patients deficiency) 7). hypothesis,...

COMPARISONOF THE EFFECTSOF MASSIVE BLOODTRANSFUSIONSANDOF LIVER EXTRACTIN PERNICIOUS ANEMIA'

By CHARLESS. DAVIDSON, JOHN C. MURPHY,R. JANET WATSON,ANDW. B. CASTLE

(From the Thorndike Memorial Laboratory, Second. and Fourth Medical Services (Harvard),Boston City Hospital and the Department of Medicine, Harvard Medical School, Boston)

(Received for publication July 10, 1946)

The features of the classical, clinical and he-matological response to the administration of liver(1) and of stomach (2) preparations, and recentlyof synthetic L. casei factor (3, 4) in patients withuntreated pernicious anemnia, are well known.The action of these therapeutic agents is, how-ever, sometimes rather carelessly regarded as oneof direct stimulation of blood production. It isperhaps more accurate to consider that liver ex-tract acts by the replacement of a nutritional de-ficiency and so, in one way or another, permitseffective response of the bone marrow to thestimulus of the anoxia common to all anemias.Moreover, despite an obvious analogy with theeffects of iron administration in patients with hy-pochromic (iron deficiency) anemia, the conceptthat liver extract therapy in pernicious anemiaalso acts by abolishing a maturation arrest leadingto decreased blood production has recently beenchallenged (5 to 7). This represents a return tothe older viewpoint that the anemia is based onexcessive blood destruction, manifest especially bythe increased output of bile pigments in the feces.Under this hypothesis, the nutritional deficiencyis assumed in some way to permit the advent of ahemolytic process (7), and the absence of ele-vated reticulocyte counts in the peripheral bloodis explained on the hypothesis that these cells aredestroyed selectively before they leave the bonemarrow (5). The megaloblastic hyperplasia ofthat organ is thus interpreted as an unusually se-vere degree of the erythroid cell immaturity seenin the bone marrows of severe hemolytic anemiasin animals (8) and in man (9, 10). It appearedto be desirable to secure, if possible, additional in-formation on some of these points of view, and toattempt to distinguish between the direct effectsof liver extract therapy and those due to elevations

1 The expenses of this investigation were defrayed inpart by grants from the J. K. Lilly Gift to the HarvardMedical School.

in hemoglobin levels following its use. Accord-ingly, observations were made of the comparativehematological and clinical effects of rapidly re-peated transfusions, and of the administration ofliver extract in pernicious anemia.

METHODS

Five patients with classical Addisonian pernicious ane-mia in relapse were selected, and were given daily trans-fusions of whole blood or red cell concentrates until, in2, the red cell counts reached 3 million per cu. mm., andin 3, the red cell counts reached 5 million per cm. mm.Liver extract was then administered to the 2 patientswith red cell counts of 3 million per cu. mm., and to 2of the 3 patients with 5 million red cells per cu, mm. Theremaining patient with a normal red cell value was givenno further therapy for 46 days, when his red cell counthad fallen to 3 million per cu. mm. Liver extract wasthen administered. Both during the period of transfusion,and after liver extract administration, the changes in clini-cal condition were observed, as well as changes in redcell count, hemoglobin concentration, corpuscular indices,reticulocytes, white cell and platelet counts, and icterusindex. Sternal marrow puncture and biopsy were per-formed at appropriate times in order to determine thecomparative effects on the marrow cytology of the redcell transfusions, and of the administration of liverextract.

The blood cell counts were made on oxalated venousblood with U. S. Bureau of Standards pipettes and count-ing chambers. The hemoglobin was determined usingthe Klett-Summerson colorimeter, using 15.6 grams ofhemoglobin per 100 ml. of blood as the standard. Thered cell indices were obtained by the method of Wintrobe(11). Blood platelet counts were performed by a modifica-tion of the method of Rees and Ecker (12). The icterusindex was determined by comparing plasma with dichro-mate standards (13). Bone marrow puncture was madein the mid-sternum at the level of the second interspace,using the needle designed by Turkel (14). Sternal mar-row biopsies were done by the surgical service, using aMA-inch trephine and standard surgical technique.

