Hemolytic reactions produced in dogs by transfusion of incompatible dog blood and plasma; serologic and hematologic aspects

1. Dogs injected intravenously with dog erythrocytes containing one or moreantigenic factors lacking in their own red cells developed iso-hemagglutinins andhemolysins exhibiting characteristics of immune antibodies.

2. Transfusions of incompatible whole dog blood and plasma were carried outunder controlled conditions. Pretransfusion observations were made and followedby closely spaced post-transfusion measurements of serologic and hematologicalterations.

3. The rate of destruction of incompatible donated corpuscles was determinedby tagging the cells with radioactive iron and also by employing the technique ofdifferential agglutination of erythrocytes. It was thereby shown that all of theincompatible donated cells disappeared from the recipient’s circulation withinthe first thirty to ninety minutes following transfusion. The probable mechanismsand relative importance of intra- and extravascular destruction of erythrocytes arebriefly discussed.

4. Destruction of recipient dogs’ corpuscles by donated immune plasma wasrelatively slow, and spherocytosis and increased osmotic fragility of the recipients’ cells were evident for periods as long as twenty days. These observationsare compared with those made in human beings after transfusions of plasma and ofblood from dangerous universal donors.

5. The titer of complement in the sera of recipient dogs was sharply reducedfor at least five hours after all transfusions of incompatible whole blood, but isoagglutinin titers were less regularly reduced after such transfusions.

6. Other notations of interest included estimates of the concentrations of serumbilirubin, sodium and potassium, determinations of clotting time, prothrombinconcentration, and observations on red cell morphology, intravascular erythrophagocytosis, and shifts in distribution of leukocytes and in the electrophoreticpatterns of plasma.

Note: ACKNOWLEDGMENTSIt is a pleasure to acknowledge the technical assistance of Mrs. Jane Peters, Miss Mary Jane Izzo andMiss Shirley Deshon.

     

MONOCYTIC LEUKEMIA AND TUBERCULOSIS

A case of monocytic leukemia associated with active tuberculosis of pleura,pericardium, and lymph nodes is reported.

The criteria for the diagnosis of monocytic leukemia and the factors excludinga leukemoid reaction are presented.

The rarity of monocytic leukemia in contrast with the frequency of tuberculousinfections and the rarity of active tuberculosis in the reported cases of monocyticleukemia indicate that the association of the two diseases is probably coincidental.

Note: Grateful acknowledgment is due to Miss Anne Shiras and Mortimer Cohen, M.D., for the photo-micrographs.

     

Hypoplastic anemia due to atabrine

Seven patients with severe hypoplastic anemia were studied at an army TropicalDisease Center. Four of the 7 patients had concomitant dermatitis. The relationship of the prolonged administration of atabrine to the anemia and dermatitis ispresented. A hematologic remission could not be induced by specific therapeuticmeasures. Four of the 7 cases recovered spontaneously.

Note: ACKNOWLEDGMENTThe authors are indebted to Miss Mary L. Boyd for her painstaking supervision of the hematologicstudies.

     

STUDIES ON THE PHYSIOLOGY OF THE WHITE BLOOD CELL: THE GLYCOGEN CONTENT OF LEUKOCYTES IN LEUKEMIA AND POLYCYTHEMIA

The technic of determining glycogen in isolated white blood cells was appliedto the study of the different types of leukemia and of polycythemia, in order toobtain information on the physiology of the white blood cell. From this study itis concluded that the granulated leukocyte is the only carrier of glycogen in wholeblood. The "reducing substances" in lymphocytes and blast cells are not consideredas true glycogen.

The glycogen content of wet white blood cells in the rabbit amounts to about1 per cent. In the human being a range of from 0.17 to 0.67 per cent was calculated.In disease higher percentages occur, in polycythemia up to 1.64 per cent and inglycogen storage disease up to 3.05 per cent.

The glycogen concentration of normal white blood cells is within the same rangeas that of the striated muscle.

Note: I acknowledge with gratitude my indebtedness to Dr. William Dameshek for giving me the opportunity of analyzing the blood of some of the patients studied. Miss M. H. Campbell, Miss H. A. Clark,and Miss L. M. Garofalo have aided in carrying out many of the blood counts.

