- Email: [email protected]
The significance of lymphocytosis in congenital hypoplastic anemia
550
October 1975 The Journal o f P E D I A T R I C S
The significance of lymphocytosis in congenital hypoplastic anemia Two infants with congenital hypoplastic anemia had an unusual number of lymphocytes in their peripheral blood and in the bone marrow. This caused an erroneous diagnosis of acute lymphocytic leukemia to be made in the first case and inappropriate therapy to be administered for three months. The second of these cases provided an unusual opportunity to study human erythrocyte precurso r dynamics. Serial bone marrow aspirates, obtained after institution of treatment with a corticosteriod, revealed an initial increase in labeled lymphocytes, a concomitant decrease in their number, and the subsequent appearance of erythroid elements. These findings suggest that cells classified morphologically as lymphocytes may serve as erythroid precursors in human beings.
Thomas D. Miale, M.D., and Gerald E. Bloom, M.D., Gainesville, Fla.
THE morphologic identity of the earliest red blood cell progenitors in the h u m a n bone marrow and, in particular, whether lymphocytes might serve as such precursors remains unresolved, chiefly because of experimental limitations. I n contrast, various animal models have provided considerable data concerning the identity of stem ceils in other species. Insight into the evolution of erythrocytic production in m a n might be gained by the investigation of congenital hypoplastic anemia (Diamond-Blackfan syndrome), a rare disorder of unknown etiology in which there is a marked, selective reduction of all R B C precursors in the bone marrow? Since a prompt remission after corticosteroid treatment usually occurs in this disorder, ~ s e q u e n t i a l analysis of bone marrow morphology and kinetics during therapy may provide an opportunity to study erythrocyte precursor dynamics. Although initial aspirates of bone marrow are normal in C H A except for depletion of the erythroid component, striking marrow lymphocytosis has been seen occasionally and was encountered in two cases of C H A reported here.
CASE REPORTS Case 1. Patient D: B., a 2-month-old Caucasian male infant, was seen in May, 1968, because of progressive anemia. The From the Division of Hematology, Department of Pediatrics, University of Florida College of Medicine.
Vol. 87, No. 4, pp. 550-553
hemoglobin level at birth had been 11.5 gm and had fallen to 2.5 mg/l by the age of 6 weeks; a blood transfusion was required. At age 2 months, the following laboratory studies were recorded: white blood cell count 4,900/mm 3 of which 98% were lymphocytes; platelet count 200,000/mm~; Hgb concentration 5.0 gm/dl; and reticulocyte count 0.1%. Both the number of the lymphocytes and the absolute neutropenia suggested acute lymphocytic leukemia. Treatment with 6-mercaptopurine and prednisone was started with a "complete remission," including a normal bone marrow aspirate, was achieved within four weeks. Abbreviations used RBC: red blood cell CHA: congenital hypoplastic anemia WBC: white blood cell count Hbg: hemoglobin ALL: acute lymphocytic leukemia PPC: primitive proliferating cells Another examination of peripheral blood and bone marrow at 5 months of age established the diagnosis of CHA, and prednisone was continued at a daily dosage of 30 rag. Many attempts to discontinue corticosteroid therapy over the past six years have been unsuccessful because of recurrence of anemia, reticulocytopenia, and bone marrow erythroid hypoplasia. Maintenance therapy with 30 mg of prednisone on alternate days has been required; current hemoglobin range is 10.2 to 12.0 gm/dl. Case 2. Patient H. GI, a 3 1/2-month-old Caucasian female infant, was referred in March, 1972, for evaluation of anemia. The Hgb level at birth was 9.7 gm, and a blood transfusion was
Volume 87 Number 4
Lymphocytosis in congenital hypoplastic anemia
551
