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·LABORATORY AIDS
IN
ENDOCRINE DIAGNOSIS

·Publication Aumber 212
(MERICAN LECTURE SERIES
A Monograph in
The BANNERSTONE DIVISION
IVERICA LECTURES IN EDOCRINOLOGI
Edited by
TELLARO O TrouersOY AED
Clinical Professor of Medicine
University of Illinois College of Medicine
Managing Editor Journal of Clinical Endocrinology and Metabolism
Editor Journal of the Imerican Geriatrics Society
Clarcago Minors

·LABORATORY AIDS
IN
ENDOCRINE DIAGNOSIS
By
ROBERTO F ESCAMILLA MD
Associate Clinical Professor of Medicine
Unterstty of California Aſedical School
San Francisco California
CHARLES C THOMAS
Springfield Illinors
PUBLISHER
USA

·CHARLES C THOMAS PUBLISHER
BAYAERSTOVE HJOL SE
301 327 EAST LANRENCE AYUE SPRINGFIELD ILLINOIS USA
Published simultaneously in the British Commonpealth of Manons by
BLACKI FIL SCIENTIFIC PUBLICATIONS LTD OXFORD ENGLAND
Published simultaneously in Canada by
THE RERSOY PRIN TORONTO
This monograph is protected by copyright no
part of it may be reproduced in any manner
without sitten permission from the publisher
Cornight 1954 by CILARLES C Titovas
PUBLISTER
Library of Congress Catalog Card Vumber $4 6555
Printed in the United States of America

·DEDICATION To Doctor HANS LISSER-stimulatıng teacher preceptor and colleague through years of training in that exacting art and science-the practice of clinical endocrinology

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·CONTENTS 8 6 6 7 9 10 10 11 I INTRODUCTION II INFORVATION CONCERNING EDOCRIVE DISEASES FROM THE USUAL ROUTINE LABORATORY TESTS Blood Counts Complete Urinalysis Serological Tests for Syphilis III SPECIAL TESTS OF Biood Eosinophile Count The Four Hour Corticotropin (ACTH) Test The 48 Hour Corticotropin (ACTH) Test Blood Sugar Determinations and Glucose Tol erance Tests Folin Tiu method Somogyı method Glucose tolerance tests The Insulin Tolerance Test Plasma Cholesterol Blood Iodine Serum Carotin Serum Calcium Phosphorus and Alkaline Phos phatase Vethod for serum calcium Vethod for serum inorganic phosphorus Vethod for alkaline phosphatase Serum Electrolytes (Sodium Potassium Chloride) Serum sodium method Serum potassium method Serum chloride method Nitrogen Metabolism Plasma CO Combining Power I SPFCIAL TESTS OF URIVE (EXCLUDING HORVOVE Issays) Urine Chlorides 12 12 13 15 17 17 21 23 24 24 2. 26 28 28 29 29 32 32 31 31 111 ·LABORATORI AIDS | IPOCRIVE DÍACTO$1$ 33 36 37 38 40 10 Curler Poner 11 ilder test Robinson Poner hepler water test The Carter Robbins Test Urine Concentration Test Sulhon scE1 Test Phosphorus Excretion Flisuorth Hossard test for pseudohıypopara thyroidism Acetone Bolies Creatine in Urine V BASAI METABOLIC RATF RADIC7E JOONI. UP TANF GASTRIC AVAI1995 EI FITROCARDIOCRATE Bioon I'RISSURE TESTS Ij8n1 Verbolic Rate Somnolent metabolic rate kudsorcine lodine Upiake (estric Inalyses Flectrocutogram Blood Pressure Tests Benzodioxine test Regitine test Dibenamine test Histamine test Vechoki test Tetrreth ammonium test Cold pressor test V ROHSTOF RAT EXAVIVATIONS ral of Skuti Lstuition of Bone lge Dental Tals ray of Chest ray of Abdominal Region Gastrointestina! Stay Intratenous and retrograde pjelogrutis Perurenal air or gas injection Extraperitoneal pneumography frienography 50 51 51 51 53 23 57 39 59 59 61 63 ·CONTENTS 18 63 63 61 65 65 66 66 66 66 67 67 Other \ray Studies rays of extremities Osteoporosis Osteitis fibrosa cystica Polyostotic fibrous dysplasia VII URINE HORVONE TESTS Urine Hormone Tests Il oxysteroids Glycogenic corticoids Compound F and F excretion Allen test for dehydrorsoandrosterone Lutemızıng hormone (interstitial cell stimulat ing hormone) Urine estrogens Urine Pregnancy Tests Ascherm Zondek test Friedman test Gallı Mainini test Male frog test Female South African claued frog test Guterman test 17 hetosteriods Follicle Stimulating Hormone Excretion (FSH) Pregnandiol Excretion VIII VAGINAL SMEAR-CERVICAL MUCLS Vaginal Smear Method Normal variations Estrogen Deficiency Pregnancy Threatened abortion Hyperestrinism Detection of Cancer Iodine Vapor Stain Cervical Mucus \ ENDOVETRIAL BIOPS) Method 67 67 67 67 68 68 68 69 69 69 75 78 82 82 82 84 85 86 86 86 87 89 88 90 90 ·LABORATORY AIDS IN TWDOCRIN DIAVOSIS 92 91 91 92 92 93 93 Interpretation of Specimens Varmil Venstrual Cycle Preosulatory pliase Postortilatory plinse I'rogesterone Deficiency Estrogen Deficiency Imenorrhea Hyperestrinism Excess of Progesterone Folloning kiministration of Androgens or An drogenic Tumors Hypothyroidism Other Conditions Fndometrial cincer Endometrial ruberculosis SEMIN FAVINATION Vethod of collection Ilumination of the Specimen Volume Turbidity Viscosity Chemical rection Mounty Count of sperm itozoa Tipes of sperm110101 and incidence of pati ological forms Blom stain for viability Other internal found in semen Correlation with Bar! Vet ibolic Rate Fructose (oncentration VI Cestis BIOISY Method Examination of specimen Normal testicular tissue Defective or abnormal tissue Classification of in, le Clinical application 91 94 91 91 94 96 96 97 97 97 97 98 98 99 99 101 101 102 102 104 104 IOR 106 106 107 109 ·CONTENTS 1 Classification of Howard et al Value in the Klinefelter syndrome Value in boys with sexual precocity VII ENDOCRINE DISEASE INDEX Pituitary Diseases Acromegaly Gigantism Anterior pituitary insufficiency-adult Simmonds disease Hypogonadotropic eunuchoidism Hypophyseal infantilism Froehlich syndrome Diabetes insipidus Thyroid Diseases Hyperthyroidism Hypothyroidism (adult-childhood) Parathyroid diseases Hyperparathyroidism Hypoparathyroidism Pseudohypoparathyroidism Diseases of the Pancreatic Islets Hyperinsulinism Diabetes mellitus Diseases of the idrenals Cushing s syndrome Adrenal cortical tumor Adrenal cortical hyperplasia Adrenal medullary tumor Addison disease Simple hirsutism Gonads-Diseases and Issociated Conditions I regnancy Endocrine ovarian tumors Venopause syndrome Ovarian aplasia Cancer of the lower female genital tract Tuberculosis of endometrium 109 110 110 112 113 113 113 114 114 114 115 116 116 117 117 117 118 118 119 120 120 120 120 121 121 122 123 123 124 125 129 125 126 126 127 127 127 ·Y14 LABORATORI AIDS IT ENDOCRINE DIAGNOSIS Hypogonadism (male and female) Includes eunuchoidism Infertility (mile and female) Male climacteric Cincer of the male genital orgins l'recociolis puberty (of pineal hypothalamic thymus or idiopathic origin) Miscellaneous syndromes-not necessarily endo- 127 127 128 129 129 129 Crinc 129 129 hlinefelter syndrome Albright s syndrome (polyostolic fibrous dys plasia) Osteoporosis I aget s dise of Vitamin D intoxication Anorexia nervosi 130 130 130 130 131

·LABORATORY AIDS
IN
ENDOCRINE DIAGNOSIS

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·Chapter 1 I INTRODUCTION *NCHINICAL practice laboratory tests frequently ud in the drgnosis ind treatment of endocrinoprtlues Many of the tests to be described are widely used-at times per haps os crused and given undue emphasis in the manage ment of the patient Therefore the word Aids has been purposely incorporated in the title in order to relegate laboratory procedures to their proper rank in relation to a thorough clinical examination Laboratory findings are for the most part only aids or adjuncts not only in diagnosis but also in following the effects of treatment in endocrine diserse They do not replace or transcend a proper and complete inimnesis and plıysical examination of the patient Furthermore the fillibility of many of the tests is nell Anoun to anyone who fas worked for any length of time in the laboratory Many of the procedures are complicated and some of the steps may be uncertain even though the final results are expressed in exact figures This persuasive preciseness may lead to a false sense of security so that it times serious errors can occur unless laboratory findings are correlated "Atli clinical impressions If the laboratory results indicate conclusions at variance with clinicıl facts it is prudent to place more credence in the clinical observations and either repeat the tests (perhaps in another laboratory) or if necessary repeat or re evaluate entire experiments At limes it is advantageous with some of the more complı cated tests to confine them to one particular laboratory or even to repose confidence in one specially trained tech nician in interpreting results for clinical use 3 ·1 LABOR 1TORI AIDS Ir bOCRIVI DIAGNOSIS On the other brand lest tlie de emplizsis bc curricd 100 for Sborutory results occasionally can be most helpful and cien definitie in lignosis is the level of TSH cxcrc cion in the urine is of critical importance in distingishing betricen pituitary inſinulism ind or arian apl7s11 (being elevated in the latter) The laboratory may also be helplul in checking results of treatment is in following the level of plasma cholesterol or ile basal metabolic rite during treatment of hypothyroxlisia with desiccated thyroid sub Stuce Also laboratory studies have increased on anon1 cdge about physiological changes in several of the endocrine diseases and thus embellish and clarify the clinicians understanding of the parent and his disease in a manner beneficial to both physican and Patient In the following pages the various tests will be listed noting the principal abnormalities found in the presently acknowledged endocrinopathies in the final chapter (VII) tlus datis recapitulated in the form of 1 disease index fiscong the rious endocrine diseases and collecting under thiese lehmgs the appropriate laboratory findings Thuis opposite "pproach offers the reader 7 cross reference to the inuterial presented and proudes a substitute for the cus tomary indes The tests listed are those which hue been proved to have some clinical value plus a fex that seem particularly promising at this time Laboratory procedures auch still ire lughly experimental or are confined to research proj ects hue been purposely omitted in some instances more than one method is in use for the same test The method ollen will be the one currently used in the laboratories in or connected with the Unni ersity of California Hos pital or Vedical School In compiling 7 revient of this type one must rely herily on the generous help of friendly colleagues Sin

·INTRODUCTION
5
cere thanks are due to the following individuals who have
been most kind in giving advice reading sections and
cooperating in general Dr James Hopper Dr Morris
Durley Dr Peter Forsham Dr Felix kolb Dr Maurice
Soholow and Dr Hans Lisser of the Department of Medi
cine Dr Ernest Page Dr Ralph Benson and Dr M James
Whitelax of the Department of Obstetrics and Gyne
cology Dr Donald Smith and Dr Franh Hinman Jr of
the Department of Urology Dr A Justin Williams and
Dr Earl R Miller of the Department of Roentgenology
and Dr Norman Freeman of the Department of Surgery
Others have been most helpful in allowing the use of
photographs and charts for some of the illustrations Each
will be acknowledged at its point of appearance in the
text
RFE