     

A method for obtaining living leukocytes from human peripheral blood by acceleration of erythrocyte sedimentation

A simple method for obtaining living leukocytes from human peripheral blood ispresented. It consists of the addition of fibrinogen to heparinized blood. Essentiallyall the leukocytes remain in plasma suspension and essentially all the erythrocytesare sedimented out at the end of one hour.

Note: ACKNOWLEDGMENTThe authors wish to express their thanks to Miss Alma Manieri, who did all the blood counts forthis study, and to Mr. Antol Herskovitz, who prepared the photomicrographs.

     

The mechanism of petechial hemorrhage formation

1. The capillary resistance test has been studied by a special technic of capillarymicroscopy.

2. Seventeen cases of hemorrhagic diseases of differing etiology have been thusstudied.

3. The site of capillary hemorrhage has been localized to the arteriolar end ofthe capillary loop.

4. Selective damage to the arteriolar-capillary junction will explain manytypes of hemorrhagic syndromes.

Note: ACKNOWLEDGMENTI wish to thank the honorary staff of the Westminster Hospital for permission to study cases undertheir care. I am much indebted to Dr. P. Hansell and Miss F. E. McAdoo of the Westminster HospitalDepartment of Medical Photography for assistance with the illustrations.

     

FURTHER OBSERVATIONS ON THE EFFECT OF BENZENE ON A STRAIN OF MYELOID CHLOROLEUKEMIA IN MICE AND ON CHANGES PRODUCED IN THE LEUKEMIC CELLS BY THE CHEMICAL

Oral administration of 5 mg. of benzene six times weekly to 46 mice that hadbeen injected 24 hours before with the cells of the myeloid chloroleukemia 1394,considerably prolonged the survival of the animals, and in 16 of these mice itseemed to prevent the development of the disease. The spleens of the animals sotreated increased in size at a much slower rate than those of the untreated animals,and the leukemic infiltration of the organs was delayed.

Five mg. of benzene given in a similar manner to mice with advanced chloroleukemia likewise prolonged the survival time of the mice and also slowed therate of increase of the size of the spleens; 25 mg. given five times in a 6 day periodto mice with this leukemia brought about a marked reduction in the size of thespleens. Further treatments slowed the rate of enlargement of the spleens andsurvival was prolonged.

In 8 mice bearing subcutaneous tumors composed of the cells of the chloroleukemia the oral administration of 25 mg. of benzene five times in a 6 day periodfollowed by 5 mg. given six times weekly brought about the disappearance of thelocal tumors as determined by palpation, but in 6 mice the tumors recurred andkilled the animals. Microscopic examination of tumors of mice treated with benzene showed advanced degenerative changes in leukemic cells.

Note: The authors with to thank Miss Pauline Pope, Miss Mary Boon, and Miss Lucille Wolf for theirtechnical assistance.

     

Preparation of stabilized solutions of hemoglobin

It is possible to prepare self-stabilizing solutions of hemoglobin from humanerythrocytes by the use of dextrose, nicotinic acid amide and ammonia during thepreparation and in the final solutions themselves. Co⁺⁺, Mn⁺⁺ and Mg⁺⁺ ions,nile blue and hexose diphosphate contribute to the speed of stabilization of thesesolutions. Stabilization is obtained by the faculty of the solutions, presumablyby enzymic action, to convert the hemoglobin to the reduced form and to maintainit in this form. The hemoglobin solutions described are suitable for intravenousadministration.

Note: ACKNOWLEDGMENTWe wish to acknowledge our deep indebtedness to Miss Lois Priester and to Mr. Edward Smith fortheir technical assistance in this work. We wish also to express our gratitude to the Biological ProductionDivision and to the Biological Control Division of Sharp and Dohme for assistance in many phases of thework.

     

The effect of radiation on hemopoiesis; is there an indirect effect?

1. The general aim of the investigations here reported has been to obtain evidence for or against "indirect" radiation effects.

2. To this end, twenty-six successful cross circulation experiments (carotid tocarotid anastomoses) have been performed between normal cats and radiated cats.

3. Cross circulation was established in most instances at some specified timeinterval after the radiation of one partner. All intervals up to eighty-two hoursafter radiation of one partner were covered.