given. Additional transfusions were required at 6 and 12 weeks of age, respectively. The pretransfusion Hgh levels on these latter occasions were 5.4 gm/dl and 6.9 gm/dl, respectively; no reticulocytes were seen on the blood smear. The bone marrow aspirate at 3 1/2 months of age established a diagnosis of CHA; treatment with prednisone was initiated at a daily dosage of 30 mg. At this time the WBC numbered 7,900/ mm 3, with 74% lymphocytes; lymphocytes comprised 94% of the marrow cellular elements. A concomitant lymphocytosis was present in both peripheral blood (74%; WBC 7,900) and bone marrow (94%). Six days after onset of treatment, erythroid precursors appeared in bone marrow aspirates. The reticulocyte count increased from 0 to 2.4% at 7 days and to a maximum of 22% at 12 days after therapy was started. The patient's Hgb level increased steadily over the ensuing months. However, over the past 36 months she has remained dependent on alternate-day therapy with 40 mg of prednisone to maintain remission. Current hemoglobin range is 10.2 to 12.2 gm/dl. MATERIALS
AND METHODS
Serial aspirations of bone marrow were obtained from the anterior tibia or posterior superior iliac spine. 3Hthymidine (specific activity, 2.0 C / m M ) was added to 1 ml of bone marrow specimen in nutrient m e d i u m TC-199 (Difco Laboratory, Detroit, Mich.) to a final concentration of 1.0 #C/ml. After incubation at 37 ~ C for 60 minutes, the bone marrow cells were washed in isotonic saline, resuspended, and centrifuged. Smears were obtained from the sediment and examined by autoradiography utilizing Kodak AR-10 photographic f i l m as previously outlined. TM After the labeled cells were identified, counted, and photographed, the photographic emulsion was removed with trypsin. The slides were then restained with Wfight's stain, and the labeled cells were found to be well-differentiated lymphocytes. RESULTS Although marked initial marrow lymphocytosis with a mixed population of small and medium-sized lymphocytes was present in each patient, lymphocytes were predominantly small in Case 1 in contrast to a preponderance of medium-sized lymphocytes in Case 2. In each patient, the lymphocytes were nonvacuolated with a high nuclear-cytoplasmic ratio and contained two or less nucleoli per cell. In addition to lymphocytes, both initial bone marrow aspirates contained a small n u m b e r of myeloid elements, a normal proportion of megakaryocytes, and an increased over-all cellularity (Fig. 1). Sequential changes were followed in bone marrow morphology after the initiation of corticosteroid therapy in Case 2. Lymphocytes decreased from 94% to 25%, and erythroid precursors increased from 0 to 19% after ten days of treatment (Fig. 2). The percentage of labeled
Fig. 1. Initial bone marrow aspirate (Case 2).
lymphoid cells incorporating 3H-thymidine also increased from 0 at the onset of treatment to 13% and 19% after 117 and 141 hours, respectively. The intensity of labeling within lymphocytes ranged from 2 to 26 grains per cell. Since occasional nonlymphoid cells also became heavily labeled, the morphology of the cells counted as lymphocytes was confirmed by removing the photographic emulsion and identifying the cells after restaining, DISCUSSION Although other studies have shown that m a r r o w lymphocytosis is occasionally encountered in both normal infants and C H A patients, no previous study has indicated that the n u m b e r and morphology of lymphocytes in bone marrow in C H A may resemble ALL (Fig. 1). Additional clinical and laboratory features, however, may permit the differentiation of these disorders even when bone marrow aspirates are similar. For example, onset in infancy, reticulocytopenia, absence of thrombocytopenia, leukocytosis, and no significant hepatosplenomegaly are more often found in C H A than in ALL. A better understanding may also be derived from a
552
Miale and Bloom
The Journal of Pediatrics October 1975 9 I00
2o1 I~
/
,
~k~k ~k
" ' "Jl
' t '~
,
m
1 1
~
i
o