·Chapter II INFORMATION CONCERNING ENDOCRINE DISEASES FROM THE USUAL ROUTINE LABORATORY TESTS E Fy the most common tests shot important abnor malities in some endocrine discuses BLOOD COUNTS A complete count including estimation of amount of hemo,lobin numbers of red cells and white cells differ ential coum of the isliste carpuscles and examination of the stuned smerr is a routine procedure with most phy sulans and certainly with most hospital entries In patients with Cuslungs syndrome, polscythemia with increase in hemoglobin (90 to 1200%) and number of red blood corpuscles (5 to 6 500 000) is a characteristic finding Polycythema is also seen in diabetic acidosis and in para thyroid telang Moderate secondary anemia is found frequently in my redema In this condition the hemoglobin is low in relation to the red cell count so that the calor index 15 less than one (color index can be estimated by doubling the first to numbers of the erythrocyte count and dividing this into the liemoglobin percentage) Occasional instances have been reported of the association of a primary anemia fith myrede ma In these the liemoglobin is high in rela tion to the red cell count sa that the color index is greater than one Secondary anemia is also found with some frequency in Simmonds disease Addison s disease severe hyperthyroid ism and late in the course of hyperparathyroidism 6 ·INFOR\IATION FROT THE USUAL I ABORATORY TESTS 7 Eosinophilia-an increase in percentage of eosinophiles in the differential count of white blood corpuscles to above the usual normal level of 1 to 3% is found with some frequency in many endocrine diseases It has been reported in more than average incidence in both Simmonds disease and acromegaly which are plıysiological opposites as re gards pituitary function It is also seen occasionally in Addison s disease severe hyperthyroidism and late lyper paruthyroidism Count of the circulating eosinophiles as de scribed in Chapter III is now used as a test of adrenal corti cal function 4 relative lymphocytosis so called when the percentage of lymphocytes in the differential count is above the usual average of 25 to 300% with a normal total hite cell count is another frequent finding in many endocrine diseases It has been reported in acromegaly pituitary infantilism hyperthyroidism my edema parathyroid tetany Addison s disease and hypogonadism As was noted in discussing eosinophilia many of these conditions are physiological opposites It is of interest to recall that before basal metabolic rate determinations were available the finding of a leucopenia sith a relative lymphocytosis was con sidered an important drgnostic sign in hyperthyroidism An increase in the total white blood cell count is seen in diabetic acidosis leucocytosis to a level as high as 75 000 white cells per cu mm is not uncommon COVIPLETE URINALYSIS This is another commonly performed test Usually the color curbidity specific gravity and degree of acidity or alkalinity are noted Tests are performed for albumin and glucose and frequently for acetone and the sediment is examined microscopically In some laboratories a Sulko witch test for rough estimation of the amount of calcium ·8 LAPORATORI AIDS I \DOCRINT DIACNOSIS in the urine is done routinely as is discussed in Chap ter IV Thic finding of glucose in the urine with associated high specific gravity is important in the dignosis of diabetes mellitus It must bi remembered glit diabetes mellitus is in occasional complication of acromegaly and it las been Variously estimated that 25 to 10% of acromegalıcs become durbetic In Cushings syndrome, most of the untreated pitsents eventually develop diabetes Glycosunt is also scen occasionally sn gigantısm hyperthyroidism and with adrenal cortical tumors (especially the Achard Tluer Syn drome- Diabetes of Beirded Women ) Diabetes insipidus may be suspected from urinary find ings alone The 24 hr volume (normally one to tho liters) will be increased to over three hters and up to a reported maximum of 56 liters Specific gravity is low-isually 1001 or 1 002-and the urine looks pole watery and tastes insipid Neither albumin nor sugar are found to be present the condition is a disease entity but may occa sonally be complication of tumors or other lesions in volving the picuscary or hypothalamus Such tumors may also cause various endocrine diseases including gigantism, acromegaly turtary infantılısm Froehlich s syndrome Simmonds discase or precocious puberty and thus diabetes insipidus may be associated with all of these clinical syn dromes Evidence of kidney damage with albumin casts and fired specific gravity is seen in the late hypertensie phase of Cushings syndrome ind occrsionilin diabetes mellitus when the kimmelstiel Wilson syndrome of intercapillary glomerular sclerosis has appeared Ulbumin may also be found during hypertensive attacks with pheochromocy toma Hiperparathyroidism is characterized by increased ·INFORMATION FROVE THE USUAL LABORATORY TESTS 9 urinary excretion of calcium so that in untreated cases lithiasis nearly always develops and eventually is further complicated by infection This results in the appearance of red cells white cells albumin casts and particularly calcium casts in thie urine Untreated myredema frequently is complicated by atony of the bladder This results in incomplete emptying stasis and residual urine which usually becomes infected so that pus cells and albumin are frequently found Acetone bodies is acetone or diacetic acid are found in diabetic acidosis or may also be present if the patient is on a seini starvation diet SEROLOGICAL TESTS FOR SYPHILIS These are performed with enough frequency to be con sidered a routine test They may be of some importance in diabetes insipidus which can be caused infection or gumma in the hypothalamus or pituitary region Also some instances of Simmonds disease have been reported to result from 2 gumma replacing and destroying the an terior lobe of the pituitary gland ·8 LABORATORI AIDS INDOCRIVF DIAGNOSIS If tlic (Irine 13 clone routincly as is discussed in Chap ter IV The finding of glucose in the urme with associucd high specific gravity is important in the diagnosis of diabetes »ellins le must be remembered that diabetes mellitus is in occasional complication of acromegal, and it has been liriously estimated th: 25 !0 40% of rcromegalics become drivetic in Cushings syndrome most of the untreated patients esentially deselop diabetes Glycosuria is also seen occasionally in gigantism lis perthyroidism and sath adrenal cortical tumors (especially the Achard Thier Syn drome, Diabetes of Beardeii Women ) Danbetes insipidus stay be suspected from uriniry find ings alone The 24 hr volume (normally one to two luers) will be increased to over three liters and isp 10 ? reported maximum of 56 Irters Specific gravity is lon-sually 1001 or 1 002-18 the urine tools prle watery and tastes irisipid Nesther albumin nor sugar are found to be present The condition is ? disease inuty but may occa sionally be a complication of rumors or orher lessons 151 Loksing the pituitary or hypothalamis Such tumors say also cruse various endocrine diseases including gigantism acromegaly prtutary infantılısm Froehlich s syndrome Simmonds disease or precocious puberty and thus diabetes insipidus may be 155ociated with all of these clinical syn dromes Fsidence of kidney damage with albumin casts and fixed specific guy is seen in the late hypertensne phase of Cushing's si sidrome and Occasionally ik drabetes melltus when the himmelstiel Wilson syndrome of intercapillary glomerular sclerosis has appeared flbumin may also be found during hypertensive attacks it Ith pheochromocy 10m Hyperparathyroidism is characterized by increased ·SPECIAL TESTS OF BLOOD 1 Stimulation of the adrenal cortical function in normal individuals will cause a fall of at least 50% in the number of circulating eosinophiles However in Addison s disease the count is high or high normal and does not drop after efforts to stimulate the adrenal cortex with adrenocortico tropic hormone (ACTH) or with epinephrine THE TOUR HOUR CORTICOTROPIN (ACTH) TEST The four hour corticotropin (ACTH) test of adrenal cortical function is based on the above Atechod An eosinoplule count is made with the patient in a fasting state and then 25 units of corticotropin (ACTH) are administered intramuscularly Four hours later another blood sample is drawn and the eosinophiles again counted Brerllast may be eaten after the first count buc lunch should be withheld us the second count is taken Normnlly the count should drop at least 50% from the stimulation of the adrenal cortex by the Cornicotropin If it does not adrenal cortical insufficiency is a possibility Recently Bonner has suggested that the test may be more sensitive if done in the non fasting patient from one to five pm THE 48 HOUR CORTICOTROPIN (ACTH) TEST Thuis variation is used when the four hour test does not result in clear differentiation between primary and sec ondary adrenal cortical disease Method A fasting count of circulating eosinophiles is done and then 10 units of corticotropin ( CTH) are add ministered intramuscularly at 10 am on the first day The dose of 10 units is repeated every six hours for 48 hours (eight injections)-giving the last injection at fam of the and day Another eosinophile count is taler four hours ·Chapter III SPECIAL TESTS OF BLOOD EOSINOPHILL COUNT Τα WIE DIRECT count of circulating eosinophiles has been found to be of value in estimation of adrenal cortical function is reported by Thorn Forshim and co-workers ? Method Fue cc of venous blood is collected with 05 cc of dried balanced ovalate solution in the tube A standard white cell pipette is used drawing the blood to the 05 nark This is diluted with a special stain to the 11 mırk The stain is prepired by mixing 5 cc of 2% aqucous eosin 5 cc of acetone and 90 cc of distilled water This should be hept in a refrigerator and filtered before use It should be prepared fresh every two weeks Origin ally a special counting chamber was used with a depth of 02 mm but it has been found that a standard counting chamber with a depth of 01 mm is satisfactory The pipette is shaken for only 30 seconds and the counting chamber filled immediately and alloned to stand for 3 minutes Most of the red blood cells and other white cells are hemolyzed while the eosinophiles are identified by the deeply stained red granules Four chanıbers of 16 sq mm are counted The total is divided by 16 and multi plied by 100 to gne the circulating eosinophiles per cu mm Cleanliness is essential as the cells tend to cluster around foreign bodies and timing is important as they are unstable in the diluent The normal range is 150 to 350 per cu mm Recently Ponner has suggested th it in eosinophile estimation from the differential count on a smear is as satisfactory as the Chamber method if 800 cells are counted 10 ·SPECIAL TESTS OF BI OOD 11 Stimulation of the adrenal cortical function in normal individuals will cause a fall of at least 50% in the number of circulating eosinophiles However in Addison s disease the count is high or high normal and does not drop after efforts to stimulate the adrenal cortex with adrenocortico tropic frormone (ACTH) or with epinephrine THE FOUR HOUR CORTICOTROPIN (ACTH) TEST The four hour corticotropin (ACTH) test of adrenal cortical function is based on the above Method An eosinophile count is made with the patient in a fasting state and then 25 units of corticotropin (ACTH) are administered intramuscularly Four hours later another blood sample is drawn and the eosinophiles again counted Breakfast may be eaten after the first count but lunch should be withheld until the second count is taken Normally the count should drop at least 50% from the stimulation of the adrenal cortex by the corticotropin If it does not adrenal cortical insufficiency is a possibility Recently Bonner has suggested that the test may be more sensitive if done in the non fisting patient from one to five pm THE 48 HOUR CORTICOTROPIN (ACTH) TEST This variation is used when the four hour test does not result in clear differentiation between primary and sec ondary adrenal cortical disease Method A fasting count of circulating eosinophiles is done and then 10 units of corticotropin (ACTH) are ad ministered intramuscularly at 10 am on the first day The dose of 10 units is repeated every six hours for 48 hours (eight injections)-giving the last injection at #am of the third day Another cosinophile count is talen four hours ·12 LABOR STORT SIDS DOCRIVE DIAGNOSIS later (al 8 m) before alloning breakfast Normal ande viduals shou a fall of over 50% while those with Addı son s disease consistently fail to fall to 50% of the original level 17 hetosteroid excretion can also be checked before and alter the course of corticotropin (ACTH) and nor mally rises to and above the upper limits of normal er cretion Patients with lapopituitarism and secondary adrenal cortical atrophy slıou an eosinoplule drop greater than the Addisonnin and usually Over 50% It is of interest to note that the lymphocytes respond in the sime manner as the eosinophiles in the above testsbut the eosinophule count is at present the more accepted BLOOD SUGAR DETERMINATIONS AND GLUCOSE TOLERANCE TESTS These are occasionally of considerable import unce in endocrine diseases The method of measuring the blooci sugar is now in 7 state of flux The older as the classical Folin 11 u method and is the one still used in most labora tories because of its proven dependability Honcier it does measure some reducing substances other than glucose so the Somogy method which measures only glucose is being set up in some laboratories and is the present one in use at the University of California Hospital Values with the Somogyi procedure are generally about 20 mg belon those ſound with the older Folin l’u so I knowledge of which method is used may be important in the clinicii interpre tation Both methods follow Folin Vu method Venous blood is dann into an ora Inte tube A protein frei filtrate is prepared as fol}ons One cc of blood is diluted with 7 cc of distilled water and mixed One cc of 10% sodium tungstate is then added and after mixing lat of 2/3 N sulfuric acid is ·SPECIAL TESTS OF BLOOD 13 added This results in a dark brown mixture which is filtered yielding a water clear filtrate The sugar test is dependent upon the reaction of the sugar with an alkaline copper solution-this results in a blue color the depth of which is in proportion to the amount of sugar Two cc of the clear filtrate is placed in a blood sugar test tube (graduated at 27 cc) and 2 cc, of two standard sugar solu lions are placed in each of two other tubes The standard solutions contain respectively 1 mg of sugar per 10 cc and 2 mg of sugar per 10 cc thus the 2 cc. of each contain 02 mg and 0 4 mg of dextrose To each of the three tubes is added 2 cc of alkalıne copper solution (prepared by dissolving 40 gm of anhydrous sodium carbonate in 400 cc of water adding 75 gm of tartarıc acid and then 45 gm of crystallized copper sulfate made up to a volume of one liter) The three tubes are boiled in a water bath for 6 minutes and then cooled in cold water Tuo cc of molybdate phosphate solution is then added to each tube (This is prepared by mixing 35 gm of molybdıc acid and 5 gm of sodium cungstate in 200 cc of 100% sodium hydroxide and 200 cc of water The mixture is boiled for 20 to 10 minutes to remove the ammonia cooled diluted to 330 cc and then mixed with 120 cc of concentrated (85%) phosphoric acid Final dilution to 500 cc is then performed ) A blue color forms and the volume is then made up to 25 cc with distilled water After mixing the unknown and the weaker standard are compared in the colorimeter The depth of the standard (in mm) is multiplied by 100 and divided by the reading of the unknown giving the sugar content in mg per 100 cc of blood If the stronger standard is used the depth of the standard is multiplied by 200 Somogyı method (blood glucose only) Venous blood is drawn and placed in an oxalate tube In preparation of the filtrate 1 cc of the oxalated blood is mixed with lo cc. of distilled water and 2 cc of barium hydroxide solution ·19 LABORATORY AIDS IFDOCRINE DIAGNOSIS later (7 8 nm) before allowing breakfast Normal indi viduals shou a fall of oser 50% while those 11th Addi son s disense consistently fail to fill to 50% of the original level 17 hetosteroid excretion can also be checked before and after the course of corticotropin (ACTH) ind nor mally rises to and above the upper limits of normal ex cretion Patients with his popitutarısın and secondary adrenal cortical atrophy shon an eosinoplule drop greater than the Addisonian and usually over 50% It is of interest to note that the lymphocyles respond in the same manner as the cosinophiles in the above testsbut the eosinople count is at present the more accepted BLOOD SUGIR DETERMINATIONS AND GLUCOSE TOLERANCE TESTS These are occasionally of considerable import ince in endocrine diseases The method of measuring the blood sugar is nou in a state of flux The older is the classical Folm Ilu method ind is the one still used in most labora torics because of its proven dependability However it does measure some reducing substances other than glucose so the Somogy: method which measures only glucose is bein, set up in some laboratories and is the present one in use at the University of California Hospital Values with the Somogyi procedure are generally about 20 m; belon those found with the older Folin lu so a knowledge of which method is used may be important in the clinical interpre tation Both methods follot Folin Wu method Venous blood is drun into an oxa late cube A protein free filtrate is prepared as follows One or of blood as diluted with 7 of distilled water and mixed One cc of 10% sodrum tungstate is then added and after mixing 1 cc of 2/3 N sulfuric acid is ·St'ECIAL TESTS OF BLOOD is adjusted to give 100% transmission through the blink An Evelyn 515 filter (green) is used which gives maximum transmission at 500 to 520 millimicians The blood glucose values tre then read directly from a graph with curie made up from standard solutions Glucose tolerance tests are generally satisfactory using the classical method as follous Fasting blood is drawn ind urine collected 337d then the patient drinks 100 gm of glucose in kater flavored with lemon Blood and urine specimens are taken at 30 minutes one two three and occasionally four he and sh hours alternards The longer tests are performed particularly if a hypoglycemic tendency is suspecued as the hypoglycemic dip in the curve may not occur unul the fourth fifth or $arth hour Some variations have been suggested in the method of performing this test such as given, the glucose according to the weight of the patsent (sustified in children) giung glucose more than once or giving the glucose intravenously (occasionally pusulied if absorprion from the gastrointesti ral tract is impaired or abnormal) Houever the method given above is generally satisfictory There has been some discussion concerning the neces sity for special diet before performing the glucose toler ance test In general the preceding diet will not grossly affect the glucose tolerance curse if the carbohydrate intale has been between 300 gm and 300 4m daily Nearly all dets aflord an enthe ot carbohidrate within these imits --the possible excepron bein, in a patient with severe noreviz nertosa Sul a patient should be fed a high carbohidrate diet several days before the test in order to obtain a tre picture of his <3pacity to handle glucose The normal glucose tolerance Chuve is as follows Fasting level between 90 and 120 mg (Folin Wu method) with a rise to a pezh of 130 to 160 m, in one half to one hour followed by a fall to the fasting level within 2 lours ·LABORATORY AIDS ENDOCRIN DIAGNOSIS added (I he latter is prepared by adding 21 gm Ba (OH) to enough water to fill a 200 « 1olumetric flash making 03B7 (OH) Tlus is adjusted with zinc sulfate solution (preparation given belon) to a pink end point using phenolphthalein is an indicator further adjustment is made by adeling water unul 4 7 to 48 cc ol buruum hy (rovide are required to citrate the zinc sulfate The solu son should be stored in a boule protected from the CO of the air by soda lume) The mature turns brown and then 2 cc of zinc sulfate solution are added (prepared by tissolving 2> gm of Inso, 7 HO in distilled water and bringing to volume 11 a 200 cc volumetric flash) This is filtered and yields a clear filtrate (lucose determination is accomplished by placing luc ol the filtrate in a sugar tube graduated at me cc In a similar cube is placed icc of distilled water One cc of copper reagent is added to each (The copper reigent is prepared by dissolung 28 gm of anhydrous disodium phosphate and 40 gm of Rochelle salt in 700 cc of water and then adding 100 cc of normal sodrum hydroxı«e Eights cc of 1 10° copper sulfate solution pre aclded with stirring and then 180 gm of anhydrous sodium sulfate This is dilured 10 a liter woned to stand for 1 to 2 days and filtered) The solutions in the tubes are mixed and then heated for 10 minutes in a boiling water bath After cooling in cold water I cc of arsenomolybdate color reagent is added to ench tube mixed and diluted to 22 cc with disulled wies They use then mixeil 333 read in a photoclectric colorimeter (I lie arschomolybd ite color reagent is pre pared by dissolving 2 sm of ammonium molybdate in 10cc of distilled water adding 25 cc of concentrated HO mexing and addın, gin of Na H4s0 7HO Clissolved in 21 cc of water This is mixed incubated at 37 C for 24 to 48 hours and then stored in a brown glass stoppereil bortle) In performing the colorimeter reading the colorimeter ·SPECIAL TESTS OF BLOOD 15 15 adjusted to give 100% transmission through the blank An Evelyn 515 filter (green) is used which gives maximum transmission at 500 to 520 millimicrons The blood glucose values are then read directly from a graph with curve made up from standard solutions Glucose tolerance tests are generally satisfactory using the classical method as follows Fasting blood is drawn and urine collected and then the patient drinks 100 gm of glucose in water flavored with lemon Llood and urine specimens are taken at 30 minutes one two three and occasionally four five and sıx hours afterwards The longer tests are performed particularly if a hypoglycemic tendency is suspected as the hypoglycemic dip in the curve may not occur until the fourth fifth or sixth hour Some variations have been suggested in the method of performing this test such as giving the glucose according to the weight of the patient (Justified in children) giving glucose more than once or giving the glucose intravenously (occasionally justified if absorption from the gastrointesti nal tract is impaired or abnormal) However the method guen above is generally satisfactory There has been some discussion concerning the neces sity for special diet betore performing the glucose toler ance test in general the preceding diet will not grossly affect the glucose tolerance curve if the carbohydrate intake has been between 100 gm and 300 gm daily Nearly all diets afford an intale of carbohydrate within these limits -the possible exception being in patient with severe anorexia nervosa Such a patient should be fed a high carbohydrate diet several days before the test in order to obtain a true picture of his capacity to handle glucose The normal glucose tolerance cure is as follows Fasting level between 90 and 120 mg (Folin Wu method) with a rise to a perk of 130 to 160 mg in one half to one hour followed by a fall to the fasting level within 2 hours ·LABORATORY AIDS IN EVDOCRINE DIAGNOSIS added (The latter is prepired by adding 24 gm Ba (OH) to enough water to fill a 500 cc solumetric flask, making 03 N B1 (OH) This is adjusted uith zinc sulfate solution (preparation given below) to 1 pink end point using phenolphthalein as an indicator further adjustment is made by adding water until 17 to 18 cc of barium hy droxide are requirel to titrate the zinc sulfate The solu tion should be stored in 7 bottle protected from the CO of the air by soul : lime) The mixture turns brown and then 2 cc of zinc sulfate solution are added (prepared by dissolung 2 gm of Znso, 7 H,O in distilled water and bringin, to volume 500 cc volumetric flash) Thuis is filtereel and yiekls a clear filtrate (lucose determination is accomplished by plaun, 1 cc of the filerate in a sugar tube graduated at 25 cc In a similar tube placed I lc of diselled water One cc of coppyr reagent is added to each (The copper reagent is prepared by dissolung 28 gm of anhydrous disodium phosphate and 40 gm of Rochelle salt in 700 cc of water and then adding 100 cc of normal sodium hydroxae Lights cc of a 100% copper sulfate solution rre deled ich stirring and then 180 gm of anhydrous sodnuni sullate This is diluted to a liter allowed to stand for 1 to 2 days and filtered) The solutions in the tubes are mixed and then hented for 10 minutes in a boiling water bath After cooling in cold nater 1 cc of arsenomolybdate color rurgent is added to eich tube mixed and diluted to %2 CC with distilled witer They tre then mixed and read in a pliotoelectric colorimeter (I le arsenomolybdate color reagent is pre pared by <lissoluing 2 sm of ammonium molybdate in 450 cc of distilled 1 tter 1dding " cc of concentrated HSO mung tu dding 3 gm of Na, H 150 7 7,0 dissolved in 20 cc of Water This is mixed incubrted at 37 C for 24 to 18 hours and then stored in 7 brown glass stoppered bottle) In performing the colorimeter reading the colorimeter ·SPECIAL TESTS OF BLOOD 15 is adjusted to give 100% transmission through the blank In Evelyn 515 filter (green) is used which gives maximum transmission at 500 to 520 millimicrons The blood glucose values are then read directly from a graph with curve made up from standard solutions Glucose tolerance tests are generally satisfactory using the classical method as follows Fasting blood is drawn and urine collected and then the patient drinks 100 gm of glucose in water flavored with lemon Blood and urine specimens are taken at 30 minutes one two three and occasionally four five and six hours afterwards the longer tests are performed particularly if a hypoglycemic tendency is suspected as the hypoglycemic dip in the curve may not occur until the fourth fifth or sixth hour Some variations have been suggested in the method of performing this test such as giving the glucose according to the weight of the patient (justified in children) giving glucose more than once or giving the glucose intravenously (occasionally justified if absorption from the gastrointesti nal tract is impaired or abnormal) However the method given above is generally satisfactory There has been some discussion concerning the neces sity for special diet before performing the glucose toler ance test In general the preceding diet will not grossly affect the glucose tolerance curve if the curbohydrate intale has been between 100 gm and 300 gin daily Nearly all diets afford an intake of carbohydrate within these limits -the possible exception being in a patient with severe unorexia nervosa Such a patient should be fed a high carbohydrate diet several days before the test in order to obtain a true picture of luis capacity to handle glucose The normal glucose tolerance curve is as follows Fasting level ben een 90 and 120 mg (Tolin Wu method) with a rise to a pezh of 130 to 160 mg % in one half to one hour folloned by a fall to the fasting level within 2 hours ·16 LABORATORI AIDS IS INDOCRINE DIAGNOSIS Such a normal curve may be importint in the diagnosis of diabeles insipidus dhgh glucose tolerance curve (indicating lou tolerance) 15 found classicılly in diabetes mellitus It is also seen when diabetes complicates Cushings syndrome acromegaly gigantism adrenal cortical lumor or hyperplasia, adrenal meilullary tumor and arrhenoblastoma Hyperthyroidism usually siions i characteristic curve with a tendency to ? quick high rise and a relatively eirly full This will cause glycosuri? but such patients may not be crue diabetics and frequently curc of the hyperthyroid ism will cause a disappearance of both the tendency to eles ated blood glucose ind the glycosuri Recently re ported observations by Berger' indicate that administra tion of 100 antes oi corticotropin (ACTH) I four before the glucose tolerance test causes lugher blood sugar levels in patients who are diabetics and also in siblings of Inow dirbetics as hose previous control glucose tolerance curies have been normal He offers the interesting sug gestion that in the latter group potential diabetics may be discos cred us this means who would other use be missed Low glucose tolerance curves (hugh sugar tolerance) are seen classically in patients with hyperinsulinism, particu Turly with a beti cell tumor of the pancreas With a mild hypoglycemic tendency it is important to run the glucose tolerance test for 5 or 6 hours as it may require that length of time for the hypoglycemic dip to be manifest Also when testing for this tendency it may be necessary to take an extra blood sugar not on the routine schedule when the patient develops signs or symptoms suggesting lıypoglycemu This may indicate a hypoglycemic fall in the curte nich will return to more normal lei els later as glycogen is freed from the liver The tendency to hypo ·SPECIAL TESTS OF BLOOD 17 glycemia is seen with varying intensities in Simmonds disease and in Addison s disease In both of these condi tions it may be very marked and deaths from lıypoglycemia have been reported Relatively lou flat glucose tolerance curves are found in pituitary infantilism myredema and cretinism THE INSULIN TOLERANCE TEST This is used occasionally as a test for diminished func tion of the anterior pituitary and demonstrates the pa tient s degree of sensitivity to insulin Method A fasting blood sugar is taken and then a dose of 1/10 unit of regular insulin per kg of ideal body weight is given intravenously Blood sugar level is again tested 20 30 45 60 90 and 120 minutes after the injection Normally the level of blood sugar will fall to about 50% of the fasting level in 20 to 30 minutes and then will rebound to the fasting level in 120 minutes Hypoglycemic reactions can occur with this test par ticularly if the patient has hyperinsulinism Simmonds disease or Addison s disease If any of these conditions are suspected it is prudent to use only one half to one third of the recommended dose of insulin and also to have glucose ready for intravenous injection in case a hypo glycemic crisis appears PLASMA CHOLESTEROL The level of the plasma cholesterol is occasionally helpful as additional information in estimation of thyroid function Method enous blood is drawn and placed in an oxa late tube One cc of plasma is transferred to a 20 cc volumetric flask and 15 cc of a mixture of equal parts of ·18 LABORATORI AIDS II\DOCRIVF DIACIONS 9. nlcolioi and acetone is added in a rapid stream while leeping the plasma in motion A finely suspended precipi the results The flash is placed in boiling water being kept in motion to 110ul bumping unul the solvent boils It is cooled to room temperature and the volume made up to 21 cc with the alcohol acetone muxture I hus is stoppered mixed thoroughly and filtered into a drı test tube (1 match glass over the funnel will minimize evapora tion of the solvent) A clear filtrate results Saponification and precipitation is the next step ind for this two to three drops of potissium hydroxide solution (10 gm of reagent grade potassium hydroxide in 20 cc of distilled water) are placed in a 15 cc graduated centri fuge tube and 3 cc of the clear filtrate added This is mused by draning up and down in the pipette unul no droplets of 11k zle can be seen the tube is then placed in a preserving jar cont uning a layer of sand about 3 cm deep previously heated in 1 water bath to 45 C The jar 1$ tightly closed and placed in an incubator al 37 10 40 C for 30 minutes The tube is then alloned to cool to room temperature and the acetone alcohol mixture added to the occ muk One drop of phenolphthalein solution (1 gm of phenolphthalein diluted to 100 cc with 90% alcohol) is added followed by 10% acetic acid drop by drop with mixing until the red color disappears One drop in excess is added folloned by 3 cc of digitonin solution (500 m, of digitonin dissolved in 100 cc of 50% alcohol) and then by thorough mixing The tube is then placed in a preserving jar covered ughtly and allowed to stand at room temperature for at least three hours After standing it is centrifuged for 15 minutes at 2800 rpm following which the supernatant fluid is decanted and drained in an inverted position The walls of the tube are then washed 11th 2 to 3 cc ot ether the precipitate mixed and again centrifuged for 5 minutes at 2000 rpm Development of the color is the next step For this a ·SPECIAL TESTS OF BLOOD 19 shallow pan containing a layer of sand 3 cm deep is heated to 110 to 115 C in an oven The tube containing the wished precipitate is placed in the pan and left in the oven for 10 minutes Then while the tube is still in the hot sand 2 cc of glacial acetic acid is added in such 7 manner that it washes the walls of the tube This is well mixed left in the hot sand for an additional two minutes and then removed and cooled to room temperature After this the tube is placed in 1 water bath at 25 C with light excluded and allowed to come to temperature equilibrium Four cc of cold acetic anhydride sulfuric acid reagent is added mixed and the tube returned to the bath for 27 minutes It is then transferred to an Evelyn tube and read within the next 10 minutes (The acetic anhydride sulfuric acid reagent is prepared just before use Twenty cc of acetic anhydride is placed in a glass stoppered cylinder and chilled in ice water One cc of concentrated sulfuric acid is added gradually with mixing and cooling The mixture is stoppered shaken vigorously and hept in an ice bath It should be used within one hour) A standard solution of cholesterol for comparison is pre pared by dissolving dry cholesterol in glacial acetic acid This stable stock solution should contain 1 mg of cho lesterol per cc It is kept in a cool temperature Before use it is warmed to room temperature and diluted with glacial acetic acid so that the dilute standard will contain 02 mg cholesterol in 2 cc This dilute standard is stable for about two months In reading the color 2 cc of the dilute standard solu tion is placed in a centrifuge tube This also is left in the water bath at 25 C and allowed to come to temperature When ready 4 a of the cold acetic anhydride sulfuric acid reagent is added This is mixed and returned to the bath for 27 minutes then transferred to an Evelyn tube and read within 10 minutes A blank is similarly pre ·20 1.ABORATORI AIDS NTDOCRINF DIAGNOSIS pared except that 2 cc of acetic acid alone is used instead of the standard cholesterol solution The specimens are reid in a photoelectric colorimeter using a 63, filter und 6 cc aperture Calculation is as follows Density of unknown X02 x 100 Total cholesterol in mg% Tensity of standard 2012 Tliere is some difference of opinion concerning the nor mal range In some texts this is said to be from 150 to 190 mg% wlule other authntities believe that the level is to be considereri normal anywhere between 100 and 300 mg % It is of some value to become ſaınıliar with the results of the test as performed in one laboratory or preferably even by one technium In the experience of the writer most normal patients have levels between 150 ind 225 mg % With lowered thyroid function (hypothyroidism my edena) the values tend to be lugh (300 to 1000 mg %) while uith increased thyroid function (hypertyronlist exophthalmıc goiter) the cholesterol levels are lor or lou normal The rest is not as dependable as the basal meta bolic rue or blood iodine but occasionally provides helpful corroboritive evidence and may be especially izluable in follon ing the effects of treatment Furthermore 1 is useful in chuldren who cannot cooperate properly in the performance of the basal metabolic test li thyroid sub stance is being administered and the cholesterol is sull above the normal range the dose of thyroid should prob ably be increased If a patient with apparent mykedemia is found to have a normal cholesterol the possibility of primary pituitary rather than thyroid my edema must be considered This is of considerable importance as the to types of my edemi should he handled differently Adminis tration of thyroid to a patient with pituitary myaedema ·SPECIAL TESTS OF BLOOD 21 may precipitate an adrenal cortical crisis and fatality The cautious use of small doses is imperative High levels of plasma cholesterol are also seen in diabetes mellitus particularly in the presence of acidosis and in canthomatosis slightly high levels are found in Cushings syndrome adrenal cortical tumors and vitilism Slightly low levels may be found in pituitary infantilism hyperinsulinism and in Addison s disease The recent work of Gofman and colleagues* in frac tionating the serum lipids by observing their flotation in the ultra-centrifuge has aroused considerable interest and discussion It is suggested that the giant molecules of the cholesterol complex are associated with the develop. ment of atherosclerosis Accumulation of correlative clini cal data is proceeding and is awaited with great interest BLOOD IODINE Estimation of the protein bound iodine content of the blood as a test for thyroid function is being employed with increasing frequency This fraction is generally con sidered to be the hormonal iodine and thus the test should be a highly accurate measure of the amount of circulating thyroid hormone Within certain limitations this is true However the test is delicate very sensitive and techni cally difficult The results are expressed in micrograms rather than milligrams and the iodine itself acts as a catalyst in the reaction so is not the measured end point There may be considerable difficulty in setting up the test but once it is working well it is usually reliable Vethod. modification of that reported by Salter and Mchay is used One cc of the unknown blooci serum is placed in an ashing tube and diluted with 2 cc of distilled water 05 cc of 10zinc sulfate and after mixing 05 cc of ·20 LABORATORY AIDS INF\DOCRIVE DIAGVOSIS pared except that 2 cc of acetic acid alone is used instead of the standard cholesterol solution The specimens are read in a photoelectric colorimeter using a 63. filter and 6 cc aperture Calculation is as follows Density of unknown x 0.2 x 100 - Total cholesterol in mz Density of standard x 012 There is some difference of opinion concerning the nor mal range In some texts this is sud to be from 150 to 190 mg % wule other authorities believe that the level is to be considered normal anyubiere between 100 and 300 mg % It is of some value to become familiar with the results of the test as performed in one laboritory or preferably even by one cechnician in the experience of the writer most normal patients have levels between 150 and 225 mg % With lonered thyroid function (hypothyroidism myx edema) the values tend to be high (300 to 1000 mg %) while with increased thyroid function (hyperthyroidism exophthalmic goiter) the cholesterol levels are lou or low normal The test is not as dependable as the basal meta bolic rate or blood lodine but occasionally provides helpful corroborative evidence and may be especially valuable in following the effects of treatment Furthermore it is useful in children who cannot cooperate properly in the performance of the basal metabolic test If thyroid sub stance is being administered and the cholesterol is still above the normal range the dose of thyroid should probably be increased If a patient with apparent myredema is found to have a normal cholesterol the possibility of primary pituitary rather than thyroid my redema must be considered This is of considerable importance as the to types of myxedema should be handled differently Adminis tration of thyroid to a patient with pituitary my redema ·SPECIAL TESTS OF BLOOD 21 may precipitate an adrenal cortical crisis and fatality The cautious use of small doses is imperative High levels of plasma cholesterol are also seen in diabetes mellitus, particularly in the presence of acidosis and in xanthomatosis slightly high levels are found in Cushing's syndrome adrenal cortical tumors and virilism Slightly low levels may be found in pituitary infantilism, hyperinsulinism and in Addison s disease The recent work of Gofman and colleagues in fruc tionating the serum lipids by observing their flotation in the ultra centrifuge has aroused considerable interest and discussion It is suggested that the giant molecules of the cholesterol complex are associated with the develop ment of atherosclerosis Accumulation of correlative clini cal data is proceeding and is awaited with great interest BLOOD IODINE Estimation of the protein bound iodine content of the blood as a test for thyroid function is being employed with increasing frequency This fraction is generally con sidered to be the hormonal iodine and thus the test should be a highly accurate measure of the amount of circulating thyroid hormone Within certain limitations this is true However the test is delicate very sensitive and techni cally difficult The results are expressed in micrograms rather than milligrams and the iodine itself acts as a catalyst in the reaction so is not the measured end point There may be considerable difficulty in setting up the test but once it is working well it is usually reliable Method A modification of that reported by Salter and Michay? is used One cc of the unknown blood serum is placed in an ashing tube and diluted with 2 cc of distilled water 05 cc of 10% zinc sulfate and after mixing 05 cc of ·22 LABOT ATOR) AIDS IS ENDOCRINE DI IGNOSIS 05 N sodium hydrovide Thuis is mixed and allowed to stund for one half hour and then the sube is centrifuged until the sediment is well packed 3 lus is Basked with 2 cc of distilled water and recentrifuged The supernatant flud is diseaded and I ac of I N sodum carbonate is added to the sediment I lie tube is then placed in a drying oven for 48 hours it 110 C or unul dry The sample is 7sherl in a muffle furnace nt 680° C for 2 hours The determination is made by adding 4 cc of IN Jy drochloric acid Ilse sides of the tube are scraped down suth 1 glass rod al alter ellers escence las ceased 2 cc of 7 N sulfuric acid and I cc of distilled water are added Alter mixing the contents are poured into a centrifuge tube and centrifuged until the sediment is well pached 730 colorimeter tubins are then used-10 one of which Is added 1 cs al castilled water I 3 cc aliquor of the supernatue dud of the unknown is pipetted into each colorimeter tube and 05 cc of 01 N sodium arsenite solution is also added to each tube The colorimeter tubes ind a 01 N ceric ammonnum sulfate solution in 10 N sulfuric acid are placed in a constant temperature nater bath at 39 for one hour or until up to temperature The amount of rodine is proportional to the rise of the reaction between the ceric ammonnm sulfate and the sodiun arsenice which causes the bright yellow color of the ceric ammonium sulfate to disappear One cc of ceric ammonium sulfite solution is added to each tube and reading, are taken at 6 and 12 minutes The reaction rates are ploited on semi log paper un colorimeter readings plotted against time in minutes Calculation is then made by the following formula at t a t equals known amount of sodme added to colorimeter tube uals time 8 minutes at interart of the rate of the tube con taining no extra iodine with designated horizontal ·SPECIAL TESTS OF BLOOD 23 + equals time in minutes at intercept of the rate of the tube con taining the added iodine with the same designated horizontal Since the colorimeter tube contains a 3 cc aliquot of the 7 cc sample the concentration of the iodine in the unknown serum will be X X 7/3 x 100 (micrograms %) The normal range in our experience is 4 to 8 micro grams % With hyperthyroidism the values are above the normal range and with hypothyroidism they are below it Any 10dine containing medications (including thyroid) must be stopped for four to five days before the day of the test No iodine should be used on the skin for sterilization at the time the blood is drawn If any organic iodine is given as for a cholecystogram intravenous pyelogram or myelo gram the values will be high for a period of weeks to months--thus rendering the test valueless as an indication of level of thyroid function Blood 10dine determinations tre as yet done in fairly few laboratories so that the available ones are frequently overworked as a consequence one is often delayed in learning the results of an individual test These factors of limited availability and slow reporting will undoubtedly be gradually eliminated particularly if a simpler method is developed Unul this occurs however measurement of the basal metabolic rate will not be supplanted by the blood iodine test Finally the test seems to be a better gauge of hyper than hypothyroidism Furthermore the narrow range in micrograms is to be contrasted with the far more striking range of percentage in basal rate (10 to 50% minus in hypothyroidism and 10 to 100% plus in hyperthy roidism) SERUM CAROTIN An increase in this factor is occasionally of interest in hypothyroidism ·LABORATORY AIDS IN ENDOCRINF DIACNOSIS Method In equil volume of serum is mixed with 93% alcolo! To this an equal volume of petroleum ether is added This is mixed by shaking to extract the carotin and alloned to stand for layering A yellow color in the upper (petroleum ether) layer indicates an excess of carolin in she serum The result may be reported as postile or the intensity of the color may be roughly indicated as 1 2 3 or 4 plus Carolinemia (increased blood carotin) has been found to be present in most patients with untreated myxedema It is also found occasionally in other conditions associated with low basal metabolic rates including Simmonds dis case and hypogonadism A dietary cause for the carors nemia (excessive intake of carotin containıng foods) must be excluded SERUM CALCIUM PHOSPHORUS AND ALLALINE PHOSPHATASC These can be considered together and are of special importance in the diagnosis of diseases of the parathyroids or other metabolit bone diseases Method (or serum calaum Thcc of serum are placed in a 15 cc graduated centrifuge tube containing 2 cc of water (The outside diameter of the tube should be 6 or 7 mm at the 01 c mark) One cc of saturated ammonium oxalate solution is added (42 gm per 100 cc of water) These are mixed with a circular motion and the mixture allowed to stand for at least 4 hours The contents are again mied and then the tube is centrifuged at 1500 rpm for 10 minutes The supernatant is poured off and 2% ammonium hydroxide is added up to 4 ct in such a manner that it washes down the skies of the tube This is mixed by a circular motion until the precipitace spirals ag in the center (all need not be mixed) The tube is then ·SPECIAL TESTS OF BLOOD 25 centrifuged for five minutes and the supernatant again removed by decantation Following this the crystals are dissolved in 2 cc of approximately normal sulfuric acid and the tube and contents heated in a boiling water bath for two minutes Titration is then performed immediately with N/100 potassium permanganate to a definite pink color that persists for at least one minute A blank is prepared fresh each day in order to stand ardıze the permanganate solution For this 2 cc of N/100 sodium oxalate is mixed with 2 cc of normal sulfuric acid heated in boiling water for two minutes and then titrated with the N/100 potassium permanganate Then 2 CC (sodium oxalate) equals correction factor - CC Vino Final calculation is then performed as follows -cc KMnO used on unknown Correction factor X 02 x 100 equals serum calcium in mg 2 (cc serum used) per 100 cc of serum The normal range is 9 to 11 mg % (However it is also important to know the level of the total serum protein for if this is low a greater percentage of the tocal calcium will be in the diffusible form-eg with a total protein of 4 gm a serum calcium of 7 mg % would be normal) Method for serum inorganic phosphorus In drawing the blood precautions are taken to prevent hemolysis One cc of serum is placed in a 25 cc volumetric flask with 5 to 6 cc of distilled water Four cc of 10% tri chloracetic acid is added and the flask filled to the 25 cc mark A pinch of kaolin is added and after standing for 10 minutes the mixture is filtered directly into ? test tubes measuring volumetrically so that there is a 10 cc alıquot in each cube Two cc of a molybdate solution is added to each tube (This solution is prepared by dis solving 125 gm of ammonium molybdate in water dilut ing to 100 ce and then mixing in 2 50 cc glass stoppered bottle with 150 cc of 10 N sulfuric acid This is brought ·20 I ABORITORI AIDS IN ENDOCRINE DI IGNOSIS to volume and is reliable for å months) Next 08 cc of sul furic teagent is added to ench tube and after standig for exactly 20 minutes the solution is transferred to an Evelyn tube and read using 2 660 filter in a photoelectric colorimeter (The sulfuric reagent is prepared by dissolving 200 mg of aminonaphtholsulfonic acki in 78 cc of 15% sodium bisulfate and 1 cc of 20% sodium sulfate After stoppering ind shaking more of the sodium sulfate may be added in loc amounts until the reagent is clear This 15 reliable for tho seeks) A blank is set up each time measuring directly into 3 test tube 7 cc of water 3 cc of 20% trichloracetic aad and mixing Tuo cc of 1 molybdate solution is then added (prepared similarly to the above except that 2.0 cc of 10 N sulfuric acid is used instead of 150 cc) lfter mix ing 08 « of the sulfuric reagent is added and the mix ture allowed to stand for (xactly 20 minutes It is then transferred to an Evelyn mbe and the colorimeter ad justed to give 100°, transmissions The seriam phosphorus salue is then send using the unknown and rending directly from a graph with curse made ир from standard solutions of pocassium dihydrogen phosphace The normal values for serum inorganic phosphorus in this laboralory are 3 long% for adults and up to 5 mg for children Method for alkaline phosphatase One half cc of un hemolyzed serum is placed in a 20 cc volumetric Hask Trith 10 cc of alkaline substrate at 37 and then incu bated for one hour a: 37 C (The alkaline substrale is prepared by adding exactly 106 gm of sodium barbstal to 12 gm sodium bezagizterophosphate and diluting to olume in 7250 < olumetric flask Snu drops of chloro form are added The substrate is reliable for one month si continuously refrigerated Before using the pH must be tested at 37 Calter incubation for one hour a: 37 C -It should be pH 93 +015) ·SPECIAL TESTS OF BLOOD 27 After incubation the mixture is cooled under tap water and 4 cc of 40% trichloracetic acid is added The solution is brought to volume (25 cc) a pinch of haolin added and after standing for 10 minutes it is filtered directly into two test tubes with a 10 cc aliquot in each tube Two CC of the molybdate solution (prepared as on page 25) is added to each tube and after mixing 08 cc of the sul furic reagent is added (prepared as on page 26) This is allowed to stand for exactly 20 minutes and then trans ferred to an Evelyn tube and read in a photoeletric colorimeter using a 660 filter This result is then read di rectly from the phosphorus curve as described on page 26 A serum phosphorus determination must be performed simultaneously and the value for the phosphatase is ob tained by subtracting the result of the phosphorus test from that obtained with the phosphatase test multiplying the phosphatase by two before the subtraction (to correct for the smaller amount of serum used in the phosphatase test) The normal values in this laboratory are from 0 to 5 units Since the method was originally described by Shino wara Jones and Reinhart these are frequently referred to as SJ and R units and are comparable to the more widely known Bodansky units The blood calcium is high (usually from 11 to 16 mg %) in hyperparathyroidism and in vitamin D intoxicationand may be slightly high in Paget s disease of bone It is low (usually under 7 mg %) in hypoparathyroidism (tetany) and has been found to be moderately lou in Sum monds disease Addison s disease and in eu choidism The blood phosphorus is ligh (usually from 4 to 10 mg %) in hypoparathyroidism (tetany) and may be slightly high in diabetes mellitus acromegaly and Addisonian crisis It is low (usually under 3 mg%) in hyperparathy Toidism and is slightly low in hyperinsulinism ·28 NOR TORT AIDS I IPOCRIVE DE IGNOSIS There is suggestie erdence that the level of serum phosphorus xay indicate the amount of circulating an terior pituitary grou th hormone A level of over 4 mg% 11$ been considered cusdence of an increased amount of growth lormone as in the active stages of acromegal and gigantism Tlus finding is still being confirmed Tlie alkalınc phosphatase is increased specifically in Pagets disease of bone and is of diagnostic importance in this condition It is also mcreased if bone is itt olsed in hyperparathyroidism (up to 20 SJR unsis) but may be low in that condition iſ there is no activity in the bones It is moderately increased in Albrights syndrome (poly ostotic fibrous dysplasia) In osteoporosis particularly the le or menopausal types the blood calcium phosphorus ad all aline phosplratise are usually within normal limits The acid phosphatase las been reported to be elevated in some patients with caremoma of the prostate especially af there is metastatic an olsement of bone SERUM ELECTROLI TIS (SODIUM POTASSIUM CHLORIDE) Study of blood levels of these constituents are pirucu larly important in diseases of the adrenal cortex These mry be primary or secondary to diseise of other endocrine glands Adrenal cortical dysfunction may also result from non endocrine diseases or Stress Serum Sodium method \ Perkins Elmer flame pho tometer is used in our laboratory Tlus is an emassion spectrometer combined with a fame source and a photo electric de tection system for the measurement of intensity of light The unknown serum is diluted 1/100 and a Inhium matrizte solution (made by dissolving 13 9 gm of lithium matrate in 2 liters of * rter) 15 «luted 1/10 The latter is used as an internal standard with the light of ·SPECIAL TESTS OF BLOOD 29 the unknown element being compared directly to the light emitted by the element lithium Standard solutions are Lised for comparison and the results read on a calibrated cune showing the milliequivalents per liter The inter mediate standard solution is prepared by adding 30 cc of a stock solution (made up by dissolung 254 gm of Maci in 1 liter of water) to 100 cc of the lithium nitrate solution and making up to 1000 cc with water A 100% standard is made up by adding 23 cc of the stock solution to 50 cc of the lithium solution and making up to 500 cc with Water The unknown is prepared by adding 1 of serum to 10 cc of the lithium nitrate solution and making up to 100 cc with water Serum potassium method. This is also accomplished with the Perkins Elmer flame photometer The unknown serum is diluted 1/10 and the lithium nitrate solution (prepared as above) is diluted 1/10 and used as the in ternal standard Standard solutions are used for com parison and the results read on a calibrated cune showing milliequivalents per liter Standard solutions are made up from a stock solution containing 191 gm of KCl per liter The intermediate standard is prepared by adding 20 cc of the stock solution to 100 cc of the lithium nitrate solution and making up to 1000 cc with water The 100% standard is made up by adding 25 cc of the stock solution to 50 cc of lithium nitrate solution and making up to 500 cc with water The unknown is prepared by mixing 1 cc of serum with Icc of the lithium nitrate solution and making up to 10 cc with water Serum chloride method A standard 4 mm (milliequus alent) chloride solution is prepared by mixing 10 cc. of 01 NaCl solution with 250 cc of a phosphoric tungstic aad reagent (prepared by dissolving 6 gm Na 110 2HO reagent grade in 1 liter of O 15 VH PO) It is standard ized by shaking 23 cc with 03 gm precipitated silver 1odate for 10 seconds and then centrifuged in a capped ·30 LABORATORI AIDS ENDOCRIVE DIAGNOSIS cube A 20 cc. portion of the clear supernatant is placed in a 100 cc Erlenmeyer flask 2 gm sodium odide is added and then titration is performed with 0 02303 N sodium thiosulfate solution adding 3 or 4 drops of 1% starch solution when the yellos rodine color has nearly disappeared The titration should take 208 cc of the thiosulfate solution and if deviation is more than 0.00 cc a cortechon factor must be used With the unknou n serum 02 cc. of the serum is added to 5 cc of the phosphoric tungstic acid reagent (prepara cion described above) in a test tube One glass spoon (60 mg = 10 mg) of sık er sodate is added and the mixture shaken for 40 seconds and filtered A 2 ce aliquot of the httrate is ntrated with the 0 02303 N sodium thiosulfate solution alter adding 02 gm sodium iodide (one glass spoon) using a 50cc Erlenmeyer flask Three or four drops of 10% starch solution are added when the yellow iodine color las nearly disappeared as an aid in arriving at al clear end point Calculation is as follons Villiequrvalents of chloride per liter of serum = 50 x < 002303 sodium thiosulfate used Grams of chloride per liter of serum = 2998 Xce of OD 303 $0 drum thiosulfate solution used If a correction factor is indicated by titraron of the standard chloride solution as mentioned above it must be included in the calculation Note that the results are expressed in mulllequn alents rather than in milligrams Comparative normal values are as follows Villiequivalents Serum Sodium ព SGO mg - 142 m/equiv - (normat range 136 to 12 m/equn ) Serum Chlorides - 103 tr/equiv- (normal range 99.3 to 1066 m/equi) $75 to 480 mg ·SPECIAL TESTS OF BLOOD 31 Serum chlorides as Maci 5,0 to 6 Omg of Serum Potassium 20 ° 7 - 35 to 53 m/equiv Results expressed in milliequr alents are appearing with increasing frequency from clinical laboratories and the change seems justified The milliequivalent figures are simpler and easier to remember and can be compared with other values including the CO, combining power sımı larly expressed The following formulae have been found useful Va Cl mor of CI m/equiv /L 5 85 CO vo] 01 HCO m/equis 3 Ci m'equiv /L + com/equiv /L + 10= Ma m/equiv /L Cm/equiv/L + 232 (0 5 HCO )= Na mequiv {L” It would appear advantageous to have standardization of reports in milliequivalents rather than milligrams % especially with ions involved in the electrolyte balance In adrenal cortical deficiencies blood levels of Na and Cl are low and the á content is high The opposite may occur in Cushings syndrome and also occasionally in acromegaly Similar changes ie slightly elevated Na and Cl and slightly lou k are encountered in adrenal cortical tirilism adrenal precocious puberty and arrhenoblastoma In Simmonds disease the factor of adrenal insufficiency may be pronounced and the electrolyte changes are similar to those in Addison s disease with low zalues for sodium and chloride and a high potassium level In hyperthy roidism the blood sodium is occasionally low and con Versely may be slightly high in hypothyroidism ·32 LABORATORY AIDS IN ENDOCRINE DIAGNOSIS NITROGEN AILTABOLISI Several tests of the blood may give information regard ing the nitrogen metabolism These include the blood uric acid, blood urea nitrogen and die non protein nitrogen The Inter is the most widely used and reported Methods for testing for these components are available in any laboratory text The non protein nitrogen has been found to be elevated in several endocrine conditions, in cluding Addisonian crisis, terminal Cushings sindrome, terminal hyperparathyroidism and diabetic coma The blood uric acid is elevated specifically during acurve attacks of gout PLISVA CO, COMBINING POWER This is a cest specifically for acıdosis or alkalosis of sarı ous origins As with the electrolytes it is becoming in creasingly frequent to report results in milliequnalents With the older manner of reporting the normal ringe was 50 to 70 volumes % Honeer with milliequualents the pera,e result is 28 m/equr with a normal range of 24 to 30 mequit Method For this test the minometric apparatus as described by Peters and Van Slyke is used " The pro cedure as outlined by these writers on pages 283 to 286 of the reference is follo ved exactly in our laboratory Blood as drawn and centrifuged anaerobically for the test The results are loc in diabetic acidosis or acidosis from other couses and are occasionally low in adrenal cortical insuficiency It may be slightly high in parathyroid telany RENERENCES I THORY GT FORSITY PH FRAMLEY TF TUILSON DL REVOLD AE FREDRICKSOY DS and JENKINS D Advances in the diagnosis and treatment of adrenal insufficiency 4m Alcd 10 595 611 May 191 ·SPECIAL TESTS OF BLOOD 33 ? BONVER D C Eosinophile levels as an index of adrenal responsive ness J.4.4 4 148 631 637 Feb 23 1952 3 SOMOGYI MA reagent for copper todometric determination of very small amounts of sugar ) Biol Chem 117 771 7 6 Feb 1937 4 NELSON N A photometric adaptation of the Somogyi method for determination of glucose J Biol Chem 153 315 380 Vay 1914 5 BERGER H Method of increasino sensitivity of glucose tolerance test J 411 4 148 361 366 Feb 9 1909 6 GOFMAN J W JOVES HB LINDGREN FTLYON T P ELLIOTT H A and STRISOWER B Blood lipids and human atherosclerosis Circulation 161 1/8 August 1930 7 SALTER W T and McLAY E A lodine in blood and thyroid of man and small animals Endocrinology 35 380 390 Nov 1914 8 SHINOWARA GI JONES L M and REINHART H C The estimation of serum inorganic phosphate and and and alkaline phosphatase activity J Biol Chem 14? 921 933 Feb 1912 9 HOPPER J J and PARTRIDCE J Anuria and oliguria California Med 72 415-491 73 59 53 June and July 1950 10 HALD P M HEINSEN HJ and PETERS J P The estimation of serum sodium from bicarbonate plus chloride J Clin Investigation 6 983 990 Sept 1917 11 PETERS J P an 1 VAN SLYAE D D Quantitative Clinical Chemistry Vol II-Methods Baltimore Williams and Walkins 1939 ·Chapter IP SPECIAL TESTS OF URINE (EXCLUDING HORMONE ASSAYS) E URINE CHLORIDES NCRLTIOV of chlorides is increased in Addison s disease, which fact is the basis of several tests for adrenal cor tical hypofunction The Cutler Pouer Nolder tcst as one of these and has been available since 1938 Method The test takes three days and hospitalization as necessary Treatment with adrenal steroids or extra salt is withheld the dry preceding a low salt dret (con taining 0 95 mg of chloride ion 059 gm of sodium and 41 gm of potassium per day) is started on the first day and free drinking of water is encouraged On the after noon of the first day extra potassium is given as potassium citrate 33 ing per kg body weight (42 mg of potassium citrate per lb) Second day-Flud intake to 40 cc per kg of body neight is given and potassium citrate is repeated Urine is collected from 8 am to 8pm -and a second sample from 8pm to 8 am of the third day Third day --Trenty cc of liquid per kg of body weight is given before 11 am and a third urine sample is collected from 8am to 12 noon The urine samples are then examined for chloride content the most significant being the four hour sample collected on the third day In this specimen the normal controls showed a mean of 57.2 mg with a range of 17 10 141 mg% uhile patients with Addison s disease 34 ·SPECIAL TESTS OF URINE 35 shoued the increased mean value of 29, 4 mg % with a range of 229 to 356 mg% The obvious disadvantages of this test are the necessity for hospitalization the length of time required (three days with one day of preparation) and the danger of with holding treatment and putting a patient with Addison s disease on a low salt diet with high potassium intake The latter can induce an Addisonian crisis and facilities must be at hand for immediate supportive treatment to prevent possible fatal outcome-considerable risk for a laboratory procedure The Robinson Power Kepler uater test described in 1916 ? is simpler and safer and has largely replaced the older procedure The method consists of two sections the first being de pendent upon the inability of patients with Addison s disease to excrete water normally because of the decreased glomerular filtration rate The second section gives data on the urea and chloride clearance in the patient with the first usually being reduced and the second increased The urea chloride ratio points up this abnormality It is not always necessary to perform the second portion and the test can be completed in one day in either case Also it can be accomplished as an office procedure provided a place is available for the patient to rest Method Tirst portion-On the day before the patient omits extra salt and takes no food or fluid after 6 pm At the bladder is emptied and the urine dıs carded All urine voided from then until 7 30 am IS collected and this might colume measured No breakfast is given and the patient is asked to void at 8 30 a m fol lowing which he is given 20 cc of water per kg of body weight (9 cc per lb) to drink within 45 minutes Urine specimens are collected at 9 30 10 30 11 30 am and 10 30 pm ·Plasma urea max 36 LABORATORI AIDS CIDOCRINE DIACVOSIS 12 30 pm (with the patient kept at rest) and the volume of each is measured separately If the volume of any of these day specimens is greater than that of the night specimen the patient probably does not have Aldisons discase and the test can be terminated However if none of the day solumes equal that of the night specimen the second part of the test is performed Second portion-specimen of blood is taken wule the patient is still fasting (at 12 30 pm) ind chemical analyses are made to set up the following equation Urinc Vrea Plasma chloride Volume of largest Night spec mg % mg" hourly urine spec (cc) Unine chloride Tolume of night night spec (mg") HIND (cc) When the above equition has been resolved the factor A will be 30 or above in normal patients and 23 or belos in patients with Addison s disease The test is a good one but still only gives a probable anster and in some instances repetition may give con tradictory results If the outcome is equivocal the Cutler Poner Wilder chloride excretion test can be done im mediately following-using this water test as the first day of the other and following the rest of the procedure 75 outlined above However the more recent availability of the eosinophile count ind corticotropin (ACTH) tests (Chapter III pp 10 11) has largely replaced the dangerous chloride excretion test An excellent survey of the present status of tests for adrenal cortical function can be found in a recent paper by Thorn et al THE CARTER ROBBINS TEST This test is useful in distinguishing true diabetes in sipidus from psychogenic diuresis and has as its basis test ·SPECIAL TESTS OF URINC 37 ing the excretory function of the kidney It is somewhat complicated but may provide the ansier to a difficult clinical decision Method Antidiuretic therapy is stopped long enough for the patients polyuria and polydipsia to reappear Then fluids are withheld for eight hours but food is per mitted Just before the test the patient is given 20 cc of water per kg of body weight to drink in one hour Thirty minutes after the start of hydration an indwelling catheter is inserted and urine specimens collected in 15 minute periods calculating the urine flow in cc per minute After two control periods with adequate flow (at least 5 cc per minute) an intravenous infusion of hypertonic salıne (25%) is started and administered for 45 minutes at the rate of 0 25 cc per kg per minute If there is no decrease in the flow of urine during the infusion or during the first two post infusion periods of 15 minutes each 01 unit of pitressin is given intravenously and the effect on the rate of urine flow observed Normal patients show a prompt and marked decrease in urine flow after the start of the hypertonic infusion while patients with true diabetes insipidus do not show any de crease until the pitressin is administered This test is not necessary in most cases of diabetes in sipidus but occasionally can be helpful when differentia tion from psychogenic polydipsia is uncertain URINE CONCENTRATION TEST This test is simpler and is occasionally helpful in the diagnosis of diabetes insipidus Method A simple modification is to collect the fasting urine specimen and then at 8 am to have the patient drink one liter of water within 1 hour Urine specimens then are collected at hourly intervals until 5 pm allow ·38 JABORATORX AIDS IN ENDOCRINE DIAGNOSIS in the patient no fluids and only a dry luncheon during thus time The volume and specific gravity of each speci men is measured Normally tlie specific grility rises as the day progresses often reachung 1022 to 1032 The commonly used Fishbcrg modification of the Volhard test which requires abstinence from any fud for a day and a night and part of tlie next day may be isipossible 11 a pacient with diabetes insipidus because of the exces SIC thirst characteristic of the disease In diabetes inupudus the urine does not concentrate to above 1001 or 1 005 The test may be dillicult to per ſoron because of intense cliscomfort resulting from great thırsı during the period of uithholding ilud SULLOWITCH TEST This is a simple qualitative test for the imount of cal cuum in the urine It is satisfactory and of considerable help clically in diseases with deranged cilesum metabo lism The test furnishes a quuch and surprisingly good indirect estimate of the level of the blood calcium Method Fue cc of Sulkowitch reagent are added to about 5 cc. of centrifuged urine The density of the cloud is toled after mixing and allowing to stand for two minutes Results are recorded as follows 0 clear I plusslight clou 2 plus-moderare ch ud 3 plus--hcaly cloud 1 plus-solid lund Normally 1 plus 2 plus or occasionally 3 plus are seen depending upon the mount of calcium in the diet It the result is hypocalcemia is present (usually under 75 mg%) as in tetany 1f the result is 1 plus or Occasionally even 3 plus luypercalcemia is indicated (usual ·SPECIAL TESTS OF URIVE 39 ly over 11 mg %) as in hyperparathyroidism The Sullo. witch reagent is an oxalate buffer solution and is prepared as follows Oxalic acid Ammonium oxalate Glacial acetic acid Distilled Water qs ad 25 gm 25 gm 50 cc. 1500 cc. Some authors suggest that the test be done on a 24 hour urine specimen However if a morning fasting urine specimen is used it is less likely to be influenced by dietary calcium intale or amount of Aud ingested-and can be compared from day to day in following the effects of therapy In some laboratories the Sulkowitch is part of the routine urinalysis thus possibly furnishing a diagnostic clue to an unsuspected instance of hyperparathyroidism or to other diseases with excessive bone destruction such as severe osteoporosis or Paget s disease The test shows a strongly positive result in hyperpara thyroidism in active Paget s disease of bone, and in severe osteoporosis When the latter two conditions are under treatment with the sex steroids reduction in the intensity of the Sulkowitch reaction is an indication of the effective ness of treatment The test is negative or low in para thyroid tetany and may be of value in suggesting hypo parathyroidism (post operative) as a cause for some of the patients symptoms which might otherwise be ascribed to hypothyroidism If vitamin D is being administered in large doses as for arthritis an increasingly positive Sullo witch test will give warning of beginning toxic effects PHOSPHORUS EXCRETION Study of the excretion of phosphorus in the urine is performed occasionally especially in differentiating hypo parathyroidism from pseudohypoparathyroidism (the so- ·40 LABORATORY AIDS IN I\DOCRINE DIAGNOSIS called Serbright bantam syndrome. which the system seems resistint to the effects of parathormonem instance of end orgin unrespons eness) The test of Ellsworth and Houards in shich the hourly excretion of phosphorus for thirce hours before and three to five lours after the injec tion of 2 cc (200 units) or parathormone is determined In pseudonypoparathyroidism slie excrction of phosphorus remains conslant or only slightly elerated-while in true My poparathyroidism the urinary phosphorus shows : marked increase after the injection ACETONE VODIES Acetone and discetic acid are easily tested for in the urine and in some laboratories one test or the other is a routine procedure They are present in diabetic acidosis or acidosis of other origin including starsation as when 1 too strenuous reduction regime is being pursued CREATINE IN URINE This test is occasionally helpful in the diagnosis of male hypogonadism Method A 21 hour urine specimen is collected! The urine volume (V) is measured The specific graszty is mersured and if above I 010 the specimen is diluted to that level with stater and the volume again measured (Vd) (only a small rolune (ie 5cc) need be diluted and cor rections can be made for total dilution volume) Fie cc are then again diluted to 100 cc in a volumetric flask II the origma} specific gravity is less than I 010 no original dilution is necessary One cc of the diluted urine is then mixed vith 8 cc of N/12 sulfuric acid and I ce of 10% sodium tungstate This Is thoroughly marked by shaking and then hiſtered if neces sary Eight cc of the filtrate is placed in an Evelyn color imeter tube and the mouth covered with tinfoil This is ·SPECIAL TESTS OF URINE 41 autoclaved for 20 minutes (115 to 120 C) at 15 lbs pressure and then alloned to cool It is necessary to run a specimen for urine creatinine at the same time and in order to do this lcc of the diluted urine is prepared exactly as in the preceding para graph except that it is not autoclaved An alkaline picrate solution is then prepared by adding I volume of 10% sodium hydroxide to 5 parts of a picnic acid solution (of which each liter contains 1175 gm of pure picric acid) This should be used within 5 minutes Four cc are added to each of the tubes and after standing for 20 minutes for development of color the readings are made in a photoelectric colorimeter using a 515 filter A blank tube is used at first containing 8 cc of water to which also has been added 4 cc of the alkaline picrate solution and allowed to stand for 20 minutes The gal anometer is set at 50 so later readings are multiplied by two before conversion Conversion is accomplished by reference to i chart previously prepared using dilutions of a stock solution containing 1 mg of creatinine per cc Calculations are as follows 1 Diluted volume of urine (Vd) x 100 x mg of creatinine (read ng from chart) Original urine volume (V) x aliquot of diluted urine in cc mg of creatinine per 100 CC- 2 Original urine volume (V) x mg of creatinine per 100 cc 100 gm creatinine in total urine specimen For creatine-the difference between the readings of the autoclaved and unautoclaved sperimens represents crea tine in terms of creatinine This is converted to creatine by multiplying by the factor 1 16 This conversion can be made after the first or second calculation aboie depending upon whether the mg per 100 cc of urine or the gm per total volume of urine is desired ·42 LABORATORY AIDS ENDOCRIVE DIAGNOSIS Aortal zalues for adults are 0 to 186 mg /21 hours and for children 10 to 15 mg/24 hours Creatine is present normally in the urine of children ind of adult women However it is very low or absent in adult men In endocrine disease it is found to be pres ent in adult men usth hypogonadism and occasionally with hyperthyroidisni ind diabetes mellitus It may be absent in children with childhood my redema The ei dence presented by the test is usually corroborative but not critical in ?rining at a clinical diagnosis REFERENCES ICFLER 33 H POWER VE H and 11 LEX R Concentration of chlorde sodies and potassium in urise and blood.Their duagnostic siguificance in adrenal insufficiency JAVA 11 117 12 July 9 1938 2 LEXY VES TOWER Y Band KEPLER E) The specificacy of the water tes2 as a diagnosese procedure in Addison s disease 1 cin Endocrinol 6 607 639 Sept 1916 3 THORS FORSHANE P H FRAI2FY TF FLOY DL RENOLD ALFREDRICASOS DS and 3F\KINS D Idvances in the diamosis and treatment of adrenal insufficiency Med 10 599 611 May 3931 # CARTER 1 ani ROBBIN} The use of fryperionic saline snlusions in the differential diagnosis of diabescs assipidus and psychogenic poly dipsta Clin Endocrinol , 753 66 \os 397 F42.108 533 R and HOW 467 F Studies on the physiology of the parathyroid glands 1 Il-Same responses of normal human hidneys and blood to intravenous parathyroid extract full Johns Hopkins Ilost 55 996 308 1931 ·Chapter V BASAL METABOLIC RATE, RADIOACTIVE IODINE UPTAKE, GASTRIC ANALYSIS, ELECTROCARDIOGRAM, BLOOD PRESSURE TESTS E BASAL METABOLIC RATE STIMATION of the basal metabolic rate has been used for years in determining the level of thyroid function Recently the test has been the subject of criticism because of the variation in results and lack of specificity in com parison with the determination of the protein bound 1odine However it has stood the test of time and if its limitations are realized and above all if there is good clinical correlation it is still of considerable value Also in its favor are the facts that it is relatively easy to perform and is widely available Method This varies considerably depending upon the machune used so will not be described However the more common factors of error will be mentioned These include poor relaxation of the patient * perforated ear drum faulty functioning or leaks in the machine worn out soda lime faulty placing of the nasal clip so that leaks can occur and the patient not being in an absolute fasting state Emotional factors also may alter the result as has been brought out in recent studies of the somnolent The rate does not chante appreciably during the menstrual flot but discomfort associated ith the per od may nterfere with prop r relaxation of the patient and result in an unsatisfactory test Therefore it is well not to perform the test on patients who are expenencing menstrual mol m na and probably neser on the first day of the men trual period 43 ·1.ABOR 1TORI AIDS IF DOCRIYE DIAGNOSIS inetabolic role by Rapport Curtis and Simcox? They per formed basal metabolic rates in the usual manner and then put the patient to sleep with intravenous Nembutal (50 mg then 23 mg per minute until asleep) The test 175 then repeated and the values were 7 so 10% loner than those then ukule the patient uas awake Howeser in nervous patients the salues were 30 to 340 loner As is well known in most normal patients values are between minus 10% and plus 10% With hyperthyroidism the figures are above plus 10% and such hypothyroidism they are belon minus 10% (Office tests usually average about 5%, lugher than those performed in the hospital) Occus1012}}) honeter an individual will be seen who has a basal rate in the vicinity of mmus 20% who feels per fectly well and who siionis no signs of laypothyroidism Administration of thyroid substance in un amount suff cient to raise the rate to usual normal levels will result in symptoms of over dosige so one must conclude that the normal level for that individual is approximately manus 20% liso xf this person should develop hyperthyroidism a basıl rate of plus 10°, may indicate a significant eleva tion Thus the need for clinical correlation is even more apparent A high basal rale is seen occasionally in leukemia the writer having encountered one instance of plus 100% OF the endocrine diseases a moderately high rate is seen olca sionally in active acromegaly gigantism Cushings syn drome tetang also usth tumors of the adrenals gonad: pineal gland or with arrhenoblastoma Non endocrine causes of increased metabolic rate are also numerous and include pregnancy fever and diseases of several of the body systems 4 recent excellent resses of the causes of extra thyroidal by permetabolism has been published by Bruger and Hollander ?

