British Journal of Haematology, 1975, 31,
$15.
Haem Synthesis in the Diamond-Blackfan Syndrome MELVIN H. FREEDMAN, DOMINICK AMATO AND E. FREDSAUNDERS
Division of Haeniatology and Department of Pediatrics, The Hospital-for Sick Chi/dren and The University of Toronto, Toronto, Ontario (Received 20 March 1975; accepttadfor publication 21 M a y 1975) SUMMARY. The response of bone marrow to erythropoietin (EPO) from five children with the Diamond-Blackfan syndrome, also known as congenital hypoplastic anaemia (CHA), was tested in tissue culture by measurement of haem synthesis. Studies of 13 control marrows indicated that the maximum EPO effect occurred at approximately 70 h incubation using an EPO concentration of 0.2-0.3 units/ml and a nucleated cell concentration of 5 x 106 per culture. Under these conditions, haem synthesis was 121% greater in EPO-stimulated than in unstimulated cultures. Patients with CHA with anaemia and diminished marrow erythroids had reduced or absent haem synthesis. In one patient, haem production became normal after a spontaneous remission, and was not inhibited by autologous plasma drawn at the time of diagnosis. Plasma from three patients did not show inhibitory activity when cultured with control marrow. In contrast, plasma from an adult with acquired pure red cell aplasia produced striking inhibition of haem synthesis when cultured with control marrow. W e conclude that, in comparison to some cases of the adult acquired condition, CHA is not due to inhibitors or antibodies. When present, erythroid precursors in children with CHA are capable of responding normally to EPO with increased haem synthesis. The Diamond-Blackfan syndrome, or congenital hypoplastic anaemia (CHA) is due to failure of red cell production. It is characterized by normochromic, normocytic red cells, absent reticulocyte response, and a normocellular marrow aspirate with diminished to absent nucleated erythroids. About one-half of these children experience a complete remission when treated with corticosteroids (Allen & Diamond, 1961).The serum and urinary erythropoietin (EPO) levels are consistently elevated (Hammond & Keighley, 1960) in CHA which suggests that the mechanism of the disorder is due to an end-organ refractory state. Another possible factor is the presence of an inhibitor to erythropoiesis. This has been demonstrated in adults with acquired pure red cell aplasia (Krantz & Kao, 1967, 1969), and possibly in children with the congenital form as well (Ortega et a / , 1975). We have studied five patients with CHA by using a cell culture method in which normal human marrow responds to the addition of EPO with an increase in haem synthesis. The data indicate that normal synthesis occurs when erythroid precursors are present, but not otherwise. Moreover, plasnia inhibitors of erythropoiesis cannot be demonstrated. Correspondence: Dr M. H. Freedman, $ 5 5 University Avenue, Toronto, Ontario, Canada.
SIS
516
M. H. Freedman, D. Amato andE. F. Saunders MATERIALS AND METHODS
Subjects. Control bone marrow was obtained from 13 children with normal erythropoiesis on whom aspirates were performed during investigation for malignancy, storage disease or idiopathic thrombocytopenic purpura. (This study was approved by the Human Experimentation Committee of The Hospital for Sick Children.) The clinical and haematologic data on the five patients withSCHA are summarized in Table I. All fit the classic clinical description for this disorder. The only haematologic abnormality was anaemia due to failure of red cell production. The other cell lines were normal. None had physical or haematologic stigmata of Fanconi's aplastic anaemia or other forms of congenital marrow failure states. Patient 5 was studied twice, at the time of diagnosis and during a spontaneous remission I year later (:A' and 'B' respectively). Cultures. Marrows were cultured by the method of Krantz (1965). Buffy coat from 4 nil of heparinized