' ' ~ ' ' LYMPHOID ERYTHROIO .... LABELLED LYMPHOID
\ \
',0~
g'~
x
\,
o
'.-"
"75
..,
,,
o
,
e
'"'"-.. \
-a
t
9
:50
0 Z u w o I
N
-25
5'
0
20
60
~
IOO
14o leo TIME ( h o u r s )
z2o
e~o
Fig. 2. Sequential changes in serial bone marrow aspirates (Case 2). Table I. Initial bone marrow differential counts (percentage of 500 cells counted)
Case
Small- Mediumsized sized Total lympho- lymphocytes Myeloid ] Erythr~id lymphoid cytes
1
9
0
2
6
0
91 94
69 10
22 84
cumulative analysis of previously reported data. In 183 bone marrow aspirates in 149 normal infants under the age of 1 year, the percentage of lymphocytes reached a maximum mean value of 47% (range 31 to 81%) at 12 weeks of age. '7-'9 "Immature" marrow lymphocytes or percentage of lymphoblasts reached a peak value of 2.0% (range 0 to 5.5%) at this age. '7 Marrow lymphocytosis was present in only 2 of 68 previously reported CHA patients whose bone marrow morphology was described in qualitative terms. The mean percentage of lymphocytes was 42.1% (range 5 to 80%) in 62 bone marrow aspirates in 24 additional CHA patients? -7 The marked marrow lymphocytosis in the two CHA patients observed here is in contrast to prior reports in both untreated CHA patients and normal infants? -~, ~-~9
In addition, this unusual observation facilitated the identification of a subgroup of marrow lymphocytes with augmented 3H-thymidine uptake after the administration of a corticosteroid. It is unlikely that this occurrence represents a nonspecifie effect since decreased incorporation of 3H-thymidine is to be expected in bone marrow ..... and lymphatic tissue 2~-~8 of normal animals after treatment with a corticosteroid. In fact, this enhanced labeling could be interpreted as additional evidence for the existence of lymphoerythropoietic stem cells in human beings. The existence of such stem cells has been suggested by previous studies in human beings and experimental animals. For instance, the earliest bone marrow in human fetuses consists of loose connective tissue interspersed among dilated, thin-walled blood vessels? .... This is followed by infiltration with lymphoid cells and the formation of red marrow, frequently in cords surrounding thin-walled arteries and large veins. 2~ Cytologic,~, 10 autoradiographic,8 and transplantation studies 11-1~ in man and experimental animals have also suggested that lymphocytes may serve as RBC precursors. For example, Cronkite and colleagues 8 studied the dynamics of hematopoiesis in man and in mice by means of in vivo ~H-thymidine labeling and identified primitive proliferating cells. These ceils incorporated ~H-thymidine earlier than any other marrow component. PPC's were morphologically lymphocytes; the term PPC was used "in order to avoid semantic difficulties." Subsequently erythroid, myeloid, and megakarocytes became labeled. Studies involving the transplantation of bone marrow cells have shown that protection from fatal marrow suppression in lethally irradiated rats and mice is a function of the number of small lymphocytes inoculated. 1'~'1'~ Trarrsplanted cells with identical radiation-induced chromosome markers have constituted erythroid colonies in one experimental system (mouse spleen) and behaved as immunocompetent lymphocytes in another (mixed-leukocyte culture). 21 Another indication that lymphocytes may have an erythropoietic function is the known effect of antilymphocytic serum in decreasing the hematopoietic activity of human bone marrow. ~-~a In the present study (Case 2), an increase in labeled lymphocytes, a concomitant decrease in their number, and the subsequent appearance of erythroid precursors (72 hours later) suggest that cells classified morphologically as lymphocytes might serve as erythroid precursors in human beings. We wish to acknowledge the assistance of A. Ashley Weech, M.D., for critical review of this manuscript; Larry Haugh, Ph.D., for statistical assistance; and Ms. Marybruce Dowd and Ms. Candace Crowder for manuscript preparation.