·BISAL METABOLIC RATE 15 Iery low rates are found in Simmonds disease with lev els of minus 45% and minus 50% being reported Similar low levels are found in anorexia nervosa in which the clinical picture is almost identical although the funda mental causative factor is psychological rather than endo crine Moderately low basal rates are found in the late burned out phases of acromegaly and gigantism They are also encountered in infantilism Addison s disease and hypo gonadism Other less specific tests of thyroid function including the specific dynamic action test the galactose tolerance test and the acetonitrile test are not in general use at this time RADIOACTIVE IODINE UPTAKE The rate of uptake of a tracer dose of this material is occasionally of considerable help in the diagnosis of hyper thyroidism However it does require a setup for handling the radioactive material and measuring its concentration and is therefore available only in relatively few properly equipped centers Method The patient must not have had Lugol s solu tion antithyroid drugs or any other source of iodine for two to four weeks before the test Organic iodine prepara tions as used for cholecystograms pyelograms or myclo grams may interfere with the test for a longer period On the morning of the test the patient is given 50 microcuries of J131 by mouth and then allowed to eat breakfast Counter readings are taken over the neck and just above mid thigh ("there the diameter is similar to the neck but without thyroid tissue) at one hour three hours and five hours A scintillation counter is used at a dis tance of 40 cm In constructing uptake cusses the thigh reading is subtracted from the neck reading ·wum LABORITORY AIDS IN ENDOCRIVP DIACIOSIS Normally the uptake at fre hours is from 5 to 30% in the gland at the perk of a gradual curve In hyperthyroudlism the oseractive gland takes up the 1998 much more spilly md completely making a steeper 100 UPTAKE OF T IN THE THYROID in the stw hou . alt Ito al editation 80 70 OVERALL of nomin terar dose of in the cayene * 50% SWAS FIGURE Radio 1odine uptake studies in normals and in pa trents with hyperthyrardism and hypothyroidsst Cunes are aver age levels as idadual tests show considerable spread on the graph (Courtesy of Dr Earl R Miller) 50 W CINTA 30 201 SP TYA 30 ry A NYA 4 6 T 2 ft adm not at 15 curve The 5 hour readings sary from 45 to 90%, However any reading above 30% is considered to indicate possible hyperthyroidism and a search is made for clinical and other evidence of the disease Low levels (1 e 5 to 10% uptake at five hours) are not as helpful in the diagnosis of hypothyroidism and may be found without this conduion being present G ISTRIC INALYSIS The gastric acidity has been found to be los or absent in Simmonds disease and in 500 of a series of proven cases achlorhy dra was present it may occur also in my redema and in Addison s disease ·BASAL METABOLIC RATE 47 ELECTROCARDIOGRAM Electrocardiographic changes are occasionally seen in endocrine diseases and may be characteristic though not pathognomonic In myredema typical changes are bradycardia pro longed P R interval low voltage urth small complexes and changes in the ST segments in the left precordial leads The 7 uaves may be depressed or inverted and the changes can be compared with those seen in coronary disease All of these may return to normal as the patient improves under treatment with desiccated thyroid sub stance In Simmonds disease the lowered metabolism may also result in small complexes and bradycardia occurs with some frequency Hyperthyroidism may result in characteristic changes including a rapid rate tall R waves tall T u aves and occa sionally auricular fibrillation or flutter These arrhythmias may be paroxysmal in nature Later in the course of the disease signs of left ventricular hypertrophy may appear In late Cushing's disease hypertensive cardiovascular disease may develop and signs of left ventricular hyper trophy and myocardial damage will appear During attacks of hypertension with pheochromocytoma rapid rate and extra systoles may appear Electrocardiographic changes can indicate changes in the level of the blood potassium and have been of consid erable importance and help clinically In diabetic coma treatment with large amounts of fluids may cause an ex cessite diuresis of potassium to the extent that serious or even fatal potassium deficiency can occur This should always be suspected when the patient in diabetic coma is ·11 Itt 1 to the HI73 4219 OVE Au F and luua. ith ਸ ਸ ਸੁਖ ZEE nie 玉与叫一在 TL *! We ដង E Thyroidectomy Sept 1948 19 BMR -51% 11 1.49 Thyroid therapy started 1929 MWBttttt PETE 1:41 REAR mete 31 p 19 理/主達基 HER PRESSE HULU Hanul RIGLR1 2 [lectrocardiographic changes in post operative hypothyroidism Thyroidectomy in Sep tember 1918 and in March 1919 BVR was minus 51% 1 hyroid then started-note progressive improvement in I wases latent was 2 female age 1 47 L'ye tena (Co tte y of Den S kolow) ·BUVL. VETABOLIC RATE 19 not responding properl to theraps In such arcumstances when a determination of blood serum potassium is not easily available the electrocardiogram may be of con siderable help in furnishing a quick estimate of the snuation is the blood! potassiu in falls the collage de 2. LangbQT Leer 2 Draw STS Time FKLRE Electrocardiographic changes in progressive hypopouas Son (dealized honlod of the rate s also helpful as a fast 12 B characteristic of brpopotassemiz, while similar dunges may be cucoan red in poxal ema, with the difference that the rate sw (frey Phorts B. leter-Eli Lilly & Co 17 th May June 19. courtesy of Dr DC Huney) creases the Teres becorre fial and a prolonged QT m ier al develops If the level is een loz the Taces be corre arrested the S-T inter al is depressed and the QT 1** Tulus further prolonged The prolonged QT intercul w probabh the most tulucble and characteristic of the changes In hiperpotasemua (potassium Intoxication) progressive elevation of serum potassium causes tall peaked T wares widening of the QKS completes depression of the ST segments and finalls zuncular standstill with disappeenne of Pwaves.

