Studies on analogues of L-cysteine and L-cystine. I. Some structural requirements for inhibiting the incorporation of radioactive L-cystine by leukemic leukocytes

The amino acids L-cysteine and L-cystine appear to have an important rolein the metabolism of leukocytes. Decreased availability of these amino acidsmay therefore have important effects on leukocytes.

The possibility of decreasing the influx of radioactive L-cystine into leukemicleukocytes was investigated by exposing the leukocytes to various analogues ofcysteine (cystine) prior to incubation with S³⁵ L-cystine. It was found that ahighly specific structural and spatial configuration is required to decrease theinflux of S³⁵ L-cystine. Thus unlabeled L-cysteine is effective in decreasing theincorporation of radioactive L-cystine. However, analogues of cystine in whichthere is modification or substitution of the sulfhydryl, amino or carboxyl groupdo not decrease the influx of S³⁵ L-cystine. Furthermore, any alteration in thespatial relationship of the sulfhydryl and amino groups of L-cysteine also resultsin a loss of the ability of an analogue to decrease the incorporation of S³⁵ L-cystine.

Of the compounds studied and in the concentrations employed, only unlabeledL-cysteine, selenium cystine and phenyl selenium cysteine were effective. Selenium cystine is identical with cystine except that selenium replaces the sulfurin the molecule. Phenyl selenium cysteine is also closely related structurallyto cysteine.

The mechanism of action of selenium cystine and phenyl selenium cysteinein decreasing the influx of S³⁵ L-cystine is not known. Other selenium compoundstested were ineffective. These compounds may exert their inhibitory effect by(a) competitive combination with specific intracellular receptors for L-cysteine(L-cystine), (b) inactivation of enzymes or compounds essential for normalcellular function, (c) alteration in membrane permeability or (d) a toxic effectof selenium.

Since selenium cystine and phenyl selenium cystine are inhibitory in lowconcentrations in vitro, these compounds may have important effects on leukemicleukocytes in vivo.

Submitted on July 1, 1955 Accepted on August 30, 1955     

Studies on Agranulocytosis. III. The Reduced Glutathione (GSH) Content of Leukocytes of Normals and Patients Recovered from Agranulocytosis

1. A method for the direct determination of GSH in leukocytes is described.Treatment with alkali (0.5 M. NaOH) effects complete solution of thewhite cells and after deproteinization (5 per cent HPO₃), the GSH is determined by the sensitive "alloxan 305" procedure. Control studies showedthat under the conditions of the test, GSH is not affected by the alkali andno splitting of soluble minus SH from protein occurs.

2. Using this method, the amount of reduced glutathione content of normal leukocytes was found to be 5.2 ± 1 mg, per 10¹⁰ WBC.

3. No differences from normal levels were detected for the leukocyticGSH of patients with mental disease and those susceptible to agranulocytosis. Incubation of whole blood with the drug which caused agranulocytosis had no effect upon the GSH content of leukocytes.

Submitted on March 24, 1960 Accepted on July 13, 1960     

Detection of Leukocyte Antibodies by Means of I131-Labelled, Purified Anti--Human-Globulin Antibody. Problems of Nonspecific Adsorption of Globulin by Leukocytes

Initial studies of a quantitative method for detecting sensitization of whiteblood cells by antileukocyte antibodies are presented. The method entailsisolating I¹³¹-labelled, anti—human-globulin antibody (prepared in rabbits) byadsorption and elution from insoluble antigen, globulinazobenzylcellulose.The degree of adsorption of the labelled antibody by presumably sensitizedas opposed to nonsensitized leukocytes is measured by radioactive isotopecounting technics. The vexing problem of nonspecific adsorption of human orrabbit globulin by leukocytes is considered, and procedures for partially circumventing this problem are set forth. Seligmann's solution with normal rabbit serum added to a final concentration of 10 per cent appeared to be themost effective washing fluid in most instances for removing nonspecifically adsorbed globulins from leukocytes. The use of purified anti—human-globulinantibody, as opposed to whole Coombs serum, also very favorably affectedthe ratio between immunologically specific adsorption and nonspecific adsorption. Results of the over-all I¹³¹ method as applied to selected positiveand negative human sera are detailed.