4. In seven experiments cross circulation was established and then one animalradiated while the other was shielded. These were considered the most criticalexperiments of the group.

5. Detailed data on leukocyte and lymphocyte counts in the normal animals obtained during an approximately twenty-eight day period of follow-up are presented.

6. These data are not considered to support the thesis of indirect effects peculiarto radiation. A trend toward slightly lowered absolute lymphocyte counts innormal animals after cross circulation was not considered significant, and in noinstance did leukopenia develop in the normal animal.

7. The literature is reviewed and discussed.

Note: ACKNOWLEDGEMENTSThe authors gratefully acknowledge the assistance of Mr. Morey J. Wantman and the Division ofStatistics for the statistical analyses and the valuable assistance of Miss Wilma Kujowski in translatingthe foreign literature.

     

THE EFFECT OF DIET ON THE HEMOGLOBIN, ERYTHROCYTE, AND LEUKOCYTE CONTENT OF THE BLOOD OF THE RHESUS MONKEY (MACACA MULATTA)

1. Rhesus monkeys fed purified rations supplemented with adequate amountsof the B vitamins, ascorbic acid, and whole liver substance maintained the following average blood picture:

See PDF for Table

2. Natural diets or purified rations supplemented with liver extract do not support the above blood picture. The hemoglobin is lower and there is an increase inthe range of the total leukocyte count and in the neutrophil-lymphocyte ratioto 2.0 ([unknown]). These figures are similar to the values in the literature and generallyaccepted as the normal.

3. Previous reports have shown the characteristic blood dyscrasias which develop when monkeys are fed certain B vitamin-deficient diets. These changes aresummarized graphically in this paper.

4. The importance of determining the concentration of hemoglobin and theformed elements of the blood as a diagnostic test in nutritional studies has beenshown.

Note: We wish to acknowledge our indebtedness to Merck and Co., Rahway, N. J., for some of the crystalline vitamins; to Wilson Laboratories, Chicago, Ill., for the various liver preparations; and to LederleLaboratories, Inc., Pearl River, N. Y., for synthetic folic acid.The authors are grateful to Miss Ethel Thewlis for aid in determining cellular elements and to Drs.Harry A. Waisman and James H. Shaw for assisting in early parts of the work.

     

The effect of artificial surfaces on blood coagulability,with special reference to polyethylene

1. The reaction to small pieces of polyethylene and lucite in the subcutaneoustissues of 30 rats was studied at intervals up to three months after insertion. Polyethylene was shown to compare favorably in respect to minimum tissue reaction,with the well-tolerated lucite.

2. A series of clotting times was performed in polyethylene, paraffin, collodionand glass tubes. The clotting time in polyethylene tubes was about twice as longas in glass, and nearly as long as in paraffin and collodion-lined tubes. These dataare similar to Hirschboeck’s findings for lucite tubes.

3. Clot retraction was found to be moderate and essentially similar in polyethylene, paraffin, and glass tubes. It was slight or absent in collodion tubes.

4. Capillary tubes of polyethylene were shown to repel water initially and thengradually to attract water over a period of days to a maximum height about one-half of that in glass tubes. Thus polyethylene follows Lampert’s rule, which statesthat the effect of a surface in delaying the coagulation of blood is proportional tothe capacity of that surface for repelling water.

Note: ACKNOWLEDGMENTSThe authors wish to express appreciation to Drs. H. G. Saroff, J. Sendroy, Jr., J. L. Tullis, E. P.Cronkite, and Miss M. E. Tarver, for helpful advice and cooperation in these studies.

     

The response of eosinophils in the guinea pig to sensitization,anaphylaxis and various drugs

1. The eosinophilic response of the guinea pig sensitized and reinjected with thespecific antigen varies with the nature of the antigen used, but also with theindividual guinea pig in any groupsensitized and reinjected with the same antigen.

2. Certain antihistamine drugs which abolish anaphylactic symptoms, do notabolish the eosinophilic response.

3. The severity of anaphylactic "shock" symptoms has no influence on theeosinophilic response.

4. Histamine phosphate has no effect on the eosinophil count of nonsensitizedguinea pigs protected by benadryl; it causes a distinct eosinophilic response insensitized animals.