·LABORATORY AIDS IN ENDOCRINE DIAGNOSIS BLOOD PRESSURE TESTS Tests which influence the level of blood pressure may be of considerable value in the diagnosis of adrenal medul lary tumor (pheochromogloma) The most helpful is the benzodioxane test which lowers the pressure in this con dition if it is elevated Recently Gifford Roth and hvaler hure reported the use of the Regitine test wluch also lovers the pressure and which they believe to be safer pleasınter and easier to administer The Dibenamine lest also is occasionally helpful in the presence of an ele kated blood pressure in ruling out pheochromocytoma If the blood pressure is normal the tests using histamme or Alecholl or letraethylammonium may be employed to induce a paroxysm The cold pressor test is also used occa sionally to help rule out pheochroinocytoma However these latter tlısee tests which may induce a sudden Tise in blood pressure carry the risk of inciting dangerous com plications including central nervous system hemorrhage etc Nethods Bensodioxone test Goldenberg Snyder ind Aranous noted that the injection of piperidyl methyl benzodioxane in doses of 10 mg per sq meter of body sur face gren intravenously over a period of two minutes resulted in a fall of blood pressure lasting up to 15 minutes in the presence of pheochroinocytoma The fall did not occur in other types of hypertension ll hile the rest has not proved uniformly reliable it is still a valuable one Regiline test. This is performed by injecting 5 mg of Regitine (C-7337) intravenously in the presence of phe ochromocytoma there is a marked fall in blood pressure if hypertension is present The blood pressure may drop in panents with essential hypertension but not to the saine degree usually less than 35 mm mercury 51 tolic and 2 min diastolic ·PASAL METABOLIC RATE 51 Dibenamine test Spear and Griswolds reported that following the administration of 7 mg of dibenamine per kg of body weight given intravenously in 300 cc of 5% glucose in normal salıne solution over a period of one hour significant decrease of blood pressure occurred in patients with pheochromocytoma In patients with mod erate essential hypertension the test usually induces only a slight postural hypotension Histamine test Roth and hvale reported that in sus pected cases of pheochromocytoma the injection of 0 05 mg of histamine base intravenously would induct an attack of hypertension usually within 2 minutes This was in contrast to a slight rise in normal individuals or patients with other types of hypertension Alecholyl test arneri and Evans' suggested the use of Mecholyl giving 25 mg subcutaneously to induce a hypertensive attack in patients with adrenal medullary tumor They believed it to be more accurate than the histamine test Tetraethylammonium test. This is given intravenously in a dose of 400 mg In hypertension the blood pressure will fall markedly and with normal tension a mild hypotensive effect will result Honever with pheochromocytoma the injection will frequently (but not invariably) indtice a hypertensive attack This lasts longer than that induced by histamine but can be controlled by placing the patient in the erect position Roth and Kvale' have reported ex perience with this test Cold pressor test This test which is performed by im mersing one of the patient shands in ice water for 1 minute is not as likely to produce hypertension with pheochromo cytoma as with other types of labile hypertension REFERENCES 1 RAPPORT RL CURTIS GM and SIMOOY S J Somnolent metabolic rate in da nosis of thyroid dysfunction J Clin Endocrinol 11 1519 1963 Dec 19.1 ·51 LABORATORY AIDS IN ENDOCRINE DIAGNOSIS are open or closed An estimation can be made from the wrist film alone using the tables compiled by Shelton' and the Atlas of the hand by Todd-or by a more complete study of the joints of the extremities using the tables com FIGURE 4 Acromegalic skull-note enlarged sella turcica ballooning of frontal sinuses thickened calvarium elongated mandible with prognathism and prominent occipital protuberance (After R F Escamilla Diseases of the Pituitary in Musser llohl Textbook of Internal Medicine 5th Editon Philadelphia Lea and Febiger 1951) piled by Hodges ? The second method is preferred by the writer as it gives a more complete picture of the develop ment and allows both the physician and the radiologist to estimate better the potentialities of further growth A good routine is one in which films are made of one elbow one wrist and hand pelvis including lups one knet one ·ROENTGEN RIT FEAMINATIONS 55 my ankle one foot and a lateral view of the skull If the patient is near bone maturity (beyond 16 to 17 years) it may be valuable to include the shoulder and clavicles Alarked relardation of the bone age is seen in childhood redema A good example of thus was seen in a child cited personally to the writer by Dr C Canelo of San Jose California This my redematous child of two years had a bone age equivalent to that seen in the seventh month of fetal lıfe Thus at tro years the bone age was minus two months Mloderate retardation is seen in pituitary infantilism and in eunuchoidism Surprisingly some relardation is occasionally seen in gigantism particularly if there is a factor of secondary eunuchoidism Mild retardation is the rule in cases of ovarian agenesis Advanced or accelerated bone age well beyond the chronological age is seen in pubertas praecox of adrenal, gonadal or pineal ongın Rapid premature closure of the epiphyseal growth zones accounts for the eventual short scature in these patients in spite of the spurt in growth to above normal levels (transitory gigantism) which usually characterizes the early stages of the disease DENTAL RAYS Studies of the teeth may show characteristic changes in certain of the endocrine diseases. In hyperparathyroidism there is characteristically a disappearance of the lamına dura around the roots of the teeth With hypoparathyroid ism enamel defects including transverse furrows and hori zontal grooves may be seen as well as blunting of the tools These changes occur particularly in the incisors and ca nines Increased spacing between the teeth is seen in acromegaly and gigantism and prognathism with change in bite may ·ICT 8cm (cm 102cm Figure 5 Chest ray showing myxedema heart The enlarged Aabby atonic heart returned to normal size under creatment with thyroid (After R F (suamilla H Lisser and H C Shepardson Ann Int Med 9 308 1935) ·ROCNTGEN RAY EXAMINATIONS 57 be apparent in acromegaly Conversely crowding of the teeth is frequent in hypophyseal infantilism because of the sinall dental arches Dental age can be estimated in childhood by noting ad vance or delay in the usual sequence of eruption It is usual ly retarded with hypothyroidism childhood my redema or cretinism or may be advanced with precocious puberty Dental rays also may emphasize the presence of osteo porosis The teeth do not lose their calcium readily while the maullary and mandibular arches may participate in a generalized osteoporotic process so that the contrast be tween the two is particularly marked-a fact which may be helpful in diagnosis XRAY OF CHEST This may demonstrate the large flabby atonıc heart dılated in all its chambers which is characteristic of myredema Occasionally a similar appearance is caused by an accumulation of pericardial fluid in the same disease and pleural fluid is also seen at times The heart also may be enlarged as part of the general splanchnomegaly of acromegaly and conversely may be of small size as part of the splanchnomicria which is characteristic of advanced Simmonds disease in late Cushing's disease and late hyperparathyroidism the development of hypertensive cardiovascular disease may result in cardiac hypertrophy This enlargement also is found occasionally in late hyper thyroidism In lateral view chest films may demonstrate acromegalic changes such as a prominence of the sternum and enlarge ment of the vertebrae with cervico dorsal kyphosis In Cushings syndrome the marked osteoporosis may result in collapse of the vertebrae causing the typical kyphosis Chest films may reveal tuberculosis as the causative ·FIGURE 6 Colon in myxedema Films taken after voluntary emptying of bowel following barium enema Note atonic bonel before treatment and improved tone and emptying after treatment with thyroid (After R F Escamilla H Lisser and HC Shepardson inn Int Med 9 310 1935) ·ROFTCE BAI FLAVIATIONS 59 factor in Addison s disease Ividence of substernal gouter also may be seen and occasionally in precocious puberty a thymic tumor is found RAY or 1rDOVINAL REGION Films may reveal calcification of the adrenal glands in Addison s disease Studies of the gastrointestinal tract oc casionally demonstrate the atony and sluggish function characteristic of my redema and studies of the urinary Tract may also reveal atony of the bladder in this condition Other urinary tract findings include signs of renal lithiasis or pyelonephritis in late hyperparathyroidism ray studies to demonstrate tumors of the adrenal Clands have been used for several years Intrar enous uro grams may demonsirate a donnnard displacement of one kidney by such a tumor or an occasional deformity of the upper calyx because of pressure from above Occasionally retrograde pelograms will demonstrate these changes more clearls In eich instance the roentgenologist should be adused of the reason for the study so that films will be taken to include the entire adrenal region Occasionally the tumor will produce a definite soft tissue shadox which hoseser must not be confused with the outline of the fundus of the stomach or a lesion in the tail of the pan creas However some good sized adrenal tumors do not markedly displace the kidney and may be anterior or posterior to it The writer recalls one adrenal tumor half the size of a football which is not located definstely as to either right or left side by ray studies (including pyelog grams) before operation Contrast studies have been used for several years as a further step in unvestigating the adrenal ver The introduction of air or gas into the perirenal space has been utslized and 15 still being used in some clinics However it is a dangerous procedure and has ·60 LABORATORY AIDS IV ENDOCRINE DIAGNOSIS occasionally resulted in sudden deaths This tragic accident has occurred in our experience and has resulted in the abandonment of thus procedurc by the writer and the con FICIRE 7 Apparatus for infection of oxygen into retroperitoneal space for extraperitoneal pneumography Note that the oxygen is injected under pressure of 20 cm of water Courtesy of Dr Donald R Smith) clusion that surgual exploration of the abdomen is safer Since the procedure is still used in some centers it is only fair to admit that refinements of technique or the use of gases other than air (oxygen carbon monoxide Carbon dioxide) may increase the safety factor Alore recently the introduction of gas retroperitoneally ·ROLIGE, RAY ELASIVATIOVS 61 has been suggested bs Rivas and further reported on by S einbach Lson, Ville ard Smuth' Thu has come to be known as "extrattorecl pneurography and is accon plshed as follos Method. The paten is placed on the rgbe side and the ste beween the ap of the cocons and the rectum is 12- Elc.ed with 1 Corozine. Anesthe. ortment is ap pled to the apes. V. allowing use for the anesthesia to tale efoc, 2 =19 spisal reede is inserted 1 or 2. below the up o the Cocess in the midline lo index ET ES placed in the as to gaide the ceedle Khich is curaduced close to the scococozeum 19 order to avoid Purcturing the recur. The up of the ceedle should reach the retroperitocal Essus 110, abose ske up of the COCOVI 1. aspiration with a surge to be sure that the poci a N in 2 blood sessel che aparatus for delivery oʻ17-1 u 24u2ched br stezie taberg with a co con fil.er trobo ile system is used the presiosly has been filled W. IN 0 to 190 C of onroer. Bs adjating the levels so th? a pressure of mom of * 13 exerted the gas will £niwibad any reat discomfort 1 coral amount of 15 cc of oxygen per kz o bod wezght is usually sats £2C1OT If this pres we does col cause tke zas to enter the rezzopeu.ceal solace the position of the prodle should be dung. The OTVE.ends to seek the bizhest level, and ore ha of the arzen a given with the patres on the Txz de 4 mmp coce os person of the fans 1672.5 tbe the za has reached that are2. Then the patient Esed to the lef.sde and the second half of the orzec ced mi boc Koning the neede-ho ascends to the ceber EnŁ Teps then left p ose tau Elms are ako, 2 came the bad of the table is fixed 15 Rrezeror25 ms be tusena once and if cot situs 42. Cep 10 of the paper co be charged as od: 220d the films repen. Y oce or two both. Sssscoge z.5090M L2.921 rems of the ab ·LABORATORY AIDS IN CNOOCRINC DIAGNOSIS occasionally resulted in sudden deaths This tragic accident has occurred in our experience and has resulted in the abandonment of this procedure by the writer and the con FIGURE 7 Apparatus for injection of oxygen into reiroperitoneal space for carraperitoneal pneumography Note that the oxygen is injected under pressure of 20 cm of water (Courtesy of Dr Donald R Smith) clusion that surgical exploration of the abdomen is sfer Since the procedure is still used in some centers it is only fair to admit that refinements of technique or the use of gases other than air (oxygen carbon monoxide carbon dioxide) may increase the safety factor More recently the introduction of gas retroperitoneally ·ROENTGES RAY EVAVITATIONS 63 Arteriography offers another method for identification of adrenal tumors by x ray The translumbar approach can be used and is performed as follows 7 Method After preparation of the patient with enemas and sedation a scout x ray film of the lower thorax is talen A general anesthetic is then administered, usually pentothal supplemented by intratracheal gas and oxygen A 17 gauge 6 inch needle is then introduced just beneath the twelfth rib posteriorly about 8 cm to the left of the spinous process It is directed medially and cephalad along the body of the 12th thoracic vertebra to enter the aorta Once the needle is in place the injection is begun im mediately using 12 cc of contrast medium (70% diodrastpatient previously tested for sensitivity Pressure for the injection can be manual from a syringe & or from an in jection apparatus to give greater pressure' (Two 18 gauge needles have been used by some workers allowing simultaneous injection of the contrast medium by two operators ) The first x ray film is taken as the last cc of the material is injected and a second as soon as the cassette can be changed. However if a mechanical cassette changer is available multiple films can be exposed in a few seconds? The renal circulation usually is well demonstrated and any extension to an adrenal tumor would be seen Also any variations in origin of the adrenal circulation may be noted This method is still in the stage of development but shows promise of occasional usefulness ihen other simpler measures have failed to demonstrate a suspected adrenal tumor OTHER RAI STUDIES Films of the hands and also of the feet may demonstrate early changes in acromegaly Lateral tufting of the terminal ·62 LABORATORI AIDS IF\DOCRINE DIAGNOSIS dominal and adrenal region are usually taken and tomog rphy may be helpful if iny con(usion is caused by gas in the stomach or intestinal tract Satisfactory films are ob tained up to 3 hours after the oxygen is introduced but after that length of time absorption begins and at 18 FIGURE 8 ray film following extraperitoneal pneumogrim Shous tumor in right adrenal area which was proved by operation to be a pheochromocytoma (Courtesy of Dr Donald R Smith) hours only about 30% remuns in six days only a trace can be found Experience with this has been so satisfactory that it is now frequently performed as a come and go procedure without hospitalizing the patient The gas be hind the adrenal usuully furnishes a satisfactory contrast so that the gland can be ouzhned definitely ·ROENTGEN RAY EXAMINATIONS 65 syndrome and may be so pronounced that vertebral col lapse occurs causing the typical hyphosis Osteoporosis may also be seen in senile or post menopausal states and occa sionally in hyperthyroidism hyperparathyroidism or ovarian aplasia rays in advanced hyperparathyroidism also may show characteristic sub periosteal cysts which can be widespread This osteitis fibrosa cystica originally described by von Recklinghausen may result in spontaneous fractures or even softening and bending of the skeleton Occasionally disseminated osteitis fibrosa cystica may be encountered as part of the clinical picture of polyostotic fibrous dysplasia This rare condition more recently called Albright s dis ease is characterized by the above bone changes with 1 segmental distribution plus areas of cutaneous pigmenta tion in somewhat related distribution and also sexual and somatic precocity when it occurs in the female REFERENCES 1 SUELTON E K Roentgenographic studies in normal osseous develop ment J 4MA 96 759 66 March 7 1931 2 HODGES PC An epiphyseal chart-Am J Roentgenol 30 809 810 Dec 1933 3 ESCAMILLA RF Interrelationships in dentistry and endocrinology I California State Dental A 7 200 204 July Au1991 4 Rivas MR Roentgenological diagnosis Generalized subserous emphy sema through a single puncture Am J Roentgenol 64 723 734 Nov 1950 5 STEINBACH HL LYOY RP MILLER ER and SMITH DR Extra peritoneal pneumography California Med 75 209_06 Sept 1951 6 SMITH P G RUSH TW and EVANS A T The technique of trans lumbar artenography J.AM 1 148 995 258 Jan "6 1952 7 FREEMAN NL with GILFILLAN RS FULLENLOVE T1 and LZEDS FH Surgery of the Large Artenes Monographs on Surgery Balti more Williams and Wilkins 1952 p 412 8 ALBRICHT F Polyostotic fibrous dysplasia A defense of the entity J Clin Endocrinol 7 307 324 May 1917 ·61 LABORATORY AIDS IN FVDOCRINE DIAGNOSIS phalanges giving a cauliflower like appearance to the phalangeal tips is a pathognomonic sign of acromegaly and may be lielpful in the early diagnosis of the disease Indı viduals who liave done hard work with their hands may Inve widening suggesting this lateral tufting but not to FICURE 9 \ray study of hands in acromegaly Note characteristic cauliflower like appearance of terminal phalanges caused by lateral tufung There is also some general widening of the bones (Ifter RF Escamilla Diseases of the Pituitary in Musser Wohl Textbook of Internal Afedicine 5th Edition Philadelphia Lea and Febiger 1951) the degree seen in acromegaly The clanges in the toes are similar though not as marked Among changes in the skeleton generally osteoporosis is occasionally important although not limited to endo crine disease This is a characteristic findin; in Cushings ·ROENTGEN RAY EXAMINATIONS 65 syndrome and may be so pronounced that vertebral col lapse occurs causing the typical hyphosis Osteoporosis may also be seen in senile or post menopausal states and orca sionally in hyperthyroidism hyperparathyroidism, or ovarian aplasia Y rays in advanced hyperparathyroidism also may show characteristic sub periosteal cysts which can be widespread This osteitis fibrosa cystula, originally described by von Recklinghausen may result in spontaneous fractures or even softening and bending of the skeleton Occasionally disseminated osteitis fibrosa cystica may be encountered as part of the clinical picture of poly ostotic fibrous dysplasia This rare condition more recently called Albright s dis ease is characterized by the above bone changes with a segmental distribution plus areas of cutaneous pigmenta tion in somewhat related distribution and also sexual and somatic precocity when it occurs in the female REFERENCES 1 SHELTON E K Roentgenographic studies in normal osseous develop ment J.AMA 96 759 766 March 7 1931 2 HODGES PC An epiphyseal chart-Am J Roentgenol 30 809 810 Dec. 1933 3 ESCAMILLA RF Interrelationships in dentistry and endocrinology California State Dental A 27 200 204 July Aug 1951 4 Rivas M R. Roentgenological d agnosis Generalized subserous emphy sema through a single puncture Am J Roentgenol 64 723 734 Nov 1950 5 STEINBACH HL LYOV R P MILLER ER and SMITH DR Extra peritoneal pneumography California Afed 75 "02 06 Sept 1931 6 SMITH P G RUSI TU and EVANS A T The technique of trans lumbar artenography J.A.M.A 148 955 258 Jan 26 1952 7 FREŁNIAN NE with GILFILLAN RS FULLENLOVE TV and LEEDS FH Surgery of the Large Arteries Monographs on Surgery Balti trore Williams and Wilkins 19 2 p 412 8 ALBRIGHT F Polyostotic fibrous dysplasia A defense of the cauty J Clin Endocrinol 7 307 324 May 1947 ·Chapter l'IL TH URINE HORMONE TESTS 11C FICLD of measurement of hormones or their excre tion products in the urine has been one of considera ble interest and activity in recent years Such investigations Inte helped to clarify the pathologic physiology in some of the endocrine syndromes Occasionally one of the tests is of critical importance in diagnosis Many are complı cated biological issays and are not available except in re search centers Honever increasing, interest has resulted gradually in greater availability of several of the tests Those most generally in use at present are 1 The urine pregnancy tests 2 Measurement of 17 hetosteroids 3 Measurement of pituitary gonadotropins (follicie stimulating hormone-FSH) 4 Measurement of pregnandıol excretion In addition to the above tests for excretion of cortiroids from the adrenal cortex may be helpful clinically and are becoming more widely available These include the mea surement of the norysteroids-a chemical test which can be performed by the copper reducing method of Talbot and co workers' or the formaldehyde producing method of Daughaday Jaffe and Williams? A biological test is also available as described by Venning hizmin and Bell whuch measures the cortical hormones having a glycogenic action Adrenalectomized mice are used for this test and the factor measured is the amount of glucose taken up bv the buyer as a result of the glycogenic activity of the un Anown A more recent test for corticoids using the phenyl ·URIVE HORVONE TESTS 67 hydrazine color reaction has been described by Porter and Silber * This has been applied to the measurement of urinary compound E and F output by Carroll et als using a chloroform extraction and by Thorne using a butanol extraction The 11 oxysteroids glyconic corticoids and compound E and F steroids all show increased excretion in Cushings syndrome Another test for an adrenal cortical steroid in the Allen test? for dehydroisoandrosterone This has been reported to be of particular value in differentiation of adrenal corti cal tumor from hyperplasia being positive in the presence of tumor and negative with hyperplasia A more recent method utılızıng the Pettenkofer reaction has been re ported by Landau and co-workers & All of these tests are promising and deserve more extensive trial and utilization Another promising urine hormone test is that for the second pituitary gonadotropic hormone (the luteini-ing hormone (LH) or interstitial cell stimulating hormone (ICSH) MacArthur' has reported a method based upon the effect of the extract on the weight of the ventral pros tate in hypophysectomized rats Measurement of urine estrogens is occasionally per formed but an entirely satisfactory method is not yet avail able Two types of tests are used The first is a biological test using the effect on the vaginal smear of castrated rats as an end point as reported by Palmer 10 The second is the fluorometric test as suggested by Jailer and modi fied by Engel et al URINE PREGNANCY TESTS The classical Ascheim Zondek test was the earliest of this group of tests which are dependent upon the increased excretion of chorionic gonadotropin producing the charac ·68 LABORATORY AIDS IN ENDOCRINE DIAGNOSIS teristic reaction Originally immature white female mice were used and the urine injected twice daily for three days At 96 hours the mice were autopsied and the ovaries er amined for the hemorrhagic follicles which indicated a positive result A fater modification used immature female rats After a single urine injection the maturity of the vaginal smear indicated the positive reaction 13 However this also required 96 hours for completion The Friedman test was found to give a more rapid answer with comparable reliability and has been widely used An adult female rabbit is used which has been isolated from the niale for at least three weeks Ten cc of urine to be tested is administered intravenously in the ear vein and repeated in 24 hours In 48 hours the rabbit is sacrificed and the oiries examined The presence of macroscopic hemorrhagic follic les indicates a positive test The Gall Mainını test using the male South American coad is the most recent and most rapid test available The male North American frog (Rana pipiens) las also been found to be a satisfactory test animal as reported by Robbuns and Parker 14 Method The patient is advised not to drink any fluid after lunch of the day before The inorning urine specimen is collected the next day and used for the test Five cc of filtered urine is injected subcutaneously into the dorsal lymph sac of each of two male frogs A drop of urine 15 taken from the cloacal openings of the frogs by pipette one and two hours later and examined microscopically as a hanging drop preparation in reduceri light If sperma tozoa are present the test is positive as the increased amount of the chorionic gonadotropin in the pitients urine has caused a seminal discharge This test wall undoubredly become increasingly popular since frogs are inexpensive easy to maintain need not be ·LRIVE HORVOVE TESTS 69 sacrificed and an be used repeatedly if rested for about one week between tests The result is quickly available (in two hours) and is 96 to 98% reliable Other tests have been advocated but are not in general use These include that using the female South African clawed frog (Shapiro and Zwarenstein) with the positive reaction being the extrusion of ova -and the Guterman test? : a colorimetric test depending upon increased uri nary excretion of pregnandiol with pregnancy In the male, a testicular chorionepithelioma will cause a positie pregnancy test because of the increased excretion of gonadotropins 17 LETOSTEROIDS These are excretion products of hormones originating in the testes of the male and in the adrenal cortices of both sexes They are considered to be an index though not a true measure of androgenic function in the individual being tested The test is a chemical one with measurable colorimetric end point as a result of the Zimmermann reaction and has added considerably to our knowledge of testicular and adrenal function in certain endocrine diseases Accumulated experience has shown that the results parallel the androgen excretion when measured biologi cally by the effect on the size of the comb of a capon Even though all of the 17 ketosteroids are not androgens and all androzens are not 17 Letosteroids the test is helpful and is assuming an important place in the practice of clinical endocrinology Vethod. That in use at present in the Vetabolic Labo Tatory of the Unrversity of California Hospital is a modı fication of that desaibed by Holtorff and hoch is A 24 hour urine sample is collected with the jug used ·68 LABORATORY AIDS IN ENDOCRINE DIAGNOSIS teristic reaction Originally immature white female mice were used and the urine injected twice daily for three days At 96 hours the mice were autopsied and the ovaries ex amined for the hemorrhagic follicles which indicated a positive result A later modification used immature female rats After a single urine injection the maturity of the vaginal smear indicated the positive reaction 13 However this also required 96 hours for completion The Friedman test 11 as found to give a more rapid answer with comparable reliability and has been widely used An adult female rabbit is used which has been isolated from the malt for at least three weeks 1 en cc of unne to be tested is administered intravenously in the ear vein and repeated in 24 hours in 48 hours the rabbit is sacrificed and the ovaries examined The presence of microscopic hemorrhagic follicles indicates a positive test The Gallı Aſarnını test using the male South American toad is the most recent and most rapid test available The male North American frog (Rana pipiens) has also been found to be a satisfactory test animal as reported by Rob bins and Parker 14 Method. The patient is advised not to drink any fluid after lunch of the day before The morning urine specimen is collected the next day and used for the test Five cc of filtered urine is injected subcutaneously into the dorsal lymph sac of each of two male frogs A drop of urine 15 taken from the cloacal openings of the frogs by pipette one and wo hours later and examined microscopically as a hanging drop preparation in reduced light If sperma toroa are present the test is positive as the increased amount of the chorionic gonadotropin in the patients urine bas caused a seminal discharge This test will undoubtedly become increasingly popular since frogs are inexpensive easy to maintain need not be ·URI F HORVOVE TESTS 71 pump Any remaining water can be evaporated slowly at relatively low temperature The cooled flask may be placed in a desicator overnight and the dried extract can be stored indefimtely Determination by Zimmermann reaction is accomplished as suggested by Callos Callou and Emmens'' and Holt orff and hoch" with some modification The dried extract Is dissolved in 2cc of absolute alcohol heating in a pan of hot water at 50 C to aid solution if necessary 02 cc of this extract is used for the reaction hlett tubes are set up as follows 4- Unknown 02 cc of the urine extract 0.2 cc. metadınitrobenzene solution (prepared fresh daily by dissols ing 315 m, of purified metadını trobenzene crystals in 3 cc of absolute alcohol) 0.2 cc 5 aqueous potassium hydroxide b-Pigment Blank-0cc absolute alcohol 0cc alcoholic extract of urine 02 cc potassium hydroxide (as above) C-Standard 02 cc. dehydroisoandrosterone solution (prepared by dissolsing 23 mg of dehydroisoan drosterone in 100 cc of absolute alcohol-keeps relatively well in refrigerator) 0.2 cc metadinitrobenzene solution (as above) 02 cc. potassium hydroxide (as above) d-Reagent Blank-02 cc. absolute alcohol 0.2 cc, metadinitrobenzene soluuon (as above) 0.2 cc potassium hydroxide (as above) lett Blank 04 cc. absolute alcohol 0.2 cc potassium hydroxide (as above) All tubes are then stoppered shaken and placed in the dark in a constant temperature water bath at 25° C for 105 minutes This causes the Zimmermann reaction producing a characteristic red color which can be measured quantitatively immediately thereafter 8 cc of 80% alcohol is added to each tube diluting in the order in which the cubes are to be read. They are mixed by in version and read on the Klett Summerson photoelectric colorimeter within 10 minutes using a 540 filter ·70 LABORATORY AIDS IN ENDOCRINE DIAGNOSIS contattung lá ce of concentrated hydrochloric acid or 5 cc. of 500% sulfuric acid as preservatile (Frre cc 20% thy mol in ghcial acetic and can also be used and if the spect men is kept refrigerated not only the 17 Letosteroids but also the compound L and F content can be run from the same urine collection) On arrival at the laboratory the total volume is measured and a 50 cc ahquot is taken for the cost Hydrolysis is accomplished with some modifica tion of the method suggested by Drekier et al 14 and Ves tergaard" by acidifying the sample with 10 cc of concen trated hidrochloric acid in a 120 cc Frenmeyer Bask with ground glass stopper This is placed in a constant temperature water bath at 80 C for 1 minutes and then cooled After cooling the sample can kept in a re frigerator for 24 hours at this stage Extraction is performed by a modification of the meth nds of Drekter el al is and Holtorff and hoch v The cooled sample is poured into a 250 cc separatory funnel and mixed twice with 20 cc portions of ether and once with a 10 cc portion shaking 11/2 minutes each ume (a pinch of detergent such as Vel or Dreit may and the separation of ether lavers--not necessary routinely) (Vashing is accomplished by combining the 3 ether ex tracts in a 250 cc separatory funnel and adding 10 cc portions of 1 freshly made sodium hydroxide sodrum hydrosulfite solution as suggested by Talbot et al s This is freshly prepared by adding 10 gm of sodium hydro sulfite porder to 180 cc of 10% sacuum hydroxide The washing is performed 3 times shaking 1 minute each time This removes the phenolic fraction which contains estrone (also a 17 ketosteroid) The extract is then washed 3 times with distilled water or until the washing is rieutra? Evaporation is the next step and for this the ether ex tract is transferred 10 a 50 cc Erlenmejer flask and evap orated to dryness over a hot plate using grooved corks Reduced pressure for this is obtained by using a water ·URIVE HORVOVE TFSTS 71 pump Any remaining water can be evaporated slowly at relatively low temperature The cooled flask may be placed in a desiccator overnight and the dried extract can be stored indefinitely Determination by Zimmermann reaction is accomplished as suggested by Callou Callow and Emmens19 and Holt orff and kochis with some modification The dried extract is dissolved in 2 cc of absolute alcohol heating in a pan of hot water at 50 C to aid solution if necessary 02 cc of this extract is used for the reaction Klett tubes are set up as follows a-Unknoun 0.2 cc of the urine extract 0.2 cc metadinitrobenzene solution (prepared fresh daily by dissolving 345 mer of purified metadını trobenzene crystals in 3 cc of absolute alcohol) 09 c 5 N aqueous potassium hydroxide b-Pigment Blak-02 cc absolute alcohol 0.2 cc alcoholic extract of urine 02 cc potassium hydroxide (as above) C-Standard 02 cc. dehydroisoandrosterone solution (prepared by dissolving 25 mg of dehydroisoan drosterone in 100 cc of absolute alcohol-keeps relatively well in refrigerator) 02 c metadınitrobenzene solution (as above) 02 cc. potassium hydroxide (as above) d-Reagent Blank-02 cc absolute alcohol 02 cc metadınitrobenzene solution (as above) 02 cc potassium hydroxide (as above) e-Klett Blank 04 cc. absolute alcohol 0.2 cc potassium hydroxide (as above) All tubes are then stoppered shaken and placed in the dark in a constant temperature water bath at 25° C for 105 minutes This causes the limmermann reaction producing a characteristic red color which can be measured quantitatively Immediately thereafter 8 cc of 80% alcohol is added to each tube diluting in the order in which the tubes are to be read They are mixed by in version and read on the Klett Summerson photoelectric colorimeter within 10 minutes using a 540 filter ·72 LABORATORY AIDS IN ENDOCRINE DIAGNOSIS The following calculation gives the result 17 ketosteroids in mg per 21 hourscorrected reading of unknown 21 hour volume X corrected reading of standard 100 The normal range for adults is considered to be males 10 to 20 mg average 15 mg per 24 hours and females mg/24hrs sem - Non ketonic or non-specific chromogen fraction 20 +-Ketonic or 17 ketosteroid fraction N3 of 22 15 10 12 5 1 0 2 12 22 11 21 March 1941 April FIGURE 10 Graph demonstrating variation in 17 hetosteroid output of a normal man over a period of 2 months 48 hour urines were tested Separation into ketonic and non ketonic fractions also indı cated (Courtesy of Dr Sidney C Werner J Clin Endocrinol 1 952 Dec 1941 and Bull Univ Calif Med Center 2 399 Feb 1951) 5 to 15 mg average 10 mg per 24 hours In children the values are near 0 until the age of 8 By 12 they are 15 to 5 mg and then increase gradually to adult levels at about the age of 16 No marked sex difference is apparent until this time There is also moderate day to day variation in excretion as demonstrated by Werner?1 (see Figure 10) * Corrected reading is colorimeter reading corrected for pigment and reagent blanks ·URINT 3 ORTOVE TESTS 73 thus rendering meaningless the reporting of results to several decimal places and diminishing the significance of slightly high and slightly low levels Also the result expressed above represents total neu tral 17 Letosteroids-the end point for most clinical tests Further fractionation is possible with separation into Letonic and non Letonic fractions using Girard s reagent T The non keronic is usually 10 to 15% of the total neutral though occasionally it may be 25% This separa tion has not added to the clinical usefulness of the test and 1$ snggestre at best Honever the Letonic fraction may be further separated into alpha and beta fractions with digitonin The veta fraction is usually under 5% buc may be increased under certain conditions and if increased lo over 50% with a total excretion of over 50 mg per day is considered to be evidence for the presence of an adrenal cortical carcinoma The simpler tllen test? (page 67) is also reported to be positive in malıgnant adrenal tumors Studies of 17 ketosteroid urinary excretion in various endocrine diseases have shown the folloning lou levels are seen sn hypophyseal infantılısm Srmnaonds disease Addison s disease (especially in women wliere excretion should be close to O) my redema and occasionally in anorexia nervosa The last is unfortunate as it was orginally hoped that this test would be of critical importance in differentiating Simmonds disease and anorexia nenosa 11hile it is true that values in Summonds disease are generally lower than those in anorexia nervosa there is Oferlapping of the ranges in the two conditions so that in an indı dual patient the test may be helpful but not decisie High 17 kelosterolds are found 1ith adrenal cortical carcinoma or malignant adenoma and moderately hugh ·LABORATORY AIDS IN INDOCRINE DIAGNOSIS figures are found in adrenal cortical hyperplasia, especially 13th the adrenogenital syndrome Excretion in patients with carcinoma is usually oser 50 mg per day and may be 1s high as 1,000 mg (1 gm) plus per day 4s already stared of the excretion is over 50 ing per day and if further fractionation shows that over 50% of this is the beta fraction a tentative diagnosis of adrenal cortical carcinoma seems justified In adrenal cortical hyperplasia, excretion of from normal to 100 mg per day is seen The Allen test (p 67) mu also help to differentiate berncen adrenal carcinoma and hyperplasia Occasional instances of verified adrenal cortical carcinoma have been reported in which the 17 ketosteroid cncreton lias been normal In spite of this the test lias its greatest clinical importante in this condition An ocasional patient with adrenal cortical carcinoma will be found among the many women complaining of increased hirsutisin (heterosexual hypertrichosis) and this test should be run routinely in such pauents This has been emphasized in a recent report by Kinsell and Lisser' of a patient with hirsutism of 11 years duration but without other evidences of dcfeminization or masculinization such as amenorrhe? infertility trophy of the breasts dcepen ing of the voice or enlargement of the clitoris A 37 Jetosteroid test done as a matter of routine interest słoned a surprisingly high excretion of 82 mg per 24 hours of which 5% 115 the beta fraction / urther in vestigation revealed an adrenal cortical tumor approxi marely four inches in diameter This was removed surgi cally and prosed to be a malignant adenoma In such a patient the tumor probably would not have been suspected had the 17 Letosteroid erition not been measured Therefore the test with all of its imperfectionis had critical clinical value ·URIVE HORVOVE TESTS 75 Slightly lou levels of excretion are found in male eunuchoidism male castrates burned out gigantism and acromegaly, diabetes mellitus in the male climacteric and in chronic debilitating diseases The level tends to fall in old age Slightly high levels are found in Cushings syndrome, simple hirsutism (occasionally to 25 to 30 mg per day) active acromegaly and gigantısm arrhenoblastoma preco cious puberly pregnancy and in hyperthecosis However the variation of daily excretion in the same individual (as illustrated in Figure 10) and the occasional variation from laboratory to laboratory depreciates the clinical value of these slightly high and slightly low levels Indeed these minor changes are by no means in variable in the diseases mentioned FOLLICLE-STIMULATING HORMONE CXCRETION (FSH) This test also is occasionally helpful clinically The measurement of this pituitary gonadotropin (also called Prolan A thylakentrın or pituitary gonadotropin A) gives some indication of the level of anterior pituitary function Clinically it may aid in arriving at the proper diagnosis or the correct treatment in an individual patient The test is a biological one and requires a colony of either rats or mice depending upon the method used The method using rats has been advocated by Jungck Maddock and Heller and is in use in several centers In this tlie urine concentrate is prepared by ultrafiltration and then is injected six times into female rats at 12 hour intervals Twenty four hours after the last injection the uterus and ovaries are removed and weighed--the weights being recorded as end points However the method using mice and reco ding the ·76 LABORATORY AIDS IN ENDOCRINE DIAGNOSIS results in mouse units is in more general use at present and is the one used in the Indocrine Laboratory of the Division of Obstetrics and Gynecology at the University of California Hospital It was originally described by Zondeh 2s and modified by hlinefelter Albright and Gris wold 24 Method The present method used is a modification of that of Dekanske and Brudbury Brown and Broun ? A 21 hour urine specimen is collected and kept under re Irigeration After measuring if there is any toluene pre servative present it is filtered the pH is then adjusted to 10 sath a Bechman pH meter using 20% hydrochloric acid Fifteen cc of 20% holin in unter us then added and after thorough stirring is allowed to settle for 1 hour The supernatant fluid is removed by suction and dts corded the reinander is transferred to a centrifuge tube and centrifuged for 10 minutes at moderate speed The supernatant ſlud is then decanted and 10 cc of OIV sodium hydroxide re added to the kaolin with thorough mixing The misture is again centrifuged for 10 minutes and the supernatant transferred to a small bezker Using 5% hydrochloric acid tlus is brought to pH of approx imately 7 as determined by nutrizine paper it is then made up to a solume of 125 cc with distilled water and placed in a tube to be kept in the deep freeze unul ready for the test Test The concentrate as prepared above represents a level of five mouse units and is injected subcutaneously undiluted into two female mice 18 to 21 days of age They are ghen 05 cc twice daily for 2 days and once on the morning of the third day making the total dose 25 cc On the fourth day the mice are autopsied and the uterus stripped of fat blotted and weighed if the weight is above 15 mg the test is positive (the normal weight being from 3 to 8 mg) The level to be tested must be decided in advance The ·URINI HORMONE TESTS 77 concentrate can be prepared for testing at other levels by dilution with distilled water the amount being fi, ured by dividing the mouse unit lesel by 5-10 40 mouse units-dilution 18 (1 ac concentrate plus 1(c distilled water) BO mouse units-dilution I 16 (1 cc concentrate plus 15 cc distilled water) The normal range in adults is considered to be 5 to 50 mouse units per 24 hours It has been found satisfactory to run an unknown urine at two levels-5 and 80 mouse units If the test proves negative at both levels the ex cretion is below normal Should the result be positive at 5 mouse units and negative at 80 units the excretion is probably within the normal range of 5 to 50 mouse units If positive at both 5 and 80 unit levels then the excretion is above the normal range If desired later specimens can be tested at higher lower or intermediate levels as indı cated by the results of the original test Thus the test is moderately complicated and requires an animal colony-but is occasionally of sufficient useful ness and importance to justify the effort of setting it up It is of crucial importance in the differentiation between hypophyseal infantılısm (pituitary dwarfism) (low ercre tron) and ovarinn aplasia (high excretion) In the latter condition it is important that the patient not receive any estrogenic therapy for about 1 month before the test as estrogens depress the high levels (occasionally 300 to 400 mouse units) to normal In addition to orarian aplasia high levels of excretion are found in the climacteric in both sexes This finding is more frequent in females but normal levels may be found Thus it is occasionally helpful in diagnosis of the climacteric but unfortunately is not an invariable finding Lunuchus castrates and certain types of eunuchoidism also show increased FSH excretion as do patients with Klinefelter s syndrome The latter syndrome ·78 LABORATORY AIDS IV FNDOCRINE DIAG\OSIS 29 was described by Klinefelter Reifenstein and Albright in 1912 It occurs in males and is characterized by gyne comastia small testes with azoospermii and increased excretion of FSH On testicular biopsy the tubules are found to be hyalınızed but the interstitial cells retain normal appearance Low levels of ESHI excretion are found in hypopituitar ism including hypophyseal infantılısm lig pogonadotropic eunuchoidism and Simmonds discase Unfortunately low levels are occasionally found also in anorexia nervosa, so once gain in the individual patient the test may not be of crucial differential importance A summary of experiences with this test in various diseases was published by the writer in 1949 PREGNANDIOL ETCRETION This is an excretion product of progesterone and has been studied considerably in centers where there is active investigation in the field of obstetrics and gynecology A fall in pregnandiol excretion during pregnancy has been considered an indication of threatened abortion The method of Venning'o measures the excretion of sodium pregnandiol glucuronidate In this the urine is extracted with butyl alcohol and the residue treated so that it can be weighed Use of a consersion factor (0 597) gives the amount of pregnandrol The method of Guterman1 3 is a colorimetric one and is considered of sufficient accuracy to be used as a test for pregnancy In habitual abortion the lowered excretion of preg nandiol may indicate the need for prophylactic use of progesterone and may be 1 guide to proper dosage An increased output has been reported with polystic ovaries (Stein Leventhil syndrome) by Fischer and Rileys ·URINE HORMONE TESTS 79 Despite the above indications the test is not as widely used as in the past It is possible that the clinical help derived has not been sufficient REFERENCES 1 TALBOT WB SALTZMAN AH Wrov RL and HOLFE J K The colorimetric assay of urinary corticosteroid like substances | Biol Chem 160 535 516 Oct 1945 DALCIADAY WH JAFFE H and WILLIANS R H Chemical assay for COTIN Determination of formaldehyde liberated on oxidation with perındıc acid J Clin Endocrinol 8 167 1,4 Feb 1918 5 VENNIY EH KAZMIN VE and BELL J C Piological assay of adrenal corticoids Endocrinolog, 38 1989 Feb 1916 4 PORTER CC and SILBER RH A quantitative color reaction for cortisone and related 17,21 dehydro ? ketosteron is J Biol Chem 195 01 207 July 1950 5 CARROLL KK MCAUTINE H T and MOBLE RL Studies on the excretion of cortisone and compound F using the Porter Silber method Canad M A J 65 363 367 Oct 1951 6 THORN GW Studies on the metabolism of cortisone T A im Physicians 1952 in press 7 ALLEN V M HAYWARD S J and PINTO A A color test for dehy droisoandrosterone J Clin Endocrinol 10 54 70 Jan 1950 8 LANDAU R L KNOWLTOV K LUGIBIHL K and MUNSON PL The determination of urinary dehydrousoandrosterone in normal men and women and in adrenal disease Endocrinology 48 506 517 May 1951 9 MACARTHUR JW The identification of pituitary interstitial cell stimulating hormone in human unne Endocrinolog 50 301 310 March 1952 10 PALMER 4 The quant tative determination of urinary estrogenic hormone (biological method) I Lab Clin Med 24 643 619 March 1939 11 JAILER J V A fluorunett c method for the determination of estro gens Endocrinology 41 198 01 Aug 1917 ENGEL L W SLAUNWHITE IR JR CARTER P and N4TH 4SON I T The separation of natural estrogens by counter current dtstribution 1 Bol Chem 185 255 963 July 19 0 13 ASCHEN S mplified method of Ascheira Zondeks test of pregnancy JAWA 19 1010 Dec 8 1915 14 ROBBINS SL and PARKER F The use of the rate orth Amer can frog (Rana pipiens) in the diagnosis of pregnancy Endocrinology 4 937 13 Apr 1 1948 15 HOLTORFF AF and Loat FC The colorimetric estimation of 17 ·80 LAVORATORI AIDS IF\DOCRIVE DIAGNOSIS Letosteroids and their application la unine Extraces 5101 Chem 135 377 392 Scpt 1910 16 DRERIER 15 l'EARSOY S BARTCZAK E and VATACK THA rapid method for the determinauon of total urbary 17 Letosteroids Chin Endocrinal 7 79 800 Dec 2017 17 ESTERGAARD P Rapid Micro modification of the Zimmermann Callow procedure for the determination of Ictosterods un utice Ada endocrinol $ 193 214 VOY 1992 18 TALBOT BBUTIIR A V VACLACHLAN EA and Jones BV Definition and elimination of certain errors in hydrolysu extraction and spectro chemical assay of alpha and beta neutral untary 17 ketosterosds ) Biol Chem 136 363 917 \ov 1910 19 CALLOWS CALLOW RK and Exess C11 17 Aerosteroid androges and acstrogen excretion in cases of gonadat or adrenal corucal deficiency } Endocrinol 88 98 Vay 1910 20 CALOW H Carrow R K and EVENS C1 Colarimetric determination of substances containing the grouping CIŁ CO in urine extraces as an indicatson of androgen moosent Brachern ] 3. 1312 1337 1998 WERSFA SC The daily sariation in 17 Letosterod excretion of men and namen J Clin Endocrinol 1 951 924 Dec 1911 2 ESCAMILLA RF Diagnostic significance of urnary horixonal assays Repart of experience with measurements of 17 ketosteroids and foibcle stmulating harmone » the urine Ann Ini Ted 30°49 290 Feb 1919 23 KINSELL LH and LIXSEX I Adrenocortical tumor causing hirsutism without other evidence of rulista I C Endocrinol Metab 12 50 5! jan 1952 21 JUNGCK EC MADDOCK WO and HELLER CG Gonadotropic hormone Comparison of ultrafiltration and alcohol precipitation tacthods of recosery from urine / Class Endocrinol 71 10 Jan 1917 23 ZONDEK B Uber die Hormone des llypophysensordertappens 11 Darstellung des Follikelbrerluagshormons (Prozart 1)--Methodik der klinischen Harnanalyse zum Wachnets des Prolan Alun 1 chnsch 9 1207 1209 June 28 1930 26 LINEFILTER IEF J* 41 ARIGEIT and GRISKOLO G C Experience with a quantitatie test for normal or decreased amounts of follicle stimulating hormone in the umme in endocrinolagical diagnosis J Clin Endocrinol 3 529 514 Oct 1919 27 DELAYSXI } The kaalis adsorption method of the assay of urinary gonadotropin Ont 1 Exper Path 30 2 2 282 Aum 1919 28 BRADBURY T PROI και E 5 and BROI 1 E Adsorption of arinary gonadotropins on kao! Proesor Exper Biol ved 71 28 252 June 1919 ·URINE HORVOVE TESTS 81 9 KUNEFELTER H F JR REIFENSTEIN ECR and ALBRICHT F Syn drome characterized by gynecomastia aspermatogenesis without a Leyd gisto and increased excretion of follicle stimulating hornone J Clin Endocrinol 619 6 7 Nov 1919 30 VENING EH Gravimetric method for determination of sodium pregnandiol glucuronidate (an excretion product of progesterone) J Biol Chem 119 473 480 July 1937 31 GUTER VAN HS A human pregnancy test based upon a color reaction of pregnandiol in the urine / Clin Endocrinol 4 262 267 June 1914 39 GUTERMAN HS Further observation on the value of the pregnandiol test for pregnancy J Clin Endocrinol 5 407 411 Dec 1915 33 FISCHER RH and RILEY L L Pregnandul excretion in a mascul n izing syndrome J Clin Endocrinol 1 890 900 July 19,?