Submitted on April 28, 1960 Accepted on July 3, 1960     

Leukokinetic studies. I. A method for labeling leukocytes with diisopropyl-fluorophosphate (DFP32)

1. A method has been presented by which granulocytes can be labeledin vivo with diisopropylfluorophosphate containing radioactive phosphorus.The leukocytes are isolated from blood by dextran sedimentation of erythrocytes and are then treated with gramicidin and lysolecithin to remove remaining red cells. Platelets are removed by differential centrifugation. Theisolated leukocytes are placed between two squares of scintillating plasticand counted with a scintillation counter.

2. Leukocytes essentially free of erythrocytes and platelets can be obtainedby the method outlined. The efficiency of the plastic scintillation countingmethod for radioactive phosphorus is about 74 per cent and leukocyte samples obtained from 20 ml. samples of normal blood can be counted with areproducibility of ±10 per cent.

3. The administration of 2 mg. of diisopropylfluorophosphate either intramuscularly or intravenously is without significant toxic side effects.

4. No evidence has been obtained that the label damages the leukocytes.

5. No evidence has been obtained that the label elutes from leukocytesunder the conditions of these studies.

6. Diisopropylfluorophosphate labels granulocytes for a brief period oftime following injection. The label is not reutilized after death of the cells.

Submitted on June 16, 1958 Accepted on July 17, 1958     

Improvement of Leukocyte Bactericidal Activity in Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) is characterized by an inability ofpatients’ leukocytes to generate hydrogen peroxide and to kill non-peroxide-forming bacteria, such as staphylococci and serratiae. We have introducedglucose oxidase into CGD leukocytes in order to generate peroxide and therebykill S. aureus and S. marcescens. These results support the concept that a leukocyte oxidative deficiency is primary to the pathogenesis of CGD.

Submitted on June 23, 1969 Accepted on September 8, 1969     

Busulfan in the Treatment of Chronic Myelocytic Leukemia. The Effect of Long Term Intermittent Therapy

1. The observations made during the administration of 114 courses of busulfan to 30 patients over a period of seven years are recorded. The responses toinitial courses of therapy corresponded with those reported previously. Withrepeated courses of therapy, subjective improvement, the decline in thenumber of leukocytes, decrease of anemia and the subsidence of physicalsigns of the disease were as satisfactory as during the first course but tendedto appear later. Patients were ambulatory and most were symptomatically andobjectively improved during and after repeated courses as compared to theirstatus beforehand.

2. Remissions were longest when the leukocyte values were 10,000 per cu.mm., or less, at the time busulfan was discontinued. Among such patients,remissions of six months or longer were seen in 85 per cent after the initialcourse of busulfan but in only 19 per cent after fifth or later courses.

3. Evidence of partial resistance to busulfan was seen in some cases aftermultiple courses of treatment. Complete resistance occurred in 15 patients upondevelopment of the acute, myeloblastic phase of chronic myelocytic leukemia.When busulfan failed, 6-mercaptopurine and colcemide were temporarily ofvalue; x-ray was not of benefit. After the onset of the acute phase. the mediansurvival was three months. This mode of termination is probably no morefrequent (50 per cent of cases) since the advent of busulfan therapy thanbefore.

4. If anemia was not relieved, or a large spleen was not reduced 50 per centin size following a course of therapy, the prognosis was poor and the acutephase imminent.

5. Thrombocytopenia in early, untreated cases was not necessarily a badsign, nor was the use of busulfan precluded because of it. However, whenthrombocytopenia appeared in a previously treated patient, without otherevidence indicating busulfan toxicity, or when it occurred in a patient withthree or more years of known disease, it usually presaged the development ofthe terminal acute phase.

6. Side effects of busulfan were significant in only one patient; this manreceived 8 milligrams daily, double the usual dose, for eight months anddeveloped glossitis, anhydrosis and alopecia totalis. The major hazard in theuse of the drug was the occurrence of pancytopenia. This could be related toexcessive dosage.

