Congenital refractory normoblastic anemia with jaundice and ineffective erythropoiesis

Case Reports Congenital Refractory Normoblastic Anemia with Jaundice and Ineffective Erythropoiesis* CAPT . ROBERT M . GREENDYKE, MC, USAt

Fort Sam Houston, Texas

C

onsIDERAnLE attention has been paid in re-

clinical course of certain of these patients seems to substantiate such an interpretation [2,28] . It is evident from even casual study of the reported cases of refractory anemia that a heterogeneous group of diseases is under consideration . It is the purpose of this report to record the results of an investigation recently carried out on a young man with a therapeutically resistant, partially compensated normoblastic anemia characterized by jaundice and significant ineffective erythropoiesis, and to note some of the possible relationships between his disease and certain other types of anemia .

cent years to the ill defined group of "refractory anemias ." Since the subject was reviewed by Bomford and Rhoads [3] in 1941, appreciable progress has been made in the study and categorization of therapeutically resistant anemia . The use of radioisotope labels has contributed greatly to our understanding of the abnormalities of erythrocyte production and destruction in this group of diseases . Recent contributions by Dacie et al . [10], Heilmeyer [25], Crosby and Sheehy [8], and Vilter et al . [51] have summarized much of the newer knowledge on the subject . As judged by current standards, many of the older descriptions of patients with "refractory anemia" lack sufficient information to permit evaluation, and patients with hyperplastic bone marrow were considered under the same heading as those with hypoplastic marrow . Recently a group of patients with an hereditary disorder characterized by normoblastic crythroid bone marrow hyperplasia with marked iron loading ("anemia sideroachrestic hereditaria" of Heilmeyer) has been separated out [14,25,27,38,39] and contrasted [24-26] with the acquired form of refractory anemia with iron loading described by several authors and reviewed by Dacie et al . [10], Heilmeyer [25], and Crosby and Sheehy [8] . Similarly, cases of pyridoxine responsive hypochromic anemia have been discovered [1,12,15, 22,36,50], one of which had been previously relegated to the refractory group [44] . In two similar cases the patients were found to he responsive to an orally administered liver extract [30] . It has also been suggested that certain of the cases in this group in reality represent chronic forms of erythremic myelosis [11,23,25], and the

METHODS

Routine hematologic studies were carried out by the methods outlined by Ham 120] . Platelet counts were made by the phase method of Brecher and Cronkite [4] . Marrow sideroblasts were sought by the staining method of Kaplan et al . [32] . Leukocyte alkaline phosphatase was estimated histochemically by the method of Kaplow [33] . Iron deposits in the liver biopsy were sought using the Gridley modification of the Mallory teshnic [78] . Hepatic function tests were performed by standard procedure [21,34,40,47, 43,16] . Serum iron was determined by the method of Walker [52], serum hemochromogens by the method of Hunter et al . [31], quantitative urobilinogen determinations by the technic of Schwartz et al . [45], erythrocyte protoporphyrins by the method of Grinstein and Wintrobe [19], electrophoresis of hemoglobin as described by Goldberg [17], and alkali-resistant hemoglobin by the method of Singer et al . [47] . Osmotic fragility studies and serologic investigations were carried out according to Dacie [9] . The radioisotope procedures, including red cell halflife estimation using Cr", plasmaa iron turnover and red cell radioiron incorporation employing Fe 69, plasma volume using RISA, and the Schilling test with Co 80-labeled vitamin B IZ were performed with

* From the Department of Pathology, Brooke General Hospital, Brooke Army Medical Center, Fort Sam Houston, Texas . Manuscript received May 5, 1961 . t Present address : P . 0 . Box 152, Pittsford, New York. VOL .