5. Heparin—in the dose injected—produced only an insignificant rise in theperipheral eosinophil count of sensitized guinea pigs; adenosine had no effect.

6. Attempts were made to correlate the eosinophilic response in bone marrow,blood and shock tissue of guinea pigs sensitized and reinjected with a specificantigen. The variation within a wide range of the number of eosinophils in thebone marrow of nonsensitized and of sensitized, reinjected guinea pigs is emphasized.A definite correlation seems to exist between the presence of a large number ofeosinophils in blood and lungs; it is shown, however, that this observation permits only limited conclusions.

7. The factors which account for discrepancies in the interpretation of theeosinophilic response, e.g., nature of antigen, route of administration and characteristics of species, are analyzed.

8. The significance of the findings is reviewed in the light of previous work.

Note: ACKNOWLEDGMENTWe wish to express our appreciation to Miss Alice Sprenger for her patient and efficient technicalassistance.

     

Experimental anemias in the rat; I. Macrocytic anemia in chronic pteroylglutamic acid deficiency and after splenectomy in Bartonella muris infection

1. In agreement with findings by other workers, rats in acute pteroylglutamicacid deficiency showed leukopenia and growth depression followed by death, without any significant change in the red cell picture.

2. In chronic deficiency, however, produced by the addition of small pteroylglutamic acid doses given intermittently, a severe anemia was obtained afterseventy days.

3. The anemia was macrocytic and "normochromic." Price-Jones curves showeda preponderance of macrocytes with anisocytosis. This agreed with findings byother workers for other species.

4. The anemia could be cured by single doses of 40 µg. or more of pteroylglutamicacid.

5. There was no significant difference between sexes to pteroylglutamic aciddeficiency. Reduction in the protein content of the diets, containing 1 per centsulfasuxidine, from 18 per cent to 10.5 per cent, produced no significant differencein the time of onset and severity of the blood symptoms.

6. These results were not due to infection with Bartonella muris. This infectionproduced a macrocytic anemia of a different type, and was curable by treatmentwith neoarsphenamine.

Note: ACKNOWLEDGMENTSWe are grateful to Dr. T. H. Jukes of the Lederle Laboratories for generous supplies of aldehyde-freePGA; and to Dr. K. Folkers of Merck Laboratories for the biotin used in these experiments. We wish tothank Dr. W. Jacobson for his advice during the course of this investigation. Valuable technical helpwas provided by Mr. D. R. Ashby, Mr. S. G. Impey, Miss M. J. Kemp and Mr. P. W. Wilson, to whomthe authors are indebted.

     

Changes in circulating leukocytes induced by the administration of pituitary adrenocorticotrophic hormone in man

1. Pituitary adrenocorticotrophic hormone (ACTH), when administered in asingle intramuscular dose of 25 mg. to human subjects with unimpaired adrenalfunction, results in a characteristic alteration of the leukocytic pattern. This consists of an increase of circulating neutrophils and a decrease of circulating lymphocytes and eosinophils.

2. The decrease in circulating lymphocytes and eosinophils is contingent uponthe stimulation of a functionally competent adrenal cortex, and does not occur inits absence. The neutrophilic response is present but somewhat diminished inadrenal insufficiency.

3. The entire pattern of leukocytic alterations found in normal subjects after administration of ACTH can be induced in patients with Addison’s disease by 17-hydroxycorticosterone (20 mg.) but not with desoxycorticosterone glucoside (30mg.).

4. Prolonged adrenal stimulation by ACTH, given over a four day period in adose of 10 mg. every six hours, results in a sustained and striking elevation ofneutrophils and depression of eosinophils; the lymphocytes, after an initial depression lasting not more than twenty-four hours, may increase above their initial levels in spite of the continued increased secretion of adrenal hormones.

5. The relation of the adrenal cortex to the characteristic nonspecific leukocytepattern, observed as a response of the organism to any type of insult, is discussed.

Note: ACKNOWLEDGEMENTSWe are indebted to Mrs. Elizabeth Dell, B.S., Mrs. Constance Forsham, B.S., M.T. (A.S.C.P.), andMiss Loretta Conroy, B.S., for their technical assistance, and to Miss Mary Kascht, B.A., Research Dietitian, who kindly took over the secretarial management in addition to the dietary work.