Volume 87 Number 4
Lymphocytosis in congenital hypoplastic anemia
REFERENCES 1. Diamond LK, Allen DM, and Magill FB: Congenital (erythroid) hypoplastic anemia. A 25-year study, Am J Dis Child 102:403, 1961. 2. Allen DM, and Diamond LK: Congenital (erythroid) hypoplastic anemia. Cortisone treated, Am J Dis Child 102:416, 1961. 3. Cathie lAB: Erythrogenesis imperfecta, Arch Dis Child 25:313, 1950. 4. Kass A, and Sundal A: Anaemia hypoplastica congenita (anaemia typus josephs-diamond-blackfan). Report of a case treated with adrenocorticoptopin with effect, Acta Paediatr 42:265, 1953. 5. Smith CH: Hypoplastic and aplastic anemias of infancy and childhood: with a consideration of the syndrome of nonhemolytic anemia ~of the newborn, J PEDIATR 43:457, 1953. 6. Burgert O Jr, Kennedy RLJ, and Pease GL: Congenital hypoplastic anemia, Pediatrics 13:218, 1954. 7. Starling KA, and Fernbach D J: Congenital hypoplastic .anemia. Review of eight cases, Texas Med 63:63, 1967. 8. Cronkite EP, Fliedner TM, Bond BP, Rubini JR, Brecher G, and Quastler H: Dynamics of hemopoietic proliferation in man and mice studied by .'H-thymidine incorporation into DNA, Ann NY Acad Sci 77:803, 1959. 9. Harris PF: Quantitative examination of bone marrow in guinea-pigs after gamma irradiation, Br Med J 2:1032, 1956. 10. Yoffey JM: Quantitative cellular hematology, Springfield, Ill, 1960, Charles C Thomas, Publisher. 11. Brecher G, Endicott KM, Gump H, and Brawner HP: Effects of X-ray on lymphoid and hemopoietic tissues of albino mice, Blood 3:1259, 1948. 12. Cudkowicz G, Bennett M, and Shearer GM: Pluripotent stem cell function of the mouse marrow "lymphocyte," Science 144:866, 1964. 13. Cudkowicz G, Upton AC, Shearer GM, and Hughes WL: Lymphocyte content and proliferative capacity of serially transplanted mouse bone marrow, Nature 201:165, 1964. 14. Kurnick NB, and Nokay N: Repopulation of bone marrow in mice: number and type of ceils required for post-Xradiation protection, Radiat Res 25:53, 1965. 15. Morrison JH, and Toepfer JR: Survival of lethally Xradiated rats after treatment with isogenic marrow lymphocytes, Am J Physiol 213:923, 1967. 16. Rich MC, and Bloom GE: An analysis of selected factors involved in the transformation of normal human peripheral blood lymphocytes, Ann Clin Lab Sci 2:404, 1972. 17. Vogel P, and Bassen FA: Sternal marrow of children in
18.
19.
20. 21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
553
normal and in pathologic states, Am J Dis Child 57:245, 1939. Shapiro LM, and Bassen FA: Sternal marrow changes during the first week of life. Correlation with peripheral blood findings, Am J Med Sci 202:341, 1941. Gairdner D, Marks J, and Roscoe JD: Blood formation in infancy. Part I. The normal bone marrow, Arch Dis Child 27:128, 1952. Yoffey JM: The stem cell problem in the fetus, Isr J Med Sci 7:825, 1971. Nowell PC, Hirsch BE, Fox DH, and Wilson DB: Evidence for the existence of multipotential lympho-hematopoietic stem cells in the adult rat, J Cell Physiol 75:151, 1970. DeMeester TR, Anderson ND, and Shaffer CF: The effect of heterologous anti-lymphocyte serum on mouse hemopoietic stem cells, J Exp Med 127:731, 1968. Field EO, and Gibbs JE: Cross-reaction of anti-lymphocyte serum with haemopoietic stem ceils, Nature 217:561, 1968. Cardinali G, Cardinali G, DeCaro BM, Handler AH, and Aboul-Enein M: Effect of high doses of cortisone on bone marrow cell proliferation in the Syrian hamster, Cancer Res 24:969, 1964. Hunstein W, and Strey M: Effect of prednisone on the H 3thymidine-uptake o f bone marrow ceils in rats, Klin Wochenschr 43:52, 1965. Stevens W, Coelssides C, and Dougherty TF: Effects of cortisol on the incorporation of thymidine-2-~'C into nucleic acids of lymphatic tissue from adrenalectomized CBA mice, Endocrinology 76:1 I00, 1965. Stevens W, Colessides C, and Dougherty TF: A time study on the effect of cortisol on the incorporation of thymidine-214C into nucleic acids of mouse lymphatic tissue, Endocrinology 78:600, 1966. Knutson F, and Lundin PM: The effect of hypophysectomy, adrenalectomy and cortisone on the incorporation of tritiated thymidine in rat organs, with special references to the lymphoid tissues, Acta Endocrinol 53:519, 1966. Glasar K, Limarzi LR, and Poncher HG: Cellular composition of the bone marrow in normal infants and children, Pediatrics 6:789, 1950. Sturgeon P: Volumetric and microscopic pattern of bone marrow in normal infants and children. II. Cytologic pattern, Pediatrics 7:642, 1951. Unanue ER, Grey HM, Rabellino E, Campbell P, and Schmidtke J: Immunoglobulins on the surface of lymphocytes. II. The bone marrow as the main source of lymphocytes with detectable surface-bound immunoglobulin, J Exp Med 133:1188, 1971.
