790
May 1976 The J o u r n a l o f P E D I A T R I C S
Granulopoiesis in childhood aplastic anemia The soft agar technique was employed to investigate factors involved in the regulation o f granulopoiesis in ten children with aplastic anemia. Children with A A had greatly reduced numbers of granulocytic colonyforming cells in their bone marrow and in their peripheral blood when compared to "control" children. Colony-stimulating activity was decreased in five o f the ten children tested. Serum from eight children with A A did not inhibit colony formation when added to normal adult bone marrow cells in culture. The defect in A A resides in the stem cell, with involvement o f the CSA-produeing cells in some cases. The serum o f these patients does not contain an inhibitor o f granulopoiesis.
Abdelsalam H. Ragab, M.D., F.R.C.P.(C),* Ellen Gilkerson, William M. Crist, M.D., and Elna Phelan, M.D., St. Louis, Mo.
APLASTIC ANEMIA is a syndrome of varying severity caused by a variety of etiologic factors ultimately leading to the development of pancytopenia and hypoplasia of the bone marrow. Although the final clinical picture is similar, the pathophysiology of this condition may vary from case to case. In a recent editorial Stohlman I speculated that the syndrome of aplastic anemia might result from one of several mechanisms including abnormality of stem cells or of the bone marrow microenvironment, or the presence of inhibitors to specific regulators. Understanding of these pathophysiologic factors is of paramount importance in the treatment of this disorder. Thus if it were demonstrated that certain cases of aplastic anemia were due to defective stem cells, patients so affected would benefit from a bone marrow transplantaFrom the Edward Mallinckrodt Department o f Pediatrics, Washington University, and the Division o f Pediatric Hematology and Oncology, St. Louis Childrens Hospital. Supported in part by National Institutes o f fIealth Grant 5RlO CA 0587-14, The American Cancer Society Grant CI-105, and the Children's United Research Effort of St. Louis (CURE). Presented in part to the Plenary Session o f the American Pediatric Society, Denver, Colo., April 17, 1975. Dr. Ragab is the recipient of a Faculty Research Award from The American Cancer Society. *Reprint address: Department of Pediatrics, Emory University School of Medicine, 69 Butler St., S.E., Atlanta, Ga. 30303.
Vol. 88, No. 5, pp. 790-794
tion. Patients who had inhibitors detectable in their serum to specific regulators and patients who had defective microenvironment in their bone marrow probably would not benefit from such a transplantation. In this study we have investigated the factors involved in the regulation of granulopoiesis by the agar culture technique in ten children with aplastic anemia. These included: three children with Fanconi's anemia, one postvaricella patient, one post-hepatitis patient, and five children with idiopathic aplastic anemia. Aplastic anemia is defined as pancytopenia with hypoplasia of the bone marrow in the absence of enlargement of the liver and spleen. The safient features are summarized in Table I.
Abbreviations used AAi aplasticanemia CFC: colony-forming cells CSA: colony-stimulating activity
MATERIALS AND METHODS
Bone marrow colony-forming cells. Bone marrow samples from children with AA were obtained and cultured in the soft agar system according to the method of Robinson and Pike. 2 Briefly, 1 • l0 B nucleated bone marrow cells were cultured in an overlayer containing 0.3% agar on top of "standard" underlayers prepared from the peripheral blood of two individuals who had similar colony-stimulating activity. T h e patients' bone
Volume 88 Number 5
Granulopoiesis in anemia
79 1
220
140 2OO ,q, 120 180
r~ I00 U.l
u Z
80
160
o
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b 40
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,
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.~ ]o
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o
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Fig. 2. CFC from the peripheral blood of children with aplastic anemia and normal children. Horizontal bar denotes median for normal children.
marrow cells were also cultured on u n d e r l a y e r s p r e p a r e d from their own peripheral blood cells. T h e u n d e r l a y e r s contained 1 X 106 peripheral blood n u c l e a t e d c e l l s / m l in 0.5% agar. The plates were i n c u b a t e d at 37 ~ C i n a humidified atmosphere with a 7.5% flow o f COs for 14 days; at the end o f this period, colonies c o n t a i n i n g 50 or more cells were scored undei" a n i n v e r t e d microscope. Duplicate, a n d later quadruplicate, cultures were prepared from each sample. In Cases 9 a n d 10 the b o n e marrow cells were also cultured at ten times the n o r m a l concentration (1 x 100. The n u m b e r o f C F C was corn-
Fig. 3. The effect of addition of 0.2 ml of normal serum and serum from children with' aplastic anemia on the CFC of normal bone marrow cells. The number of colonies formed by normal bone marrow cells cultured without serum is arbitrarily designated as 100%.
p a r e d to those o b t a i n e d from the b o n e m a r r o w o f 25 "control" children cultured on the same underlayers. T h e " c o n t r o l " children h a d b o n e m a r r o w aspirations perf o r m e d for a variety o f reasons (solid t u m o r s at time of diagnosis, for the exclusion o f storage disorders, etc.); they had normal hem 9 and normal bone marrow morphology. Peripheral blood CFC. T e n milliliters o f peripheral b l o o d were aspirated from each child w h o h a d AA and cultured in agar on top o f the " s t a n d a r d " underlayers at a c o n c e n t r a t i o n o f 4 x l0 s nucleated cells. In Case 10 the
792
Ragab et al.
