 ·RIOOD CROUTINO AD BLOOD TKASTUS101 107 llamagglutination and blood groups The transfusion of blood requires the use of compatible blood that » Wood in which the corpuscles of the donor are not agglutinated or harmoly ") by the plasma of the recipient (the patieni) and the corpuscics of the Titipicnt are not agglutinakd or hemolysed by the plasma of the donor The plicnomeson of fut magglutination is due to the fict that thert use in the blond of man intermigglutinogens in the sed corpu cies in<l iso humag, lutinin in the plasma there are fun myip ya lirmgglutinogens A and B that occur in the red cells and two corresponding arginiinins a anu A) and (anlı 1) in the pla ma * * possible to dnede mankind into four main group according to the apglutınıns and agglutinogens that are found in theis plasmi and red cells respec Axely Agglutinogens are present in the red cells of an infant from buih but pecific agglutining only make their appearance after some time Both increase in hire up to the 16e of puberty alter which the titre of the agglutinogens remains stationary but that of the agglutins gradually diminishes ind i sery low in old agr Both the agglutinogens and agglutinins remain contant qualitatively throughout life These four groups are as follows Coup Agglutinogens shr celle (internal ons? nomenclature) Agglutining in 112 m2 or struin) AB Aan B hone A A 8 (anla 13) (ants A) 0 nonc and B (2001 A ani anti 13) It is thus the agglutiningens in a person s red cells that give the trame to his Ayour though by simple deduction st 25 possible to ascertain his group from the agelutinins in his plasma (or stum) When A agglutinogea is the red cells) tomes in contact with a (an! A) agglutinin in the plasma) agglutination takes para similarly FanUtination takes place when 1 asglutinogen comes in contact >>3t) B (ants }}) agglutinin The arrangem nt of agglutinasons and agglutitimas. As always such that. 10. any Single individua) auto barmagglutination will not take place but the fact that in the HUTIT TOCC Mere are four different groups of indu iduals precludes the indis criminate mixing of blood of different individuals en ervo Provided bloods are of the same group they can usually be mixed with safety for example group AB blood can be given to an AB individua) because neither the donor nor_the_tecipient has a (anti A) or B (ants B) agglutinins in his plasma to act with the A and B agglutinogens in the red cells and cause agglutination but AB blood cannot be gren--to an A individual because he will have 8 (antı B) a glutinins in his plasma ·106 HÆVATOI OGICAL TECHNIQUE 19 BLOOD GROUPING AND BLOOD TRANSFUSION Historical -After Harvey s discovery of the circulation of the blood Sir Christopher Wren in 1650 injected opium beer etc into the veins of dogs using quills as cannulæ but the first transfusion of blood was given by Richard Lower in 1665 he transfused blood from one animal to another of the same species using dogs mostly as his experimental animals The credit however for performing the first transfusion in man is usually given to Denys de Montpellier physician to Louis XIV After this transfusion was indiscriminately used as a curative agent in all diseases and in many cases animals were used as donors So many deaths followed this indiscriminate use of transfusion that the operation was banned by the Royal Society in England and by the Académie de Medecine in France and was con demned by the Pope as immoral Blood transfusion then fell into disuse as a therapeutic measure for about 150 years being revived in 1818 by James Blundell an obstetrician in Guy s Hospital for the first time he gave transfusion through a syringe in cases of post partum hæmorrhage and claimed immediate success He was of the opinion that transfusion by the new technique was a very teasible operation which after undergoing the usual ordeal of neglect opposition and ridicule will hereafter be admitted into general practice By this method whole blood was used for transfusion until 1835 when Bischoff first introduced defibri nated blood for this purpose and showed that it was safer as it obviated the dangers of coagulation However the transfusion both whole and of the defibrinated blood still resulted in death very frequently and it was not until early in the present century that transfusion of blood was firmly established as a dependable therapeutic agent In r9oo Landsteiner an Austrian brochemist made the crucial discovery of the presence of iso agglutinins and 10 agglutinogens in the blood On the basis of Landsteiner s discovery Jansky a Czecho slovakian and Moss an American independently showed (1907 that the blood of the human race belonged to one of four groups-I II III and IV-with this difference however that Jansky s groups I and IV corresponded respectively to groups TV and I of Moss groups II and III being identical in the two cases To do away with the confusion caused by the two different nomenclatures an international committee of the Health Organization of the League of Nations introduced a simplified and rational nomenclature for the four main blood groups which they called AB A Band o The international nomenclature of the blood groups and those of Jansky and Moss are shown in the table-below International nomenclature Moss nomenclature Jansky nomenclature AB A II 111 IV IV II 1) 1 0 ·BLOOD GROEPING AND BLOOD TRANSFLSIOY 109 may be fatal Ilash the red cells in the citrated saling tube three tunes with normal saline Finally make a 5 per cent cell suspen on in normal saline The blood in the other tube is abo centrſuged and the clear scrum is then transferred to another labelled tabe- this is an important detail neglect of which is lable to lead to serious mistakes and it mu t be emphasized that such mistakes The name should at the same time be entered in a register and a serial number given which should also be written on the label Astaratus a d sate al requr ed- (1) hnown high titre (antı ) and B (loti B) serat (11) hdown Aan B cells-wash! but not litate (11) Class slides 3 inch x I inch (t) Test tabes 2 x preſ rably without lips (u) Glass penal (ti) Capillary pipettes with rubler teats (1) Sall solid glass rods with a dimeter of Rot more than inch and with the en is rounded off (UNI) Black an I white tile (figure 32) (1) Iland lety (x) Voist chamber (f gure 33) Thi can be prepared by lacing a piece of filter paper at the bottom of a pein-dish and keeping it wet with water Tuo glass rods of equal calibre are placed across the duameter of the perrı-dish about 1 inch apart with a piece of glass mc of a small r calibre inserti in between the two long glas rods to keep them apart It is then kep covered (1) The unknown washel 5 per cent red cell su pension in normal saline (+) The unknown serum Procedure To ascertain the group of an unknown sample of blood mix the unknown red cell separately with cach known high titre serum which contain . (antı A) and B (anti B) agglutinins respectis ely wait for a few minutes and The washing of the red cells is done in the following way --Centrifuge the blood in the citrated saline tube until the supernatant Rurd is clear carefully pipette off the supernatant fluid add an equal volume of normal salse centrifuge again and again discard the supernatant fluid Tepeat the process a third time 1. When doing groaping of blood for th first time known high titre and B sera sh yuld be of tained from any reliable institution where blood grouping is done as a foutine Provided the whole process carried out with the strictest aseptic precaution serum kept sealed w a refngerator will undergo do changes for a year or more For subsequent us hugh titre « and Asta hoald be collected from time to time to r plenish the stock from the subjects whose lood are groupe In places where blood grouping 1 dore only occasionally the groups of the personn 1 of the lat.oratory hospital or dispensary should be ascrrtained so that in an emergency grouping of an u known samp! of blood can be carried out wth and 8 sera collected from among them even if the agglutinins are not present in in b gb titre A gh