·Chapter VII VAGINAL SMEAR-CERVICAL MUCUS XAVIINATION of the vaginal smear is simple and easily E as able assistance to the clinician in estimating the level of Ovarian function and also can be used as a guide in deter mining the adequacy of therapy with estrogens The cells collected for examination are those being desquamated from the vaginal epithelium and the upper genital tract Consideration of a few fundamental facts will indicate the basis for interpretation Estrogens cause an increase in the layers of cells in the vaginal epithelum If a large amount of estrogen is pres ent there are many rous of epithelial cells the most superficial of which are squamous flattened and cornified If the level of estrogen falls the number of lasers dımın ishes and the flattened cornified cells tend to disappear with precornified cells of the intermediate layers predom nating These are moderately flat but not the true squa mous type If the estrogen level is low the epithelium is thinner and cells of the basal layer appear These are small of round or oval shape with relatively large nuclei The nuclei of the deeper cells are distinctly hyperchromatic The picture seen in smeurs varies from the extreme ly poestrın picture of the atrophic postmenopausal state to that seen during pregnancy when the system is surfeited with estrogens Honever the change is sensitive enough to indicate the differences in estrogen level during the normal menstrual cycle Method The smear must be taken before the usual 82 ·VAGINAL SVEAR-CERVICAL MUCUS 83 vaginal examination with lubricant is performed and at least 24 hours after the last douche A slightly curved glass pipette sıx inches (15 cm) in length and 05 cm in di ameter is used At one end is a rounded cip with relatively small opening and at the other is attached a rubber bulb of one to two ounce capacity The bulb is compressed and the tube inserted deep to the posterior fornix of the vagina and turned from side to side The suction created by the bulb is allowed to take up the vaginal secretion The tube 1$ withdrawn and the contents expelled onto a clean glass slide spreading the secretion and fixing immediately for drying causes distortion of the cell picture Swabbing or scraping of the mucous surfaces has been recommended but is generally thought to distort the cells as well as to cause false exfoliation However if the vaginal contents are very atrophic and dry this procedure may be necessary (the fact that fluid is unobtainable is an important indica tion of hypoestrinism) The fixative used is made up of equal parts of 95% ethyl alcohol and ether and should be in a Coplin jar or receptacle of similar size and shape beside the examin ing table for immediate use A piper clip on each glass slide is sufficient to keep the slides separated in the solu tion Fixation is accomplished in a few minutes but is more certain if continued for one hour The slides can be left in this solution without harm for one week Staining- The Short Trichromel stain is satisfactory for office use It can be made up as follows Biebrich Scarlet (aqueous solution) 05 Orange G Fast Green FCF Aniline Blue (aqueous solution) Phosphotungstic Acid 0 mg Phosphomolybdic Aad 05 go Glacial Acetic Acid 10 CC All dissolved in 500 ethyl alcohol 100 CC This stain 15 applied to the slide by dropper or in Copgr Ony gm 0070 gm 004 go ·81 LABORATORY AIDS IN ENDOCRINE DIAGNOSIS lin jar and allowed to stand for 1 minute Then the slide is dipped 10 times in 70% alcohol and 10 times in 95% alcohol well cleaned with xylol and mounted the fully cornified cells in addition to their typicai slope with small nuclei stain a reddish orange while the cells from the deeper layers stain green or blue A differential count can be made counting 100 to 300 cells and reported as the percentage of acidophilic cornified cells in the smear Normal variations These are seen during the normal menstrunl cycle as follows Menstrual phase Many red cells are present The vaginal cells chefly folded thickened and mucified superficial layer cells stun poorly Dark pigment like granules may be present After a day or two more basophilic cells appear Post menstrual and errly follicular phase The estrogen level is rising and cells of the intermediate levci appear in increasing numbers Cornified elements increase gradually (up to 30%) and are more or less separate with folded cyto plasm Pre ovulatory phase This lasts for 24 to 48 hours and is characterized physiologically by a rapid rise in estrogen production The vaginal smear reflects this with a rise in the cornified elements to 40 to 50% Doderlein s bacıllı are in evidence Ovulatory phase This lasts only about 21 hours and shows the intermediate peak of cornification with cornified elements at levels of from 50 to 75% and very few leuco cytes The vaginal cells stain brightly many occur in shingle like plaques and the nuclei are pyknotic and may be fragmented Post-ovulatory phase At this time there is a full in estro gen production for two to three days Progesterone effect also appeurs and opposes somewhat the cornifying effect of the estrogens The cornified cells begin to show folding ·PERINUCLEAR CERVICAL CELL PATHOLOGY IN SQUAMOUS TISSUE GRADES AND CELL TYPES IN "SURFACE BIOPSIES" OF CELLS INFLAMMATORY NORMAL ☺ CORNUFIED HYPERCORNIFIED SUPERFICIAL INFLAMMATORY INTERMEDIATE DO PRE CORNIFIED INTERMEDIATE PARABASAL BASAL BASAL DUMB BELL TYPE LINDIFFERENTIATED PRECOCIOUS MULTILOBULATION MULTINUCLEATION DIFFERENTIATED CELL BASAL TYPE PRE INVASIVE GIGANTISM BASAL CANCER PRE-CANCER (NEARO CANCER FIGURE 11 Cell types seen in vaginal swears (After Cuba Clinical Symposio Vol 3 51 June 1931 Courtesy of Ds E Oppenheimer and Dr E Isrc Cancer Cytology of the uterus lew York Grune and Stratton 1991) ·3 I'IGURE 12 Vaginal smear taken in preovulatory phase (Short stun) Note htglı percentage of cornihed cells (After Cuba Clinical Symposia Vol 1 4 p 107 April Vay 1949 courtesy of Dr R Greenblatt and Dr E Oppenheimer) ·85 VAGINAL SVEAR-CERTICAL WUCUS and curling of the edges and the proportion of basophılıc cells increases Percentage of acidophulic corufied cells is usually about 50% Leucocytes and more densely staining mucus also appear in the smear Luleal phase This lasts for eight to 10 days during which the progestational effects are more marked The acı dophilic cornified cells become more curled and folded and the outlines may be irregular There is some tendency to clumping The nuclei remain pyłnotic Percentage of acidophilic cornified cells gradually drops from 50% to the 20 to 30% level The leucocytes increase further and the basophılıc staining of the mucus is more prominent Mixed bacterial flora is seen with cocci predominating Pre menstrual phase This is caused by the drop in levels of both estrogen and progesterone one to five days before the onset of menstrual flow The percentage of cornified cells drops rapidly to 20% or under and those remaining are folded irregular and stain poorly Leucocites and mucus are abundant ESTROGEN DEFICIENCY Varying degrees cause different findings in the smear as follows Marked deficiency (seen is castrates and senile women moccasionally with severe menopause syndrome and with androgen therapy) Nearly all the cells are from the basıl layer and the percentage of conification is very low Aitotic activity may be seen in the basal cells Leucocytes are numerous 11th many bacıllı and coach and may be excessue with associated atrophic vaginitis Moderate deficiency (most common in the menopause or post menopausal statesmalso with long standing ovarian deficiencies in Jounger women) Most of the cells are from the basal layer but some intermediate layer cells are seen ·86 LABORATORY AIDS IV EPOCRINE DIAGNOSIS Occasional busophilic squamous cells are noted but the percentage of acidophilic cornified cells is low Leucocy tes are present and a moderate amount of mucus Is in evidence Slight deficiency (seen in mild menopause syndrome (r in younger women with moderate ovanan deficiency) Most of the cells are from the intermediate layer with some cells from the basal layer Cornified cells when counted are usually at levels of 20% or under A moderate number of leucocytes and some mucus are present PREGNANCY In this condition the findings are the result of progres sive increase of estrogens and progesterone After to to four wechs the number of acidophulic cornified cells de creuses and the smear shons increasing numbers of folded bisophilic cells and mıdzonal cells wuch are grouped and show accentuation of the margins with curling of the na vicular shapes If abortion is threatened red cells are seen and if incomplete leucocytes and histiocytes appear often in clusters or Also evidences of infection and thick strands of ropy mucus are seen rosettes HIPERESTRINISM This condition is seen with granulosa or the cell tumors of the ovary and tuith the administration of large doses of estrogens The smear shows a high percentage (up to 90°C) of the acidophilic cornified cells These cells may be small ind the nuclei fragmented or even absent Leucocytes are rare and the mucus though abundant stuins poorly An important confusing factor in the interpretation of 12,inal smears is the presence of injection, particularly ·VAGINAL SVEAR-CERVICAL SUCUS 87 cervicit s or vaginitis Some cornification may result with Irichomoniasis which is not due to estrogen effect Also basal layer cells may appear from ulcerated areas when they would not otherwise be present The presence of an unusually large number of leucocyces or of trichomonas should alert the observer to the possibility of these effects DETECTION OF CANCER The Shorr polychrome stuin as described a boue is salls factory for determmmz the estrogen level of the patient and also for following the results of estrogenic therapy However if the smear x to be used for the detection of cancer cells the Papanicolaou stair is preferable It cakes somes bar longer but has the advantages of greater penetra cson and differential staining of chick mucoid or bloody smears Method Alter fixing in 91ethyl alcohol and ether (equal parts) the smear is run through 70% and 50% al cohols and then stained in hematoxylın for 5 minutes It 18 rinsed in distilled water talloned by 053queous hydrochloric acid and then washed thoroughly in tap water Next 11 is run througe distilled water and 50% 70% 80% and 93% alcohols and stained for one minute in 05% solution of Orange G in 95% alcohol occasion ally 0015 gm per 100 cc of phosphatungstic acid is added) The smear is then washed in 2 chariges of 9» alcohol and stained in inchrome solution for two minutes This stain contains 45 cc of 05" solution of Light Green SE Yellowish in 99% alcohol 45 cc of 57 solution of Eosin Yellowish in 930alcohol 10 cc of gee solucion of Bismark Brown in 9. alcohol 02 gm of acid phos photungsric and I drop of a sajurated aqueous solution of lithium carbonate Atter startung the slide is rinsed $ umes in 95% alcohol run through absolute alcohol and xylol and mounted With this stain the sadophilic cells ·88 LABORATORY AIDS IN ENDOCRINE DIAGNOSIS appear red to orange and the basophilic cells green or green blue The epithelial cells and erythrocytes though stained are relatively transparent The diagnosis of cancer by thuis method has been found accurate in proper hands with diagnostic error of only 1% for squamous cell carcinoma as reported by Fremont Smith and Graham With endometrial or fundal carcı noma the accuracy is lower but is still satisfactory in al most 80% of cases IODINE VAPOR STAIN This stain is simple rapid and is occasionally helpful It gives an indication of the vaginal gh cogen index Method A simple dried smear is placed face down over a shallos dish containing a small amount of Lugols solu tion The iodine vapors which arise insensibly stain the glycogen containing cells in 2 to 3 minutes They should be examined immediately but can be stained in the same manner after 24 to 48 hours The amount of glycogen in the cells of the smear indı cates the degree of estrogenic response and can be classified in 4 grades Grade 1 shows complete glycopenia while grade 1 shows exclusively large flat deeply stained brown nodophılıc cells singly or in clumps This latter corresponds to the smear of the normal proliferative phase CERVICAL MUCUS Examination of mucus aspirated directly from the ceruix is occasionally helpful in sterility studies Cohen Stein and haye have pointed out that the cervical mucus becomes profuse and thin at the time of greatest fertility and they feel that this is a more accurate indication than observa tion of the basal body temperature curve The specimen