marrow was washed three times in NCTC- r og medium containing penicillin roo unitslml and streptomycin 50 rg/nil by spinning at 1200 rpm at 4°C for 10 min per wash. The cells were resuspended in a medium of 60% NC TGIW and 40% human group AB heat-inactivated (56"C, 30 min) plasma obtained from a haematologically nornial adult. I ml of marrow suspension containing 5 x I O ~nucleated cells was plated in 35 x 10 mni plastic tissue culture dishes (Falcon) and incubated for varying times with and without 0.2 unitslml EPO in 5% C 0 2 in air, in high humidity, at 37°C. EPO was obtained from anaemic sheep plasma (Connaught Laboratories, Toronto) and had a potency of 2.8-4.7 units/mg dry weight (Step 111). It was dissolved in NCTC-rog in a concentration of 2 unitslnil and frozen until used. NQ other substrates, such as protoporphyrin, were added to the incubation mixture. 4-6 h prior to harvesting, I PCi of "FeCI,, preincubated at 37°C for at least I h with heatinactivated AB plasma as a source of transferrin, was added to each culture. At the end of incubation, the dishes were chilled at 4°C for 10 min. The contents were transferred to plastic centrihge tubes and washed thrice with cold phosphate-buffered saline. 2 ml of distilled water was added to each tube, mixed with the cells to lyse them, and allowed to stand for 5 min. 2 mllof Drabkin's solution was added, followed by 1.5 nil of 0.2 N hydrochloric acid, and then 6'ml of methylethylketone to separate the haem fraction (Teale, 1959). The tubes were stoppered, shaken vigorously, and spun at r o o 0 rpm at 4°C for 10 min. An aliquot of the upper haem layer was removed from each specimen and the radioactivity counted. The haem was not recrystallized, and the degree of purity not determined; however, the separation of haem and protein by this method is felt to be complete (Teale, 1959).Each culture was done in duplicate or triplicate and the results averaged. Haem synthesis was expressed in counts per minute (cpm) or as a percentage increase over control cultures without EPO. To detect plasma inhibitors of erythropoiesis patients' plasma was substituted for normal AB plasma in certain experiments. Plasma from an adult with acquired pure red cell aplasia was also tested. RESULTS A haem synthesis study on normal marrow was performed at four separate incubation times.
Haem Synthesis in Diamond-Blackfan Syndrome 125 +
517
5
{ 100-
*' /
/
E
8 75-
2=
.-C
t
8
"I /;/ 50-
.-C
3
?i 5
0
-0
I
I
I
I
TABLE I. Data on patients with Diamond-Blackfan syndrome Retic
1
0.4
0.6 0.0 0.5 0.0 1.5
l
1
l
8.2 6.4 6.9 4.5
200.0
I0
420.0
7
5.2
225.0
7.0
220.0
220.0
I
481.0
2
TABLE 11. Haem synthesis in Diamond-Blackfan syndrome
I
I
% increase
Patient
in synthesis
EPO
NoEPO -
~
I
50
50
2
I so
3
83 48
165 95 53
4
SA
50
52
SB
I20 I 60
3 60 450
363
622
5c Control (mean)
1
Marrow
I
I
0
IS
I
518
M. H. Freedntan, D.Atnato and E. F. Saundcrs
Synthesis occurred in all cultures whether stimulated with EPO or not, and generally dcclined for the duration of the incubation. The synthesis in those cultures with EPO was greater and declined slower than those without. At any time point in the study, haem synthesis could be expressed as percentage increase in cultures with EPO compared to those without EPO. Eleven control marrows were cultured for various incubation times to identify thc peak EPO effect (Fig I). The mean maximum increase in haem synthesis of 121% (range 15-332%) occurred at 70 h at which time the mean cpm 59Feuptake into haem was 622 and 363 in the EPO-stimulated and unstimulated cultures respectively. Dose-response studies were performed 011 two control marrows varying either thc conceiitratioiis of EPO or the number of nucleated marrow cells pcr culture. Increasing the EPO
n without EPO
T
With EPO T
AB
Type of
Pt
Pt
2
3
PRCA
PlOSma
FIG 2. Effect of plasma from three children with CHA and one adult with acquired pure red cell aplasia (PRCA) on haem synthesis of control marrow.