     

Dangerous universal donors; II. Further observations on in vivo and in vitro behavior of isoantibodies of immune type present in group O blood

1. Severe hemolytic reactions were observed in 3 group A (subgroup A₁) recipients transfused with group O whole blood or plasma. In one case, 10 ml. of acommerical preparation of soluble A and B factors had been added to 500 ml. ofwhole blood prior to the transfusion and it is believed that the reaction mighthave been even more serious had not this material been added.

2. The anti-A antibodies in the serum of the dangerous universal donors causingthe hemolytic reactions fixed complement, acted as hemolysins, were difficult toneutralize with soluble A and B factors, were capable of giving positive Coombstests and their ability to agglutinate A cells was enhanced by the presence of normal human serum. These characteristics were similar to those observed in serumfrom donors known to be actively immunized against the A factor, but the stimulusfor development of "immune" anti-A antibodies in the dangerous group O donorswas not apparent.

3. Small amounts of immune A antibody were consistently demonstrated in 12of 100 random group O sera which, after neutralization, produced indirect Coombstests with A₁ cells and agglutinated A₁ cells suspended in compatible normal humanserum.

4. Screening procedures for elimination of dangerous group O donors are discussed.

Note: ACKNOWLEDGMENTSIt is a pleasure to thank Mrs. Jane Peters, Mrs. Nieves Dole and Miss Jean Dorothy for technicalassistance and Dr. R. Wendell Davis for collaboration in the study of Case 1.

     

Results of therapy of erythroblastosis with exchange transfusion

1. In the authors’ technic of exchange transfusion, citrated blood is introducedinto the saphenous vein at the ankle and the infant’s blood simultaneously withdrawn from the radial artery at the wrist, coagulation being prevented by the administration of small amounts of heparin. The procedure besides being simple, issafe, there having been no operative mortality in more than 40 transfusions.

2. The results of exchange transfusion therapy in erythroblastosis in our first28 cases are presented. Of these 28 cases, 16 were very severe and almost certainlywould have been lethal if left untreated, 6 were of moderate severity, and 6 weremild. Only 7 of the infants died, and the available data indicate that the mortalitywould have been at least twice as high had the usual treatment with simple transfusions been given.

3. Aside from its greater efficacy in reducing mortality, exchange transfusion ismore efficient, so that supplementary treatment is not required as a rule.

4. Fresh blood should be used instead of bank blood because of its greater survival time and smaller likelihood of introducing infection.

5. All infants who have survived have developed normally both physically andmentally and have shown no sequelae of liver or brain damage.

6. The most reliable index of the severity of the disease in the erythroblastoticinfant is provided by antenatal titrations of the maternal univalent Rh antibodies,as well as by tests for the presence of univalent antibodies in the infant’s blood.

     

Response of tropical sprue to vitamin B12

These findings show that the administration of vitamin B₁₂ to patients with tropical sprue was followed by general clinical and hematologic improvement providedthe dosage was adequate. A single dose of 4 micrograms administered in case 4produced little or no change. The larger dosage of 10-25 micrograms administeredin the other cases was accompanied by striking increase in strength and vigor anda decrease in the diarrhea; however, in no instance was a maximal dose given andthese patients quickly tended to relapse clinically and hematologically. Theycould be relieved promptly again either by another injection of vitamin B₁₂ orby a compound of folic acid. (The conjugated compounds of folic acid used in thesecases were used for experimental purposes, and they produced the same hematologicresponse as that of folic acid per se.) Case 3, who had an excellent hematologicresponse after eating one serving of 400 grams of liver, is regarded as especiallysignificant in that it suggests that, as powerful as vitamin B₁₂ is as a therapeuticagent, it is more effective when given with liver. It is especially noteworthy thatcases 1 and 2, who had three injections of vitamin B₁₂, have had steady clinicaland hematologic improvement. The reader should have in mind that a singleinjection of approximately 100 micrograms of vitamin B₁₂ probably would beneeded to produce a full hematologic response in persons so ill. This tentative appraisal would suggest that this therapeutic compound, per unit of weight, is moreeffective in treating human disease than any compound that yet has been used.