The Journal of Pediatrics May 1976
Table I. Hematologic values at time studies were performed
Case No.
Age (yr)
Sex
Race
Etiology
Outcome
1 2 3 4 5 6 7 8 9 10
5 10/12 5 2/12 1 9 2/12 10 1/12 11 6 13 13 8
F F M M M M F M F F
White White Black Black White White White White White White
Idiopathic Idiopathic Idiopathic Post-hepatic Idiopathic Fanconi Fanconi Fanconi Idiopathic Post-varicella
Died Died Died Alive Alive Alive Alive Alive Alive Alive
Time from Dx
Hgb (gm/dl)
66rno Dx 21mo 8mo 35mo 28mo Dx 24mo Dx Dx
6.5 6.3 12.2 13.7 11.7 13.9 9.7 6.4 5.2 5.5
Platelets (/mm 3) 13,000 14,000 5,000 120,000 15,000 65,000 36,000 73,000 12,000 4,000
% WBC Lymph(~ram~) ocytes 5,600 2,500 3,500 4,070 3,200 3,600 4,500 2,900 1,300 2,600
39 90 62 35 66 79 27 69 60 67
% Polymorphonuclear cells
% Monocytes
BM eellu. larity
56 9 38 62 34 20 4 29 24 25
2 0 0 2 0 1 9 0 t2 7
J, $ $ $ J, J, $ $ ,~ ~, ,L ,L ,L ,L $ $ ,L ,~ J, J, ~, $ ~, ~. ,~
Abbreviations: Dx = diagnosis, BM = bone marrow, BM CFC = bone marrow colony forming cells/10s nucleated cellsplated, PB CFC = peripheral blood colony forming cells/4 x 10~nucleated cells plated, Ox = oxymetholone, P = prednisone, ND = not done.
peripheral blood cells were also cultured at a higher concentration (1 x 106 nucleated ceils). These were compared to the C F C obtained from the culture of the peripheral blood cells of 14 normal children. Peripheral blood CSA. Underlayers were prepared from the peripheral blood cells o f children with AA. Bone marrow cells from children with acute lymphocytic leukemia in remission were cultured on the " s t a n d a r d " underlayers as well as on underlayers from the cells of A A patients. A m i n i m u m of two and a m a x i m u m of nine bone marrow samples were cultured simultaneously on each underlayer; the colonies formed on each were compared. The colony counts obtained with the "standard" underlayers were taken as 100%. The colony-stimulating activity of the peripheral blood cells of children with A A was compared by expressing the colony counts obtained with the aplastic anemia underlayers as a percentage o f those obtained with the "standard" underlayers. Serum (humoral factors). The children with A A were investigated for the presence of inhibitors of granulopoiesis in their sera. Sera from eight children with A A were added to normal bone marrow cells obtained from normal adult volunteers and left to incubate for one hour, then mixed with agar, and cultured on the " s t a n d a r d " underlayers. The colonies were counted and expressed as a percentage of the control colony counts obtained without the addition of serum. This was compared to the addition of h u m a n sera, obtained from 18 normal individuals, which were added to normal adult bone marrow cells and cultured in agar in the same manner. RESULTS
Bone marrow CFC. All children with A A had greatly decreased C F C in their bone marrow when c o m p a r e d to
"control" children (Fig. 1). Utilizing the Wilcoxan rank sum test, this is statistically significant (P 0.001). In the tast two patients, the marrow cells were also cultured at a concentration of 1 x 106 nucleated cells; even with this stimulation, very few colonies (1 to 4) were observed. When the cells of the patients' bone m a r r o w were cultured on their own underlayers, no colonies were observed. W e have attempted to quantitate the dilution o f the bone marrow sample with peripheral blood. This was performed by observing the difference in percentage of myelocytes in the morphology marrow sample in comparison with the percentage of myelocytes in the cultured marrow sample. ~ The marrow sample was never diluted more than twofold with peripheral blood. Peripheral blood CFC. Children with A A had decreased C F C in their peripheral blood. In seven of the ten children examined, no C F C were detected (Fig. 2). In the last patient (Case 10) no colonies were observed when the peripheral blood cells were cultured at a concentration o f 1 X l0 s nucleated cells. Utilizing the Wilcoxan rank sum test, the difference in the peripheral blood C F C between normal children and children with A A is statistically significant (P 0.001). Colony-stimulating activity. The CSA of the peripheral blood cells o f children with A A was decreased in five o f the eight children tested. It was greatly reduced in Cases 7 and 8 (both children had Fanconi's anemia) (Table I). The variability i n CSA among the peripheral blood of normal individuals varies in our laboratory from 80 to 220% of the "standard" underlayers. Effect of serum. When normal h u m a n serum was added to cells of normal adult bone marrow in culture, it either had no effect: (6/18) or it stimulated ( 1 0 / 1 8 ) - o n l y two sera inhibited colony formation (Fig. 3). W h e n serum