Whole blood for transfusions was collected in sodiumcitrate solution by the hospital blood bank. Red cell con-centrates were prepared by centrifuging fresh citratedblood and withdrawing most of the plasma. A smallamount of saline (50 to 75 ml.) was added and well

858

MASSIVE BLOODTRANSFUSIONSIN PERNICIOUS ANEMIA

mixed with the cells before infusion. Cell concentrateswere used before they were more than 3 days old.

RESULTS

(a) Red blood cell values and indices.During the period of transfusions, there was a

rapid rise of the red cell count, hemoglobin con-centration and hematocrit.

Case No. 1 received the red cell equivalent of9,750 ml. of blood, 7,900 ml. of which were neces-sary to bring the red cell values to normal in 13days. Case No. 2 received the red cell equivalentof 6,500 ml. of blood, all as red cell concentrates,

bringing the red cell values to normal in 7 days.Cases No. 3 and No. 4 received the red cellequivalent of 3,000 ml. of blood in 3 days, with aresultant red cell count of about 3 million per cu.mm. Cases No. 5 received the red cell equivalentof 8,000 ml. of whole blood in 8 days, resulting inan essentially normal red cell value.

(b) Reticulocytes.During the period of blood transfusions there

was no significant rise in the reticulocyte count inany of the patients.

In the 2 patients given multiple U.S.P. units ofliver extract by intramuscular injections immedi-

FIG. 1. CAsE No. 1

859

C. S. DAVIDSON, J. C. MURPHY, R. J. WATSON, AND W. B. CASTLE

ately after the end of the series of transfusionswhen their red cell values were normal, no rise inreticulocytes occurred in Case No. 1 (Figure 1),and only a slight rise, reaching 60,000 per cu. mm.,in Case No. 2. The peak of this response was on

the 7th day after liver extract was first injected,and after the red cell count had fallen to about4 million per cu. mm. (Figure 2).

The 3 patients given liver extract when theirred cell counts were near 3 million per cu. mm.

FIG. 2. CASE No. 2

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had rises in reticulocyte counts reaching maximaof 123,000, 116,000 and 326,000 per cu. mm., re-

spectively, in Cases Nos. 3, 4, and 5.(c) White blood cells and platelets.Leukopenia was present in all patients before

and during the period of blood transfusions. Infact, it was only after the administration of liverextract that a significant rise in the white cellcount occurred. That this rise was not due to a

delayed response to previous transfusions is madeclear by the observations in Case No. 5 (Figure 5),

in which a period of 55 days separates the trans-fusions from the administration of liver extract.

Thrombocytopenia was still evident during thetransfusions in the 4 patients in whom plateletcounts were made. Here again, it is evident thatno significant rise in blood platelets occurred untilafter liver extract was administered. This isparticularly well shown in Cases Nos. 1, 2, and 5(Figures 1, 2, 5), and is suggestedin Case No. 4(Figure 4), in whom but 2 platelet counts were

made.

861

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1

4

4

ICASE NO. 3 j


(d) Bone marrow.A summary of the differential bone marrow nu-

cleated cell counts is presented in Table I. Thepolymorphonuclear and band form granulocytesremained about the same throughout, except inCases Nos. 1 and 2 where a definite increase wasseen after liver extract therapy.

More striking, however, is the change in megalo-blast percentage following blood transfusions. Noexamination of the bone marrow was made inCase No.' 1 before transfusions were begun, but

undoubtedly megaloblasts were present in thequantity usual for pernicious anemia in rel;apse.Following transfusions they almost disappeared.The effect of transfusions in sharply decreasingthe percentages of megaloblasts is seen in the dif-ferential counts of marrow cells obtained fromCases Nos. 2, 3, and 5, and is illustrated by thephotomicrographs (Figures 6, 7). Furthermore,in Case No. 5, the megaloblasts reappeared dur-ing the 1% months after transfusions were dis-continued, at the end of which time the red cell

FIG. 4. CASE No. 4

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FIG. 5. CASE No. 5

count had dropped from 5 to 3 million per cu.mm.

(e) Clinical course.Case No. 1, a 67-year-old white female, known to have

had pernicious anemia in the past, suffered a relapse fromfailure to continue liver extract therapy. Before transfu-sions were begun she complained of weakness, anorexia,"indigestion," and a recurrence of nervous system symp-toms. Inspection showed marked pallor, white hair, blueeyes, and a pale, smooth tongue. There was cardiac en-largement and dependent edema. Drowsiness with eupho-ria were the chief mental symptoms.