titre serum 15 one which will agglutinate the appropriate red c L ua a lilation of at last I in 10 In some countries dried high titre grouping seta e ani p an Dow available uch xra are supplied in pou ler form and ar reconstitated by the addit on of salin Th advantage of these is that they are easy to send any distance they keep almost and fintely and they can be reconstituted to forn a hyper-concentrated solution Known A and B cells ma t always be collected freshly from persons kao o to belong to A and B groups for even when collected aseptically and kept in a refrig rator the cell bæmolyse in a few days and are pot sustal le for use There is also a danger in u ing tored cells as bæmagglut nation has been found to occur when stored cell come in contact with bacteria especially of the coryn bacterium group which may be present in the glassware used ja performing the test (Tlo nsen s ha naggi talion phenomenox) ·108 HEMATOLOGICAL TECHNIQUE which will act with the B agglutinogen in the donor s red cells and agglutinate them to a B individual because of the a agglutinins or to an O individual because he has both a and B agglutinins On the other hand in an O blood there are no agglutinogens so that such a blood can be given with safety to any person of his own group or as far as the donor s red cells are concerned to a person of any other group and for this reason such an individual is sometimes called a universal donor However in the plasma of the universal donor there are agglutinins both a (antı A) and ß (anti B) and these are capable of acting on the appropriate agglutinogens in the recipient s red cells agglutinins are always weaker than the agglutinogens so that in practice of the blood is given slowly the agglutinins in the donor s plasma become diluted with the recipient s plasma and no agglutination of the recipients red cells takes place unless as sometimes happens the agglutinins are present in exceptionally high concentration (high titre I in 32 or higher) a person who has agglutının present in high titre is known as a dangerous tensversal donor and his blood should only be given to an individual of his own group Now let us consider the position from the point of view of the recipient A B and O persons have in their plasma ß (antı B) (antı A) and both a and B agglutinins respectively and can therefore receive blood only from persons of their own groups that is persons who have not the corresponding agglutinogens in their red cells but an AB person has no agglutinins in his plasma so that he can receive red cells of any group and consequently an AB person is known as a universal recipient however his red cells contain both A and B agglutinogens and therefore if plasma from an A a B or an O person which happens to contain a high concentration of agglutinins is given his red cells may be agglutinated a For these reasons it is always essential that direct cross matching should be done before the blood of a universal donor is given to anyone except a person of his own group or the blood of anyone except a person of his own group is given to a universal recipient Further as there are a number of sub groups in the main blood groups-especially in groups A and AB and as an extra check against mistakes even when blood is given to a person of the same group it is advisable except in cases of great urgency to do a direct cross matching that is a matching of the donor plasma with the recipient s red cells and vue versa A GROUPING Collection of blood for grouping –Grouping of blood can be done with a drop of blood obtained by pricking a finger or the ear lobe but it is best done with washed red cells and the result checked by the cross identification of the agglutınıns in the serum Collect about 3 c cm of blood aseptically in a dry syringe put 2 c cm in a dry labelled test tube and r с cm in another labelled tube containing 3 to 4 c cm of citrated salıne (1 5 per cent sodium citrate in normal saline) IVnite the name and full description of the individual or the labels of ti e tubes ·BLOOD GROC PING AND BLOOD TRANSFUSION 109 Mash the red cells in thil citrated saling tube three times with normal salıne* Finally make 15 per cent cell suspension in normal saling The blood in the other tube also centrifuged and the clcar serum is then transferred to another labelled tube this is an important detal neglect of which is liable to lead to serious mistakes and it must be emphasized that such mistakes may be fatal The name should at the sime time be entered in a register and a serial number given which should also be written on the label Arpa alws and rateral required (1) hnown high titre . (ant: 1) and ß (ants B) serat (11) hnown A an! Bc11suashed but not dilutedt (1) Glashi les 3 inch x I inch (iv) Test tubes 2 xt" preferably without lips (u) Glass pencil (ti) Capillary tupettes with ruller tents (16) Small solul glass rols with a diameter of not more than inch and with the ends rounded oft (v11) Black and white tile (figure 32) (*) Hand lens (*) Voist chuber (figure 33) This can be prepared by placing a piece of filter paper at the bottom of a petra-dish and keeping it wet with water glass rods of equal call me are placed across the diameter of the pendi h about I och apart with a piece of glass rol of a smaller calibre insertech in between the two long glass rols to keep them apart Its then kep covered (xi) The unknown washed 5 per cent red cell suspen on in normal saline (v) The unknown scrum TW) Procedure -To ascertain the group of an unknown sample of blood mix the unknown red cells separately with cach known high titre serum which contain a (anti A) and B (antı B) agglutinins respectively wait for a few minutes and The washing of the red cells is done in the following way --Centrifuge the blood in the citrated saline tube until the supernatant fluid is clear carefully pipette off the superatant fluid add an equal volume of bormal sal de centnfuge again and again discard the supernatant fund repeat the process a third time 1. When do ng grouping of blood for the first tim known h gh titre and B stra should be obtained from any relił le institution where blood group ng is done as a routine 1 sovided the whol process is carried out with the strictest aseptic precaution 5 rum kept ealed a refngerator ill undergo no changes for a year or more For subsequent we high titre and sa should be collected from time to tim to replenish the tock from the subjects whose I loods are gro ped. In places where blood grouping is done only Xccasionally the groups of the personnel of the lalytatory to pital or dispensary should be ascertained so that in an emergency grouping of an unknown sample of blood can be camed w th apd sera collected from am ng them even if the agglutinins are not I resent in h gh titre A high titre serum is one which will agglutinate the approprnte red cells in a diution at 1 In 10 In some countries and high ttr grouping sera an1 Bart now ay luble such bera are supplied in powder form and are reconstituted by the addition of sal nc The advantage of these is that they are easy to sen1 any distance they k ep almost indefinitely ba reconst tuted to form a hyper-concentrated soluton i nown A and B cell mu t always be collected freshly from persons known to belong to A and B groups for even when collected aseptically nd kept in a refngerator the c 11 harnolyse in a few days and are not suitabl for u e There is also a danger in in contact with bacteria especially of the corynebacterium group which may be present using sto ed cells as hænagglutinat on has been found to occur when stored cells come in the glassware used to performing the test (Thomsen s hæ nagglutsatton phenomenon) out of least and they can ·110 }} CYATOLOGICAL TECHNIQUE then examine for the presence or absence of agglutination which will indicale the group of the unknown cell as shown below W P 10 Tig 33 F g 32 I 4 + + + B Example 2 3 a (ant A) serum (from group B)+unkUown cells A (anti B) serum (from group A) + vakoown cells + Group to which unknown blood belongs A3 A + denotes agglutraatsob - denotes no agglutination