32, APRIL 1962

611



612

Ineffective Erythropoiesis-Greendyke

slight modifications of the technics described by Veall and Vetter [49] . CASE REPORT

A seventeen year old white man was referred in January 1960 with chief complaints of mild anemia present since birth and progressively increasing jaundice observed over the past five years . The patient was born at term after an uneventful pregnancy . Birth weight was 7 pounds and 14 ounces (3,600 gm .) . Because of pallor at three weeks of age, the patient was hospitalized . No jaundice was noted at this time . The red cell count was 1 .75 million per cu . mm ., the white blood cells 6 thousand per cu . mm. with a normal differential count . Investigation revealed both parents and patient to be of blood group A, Rh positive . The patient was given a transfusion of 50 ml . of whole blood and discharged on a regimen of oral dietary supplements of ferrous sulfate and powdered liver . Follow-up over the next five months revealed a progressive rise in red blood cells to 3 .5 million per cu . mm . and weight gain to 14 pounds and 8 ounces (6,600 gm .) . Over the ensuing seven years the patient's growth and development was normal . Measles and tonsillitis constituted the only significant illnesses . Jaundice was never observed . During this time he was hospitalized on several occasions for investigation of mild asymptomatic anemia . Slight pallor was the only physical finding that could ever be demonstrated . At two and a half years of age the red cells ranged between 3 .7 and 3 .8 million per cu . mm ., and the hemoglobin between 11 .2 and 12 .4 gm. per cent. Red cell anisocytosis and poikilocytosis were described in peripheral blood mews, and treatment with ferrous sulfate and liver was again prescribed . At five and a half years of age the patient was admitted to the hospital for tonsillectomy, at which time a red cell count of 3.8 million per cu . mm. and hemoglobin of 11 gm . per cent were recorded . At seven years of age the patient was admitted to a second hospital for evaluation of persistent mild anemia . The red cell count at this time was 3 .2 million per cu . mm. and hematocrit 36 volumes per cent . The patient was given a transfusion of 500 ml . of whole brood and discharged . Readmission workup seven months later disclosed : red blood cells 3 .8 million per cu. mm ., hemoglobin 13 .9 gm . per cent, hematocrit 40 volume per cent, and reticulocyte count 0 .9 per cent . Determinations of serum bilirubin, non-protein nitrogen, red cell osmotic fragility, platelet and white cell counts, Wassermann test, urinalyses, intravenous pyelogram, and radiographic estimation of bone age were normal . The patient was first seen in this hospital in January 1955, at twelve years of age, for evaluation of his anemia . Physical examination showed normal development and only slight pallor, with no other abnormal findings . The red cell count was 3 .8 million per cu

., ,m hemoglobin 12 .2 gm . per cent, hematocrit 37 volume per cent, and reticulocyte count 0 .2 per cent . Anisocytosis and poikilocytosis were noted in the peripheral blood smear . Smear of sternal bone marrow revealed hypercellularity with promincnt but orderly normoblastic erythroid hyperplasia and a myeloid : erythroid ratio of 1 : 2 .3 . The patient was followed up for the next two years . Mild jaundice was observed for the first time at twelve and a half years of age and persisted thereafter . He was treated with courses of oral folic acid, oral and parenteral vitamin B,z, and oral multiple vitamins without response . Repeated determinations over this period disclosed red cell counts in the range of 3 to 3 .9 million per cu . mm., hemoglobins of 10 to 12 .8 gm. per cent, hematocrits of 30 to 40 volume per cent, and reticulocyte counts of 0 .2 to 1 .7 per cent . The patient remained asymptomatic during the period of observation . The patient was referred to the author in January 1960, at the age of 16 years, for re-evaluation of continuing mild anemia and jaundice which in the past three years had become increasingly conspicuous . There was no history of dietary deficiency, exposure to potentially toxic chemicals, or symptoms of gastrointestinal or biliary tract disease . The patient was asymptomatic . Physical examination revealed a lean, well developed, intelligent white man, 6 feet 3 inches tall (189 cm .), and weighing 164 pounds (74 .5 kg .) . Moderate icterus was apparent, but no other significant physical findings were demonstrated . There was no palpable enlargement of liver or spleen, and no neurologic deficit . Initial laboratory data were as follows : red blood cells 4 .1 million per cu . mm ., hemoglobin 11 .6 gm. per cent, hematocrit 35 volume per cent, reticulocyte count 0.4 per cent, white blood cells 6 .2 thousand per cu . mm. with a normal differential count, and platelet count 240,000 per cu . mm . Mean corpuscular volume was 85 cu . p ., mean corpuscular hemoglobin 28 µµg. per cent, and mean corpuscular hemoglobin content 33 per cent. Peripheral blood smear (Fig . 1) showed moderate anisocytosis and poikilocytosis, scant polychromatophilia, and no basophilic stippling, spherocytes or target cells . Most of the erythrocytes were notmochromic, but some of the abnormal forms appeared deficient in hemoglobin content . There were no morphologic abnormalities of leukocytes or platelets . Urinalysis was normal, with no detectable protein, sugar, bile, or abnormality in the sediment. Smears of aspirated sternal bone marrow (Fig . 2) revealed an appearance similar to that of the examination carried out five years previously, exhibiting hypercellularity and brisk normoblastic erythroid hyperplasia, with a myeloid : erythroid ratio of 1 :1 .7 . There were no megaloblastic features, but occasional degenerating or abnormal erythrocyte precursors were noted . Leukocyte abnormalities were not seen . Stainable marrow iron was 2 plus. Examination for sideroblasts revealed no increase in amount or coarse-