·a.
$
FIGURE 13 Group of cancer cells in a vaginal smear (Papanicolaou
method) (After Civa Clinical Symposta Vol 1 #1 Spring 1948
courtesy of Dr L Oppenheimer)

·No text on this page.

·VACINAL SVCAR--CERVICAL MUCUS 89 is obtained by inserting the tip of a glass cannula into the endocervix and aspirating mucus by means of gentle suc tion from a 10 cc syringe attached to the other end The mucus is blown out on a glass slide and a glass coverslıp placed over at The thread formation (Spinnbarheit) is then tested by lifting the coverslıp unul the thread of mucus breaks During the fertile period the coverslıp can be lifted 10 to 20 cm wlule during the infertile period only 0 to 1 cm lift is possible before the thread breaks The mucus during the infertile period is also thuch scant and difficult to aspirate Small doses of stilbestrol (01 to 0 2 mg daily) may improve the unount and thinness of the cerncal mucus Examination of the cervical mucus is es pecially valuable in selecting the optimal time for artificial insemination Profuse clear mucus is also seen after ad ministration of estrogens to the castrate and occasionally in patients sith bilateral polycystic ovaries REFERENCES 1 SHORR E Evaluation of the clinical application of the vaginal smear method J Vt Sinai Hosp 12 607 688 Vay June 191, 2 PATANICOLADU ON A general survey of the vaginal smear and its use in research and dia, nosis. Am J Obst Ginec 51 316 S 8 March 1916 3 FREMOVT SVIH V and GRANAV RM The Dio, posts of Ulerine Cancer by laginal Smear Progres in Gynecoloy (Ed Meins and Sturgis) \ew York Grune and Stratton 1916 PP 92 94 4 MACK H C and THOMPSOV A Appraisal of estrogenic activity by the 1a inal glycogen index J Clin Endocrinol = 961 564 une 1912 5 COHEN MR STEN I F and LAYE BV Spinnbarkeit A character 1 fic of cervical mucus S1 suificance at ovulation time Ferul - Steri! 3 201 209 May June 1992 ·Chapter IX H Η ENDOMETRIAL BIOPSY PISTOLOGICAL examination of a small portion of the endometrium can be accomplished as an office pro cedure It is more complicated than the vaginal smear and should be done by one with training in gynecologic methods since the cervix must be dilated and the uterine cavity entered to obtain fundal specimens these being the most satisfactory In proper hands it is safe and usually not uncomfortable for the patient and yields in formation regarding ovarian and corpus luteum function that cannot be obtained by other methods including vaginal smears and hormone estimations in blood and urine Method The patient is placed in the lithotomy posi tion and the position of the uterus determined Under dnect speculum observation the cervix and the external os are cleansed with an antiseptic (70% alcohol is satis factory) The anterior cervical lip is grasped by a single toothed cervix tenaculum and held steady while a suction curette is introduced in the direction of the fundus A circular stripping motion is made while exerting suction to obtain the specimen This is placed immediately in fixative solution (Bouin s solution Zenker s solution or 100% alcohol for glycogen staining) Sections are run through as with other histological or pathological speci No after care of the patient is necessary Usually only slight spotting occurs and there may be a few cramps 90 mens ·FIGURE 14 Endometral biopsy during normal proliferative phase The glandular lumen is narrow and is surrounded by a single layer of columnar epithelium (Aſter Ciba Clinical Symposia Vol 1 p 138 June July 1919 courtesy of Dr E Oppenheimer ) ·FIGURE 15 Endometrial biopsy-early secretory phasc-Notc gly cogen in base of cells (After Cuba Chnical Symposia tol 1 June July 1919 Courtesy of Dr E Oppenheimer) ·CYDOVCIRIAL BIOPSY 91 The possibilities of perforation of the uterus and spread of infection with pelvic peritonitis have been pointed out but are unlikely and can easily be avoided Undue force should not be used the course of the cervix and direction of the body of the uterus should be ascertained before hand aseptic technique should be observed and patients with gonorrhea and acute cervicitis should not be sub jected to the procedure INTERPRETATION OF SPECIMFNS NORMAL MENSTRUAL CICLE Preovulatory phase The estrogenic effect is dominant with typical proliferation of the endometrium which has been called the proliferative phase The endometrium gradually thichens and the glands increase in number and size and become more crowded and coiled The lumen dı lates but the epithelium muntains a well defined even con tour Glandular epithelial cells are tall and columnar and are crowded with large dark staining nucler in a scanty pale cytoplasm--mitoses are frequent Postovulatory phase This is dominated by the effect of progesterone and secretory changes appear giving the characteristic picture of the secretory phase Mucus and glycogen appear in the dilated and coiled glands and the cell nuclei are pushed to a central position The glycogen vacuoles move from a basal to a peripheral position and the nuclei now round and pale migrate to the base of the cell Coiled arterioles ppear with congestion and small hemorrhages in the superficial layers The cells become turgescent and pyramidal in shape and the epithelial layer becomcs folded Just before menses the glunds have discharged their ·92 LABORATORY AIDS IN ENDOCRINC DIAG\OSIS secretions and leucocytes and scattered hemorrhages are prominent With normal function an experienced observer can usually tell nithin a few days the time of the cycle when the biopsy was taken Horsever in pathological conditions it may be extremely important to know exactly the timing in the cycle when the specimen was obtained Therefore, the dates of the biopsy and the dates of two or three pre ceding menstrual periods should always be noted for cor relation and to aid in interpretation For diagnostic purposes the biopsy is best taken betu een the twenty first day of the cycle and the day of onset of bleeding With normal estrogenic and progestational effects the endometrium will slow good development and secretory activity This is also the best eudence that can be secured that ovulation has taken place-important knowledge in sterility studies PROGESTERONE DEFICIENCX Moderate degree is indicated by secretory changes less than normal in the specimen However quantitation is difficult and the possibility of a refractory endometrium must be considered Progesterone lack and absence of ovulation (anovulatory cycle) This is indicated by persistence of the proliferative phase and absence of secretory changes up to the time of menstruation Glandular cystic hyperplasia may also be present due to prolonged unopposed estrogenic effect ESTROGEN DEFICIENCY Complete ovarian deficiency (also seen in the meno pause)-This is indicated by a specimen of the castrate or ·ENDOMETRIAL BIOPSY 93 postmenstrual type occurring just before menstruation It is characterized by little or scant proliferative change and absence of secretory changes It should be remembered that the above findings change gradually from one to the other and that there may be transitional pictures and mixed types between those absolutely characteristic Also endometrial biopsy does not help to distinguish between primary ovarian failure (fe male eunuchoidism ovanan aplasia) and that resulting secondarily from changes in function in other glands as with the severe hypopituitarism of Simmonds disease AMENORRHEA Biopsies can be taken at any time and if serial studies are made at weekly or biweekly intervals there will be ev dence of any cycle that is occurring including ovulation without bleeding HYPERESTRINISM This is seen with granulosa theca cell tumors of the ovary and with large doses of estrogens A marhed prolifera tive picture is seen and occasionally endometrial cystic hyperplasia is present-(the Suiss cheese endometrium of Novak) Decidua like changes have been reported with granulosa cell tumors EXCESS OF PROGESTERONE This also may be present with granulosa theca cell tu mors which have undergone luteinization-occasionally to the degree that they can be called luteomas It will be reflected in the biopsy specimen by varying degrees of secre tory changes usually in a hyperplastic type of endome triumn ·92 LABORATORY AIDS IN ENDOCRINC DIAGNOSIS secretions and leucocytes and scattered hemorrhages are prominent With normal function an experienced observer can usually tell within a few days the time of the cycle when tlie biopsy was taken Honeer in pathological conditions it may be extremely important to know exactly the timing in the cycle when the specimen uas obtained Therefore the dates of the biopsy and the dates of two or three pre ceding menstrual periods should always be noted for cor relation and to aid in interpretation Tor diagnostic purposes the biopsy is best taken between the twenty first day of the cycle and the day of onset of bleeding With normal estrogenic and progestational effects the endometrium will show good development and secretory activity This is also the best evidence that can be secured that ovulation has taken place-important knowledge in sterility studies PROGESTERONE DEFICIENCY Moderate degree is indicated by secretory changes less than normal in the specimen However quantitation is difficult and the possibility of a refractory endometrium must be considered Progesterone lach and absence of ovulation (anovulatory cycle) This is indicated by persistence of the proliferatie phase and absence of secretory changes up to the time of menstruation Glandular cystic hyperplasia may also be present due to prolonged unopposed estrogenic effect ESTROGEN DCFICIENCY Complete ovarian deficiency (also seen in the meno pause) - This is indicated by a specimen of the castrate or ·ENDOVIFTRIAL BIOPSY 95 REFERENCES I BURCH J C and PHELPs D Endometrial biopsy J Clin Endocrinol 3 4,5-479 Aug 1943 ? HILLJAMS С PHELPS D and BURCH JC Observations on the effect of hypothyroidism on ovarian function in the guinea pig Endo crinology 9 373 38. Sept. 1H1 3 H4MBLEX EC PULLEN RL and CL YLER YK Clinical studies of thyro gonadal correlates I Basal metabol c rates and endometrial responses ) Clin Endocrinol 1 593 527 June 1941 4 GOLDSVITR R STURCIS S STANBURY JB and LERMAN J Ovanan function in thyrotoxicosis and hypothyroidism J Clin Endocnnol 11 160 July 1931 5 TURCH J Cand PRELPs D Endometrial Biopsy-Progress in Gyne cology (Ed Megs and Sturgis) New York Grune and Stratton 1916 Pp 77 80 ·91 LABORATORY AIDS | ENDOCRINE DIAGNOSIS FOLLOWING ADVINISTRATION OF ANDROGENS OR ANDROGENIC TUMORS (Including adrenal cortical tumors and hyperplasia and arrhenoblastoma) Varying degrees of atrophy of the endometriunt are seen With lugh doses of andro,ens it may be reduced to a thickness of only one layer of cells Ovulation will usually be suppressed so that no secretory changes are seen HYPOTHYROIDISM This condition is often characterized clinically by ex cessive uterine bleeding in animal studies Williams Phelps and Burch have demonstrated that hypothyroidism can induce glandular cystic hyperplasia Himblen Pullen and Cuylers after investigations in humans concluded that the change in hypothyroidism svas a quantitative slowing down of ovarian activity rather than a qualitative effect and that such patients bled more frequently from a pro gestational endometrium Goldsmith Sturgis et alhave recently studied this problem and report bleeding more frequently from proliferatite endometrium but also occa sionally from that showing both the early and late secre tory phase All abnormalities reverted to normal following adequate treatment with thyroid substance OTHER CONDITIONS Endometrial concer This can be diagnosed by biopsy but the incidence of positre diagnosis has been estimated only at 75%Therefore the greater certaincy of a dilata tion and curettage with examination of the complete endometrium is preferable Endometrial tuberculosis This riso is occasionally on countered in this type of biopsy Typical histopathologıcı} findings of tuberculosis are seen in the specimen ·SEVEN EXAMINATION 97 temperature until delaered to the laboratory preferably within two hours The laboratory should be apprised of the time interval when the specimen is deluered Cleanlı ness and dryness of the bottle are important as water or other material present may radically affect the sperm I auients usually cooperate in this manner of obtaining the specimen if the reason is explained but should they demur or deem it offensive it can be obtained by coitus interruptus This may be satisfactory but occasionally the first few drops are lost and these contain a high per centage of the spermatozoa Objections on religious grounds occasionally preclude either of these methods in which case it becomes necessary to obtain semen from the Vagina as soon as possible after intercourse This method is not satisfactory as dilution with the normal acid vaginal secretion can result in a completely distorted picture It may be more satisfactory to perform a testicular biopsy Collection in a condom or sheath is usually not satisfactory as it is difficult to retrieve the full specimen and also the powder present may have a toxic effect upon the spermatozoa EXAMINATION OF THE SPECIMEN1 1 Gross Examination a Tolume Normally this is 25 to 5 cc with an average of 4 ccDiminished volume occurs in primary or sec ondary male hypogonadism and when this is severe it may be impossible to obtain a specimen McCullagh and Schaffenburg consider this a fairly good index of androgenic function sah a volume under 2 cc indicating a deficiency b Turbidity The semen is usually opaque especially when sperm concentration is normal If more transparent than normal decreased sperm concentration is usually present c Tiscosil) Normally the semen is moderately suscid though by the time it arrives at the laboratory it is usually an ·Chapter 1 A SEMEN EXAMINATION NALYSIS of the seminal fud is of prime importance in evaluating the spermatogenic function of the testes It also verifies patency of the tubular apparatus nhich conducts spermatozoa to the prostate and urethra If the seminal fluid is normal in amount and content it is safe to assume that both the spermatogenic and secretory functions of at least one tests are normal The examina tion is of particular value in the study of male infertility but may reveil typical changes in primary or secondary Irypogonadism Some inferuile men may have decreased or impaired spermatogenic function with normal secretory function so that there are no outliard manifestations of en docrine diseise An exception is the hiinefelter syndrome which is characterized by other endocrine changes especial ly gynecomastia Examination of the semen is relatnely simple but of course requires complete cooperation of the patient It should always be done before testicular biopsy is performed as it may eliminate the necessity for this more radical pro cedure Method of collection The patient is advised not to hare intercourse for at least five days before and not to collect a specimen during or shortly after a febrile illness It is recommended that it be obtained by masturbation and collected in a clean dry wide mouthed bottle Foi lowing collection it should be lept at moderate room 96 ·SEMEN EXAMINATION 99 may vary considerably in patients with oligospermia and it is advisable to examine more than one specimen 4 Types of spermatozoa and incidence of pathological forms For this portion of the examination a drop of semen is placed on a microscope slide and spread with another slide as in mahing a blood smear The smear is fixed with heat and then covered with 1% solution of chloramine for several minutes (This removes mucus and overlying spermatozoa so should not be used if very few are present) The slide is then washed with water and 95% alcohol and dried by blotting with filter paper It is then stained for two to five minutes using Ziehl Neelsen s carbol fuchsin tro parts concentrated alcoholic solution of eosin one part and 95% alcohol one part Following this it is washed with water and counterstained with Loefflers methylene blue for a few seconds It is then washed dried and examined under the oil immersion lens The heads stain a purplish color while the tails and middle portions are red Usually 300 spermatozoa are counted noting the following charac teristics for classification 1 Normal spermato-oa-usually over 70% of the total FIGURE 16 Normal spermatozoa showing head with full oval shape (After R S Hotchkiss Infertility in Alen American Lecture Series No 53 Ed Willard O Thompson MD Springfield Ill Charles C Thomas Publisher 1952) ·98 LABORATORY AIDS IN ENDOCRINE DIAGNOSIS easy flowing liquid If watery a low sperm count may be associated wlule a high riscosity may impair sperm mobility d Chemical reaction It is always alkalıne and nitrazine paper will usually demonstrate a pH of 75 to 8 2 Motility This should not be impaired within six hours if kept at moderate room temperature It can be observed in a drop of semen placed on a slide under a cover slip and examined with low power of the microscope A satisfactory part of the field toward the end of the cover slip is then brought under high power and 200 cells counted noting percentage of absolute nonmotility This is occasionally repeated at one to four eight and 24 hours Normally 70 to 80% of the sperm are motile for six hours 3 Count of spermatozoa This is accomplished by dilution and then using a count ing chamber as in counting blood cells A 1 to 20 dilution is usually satisfactory and is prepared by measuring 05 cc in a pipette and adding 95 cc of diluting fluid (saturated sodium carbonate and 1% phenol) After mixing the sperm suspension is transferred to a counting chamber using a dropper pipette Five blocks of 16 squares are counted as in a red blood cell count and the number of spermatozoa per cc is calculated by adding 6 ciphers to the total number counted If the count is low the mixture may be shahen with 1 drop of HCl and the entire RBC held (25 squares-1 square m ) counted and 4 zeros added Normally 90 000 000 to 200 000 000 per cc are found Counts of 20 to 60 million indicate relative infer tility although occasionally pregnancies do occur with counts of from 2 to 15 million per cc The sperm count ·SEVEN EXAMINATION 99 may vary considerably in patients with oligospermia and it is advisable to examine inore than one specimen 4 Types of spermatozoa and incidence of pathological forms For this portion of the examination a drop of semen is placed on a microscope slide and spread with another slide as in making a blood smear The smear is fixed with heat and then covered with 1% solution of chloramine for several minutes (This removes mucus and overlying spermatozoa so should not be used if very few are present ) The slide is then washed with water and 95% alcohol and dried by blotting with filter paper It is then stained for two to five minutes using Zienl Veelsen s carbol fuchsin two parts concentrated alcoholic solution of cosin one part and 95% alcoliol one part Following this it is washed with water and counterstained with Loefflers methylene blue for a few seconds It is then washed dried and examined under the oil immersion lens The heads stain a purplish color while the tails and middle portions are red Usually 300 spermatozoa are counted noting the following charac teristics for classification 1 Normal spermato-oa-usually over 70% of the total FIGURE 16 Vormal spermatozoa showing head with full oval shape (\fter R S Hotchk15 Infertility in Men American Lecture Series No 53 Ed I'llard Thompson VD Springfield III Charles C Thomas Publisher 1952) ·100 LABORATORY AIDS IN EVDOCRINE DIAGNOSIS These present ? head with full oval shape Some ari ations in size and shape may occur ? Abnormal nucleus-the nucleus is at the base of the head of the sperm and slould present a rounded posterior angle Varying degrees of acuteness of this angle indicate abnormality These are significant if over 10% of the number counted FIGURE 17 Abnormal spermatozoa showing duplicate forms (above) giant heads (megalospermia) and pen heads (microspermia) (belou) (After R S Hotchkiss Infertility in Alen American Lec ture Series No 53 Ed 11 tard o Thompson MD Springfield III Charles C Thomas Publisher 1932) 3 Alicrospermia--thuis is caused by deficient acrosome which forms the anterior portion of the head of the spermatozo1 They are occasionally so small as to be classified as pin heads Normally they should not be more than 15% of the number counted 4 Megalospermua-usually not over 5% Many large forms may indicate pathologic spermatogenesis 5 Abnormal acrosomia-this reters to other abnormalı ties of the head besides those noted in microspermia and should not exceed over 5% of the total count ·SEVEN EXA NATION 101 6 Miscellaneous forms-tliese refer to abnormalities in staining cytoplasmic extrusions and abnormalities of the central part of the sperm or tail They should not exceed 5% Blom' has suggested a stain which is occasionally of value in indicating viability of sperm even though they are not motile For this a small drop of semen about the the size of a pinhead is placed on a clean slide Next a drop of 5% eosin (bluish) about double the size is placed beside it and finally a drop of 10% nigrosin double the size of the eosin drop With a glass rod the semen and eosin drops are mixed and then the nigrosin drop is included After a fer seconds the mixture is spread as a thin layer with the glass rod held flat The mixing and smearing should not tale oser 1 minute The slide is then dried over a fame and examined under the oil immersion lens Dead cells will appear red having taken up the eosin stain while the live cells re main un colored For a differential enumeration 200 to 500 spermatozoa may be counted Other material found in semen a II hite blood cells--these are usually associated with infection in the urinary tract either in the prostate or the tirethra b Tissue cells-cells from the bladder urethra or other parts of the genito urinary tract are occasionally seen c Crystals-seteral types are observed and in the pres ence of germinal lypoplasia they may be abundant In any type of hypogonadism whether primary or second ary the volume and sperm concentration will be dimin ished As noted abose in severe hypogonadism it may be impossible to obtain a specimen It is of interest to recall thit in alinejelter s syndrome there may be spermatogenic failure with normal Leydig-cell function (Other charac ·102 LABORATORY AIDS IN ENDOCRINC DIAGNOSIS teristics of the syndrome include gynecomastia and in creased excretion of FSH in the urine) Local pathology within the testicle may be the cause of deficient sperm production and represents a major portion of the problem in the study of infertility in men Hamblen Pullen and Cuyler reported a study of the correlation of findings in seminal fluid with the level of basal metabolic rates done in males undergoing routine studies because of presumed sterility They found no consistent relationship indeed patients with basal meta bolic rates below minus 15% generally produced better seminal specimens than those within the normal range The estimation of fructose concentration in semen has recently been a subject of some interest as an index of androgenic activity Originally the estimation was found to be of value as an indicator of the fertility of bull semen Recently Landau and Lougheadhne studied its concen tration in man--noting that fructose apparently is secreted by the seminal vesicle and metabolized by spermatozoa They reported that in normal men the seminal fructose concentration ranged from 200 to 800 mg per 100 cc In hypogonadism the concentration was less than 135 mg per 100 cc and became elevated after the administration of androgens However patients with oligospermia or azoospermia who otherwise were normal had normal fructose concentration REFERENCES 1 WILLIAMS WV W Semen Examination and Interpretation Progress in Gynecolo,y (Ed Veigs and Sturgis) New York Grune & Stratton 1946 pp 254 261 2 FARRIS E J Evaluation of sterility problems of the male / Urol 63 748 763 April 1950 S MCCULLACHI E P and SCHAFFEVELRG C 1 Testicular androgen de ficiency and semen volume JAVA 149 1211 1215 July 26 19