For corroboration a further test with the unknown serum may be made The unknown serum is mixed with known A and B undiluted washed cells when one of the following combsnations of reaction will occur indicating the group of the unknown serum -- J 2 3 + + Example Cells of group A+unkuown serum Cells of group B+unknot a serum Group to which the unknout belongs + + AB A B Technique (1) Tube method (a) Test for agglutinogens in the cells Place the test tubes 2 x 15 the first and second rows of a specially made rack With a glass pencil write the letters a 1 a 2 a 3 a 4 and so on os the first row of the tubes and the letters bzb 2 b3 b4 and so on on t3ze second row of the tubes Put one volume 00cc of anti A serum into the tubes on the first rou and one volume of ant B serum into the tubes of the second row and one volume of normal saline 117 all the tubes The samples of blood to be tested are numbered 1 2 3 4 and so on volume of red cell suspension from each sample of blood is put into the tubes 2 ·BIOOD crouri CAD proop TRANSFUSIOY III Dearing identical numbers 1 ach tube is rotated between the palms of the two hands to mix the contents and is capped to present er aporation The tubes are liſt for two hours Reading the results -The tubes are picked up one by one the cap: removed and the tube gently Micked with a finger to disperse the cell deposit from the Lottom of the tube If definste agglutination is not visible to the naked eye in any tube the contents of that tube are examined miroscopically The result art recorded as follows A I usible to the naked eve C (complete) Di tinct clump of cells v (v ual) Weaker but distinct clumping 13 Seen under the microscope ++ Iery big clumps + Smaller clumps less than + clumps of 8 12 cell 1 (Heah) Small clumps of 4-6 cells No definite clumps Any reaction less than + is unlikely to be due to true ha magglutination In such cases the cells must be tested again and the diagnosis confirmed by testing 2 the tum (6) Test for agglutinias in serum -The test is set up exartly in the same *?y as for tests for agglutinogens but in place of known antı A and anti B sera one volume of known A and B cells are put in each pair of tubes to which is added one volume of unknown serum and one volume of salinc The readings are taken after 2 hours and recorded as in (a) seaker reactions are more frequent and even a V reaction might be taken as a positive reaction (11) Slide method (the method of choice) (a) Test for agglutinogens in the cells -Place a clean glass slide lengthwise on the white portion of the ule in front of you With a glass pencil put the mark across the slide half an inch from the right end of the slide and with another mark divide the remaining portion of the slide into two equal portions Write the letter on the upper right hand corner of the extreme left portion the letter ß on the upper nght hand corner of the middle portion and the number of the unknown sample of blood on the extreme right hand portion of the slide (sce figure 32) Place a drop of anti A serum on the slide in the area marked a and a drop of antı B serum in the area marked A Place a drop of salıne in both the areas Rotate the tube containing the unknown cells between the palms of your hands to set a uniform suspension of the cells Withdraw a little cell suspension with 2 pipette and a put small drop of it by the side of but not in contact with the antı A serum and another drop by the side of ant B serum Mix each combination The signs used by the Blool Transfusion Rearch Committee (appointed hy the Meducal Research Council) bave been followed ·112 HEMATOLOGICAL TECHNIQUE -- cases ACT well with the glass rod using one end for each and then tilt the slide backwards and forwards to get an intimate mixture of the cells and the serum Place the slides on the glass rods of the petri dish over the moist filter paper and keep them covered (see figure 33) Serum Serum ß Examine for the pre sence of agglutination after GROUP AB BLOOD 10 minutes In most clear cut results are obtained (See figure 34) but if there is any doubt about the presence of agglutination examine with a hand lens or under a micro GROUP A BLOOD scope and record the results as in the tube method (b) Test for agglutınıns in the serum -The test with the unknown serum is made GROUP B BLOOD exactly in the same way as for tests for agglutinogens The unknown serum is mixed with known A and B washed cells and the readings are taken as GROUP O BLOOD in (na) For routine work prefer the slide method to the Fig 34 tube method for only a very small amount of testing serum is required for each test and in most cases clear cut results are obtained in a much shorter time Pseudo agglutination is occasionally seen in certain pathological states In hämagglutination real clumping of the cells occurs whereas there is on rouleaux formation of the cells in pseudo agglutination If there is still any doubt add a drop of salıne and shake the preparation gently by tilting the slide backwards and forwards when clumping due to hæmagglutination becomes marked but that due to rouleaux formation will often tend to disappear If there still any doubt the test should be repeated with a more diluted cell suspension when clear cut results will be obtained Prozone phenomenon -Very rarely undiluted serum will exhibit a peculiar inhibitory property and agglutination will not occur but agglutination becomes quite evident when the serum is diluted this is known as the prozone phenomenon B DIRECT CROSS MATCHING BEFORE TRANSFUSION This should be done not only the first time but on each occasion that a donor gives blood to a particular recipient because instances have occurred when bloods that were compatible at the time of the first transfusion have on a subsequent occasion shown a degree of heterogeneity and some agglutination has resulted ·TRANSFUSION 113 Technique -One drop of 5 per cent washed cell suspension of the donor us plared by the side of one drop of the serum of the recipient in one section of the lide and in the other one drop of serum of the donor by the side of one drop of 5 per cent washed cell suspension of the recipient Each pair » mixed as in the onginal grouping procedure and in due course the two mixtures are examined for agalutination II in neither case there is any agglutination thi confirms the fact that the blood are compatible 20 TRANSFUSION otic I Sources of blood Donors blood Healthy adults upto 50 years of age can donate 230 to 500 c cm of blood at a time once every two or three months according to the constitution of the donor Placental blood --It has been suggested that the blood left in the placenta at Jelnery should be utilized for transfusion Not only is it very difficult to ensure the sterility of this blood but any procedure that encourages the deprivation of the infant of csen a drachm of the blood that is its due is in our opinion an immoral Moreover the blood collected from a single placenta is quite insufficient even for a small tran fusion and is therefore not of great practical values Cadauer blood This has been used in Russi3 and in certain special circumstances the utilization of the blood of a body that has no further use for it seems justifiable in a hot climate the difficulties would be o great that the method need not be discussed here Transfuston of diferent elements of blood II hole blood Whole blood which acts as a complete tissue graft is Ideal for traosfusion where all the elements of blood are required The whole operation of withdrawing and trans the ite amount of blood must be completed within 3 minutes as otherwise the blood will clot in the syringe The transfusion therefore has to be given very rapidly which may not be advisable Fien then the transfusion of whole blood desnands a special apparatus or a team of trained workers with considerable dexterity when the ordinary syninge is used For at is seldom practicable to use a syringe larger than 50 ccm and of these three will be required one to withdraw the blood and one to give it and the third one being washed out with sterile cifrate salive and kept