AMERICAN JOURNAL OF MEDICINE

Ineffective

Erythropoiesis-Greendyke

Ftn . 1 . Peripheral blood exhibiting anisocytosis and poikilocytosis with scattered hypoehromic cells . Wright's stain, original magnification X 475 .

613

Fm . 2 . Bone marrow displaying normoblastic hyperplasia. Wright's stain, original magnification X 475 .

ness of granulation . Siderocytes were absent from the peripheral blood smear . The leukocyte alkaline phosphatase score fell in the middle of the normal range for this laboratory . Further laboratory testing showed a serum total bilirubin of 7 .4 mg ., direct-reacting bllirubin of 0 .2 mg., and serum iron of 144 µg . per 100 ml . Hemoglobin electrophoresis on paper and on starch block* showed only type A present . Starch block electrophoresis revealed the A2 fraction to be 2 per cent . The alkali-resistant fraction was 0 .9 per cent . Reactions to direct and indirect Coombs' test were negative . Red cell osmotic fragility, using fresh cells and after twenty-four hours incubation, was normal . Normal roulcaux formation was observed in wet preparations of the patient's erythrocytes . Test results of hepatic function, including bromsulfalein retention, serum glutamic oxalacetic transaminase, thymol turbidity, cephalin-cholesterol flocculation, and serum alkaline phosphatase were normal . Other studies carried out with normal or negative results included a gallbladder series ; repeated urinalyses; white blood counts; stool examinations for ova, parasites, and occult blood ; Schumm test ; determination of serum hemochromogens ; Ham acid serum test ; tests for Donath-Landsteiner antibody, cold agglutinins, and cold hemolysins ; lupus erythematosus tests ; tests for stainable iron in urine sediment; determination of blood urea nitrogen ; an oral glucose tolerance test ; determination of total serum proteins ; and paper electrophoresis of serum proteins. Quantitative determinations of fecal urobilinogen excretion were carried out over five periods of seventytwo to 120 hours over a two month period with the following results : period 1 (ninety-six hours), 1,170 mg . ; period 2 (ninety-six hours), 1,209 mg . ; period 3

(120 hours), 1,471 mg . ; period 4 (seventy-two hours), 830 mg . ; and period 5 (seventy-two hours), 1,148 mg . Two quantitative determinations of twenty-four hour excretion of urine urobilinogen during period 1 were 9 .8 and 6.9 mg . and two additional twenty-four hour specimens during period 3 yielded 4 and 6 .2 mg . Determinations of twenty-four hour urinary porphyrin excretion during periods 1 and 3 showed no detectable porphobilinogen or uroporphyrin, and corproporphyrin excretion of 108 and 94 mg . (normal : 2.0 to 100 mg.) . Determination of erythrocyte protoporphyrins yielded a value of 18 µg . per 100 ml . of packed erythrocytes compared with control values of 31 and 34 µg . (normal :-30 to 35 pg . per 100 ml . packed erythrocytes) 1191A needle biopsy specimen of the liver was entirely normal, with no evidence of inflammatory or degenerative changes, fibrosis, cholestasis, or pigment deposition . No stainable iron was present . Radioisotope studies were carried out* to determine the life span of the patient's erythrocytes and to investigate his iron metabolism. Using Cr'' tagging, the apparent half survival of the patient's red cells in his own circulation was twenty-nine days (normal in this laboratory is twenty-six to thirty-eight days), corresponding to an actual mean cell life span of approximately 100 days . External surface counting over the liver, spleen and heart during the sixty day period of study showed no evidence of abnormal accumulation of radioactivity in the spleen or liver . Studies of the patient's iron metabolism using Fess revealed a plasma clearance half-time of forty-two minutes (normal : sixty to 120 minutes) . Using a plasma volume determined by the RISA method, the plasma iron turnover was 0 .99/mg./kg./day (normal : 0 .46 to 0 .75 mg ./kg./day) . Iron reincorporation in the red blood cells was diminished in comparison to