7. Morbidity was clearly decreased. Longevity (median 42 months) wasgreater than in Minot’s untreated cases (median 31 months) and at least asgreat as that achieved by treatment with radioactive phosphorus and x-ray(median 32-41 months). Repeated courses of busulfan are considered to offeran effective and practical palliative form of therapy for chronic myclocyticleukemia, up to the time of appearance of the terminal acute myeloblasticphase.

Submitted on August 29, 1960 Accepted on October 15, 1960     

Studies on analogues of L-cysteine and L-cystine. III. The effect of selenium cystine on leukemia

Selenium cystine was administered orally to 2 patients with acute leukemiaand to 2 patients with chronic myeloid leukemia. In all patients there was arapid decrease in the total leukocyte count as well as a decrease in spleen size.This effect was observed in patients refractory to other chemotherapeutic agentsas well as in the usually resistant types of leukemia. In patients with chronicmyeloid leukemia the immature granulocytes disappeared much more rapidlythan the mature granulocytes. The most striking and consistent effects wereobserved in acute leukemia. One patient who had become resistant to 6-mercaptopurine appeared to reacquire sensitivity to this compound after receivingselenium cystine.

These effects of selenium cystine on leukocytes correlate with the ability ofselenium cystine to decrease the influx of S³⁵ L-cystine by leukemic leukocytesin vitro. Other potentially effective analogues of cystine (or cysteine) maytherefore be selected by this technic.

No changes were detected in any of the organs attributable to selenium cystinetoxicity. The nausea and vomiting associated with the oral administration ofselenium cystine was so severe that it was not possible to administer seleniumcystine for a sufficient period of time to determine whether an appreciableremission can be obtained in leukemia. Further study is necessary to determinewhether selenium cystine has any practical applicability in the chemotherapyof leukemia.

Although the mechanism of action of selenium cystine is not known, thesestriking effects of an analogue of cystine on leukemia are further suggestive ofthe importance of cystine (cysteine) in the metabolism of leukocytes.

Submitted on July 1, 1955 Accepted on August 30, 1955     

The Antigenicity of Normal and Leukemic Human Leukocytes

1. A leukoagglutinin was formed in the serum of a normal human subjectwho received 10 intravenous injections of blood from a single patient withchronic myelogenous leukemia over a period of 20 weeks.

2. Coincident with development of the leukoagglutinin, first detectable oneweek after the fifth injection of leukemic blood, the normal subject experiencedprogressively more severe febrile reactions to the infusions and exhibited acharacteristic pattern of leukocyte response—namely, an immediate transientleukopenia, followed by a leukocytosis which reached its peak around 3 hoursand subsided to normal within 12 hours.

3. During the early part of the investigation immature leukocytes, presumably from the leukemic donor, could be identified in the recipient’s circulationduring the first hour immediately following injection, but none could befound following the tenth infusion of leukemic blood.

4. The leukoagglutinin which appeared in response to the injections of bloodfrom the single leukemic donor was a typical iso-antibody, showing a broadpattern of reactivity against normal leukocytes from 127 of 129 donors, leukemicleukocytes from 5 of 5 patients with chronic myelocytic leukemia and 6 of 6patients with chronic lymphocytic leukemia. No reactivity was observed againstthe recipient’s own leukocytes, and little or no reactivity was demonstrableagainst the immature leukocytes from 3 patients with acute leukemia.

5. Eighteen months after the last injection of leukemic blood, restimulationof a leukocyte iso-agglutinin in the previously immunized recipient was provoked within one week of commencing a series of intravenous infusions ofblood from a single normal donor.

6. The volume of normal leukocytes employed for the restimulation was 1/10to 1/100 the volume of leukemic leukocytes employed for the primary immunization.

7. The concept of antibody excess was demonstrable in the sensitized recipient. No evidence of in vivo absorption of leukoagglutinin activity was observedafter transfusion of 500 ml. of blood from the normal donor. The severity ofthe recipient’s reaction to the transfused blood was clearly related to the doseof donor leukocytes administered, 0.47 billion cells causing no reaction but4.16 billion causing a moderately severe reaction.