ready for use again Blood to which some anti coagulant has been added (1) Citrated blood. As an anti-coagulant various strengths of sodium citrate from 25 per cent to 38 per cent have been suggested the final strength of the sodium citrate in these mixtures is from 38 per cent to o5 per cent We prefer to use 38 per cent sodium citrate arriving at a final dilution of 38 per cent with this strength the dilution of the blood is not great and it is easy to calculate the amount of sodium citrate solution required viz 10 c cm for each 100 c cm of the mixture IS 2 ·114 HÆMATOLOGICAL TECHNIQUE Citrated blood can be used immediately or 1t collected with certain precautions it may be preserved* in a refrigerator between 4 to 6 C and used later ten days is usually considered the maximum safe storage time A great advantage in using citrated blood is that the time factor in unimportant The flow can be regulated and if necessary it can be given by the drip method Transfusion of citrated blood is however often followed by a mild reaction (11) The use of heparin as an antı coagulant (a) In vitro heparin may be used in place of sodium citrate solution 4 mg for each 100 c cm of blood is recommended the high cost of heparin limits its use (6) In vivo 1 mg per kilogram body weight is gren intravenously to the donor After the injection the coagulation time is greatly lengthened although this relative incoagulability of the blood persists for 30 to 60 minutes or more It is recommended that the blood be collected from the donor 7 minutes after the injection that the transfusion be given through a wide bore needle and that at be completed within 15 minutes Injection of such a blood is not injurious to the patient in any way and does not alter the coagulation time of his blood Transfusion by this method can be given with a 50 or a 100 c cm syringe and no special apparatus is required 3 Plasma or Serum Plasma -If the citrated blood is allowed to stand undisturbed in the refrigerator the corpuscles completely settle to the bottom of the bottle in two or three days time and leave the clear plasma at the top The plasma can now be pipetted off without disturbing the corpuscles at the bottom Serum If blood is collected without the addition of any anti coagulant the serum separates out in about 24 hours The serum can now be pipetted off without disturbing the clot Thus separated the plasma or serum after being filtered can be used at once stored in a refrigerator for future use or dried by various processes and Inter redissolved and used The great advantages of dried plasma or serum are reduction in bulk and ease of storage as it is not necessary to keep dried plasma or serum in the cold Properly dried plasma or serum can be u ed for at least two years from the date of its collection Transfusion of plasma or serum is not usually attended with any reaction at does not transmit malana and grouping of the blood is made unnecessary of the blood of several persons of different groups is mixed before or even after the plasma or serum is eparated They are ideal therapeutic agents to combat shock and are being very extensively used in the as their small bull facilitates transport 4 Resuspended Corpuscles The concentrated corpuscles remaining at the bottom of the bottle after the plasma is separated off can be used for transfusion in suitable cases of anæmia According to holmer (1940) these red cells can be war * Following preservative is recommended (Vollison and Young 1941) for store ! blood Sodium citrate 3 per cent 185 ccm Glacose 30 per cent 1 85 ccm For each TOO c cm of blood to be takep ·TRAVSFUSION 115 used with satisfactory results up to about eight days after which their orygen carrying capacity and other properties begin to diminish gradually The concentrated red corpuscles are diluted with glucose saline-to ench 400 ccm of sedimented red cells 50 c cm of 1 per cent saline with 4 5 grammes of glucose is added so that the final strength of the glucose in the mixture i 10 per cent The chances of a reaction following the transfusion of the concentrated red cells will be reduced if the leucocytes and platelets which form a white laver on the top of the red cells are discarded and if the mixture is filtered before administration (Vacquaide and Vollison 1940) 5 Defibrinaled blood The use of defibrinated blood is nou almost obsolete for the process of defibrination temos es many vital properties of the blood and also renders it more toxic Indications for transfusion After acule hæmorrhage -To make good loss su tained by an acute hæmorrhage the transfusion of whole or citrated blood either preserved or fresh is the most effective The amount to be transfu ed will be dependent on the amount lost but anything less than 500 c cm is unlikely to be effective 2 Shock From whatever causes In shock without hæmorrhage plasma or serum is better than whole blood two or more litres may be nece sary In shock with hæmorrhag in addition to plasma or serum transfu ion of citrated blood 300 to 500 ccm depending on the amount lost should also be given 3 As a prophylactic measure against shock-In weak or exsangurated patients even if their condition would not ordinarily demand transfusion this is often a very valuable procedure to prepare them for a surgical or obstetric operation blood or plasma is given according to the blood state of the patient 4 In anæmia -Blood transfusion is indicated to replace blood lost by hæmorrhage or destroyed by other processes to maintain life in the case of aplastic anæmia and/or to stimulate blood formation Whole blood citrated blood or resuspended corpuscles can be used In ancemia repeated small transfusions of 300 to 400 ccm are usually better than a single big tran fusion of say I 000 ccm in either case slow administration is to be recommended For supplying other deficit elements riz platelets in thrombocytopenic purpura leucocytes in agranulocytosis or fibrın in some cases of clotting deficiency transfusion of whole blood will sometimes be effective 6 For counteracting to tins of increasing passile immunity --This is procedure that was at one time in fasour particularly in America the bloods of convalescents or of others who had recen ed injections in order to tale the antitoxic value of their blood or to produce active immunity were used as specific transfusions in certain cases However the new chemotherapeutic agent introduced in the last few years have put this procedure into the background 5 a ·116 HÆMATOLOGICAL TECHNIQUE 'Technique of collection and transfusion of blood Apparatus and solutions requsred () Sphygmomanometer (11) Stethoscope (111) Bottle for collection of blood (Egure 35) (iv) Standard bottle for collection and transfusion of blood (Fig 36) (v) Potain s aspirator or a Higginson s syringe (vi) Glass container with an opening at the bottom to serve as a resery Gir for the blood (Fig 37) (011) Two moderate size serum synnge needles in a beaker containing liquid paraffia and a suitable adapter (figure 36) It is better to have two sizes as a smaller bore 19 required for giving than for drawing blood (vsus) Rubber pressure tubig (3x) Surgical silk thread (*) Pair of scissors (vi) Two enamel bowls (*1) Lifter (xui) Drum of sterilized dressings and towels (ru) Thermometer (*v) Rubber cloth (XVI) Measuring cylinder (*UN) Examination couch or easy chair for the donor (xvui) 3 8 per cent sodium citrate solution and normal salide both freshly prepared with pyrogenfreet distilled water and sterilized (XIT) Tincture of iodine (**) Tincture of benzoin (xxi) Absolute alcohol (*x+1) Bowl of antiseptic lotion (**111) Glass of water or some beverage to counteract any temporary psychological shock experienced by the donor Addtional apparatus of the transfusion is given by the open method (1) A splint to immobilize the Tumb tu which th transfu 100 is given • A Potaja s aspiratıng bottle makes a very useful receptacle for taking biood from a donor Pieces of pressure tubug are attached to the inlet and outlet taps to the other end of the inlet tube a record adapter is inserted ready to receive