During the course of transfusions, the patient became

even less active and responsive, and dozed most of thetime. Her tongue became fiery red, with fissures, but nogrowth of papillae became evident. Her pallor was, how-ever, replaced by normal skin color. This effect, whichwas presumably due simply to the return to normal hemo-globin concentration, was the only sign of clinical improve-ment on the 14th day, when liver extract was administered.On the 21st day, 7 days after liver extract was begun,the patient seemed for the first time definitely better. Sheappeared more alert, and took a more active interest inher surroundings. This improvement progressed rapidly,so that a week later she was up in a chair most of theday. The tongue had become less red, and papillae werebeginning to appear. There was no significant change in

863


symptoms or signs of spinal cord disease during the periodof observation.

Case No. 2, a 45-year-old white male, was admitted inhis first attack complaining of fatigue and weakness ofseveral months' duration. He had had constipation andattacks of "indigestion," together with progressive ano-

rexia. He had noticed numbness and tingling of his fingertips for about 1 week He was poorly nourished and pale;there was moderate loss of papillae on the tongue, espe-

cially along the margins. A definitely impaired vibrationsense was present in both lower extremities. Mentally hewas clear but a little lethargic. His pallor was replacedby normal color during the period of transfusion. How-ever, although ambulatory since admission, his lethargy

continued, and anorexia, "indigestion," and tongue changeslikewise remained the same.

Beginning about 7 to 10 days after the administrationof liver extract, there was a distinct change in the pa-

tient's condition. He became brighter and more cheerful.His appetite improved rapidly, and the tongue showedsome regrowth of papillae. His paresthesiae disappearedin the course of about 2 weeks.

Case No. 3, a 59-year-old white female, in whoma diag-nosis of pernicious anemia had been made 2 years previ-ously, suffered a relapse after discontinuing liver therapy.Before blood transfusions were initiated, she complainedof weakness, "indigestion," anorexia, and "gas" aftermeals. She noticed pounding in the head and had palpita-

TABLE I

Distribution of nucleated bone marrow cells

Status Day Polymorphs. Metamyelocytes Normoblasts Erythroblasts Megaloblastsand bands and myelocytes

per cent per cent per cent -,Per cent per centCase no. 1

After transfusionBefore liver extractR.B.C. 4.70 million 14 22 36 6 8 3

35 daysAfter liver extractR.B.C. 5.1 million 49 40 24 10 6 1

Case no. 2Before transfusionR.B.C. 1.03 million 1 29 22 7 10 25



Case no. 3Before transfusionR.B.C. 1.18 million 3 16 18 11 15 28



Case no. SBefore transfusionR.B.C. 1.02 million 2 21 8 11 19 21

During transfusionR.B.C. 2.0 million 5 27 18 9 12 11

During transfusionR.B.C. 4.0 million 8 22 11 18 8 2

End of transfusionR.B.C. 4.5 million 12 11 40 9 14 2

No therapy 46 daysR.B.C. 3.0 million 58 27 13 10 20 10

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FIG. 6. PATIENT No. 2-DAY 1. BEFORETRANSFUSIONRed cell count, 1.03 million per cu. mm. Bone marrow film with large proportion

of megaloblasts. (Wright's stain, oil immersion.)

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FIG. 7. PATIENT No. 2-DAY 8. TRANSFUSIONSFINISHEDRed cell count, 5.17 million per cu. mm. Bone marrow film is relatively normal.

Megaloblasts gone. (Wright's stain, oil immersion.)

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tion on exertion or on becoming disturbed emotionally.Her skin was waxy-yellow in appearance; the hair gray,the eyes blue, and the tongue papillae atrophic. Cardiacenilargement was moderate, but there was no dependentedema.

During anld immediately after the course of red celltransfusionis the patient noted disappearance of the lheadp)ouniding anid palpitation, an(d her color improved. Therewvere nio other subjective or objective chaniges. Soon aftertlle retictilocyte peak, followinlg liver extract administra-tioin, the patienlt began to improve markedly. Her appe-tite inicreased, gastro-intestilnal symptomns dniiiniislhed, andweakniess gradually disappeared.