dose from 0.I to 0 . 3 unitslml produced a linear iiicreasc in haein synthesis. At higher doscs inhibition appeared. There was also a linear increase with iiucleated cell concentrations from 2.5 x 106to I.OX 10'/ml. Optimum coiiditions appeared to be a 70 h iiicubatioii using 0.2-0.3 units per ml of EPO and 5 x 106iiucleatcd marrow cells. When the marrows of the five children with CHA were cultured there was a consisteiit pattern of response (Table 11). The patients failed to increase haem synthesis in response to EPO. All had moderate to marked anaemia and reduced iiunibers of marrow erythroids (Table I). Patient 5B was restudied at a time when the haemoglobin concentration and marrow crythroids werc normal, and .thc haciii synthesis.rcspotisc had also become normal. Marrow from patient 5B was also cultured in autologous plasma, which had been drawn at the time of diagnosis. The h e m synthesis response (5C)was essentially uiichangcd. Certainly, there was no evidence of inhibition from the autologous plasma. Using a control marrow (Fig 2 ) , plasma from patients,I, 2 and 3 had no significant effect
Hneirr Syiithesis in Diatnotrd-Blackfan Syndrome
519
011 hacm synthcsis in thc EPO-containing cultures, when compared to normal type AB plasma. In comparison, plasma from an adult with acquired pure red cell aplasia resulted in marked supprcssion of haem synthesis.
DISCUSSION The haem synthcsis technique using short-term marrow cultures has been used in the investi-a gation of a variety of disorders including aplastic anaemia (Mizoguchi et a/, 1971), polycythaeniia vcra and chronic granulocytic leukaemia (Zucker ct a/, 1972), acquired pure red cell aplasia (Krantz & Kao, 1967), and congenital hypoplastic anaemia (Ortega et a/, 1975). The nicthod nicasurcs thc ability of erythroid tissue to produce haem and assesses the effect of added EPO in enhancing haem synthesis. O u r control data illustrate this stimulatory effect of E P O on haeni synthesis. In the cultures of I I control marrows, the range at the point of maximum increase in haem synthesis in response to EPO was very wide (15-332%). Th’IS was due to at least two factors : biologic differences in numbers of haeni-producing erythroids from marrow to marrow, and daily variables in laboratory culture conditions. O u r data indicate that little or 110 increase in haem synthesis occurs in response to E P O in marrows ofchildrcti with CHA when they are anaemic and have reduced numbers of marrow crythroids. In rcmission, thc response is normal. This short-term culture obviously has liniitatioils in thc study o f marrow failure states because if the nucleated marrow cells plated arc diminished or absent, as in our patients with CHA, then oiic would logically expect a dccrcased EPO effect. The technique provides 110 information about erythroid steni cells. A current and widely-accepted concept of erythropoiesis is that red cell differentiation starts with a pluripoten t stem cell, differentiates into a unipotent EPO-responsive or ‘committed’ stem ccll, and ends as progeny of morphologically identifiable erythroids. O u r data suggests that erythroid viability, and therefore haeni synthesis, declines for the duration of incubation of tlic cultures. The systcm does not support erythroid growth long enough to detect the cmergcncc of tlic differentiating crythroid stem cclls, and does not allow insight into early cvents ill crythropoiesis in CHA. Thc incorporation o f 59Fe into haeni is not a dircct index of red cell proliferation. The tcchniquc is of niorc valuc in dctccting any hunioral inhibitor of crythropoiesis and fornicd the basis of Krantz’s findings of tlic anti-crythroid antibody in adults with acquired purc rcd cell aplasia (Krantz & Kao, 1967, 1969). The one adult that we studicd also had plasma inhibitory activity to haem synthcsis from control marrow. 111 sharp contrast, plasma from thrcc of our paticiits with CHA had 110 inhbitory effect 011 haem synthesis from control marrow. Morcovcr, thc plasma drawn from one of thc paticnts at thc tinic of diagnosis had no inhibitory cffcct whcn tcstcd in autologous marrow obtaincd a ycar latcr during a spontaiicous rcmissioii of the discase. O u r data, therefore, arc opposcd to Ortega’s (Ortega c’t nl, 197s) aiid do not allow us to coi~cludcthat inhibition of hacm syntliesis is the nicchanisni of crytliropoictic failurc in CHA. CHA would secni to bc due to a problem intrinsic to the erythroid stem ccll. Growth of crythroid colonies in tissue culture (Stephenson at a!, 1971) arising from precursor erythropoictin-responsivc stem cells may be a more promising tool for further investigation of CHA.