the needle and the other end of the outlet tube 19 attached to the Potain s syringe As an extra precaution to prevent micro-organisms passing into the bottle dunng the back stroke of the Potain s aspirator a piece of glass tubing containing a plug of stenle cotton wool may be inserted 327 the length of the pressure tubing The Potain s aspirating bottle is graduated on its outer surface so that the amount of blood collected can be seen at a glance The joints taps etc must be absolutely air tight *There are many standardized bottles now use which can be used for collecting storing and giving blood without transferring it the chances of contamination are thus reduced to a minimum The bottles are so made that for giving blood a transfusion tube with filter is inserted and is hung in an inverted position Such a bottle 19 described by Bail y (1940) (figure 38) See also Hayes et al (1941) + Water may be made pyrogen free up the following way In a clean glass stull re-distil some freshly distilled water to which a little sulphuric acrd and one or two crystals of potassium pertoanganate have been added to give it a fast pick colour If during the process of distillation the pink colour disappears froin the water in the still a little more sulphuric acid and potassium permanganate must be added The distillate is collected in a closed glass flask which has been previou ly prepare i by rinsing first with a solution of potassium bachromate and sulphuric acid they washing out first with distilled water and then with pyrogen free water and finally sterilized by autoclaving The pyrogen free water is sterilized in an autoclave and may be used for about three to four days ·TRANSFUSIOS 117 (11) Dis acting instruments viz scalpel scissors probe forceps and an 1919m necdl (11) Sterlized silk (v) Curved needle and bulkworm gut Selection of donors Strong healthy persons between the ages of 20 and 45 are the best donors The donor must be free from any infectious disease and his blood must always be examined for syphilis (Wassermann or hahn reaction) He should be questioned about allergic manifestations eg asthma urticaria etc as a temporary sensitivity to allergens may be transmitted through transfusion Malaria in a donor is a special danger in this country especially if the recipient has never had malaria Preferably the donor should be of the same group as the recipient but a safe universal donor group 0 (tide p 108) may be used for giving blood to people of all four oups and a universal recipient (group AB) may receive blood from donors of all four groups The following table shows the groups of suitable donors for different recipients If recipient belongs to group Dogor may be of group AB AB A B or 0 A A or o B B or O 0 In all cases the cells and serum of the recipient must be cross matched with the serum and cells of the selected donor even when the recipient and the donor art of the same group After 1 transfusion this cross matching of blood between the donor and the recipient must always be done again before another transfusion is given because the previous transfusion may produce some change in the blood of the recipient which will render it incompatible with the blood of a donor whose blood was found to be suitable before a transfusion was given The same donor must never be used for the same recipient after an interval of more than ten days after the last transfusion on account of the dangers of anaphylactic like reactions that have been reported A healthy donor of average size can given 500 ccm of blood once a month without any detrimental effect to his health After such bloodletting it takes seven days to make good the loss of blood but the compensatory mechanisms of the body are so efficient that whilst this deficit is being made up the donor will not be conscious of the loss in any way and will be capable of performing his full days work as usual Collection of blood The donor should u possible fast for 2 to 3 hours before the blood is taken because some articles of dret though innocuous to the donor may produce allergic symptoms in the recipient ·118 HÆMATOLOGICAL TECHNIQUE 2 300 | 700 The donor should be made to lie comfortably on the couch and there should be sufficient light on the selected arm A sheet of rubber cloth should be placed under the arm (1) Collection in a Potain s aspıralıng bottle (fig 35) Put the requisite amount of 38 per cent sodium citrate solution (10 c cm for 90 c cm of blood to be taken or for every too c cm of the mixture) in the bottle from which the air is now partially exhausted by pumping with a Potain s aspirator syringe or with a reversed Higginson s syringe if the former is not available Fit a needle of medium calibre on to the tube attached to the Inlet tap and test the negative pressure in the bottle by immersing the needle in sterilized saline and partially opening the stop cock in the inlet tap when the saline will be Fag 35-Potain s aspirating bottle sucked into the bottle The bottle is now used fo- receiving blord ready for use In the meantime an assistant has taken the systolic and diastolic pressures of the donor The skin over the vein is sterylized with iodine and then swabbed with alcohol to remove the stain of the iodine Raise the pressure to about the level of the systolic readings puncture the vein with the needle* and when it 15 in the vein open the stop cock in the inlet tap and lower the pressure to about the level of the diastolic reading when the blood will flow freely into the bottle The bottle is shahen gently so that the blood mixes thoroughly with the citrate solution and does not clot When the requisite amount of blood has been collected let out the air from the sphygmomanometer close the stop-cock and then take out the needle from the vein Apply digital pressure over the puncture for a minute or two seal with tincture of benzoin apply a sterile dressing and put on a firm bandage (1) Collection in a standard bottle (fig 36) Put the requisite amount of sodium citrate solution (VS) or if the blood is to be stored a mixture of sodium citrate and glucose (p 112) in the bottle With the cap lightly fitted the bottle with the solution is sterilized in an Fig 36 autoclave The tandard and bottle for col When still very hot bring out the lection of blood C-The cap with bottle from the sterilizer and screw the cap a piece of rubber inside cf-The collecting tube wath tightly This will produce a quite good vacuum needles N for the donor and N for in the bottle The bottle is now ready for use the bottle 0i If the vein is not prominent or the operator is inexperienced it is not advisable for him to attempt to puncture the vet with the needle alone but to attach it to a syninge a little blood is drawn into the syringe to make certain that the needle is in the vein the syringe is then detached from the peedle to which the adapter in the end of the collecting tube is quickly fitted The vein seeker which is sometimes recommended achieves much the same end ·TRANSFUSION 119 Now proceed as in (1) using the collceting tule (fih 36) in place of the tube attached to the inlet tule of the Potains bottle Puncture the icin with the needle (Ni) attached to one end of the collecting tube and when it is in t'u vein pierce the rubber through one of the two holes in the cip of the bottle (fio 30) with the stout necdle (1) attached at the other end of the tube when the blood wall flow freely into the bottle When the requisite amount of blood has been collected let out the air from the sphygnonanometer and take out the needle from the vein Seal and dress the puncture \fter the collection of the blood give the donor a drink of cold water or milk or if the donor shows any s.119 of psychological shock some stimulart eg sal volatile or soluc suitable form of alcohol Transfusion of blood Jake certain that all the apparatus for transfusion is rendy and sterilize 1 Iscfore starting the operation I or the method of sterilizing the different parts see below Select a good sem and arrange the patient comfortably on the bed with his ar restin, on? pillow coscred with rubber cloth (1) Transfusion trith the ordinary apparatus (fig 37) Sterilize your hands and fit one end of a piece of sterilized rubber tubin, to the bottom of the 6,115* reservoir record adapter to which is fitted a needle of small bore is now firmly fitted to a narrou tube about 9 inches long the other end of this tube is fitted by means of a klasy connection to the en l of the long rubber tube attached to the flass rosei voir (figure 37) The glass and the rubber conner tions the rubber tubing with the reservoir glass tubing and the adapter are now firmly tied with silk If the transfusion is to be 611 en very slowly the drip apparatus is incorporateel at about tha middle of the long rubler tube attached to the klass resert orr To tlic rubber tube a little above the drip ypparatus is attached pinch cock to regulate the flow of the transfuscl fluid llisht out the apparatus hy pouring sterilized normal silme into the reser or drive out the air bubbles in the tube by quezing the rubber tubing and see that the saline flows freely through the necdle, leiving a little about 10 cm of siline 11 the glass reservoir but when a drip apparatus is uscd nanipulate the pinch cock above it so that Fig 37—Transfus on appara tug for 51110g blool or sale the salıne flows only in drops throu,l the drip with the dr p fred apparatus apparatus and consequently also at the same rate It orco ated in the tube (10 this figure the leogth of the through the needle at the citrume end tuli & bet een the dr p feel The skin over the selected rein is sterilize 1 Anithe ne lle is shorter that it would be in practice) with tincture of iodine and an assistant applies pressurc with one hand just above the selected vein in order to make ·120 HEMATOLOGICAL TECHNIQUE it stund out prommently The operator nou puncttires the Telll sth a success ful puncture the blood from the patient will flow hack into the glass tubin, but, when the pressure above the vem is released the flow is thereby reverse ancl the saline will pass mto the vem (As m the (?se of the donor the puncture may first I made with the ner dle attached to 1 sma'l Svimme 1 little blooll with 14 the syring detachell from the necillind the adapter at tha end of the rubber tubin), from the reservoir fitted to the needle ) il hen it is seen that the saime is pissin, into the vem the (tinted blood in the I ottle 15 pent) shuhen and then poured into the glass respion throu,h few layers of stealizel 471,-placed on the top of it When most of the blood has been len z lattle wirin salme is addesi to wash out the residual blood in the reservoir ind the tube the needle 35 taken out ind for a minutu o » pressure 14 Ipplied to the punctue which is then seal with tinctmt of benzoin and a bena.e applied o el 7 steule des (1) Transfusion with the standard apparatus (fig 38) when stored blood is used, the tenpei ture is slowly rusend to 37°C hy puttı, the bottle ist? the blood in 1 vessel containin, hot water the tempuintuie ut which must not be very high When the temperature has been raised to about 37°C th cap is taken out and the transtusion outfit is fitted 1g 35 into the bottle--the outer rubber cork will fit in The tai lud Lottic micely into the mouth of the bottle but is in 11 matli ti in fu in out piee uution the coil is hept fived to the nec) of fit I -- lulit tube for at the rottle with adhesive plister Filter for DI ) I LLU The bottle is now insertid ud with little mani of fine wire gauze C-Tube cork to Joll pulation ? continuous colun 1 of blonxl without the filter in ustion my ait pubble is made to fill up the whole len, in Clubber ork fit of the tuba I he pinchcock above the clip ir into the auth the hottic piritus s brampil ted so that the Wood floss in IT-Trinsfusien tul> dtops thrudi b the dripipputus flu veins Lok D_Dun feed 11 lr itu pun tuled and the intc of flow is regulated hos manipulition of the pinchcock Wien all the blood his been plan the needle is taken out and the punctul scalert is in (1) D it Rate of transfusion The inte at which blood is transfused ]lepend entirely on the crcunstant In 1 case of acute and serious blood loss a rapid transfusion is indicated 200 cent being ellen in 10 to 17 minutes but in chronic conditions especulls cases of sever anæmia the drip feest method by which I cem per pound weight of patient per ·TRANSFUSION 121 veins hour is given or an even sloner rate in which it takes 24 hours to g11e 500 ccm s preferable The open melhod --The trn fusion by the closed method is possible in all but extremely debilitated patients with very low blood pressure and collapsed in those cases tran fusion by the open method t resorted to The sein used for open transfusion is subsequently ligatured and for that reason and on account of the greater contenience of heeping the leg Timobilized for a long time the great saphenous sein is usually chosen 1 immobilized with a splint the vein is opened and a Rogers cannula which is u od in place of the ordinary necdle is inserted into the vein The cannula i hept in position with adhesive plaster With this technique blood can be glen by the dnp method at a very slow rate over a long period 24 hours or more without tiring the patient or interfering seriously with his movements in bed The leg Cleaning of the apparatus As soon as the transfusion is finished separite all the parts of the apparatus wash in tap water and soak in water for ses eral hours The different parts are then cleaned in the following way - Bottles --Thoroughly scrub the inside of the bottle with a stiff test tube brush wash in hot water Needles - Clean with stillete sah sith hydrogen peroxide and then with distilled water dry in alcohol and ether and keep in a vessel containing liquid paraffin Other parts --Thoroughly clean all the parts of any restige of blood with water using a stiff wire for the rubber tubing Next wash with dilute solution of soap and rinse in water to remote the soap Place all the parts in a large pan containing OI per cent Maoll and boil for 5 minutes Transfer to another bowl and wash first with di tilled water and finally with pyrogen free water Allow to dry re assemble thet different parts and pack for sterilising Stenlization The different parts of the transfusion set packed in a cloth bag and the glasswares eg bottle for collection of blood and the glass reseri or for transfused blood are sterilized in an autoclase at 120 C for half an hour Dressings are also sterilized in the same way (16) Needles-by boiling in liquid paraffin for about 10 minutes (111) Cutting instruments in strong lysol lotion and washing in sterilized distilled water before use Reactions after transfusions Transfusion of citrated blood is very often accompanied by light reactions which are sometimes due to pyrogenic substances present in the apparatus and in the distilled water used for preparing solutions There is seldom any reaction after transfusion of plasma alone 16 ·122 HEMATOLOGICAL TECHNIQUE chloride may Reactions after transfusion may be divided into three groups due to defects in the donor (11) due to technical causes and (11) due to autogenous causes in the recipient These reactions will therefore to some extent be avoided by (1) careful selection of donors particularly avoiding allergic subjects (11) following carefully the instructions given above and giving particular attention to the following-washing all the apparatus with pyrogenfree normal or citrated salıne so that no trace of distilled water i left in any of them and avoiding excessive shaking overheating and rapid administration of the blood and (111) taking action according to the circumstances as follows Autogenons causes in the recipient Remedt s (a) Susceptibility of the patient Lower the head end of the bed cover to shock with blanket and apply heat to the extrem ities If required 05 ccm of adrenalin be given hypodermically (b) Allergy Adrenalin chloride 05 ccm hypoderm ically calcium by the mouth and by injec tion and calamine lotion locally in the case of urticaria (c) Lysis of recipients blood An alkaline mixture should be taken (generally seen in cases of before