Note: ACKNOWLEDGMENT We are very much indebted to others who have aided us in selecting cases and in observing results.Especially we wish to thank Dr. F. Hernandez-Morales, Dr. Hector Marchand, Miss Clemencia Benitez-Gautier, and Miss Sara Torres.

     

Thiamin deficiency in the rhesus monkey; clinical,metabolic and hematologic observations

Seven rhesus monkeys were subjected to one or more episodes of acute thiamindepletion. It is clear that significant metabolic inadequacies preceded demonstrablestructural changes. Diminished food consumption and weight loss were manifestabout two weeks after thiamin was removed from the diet. When the deficiencywas prolonged the animals became apathetic, inactive and progressively weaker.This was followed by ataxia and at times ptosis and tremors. Even in such advancedstates of depletion, administration of thiamin produced dramatic improvement inlocomotion, appetite and reactivity. The blood thiamin content of normal monkeysranged from 5.5 to 10 per 100 ml. of whole blood, values which are comparableto those of healthy human beings. Following withdrawal of thiamin the bloodconcentration fell to values of 4 or less. The tissue content of thiamin was correspondingly reduced in depleted animals. The minimum daily requirement forthiamin calculated on the basis of the time required to redeplete a deficient monkeyfollowing a small dose of thiamin was approximately 15 per kilogram bodyweight. Characteristic degenerative changes in the heart muscle and severe retrogressive changes in the nuclear structures of the central nervous system previouslyreported were noted. Based on careful hematologic studies in 4 animals it isconcluded that thiamin is essential for normal erythropoiesis. Acute or chronicdepletion results in anemia due to suppression of red blood cell formation asindicated by severe depression or absence of reticulocytes in the blood.

Note: ACKNOWLEDGMENTWe are indebted to Miss Mariette Quigley for the blood counts, and to Miss Ruth Johnson for assistance in thiamin assays.

     

The relationship between carbonic anhydrase activity and zinc content of erythrocytes in normal,in anemic and other pathologic conditions

A good correlation exists between zinc content and carbonic anhydrase activityof the red blood cells under all conditions studied, including anemia and polycythemia. In almost all patients with anemias other than pernicious anemia, bothzinc and carbonic anhydrase levels were lowered in parallel fashion. These changeswere proportional to decreases in hematocrit and hemoglobin levels and erythrocyte counts so that both zinc and carbonic anhydrase values per unit of RBC werein the normal range. In a few instances of anemia associated with leukemia andin one of sickle cell anemia, neither zinc content nor carbonic anhydrase activitywas decreased in proportion to the anemia; in these cases the zinc and carbonicanhydrase levels per unit of blood were both elevated to the same degree.

Patients with pernicious anemia showed no decrease in absolute values for zincand carbonic anhydrase activity in spite of marked lowering of hematocrit andhemoglobin levels and of erythrocyte count. Accordingly, both zinc concentrationand carbonic anhydrase activity per unit of blood were elevated, often to a markeddegree. These increases were parallel, varying inversely with the degree of anemia;when they regressed under treatment, both did so at the same rate.

There are no methods available for estimating carbonic anhydrase concentration;all methods now in use measure only the activity of the enzyme. It is suggestedthat zinc concentration could be used as an indicator of carbonic anhydrase contentof the red blood cells.

Note: ACKNOWLEDGMENTSDrs. Joseph C. Aub and Ira T. Nathanson were kind enough to refer several patients for study. Dr.Byrl J. Kennedy was most helpful in regard to obtaining samples of blood. The technical work wasperformed by Miss Mary Lou Roney, Betty Hickey and Marion Taylor.

     

Tuberculous splenomegaly with the hypersplenism syndrome; a case report

A case of tuberculous splenomegaly with leukopenia and anemia followingmiliary tuberculosis has been presented. Splenectomy was required after streptomycin failed to control the cytopenias, progressive emaciation, and splenic infection. However, following what appeared to be six weeks of marked improvement,the patient developed a fulminating tuberculous meningitis and died.

Note: ACKNOWLEDGMENTThe authors are indebted to Dr. Byrd S. Leavell for his suggestions in the preparation of this paper.