* This study was conducted through the courtesy of Mrs . Lily Singer, Hematology Department, Michael Reese Hospital, Chicago, Illinois .

* These studies were conducted by Capt . Perry C. Smith, MC, Chief, Radioisotope Clinic, Brooke General Hospital .

VOL . 32, APRIL 1962

614

Ineffective Erythropoiesis-Greendyke

normal, with 12 per cent incorporated by the second day, 46 per cent by the sixth day, 68 per cent by the twelfth day, and 64 per cent on the fourteenth day (normal maximum : eighty to 100 per cent) . A Schilling test to demonstrate absorption of Cc"labeled vitamin Bls was normal, 36 per cent of the administered radioactivity being recovered in the twenty-four hour urine . Family history revealed that the patient's parents are both of northern European extraction . There is no family history of jaundice, anemia or splenomegaly . Both grandmothers are said to have gallbladder disease . Investigation of the family, including the father, mother, paternal grandmother and two sisters revealed normal values for hemoglobin, hematocrit and reticulocytes, and normal peripheral blood smears, except initially in the case of the patient's mother . She was found to be suffering from moderate typical iron deficiency anemia which responded readily to administration of oral ferrous sulfate with rapid restoration of blood values and erythrocyte morphology to normal . During the year in which the patient has been followed up, mild fluctuations in blood values have occurred unrelated to any discernible factor . Values for hemoglobin have ranged from 11 .5 to 13 .4 gm. per cent, hematocrit from 35 to 40 volume per cent, reticulocytes from 0 .2 to 1 .7 per cent, serum bilirubin (total/direct) from 8/0 .8 to 3 .8/0 .7 mg. per cent, and serum iron from 109 to 202 pg . per cent . Separate therapeutic trials of a number of agents have been conducted empirically . These have included ferrous sulfate (1 gm . given orally daily for two months), vitamin B11 (100 mg . given intramuscularly three times a week for four weeks), folic acid (15 mg . given orally daily for three weeks), crude liver extract (45 cc . given orally daily for four weeks), crude liver extract (5 cc . given intramuscularly three times a week for four weeks), and pyridoxine hydrochloride (100 mg. given intramuscularly three times a week for three weeks) . No consistent alteration in the subjective state of the patient or in hemoglobin, hematocrit, reticulocyte, serum iron or serum bilirubin levels, or in peripheral blood morphology, could be demonstrated during treatment . COMMENTS A number of points in connection with this case seem worthy of emphasis . The congenital nature of the disorder seems established in light of the documented history of anemia since birth . The basic abnormality appears to be one of faulty erythropoiesis, although enough functionally normal cells are being produced by the compensatory hyperplasia of the erythroid marrow elements to result in almost normal peripheral blood values .