Volume 88 Number 5
Medication
Granulopoiesis in anemia
BM CFC
Ox + P Ox + P Ox + P None Ox + P Ox None None Transplant Ox + P
0 3 0 9 0 1 0 0 0.5 0
PB CFC
% CSA
Serum tested
0 4 0 0 1 2 0 0 0 0
81 129 83 69 42 56 11 12 133 87
ND ND + + + + + + + +
samples from eight children with AA were added to normal bone marrow cells in culture, there was stimulation of colony formation by seven of the eight sera tested. one serum sample had no effect. It was concluded that no inhibitor of granulopoiesis exists in the serum of these children. DISCUSSION The basic mechanism involved in the pathogenesis of AA still is unknown. Based on animal models, two alternative hypotheses are put forward to explain the marrow hypoplasia.' It has been determined that the defect in genetically anemic S1/SP mice resides in the microenvironment of the marrow. Transplantation of normal pluripotent stem cells in this case will not correct the anemia? In contrast, the anemia in W / W v mice can be corrected by transplantation of normal bone marrow stem cells, demonstrating that in this case the defect resides in the stem cell? In man, however, there is to date no system available which can measure the functional activity of the pluripotent stem cell. An in vitro agar culture system is available which measures a committed granulocytic progenitor or stem cell (CFC). In this culture system, two groups of cells are required: a colony-forming cell, which now is believed to resemble a lymphocyte on light microscopy (but is indistinguishable on electron microscopy),~ and a "helper" cell that produces CSA. The latter cell is believed to be a monocyte7 or macrophage? This culture system has been used to study granulopoiesis in a variety of disorders. Thus it has been demonstrated that adults with acute myelogenous leukemia"' ,6 and children with acute lymphocytic leukemia have decreased CFC in their bone marrow," but increased CFC in their peripheral
793
blood?, 1~These observations suggest a perturbed condition in the bone marrow of these patients in which their CFC migrates into the peripheral blood. The peripheral blood CFC is increased in adults with myelofibrosis13 and in children with osteopetrosis." The CSA is decreased in children with acute lymphocytic leukemia." Senn and colleagues1~ studied CFC in bone marrow and CSA production in peripheral blood of six patients with neutropenia, and noted that the defect varied from patient to patient: three patients had normal CFC and CSA production (suggesting peripheral destruction of white cells), two patients had decreased CFC and CSA production, and one patient had decreased CFC but normal CSA production. Greenberg and Schrier ~6 studied three adults with drug-induced neutropenia and two adults with neutropenia associated with hypoplastic bone marrow and noted that they had decreased bone marrow production of CFC. In the latter study, production of CSA was not examined. Our results demonstrate that there is a decrease in colony-forming cells in the bone marrow and peripheral blood of children with aplastic anemia. This reduction of CFC is absolute since plating with bone marrow cells at ten times the normal concentration did not increase the number of colonies observed. This decrease in bone marrow CFC was observed in all children examined, including three children who were not neutropenic at the time of study, suggesting perhaps that some areas in the bone marrow were still functional and capable of proliferation. Production of CSA also was decreased in five of the eight children studied. It was very much reduced in two of the three children with Fanconi's anemia who were investigated. This finding indicates that in some cases of AA, both the CFC and the CSA-producing cells are defective. It is interesting to note that in seven out of ten children studied there was a concomitant severe monocytopenia. One child (Case 7) had a normal number of m0nocytes in the peripheral blood, but had decreased CSA. From these data it is difficult to decide whether there is decreased CSA production or whether the CSA produced is defective. No inhibitor of in vitro granUlopoiesis could be detected in the sera of the eight children with aplastic anemia tested. It is generally believed that the sulfur colloid scan (99mTc) measures uptake by the reticuloendotheliat system, whereas the ' " I n d i u m chloride scan measures uptake by the erythroid elements of the bone m a r r o w y Both patients (Cases 9 and 10) had normal colloid scans, although one patient had a normal indium scan despite the biopsy-proved hypoplasia. It therefore is difficult to interpret the value of these scans in children with AA? 8 Although we have studied only the granulocytic pro-
794
Ragab et al.