M.H. Freedninti, D.Amato andE. F. Saunders ACKNOWLEDGMENTS
The. technical assistance of Wilma Hiddleston is gratefully acknowledged. We thank Dr P. D. McClure for allowing us to study his patient. This study was supported by Medical Research Couiicil of Canada Grant Number MA-4982. REFERENCES
ALLEN,D.M. & DIAMOND, L.K. (1961) Congenital (erythroid) hypoplastic anemia. Atnericatr Joirrnal of Diseases of Children, 102, 416. HAMMOND. D. & KEIGHLEY, G. (1960) The erythrocyte-stimulating factor in serum and urine in congenital hypoplastic anemia. American foitrtial of Diseases of Children. 100, 466. KRANTZ, S.B. (1965) The effect of erythropoietin on human bone marrow cells in uitro. L$ Sciences, 4, 2393.
KRANTZ, S.B.& KAO,V. (1967) Studies on red cell aplasia. I. Proceedings of the National Academy .f Sciences of the United States of America, 58, 493. KRANTZ, S.B. & KAO, V. (1969) Studies on red cell aplasia. 11. Report of a second patient with an antibody to erythroblast nuclei and a remission after immunosuppressive therapy. Blood, 34, I. MIZOGUCHI, H., MIURA,Y., TAKAKU. F., SASSA, S., CHIBA,S. & NAKAO,K.(1971)The effect of erythro-
poietin on human bone marrow cells in vitro. I. Studies of nine cases of bone marrow failure. Blood, 37, 624. ORTEGA, J.A., SHORE, N.A., DUKES, P.P. & HAMMOND, D. (1975) Congenital hypoplastic anemia inhibition of erythropoiesis-by sera from patients with congenital hypoplastic anemia. Blood, 45, 83. STEPHENSON, J.R., AXELRAD, A.A., MCLEOD,D.L. & SHREEVi!, M. (1971) Induction of colonies of hemoglobin-synthesizing cells by erythropoietin in vitro. Proceedings of the Ndtional Academy of Sciences qf the United States ofAmerica, 68, 1542. TEALE,F.W.J.(1959) Cleavage of the haem-protein link by acid methylethylketone. Biochimica et Biophysica Acta, 35, 543. ZUCKER, S., HOWE, D.M. & WEINTRAUB, L.R. (1972) Bone marrow response to erythropoietii in polycythemia Vera and chronic granulocytic leukemia. Blood, 39. 341.
$15.
Haem Synthesis in the Diamond-Blackfan Syndrome MELVIN H. FREEDMAN, DOMINICK AMATO AND E. FREDSAUNDERS
Division of Haeniatology and Department of Pediatrics, The Hospital-for Sick Chi/dren and The University of Toronto, Toronto, Ontario (Received 20 March 1975; accepttadfor publication 21 M a y 1975) SUMMARY. The response of bone marrow to erythropoietin (EPO) from five children with the Diamond-Blackfan syndrome, also known as congenital hypoplastic anaemia (CHA), was tested in tissue culture by measurement of haem synthesis. Studies of 13 control marrows indicated that the maximum EPO effect occurred at approximately 70 h incubation using an EPO concentration of 0.2-0.3 units/ml and a nucleated cell concentration of 5 x 106 per culture. Under these conditions, haem synthesis was 121% greater in EPO-stimulated than in unstimulated cultures. Patients with CHA with anaemia and diminished marrow erythroids had reduced or absent haem synthesis. In one patient, haem production became normal after a spontaneous remission, and was not inhibited by autologous plasma drawn at the time of diagnosis. Plasma from three patients did not show inhibitory activity when cultured with control marrow. In contrast, plasma from an adult with acquired pure red cell aplasia produced striking inhibition of haem synthesis when cultured with control marrow. W e conclude that, in comparison to some cases of the adult acquired condition, CHA is not due to inhibitors or antibodies. When present, erythroid precursors in children with CHA are capable of responding normally to EPO with increased haem synthesis. The Diamond-Blackfan syndrome, or congenital hypoplastic anaemia (CHA) is due to failure of red cell production. It is characterized by normochromic, normocytic red cells, absent reticulocyte response, and a normocellular marrow aspirate with diminished to absent nucleated erythroids. About one-half of these children experience a complete remission when treated with corticosteroids (Allen & Diamond, 1961).The serum and urinary erythropoietin (EPO) levels are consistently elevated (Hammond & Keighley, 1960) in CHA which suggests that the mechanism of the disorder is due to an end-organ refractory state. Another possible factor is the presence of an inhibitor to erythropoiesis. This has been demonstrated in adults with acquired pure red cell aplasia (Krantz & Kao, 1967, 1969), and possibly in children with the congenital form as well (Ortega et a / , 1975). We have studied five patients with CHA by using a cell culture method in which normal human marrow responds to the addition of EPO with an increase in haem synthesis. The data indicate that normal synthesis occurs when erythroid precursors are present, but not otherwise. Moreover, plasnia inhibitors of erythropoiesis cannot be demonstrated. Correspondence: Dr M. H. Freedman, $ 5 5 University Avenue, Toronto, Ontario, Canada.