The concept of ineffective erythropoiesis, as developed by Finch and his co-workers [13], involves the production of defective erythrocytes which are destroyed either as precursors or mature cells within the marrow, or at least almost immediately upon release into the peripheral blood . This process is in contrast to effective erythropoiesis, which results in delivery of normal erythrocytes to the circulation . Although occurring normally to a slight degree, significant ineffective erythropoiesis is characteristic of a number of anemias . Proof of ineffective erythropoiesis derives from the demonstration of a significant discrepancy, as in the case reported, between measures of total and effective erythropoiesis . Among the measurements used to determine total erythropoiesis are the enumeration of total erythroid precursors in smears of aspirated bone marrow (myeloid : erythroid ratio), determination of total excretion of breakdown products of hemoglobin, and measurement of plasma iron turnover using Fe" . Effective erythropoiesis is estimated from reticulocyte counts and from percentage erythrocyte uptake of injected radioiron . It is immediately appreciated that quantitative measurements using these technics may entail considerable technical error, but when in agreement among themselves, and when significance is attached only to values several times the expected, such measurements are of value . In the case presented, total erythropoiesis as estimated by the myeloid :erythroid ratio in the bone marrow smears of 1 : 1 .7 is roughly three to four times normal . Several determinations of fecal urobilinogen excretion revealed an average value of 300 mg, per twenty-four hours, yet based on the red cell mass determined as 700 gm., the estimated red cell life span of 100 days, and the conversion ratio of 35 mg . of urobilinogen excreted for each gram of hemoglobin catabolized, the theoretic maximum urobilinogen excretion in this patient should be only in the range of 250 mg, per twenty-four hours . In light of the fact that determination of fecal urobilinogen is notoriously incomplete [7,10,16], it is evident that excretion of urobilinogen is probably at least twice the theoretically expected value . Further, the plasma iron turnover of 0 .99 mg ./kg ./day is approximately twice the usual normal value encountered in this laboratory . These values lie in sharp contrast to the measures of effective erythropoiesis : the reticulocyte counts below 1 per cent, and the subnormal AMERICAN JOURNAL OF MEDICINE



Ineffective Erythropoiesis-Greendyke radioiron incorporation maximum of 68 per cent . The discrepancy between the indices of total and effective erythropoiesis thus demonstrated is interpreted as representing ineffective erythropoiesis . Inasmuch as the Cr" studies indicated a normal life span for the patient's peripheral blood erythrocytes, the excess pigment excretion must be accounted for by hemoglobin breakdown from cells not included by this technic ; i .e ., bone marrow erythrocyte precursors or peripheral blood cells disappearing from circulation shortly after entering . Similarly, the brisk erythropoiesis suggested by the bone marrow smears and plasma iron turnover value is not reflected in the peripheral blood by reticulocyte values or erythrocyte radioiron uptake and is best explained in like fashion . The reason for the patient's jaundice is not apparent . Crosby [7] is of the opinion that the normal liver is capable of metabolizing two to four times the normal load of bile pigment without the appearance of jaundice . In light of the measured rates of total production and destruction of two to four times normal in this patient, significant jaundice would not be anticipated . A similar discrepancy between degree of bilirubinemia and rate of hemoglobin breakdown was noted by Conrad et al . [6] in their study of hereditary non-spherocytic hemolytic disease . These authors noted patients with mild hemolysis whose serum bilirubin levels frequently exceeded those in patients with pronounced hemolysis, and were forced to postulate a defect in bilirubin metabolism in addition to the intrinsic erythrocyte abnormality . Similarly, some defect in bile pigment metabolism may be postulated in this patient, but must exist in the face of the normal liver biopsy and normal hepatic function tests . Uncomplicated constitutional hepatic dysfunction (Gilbert's disease), of course, does not exhibit hematologic abnormalities such as those described in this patient . In view of the recent suggestion by Kent and Popper [35] that secondary hemochromatosis is a possible complication of anemias characterized by defective iron metabolism (ineffective erythropoiesis) and especially because of the frequency with which hemochromatosis has been reported in certain refractory anemias (as emphasized by IIeilmeyer [25,26,29]), hemosiderin deposition was sought in the liver of this patient, without success. VOL . 32, APRIL 1962