genitor cell (or CFC), the defect may reside in the stem cell, inasmuch as all bone marrow elements (erythroid, granulocytic, and megakaryocytic) are affected. Freedman and associates TM have followed eight children with A A for periods up to nine years. They noted that none of their patients ever achieved hematologic normality. Our surviving patients still have abnormally 10w granul0cyte and platelet counts. This would suggest that the use of androgens, by increasing erythropoietin production 2~ or by acting directly on the stem cells, increases red cell production, but does not affect the megakaryocytic or myeloid elements. 2.... Recently, this hypothesis has been disptlted. 23 The prognosis of AA, with the exception o f druginduced A A in children, 24 is generally poor. Recently, Storb and associates 25 published their results on bone marrow transplantation in 24 patients with AA. Twentyone patients showed prompt hem0poietic engraftment, and 13 patients (54%) demonstrated marked i m p r o v e m e n t after the transplant. This study would suggest that the stem cell is defective in AA, and that engraftment by normal stem cells from related donors corrects the disorder in most instances. Further refinement in the immunosuppression and support of patients during bone marrow transpl~mtation may result in this m o d e of therapy becoming the treatment of choice in AA. Inasmuch as there is an ongoing International Aplastic Anemia Transplantation Study, it m a y be beneficial to study bone marrow C F C and other in vitro factors in all A A patients. We would like to thank Dr. Philip R. Dodge (Chairman, Department of Pediatrics, Washington University) and Dr. Stuart Kornfeld (Professor of Medicine, Washington University) for their criticai review of this manuscript. REFERENCES
1. Stohlman F Jr: Aplastic anemia, Blood 40:282, 1972. (Editorial.) 2. Robinson WA, and Pike BE: Colony growth of human bone marrow cells in vitro, in Stohlman F Jr, editor: Hemopoietic cellular pl?oliferation, New York, I970, Grune & Stratton, Inc, pp 249-259. 3. Ragab AH, Gilkerson E, Myers M, and Choi SC: The culture of colony forming units from the peripheral blood and bone marrow of children with acute lymphocytic leukemia, Cancer 34:663, 1974. 4. McCull0ch. EA, Siminovitch L, Till JE, Russell ES, and Bernstein SE: The cellular basis of the genetically determined hemopoietic defect in anemic mice of genotype S1/S1d, Blood 26:399, 1965. 5. McCulloch EA, Siminovitch L, and Till JE: Speen-colony formation in anemic mice of genotype W/W ~, Science 144:844/1964.
The Journal of Pediatrics May 1976
6. Zucker-Franklin D, Grusky G, and L'Esperance P: Granulocyte colonies derived from lymphocyte fractions of normal human peripheral blood, Proc Natl Acad Sci 71:2711, 1974. 7. Chervenick PA, and LoBuglio AF: Human blood monocytes-stimulators of granulocyte and mononuclear colony formation in vitro Science 178:164, 1972. 8. Messner HA, Till JE, and McCulloch EA: Interacting cell populations affecting granulopoietic colony formation by normal and leukemic human marrow cells, Blood 42:701, 1973. 9. Brown CH, and Carbon e PP: In vitro growth of normal and leukemic human bone marrow, J Natl Cancer Inst 46:989, 1971. 10. Iscove NN, Senn JS, Till JE, and McCulloch EA: Colony formation of normal and leukemic human marrow cells in culture-effect of conditioned medium from human leukocytes, Blood 37:1, 1971. 11. Ragab AH, Gilkerson E, and Myers ML: Granulopoiesis in childhood leukemia, Cancer 33:791, 1974. 12. Robinson WA, Kurnick JE, and Pike BL: Colony growth of human leukemic peripheral blood cells in vitro, Blood 38:500, 1971. 13. Chervenick PA: Increase in circulating stem cells in patients with myelofibrosis, Blood 41:67, 1973. 14. Ragab AH, Ducos R, Crist WM, and Duck SC: Granulopoiesis in oste0petrosis, J PEDIATR87:422, 1975. 15. Senn JS, Messner HA, and Stanley ER: Analysis of interacting cell populations in cultures of marrow from patients with neutropenia, Blood 44:33, 1974. 16. Greenberg PL, and Schrier SL: Granulopoiesis in neutropenic disorders, Blood 41:753, 1973. 17. McNeil BJ, Holman BL, Button LN, and Rosenthal DS: Use of Indium chloride scintigraphy in patients with myelofibrosis, J Nuel Med 15:647, 1974. 18. Merrick MV, Gordon-Smith EC, Lavender JP, and Szur L: A comparison of XHInwith 52Fe and "~mTc-sulfur colloid for bone marrow scanning, J Nucl Med 16:66, 1975. 19. Freedman MH, Saunders EF, Hilton J, and McClure PD: Residual abnormalities in acquired aplastic anemia of childhood, JAMA 228:201, 1974. 20. Fried W, and Gurney CW: The erythropoietic response to testosterone in male and female mice, J Lab Clin Med 67:420, 1966. 21. Shahidi NT: Androgens and erythropoiesis, N Engl J Med 289:72, 1973. 22. Gorshein D, Hait WN, Besa EC, Jepson JH, and Gardner FH: Increased stem cell response to erythropoietin induced by androgens, Endocrinology 93:777, 1973. 23. Reissmann KR, Udupa KB, and Kawada K: Effects of erythropoietin and androgens on erythroid stem cells ~/fter their lelective suppression by BCNU, Blood 44:649, 1974. 24. Heyu RM, Ertel IJ, and Tubergen DG: Course of acquired aplastic anemia in children treated with supportive care, JAMA 208:1372, 1969. 25. Storb R, Thomas ED, Buckner CD, Clift RA, Johnson FL, Fefer A, Glucksberg H, Giblett ER, Lerner KG, and Neiman P: Allogeneic marrow grafting for treatment of aplastic anemia, Blood 43:157, 1974.