Case No. 4, a 73-year-old white muale in wlhom a diag-niosis of perniicious anemia had been made a year previ-ously, later discontinued treatment with liver extract andwas admitted after having collapsed. He confessed tohaving orthopnea and anginal paini onl slight exertion.Marked pallor was evidenit on examinationi. The hair was"gray, the eyes brown, and the tonlgue papillae moder-ately atrophic. No cardiac enlargement was observed, butdependent edema was presenlt. The spleen was palpableat the left costal margin. There was nio evidence of com-bined system disease.

Following red cell transfusions, orthopnea and anginalpain entirely disappeared, but anorexia and a listless facialexpression continued. Following the administration ofliver extract these also gradually disappeared, althoughnot as dramatically as in the foregoing 3 patients.

Case No. 5, a 75-year-old white male without a previoushistory of pernicious anemia, on admission complained ofincreasing weakness, anorexia, and sore tongue of about1 month's duration. On examination he was found to bemarkedly pale and emaciated, with an atrophic tongue.There was no cardiac enlargement nor dependenit edema.No evidence of combined system disease was observedexcept for diminished vibratory senise in the lowerextremities.

Following transfusions, his weakniess decreased some-what, but, in general, he was symptomatically the samefor the 11/½z months between blood transfusions and liverextract therapy. However, following the latter therewas striking subjective and objective improvement. Espe-cially remarkable was the regrowth of papillac on1 thetonigue.

DISCUSSION

The a(liniistration of liver extract, in a deci-sively effective dose, to patients witlh perniiciousanieimiia in relapse, prodtuces certain constant physi-ological effects. After as little as 6 hours, thel)rimIlitive miiegaloblasts of the bone marrow beginto decrease while a corresponding increase in themiiore miiature cells takes place (15). Within 3 to4 days the predominant erythroid cell is the nor-moblast. At about this time, the characteristicreticulocyte rise beginls in the peripheral blood,and(l relief of aniorexia witlh inicrease in strength

and sense of well-being are experienced by thepatient. The leukocytes, and especially the plate-lets, also increase in the peripheral blood. It isnoteworthy that clinical improvement is oftenstriking before any significant increase occurs inthe levels of red cells and hemoglobin.

In none of the cases reported here, however, didtransfusions modify the blood findings or clinicalstattis, other tllain to abolish the anemia and thesymllptomiis directly related to decreased lhemoglobinconcenitration in the peripheral blood: pallor, pal-pitation, or angilna. Anorexia, apathy, and diges-tive symiiptolmis were not affected. Transfusionisalso failed to cause any increase in reticulocytes,or in leukocytes or platelets.

Although, in contrast to transfusionis, liver ex-tract therapy apparently caused remarkable clinicalimprovement and increases in leukocytes andplatelets, the response of the reticulocytes wasgreatly modified by previous transfusions. Thus,reticulocyte response to liver extract therapy wasabsent in Case No. 1 with 5 million red cells percu. mm., was slight in Case No. 2, whose red cellshad dropped to about 4 million per cu. mm., wasmoderate in Cases No. 3 and 4 which were trans-fused to only 3 million per cu. mm., and wasmarked in Case No. 5, in which the red cells wereallowed to drop from 5 to 3 million per cu. mmni.Tlhus, the reticulocyte response to liver extractNas roughly inversely proportionate to the heiglhtof the artificially elevated red cell counlt anidhemiioglobin present at the time of its occurrence,and not to their low values before transfusionswere given.

Such depression, or even failure, of the reticulo-cyte response to occur after liver extract therapyat artificially elevated red cell and hemoglobinlevels could theoretically be the result of at least2 effects of tlle transfusions. First, the abolitioniof the anemiiia, and consequently of the general ac-cepted stimiiulus to red cell production, anemiicanioxia. Second, the introduction of substancesin the transfused normiial blood equivalent in plhysi-ological action to liver extract, with resulting par-tial or complete inability of the patient to respondto a subsequent injection of liver extract.

Experiments on animals as well as observationsin man have shown that the number of reticulo-cytes in the periplheral blood becomes (lecreased

vhen the tissue oxygen tension is increased either

866


by transfusions or by breathing high concentra-tions of oxygen (16 to 18). In pernicious anemiain relapse, the inverse relationship between heightof reticulocyte response to liver extract and initialred cell (or hemoglobin) level (19) is well known.For red cell levels above 3.5 million per cu. mm.,reticulocyte responses are slight or absent. Thisdiminished response is conceivably due either tothe relatively slight deficiency of the active prin-ciple of liver extract presumably existing in pa-tients with such relatively high blood levels, orto the comparatively slight degree of anemic anoxiaof their bone marrow. The present observations,in which for reasons given below it is probablethat the existing deficiency of liver extract wasnot modified by the previous transfusions, suggestthe latter explanation as the correct one.