615

Inasmuch as the usual hemolytic diseases are excluded in this case by the normal red cell life span, the differential diagnosis is essentially confined to a number of conditions characterized by significant amounts of ineffective erythropoiesis . Erythrokinetic studies similar to those obtained in this patient have been reported in cases of thalassemia major [48] and minor [421, pernicious anemia [13,37], pyridoxine deficiency [50], erythremic myelosis [11], and refractory anemia [10,14,25,26,51], including the previously mentioned "sideroachrestic anemia ." Thalassemia is ruled out in this case by a number of atypical findings, including the family studies, normal osmotic fragility, and absence of an elevated A2 or alkali-resistant hemoglobin fraction . The absence of a megaloblastic marrow, the lifelong history, and the failure to respond to usually effective therapeutic agents argue against megaloblastic anemia . A therapeutic trial of pyridoxine was ineffectual . Despite the modest morphologic abnormalities of the crythroid series in the bone marrow, one would expect erythremic myelosis to declare itself in the course of seventeen years . The usual reported cases of refractory anemia are not characterized by jaundice of the degree present in this patient, and the coarse iron granulation of the normoblasts and the tissue siderosis typical of sideroachrestic anemia are absent . In many other respects, however, this case bears similarity to the hereditary form of sideroachrestic anemia described by Garby et al . [14], by Heilmeyer [24-27], and by Ludin [38,39] . IIeilmeyer has particularly commented on the "cryptogenic hemolysis" found in his cases of siderochrestic anemia [25] . He separates the condition into congenital and acquired forms on the basis of the erythrocyte protoporphyrin values, which are low in the congenital form and normal in the acquired disease . He suggests that the acquired disease, which is an affliction of the elderly, may bear a close resemblance to the condition otherwise known as chronic erythremic myelosis, whereas the congenital form bears no such relationship and has a favorable outlook . Of note are the low erythrocyte protoporphyrin values obtained in our patient . In this connection, however, it should be noted that low erythrocyte protoporphyrins have also been reported in pyridoxine-deficient animals [5] and also in human subjects with pyridoxine deficiency anemia [50] . The possible interrelationship between the



616

Ineffective Erythropoiesis-Greendyke

case reported here and other forms of refractory anemia remains unclear . Further reporting and study of such cases is desirable in an attempt to distinguish among the various entities described in the category of "refractory anemia ." SUMMARY A case of congenital, partially compensated, therapeutically refractory, normoblastic anemia is presented . The patient's disease is characterized by jaundice, marked ineffective erythropoie-

sis

and absence

of siderosis . The differential

diagnosis is discussed . REFERENCES 1 . BISHOP, R . C . and BETHELL, F . H . Hereditary hypochromic anemia with transfusion hemosiderosis treated with pyridoxine . New England J. Med., 261 : 496, 1959 . 2 . BJoRKMAN, S . E . Chronic refractory anemia with sideroblastic bone marrow . Blood, 11 : 250, 1956 . 3 . BoMFORD, R . R . and RHOADS, C. P . Refractory anemia. Quart. J. Med., 10 : 175, 1941 . 4 . BRECHER, G. and CRONKITE, E . P . Morphology and enumeration of human blood platelets . J. Appl. Physiol., 3 : 365, 1950 . 5 . CARTWRIGHT, G. E . and WINTROBE, M . M. Studies on free erythrocyte protoporphyrin, plasma copper, and plasma iron in normal and in pyridoxinedeficient swine. J. Biol. Chem ., 172 : 557, 1948 . 6 . CONRAD, M. E ., JR ., CROSBY, W . H, and Howig, D . L . Hereditary nonspherocytic hemolytic disease . Am . J. Med., 29 : 811, 1960 . 7 . CRosuv, W . H . The hemolytic states . Ball . New York Acad. Med., 30 : 27, 1954. 8. CROSBY, W. H. and SHEEHY, T. W. Hypochromic iron loading anemia : studies of iron and hemoglobin metabolism by means of vigorous phlebotomy. Brit . J. Hemat., 6 : 56, 1960 . 9. DACIE, J . V. Practical Hematology, 2nd ed . New York, 1956 . Chemical Publishing Co . 10. DAGIE, J . V., SMITH, M . D., WHITE, J. C. and MOLLIN, D . L . Refractory normoblastic anemia : a clinical and hematological study of seven cases . Brit . J. Hemat., 5 : 56, 1959 . 11 . DAMASHEK, W . and BALDINI, M . The DiGuglielmo syndrome . Blood, 13 : 192, 1958 . 12. ERSLEV, A . J ., LEAR, A. A. and CASTLE, W . B. Pyridoxine responsive anemia . New England J. Med., 262 : 1209, 1960 . 13. FmcH, C . A ., COLEMAN, D . H ., MoTuLsxy, A . G ., DoNOHUE, D . M . and REIFF, R . H . Erythrokinetics in pernicious anemia . Blood, 11 : 807, 1956 . 14 . GARBY, L., SJ6LIN, S . and VAHLQUIST, B . Chronic refractory anemia with disturbed haem metabolism . Brit. J. Hemat., 3 : 55, 1957 . 15 . GEHRMANN, G . Pyridoxinmangel-Anomie beim Menschen . Folia Hemat., 2 : 225, 1958 . 16 . GIBLETT, E . R ., COLEMAN, D . H ., PIREIo-BIROLI, G., DONOHUE, D . M. MOTULSKY, A . G. and FINCH, C. A. Erythrokinetics : quantitative measurements of red cell production and destruction in normal