October 1975 The Journal o f P E D I A T R I C S
The significance of lymphocytosis in congenital hypoplastic anemia Two infants with congenital hypoplastic anemia had an unusual number of lymphocytes in their peripheral blood and in the bone marrow. This caused an erroneous diagnosis of acute lymphocytic leukemia to be made in the first case and inappropriate therapy to be administered for three months. The second of these cases provided an unusual opportunity to study human erythrocyte precurso r dynamics. Serial bone marrow aspirates, obtained after institution of treatment with a corticosteriod, revealed an initial increase in labeled lymphocytes, a concomitant decrease in their number, and the subsequent appearance of erythroid elements. These findings suggest that cells classified morphologically as lymphocytes may serve as erythroid precursors in human beings.
Thomas D. Miale, M.D., and Gerald E. Bloom, M.D., Gainesville, Fla.
THE morphologic identity of the earliest red blood cell progenitors in the h u m a n bone marrow and, in particular, whether lymphocytes might serve as such precursors remains unresolved, chiefly because of experimental limitations. I n contrast, various animal models have provided considerable data concerning the identity of stem ceils in other species. Insight into the evolution of erythrocytic production in m a n might be gained by the investigation of congenital hypoplastic anemia (Diamond-Blackfan syndrome), a rare disorder of unknown etiology in which there is a marked, selective reduction of all R B C precursors in the bone marrow? Since a prompt remission after corticosteroid treatment usually occurs in this disorder, ~ s e q u e n t i a l analysis of bone marrow morphology and kinetics during therapy may provide an opportunity to study erythrocyte precursor dynamics. Although initial aspirates of bone marrow are normal in C H A except for depletion of the erythroid component, striking marrow lymphocytosis has been seen occasionally and was encountered in two cases of C H A reported here.
CASE REPORTS Case 1. Patient D: B., a 2-month-old Caucasian male infant, was seen in May, 1968, because of progressive anemia. The From the Division of Hematology, Department of Pediatrics, University of Florida College of Medicine.
Vol. 87, No. 4, pp. 550-553
hemoglobin level at birth had been 11.5 gm and had fallen to 2.5 mg/l by the age of 6 weeks; a blood transfusion was required. At age 2 months, the following laboratory studies were recorded: white blood cell count 4,900/mm 3 of which 98% were lymphocytes; platelet count 200,000/mm~; Hgb concentration 5.0 gm/dl; and reticulocyte count 0.1%. Both the number of the lymphocytes and the absolute neutropenia suggested acute lymphocytic leukemia. Treatment with 6-mercaptopurine and prednisone was started with a "complete remission," including a normal bone marrow aspirate, was achieved within four weeks. Abbreviations used RBC: red blood cell CHA: congenital hypoplastic anemia WBC: white blood cell count Hbg: hemoglobin ALL: acute lymphocytic leukemia PPC: primitive proliferating cells Another examination of peripheral blood and bone marrow at 5 months of age established the diagnosis of CHA, and prednisone was continued at a daily dosage of 30 rag. Many attempts to discontinue corticosteroid therapy over the past six years have been unsuccessful because of recurrence of anemia, reticulocytopenia, and bone marrow erythroid hypoplasia. Maintenance therapy with 30 mg of prednisone on alternate days has been required; current hemoglobin range is 10.2 to 12.0 gm/dl. Case 2. Patient H. GI, a 3 1/2-month-old Caucasian female infant, was referred in March, 1972, for evaluation of anemia. The Hgb level at birth was 9.7 gm, and a blood transfusion was
Volume 87 Number 4
Lymphocytosis in congenital hypoplastic anemia
551