8. Fifteen months after completion of the injections of normal blood, reexposure of the normal subject to injections of blood from a second leukemicdonor resulted in prompt restimulation of leukoagglutinin activity in therecipient’s serum.

9. The leukoagglutinin could be completely absorbed in vitro by incubationwith donor leukocytes.

10. The leukoagglutinin was concentrated in the gamma globulin fraction ofthe recipient’s plasma.

11. The recipient exhibited typical symptomatic reactions and transient hematologic changes following the infusions of leukemic blood.

12. It was possible to correlate the severity of the recipient’s clinical reactionsboth with the strength of the recipient’s leukoagglutinin, as well as with thedose of donor leukocytes transfused.

13. Serologic observations, plus the results of fractionated transfusion studies,indicated that the recipient’s transfusion reactions were related to sensitivityto the donor’s buffy coat (Part II), and more specificially to donor leukocytes(Part III), rather than to donor plasma, platelets or erythrocytes.

14. Sustained stimulation of the recipient’s white cell count as a result of theinjections of leukemic blood was not observed.

15. There has thus far been no evidence of transmission of leukemia to therecipient (now 6 years after the first course of injections of leukemic bloodand 2 years since completion of the present study).

Submitted on July 15, 1960 Accepted on November 20, 1960     

The effect of quantitative and qualitative protein deficiency on blood regeneration; white blood cells

1. The effect of diets, varying in quantity or quality of protein, on white bloodcell regeneration was studied in leukopenic rats, the leukopenia having been induced by a protein-free diet.

2. Diets containing different amounts of casein (3, 6, 9 and 18 per cent, respectively), were fed ad libitum. At the 3 per cent level, a further decrease occurred ofwhite blood cells, whereas the other three diets initiated a regeneration of leukocytes, its degree being more or less in proportion to the casein content.

3. In experiments with diets containing 18 and 30 per cent of casein, the amountof protein eaten and not its level in diet was the decisive factor in the regenerationof leukocytes. The white blood cell regenerating effect of a diet containing anoptimal level of protein, may be neutralized when given in restricted amounts.

4. Diets containing nutritionally inferior proteins, fed at 9 per cent level, alsoimpaired normal regeneration of leukocytes. The white blood cell regenerationafforded by the proteins investigated was found to increase in the following order:maize, gelatin, wheat, casein, processed soya, peanut, meat, egg.

5. In white blood cell regeneration promoted by dietary protein, granulocyteswere found to react to a greater degree than lymphocytes and monocytes.

     

Studies of in Vivo Phagocytosis. I. Inhibition of Phagocytosis by Intact Neoplastic Body Fluids

1. By means of a modified skin window technic, the phagocytic response ofrabbit leukocytes to external stimuli has been observed.

2. Suspensions of particulate antigens, such as rice starch and Candidaalbicans, made in serums or urines from patients with leukemias and lymphoproliferative disorders, were not phagocytized normally when compared tosimilar suspensions in normal media.

3. These results suggest that exogenous or endogenous factors, individuallyor in combination, interfere with normal phagocytic activity in the rabbitand, by analogy, may exercise a similar function in the human.

Submitted on May 25, 1962 Accepted on December 3, 1962     

Studies on analogues of L-cysteine and L-cystine. II. The effect of selenium cystine on Murphy lymphosarcoma tumor cells in the rat

Selenium cystine decreases the incorporation of S³⁵ L-cystine by rat Murphylymphosarcoma tumor cells both in vitro and in vivo. Selenium cystine alsodecreases the growth of the tumor in the intact animal.

Benzyl selenium cysteine does not inhibit the incorporation of S³⁵ L-cystineby Murphy lymphosarcoma tumor cells in vitro nor does it affect tumor growthin the intact animal.

Thus the ability of selenium cystine to decrease the influx of S³⁵ L-cystineinto tumor cells in vitro is associated with the ability of this compound to inhibit tumor growth in the intact animal. Furthermore, the inhibitory effect ofselenium cystine is not due solely to the presence of selenium within the molecule but is related to its structural identity with cystine.