subjects and patients with anemia . Blood, 11 : 291, 195617 . GOLDBERG, C . A . J . Identification of human hemoglobins. Clin . Chem ., 3 : 1, 1957. 18 . GRIDLEY, M. F. Manual of Histologic and Special Staining Techniques, p . 149 . Washington, D . C., 1957 . Armed Forces Institute of Pathology . 19. GRINSTEIN, M . and WINTROBE, M . M . The free erythrocyte protoporphyrin . J. Biol. Chem ., 172 : 459,1948 . 20. HAM, T . H ., Ed. A Syllabus of Laboratory Examinations in Clinical Diagnosis . Cambridge, 1951 . Harvard University Press . 21 . HANGER, F. M . Serological differentiation of obstructive from hepatogenous jaundice by flocculation of cephalin cholesterol emulsion . J. Clin. Invest., 18 : 261, 1939 . 22 . HARRIS, J. W., WHITTINGTON, R . M., WEISMAN, R ., JR . and HORRIGAN, D . L . Pyridoxine responsive anemia in the human adult . Proc . Soc. Exper . Biol. & Med., 91 : 427, 1956 . 23 . HAYHOE, F. C . J . and QUAGLINO, D . Refractory sideroblastic anemia and erythremic myelosis : possible relationship and cytochemical observations . Brit. J. Hemat ., 6 : 381, 1960. 24 . HEILMEVER, L. Les anemies sideroachrestiques . Le Sang, 29 : 465, 1958 . 25 . HEILMEVER, L. Die sideroachrestischen Anamien . Deutsch . med. Wchnschr., 84 : 1761, 1959 . 26 . HEILMEVER, L . Die sideroachrestischen Anamien . In : Proceedings of the Seventh Congress of the European Society of Hematologists, London, 1959 . Arta hemat ., 24 : 19, 1960 . 27 . HEILMEVER, L ., EMMRICH, J ., HENNEMANN, H . H ., KEMERLING, W ., LEE, M ., BIGGER, R . and ScHOBOTHE, H. Uber ein chronische hypochrome Anomie bei zwei Geschwistern auf der Grundlage einer Eisenverwertungsstorung (Anemia hypochromica sideroachrestica hereditaria) . Folia 11emat ., 2 : 61, 1958 . 28 . HEILMEVER, L., KEIDERLING, W ., BIGGER, R . and BERNAUER, II . Uber chronische refraktare Anamien mit sideroblastischen Knochenmark . Folia Hemat., 2 : 49, 1958 . 29 . HEILMEVER, L ., KEIDERLING, W., MERKER, H ., CLOTTEN, R . and SCHUBOTHE, H . Die Anemia refractoria sideroblastica and ihre Beziehungen zur Lebersiderose and Hamochromatose . Acts hemat ., 23 : 1, 1960 . 30 . HORRIGAN, D . L ., WHITTINGTON, R . M ., WEISMAN, R ., JR. and HARRIS, J . W . Hypochromic anemia with hyperferricemia responding to oral crude liver extract. Am. J. Med., 22 : 99, 1957 . 31 . HUNTER, F . T., GROVE-RASMUSSEN, M . and SOIrrrER, L . A spectrophotometric method for quantitating hemoglobin in plasma or serum . Am . J. Clin . Path., 20 : 429, 1950 . 32 . KAPLAN, E ., ZUEIZER, W . W. and MOURIQUANT, C . Sideroblasts : A study of stainable non-hemoglobin iron in marrow normoblasts . Blood, 9 : 203, 1954 . 33 . KM-Low, L . S . A histochemical procedure for localizing and evaluating leukocyte alkaline phosphatase activity in smears of blood and bone marrow. Blood, 10 : 1023, 1955 . 34. KARMEN, A . A note on the spectrophotometric assay AMERICAN JOURNAL OF MEDICINE