May 1976 The J o u r n a l o f P E D I A T R I C S
Granulopoiesis in childhood aplastic anemia The soft agar technique was employed to investigate factors involved in the regulation o f granulopoiesis in ten children with aplastic anemia. Children with A A had greatly reduced numbers of granulocytic colonyforming cells in their bone marrow and in their peripheral blood when compared to "control" children. Colony-stimulating activity was decreased in five o f the ten children tested. Serum from eight children with A A did not inhibit colony formation when added to normal adult bone marrow cells in culture. The defect in A A resides in the stem cell, with involvement o f the CSA-produeing cells in some cases. The serum o f these patients does not contain an inhibitor o f granulopoiesis.
Abdelsalam H. Ragab, M.D., F.R.C.P.(C),* Ellen Gilkerson, William M. Crist, M.D., and Elna Phelan, M.D., St. Louis, Mo.
APLASTIC ANEMIA is a syndrome of varying severity caused by a variety of etiologic factors ultimately leading to the development of pancytopenia and hypoplasia of the bone marrow. Although the final clinical picture is similar, the pathophysiology of this condition may vary from case to case. In a recent editorial Stohlman I speculated that the syndrome of aplastic anemia might result from one of several mechanisms including abnormality of stem cells or of the bone marrow microenvironment, or the presence of inhibitors to specific regulators. Understanding of these pathophysiologic factors is of paramount importance in the treatment of this disorder. Thus if it were demonstrated that certain cases of aplastic anemia were due to defective stem cells, patients so affected would benefit from a bone marrow transplantaFrom the Edward Mallinckrodt Department o f Pediatrics, Washington University, and the Division o f Pediatric Hematology and Oncology, St. Louis Childrens Hospital. Supported in part by National Institutes o f fIealth Grant 5RlO CA 0587-14, The American Cancer Society Grant CI-105, and the Children's United Research Effort of St. Louis (CURE). Presented in part to the Plenary Session o f the American Pediatric Society, Denver, Colo., April 17, 1975. Dr. Ragab is the recipient of a Faculty Research Award from The American Cancer Society. *Reprint address: Department of Pediatrics, Emory University School of Medicine, 69 Butler St., S.E., Atlanta, Ga. 30303.
Vol. 88, No. 5, pp. 790-794
tion. Patients who had inhibitors detectable in their serum to specific regulators and patients who had defective microenvironment in their bone marrow probably would not benefit from such a transplantation. In this study we have investigated the factors involved in the regulation of granulopoiesis by the agar culture technique in ten children with aplastic anemia. These included: three children with Fanconi's anemia, one postvaricella patient, one post-hepatitis patient, and five children with idiopathic aplastic anemia. Aplastic anemia is defined as pancytopenia with hypoplasia of the bone marrow in the absence of enlargement of the liver and spleen. The safient features are summarized in Table I.
Abbreviations used AAi aplasticanemia CFC: colony-forming cells CSA: colony-stimulating activity
MATERIALS AND METHODS
Bone marrow colony-forming cells. Bone marrow samples from children with AA were obtained and cultured in the soft agar system according to the method of Robinson and Pike. 2 Briefly, 1 • l0 B nucleated bone marrow cells were cultured in an overlayer containing 0.3% agar on top of "standard" underlayers prepared from the peripheral blood of two individuals who had similar colony-stimulating activity. T h e patients' bone
Volume 88 Number 5
Granulopoiesis in anemia
79 1
220
140 2OO ,q, 120 180
r~ I00 U.l
u Z
80
160
o
d~
Oc b.I (/)
60 L).
b 40
z
-k
>I->
20
140
izo
,
Z
o_.1 o r
o~
6O
4O
k)
,,q
30 20
uJ
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o
NORMAL
.~ ]o
APLASTIC ANEMIA
o
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o ~ o - API.ASTIC ANEMIA
Fig. 2. CFC from the peripheral blood of children with aplastic anemia and normal children. Horizontal bar denotes median for normal children.
marrow cells were also cultured on u n d e r l a y e r s p r e p a r e d from their own peripheral blood cells. T h e u n d e r l a y e r s contained 1 X 106 peripheral blood n u c l e a t e d c e l l s / m l in 0.5% agar. The plates were i n c u b a t e d at 37 ~ C i n a humidified atmosphere with a 7.5% flow o f COs for 14 days; at the end o f this period, colonies c o n t a i n i n g 50 or more cells were scored undei" a n i n v e r t e d microscope. Duplicate, a n d later quadruplicate, cultures were prepared from each sample. In Cases 9 a n d 10 the b o n e marrow cells were also cultured at ten times the n o r m a l concentration (1 x 100. The n u m b e r o f C F C was corn-
Fig. 3. The effect of addition of 0.2 ml of normal serum and serum from children with' aplastic anemia on the CFC of normal bone marrow cells. The number of colonies formed by normal bone marrow cells cultured without serum is arbitrarily designated as 100%.
p a r e d to those o b t a i n e d from the b o n e m a r r o w o f 25 "control" children cultured on the same underlayers. T h e " c o n t r o l " children h a d b o n e m a r r o w aspirations perf o r m e d for a variety o f reasons (solid t u m o r s at time of diagnosis, for the exclusion o f storage disorders, etc.); they had normal hem 9 and normal bone marrow morphology. Peripheral blood CFC. T e n milliliters o f peripheral b l o o d were aspirated from each child w h o h a d AA and cultured in agar on top o f the " s t a n d a r d " underlayers at a c o n c e n t r a t i o n o f 4 x l0 s nucleated cells. In Case 10 the
792
Ragab et al.