The data are further suggestive of the importance of SH compounds in thegrowth of malignant cells.

Submitted on July 1, 1955 Accepted on August 30, 1955     

Effect of Ascorbic Acid in Vivo on Labeling of Red Cells by Radioactive Sodium Chromate

Fifty-two whole blood samples from 17 patients were analyzed for ascorbicacid concentration and Cr⁵¹ tagging. Physiologic concentrations of ascorbicacid in whole blood in vivo reduced radioactive sodium chromate in vitro andimpaired tagging.

Submitted on December 5, 1963 Accepted on April 23, 1963     

A Case of Juvenile Pernicious Anemia: Study of the Effects of Folic Acid and Vitamin B12

Observations on a 4-year-old boy with Addisonian pernicious anemia havebeen presented. Noteworthy clinical features included the onset of glossitisat the age of 4 months, followed by anemia severe enough to require hospitalization at the age of 1 year. Relapse occurred in the absence of specific therapywith vitamin B₁₂ and was completely unaffected by the administration of folicacid.

Studies with radioactive vitamin B₁₂ demonstrated that almost all of thecompound administered by mouth was unabsorbed and was recovered in thestools. When the vitamin was given simultaneously with a concentrate of intrinsic factor, however, approximately 70 per cent was absorbed. Furthermore, the child’s gastric juice, when mixed with radioactive vitamin B₁₂ andfed to an adult with pernicious anemia in relapse, failed to enhance the latter’s absorption of the vitamin. The failure of our patient to absorb the vitaminalone, but his ability to do so when it was administered with intrinsic factorconcentrate, was also confirmed by the "Schilling test," in which a proportion ofthe absorbed radioactive vitamin was "flushed" into the urine by parenteralinjection of one milligram of conventional vitamin B₁₂.

Of special interest was the occurrence in the urine of an unidentified derivative of tetrahydrofolic acid, derived from orally administered pteroylglutamicacid. The presence of this compound in the urine was demonstrated chromatographically when the patient was critically ill with his disease prior to treatment with vitamin B₁₂. Subsequent to therapy with vitamin B₁₂, while theadministration of folic acid was continued, the abnormal metabolite of folicacid could not be found in the urine. Similarly, the administration of folicacid did not lead to the appearance of this metabolite in the urine at a timewhen, after more than two years without specific therapy, a hematologicalrelapse occurred that was much less severe than that previously observed. Theimplications of these observations, with respect to the metabolic interrelationships of folic acid and vitamin B₁₂, are discussed.

Of further interest were the findings of strongly acid gastric juice containingmuch mucus and free hydrochloric acid. A fairly normal gastric mucosa wasdemonstrated by biopsy. The meaning of these unusual findings is discussedand an hypothesis to account for them is offered. The probable sequence ofevents in these patients from childhood to the development of anemia, usuallyin later life, is set forth.

Submitted on October 26, 1960 Accepted on February 9, 1961     

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.

     

Decreased Erythrocyte Survival in Hemoglobin H Disease As a Result of the Abnormal Properties of Hemoglobin H: The Benefit of Splenectomy

Two of three siblings with hemoglobin H disease were splenectomized. Bothwere benefited as judged by improved feeling of well-being, exercise tolerance and improvement in hemoglobin level and in erythrocyte mean lifespan.However, they were still susceptible to hemolytic crises, and their erythrocytemean lifespan was below the normal range. Erythrocyte survival studies by theCr⁵¹ method indicated a finite lifespan of 40-45 days before splenectomy, whichincreased to normal following splenectomy. In addition, there was randomdestruction of the erythrocytes, which was slightly reduced following splenectomy. The erythrocyte survival of a member of this family who had the hereditary leptocytosis trait, but no hemoglobin H, was normal.