SIS
516
M. H. Freedman, D. Amato andE. F. Saunders MATERIALS AND METHODS
Subjects. Control bone marrow was obtained from 13 children with normal erythropoiesis on whom aspirates were performed during investigation for malignancy, storage disease or idiopathic thrombocytopenic purpura. (This study was approved by the Human Experimentation Committee of The Hospital for Sick Children.) The clinical and haematologic data on the five patients withSCHA are summarized in Table I. All fit the classic clinical description for this disorder. The only haematologic abnormality was anaemia due to failure of red cell production. The other cell lines were normal. None had physical or haematologic stigmata of Fanconi's aplastic anaemia or other forms of congenital marrow failure states. Patient 5 was studied twice, at the time of diagnosis and during a spontaneous remission I year later (:A' and 'B' respectively). Cultures. Marrows were cultured by the method of Krantz (1965). Buffy coat from 4 nil of heparinized marrow was washed three times in NCTC- r og medium containing penicillin roo unitslml and streptomycin 50 rg/nil by spinning at 1200 rpm at 4°C for 10 min per wash. The cells were resuspended in a medium of 60% NC TGIW and 40% human group AB heat-inactivated (56"C, 30 min) plasma obtained from a haematologically nornial adult. I ml of marrow suspension containing 5 x I O ~nucleated cells was plated in 35 x 10 mni plastic tissue culture dishes (Falcon) and incubated for varying times with and without 0.2 unitslml EPO in 5% C 0 2 in air, in high humidity, at 37°C. EPO was obtained from anaemic sheep plasma (Connaught Laboratories, Toronto) and had a potency of 2.8-4.7 units/mg dry weight (Step 111). It was dissolved in NCTC-rog in a concentration of 2 unitslnil and frozen until used. NQ other substrates, such as protoporphyrin, were added to the incubation mixture. 4-6 h prior to harvesting, I PCi of "FeCI,, preincubated at 37°C for at least I h with heatinactivated AB plasma as a source of transferrin, was added to each culture. At the end of incubation, the dishes were chilled at 4°C for 10 min. The contents were transferred to plastic centrihge tubes and washed thrice with cold phosphate-buffered saline. 2 ml of distilled water was added to each tube, mixed with the cells to lyse them, and allowed to stand for 5 min. 2 mllof Drabkin's solution was added, followed by 1.5 nil of 0.2 N hydrochloric acid, and then 6'ml of methylethylketone to separate the haem fraction (Teale, 1959). The tubes were stoppered, shaken vigorously, and spun at r o o 0 rpm at 4°C for 10 min. An aliquot of the upper haem layer was removed from each specimen and the radioactivity counted. The haem was not recrystallized, and the degree of purity not determined; however, the separation of haem and protein by this method is felt to be complete (Teale, 1959).Each culture was done in duplicate or triplicate and the results averaged. Haem synthesis was expressed in counts per minute (cpm) or as a percentage increase over control cultures without EPO. To detect plasma inhibitors of erythropoiesis patients' plasma was substituted for normal AB plasma in certain experiments. Plasma from an adult with acquired pure red cell aplasia was also tested. RESULTS A haem synthesis study on normal marrow was performed at four separate incubation times.