given. Additional transfusions were required at 6 and 12 weeks of age, respectively. The pretransfusion Hgh levels on these latter occasions were 5.4 gm/dl and 6.9 gm/dl, respectively; no reticulocytes were seen on the blood smear. The bone marrow aspirate at 3 1/2 months of age established a diagnosis of CHA; treatment with prednisone was initiated at a daily dosage of 30 mg. At this time the WBC numbered 7,900/ mm 3, with 74% lymphocytes; lymphocytes comprised 94% of the marrow cellular elements. A concomitant lymphocytosis was present in both peripheral blood (74%; WBC 7,900) and bone marrow (94%). Six days after onset of treatment, erythroid precursors appeared in bone marrow aspirates. The reticulocyte count increased from 0 to 2.4% at 7 days and to a maximum of 22% at 12 days after therapy was started. The patient's Hgb level increased steadily over the ensuing months. However, over the past 36 months she has remained dependent on alternate-day therapy with 40 mg of prednisone to maintain remission. Current hemoglobin range is 10.2 to 12.2 gm/dl. MATERIALS
AND METHODS
Serial aspirations of bone marrow were obtained from the anterior tibia or posterior superior iliac spine. 3Hthymidine (specific activity, 2.0 C / m M ) was added to 1 ml of bone marrow specimen in nutrient m e d i u m TC-199 (Difco Laboratory, Detroit, Mich.) to a final concentration of 1.0 #C/ml. After incubation at 37 ~ C for 60 minutes, the bone marrow cells were washed in isotonic saline, resuspended, and centrifuged. Smears were obtained from the sediment and examined by autoradiography utilizing Kodak AR-10 photographic f i l m as previously outlined. TM After the labeled cells were identified, counted, and photographed, the photographic emulsion was removed with trypsin. The slides were then restained with Wfight's stain, and the labeled cells were found to be well-differentiated lymphocytes. RESULTS Although marked initial marrow lymphocytosis with a mixed population of small and medium-sized lymphocytes was present in each patient, lymphocytes were predominantly small in Case 1 in contrast to a preponderance of medium-sized lymphocytes in Case 2. In each patient, the lymphocytes were nonvacuolated with a high nuclear-cytoplasmic ratio and contained two or less nucleoli per cell. In addition to lymphocytes, both initial bone marrow aspirates contained a small n u m b e r of myeloid elements, a normal proportion of megakaryocytes, and an increased over-all cellularity (Fig. 1). Sequential changes were followed in bone marrow morphology after the initiation of corticosteroid therapy in Case 2. Lymphocytes decreased from 94% to 25%, and erythroid precursors increased from 0 to 19% after ten days of treatment (Fig. 2). The percentage of labeled
Fig. 1. Initial bone marrow aspirate (Case 2).
lymphoid cells incorporating 3H-thymidine also increased from 0 at the onset of treatment to 13% and 19% after 117 and 141 hours, respectively. The intensity of labeling within lymphocytes ranged from 2 to 26 grains per cell. Since occasional nonlymphoid cells also became heavily labeled, the morphology of the cells counted as lymphocytes was confirmed by removing the photographic emulsion and identifying the cells after restaining, DISCUSSION Although other studies have shown that m a r r o w lymphocytosis is occasionally encountered in both normal infants and C H A patients, no previous study has indicated that the n u m b e r and morphology of lymphocytes in bone marrow in C H A may resemble ALL (Fig. 1). Additional clinical and laboratory features, however, may permit the differentiation of these disorders even when bone marrow aspirates are similar. For example, onset in infancy, reticulocytopenia, absence of thrombocytopenia, leukocytosis, and no significant hepatosplenomegaly are more often found in C H A than in ALL. A better understanding may also be derived from a
552
Miale and Bloom
The Journal of Pediatrics October 1975 9 I00
2o1 I~
/
,
~k~k ~k
" ' "Jl
' t '~
,
m
1 1
~
i
o
' ' ~ ' ' LYMPHOID ERYTHROIO .... LABELLED LYMPHOID
\ \
',0~
g'~
x
\,
o
'.-"
"75
..,
,,
o
,
e
'"'"-.. \
-a
t
9
:50
0 Z u w o I
N
-25
5'
0
20
60
~
IOO
14o leo TIME ( h o u r s )
z2o
e~o
Fig. 2. Sequential changes in serial bone marrow aspirates (Case 2). Table I. Initial bone marrow differential counts (percentage of 500 cells counted)
Case
Small- Mediumsized sized Total lympho- lymphocytes Myeloid ] Erythr~id lymphoid cytes
1
9
0