As already indicated, the bone marrow wasfound to be strikingly altered in its morphology bytransfusions given prior to the administration ofliver extract. The percentage of typical megalo-blasts in the bone marrow became sharply reducedwithin from 4 to 12 days after transfusions werebegun, as the red cell counts rose in the peripheralblood. Because this effect is also so characteristica sequel of the administration of liver extract, itis necessary to consider whether the transfusionsof normal blood could have supplied, or have beenequivalent to, the active principle of liver extractin these patients.

In numerous observations on patients with per-nicious anemia, there has never been, in our ex-perience, any detectable elevation of reticulocytelevels in the peripheral blood from transfusions of500, or sometimes 1,000 ml. of whole blood duringcontrol periods without other therapy. The nega-tive effect of even larger transfusions upon reticu-locytes is also convincingly shown in Cases Nos. 3and 4 in which responses to the administration ofliver extract followed shortly. This evidence, to-gether with the failure of the white cells and plate-lets to increase, and especially the lack of signifi-cant clinical improvement, are in sharp contrastto the striking effects subsequently observed whenliver extract was administered.

At first thought, it seems remarkable that thecharacteristic appearance of the bone marrow inpernicious anemia, which, since Peabody's (20)work, has been interpreted by many observers asevidence of a specific "maturation arrest," should

be susceptible to rapid modification by transfusionalone. However, it is unlikely that all adult redcell production is at a standstill in untreated per-nicious anemia. Consequently, if it is assumedthat the megaloblasts are the precursors of moremature erythroblasts, and finally of adult reticulo-cytes and adult erythrocytes, the so-called "matu-ration arrest" must be a relative rather than anabsolute state. Indeed, it has been suggested thatthe extensive, highly cellular and erythropoieticallyimmature bone marrow pictuce in pernicious ane-mia is simply the response of that organ to theanemia resulting from a hemolytic process whichdestroys erythrocytes, especially reticulocytes, be-fore they leave the marrow (5). Certainly thegreatly increased output of urobilinogen (21, 22)and of coproporphyrin Type I (7) in the stoolsfind their analogy in the classical hemolytic ane-mias, such as congenital hemolytic jaundice.Moreover, in these undisputed hemolytic anemiasthe bone marrow, though it does not actually con-tain megaloblasts, is extensive and highly cellular,and the dominant erythroid cells are normoblastsor even younger erythroblasts (9, 10). In animalexperiments Steele (8) has shown that the im-maturity of the erythroid cells of the bone marrowincreases with the severity of the anemia producedby blood loss or destruction. Consequently,whether pernicious anemia is an anemia causedby diminished production or by increased destruc-tion of red cells, the stimulus to red cell produc-tion will be decreased if the severity of the anemicanoxia is diminished artificially by blood transfu-sions which do nothing more than raise the hemo-globin concentration in the blood perfusing thebone marrow. Thus, if the development of mega-loblasts in the bone marrow in pernicious anemiais partly proportional to the degree of anemicanoxia of the bone marrow, transfusions of bloodshould have the effect of causing the megaloblaststo diminish in number. The rapid disappearanceof the megaloblasts in the bone marrow as a resultof their probable conversion to move mature formsas suggested by Schwartz (23) is further evidenceagainst any specific genetic origin of these cells.