Ineffective Erythropoiesis-Greendyke

35 .

36 .

37 .

38 .

39 . 40 .

41 .

42 .

43 .

of glutamic-oxalacetic transaminase in human blood serum . J. Clirs . Invest., 34 :131,1955 . KENT, G . and POPPER, H . Secondary hemachromatosis : its association with anemia . Arch. Path., 70 : 623, 1960. LREInNG, J . T . and WH.xmsoN, J . F . Hypochromic anemia in an adult responding to pyridoxine hydrochloride . Clin . Res., 7 : 208, 1959 . LONDON, I . M . and WEST, R . The formation of bile pigments in pernicious anemia . J. Biol. Chem ., 194 : 359, 1950 . LUDIN, H . Discussion of Heilmeyer, L . : Ober ein ncuartige hypochrome Anionic bei zwie Geschw istern auf der Grundlage Liner Eisenverwertungsstorung (Anemia sideroachrestica hereditaria) . Presented at Twelfth Swiss Hematologic Congress, 1957 . Schweiz . med. Wchnschr ., 87 : 1237, 1957 . LOmN, H . Personal communication . MACLAGEN, N . F . The thymol turbidity test as an indicator of liver dysfunction . Brit . J. Exper. Path ., 25 : 234, 1944 . MALLOY, H . T . and EVELYN, K . A. The determination of bilirubin with the photoelectric colorimeter. J. Biol . Chem., 119 : 481, 1937 . PEARSON, H . A., MCFARLAND, W. and KING, E . R . Erythrokinetic studies in thalassemia trait . J. Lab . & Clin . Med., 56 : 866, 1960 . ROSENTHAL, S . M . and WHITE, E . C . Clinical application of the bromsulphalein test for hepatic function. J. A . M. A ., 84 : 1112, 1925 .

VOL . 32, APRIL 1962

617

44 . RUNDLES, R . W . and FALLS, H . F . Hereditary (sex linked) anemia . Am . J. M. Sc ., 211 : 641,1946 . 45 . SCHWARTZ, S ., SEoRov, V. and WATSON, C . J . Studies of urobilinogen . iv . Quantitative determination of urobilinogen by means of Evelyn photoelectric colorimeter . Am . J. Clin. Path ., 14 : 598, 1944 . 46 . SIGNOWARA, G . Y ., JONES, L . M . and REINHART, H. L. The estimation of serum inorganic phosphate and acid and alkaline phosphatase activity . J. Biol. Chem ., 142 : 921, 1942 . 47 . SINGER, K ., CHERNOFF, A . 1 . and SINGER, L. Studies on abnormal hemoglobins . L Their demonstration in sickle-cell anemia and other hematologic disorders by means of alkali denaturation . Blood, 6 : 413, 1951 . 48 . STURGEON, P . and FINCH, C . A. Erythrokinetics in Cooley's anemia. Blood, 12 : 64, 1957 . 49 . VEALL, N, and VETTER, H . Radioisotope techniques in clinical research and diagnosis . London, 1958 . Butterworth . 50. VERLOOP, M . C . and RADEmARER, W. Anemia due to pyridoxine deficiency in man . Brit. J. Hemat ., 6 : 66, 1960 . 51 . VILTER, R . W ., JARROLD, T., WILL, J. J ., MUELLER, J . F ., FRIEDMAN, B . 1 . and HAWKINS, V . R . Refractory anemia with hyperplastic bone marrow. Blood, 15 : 1, 1960 . 52 . WALKER, B . S. The iron of human blood serum . J. Lab . & Clirs . Med., 24 : 308, 1939 .