2
6
0
91 94
69 10
22 84
cumulative analysis of previously reported data. In 183 bone marrow aspirates in 149 normal infants under the age of 1 year, the percentage of lymphocytes reached a maximum mean value of 47% (range 31 to 81%) at 12 weeks of age. '7-'9 "Immature" marrow lymphocytes or percentage of lymphoblasts reached a peak value of 2.0% (range 0 to 5.5%) at this age. '7 Marrow lymphocytosis was present in only 2 of 68 previously reported CHA patients whose bone marrow morphology was described in qualitative terms. The mean percentage of lymphocytes was 42.1% (range 5 to 80%) in 62 bone marrow aspirates in 24 additional CHA patients? -7 The marked marrow lymphocytosis in the two CHA patients observed here is in contrast to prior reports in both untreated CHA patients and normal infants? -~, ~-~9
In addition, this unusual observation facilitated the identification of a subgroup of marrow lymphocytes with augmented 3H-thymidine uptake after the administration of a corticosteroid. It is unlikely that this occurrence represents a nonspecifie effect since decreased incorporation of 3H-thymidine is to be expected in bone marrow ..... and lymphatic tissue 2~-~8 of normal animals after treatment with a corticosteroid. In fact, this enhanced labeling could be interpreted as additional evidence for the existence of lymphoerythropoietic stem cells in human beings. The existence of such stem cells has been suggested by previous studies in human beings and experimental animals. For instance, the earliest bone marrow in human fetuses consists of loose connective tissue interspersed among dilated, thin-walled blood vessels? .... This is followed by infiltration with lymphoid cells and the formation of red marrow, frequently in cords surrounding thin-walled arteries and large veins. 2~ Cytologic,~, 10 autoradiographic,8 and transplantation studies 11-1~ in man and experimental animals have also suggested that lymphocytes may serve as RBC precursors. For example, Cronkite and colleagues 8 studied the dynamics of hematopoiesis in man and in mice by means of in vivo ~H-thymidine labeling and identified primitive proliferating cells. These ceils incorporated ~H-thymidine earlier than any other marrow component. PPC's were morphologically lymphocytes; the term PPC was used "in order to avoid semantic difficulties." Subsequently erythroid, myeloid, and megakarocytes became labeled. Studies involving the transplantation of bone marrow cells have shown that protection from fatal marrow suppression in lethally irradiated rats and mice is a function of the number of small lymphocytes inoculated. 1'~'1'~ Trarrsplanted cells with identical radiation-induced chromosome markers have constituted erythroid colonies in one experimental system (mouse spleen) and behaved as immunocompetent lymphocytes in another (mixed-leukocyte culture). 21 Another indication that lymphocytes may have an erythropoietic function is the known effect of antilymphocytic serum in decreasing the hematopoietic activity of human bone marrow. ~-~a In the present study (Case 2), an increase in labeled lymphocytes, a concomitant decrease in their number, and the subsequent appearance of erythroid precursors (72 hours later) suggest that cells classified morphologically as lymphocytes might serve as erythroid precursors in human beings. We wish to acknowledge the assistance of A. Ashley Weech, M.D., for critical review of this manuscript; Larry Haugh, Ph.D., for statistical assistance; and Ms. Marybruce Dowd and Ms. Candace Crowder for manuscript preparation.
Volume 87 Number 4
Lymphocytosis in congenital hypoplastic anemia