The Journal of Pediatrics May 1976
Table I. Hematologic values at time studies were performed
Case No.
Age (yr)
Sex
Race
Etiology
Outcome
1 2 3 4 5 6 7 8 9 10
5 10/12 5 2/12 1 9 2/12 10 1/12 11 6 13 13 8
F F M M M M F M F F
White White Black Black White White White White White White
Idiopathic Idiopathic Idiopathic Post-hepatic Idiopathic Fanconi Fanconi Fanconi Idiopathic Post-varicella
Died Died Died Alive Alive Alive Alive Alive Alive Alive
Time from Dx
Hgb (gm/dl)
66rno Dx 21mo 8mo 35mo 28mo Dx 24mo Dx Dx
6.5 6.3 12.2 13.7 11.7 13.9 9.7 6.4 5.2 5.5
Platelets (/mm 3) 13,000 14,000 5,000 120,000 15,000 65,000 36,000 73,000 12,000 4,000
% WBC Lymph(~ram~) ocytes 5,600 2,500 3,500 4,070 3,200 3,600 4,500 2,900 1,300 2,600
39 90 62 35 66 79 27 69 60 67
% Polymorphonuclear cells
% Monocytes
BM eellu. larity
56 9 38 62 34 20 4 29 24 25
2 0 0 2 0 1 9 0 t2 7
J, $ $ $ J, J, $ $ ,~ ~, ,L ,L ,L ,L $ $ ,L ,~ J, J, ~, $ ~, ~. ,~
Abbreviations: Dx = diagnosis, BM = bone marrow, BM CFC = bone marrow colony forming cells/10s nucleated cellsplated, PB CFC = peripheral blood colony forming cells/4 x 10~nucleated cells plated, Ox = oxymetholone, P = prednisone, ND = not done.
peripheral blood cells were also cultured at a higher concentration (1 x 106 nucleated ceils). These were compared to the C F C obtained from the culture of the peripheral blood cells of 14 normal children. Peripheral blood CSA. Underlayers were prepared from the peripheral blood cells o f children with AA. Bone marrow cells from children with acute lymphocytic leukemia in remission were cultured on the " s t a n d a r d " underlayers as well as on underlayers from the cells of A A patients. A m i n i m u m of two and a m a x i m u m of nine bone marrow samples were cultured simultaneously on each underlayer; the colonies formed on each were compared. The colony counts obtained with the "standard" underlayers were taken as 100%. The colony-stimulating activity of the peripheral blood cells of children with A A was compared by expressing the colony counts obtained with the aplastic anemia underlayers as a percentage o f those obtained with the "standard" underlayers. Serum (humoral factors). The children with A A were investigated for the presence of inhibitors of granulopoiesis in their sera. Sera from eight children with A A were added to normal bone marrow cells obtained from normal adult volunteers and left to incubate for one hour, then mixed with agar, and cultured on the " s t a n d a r d " underlayers. The colonies were counted and expressed as a percentage of the control colony counts obtained without the addition of serum. This was compared to the addition of h u m a n sera, obtained from 18 normal individuals, which were added to normal adult bone marrow cells and cultured in agar in the same manner. RESULTS
Bone marrow CFC. All children with A A had greatly decreased C F C in their bone marrow when c o m p a r e d to
"control" children (Fig. 1). Utilizing the Wilcoxan rank sum test, this is statistically significant (P 0.001). In the tast two patients, the marrow cells were also cultured at a concentration of 1 x 106 nucleated cells; even with this stimulation, very few colonies (1 to 4) were observed. When the cells of the patients' bone m a r r o w were cultured on their own underlayers, no colonies were observed. W e have attempted to quantitate the dilution o f the bone marrow sample with peripheral blood. This was performed by observing the difference in percentage of myelocytes in the morphology marrow sample in comparison with the percentage of myelocytes in the cultured marrow sample. ~ The marrow sample was never diluted more than twofold with peripheral blood. Peripheral blood CFC. Children with A A had decreased C F C in their peripheral blood. In seven of the ten children examined, no C F C were detected (Fig. 2). In the last patient (Case 10) no colonies were observed when the peripheral blood cells were cultured at a concentration o f 1 X l0 s nucleated cells. Utilizing the Wilcoxan rank sum test, the difference in the peripheral blood C F C between normal children and children with A A is statistically significant (P 0.001). Colony-stimulating activity. The CSA of the peripheral blood cells o f children with A A was decreased in five o f the eight children tested. It was greatly reduced in Cases 7 and 8 (both children had Fanconi's anemia) (Table I). The variability i n CSA among the peripheral blood of normal individuals varies in our laboratory from 80 to 220% of the "standard" underlayers. Effect of serum. When normal h u m a n serum was added to cells of normal adult bone marrow in culture, it either had no effect: (6/18) or it stimulated ( 1 0 / 1 8 ) - o n l y two sera inhibited colony formation (Fig. 3). W h e n serum
Volume 88 Number 5
Medication