The available evidence, however, suggests thatboth the anoxia of severe anemia and a deficiencyof an essential factor for maturation are involvedin the production of the classical bone marrowpicture in pernicious anemia. According to

867

C. S. DAVIDSON, J. C. MURPHY, R. J. WATSON,AND W. B. CASTLE

to Davidson, Davis, and Innes (15) the adminis-tration of liver extract may initiate in 6, and com-plete in 72, hours the disappearance of the megalo-blasts from the bone marrow. This result clearlyprecedes any significant rise in red cell and hemo-globin values in the peripheral blood. Therefore,it is highly unlikely that the disappearance of themegaloblasts is entirely secondary to a decreaseof bone marrow anoxia, as a result of an inicreasedhemoglobin concentration in the blood perfusingthe bone marrow. Consequently, the transforma-tion of the megaloblasts cannot be considered toresult wholly from the cessation of a hemolyticprocess, or from increased blood production. In-stead, the administration of liver extract musthave a direct effect on the rate of maturation ofthe erythroid cells. It should be understood, how-ever, that this evidence of relative maturation ar-rest in the formation of megaloblasts does not ex-clude a hemolytic process as contributory to theanemia, nor does it deny the possibility that liverextract therapy also acts by abolishing a processdestructive to reticulocytes, leukocytes, andplatelets.

Because deficiency of the active principle of liverextract, as well as the severity of the anemia, canapparently cause immaturity of the erythroid cellsof the bone marrow, there is nothing paradoxicalin the observation that both liver extract adminis-tration and transfusions tend to diminish themegaloblasts in the bone marrow in perniciousanemia. In other types of equally severe anemias,however, immaturity of the erythroid cells doesnot proceed to the formation of numerous megalo-blasts (9, 10). Consequently, it is logical to as-sume that the anoxia caused by severe anemia is,by itself, insufficient to produce erythroid cellsmore immature than erythroblasts, unless an addi-tional impediment to the development of their pre-cursors is created by a nutritional deficiency of theactive principle of liver extract. Certainly, as wasshown by the failure of transfusions to cause theleukocyte and platelet counts to rise, the establish-ment of normal levels of these elements in theperipheral blood stream also requires the adminis-tration of an active principle of liver extract. Theclose dependency of the red cell, leukocyte, and.platelet responses in pernicious anemia upon acommon nutritional factor in liver extract is fur-ther attested by the similar responses of these

blood elements to pure synthetic L. casei factor(3, 4).

SUMMARYANDCONCLUSIONS

1. Rapidly repeated transfusions of blood givento 5 patients with pernicious anemia in relapse,with rises to normal red cell values in 3, failed toproduce the striking clinical improvement usuallyseen following the administration of liver extract,nor did the white cell or platelet counts increase.Thus, as in other nutritional deficiency diseases,much of the clinical improvement in perniciousanemia occurring promptly after administrationof the missing nutritional element is presumably aresult of specific effects on general cell metabolism,rather than a result of any change in blood values.

2. The previously observed negative effect oftransfusions of usual amounts of blood on reticulo-cytes, leukocytes, and platelets, was shown to ex-tend to larger amounts of blood. Clearly, trans-fusions do not have hematopoetic effects compara-ble to those of liver extract administration.

3. Subsequent intramuscular liver extract ther-apy was followed by rapid clinical improvement,but reticulocyte responses occurred only in thosepatients with red cell counts below 5 million percu. mm. A return to normal values of the whitecells and platelets was observed at all red celllevels. Thus, liver extract therapy produces areticulocyte response only when anemic anoxia,the normal stimulus for red cell production, ispresent; but the responses of white cell and plate-lets are independent of the degree of anemia.

4. Following the blood transfusions, but priorto the liver extract therapy, the bone marrowmegaloblasts characteristic of pernicious anemiadisappeared. Others have shown that liver extracttherapy also causes these cells to disappear beforeany significant change occurs in the red cell orhemoglobin levels of the blood. This proves thata state of "maturation arrest," due to a nutritionaldeficiency, exists. The effect of transfusions sug-gests that this state is relative rather than absolute,and that it can be affected by an artificial decreasein the degree of bone marrow anoxia.

5. Although relative "maturation arrest" of thebone marrow megaloblasts in pernicious anemiaappears to result in part from a deficiency of theactive principle of liver extract, this does not ex-clude increased blood destruction as a contribut-

868


ing, or even as a dominant, factor in causing theanemia.

Weare indebted to Miss Geneva A. Daland for inter-pretation of bone marrow films; to the Surgical Services,Boston City Hospital, for performing sternal marrowbiopsies; and to the Resident Staff of the Second andFourth Medical Services, Boston City Hospital, for theircooperation in caring for the patients during this study.

BIBLIOGRAPHY

1. Minot, G. R., and Murphy, W. P., A diet rich inliver in the treatment of pernicious anemia; studyof one hundred and five cases. J. A. M. A., 1927,89, 759.