REFERENCES 1. Diamond LK, Allen DM, and Magill FB: Congenital (erythroid) hypoplastic anemia. A 25-year study, Am J Dis Child 102:403, 1961. 2. Allen DM, and Diamond LK: Congenital (erythroid) hypoplastic anemia. Cortisone treated, Am J Dis Child 102:416, 1961. 3. Cathie lAB: Erythrogenesis imperfecta, Arch Dis Child 25:313, 1950. 4. Kass A, and Sundal A: Anaemia hypoplastica congenita (anaemia typus josephs-diamond-blackfan). Report of a case treated with adrenocorticoptopin with effect, Acta Paediatr 42:265, 1953. 5. Smith CH: Hypoplastic and aplastic anemias of infancy and childhood: with a consideration of the syndrome of nonhemolytic anemia ~of the newborn, J PEDIATR 43:457, 1953. 6. Burgert O Jr, Kennedy RLJ, and Pease GL: Congenital hypoplastic anemia, Pediatrics 13:218, 1954. 7. Starling KA, and Fernbach D J: Congenital hypoplastic .anemia. Review of eight cases, Texas Med 63:63, 1967. 8. Cronkite EP, Fliedner TM, Bond BP, Rubini JR, Brecher G, and Quastler H: Dynamics of hemopoietic proliferation in man and mice studied by .'H-thymidine incorporation into DNA, Ann NY Acad Sci 77:803, 1959. 9. Harris PF: Quantitative examination of bone marrow in guinea-pigs after gamma irradiation, Br Med J 2:1032, 1956. 10. Yoffey JM: Quantitative cellular hematology, Springfield, Ill, 1960, Charles C Thomas, Publisher. 11. Brecher G, Endicott KM, Gump H, and Brawner HP: Effects of X-ray on lymphoid and hemopoietic tissues of albino mice, Blood 3:1259, 1948. 12. Cudkowicz G, Bennett M, and Shearer GM: Pluripotent stem cell function of the mouse marrow "lymphocyte," Science 144:866, 1964. 13. Cudkowicz G, Upton AC, Shearer GM, and Hughes WL: Lymphocyte content and proliferative capacity of serially transplanted mouse bone marrow, Nature 201:165, 1964. 14. Kurnick NB, and Nokay N: Repopulation of bone marrow in mice: number and type of ceils required for post-Xradiation protection, Radiat Res 25:53, 1965. 15. Morrison JH, and Toepfer JR: Survival of lethally Xradiated rats after treatment with isogenic marrow lymphocytes, Am J Physiol 213:923, 1967. 16. Rich MC, and Bloom GE: An analysis of selected factors involved in the transformation of normal human peripheral blood lymphocytes, Ann Clin Lab Sci 2:404, 1972. 17. Vogel P, and Bassen FA: Sternal marrow of children in
18.
19.
20. 21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
553
normal and in pathologic states, Am J Dis Child 57:245, 1939. Shapiro LM, and Bassen FA: Sternal marrow changes during the first week of life. Correlation with peripheral blood findings, Am J Med Sci 202:341, 1941. Gairdner D, Marks J, and Roscoe JD: Blood formation in infancy. Part I. The normal bone marrow, Arch Dis Child 27:128, 1952. Yoffey JM: The stem cell problem in the fetus, Isr J Med Sci 7:825, 1971. Nowell PC, Hirsch BE, Fox DH, and Wilson DB: Evidence for the existence of multipotential lympho-hematopoietic stem cells in the adult rat, J Cell Physiol 75:151, 1970. DeMeester TR, Anderson ND, and Shaffer CF: The effect of heterologous anti-lymphocyte serum on mouse hemopoietic stem cells, J Exp Med 127:731, 1968. Field EO, and Gibbs JE: Cross-reaction of anti-lymphocyte serum with haemopoietic stem ceils, Nature 217:561, 1968. Cardinali G, Cardinali G, DeCaro BM, Handler AH, and Aboul-Enein M: Effect of high doses of cortisone on bone marrow cell proliferation in the Syrian hamster, Cancer Res 24:969, 1964. Hunstein W, and Strey M: Effect of prednisone on the H 3thymidine-uptake o f bone marrow ceils in rats, Klin Wochenschr 43:52, 1965. Stevens W, Coelssides C, and Dougherty TF: Effects of cortisol on the incorporation of thymidine-2-~'C into nucleic acids of lymphatic tissue from adrenalectomized CBA mice, Endocrinology 76:1 I00, 1965. Stevens W, Colessides C, and Dougherty TF: A time study on the effect of cortisol on the incorporation of thymidine-214C into nucleic acids of mouse lymphatic tissue, Endocrinology 78:600, 1966. Knutson F, and Lundin PM: The effect of hypophysectomy, adrenalectomy and cortisone on the incorporation of tritiated thymidine in rat organs, with special references to the lymphoid tissues, Acta Endocrinol 53:519, 1966. Glasar K, Limarzi LR, and Poncher HG: Cellular composition of the bone marrow in normal infants and children, Pediatrics 6:789, 1950. Sturgeon P: Volumetric and microscopic pattern of bone marrow in normal infants and children. II. Cytologic pattern, Pediatrics 7:642, 1951. Unanue ER, Grey HM, Rabellino E, Campbell P, and Schmidtke J: Immunoglobulins on the surface of lymphocytes. II. The bone marrow as the main source of lymphocytes with detectable surface-bound immunoglobulin, J Exp Med 133:1188, 1971.