Granulopoiesis in anemia
BM CFC
Ox + P Ox + P Ox + P None Ox + P Ox None None Transplant Ox + P
0 3 0 9 0 1 0 0 0.5 0
PB CFC
% CSA
Serum tested
0 4 0 0 1 2 0 0 0 0
81 129 83 69 42 56 11 12 133 87
ND ND + + + + + + + +
samples from eight children with AA were added to normal bone marrow cells in culture, there was stimulation of colony formation by seven of the eight sera tested. one serum sample had no effect. It was concluded that no inhibitor of granulopoiesis exists in the serum of these children. DISCUSSION The basic mechanism involved in the pathogenesis of AA still is unknown. Based on animal models, two alternative hypotheses are put forward to explain the marrow hypoplasia.' It has been determined that the defect in genetically anemic S1/SP mice resides in the microenvironment of the marrow. Transplantation of normal pluripotent stem cells in this case will not correct the anemia? In contrast, the anemia in W / W v mice can be corrected by transplantation of normal bone marrow stem cells, demonstrating that in this case the defect resides in the stem cell? In man, however, there is to date no system available which can measure the functional activity of the pluripotent stem cell. An in vitro agar culture system is available which measures a committed granulocytic progenitor or stem cell (CFC). In this culture system, two groups of cells are required: a colony-forming cell, which now is believed to resemble a lymphocyte on light microscopy (but is indistinguishable on electron microscopy),~ and a "helper" cell that produces CSA. The latter cell is believed to be a monocyte7 or macrophage? This culture system has been used to study granulopoiesis in a variety of disorders. Thus it has been demonstrated that adults with acute myelogenous leukemia"' ,6 and children with acute lymphocytic leukemia have decreased CFC in their bone marrow," but increased CFC in their peripheral
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blood?, 1~These observations suggest a perturbed condition in the bone marrow of these patients in which their CFC migrates into the peripheral blood. The peripheral blood CFC is increased in adults with myelofibrosis13 and in children with osteopetrosis." The CSA is decreased in children with acute lymphocytic leukemia." Senn and colleagues1~ studied CFC in bone marrow and CSA production in peripheral blood of six patients with neutropenia, and noted that the defect varied from patient to patient: three patients had normal CFC and CSA production (suggesting peripheral destruction of white cells), two patients had decreased CFC and CSA production, and one patient had decreased CFC but normal CSA production. Greenberg and Schrier ~6 studied three adults with drug-induced neutropenia and two adults with neutropenia associated with hypoplastic bone marrow and noted that they had decreased bone marrow production of CFC. In the latter study, production of CSA was not examined. Our results demonstrate that there is a decrease in colony-forming cells in the bone marrow and peripheral blood of children with aplastic anemia. This reduction of CFC is absolute since plating with bone marrow cells at ten times the normal concentration did not increase the number of colonies observed. This decrease in bone marrow CFC was observed in all children examined, including three children who were not neutropenic at the time of study, suggesting perhaps that some areas in the bone marrow were still functional and capable of proliferation. Production of CSA also was decreased in five of the eight children studied. It was very much reduced in two of the three children with Fanconi's anemia who were investigated. This finding indicates that in some cases of AA, both the CFC and the CSA-producing cells are defective. It is interesting to note that in seven out of ten children studied there was a concomitant severe monocytopenia. One child (Case 7) had a normal number of m0nocytes in the peripheral blood, but had decreased CSA. From these data it is difficult to decide whether there is decreased CSA production or whether the CSA produced is defective. No inhibitor of in vitro granUlopoiesis could be detected in the sera of the eight children with aplastic anemia tested. It is generally believed that the sulfur colloid scan (99mTc) measures uptake by the reticuloendotheliat system, whereas the ' " I n d i u m chloride scan measures uptake by the erythroid elements of the bone m a r r o w y Both patients (Cases 9 and 10) had normal colloid scans, although one patient had a normal indium scan despite the biopsy-proved hypoplasia. It therefore is difficult to interpret the value of these scans in children with AA? 8 Although we have studied only the granulocytic pro-
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Ragab et al.