the transfusion and the transfusion hæmolytic anæmia) should be given very slowly Still another kind of reaction may occur in a small nurnber of cases due to the difference in the Rh agglutinogen in the recipient and the donor It has now been definitely established that even when the donor and the recipient belong to the same ABO group lysis of the donor s blood may occur after the second and/or subsequent transfusions of the recipient is Rh negative and the donors are Rh positive Symptoms simulating those seen after-a-mismatched -transfusione con respiratory distress pain in the back vomiting may appear within a few minutes and later blood may appear in the urine If the symptoms appcar during the course of a transfusion the transfusion should-be stopped at oneIn an such cases every effort must be made to alkalınıse the urine and to promote simple diuresis by administration of big doses of Sodium citrate e g two drachms at once and one ouncc during the following -24 hours and subsequently tir-the urine is free of blood pigments In very severe cases 150 cc of 3 per cent Sodiutn citrate solution should be given intravenously at once which is to be followed by 450 cc of 3 per cent Sodium citrate solūtión mixed with 2400 cc of 5 per cent glucose and given-tery slowly by drip during the next 24 hour Prompt transfusion of 200 300 ccm of compatible blood from Rh negative donor es reported to give excellent results 7 ·TRANSFUSION 123 21 THE INVESTIGATION OF A CASE AND THE REPORTING OF RESULTS The question that we are frequently asked us that do you considtr the minimum erammations that should be carried out in a case of anæma Wc should be force to reply that sith the exception of tho in section 17 which apply particularly s kamoskagic diseases at least all the examinations that have been described in this little bol should be made for the proper inuestigation of a cate In actual practice nearly all the blood examinations can be carried out from a single pecimen of blood and do not take a good technician more than an hour if the Price Jones cune is crcluded A certain amount of time can be sased by omitting the Ameth count the fragihty test pod the enumeration of thrombocytes as there are many circumstances in which these tests will give httle information of practical valve This leases the hiemoglobin estimation the total red and weate cell courts the reticulocy to count cell volume percentage ratimation (hematocrit) and settimentation rate van den Bergh s reaction and while cell differential count to be done 35 a minimum routine procedure in truty case The findings will indicate whether these examinations should be supplemented Sternal puncture gives sery valuable additional information but it must not be considered as a short cut to diagnosis for it wil eldom be of any sale without a complete blood count and fartfer the identification of the various cells is a very skilled work that necessitates considerable experience There will be occasions when elcctnc current is not as silable and in these circumstances it will not be possible to estimate the cell volume percentage satisfactorily The meas corpuscular hemoglobin (MCH) houerer usually runs more-or less parallel with the mean corpuscular olume (HCV) and it will be advisable to base the diagnosis on the mean cospuscular bæmoglobin alone The determination of the cell diameter by clinical halometres though very easy and simple is in our opinion sery inaccurate specially in pathological conditions and should never be employed The inclusion of the erytkrocyte sedimentation rate (ESR) in the minimum procedures might be questioned but if a I introbe s tube is used for cell solume estimation the ESR requires no additional apparatus and entails no extra labour except the taking of a seading at the end of an hour and the informaton ut gises is often useful Gastric analysis as important when the question of pernicious anemia anises and in some cases of microcytic nizma of doubtful xtiolon but except in these cases and as a general procedsre in sestigating the function of the castro intestinal tract dysfunction of which is so often an etiological factor in apxma in India ne vould not give it a high place in the order of mportance is the investigation of cases of anæsia in this country unless there are associated gastro-intestinal symptosis ·124 HEMATOLOGICAL TECHNIQUE For the complete investigation of the æuiological factors many other examina tions will have to be carried out eg the urine for albumin blood and ova the stools for protozoa helminths and pathogenic bacteria for fat content and for the presence of occult blood and the blood for malaria aldehy de test blood grouping the Wassermann reaction cholesterol albumin/globulin ratio etc Controlling progress-Circumstances will usually dictate how often blood examinations should be carried out during treatment but there are certain points which we will consider here In the first place when a veriously ill patient is admitted into hospital he is often suffering from some degree of hæmo concentration and a second examination made a few days after admission may show a marked drop in hæmoglobin percentage whereas the patient s condition may have improved considerably It allowance is not made for this fact the effect of the first treatment that is given may be misjudged and we have always adopted the practice of making second examination before prescribing specific treatment (except in urgent cases where an immediate blood transfusion is indicated) Further there are many nutritional anæmias in India in which there is a steady improvement directly the patient is put to bed and given a good diet Allowance must also be made for this fact if the value of any special form of treatment is being estimated and in such circumstances at least two weeks of rest and dict should be allowed before the specific treatment is started We make a practice of repeating the blood examination including hæmoglobin percentage red cell count reticulocytes cell volume percentage van den Bergh s reaction and ESR and examination of the film for abnormal cells once a week or earlier if a set back is suspected or if the treatment is to be changed In special cases other examinations such as the platelet and Jeucocyte counts are also repeated When any specific form of treatment is given a reticulocyte count is done daily from the 4th until about the 12th day or in a case where there is a reticulocyte crisis until the reticulocyte percentage has fallen to its previous level The printed forms that we use in the School are shown below There is quite possibly room for improvement in these forms and we have modified them from time to time but we find that without being cumbersome they are sufficient for our purpose Form 1 is the ordinary report form which is sent to the ward and attached to the riotes Size 81 by 6 inches Form 2 is the sternal puncturo report form similarly sent to the ward and attached to the notes a résume of this is entered on the reverse of form 3 size 8 by 54 inches Form 3 is the card which we keep in the laboratory The important data on which progress I judged are entered on the face of the card and other data and records of examination that are not usually repeated on the reverse size 81 by 64 inches_stiff card ·HEMATOLOGICAL TECHIZOLE 123 FORM I ST ANTUA DENT BLOOD REPORT Nalle Bed & rer 100 ccm femu per cent NYT ceat Hamaglaban Red blood cells Retsculocytes Cell volume Meas corp solume Vman corry hemoglobin Meas com hemoglobin cone if hite blood cells CU # YY ser cent per c mm per cent per c namn eu trophis Lymphocytes Monocytes Eosinophils Baxophus Aste à count i 11 311 1 1 Weighted mcan Blood group Abnormal cells Platelets per cm Coagulat on ts MA 131 ed ug ihne raglily of red cells roth ombre Capula y resistance test S¢dwalion ale (+ hout) Obsered Corsted Direct I an den Bergh s test mg per 106 CD Sadureet Option Date ·124 HÆMATOLOGICAL TECHNIQUE For the complete intestigation of the ætiological factors many other examina tions will have to be carried out eg the urine for albumin blood and ova the stools