Evidence is presented indicating that hemoglobin H denatures and precipitates irreversibly at a cell age of 40-45 days, forming intraerythrocytic inclusions which lead to shortening of the erythrocyte finite lifespan due to rapidremoval of such erythrocytes by the spleen. In addition, hemoglobin H has alower solubility in the deoxygenated state and precipitates reversibly, regardless of cell age, in the capillary bed, causing random erythrocyte destruction.Splenectomy is beneficial because of survival of erythrocytes with inclusionswhose hemoglobin A presumably retains its function in oxygen transport.Methemoglobin-forming chemicals such as amyl nitrite and drugs such assulfisoxazole were found to denature hemoglobin H and produce in vitro inclusions in every erythrocyte of these patients but not of normal subjects. It issuggested that the shortening of the erythrocyte survival and the degree ofpostsplenectomy improvement is greater the higher the amount of hemoglobinH present in the erythrocytes.

The apparent aging of the hemoglobin H molecule which leads to shortenederythroctye finite lifespan is discussed, and a concept of aging proteins determining the lifespan and/or the function of cells is advanced.

Submitted on August 1, 1960 Accepted on November 5, 1960     

The In Vivo Differentiation of Human Leukocytes into Histiocytes, Fibroblasts and Fat Cells in Subcutaneous Diffusion Chambers

Normal human leukocytes were cultivated in millipore diffusion chamberswhich had been implanted subcutaneously in autologous and homologoussubjects. The observations were made over periods of a few days to six weeks.It was found that mature granulocytes underwent disintegration within one totwo weeks. Mononuclear leukocytes underwent differentiation into macrophages and "polyblasts" within a few days and by three weeks had assumedthe morphologic appearance characteristic of histiocytes and fibroblast-likecells. By four to six weeks extensive fibroblastic proliferation and marked collagen formation was found. In several chambers numerous fat cells were seen.

These in vivo studies demonstrate the mesenchymal potential for differentiation possessed by circulating mononuclear leukocytes of adult blood.

Submitted on July 8, 1960     

The Clinical Significance of Fever in Acute Leukemia

1. The total course of acute leukemia in 55 patients managed in one clinicdisclosed 149 febrile episodes.

2. Fever occurred virtually only when the leukemia was in relapse.

3. Infection was the cause of fever in 102 of the 149 episodes of fever.

4. With survival after the onset of symptoms of acute leukemia beyond themean duration of life characteristic of that kind of acute leukemia, infectionappeared to be more often the cause of fever than when survival was lessthan the mean for that kind of leukemia.

5. Fever due to infection could not be reliably differentiated from fever notdue to infection by either the height or character of the fever curve, or bycounting the absolute number of mature polymorphonuclear leukocytes inthe blood.

6. Infection causing febrile episodes was most successfully detected bymeans of history, physical examination, chest roentgenogram, blood cultureand other cultures as indicated by the preceding maneuvers.

7. Consideration of the fever of acute leukemia from the point of viewof present day studies of the pathogenesis of fever did not clearly implicateknown mechanisms of pyrogenesis.

8. The interplay of state of leukemia, nature of infecting microorganismand anti-infectious and antileukemic therapies is related to outcome in febrile episodes due to infections. The management of febrile episodes isdiscussed.

9. Increased survival time in children with acute lymphoblastic leukemiahas not been accompanied by increased morbidity.

Submitted on March 21, 1960 Accepted on April 21, 1960     

The electrical conductivity of blood. I: Relationship to erythrocyte concentration

1. The factors influencing blood conductivity have been noted.

2. An accurate apparatus has been designed to measure blood and plasma conductivity.

3. A new cell has been designed to measure conductivity of blood.

4. Through studies on normal blood before and after dilution, a correlation hasbeen shown to exist between blood conductivity and the red cell count.

5. The form factor for normal human red cells has been determined to be 1.393.

6. A mathematical equation is presented relating red cell count with conductivityfor normal blood.

See PDF for Equation

7. The factor C on the basis of 33 determinations has been calculated to be10.80.

     

Study of Leukopenias and Thrombocytopenias by the Direct Antiglobulin Consumption Test on Leukocytes and/or Platelets

A new test, the direct antiglobulin consumption test (direct ACT), has beendevised to be performed on the leukocytes and platelets of patients. It wasperformed in parallel with the indirect antiglobulin consumption test (indirectACT), and other serologic tests on 492 individuals comprising 24 cases of diffuse lupus erythematosus, 93 primary thrombocytopenias, 18 secondarythrombocytopenias, 48 primary leukopenias or leukothrombocytopenias, 80secondary leukopenias, 101 non-leukothrombocytopenic patients and 128 normal subjects.