Haem Synthesis in Diamond-Blackfan Syndrome 125 +
517
5
{ 100-
*' /
/
E
8 75-
2=
.-C
t
8
"I /;/ 50-
.-C
3
?i 5
0
-0
I
I
I
I
TABLE I. Data on patients with Diamond-Blackfan syndrome Retic
1
0.4
0.6 0.0 0.5 0.0 1.5
l
1
l
8.2 6.4 6.9 4.5
200.0
I0
420.0
7
5.2
225.0
7.0
220.0
220.0
I
481.0
2
TABLE 11. Haem synthesis in Diamond-Blackfan syndrome
I
I
% increase
Patient
in synthesis
EPO
NoEPO -
~
I
50
50
2
I so
3
83 48
165 95 53
4
SA
50
52
SB
I20 I 60
3 60 450
363
622
5c Control (mean)
1
Marrow
I
I
0
IS
I
518
M. H. Freedntan, D.Atnato and E. F. Saundcrs
Synthesis occurred in all cultures whether stimulated with EPO or not, and generally dcclined for the duration of the incubation. The synthesis in those cultures with EPO was greater and declined slower than those without. At any time point in the study, haem synthesis could be expressed as percentage increase in cultures with EPO compared to those without EPO. Eleven control marrows were cultured for various incubation times to identify thc peak EPO effect (Fig I). The mean maximum increase in haem synthesis of 121% (range 15-332%) occurred at 70 h at which time the mean cpm 59Feuptake into haem was 622 and 363 in the EPO-stimulated and unstimulated cultures respectively. Dose-response studies were performed 011 two control marrows varying either thc conceiitratioiis of EPO or the number of nucleated marrow cells pcr culture. Increasing the EPO
n without EPO
T
With EPO T
AB
Type of
Pt
Pt
2
3
PRCA
PlOSma
FIG 2. Effect of plasma from three children with CHA and one adult with acquired pure red cell aplasia (PRCA) on haem synthesis of control marrow.
dose from 0.I to 0 . 3 unitslml produced a linear iiicreasc in haein synthesis. At higher doscs inhibition appeared. There was also a linear increase with iiucleated cell concentrations from 2.5 x 106to I.OX 10'/ml. Optimum coiiditions appeared to be a 70 h iiicubatioii using 0.2-0.3 units per ml of EPO and 5 x 106iiucleatcd marrow cells. When the marrows of the five children with CHA were cultured there was a consisteiit pattern of response (Table 11). The patients failed to increase haem synthesis in response to EPO. All had moderate to marked anaemia and reduced iiunibers of marrow erythroids (Table I). Patient 5B was restudied at a time when the haemoglobin concentration and marrow crythroids werc normal, and .thc haciii synthesis.rcspotisc had also become normal. Marrow from patient 5B was also cultured in autologous plasma, which had been drawn at the time of diagnosis. The h e m synthesis response (5C)was essentially uiichangcd. Certainly, there was no evidence of inhibition from the autologous plasma. Using a control marrow (Fig 2 ) , plasma from patients,I, 2 and 3 had no significant effect
Hneirr Syiithesis in Diatnotrd-Blackfan Syndrome
519
011 hacm synthcsis in thc EPO-containing cultures, when compared to normal type AB plasma. In comparison, plasma from an adult with acquired pure red cell aplasia resulted in marked supprcssion of haem synthesis.
DISCUSSION The haem synthcsis technique using short-term marrow cultures has been used in the investi-a gation of a variety of disorders including aplastic anaemia (Mizoguchi et a/, 1971), polycythaeniia vcra and chronic granulocytic leukaemia (Zucker ct a/, 1972), acquired pure red cell aplasia (Krantz & Kao, 1967), and congenital hypoplastic anaemia (Ortega et a/, 1975). The nicthod nicasurcs thc ability of erythroid tissue to produce haem and assesses the effect of added EPO in enhancing haem synthesis. O u r control data illustrate this stimulatory effect of E P O on haeni synthesis. In the cultures of I I control marrows, the range at the point of maximum increase in haem synthesis in response to EPO was very wide (15-332%). Th’IS was due to at least two factors : biologic differences in numbers of haeni-producing erythroids from marrow to marrow, and daily variables in laboratory culture conditions. O u r data indicate that little or 110 increase in haem synthesis occurs in response to E P O in marrows ofchildrcti with CHA when they are anaemic and have reduced numbers of marrow crythroids. In rcmission, thc