for protozoa helminths and pathogenic bacteria for fat content and for the presence of occult blood and the blood for malaria aldehy de test blood grouping the Wassermann reaction cholesterol albumin/globulin ratio etc Controlling progress-Circumstances will usually diutate how often blood examinations should be carried out during treatment but there are certain points which we will consider here In the first place when a seriously ill patient is admitted into hospital he is often suffering from some degree of hæmo concentration and a second examination made a few days after admission may show a marked drop in hæmoglobin percentage whereas the patient s condition may have improved considerably If allowance is not made for this fact the effect of the first treatment that is given may be misjudged and we have always adopted the practice of making a second examination before prescribing specific treatment (except in urgent cases where an immediate blood transfusion is indicated) Further there are many nutritional anæmias in India in which there is a steady improvement directly the patient is put to bed and given a good diet Allowance must also be made for this fact if the value of any special form of treatment is being estunated and in such circumstances at least two weeks of rest and diet should be allowed before the specific treatment is started We make a practice of repeating the blood examination including hæmoglobin percentage red cell count reticulocy tes cell volume percentage van den Bergh s reaction and ESR and examination of the film for abnormal cells once a week or earlier if a set back 15 suspected or if the treatment is to be changed in special cases other examinations such as the platelet and leucocyte counts are also repeated When any specific form of treatment is given a reticulocyte count is done daily from the 4th until about the 12th day or in a case where there is a reticulocyte crisis until the reticulocyte percentage has fallen to its previous level The printed forms that we use in the School are shown below There is quite possibly room for improvement in these forms and we have modified them from time to time but we find that without being cumbersome they are sufficient for our purpose Form I is the ordinary report form which is sent to the ward and attached to the notes site 8f by 61 inches Forn 2 is the sternal puncture report form similarly sent to the ward and attached to the notes a résumé of this is entered on the reverse of form 3 Size 84 by 5t inches Form 3 the card which we keep in the laboratory The important data on which progress is judged are entered on the face of the card and other data and records of examination that are not usually repeated on the reverse size 81 by 64 inches--stift card

·FORM 3
AN EVIA CARD
ΑΝ ΞMIA OΓPT
STX
11 ard
Bed
Ser
Name
LEUCOCYTES
TSR
Abnormal
VUD
Irb RBC Ret
CV
HCV
vir
VICIIC
Trentment
and diet
cell
Date
Total
N
L
E
B
HEMATOLOGICAL TECHNIQUE
127
-

·126 HÆMATOLOGICAL TECHNIQUE FORM 2 S TM ANEMIA DEPT STERNAL PUNCTURE REPORT Narne Age Hard Bed Total nucleated cells Reticulocytes Endothelial cells Red cell series Pro erythroblast A Megaloblast B с * Normoblast White cell series Granular series Myeloblast Pre myelocyte Neutro myelocyte А B meta myelocyte band segmented Eosino myelocyte meta myelocy te band segmented Basophils Non granular senes Lymphoblast Lymphocyte { А B Plasma cell Monoblast Pre monocyte Monocytes Alegakaryocyte Undifferentiated Parasiles Date ·RFHIRFYT A***TH 1 (1994) BOLEY IE (1940) BARROY ES (1931) Heck RL (1933) Te ha g** BEVJAXIY A (193+) מטר BETREL F1 (193) CA TLF 3 B ans VNO CR (19f} Quote by Whatby ani Pr tror 1939 Surgery of the Mode « l arfare ant Living stone Elinburgh Sedis $0 p 77 Lalural y Wawwal Hamulwr 11 unders to l hula lelphia Red cont kc deus ry RH i don Researri EX ASSOC 1935 1 Delhi or fed soc 10T 594 Paikusog! !!! 3101 ky edi! Kul Desc iption of the da 25 () fonal In ersts Pres London The oiy nucra CI in and Co London | Path and Black 18 331 Indart ved ra T6 P 767 Buchem | 19 P1332 Ared Imern Ned 25 325 COOKE 11 F ant PONDER E (1937) 36 + 249 16 09 Indisy Vrd Cox Lancet P 249 1 * & Bact 48 211 su v od OLI BDP 97 1 CREED F (1938) DAS GUPTA CR (1941) GODFRIED G (1935) GRAY 55 C (1930) CRFIVE C 35 A W and WALTERS W (1925) HAYES Sef al (1941) WYNES 25 an WTBY L E 1 (1938) ISNAXIS MCG (1939) IVY AC SAPIRO Pant VELKICK P (1935) IYE CAR Nh SEN and VUKHERJI 13 (1911) HEMEDY P an: VACKAY (1935) holmek) A (1940) TRACKER R ant CARVER JI I (1937) LEVEYSOY SA ans VACH MIR RP (1937) MACFARLAVE R (193}} VACQUAIDE DNI and VorlesOY P L (140) VILLs 3 af 1 MAWSON C (1938) VOLLISOV PL 404 Young (1941) NAPIER LE and HILINDRIA HS (1937) NAPIER LE CHAUDRURE RN and Rar CHAUDHURE M N (19383 WAPTER LT an ! DAS Curs CR (1935) Idem (1935) Idem (1933) 1dem (1935) Idente (1936) NAPIER L F aod VASUUDAR DN (1938) Current Sc 10 p 516 Trans Ry Fur Trop d Hye P 291 Lai and CIA VA 18 82 D seases of the Blud and allu #logy ] Biri ott Co Philad Iphia (Incal Labu atury Drag us s Henry humpton London La cer P 1199 Bad Ved I 555 P 14$$ Bnt Vel) 79 ind / Vrå RS 15 R$29 Ind an Vor: 13 P5 Indax Ved Res 25 102 fod hd - P Rog 73 p 305 73 P 321 13 P 455 23 973 Na 26 P 541 ·FORM 3 (reverse) Blood Platelets Fragihty Arneth count Coagn time I II III IV V Bleeding time Wt mean Cap_res W R Prothrombin time Blood group Sternal puncture Total Gran Non gran Red Pro Erbt Mgbt BC Normo А в с Ret Spleen puncture Gastric analysis F I 3 hours Peptic d gest on Unine Ova Stool Protozoa Occult blood Price Jones curus Mean corpuscular diam (MICD) Standard deviation (Sd) Coefficient variation (0) Per cent macrocytosis microcytosis HEMATOLOGICAL TECHNIQUE Other data Spleen Clinical notes Liver Weight Epidemiological data Economic status Dietary habits Re idence-malarious of Dot Occupation Family 1 tory ·ERRATA 1 116 Fur filamenclatan 16 court 20 ditutu llutet 10 TUY ruling 203 1 รูบาน el lules propres 03l3 prepustil 33333341 raal 13 11031131 101231 El 1011 (I won 9 tfit till volumne et une autre -- ?? mulutc the st! eltrintron <tbaltus <obalt 51 scrols tse machixstie * 1 atselt Israel ? disclop I stop 1 Chtit pashtu 67 21 nomal data O al data (under Table r P3 7. tllet 11 Ciruik. 31 ) anulugulisation ·[x] REFERENCES concld Indian Med Res Mem & Directories Ltd No 33 Thackers Press Calcutta Indian J Med Res 29 P 375 Indian Ved Gaz 73 P1 NAPIER LE and NEAL EDWARDS M I (1941) NAPIER LF NEAL EDWARDS MI and Das GUPTA CR (1941) NAPIER L E and SEN GUPTA PC (1938) NAPIER SEN GUPTA and CRANDRA SEKHAR (1911) NICHOLSON D (1934) ORDWAY T GORHAM L and ISAACSR (1937) OSGOOD E E HASKINS HD and TROTMAN FE (1931) OSGOOD E E and WILHELM VN (1934) OSGOOD EE et al PEPPERO H and FARLEY DL (1933) PERKIN F S (1927) PRICE JONES C (1931) Idem (1933) Indian ) Med Res 29 October 4 Laboratory Medic ne Henry himpton London Diagnosis and Treatment of Diseases of the Blood Oxford University Press London | Lab and Clin Med 10 p 476 Ibid 19 P1129 Arch Intern Med 64 P 105 July Practical Hematological Diagnosis W B Saunders Co Philadelphia Arch Intern Med 40 P 195 Path and Bact 34 P 779 Red Blood Cell Diameters Oxford University Press London 1 Amer Ved Assoc 110 P 1658 Indian ) Ved Res 25 P 957 Ibid 25 P 741 Quart] Med 32 P 127 Quoted by Whatby and Bntton 1937 Indian Ved Res 25 p 505 Ibid 26 P 723 J 179 Deut Arch klin Ved 131 р mical Dugnosis by Iaborato y Vethods B Saunders Co Philadelphia WV QUICK A J (1938) RADHAKRISHNA RAO MV (1938) SANKARAN G and RAJAGOPAL к (1938) SCOTT RB (1939) SIMMEL H (1923) SOKHEY SS et al (1937) Iden (1938) THANNHAUSER S and ANDERSON E (19) TODD JC and SANFORD H (1939) VAUGHAN J M and HASLEWOOD G A D (1938) WESTERGREN A (1926) WHITBY LEH and BRITTON CJ C (1939) 1 HITBY L EH and HYNES M (1935) WINTROBE MM (1933) WINTROBE MM and LANDSBERG J W (1935) WVITTS LJ and HOBSON F C G (1940) ZANATY A F (1937) Lancet 1P 133 Amer Rev Tuberc 14 P 94 Disorders of the Blood J and A London Churchill Ltd | Path and Bact 40 P 219 Amey] Ved Sci 185 p 58 Ibid 189 P 102 P 862 Brit Ved J tt Lancet 15 p 958

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