A good correlation was obtained between a positive or negative result andthe presence or absence of a cytopenia in the corresponding cell series. Outof 183 thrombocytopenic patients, 42.2 per cent gave a positive result withplatelets, and out of 128 leukopenic patients, 25.8 per cent gave a positive result on leukocytes, whereas of the 299 patients with normal leukocyte andplatelet counts, 98 per cent gave negative results. The test was found positivein three categories of patients:

(1) In diffuse lupus erythematosus, tests on both leukocytes and plateletswere almost uniformly positive. The indirect ACT permits a distinctionto be made between three substances in the serum of these patients.These are antiplatelet, antileukocyte-cytoplasm and antileukocyte-nuclearsubstances.

(2) The thrombocytopenic patients were subdivided into two groups, namely primary and secondary thrombocytopenia:

(a) Out of 93 cases of idiopathic thrombocytopenic purpura, about50 per cent gave a positive result with platelets. Neither thehistory nor the clinical picture suggested any differentiation between those who gave negative results, apart from the fact thata history of infection was more frequent among the negativecases. Corticosteroid therapy did not affect the result of the test,but following splenectomy, 38 per cent of the cases become negative.

(b) Of 18 patients with a secondary thrombocytopenia, three cases(16 per cent) gave a positive result.

(3) The leukopenic and leukothrombocytopenic patients were also subdividedinto two groups:

(a) Out of 48 primary or idiopathic cases, some 50 per cent gave apositive direct ACT either on leukocytes and/or on platelets. Byusing the indirect ACT it was possible to distinguish two substances in the serum, one being antiplatelet and the other an antileukocyte cytoplasmic substance.

(b) The 80 cases of secondary leukopenia or leukothrombocytopeniagave a positive result in 15 per cent of cases with leukocytes and/or with platelets.

Of the 101 non-leukothrombocytopenic patients, only five were found togive positive tests.

All the 128 normal subjects gave negative results.

The direct ACT provides direct evidence of the presence of a -globulin,probably an auto-antibody, on the leukocytes and/or the platelets of some 99per cent of cases of diffuse lupus erythematosus, of about 50 per cent ofcases of idiopathic thrombocytopenia and idiopathic leukothromobcytopenia,and in about 15 per cent of cases of secondary thrombocytopenia and leukothrombocytopenia. No marked difference was found in the history, clinicalpicture, or hematological findings between patients giving positive and negative results.

Submitted on April 3, 1961 Accepted on August 1, 1961     

The efficiency of oxidative phosphorylation by normal and leukemic human leukocytes

1. A new modification of existing methods has been described for the separation of leukocytes from whole blood which provides a procedure for therapid isolation of uninjured cells suitable for the study of oxidative phosphorylations.

2. This method has been employed in a study of the relative efficiencyand yield of oxidative-linked phosphorylations mediated by normal and leukemic or immature leukocytes. The maximum aerobic phosphorylating capacitywas exhibited by chronic lymphocytic leukemic leukocytes, followed in decreasing order of activity by acute monocytic leukemic leukocytes and chronicmyelocytic leukemic leukocytes. Oxidative phosphorylation was not demonstrated with normal leukocytes.

3. Results of this study suggest that expression of leukocyte metabolicdata on a unit nitrogen basis more accurately reflect the morphologically obvious size differences among the various leukocytes than presentation of dataon a unit cell basis.

4. The aerobic phosphorylations mediated by leukemic leukocytes werefound to be dependent upon substrate addition and were depressed by lowlevels of dinitrophenol. Under the experimental conditions employed in thisstudy, glucose-6-phosphate was formed in stoicheiometric amounts. These results indicate that leukemic leukocytes are capable of the aerobic esterification of inorganic phosphate accompanying the oxidation of selected Kreb'scycle intermediates.

Submitted on August 2, 1957 Accepted on November 2, 1957