·SI MEN EXAMINATIOV
103
4 KOLMER J A and BOERNER F Approred Laboratory Technic Srd Ed
New Work Appleton 1911 p 817
5 BioW E A one minute live dead sperm stain Fertil & Steril 1 1.6
17 19.0
6 HAVALEX EC PLLL RL and CUYLER K Clinical studies of
thyrow gonadal correlates II Basal metabolic rates and seminal find
ings ] Clin Endocrinol 15 8 531 June 1911
7 LANDAU R L and LOLCHEAD R Seminal fructose concentration as an
Index of androgenic activity in man J Clin Endocrinol 11 1411 1424
Dec 1951

·Chapter 11 TESTIS BIOPSY 'N THE STUDY of infertile men when the seminal fluid shows azoospermia or severe oligospermia or if no specimen of semen can be obtained a microscopic evamını tion of the testicular tissile obtained by testis biopsy is indi cated The simplicity of this procedure is surprising It was first performed experinientilly by Engle and clinically by Hotchkiss as quoted by Charny? A study of the biopsy section obtained will give an absolute ansiver as to whether or not spermatozon are being produced in proper numbers in the seminiſerous tubules of the testes The procedure produces no deleterious effects on the human testis in con trast to damaging effects on testes of other mammalian species Method This is well given by Hotchkiss After prepa ration of the genital region a small area of the scrotal skin 15 anesthetized with 1% novocaine A2 to 3 cm incision is then made through the tunica vaginalis and a pair of ocular palpebral retractors inserted 11 hile holding the testicle in apposition to the sound a small V shaped incision is made in the tunica albu,iner and a piece of tissue excised using small eye scissors Hemorrhage us ar rested and the tunica and wound closed with silk or fine plain gut sutures This is repeated on the opposite side after which a suspensory us applied and the patient allowed to return to his home The tissue is fixed in Bouin s fluid Sections are prepared for microscopic examination as with other biopsy or pathological specimens ·FIGURE 18 Normal testiculir tissue (low and high power) Testis bropsy in a man with azoospermia caused by mechanical block Spermatozoa appeared in semen after vaso epididymal anastomosis and patients wile became pregnant (Courtesy of Dr L Michelson-after ll est Surg Obst and Gynec 55 120 Feb 1917 ind Surg C, nec and Obst 87 327 March 1916)

·106
LABORATORI AIDS I\DOCRINC DIAGVOSIS
FIGURF 19 Abnormal testicular tissue Testis biopsy showing tubular
degeneration and absence of spermatogenesis Except for a few sperm
atogonia only Sertoli cells remain From a man aged 21 years with
azoospermia but normal build and sexual function Had had
severc electric shock at age of five years (Courtesy of Dr Lewis
Michelson and Dr David A Wood)
Eramination of the specimen will reveal the histologic
basis for functional impairment In general 2 classes of
tissue are found
1 Normal testicular tissue This shows normally func
tioning seminiferous tubules and adequate numbers
of spermatozoa If the semen of such a patient does
not show spermatozoa or presents severe oligosper
mia there is obviously a mechanical block between
the testicle and the point of delivery into the urethri
Minor lustologic changes are occasionally seen which
may be due to toxic nutritional or constitutional
states u lucl can be corrected
2 Defective or abnormal tissue Several varitions are
seen as follouis

·TESTIS BIOPSY
107
a Disorganized spermatogenesis with few mature
sperm
b Germinal epithelium showing arrest of develop
ment at an early stage of spermatogenesis
c Tubules with little or no germinal epithelium
FIGURE 20 Abnortnal testicular tissue Testis biopsy showing
marked tubular degeneration following mumps Interstitial tissue 15
loosely cellular and Leydig cells are not prominent From a man
aged 26 with azoospermia Had normal potency but some physical
characteristics of hypogonadism (Courtesy of Dr Lewis Michelson
and Dr David 4 Wood)
(germinal aplasia) and with no spermatozoa (see
Figures 19 and 20)
d Proliferation of a large amount of fibrous tissue
about the tubules
Changes in the interstitial or Leydig cells are also
easily recognized in the sections
Engle' has suggested the following classification which
correlates the semen analysis with testis biopsy