genitor cell (or CFC), the defect may reside in the stem cell, inasmuch as all bone marrow elements (erythroid, granulocytic, and megakaryocytic) are affected. Freedman and associates TM have followed eight children with A A for periods up to nine years. They noted that none of their patients ever achieved hematologic normality. Our surviving patients still have abnormally 10w granul0cyte and platelet counts. This would suggest that the use of androgens, by increasing erythropoietin production 2~ or by acting directly on the stem cells, increases red cell production, but does not affect the megakaryocytic or myeloid elements. 2.... Recently, this hypothesis has been disptlted. 23 The prognosis of AA, with the exception o f druginduced A A in children, 24 is generally poor. Recently, Storb and associates 25 published their results on bone marrow transplantation in 24 patients with AA. Twentyone patients showed prompt hem0poietic engraftment, and 13 patients (54%) demonstrated marked i m p r o v e m e n t after the transplant. This study would suggest that the stem cell is defective in AA, and that engraftment by normal stem cells from related donors corrects the disorder in most instances. Further refinement in the immunosuppression and support of patients during bone marrow transpl~mtation may result in this m o d e of therapy becoming the treatment of choice in AA. Inasmuch as there is an ongoing International Aplastic Anemia Transplantation Study, it m a y be beneficial to study bone marrow C F C and other in vitro factors in all A A patients. We would like to thank Dr. Philip R. Dodge (Chairman, Department of Pediatrics, Washington University) and Dr. Stuart Kornfeld (Professor of Medicine, Washington University) for their criticai review of this manuscript. REFERENCES
1. Stohlman F Jr: Aplastic anemia, Blood 40:282, 1972. (Editorial.) 2. Robinson WA, and Pike BE: Colony growth of human bone marrow cells in vitro, in Stohlman F Jr, editor: Hemopoietic cellular pl?oliferation, New York, I970, Grune & Stratton, Inc, pp 249-259. 3. Ragab AH, Gilkerson E, Myers M, and Choi SC: The culture of colony forming units from the peripheral blood and bone marrow of children with acute lymphocytic leukemia, Cancer 34:663, 1974. 4. McCull0ch. EA, Siminovitch L, Till JE, Russell ES, and Bernstein SE: The cellular basis of the genetically determined hemopoietic defect in anemic mice of genotype S1/S1d, Blood 26:399, 1965. 5. McCulloch EA, Siminovitch L, and Till JE: Speen-colony formation in anemic mice of genotype W/W ~, Science 144:844/1964.
The Journal of Pediatrics May 1976
6. Zucker-Franklin D, Grusky G, and L'Esperance P: Granulocyte colonies derived from lymphocyte fractions of normal human peripheral blood, Proc Natl Acad Sci 71:2711, 1974. 7. Chervenick PA, and LoBuglio AF: Human blood monocytes-stimulators of granulocyte and mononuclear colony formation in vitro Science 178:164, 1972. 8. Messner HA, Till JE, and McCulloch EA: Interacting cell populations affecting granulopoietic colony formation by normal and leukemic human marrow cells, Blood 42:701, 1973. 9. Brown CH, and Carbon e PP: In vitro growth of normal and leukemic human bone marrow, J Natl Cancer Inst 46:989, 1971. 10. Iscove NN, Senn JS, Till JE, and McCulloch EA: Colony formation of normal and leukemic human marrow cells in culture-effect of conditioned medium from human leukocytes, Blood 37:1, 1971. 11. Ragab AH, Gilkerson E, and Myers ML: Granulopoiesis in childhood leukemia, Cancer 33:791, 1974. 12. Robinson WA, Kurnick JE, and Pike BL: Colony growth of human leukemic peripheral blood cells in vitro, Blood 38:500, 1971. 13. Chervenick PA: Increase in circulating stem cells in patients with myelofibrosis, Blood 41:67, 1973. 14. Ragab AH, Ducos R, Crist WM, and Duck SC: Granulopoiesis in oste0petrosis, J PEDIATR87:422, 1975. 15. Senn JS, Messner HA, and Stanley ER: Analysis of interacting cell populations in cultures of marrow from patients with neutropenia, Blood 44:33, 1974. 16. Greenberg PL, and Schrier SL: Granulopoiesis in neutropenic disorders, Blood 41:753, 1973. 17. McNeil BJ, Holman BL, Button LN, and Rosenthal DS: Use of Indium chloride scintigraphy in patients with myelofibrosis, J Nuel Med 15:647, 1974. 18. Merrick MV, Gordon-Smith EC, Lavender JP, and Szur L: A comparison of XHInwith 52Fe and "~mTc-sulfur colloid for bone marrow scanning, J Nucl Med 16:66, 1975. 19. Freedman MH, Saunders EF, Hilton J, and McClure PD: Residual abnormalities in acquired aplastic anemia of childhood, JAMA 228:201, 1974. 20. Fried W, and Gurney CW: The erythropoietic response to testosterone in male and female mice, J Lab Clin Med 67:420, 1966. 21. Shahidi NT: Androgens and erythropoiesis, N Engl J Med 289:72, 1973. 22. Gorshein D, Hait WN, Besa EC, Jepson JH, and Gardner FH: Increased stem cell response to erythropoietin induced by androgens, Endocrinology 93:777, 1973. 23. Reissmann KR, Udupa KB, and Kawada K: Effects of erythropoietin and androgens on erythroid stem cells ~/fter their lelective suppression by BCNU, Blood 44:649, 1974. 24. Heyu RM, Ertel IJ, and Tubergen DG: Course of acquired aplastic anemia in children treated with supportive care, JAMA 208:1372, 1969. 25. Storb R, Thomas ED, Buckner CD, Clift RA, Johnson FL, Fefer A, Glucksberg H, Giblett ER, Lerner KG, and Neiman P: Allogeneic marrow grafting for treatment of aplastic anemia, Blood 43:157, 1974.
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