2. Sturgis, C. C., and Isaacs, R., Desiccated stomach inthe treatment of pernicious anemia. J. A. M. A.,1929, 93, 747.

3. Moore, C. V., Bierbaum, 0. S., Welch, A. D., andWright, L. D., The activity of synthetic lactobacil-lus casei factor ("Folic acid") as an anti-perniciousanemia substance. I. Observations on four pa-tients: two with Addisonian pernicious anemia, onewith non-tropical sprue, and one with perniciousanemia of pregnancy. J. Lab. and Clin. Med.,1945, 30, 1056.

4. Vilter, C. F., Spies, T. D., and Koch, M. B., Furtherstudies on Folic acid in the treatment of macrocyticanemia. South. Med. J., 1945, 38, 781.

5. Dock, W., The importance of hemolysis in the patho-genesis of macrocytic anemia. Medical Papersdedicated to Dr. Henry A. Christian, p. 545 to558, Williams and Wilkins Co., Baltimore, 1936.

6. Dock, W., The ebb and flow of theories about per-nicious anemia. Am. J. Clin. Path., 1938, 8, 620.

7. Dobriner, K., and Rhoads, C. P., The metabolism ofblood pigments in pernicious anemia. J. Clin. In-vest., 1938, 17, 95.

8. Steele; B. F., The effects of blood loss and blood de-struction upon the erythroid cells in the bone mar-row of rabbits. J. Exper. Med., 1933, 57, 881.

9. T6tterman, G., Das Knochenmark bei hamolytischenIkterus mit einem Beitrag zur Frage nach derNatur der Megaloblasten. Acta Med. Scand.,1936, 90, 527.

10. Dameshek, W., and Schwartz, S. O., Acute hemolyticanemia (acquired hemolytic icterus, acute type).Medicine, 1940, 19, 231.

11. Wintrobe, M. M., Clinical Hematology, 703 pp., Phila-delphia, Lea and Febiger, 1942.

12. Pohle, F. J., Blood platelet count in relation to themenstrual cycle in normal women. Am. J. M. Sc.,1939, 197, 40.

13. Murphy, W. P., An easy method of estimating theamount of jaundice by means of the blood serum,Boston M. and S. J., 1926, 194, 297.

14. Turkel, H., and Bethell, F. H., Biopsy of the bonemarrow performed by a new and simple instrument.J. Lab. and Clin. Med., 1943, 28, 1246.

15. Davidson, L. S. P., Davis, L. J., and Innes, J., Theeffect of liver therapy on erythropoiesis as observedby serial sternal punctures in twelve cases of perni-cious anemia. Quart. J. Med., 1942, 11, 19.

16. Campbell, J. A., Prolonged alterations of oxygen pres-sure in the inspired air with special reference totissue oxygen tension, tissue carbon dioxide tension,and hemoglobin. J. Physiol., 1927, 62, 211.

17. Boycott, A. E., and Oakley, C. L., The regulation ofmarrow activity: experiments on blood transfusionand on the influence of atmospheres rich in oxygen.J. Path. and Bact., 1933, 36, 205.

18. Reinhard, E. H., Moore, C. V., Dubach, R., and Wade,L. J., Depressant effects of high concentrations ofinspired oxygen in erythrocytogenesis. Observa-tions on patients with sickle cell anemia with adescription of the observed toxic manifestations ofoxygen. J. Clin. Invest., 1944, 23, 682.

19. Minot, G. R., Murphy, W. P., and Stetson, R. P., Theresponse of the reticulocytes to liver therapy, par-ticularly in pernicious anemia. Am. J. M. Sc.,1928, 175, 581.

20. Peabody, F. W., The pathology of the bone marrowin pernicious anemia. Am. J. Path., 1927, 3, 179.

21. Eppinger, H., and Charnas, D., Was lehren uns quan-titative Urobilinbestimmungen im Stuhl? Ztschr. f.klin. Med., 1913, 78, 387.

22. Watson, C. J., Studies of Urobilinogen. III. The perdiem excretion of urobilinogen in the common formsof jaundice and liver disease. Arch. Int. Med.,1937, 59, 206.

23. Schwarz, E., The nature of the megaloblast. Hematol.Monographs, 1946, 1, 3.

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patients deficiency) 7). hypothesis,...

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