response is normal. This short-term culture obviously has liniitatioils in thc study o f marrow failure states because if the nucleated marrow cells plated arc diminished or absent, as in our patients with CHA, then oiic would logically expect a dccrcased EPO effect. The technique provides 110 information about erythroid steni cells. A current and widely-accepted concept of erythropoiesis is that red cell differentiation starts with a pluripoten t stem cell, differentiates into a unipotent EPO-responsive or ‘committed’ stem ccll, and ends as progeny of morphologically identifiable erythroids. O u r data suggests that erythroid viability, and therefore haeni synthesis, declines for the duration of incubation of tlic cultures. The systcm does not support erythroid growth long enough to detect the cmergcncc of tlic differentiating crythroid stem cclls, and does not allow insight into early cvents ill crythropoiesis in CHA. Thc incorporation o f 59Fe into haeni is not a dircct index of red cell proliferation. The tcchniquc is of niorc valuc in dctccting any hunioral inhibitor of crythropoiesis and fornicd the basis of Krantz’s findings of tlic anti-crythroid antibody in adults with acquired purc rcd cell aplasia (Krantz & Kao, 1967, 1969). The one adult that we studicd also had plasma inhibitory activity to haem synthcsis from control marrow. 111 sharp contrast, plasma from thrcc of our paticiits with CHA had 110 inhbitory effect 011 haem synthesis from control marrow. Morcovcr, thc plasma drawn from one of thc paticnts at thc tinic of diagnosis had no inhibitory cffcct whcn tcstcd in autologous marrow obtaincd a ycar latcr during a spontaiicous rcmissioii of the discase. O u r data, therefore, arc opposcd to Ortega’s (Ortega c’t nl, 197s) aiid do not allow us to coi~cludcthat inhibition of hacm syntliesis is the nicchanisni of crytliropoictic failurc in CHA. CHA would secni to bc due to a problem intrinsic to the erythroid stem ccll. Growth of crythroid colonies in tissue culture (Stephenson at a!, 1971) arising from precursor erythropoictin-responsivc stem cells may be a more promising tool for further investigation of CHA.
M.H. Freedninti, D.Amato andE. F. Saunders ACKNOWLEDGMENTS
The. technical assistance of Wilma Hiddleston is gratefully acknowledged. We thank Dr P. D. McClure for allowing us to study his patient. This study was supported by Medical Research Couiicil of Canada Grant Number MA-4982. REFERENCES
ALLEN,D.M. & DIAMOND, L.K. (1961) Congenital (erythroid) hypoplastic anemia. Atnericatr Joirrnal of Diseases of Children, 102, 416. HAMMOND. D. & KEIGHLEY, G. (1960) The erythrocyte-stimulating factor in serum and urine in congenital hypoplastic anemia. American foitrtial of Diseases of Children. 100, 466. KRANTZ, S.B. (1965) The effect of erythropoietin on human bone marrow cells in uitro. L$ Sciences, 4, 2393.
KRANTZ, S.B.& KAO,V. (1967) Studies on red cell aplasia. I. Proceedings of the National Academy .f Sciences of the United States of America, 58, 493. KRANTZ, S.B. & KAO, V. (1969) Studies on red cell aplasia. 11. Report of a second patient with an antibody to erythroblast nuclei and a remission after immunosuppressive therapy. Blood, 34, I. MIZOGUCHI, H., MIURA,Y., TAKAKU. F., SASSA, S., CHIBA,S. & NAKAO,K.(1971)The effect of erythro-
poietin on human bone marrow cells in vitro. I. Studies of nine cases of bone marrow failure. Blood, 37, 624. ORTEGA, J.A., SHORE, N.A., DUKES, P.P. & HAMMOND, D. (1975) Congenital hypoplastic anemia inhibition of erythropoiesis-by sera from patients with congenital hypoplastic anemia. Blood, 45, 83. STEPHENSON, J.R., AXELRAD, A.A., MCLEOD,D.L. & SHREEVi!, M. (1971) Induction of colonies of hemoglobin-synthesizing cells by erythropoietin in vitro. Proceedings of the Ndtional Academy of Sciences qf the United States ofAmerica, 68, 1542. TEALE,F.W.J.(1959) Cleavage of the haem-protein link by acid methylethylketone. Biochimica et Biophysica Acta, 35, 543. ZUCKER, S., HOWE, D.M. & WEINTRAUB, L.R. (1972) Bone marrow response to erythropoietii in polycythemia Vera and chronic granulocytic leukemia. Blood, 39. 341.