·108 I ABORATORY AIDS INDOCRIVE DIAGNOSIS 1 Norinn semen 2 Azoosperma i Isith demonstrable occlusion 1 norms) rubules 2 distorted spermatogenesis b without demonstrable occlusion i normil tubules 2 progressive tubular fibrosis 3 germinal pls17 4 spermatogenic arrest (to spermatocyte I) 3 Oligospermia 1 with high frequency of abnormal spermatozot incomplete maturation 2 progressive tubular fibrosis b inflammation and infection This has helped in evaluition of the fertility potential Clinical application The biopsy may aid by indicating proper therapy eg if considerable amounts of fibrosis and sclerosis are seen it is pprrent that the testicle his sus tained severe direct dimage from the etiolo,ac fictor-- seen following the orchitis of mumps or after x ray therapy Tiic frult therefore is primary in the testicle which is obuously incapable of responding to stimulation theripy so substatution therapy with androgens is indicated However if the tubules are immature and the intersti tral cells preprberal the lesion is probably secondary to rock of pituitary scumulation it is knonn from animal intestigation that the pituitary sundotropin aluch stimu Intes the follicles in the 01zry (follile stimulating lior mone-TSH) is the one which in the male restis stimulates tubular development and function This would seem to indicate treatment with prepirations of gonadotropins in these patients but unfortunately rise of those now avail able has usually been disappoinung in luman subjects ·TESTIS BIOPSY 109 Should the testicular tissue appear normal a search for the obstruction in the vas deferens is indicated with the possibility of plastic surgical relief (Figure 18) If treatment is instituted with the intent of improving the testicle repeat testis biopsies can be performed at intervals in order to follow progress from therapy Another classification of testicular deficiency has been suggested by Howard et al* which correlates the picture of testis biopsy with clinical and other laboratory findings From their interesting and thorough study they concluded that the following was satisfactory a Patients with low FSH excretion I cases without pituitary stimulation with prepuberal appearance of testes 2 cases with decreased pituitary function after a period of normal function--testes characterized by hyposperma togenesis lipoid accumulation in the Sertoli cells thickening of the tubular tunica propria deposition of peritubular collagen and elastic tissue and absence of normal Leydig cells b Patients with normal FSH excretion 1 cases with endocrine disturbances-changes thought due to lack of luteinizing (interstitial cell stimulating) hormone - acroinegaly 2 cases with spermatogenic failure only and without other evidence of endocrine disease--these include arrest of maturation hypospermatogenesis and oligospermia c Patients with high FSH excretion-this group includes patients with absent germ cells sclerosing cubular de generation mumps orchitis male climacteric and ab sence of testes Nelson and Hellers have also made careful s udies in cluding histochemical of the testicular lipides and have pointed out that men with primary lay pogonadism with ·110 LABORATORY AIDS IN ENDOCRINC DIAG\OSIS few exceptions hue high levels of excretion of urinary gonadotropins and therefore should not be treated with gondotropic substances The testis biopsy is occasionally of interest and may proude cruciul esidence in mahing the diagnosis of the Alinefelter syndrome Sections of the testis shon varying FICURE 21 Testis biopsy in hlinefelter s Syndrome Note tubular degeneration and prominence of interstitial tissue Patient aged 24 had gynecomastia normal sized genitalia and increased FSH ex cretion However sexual function was poor and improved with testosterone therapy (Courtesy of Dr Frank Hinman jr and Dr Gilbert Gordan) degrees of tubular lesions with partial to complete hyalını zation and loss of spermatogenesis The interstitial or Leydig cells are nu and prominent and are con sidered to indicate that the production of male hormone in these patients is normal Honeler occasional cases do show evidence of some degree of hormonal deficiency Recently Sobel Sniffen and Talbot have reported that testicular biopsy may be of value in boys with sexual precocity in differentiating simple premature masculinity ·TFSTIS BIOTS 111 from precocity caused by overactivity of the adrenal cortex In the patients with premature masculınty all testicular elements are stimulated-Leydig cells are seen and varying degrees of spermatogenesis are present In precocity of adrenal cortical origin Leydig cells are not activated and spermatogenesis is not seen However some tubular de Selopment does occur REFERENCES 1 CHARVY C * Tescicular biopsy its value in male sterility JAMA 115 3499 1432 Oct 26 1910 2 HOCHKISS RS Fertility in Men--A Clinical Study of the Causes Diagnos s and Treatment 3rd ed Thiladelphia Lippincott 19H p 171 3 EXCLE E T The testis biopsy in infertility I Urol 57 489 198 Vay 1917 + HOWARD EI SWIFFEN RC SANNONS FA and ALDRIGUT F Tes ticular deficiency a clinical and pathologic study J Clin Endocri ol 10 121 186 Feb 19,0 5 \ELSON 11 and HetR CG Te Testes in Taman Hypogonad 15m I ecent Porress in Hormone research (Proc Laurcntian Hor mone Cont) (Ed Gregory Pincus) Vew York Acad Press 1948 9 197 6 SOBEL EH SNIFFEN RC and TALBOT N B The testis. V-Use of testicular biopsies in the differential diagnosis of precocious puberts Pediatrics 8 01 16 Nov 19,1 ·Chapler VII I ENDOCRINE DISEASE INDEX 'N CLINICAL practice it is helpful to know what laboratory results may be expected in certain diseases and to be familiar with the tests which are of particular aid in es tablishing the diagnosis Therefore in this final chapter the material presented has been rearranged with the tests grouped under the lieedings of the various endocrinop thics The order of listing will be anatomical with the disease conditions assigned to the various glands primarily affected-starting with the pituitary Disorders caused by hyperfunction will be listed first followed by those as cribed to hypofunction A fes conditions not clearly re lated to one gland or even not primarily endocrine but which shon important findings in the laboratory tests discussed will be listed at the end The laboratory tests will be divided into the following categories 1 Those most important in the disease whuch probably should be performed in every case 2 Those of interest in the disease thich are occasion ally helpful in diagnosis or in following the effects of treatment § Abnormal laboratory findings of incidental interest in the disease The more important findings will be shown in heaver type ·ENDOCRINE DISEASE INDEX 113 PITUITARY DISEASES Acromegaly (1) MOST IMPORTANT TESTS ray of skull usually shows enlarged deformed sella turcica ballooning of frontal sinuses enlargement of maxillary sinuses jong mandible causing pro, nathism change in bite spacing of teeth thickening of calvarium and increased prominence of external occipical protuber ance (Chapter VI P 55) Glucose tolerance test may show diabetic tendency or actual diabetes mellatus (Chapter III p 15) Basal metabolic rate moderately elevated in active phase moderately lou in burned out phase (Chapter 1 p 43) Urinary 17 hetosteroids excretion elevated in active phase may be low in burned out phase (Chapter VII p 69) ( TESTS OF INTEREST-OCCASIONALLY HELPFUL Serum phosphorus may be increa ed with active phase (Chapter III P ray of hands may show lateral tufling of terminal phalanges-occa $ionally an early diagnostic sign (also occurs in toes but change is less striking) (Chapter VI p 63) Dental X rays may show spacing of teeth and change in bite (Chapter VI p 55) Xray of chest (antero posterior and lateral) may show enlarged heart (splanchnome, aly) prominence of sternum and enlarged vertebrae with kyphosis (Chapter VI p 51) Ur nalyss may show glycosuria or occasionally evidence of diabetes insip dus such as low specif c gravity and increased 24 hour volume (Chapter II P ) (3) LABORATORY FINDINGS OF INCIDENTAL NTEREST Complete blood count may show eosinophilia and relative lymphocytosis (Chapter 11 p 6) Serum sod um occasionally elevated (Chapter III p 28) Serum chlorides occasionally elevated (Chapter III p 9) Serum potass um level occas onally depres ed (Chapter III p 29) Gigantism (physiopathological process similar to that in acromegaly except that disease starts during childhood) (1) MOST IMPORTANT TESTS \ray of skull usually shons enlarged or deformed sella turcica acro- ·114 LABORATORY AIDS IN F\DOCRINE DIAGNOSIS megalıc changes alsa may occur after epiphyseal closure (Chapter VI r 53) lucose tolerance test may shoi calzetic tendeng or diabetes (Chapter NII p 13) Basal metabolic rate moderately cleated wuh autre phase moderately low in burned out phase (Chapter ! p43) Urinary 17 hetosteroids excretion clevated in acne phase may be lou in burned out phase (Chapter 1 p 69) (?) TESTS OF INTEREST-OCCASIONALLY HILLI FUL Serum phosphorus may be increased with actre phase (Chapter II P 23) Bone age may be slightly retarded especially with secondary eunuch did im (Chapter II P 3) Urinalysis inay shox glycosuria or occasionally evidence of dubetes insipidus such as low specific gravity and increased 21 hour volume (Chapter II ) Dental X rays may show spacing of teeth (Chapter 17 p 5») Anterior Prtutan Insufficiency-Adult (of varying degrees-findings typical of extreme insuficiency (Simmonds disease) will be indicated) (1) VOST IMPORTANT TESTS Yray of skull mas show enlarge or deformed sella turaca if tumor Is cause of the disease (Chapter II p 3) Basal metabolic rate los may be very low fie minus 40 10 Jor) 18 Simmonds disease (Chapter 1 p 43) Urinary 17 hetosteroids usually low (Chapter VII p 69) Unnary gonadotropins (FSH) usually lou-important in diagnosis of hypogonadotropic eunuchordis? (Chapter Vil p 7) Clucose tolerance test low fide curls with tendency to hypoglycemia latter may be marked in Simmonds disease (Chapter III P15) Insuun tolerance test shor s sensitivity to insulin may be masked to the point of danger in Simmonds disease--if this condition is suspected tise le to recommended dose of insulin and have glucose ready for parenteral entz cuin if necessary dunng course of test (Chapter Il P II) t") TESTS OF IYTER EST-OCCASIO VALLY HELPFUL Serum cholesterol normal or high in pituitars my redema type-normal Iexcls 15 a patient with my xedematous appearance should suggest ·E\DOCRIVE DISEASE INDEX 115 possibility of primary pituitary etiology (Chapter III p 1.) Eosinophile count normal or low (Chapter III p 10) Corticotropin (ACTH) test eosinophiles usually drop more than 50 (Chapter III p II) Serum sod um occasionally lok with secondary diminution of adrenal cortical function (Chapter III p 28) Serum chlorides occasionally box with secondary diminution of adrenal cortical function (Chapter III p 99) Serum potassium occasionally h gh with secondary diminution of adrenal cortical function (Chapter III P 29) (3) LABORATORY FTVDINGS OF NCIDCITAL INTEREST Serum carot n occasionally increased caus ng carotinemia (Chapter III p 23) Electrocardiogram shows bradycardia and small complexes changes more marked in Simmonds disease (Chapter 1 p 47) Chest Xray may show small heart (splanchnomica) especially in Simmonds disea e (Chapter II pu) Complete blood count may show secondary anemia and cosinophil a more frequent in Simmonds disease (Clapter II p 6) Gastric analysis may show hyp acidity or achlorhydria especially in Smmonds discase (Chapter 1 p 46) Urinalys s occa onally shows hgh 21 hour volume and low specific grav ty with diabetes ins p dus (Chapter II p 7) Serum calcium may be moderately low (Chapter III p 24) Endometrial b opsy shows ovarian failure with little proliferative and no secretory change most mashed in severe insufficiency (Chapter IX P 93) Semen examination volume small sperm ab ent or in low concentra tron fructose concentration low spe imen may be unobtainable in severe hypopituitarism (Chapter Y p 96) Hypophyseal Infanulism (physiopathological process similar to that in adult anterior pituitary insufficiency except that disease starts in childhood) (1) VOST IMPORTANT TESTS ray of skull may show enlarg d or deformed sella turcica if tomor 15 cause of the discase (Chapter VI p 53) Basal metabol c rate moderately low (Chapter 1 p 43) Urinary 17 ketostero ds low excretion for age (Chapter VII P 69) Ur nary gonadotrop (FSH) lon or absent (Chapter VII P 7) Bonc age moderately retarded in childhood (Chapter VI P 53) ·116 LABORATORI AIDS I EVDOCRINE DIAGNOSIS (2) TESTS OF INTEREST-OCCASIO ILLY HELPFUL Glucose tolerance test for flat cure with tendency to hypoglycemia (Chapter III p 15) Plasma cholesterol slightly low (Chapter III p 17) (3) LABORATORI FINDINGS OF INCIDENTAL INTEREST Blood counts may shon relatesc lymphocytosis (Chapter II p 6) Urinalysis occasionally shows high 21 hour solume and low specific gravity if diabetes insipidus is present (Chapter p 7) Dental Y rays may shoi cronding of teeth in small arch (Chapter 11 P 55) Froehlich Synıdrome (diagnosis considered only in the classical sense when condition is caused by a pituitary tumor as in Froehlich s original case) (1) VOST II/PORTIVT TESTS ray of shull shows enlarged or deformed sella turuca (Chapter VI p 53) Unnalysis occasionally shows high 21 hour olume and low specific gravity if diabetes in spidus is present (Chapter II p ) Other findings characteristic of hypopituitarism may be present Diabetes Insipidus (1) MOST IMPORTINT TESTS Urinalysis increa ed 21 hour volume (3 to 6 hiters) lor specific grasity (1 001 to 1 003) (Chapter II p 7) Xray of skull selja turcica enlarged or deformed if tumor is causanne factor (Chapter II p 53) (2) TESTS OF NTEREST-OCCASIOVALLI HELPFUL Blood serology posttre occasional case caused by central nervous system syphilis (Chapter II p 9 Urine concentration test usually does not raise urine conceniration to above 100. (Chapter II P $1) Carter Robbins urinary excretion test helps to differentiate the disease from psichogenic polidipsia (Chapter II p 30) Glucose tolerance test normal cure distinguishes the disea c from diabetes mellitus (Chapter Ili p » ·ENDOCRINE DISEASE INDEX 117 THYROID DISEASES Hyperthyroidism (includes that resulting from hyperplasia or toxic adenoma and exophthalmic gorter) (1) MOST IMPORTANT TESTS Basal metabol c rate le el increased (Chapter 1 p 43) Blood protein bound 10dine lesel increased (Chapter III p 21) Plasma cholesterol low or low normal (Chapter III p 17) (TESTS OF INTEREST-OCCASIONALLY HELPFUL Uptake of radioactive iodine high curve with rapid rise to over 307 in five hours (Chapter 1 p 45) Glucose tolerance test may show sharp rise to high level and then rapid fall (Chapter III p 15) Urinalysis may show glycosuria (Chapter II p 7) Electrocard ogram shows rapid rate with tall R and T waves-occasion ally auricular fibrillation or futter (wh ch may be paroxysmal)-late in disease signs of left ventricular hypertrophy may be present (Chapter 1 p 47) Chest Xray may shnu evidence of substernal goiter or deviation of the trachea-late in disease may show cardiac hypertrophy or dilatation (Chapter VI P 57) (3) LABORATORY FINDINGS OF INCIDENTAL INTEREST Blood counts may show leucopenia with relative lymphocytosis when disease is severe eosinoptilia and secondary anemta may be present (Chapter II P 6) Serum sod um occasionally low (Chapter III p "8) Creatine in urine occasionally present in adult men (Chapter IV p 40) Hypothyroidism (of varying degrees--in adults and children changes character istic of extreme insufficiency ( medema) will be indicated) (1) MOST IMPORTANT TESTS Basal metabolic rate lesel decreased (Chapter 1 p 43) Blood proten bound jodine low or low normal (Chapter III p 21) Pla ma cholesterol leret increased (f normal le el 1 found consider diagnos s of pitu tary myxedema) (Chapter III p 17) CH est ray may reseal large flabby atonic myxedema heart or show evidence of pericard al flv d or pleural fluid (Chapter VI P 57) ·118 LABORATORI AIDS IN ENDOCRINE DIAGNOSIS Blood counts secondary anemia frequent primary anemia occasionally seen relative lymphocytosis (Chapter II p 6) Urinalysis albumin and signs of chronic infection resulting from bladder atony and residual urine (Chapter 11 p 7) Bone age (important in childhood) shows marked retardation in child hood my redema (Chapter VI P 53) (2) TESTS OF INTEREST-OCCASIO ILLI HELPFUL Uptake of radioactive sodine lou uptake cure (Chapter 1 p 45) Serum carotin usually increased so that carolinemia is present in un treated mykedema (Chapter III p 9) Glucose tolerance test curve tends to be low and flat (Chapter III p 15) Electrocardiogram shows bradycardia prolonged I'R interal low voltage changes in ST segments and depressed or inverted T waveschanges most marked in definite my redema and are reversible with treatment (Chapter 1 p 41) Urinary 17 hetosteroids excretion usually lou (Chapter VI P 69) Dental rays may show retarded dentition in children (Chapter VI P55) Gastrointestinal x rays demonstrate atony and sluggish function of bowel (Chapter VI p 9) Pyelograms-(intrasenous or retrograde) may demonstrate atony of bladder and residual urine (Chapter VI P 9) (3) LABORATORI FINDINGS OF INCIDENTAL INTEREST Creatine in urine may be absent in childhood type of my xedema (Chap ter IV p 40) Gastric analysis acidity usually low with occasional achlorhydria (Chap ter 1 p 46) Serum sodium occasionally slightly high (Chapter III p "8) Endometrial biopsy may show excessive bleeding from proliferatic endometrium-also occasionally from secretory endometrium (Chapter IX P91) PARATHYROID DISEASCS Hyperparathyroidism (1) MOST IMPORTANT TESTS Serum calcium level increased (Chapter III p 21) Serum phosphorus lesel depres ed (Chapter III p) Serun alkaline phosphatase les cl increased with bone insolicmentlow na bonc involvement (Chapter III p 26) Urinc Sulkowich strongly positise (Chapter 11 p 38) ·ENDOERITE DISEASE INDET 119 Urmalysis calcun casts may be found late in disease renal stones usually present causing albumuried blood cells white blood cells and casts (Chapter II p ) ray bone studies may plus osteoporosis or the typical kidely dis &nbuted subperiosteal assis (osteitis fibrosa stica of von Reckling hausen) (Chapter VI pp 6163) ( ) TESTS OF NTEREST-OCCASIOVALLI NELPFUL Fyelograms (intravenous or retrograde) in Saie cases may demonstrale nephrocalcinosis tenal Isachisis or prelonephrisis (Chapter 59) Dental Xrays show di appearance of lanunta dura around roots of teeth (Chapter VI P 53) Blood non protein nitrogen may be elevated in terminal cases (Chap ter 111 P 39) (3) LABORATORI FINDINGS OF INCIDENTAL INTEREST Blood counts occassonally show secondary anemia and cosin alia (Chapter II p 6) Chest ray cardiac enlargement may be found in late cases (Chapter + P đỏ) Hypoparathyroidism (parathyroid tetany) (1) NIOST IMPORTANT TESTS Serum caictum fesel depressed (Chapter 11 p 24) Serum phosphorus level increased (Chapter III p"> Urine Suthowitch results negalise or weally positie (Chapter Il p 38) ( ) TESES OF INTEREST - OCCASIOYALLI HELPFUL Elisworth Howard test shons mcrease in urinary phosphorus (important in distcren tauon from pseudotypoparathyroidism) (Chapter 1 P 405 Dental rayu may show enamel defects and blunting of dental rooms (Chapter VIP »» (3) LABORATORY FINGS OF INCIDENTAL INTEREST Plasma CO combining poner occasionally slightly increased (Chapter 311 p $ ) Basal metabol c rate occasionally moderately cleared (Chapter 1 p 43) Blond counts may show pohothenua and relanie hmphocy to us (Chaga ter II P 6) ·120 LIBORATORI AIDS I C\DOCRINE DIAGNOSIS Pscudolypoparathyroidism (Seibright Bintim syndrome) (1) MOST IMI ORTIVT TEST Ellsworth Itoward test shows constant excretion of phosphorus (Chap ter II p 40) DISEASES OF PANCREATIC ISLETS Hyperinsulinism (1) MOST IMPORTINT TEST Glucose tolerance fest shows low fasting leic and lou cune with ten dency to hypoglycemic dip-tests should be run from 5 to 6 hours and extra blood specimen taken if hypoglycemic symptoms occur (Chapter III r 13) (°) TEST OF INTEREST-OCCASIOVALLY FIELPFUL Insulin tolerance test shows sensitivity to insulin with low blood sugar levels-may induce hypoglicemic attack and parenteral glucose should be ready for injection if necessary (Chapter 11 p 17) (3) LABOR (TOTI FINDINGS OF INCIDENTAL INTEREST Plasma cholesterol may be slightly low (Chapter III p 17) Serum phosphorus mav be slightly low (Chapter III p 21) Diabetes Mellitus (including acidosis) (1) MOST IMPORT INT TESTS Urmalysts shows sugar and high specific gravity in acido is acctone and diacetic acid are present with himmelsticl 11 on sindrome albumin and casts are found (Chapter II p 7) Glucose tolerance fest shows high curve with slow fall (Chapter 111 p 10) Plasma co combining power lw in acidosis (Chapter 17 p 39 (2) TESTS OF INTEREST-OCCASIOILLY HELPFUL Plasma cholesterol tends to be high especially in acidosis (Chapter 111 P 17) Blood non protein nitrogen may be elevatel in acidosis (Chapter III P32) Electrocardiogram may show signs of low sertim pocassium as a complı cation of acidosis (Chapter 17) Berger test 100 mg corticotropin (ICTH) before gluco e rolcrance test may indicace potential diabetes mellitus (Chapter III p 16) ·EVDOCRINF DISEASE INDEX 121 Serum phosphorus may be slightly high and does not fall normally during intravenous glucose tolerance test (Chapter Ill p 2.) (3) LABORATORI FINDINGS OF INCIDENTAL INTEREST Urine creatıne occasionally present in adult men (Chapter II p 40) Unnary 17 hetostero ds excretion may be slightly low (Chapter VII p 69) Blood counts may show polycythemia and leucocytosis in acidosis (Chap ter II p 6) DISEASES OF THE ADREN ILS Cushings Syndrome (hyperadrenocorticism) (1) MOST IMPORTANT TESTS Glucose tolerance test shows tendency to diabetic type of curve (Chap ter III P15) Urinalysis may show glycosuria in hypertensive phase may show albumin casts and fixed specific gravity (Chapter II p 7) Blood counts show character tic polycytheroia (Chapter II P 6) Urinary excretion of Iloxysteroids or glycogen c cortico ds or Com pound I & F steroids usually increased (Chapter VII P 66) Y rays of bones may show osteoporosis including collapse of vertebrae (Chapter VI P 61) () TESTS OF INTEREST-OCCASIONALLY HELPFUL Urinary 17 hetostero ds may be slightly elevated higher if condition is result of adrenal cortical tumor (Chapter VII p 69) Abdominal rays includ ng pyeloorams (either intravenous or retrograde) Causati e adrenal tumor may cast soft tissue shadow or can e down ard di placement of hidney or deform ty of upper calyx (Chap ter 1 p 59) Extraperitoneal pneumography (retroperitoneal gas) may demonstrate causative tu nor by contrast (Chapter VI P 61) Serum sodium may be elevated (Chapter Ifl p 28) Serum chloride may be elevated (Chapter III p 23) Serum potass um may be lowered (Chapter III p 29) ray of chest may show enlargement of the heart late in course of di ease with lateral view demonstrating possible collapse of vertebrae from osteoporosis (Chapter VI p 57) Electrocard ogram may show s gns of left sentncular hypertrophy and myocardial damage late in course of disease (Chapter V P47) Blood non protein nitrogen may be elevated in terminal phase of disease (Chapter III p ) ·122 TABORATORI AIDS IN INDOCRINT DIAGNOSIS (3) LABORATORI FINDINGS OF INCIDENTIL INTI REST Basal metabolic rate may be moderately clevated (Chapter 1 p 43) Plasma cholesterol mas be slightly high (Chapter III p 11) Adrenal Cortical Tumor (occasional patients with Cuslun, s syndrome are in this cate fory-chuldhood syndromes (adrenogenital) includle precocious puberty in boys precocious puberty in the masculine direction in girls and very rarely gynecomastia in boys) (I) MOST IVPORTANT TESTS Urinary 17 ketosterolds excretion usually increased reaching levels of 50 to over 1000 mg per "4 hours rarely however the excretion may be normal 1f beta fraction is over 2004 with a total crcretion of over ing carcinoma is likely (Chapter 1 p (9) Abdominal rays including pyelograms (esther intravenous or retro grade) tumor may cast soft tissue shadow or cause downward unplace ment of kidney or deformity of the upper caly (Chapter VI p 59) Extraperitoneal pneumography (retroperitoneal gas) demonstrates tumor by contrast (Chapter 11 p 61) Glucose tolerance cest may show diabetic type of curic (Chapter III P15) Urinalysas mas show glycosuria (Chapter 11 p) Allen test (for dehydroisoandrosterone in unine) positive with tumormay be helpful in differentiation from adrenal cortical hyperplasia (Chapter VII P 67) Bone age advanced with pubertus praecox in childhood (Chapter 11 p53) (2) TESTS OF INTEREST-OCCASIONALLY HELPFUL Scrum sodium may be elevated (Chapter ill p -8) Scrum chloride may be elevated (Chapter III p 29) Serum potassium may be low (Chapter Hip "3) Petisenal air (or gas) insulation may help to demonstrate the tumor by contrast a relatisely dangerous procedure (Whapter 11 P ) Arteriography may demonstrace arculation to the tumor (Chapter 11 P 65) Testus biopsy in boys with sexual precocity-shows some tubular de elopment but nu spermatogenesis and no activation of Leydig cells may help to differentiate from other types of precocious puberty (Chapter VI p 110) Deptat X rays may show advanced dentition with precocious pubert) (Chapter VI p 5)) ·ENDOCRINE DISEASE DY 123 211) (5) LABORATORI FINDINGS OF INCIDENTAL NTEREST Endomensal dsopsy mas show atrophy of endometrium (similar to pic tve seen toll ning large dares <t androgers) (Chapter 1 p 91) Basal metabol c rate may be moderately elevated (Chapter 1 p 45) Plasma cholesterol may be slightly high (Chapter III Idrena) Costical Hyperplasia (Many laboratory hindmgs are similar to those found sa adrenal cortical tumor though changes are usually less marked) (3) MOST IMPORT INT TESTS Urinary 17 hetosteroidt excretor urually increased Lusual sange 30 % 100 mg per hours (Chapter 31 p 69) Xray studies of adrenal regions (ncluding Pyelograms and contrase stud es) no evulence of tumor seer (Chapter 11 PPI & GI) Allea test (lor dehydrosseandrosterone sn the urine) negatne-may be helpsul merentiation from adsena) <ulical tumor (Chapter 1 P67) Clucose tolerance test may show draberre type of cine (Chapter Ill P 15) Ut nalysis may shos glycosta (Chapucs II p 7) Bone age adı anced nah puheria praecox in childhood (Chapter VI P-3) (TESTS OF INTEREST-OCCASIOVALLI HELPFUL Test benpsy en lays with exual precocushow some tubular develop ment but no permatogenesis and no assuon of Ley fig celts may help so d fferentiate from other topes of precoct us puberty (Chapter \p 110) Dentai rays may show ads anced dension with precocious puberty (Chapres IP (3) LABORATORY FINDICS OF INCIDENTIL INTEREST Endometnat biopsy slay show aire phy of endometr m (similar to psc ture seen following large doses of androgens) (Chapter IP 91) Adrenal Meclullary Tumor (Pheochromocitoma) (1) MOST IMPORTAST TESTS Benrod oxane est u ualto cause dr pa biul pressure during periods of hypertems on Chapter 1 p 50) Reg une te te sually cau es salel la!! in blend pressure luring peris of hiperten son (Chapser ! P 03 ·124 LABORATORY AIDS IE\DOCRINF DIAG\OSTS Abdominal rays including pyelograms (exther intravenous or retrograde) tumor may cast soft lissue shadow or cause downward displace ment of kidney or deformity of the upper caly Chapter 11 p 9) Extrapentoneal pneumography (retroperitoneal gas) demonstrates tumor by contrast (Chapter 11 p 61) Caution--pressure from gas on tumor mai precipitate attack of paroxysmal hypertension (") TESTS OF NTEREST-OCCASIOVALLY HELPFUL Dibenamine test causes decrease in blood pressure if hypertension 13 present (Chapter 1 p pl) Histamine test may cause marked increase in blood pressure and pro roke a characteristic attack (increase in blood pressure may be dangerous) (Chapter 1 pol) Vecholy test (acetyl beta methylcholine) may induce hypertensive attach (inay be dangerous) (Chapter 1 p 51) Tetraethylammonium test usually induces a paroxysmal hypertensive attach may be dangerous) (Chapter 1 p 1) Cald pressor test nje a likely to produce hypertension a differential point from other types of labtle hypertension (tay be dangerous) (Chapter 1 p) (3) TESTS OF INTEREST-OCCASIOVALLI HELPFUL Basal metabolic rate may be clcuated (Chapter 1 p 43) Glucose tolerance test may show diabetic type of cone (Chapter 111 P 15) Urinalysis may show sugar and albuiain especially during attacks (Chapter il Pi) Electrocardiograin may shon tachycardia and extrasistoles during attacks (Chapter 1 p ) Addisons Disease (1) VOST INPORTANT TISTS Serum sodium level depres ed (Chapter III p "8) Serum chloride lesel depressed (Chapter III p 29) Serum potassium level increased (Chapter 11 p 29) Robinson Power hepler water excretion test demonstrates stow excretion of ater and factor be? * (Chapter 11 p 3->) Circulating cos nophile count high or high normal (Chapter III p 10, Corticotrop n (1CTH) test (4 hours and 48 hours) shows failure of the circulating eosinophiles to drer Om or more (Chapter III p 5) Urnary 17 hetosteroids excretion low may approximate 0 in women (Chapter III p 69) ·ENDOCRINE DISEASE INDEX 125 Glucose tolerance test show's low dat curve with tendency to hypoglycemia (Chapter III p 15) Insulin tolerance test may demonstrate extreme sensitivity to insulinif this condition is suspected use to 1. recommended dose of insulin and have glucose ready for parenteral injection if necessary during test (Chapter III p 10) Blood non protein nitrogen may be elevated in crisis (Chapter III P 39) (") TESTS OF INTEREST-OCCASIONALLY HELPFUL Cutler Power Wilder test shows increased excretion of urinary chlorides (hospitalization necessary-may precipitate crass) (Chapter I p 34) Chest Tray may show tuberculosis (Chapter VI P 57) Abdominal rays may show calcification of adrenal glands (Chapter VI p59) Blood counts may show se ondary anemia eosinophilia and relaave lymphocytosi (Chapter II P 6) Gastric analysis frequently shows low gastro acidity or achlorhydria (Chapter V p 46) (3) LABORATORY FINDINGS OF INCIDENTAL INTEREST Basal metabolic rate may be moderately low (Chapter 1 p 43) Plasma cholesterol may be slightly low (Chapter III p 17) Serum calcium may be moderately low (Chapter III p 21) Serum phosphorus may be slightly high in Crisis (Chapter III p 2.) Plasma CO combining power occa ionally low (Chapter III P 3%) Simple Hirsutism (1) MOST IVIPORTANT TEST Ur nary 17 hetostero d may be slightly increased-to levels of 20 to 30 mg per 1 hours Higher lesels (7 mg or above) suggest adrenal cortical tumor (Chapter VII p 69) GONADS-DISEASES AND ASSOCIATED CONDITIONS Pregnancy (1) MOST IMPORTANT TESTS Hormone excretion tests (Aschem Zondek test Freidman test South American male toad test (Gallı Vain nu) North American male frog test South African female clawed frog test and Guterman test) rosttive (Chapter VII P 01) (2) TESTS OF INTEREST-OCCASIONALLY HELPFUL Vaginal smear shans high percentage of cortification at first but after two to four weeks increased number of folded basophilic and mid ·126 LABORATORY AIDS I F\DOCRINF DIAGNOSIS zunal cells are seen with threatened of incomplete abortion red cells appear as well as leucocytes and histiocytes often in clusters or rosettes (Chapter 1111 p 80) Urinary 17 hetosteroids excretion slightly increased (Chapter VII P 69 Pregnandol excretion elevated level falls with threatened abortion (Chapter III p 78) Endocrine Orarıın Tumors (2) TESTS OF INTEREST-OCCASIOVILLI HELPELL Urinary 17 hetasteroids may be slightly increased in his perthecosis and in arrienoblasto 1 (Chapter VI P 63) Endometrial borty with granulosa checa cell tumor may show cudences of hyperestinism such as cystsc hyperplasia or decidua like changes. occasionally secretory changes are seen with arrher oblastoma atrophic endometriun may be found (Chapter 1 pp 93 & 91) Urinary pregnandiol excrecion may be increased with polystic orantes (Stern Leichthal syndrome) (Chapter 111 p 78) Cervical mucus profuse clear and thin with fohostic ouantes (Chapter VIII P 88) (3) LABORATORI FINDINGS OF INCIDENTAL INTEREST Rasal metabolic rate may be moderately clevated in arylenollastoma (Chapter 43) Glucose tolerance fest occasionally shows diabetic type of curve i arrhenoblastoma (Chapter III pi) Serum sodium and chloride may be slightly clevated in arthenoblastoma (Chapter III pp 28 & 29) Scrum postassium may be slightly low in a henol lastoma (Chapter 111 P 29) Venopause Syndrome (1) MOST IMPORTANT TEST Vaginal smear shows varying degrees of estrogen deficiency-f mild most cells from intermediate layer and communed cells go OY tinder if moderale most cel from basal laser some intermediate cells seen and percentage of complication low f serere nearly all cells from basal laser leucocytes and bacteria promment Chapter III p Bs) TESTS OF INTEREST-OCCASIOVALLY HELPFUL Urinary gonadotropías (FSH) usually elevated (Chapter VII p 7) Urinary 17 ketostero ds slightly diminished level further depressed in old abc (Chapler VII p 69) Endometrial biopsy shows lilcc r scant proliferative change and no secretory change (Chapter 18 P 3 ·ENDOCRINE DISFASE INDEX 127 V rays of bones occasionally show osteoporosis more frequent in later years (Chapter VI P 61) Ovarian Aplasia (Ovarian agenesis Turner s syndrome) (1) MOST IMPORTANT TEST Ur nary gonadotrop ns (FSH) high-(patient should not have received estrogens for one month before test is performed) (Chapter VII p 7) ( TEST OF INTEREST-OCCASIONALLY HELPFUL V rays of bones may show osteoporosis or evidences of congenital de formities (Chapter VI p 6f) Bone age only slightly retarded (Chapter VI P »3) Cancer of the Lower Female Genital Tract (1) IMPORTANT TEST Vaginal smear (Papanicolaou stain) 96% accuracy with squamous-cell carcinoma 80 accuracy with endometrial or fundal carcinoma (Chapter VIII p 81) ( TEST OF INTEREST-OCCASIONALLY HELPFUL Endometrial b op y shows carcinoma in 7507 of cases-D & C is more certain and is therefore preferable (Chapter IX p 94) Tuberculosis of Endometrium (1) IMPORTANT TEST Endometrial bopsy occasionally shows tuberculous h stopathology (Chapter IX p 9) Hypogonadism (male and female-includes eunuchoidism) (1) MOST IMPORTANT TESTS Urinary lhetoste o ds excretion diminished in males but may be slightly elevated (Chapter VII P 69) Ur n ry gonadotrop ns (FSH) excretion increased in eunuchs female ca trates and n some eunucho ds in latter may also be normal or low (Chapter VII p 7) (") TESTS OF INTEREST-OCCASIONALLI HELPFUL Bone age moderately retarded with delayed epiphyseal clo ure in child hood and early adult life (Chapter VI p.3) Basal metabolic rate may be moderately low (Chapter 5 p 43) ·128 I ABOR ITOK) AIDS I ENDOCRIVE DIAGNOSIS Urinary creatine may be present in adult males (Chapter II p 40) Serum carotin occasionally increased causing carptinemia (Chapter III P 23) Vaginal shear shows nearly ali ba al cells with low percentage of cornification leucocytes and bacteria promuticat (simular picture seen following androgen therapy) (Chapter III p 8») lodine zapor stain shows tox glycogen index (Chapter VIII p 89) Endometrial biopsy shows little or no proliferative change and no secretory change serial studies tiery one to tho wechs will show any evidence of cycle or ovulation (Chapter II p 92) Semen examination volume is diminished and sperm concentration Jou or absent fructose concentration lou (specimen may be unobtainable) Chapter \p 96) (3) LABORATORI FINDINGS OF INCIDENTAL INTEREST Serum calcium may be moderately low (Chapter III p 21) Dlood count frequently shows a relative lymphocytosis (Chapter II p ) Infertility (male and female) (1) MOST IMPORT INT TESTS Setien examunatron zolume decreased sit hypogonadi m abnormal transparency and watcry riscosity associated with low sperm con centration (Chapter \p 91) sperm count shows Oligospermia (under million per cc) or with relatie infertility 20 to 60 million per cc or azoospermia-the latter may be from local pathology in testicle (Chapter \p 98) impaired monility of sperm may be seen (Chapter P98) abnormal forms of sperm over 300 in differential count (Chapter X p 99) high percentage of non rable cells by Blom stain (Chapter ' p 101) abundant crystals with germinal hypoplasia (Chapter X p 101) Endometrial biopsy mat show en een deficiency with little or no change (Chapter 1 p 9') or progesterone deficiency (evidence of anovulatory cycle) with no secreton change (Chapter I p 9) (2) TESTS OF INTEREST-OCCASIONALLI HELPFUL Testis biopsy inay be norial of mechanical black is present in vas (I may show the following anno nalities di organized spermatogenesis arresced development of germinal epithelttim germinal aplasia tubu Jar r brosis and sclerosis or e idence of inflammation and infection (Chapter 1 p 101) Examination of cervical mucus may be thick (as opposed to thin at time of greatest fertility) (Chapter IIII p 88) ·ENDOCRINE DISCASC I\DE 129 Vale Climacteric (° TESTS OF INTEREST-OCCASIONALLY HELPFUL Urinary gonadotropins (FSH) may be elçi ated (Chapter VI P 7») Urinary 17 hetosteroids level slightly dim nished may be further de pres ed in old age (Chapter 11 p 69) Cancer of the Male Genital Organs (1) MOST IMPORTANT TESTS Setum acid phosphatase may be elevated in carct to ia of the prostate particularly if there is metastatic involvement of bone (Chapter III p 28) Chortonepithel oma of testis gives positi e pregnancy test (Ascheim Zondek Friedman male frog male toad etc) (Chapter VII p 69) Precocious Puberty (of pineal lıypothalamic thymus or idiopathic origin) (1) VOST IMPORTANT TESTS Urinary 17 hetosteroids may be increased if clin cal picture results from O er stimulation of the adrenal cortex (Chapter VII P 69) Tone age advanced over chronological age (Chapter 1 p 3) Tests bopsy in straple premature = asculmily shows stimulation of all elements--Leydig cells are seen as well as varying degrees of spenna togenesis (Chapter 1 p 110) Xray of skull may show evidence of pineal or hypophalanaic tumor (Chapter VI P 53) Chest Xray may show cs dence of thymic tumor (Chapter 1 p 57) (") TESTS OF INTEREST-OCCASIONALLY HELPFUL Urinalysis may shoo increased ?! hour volume anI low specific gravity if diabetes insipidus is present (Chapter 11 p 7) Dental X ray may show advanced dentition (Chapter VI P 55) (3) LABORATORY FINDINGS OF I\CIDCVTAL INTEREST Basal metabil c rate may be moderately clevated (Chapter 1 43) Serim sodium and chlor le may be slightly high (Chapter III PP 28 & -9) Serum potass um may be slightly low (Chapter III p 9) VISCELL INEOUS SYNDROVIES-NOT NECESSARILY E\DOCRIF hlinefelter Syndrome (L) VIQST IVIPORTANT TESTS Unnary gonadotropins (FSH) excretion clrsated (Chapter VII p 7») ·130 LABORATORY AIDS INDOCRINE DI IGNOSIS Semen cxamination shous esidence of spermatogenic failure with oligospermia or azoospermia (Chapter \p 101) (2) TESTS OF JYTEREST-OCCASIONALLY HELPFLL Testis biopsy shons partial to complete hyalinization of tubules inter stital (Leydig) cells numerous and prominent (Chapter 1 p 110) Albrights Syndrome (polyostotic fibrous dysplasia) (1) IMPORTANT TEST Yray of bones may show characteristic disseminated ostentis fibrosa custica in segmental distribution (Chapter 11 po) (-) TEST OF INTEREST Serum alkaline phosphatase may be moderately clevated (Chapter III P 26) Osteoporosis (I) MOST IMPORTANT TESTS Urine Sulkonitch test strong's positive when condition is seycrc (Chap ter IV p 38) Serum calcium phosphorus and alkaline phosphatase normal (Chap ter II PP 21 & 26) Dental rays may emphasize presence of osteoporosis by contrast (Chap ter 11 p 5-) Pagets Disease of Bone (I) VOST WIPORTANT TESTS Urine Sulhowitch strongly positive (Chapter 11 p 38) Serum alkat ne phosphatase high (Chapter III p 26) (2) TEST OF INTEREST Serum calaum occasionally slightly high (Chapter II P.1) Vitamin D Intoxication (1) MOST IMPORTANT TESTS Urine Sulkowitch test strongly positic (Chapter 11 p 38) Serum calaum elevated (Chapter III p 24)

·ENDOCRIVE DISEASE I DEX
131
Anorexia Menosa
(may be difficult to distinguish from Simmonds disease)
(I) MOST IMPORTANT TESTS
Basal metabol c rate may be very low (Chapter 1 p 43)
Urinary 17 hetosteroids normal or low (Chapter VII 69)
Urinary gonadotropins (FSII) normal or lou (Chapter VII P »)

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·This Book LABORATORI AIDS IN ENDOCRINE DIAGNOSIS BY ROBERTO F ESCAVILLA MD was set printed and bound by The Collegiate Press of Alenasha Jl isconsin The engravings were made by The Northwestern Engraving Company of Menasha Il esconsin The page trim sin 15 51/2 S1/ inches The type page is 2, X 19 picas The type face is Linotype Baskerville set point on I, point The text paper is 70 lb White Il ood bine Folding Enamel WKS ESIMBO DERSHIPS II ith THOMAS BOOKS careful attention is given to all details of manufacturing and design It is the Publisher's desire to present books that are satisfactory as to their phys ical qualities and artistic possibilities and appropriale for their particular use THOMAS BOOKS uill be true lo those laus of quality that assure a good name and good uell