Red Cell Aplasia and Chronic Granulocytic Leukaemia

S ummary. Two patients with chronic granulocytic leukaemia developed red cell aplasia during the course of their disease. In one of them, cell culture studies demonstrated the presence in the patient's serum of an IgG inhibitor of haemoglobin synthesis by his own mature erythroblasts and erythroblasts grown in vitro from his erythroid colony forming cells. The IgG fraction was also found to be cytotoxic for the patients' marrow erythroblasts that were present after disappearance of the red cell aplasia. Treatment with corticosteroids resulted in reappearance of the erythroblasts in the marrow and decrease in the transfusion requirement. Red cell aplasia can occur before, at the same time or after the onset of chronic granulocytic leukaemia and may have the same immune pathogenesis as chronic idiopathic pure red cell aplasia. It occurs without busulphan treatment and seems to have no direct relation to the terminal metamorphosis. Treatment of the red cell aplasia with corticosteroids would appear worthwhile as it may reduce the transfusion requirement without affecting the course of the underlying leukaemia.     

Transient erythroblastopenia of childhood: Varied pathogenesis

The mechanism of anemia in four patients with transient red cell aplasia of childhood (“erythroblastopenia”) was studied at the time of diagnosis by assessing the colony growth of marrow erythroid progenitors in methylcellulose tissue cultures. Marrow from Patient 1 yielded high normal numbers of BFU-E colonies that were completely abolished on addition of autologous serum or IgG. Patient 2 had normal BFU-E growth that markedly declined when autologous serum or IgM was added to the cultures, but growth remained unchanged with added autologous IgG or peripheral blood mononuclear cells (PBMC). Marrow from Patient 3 yielded low CFU-E and BFU-E numbers with standard plating techniques, but colonies strikingly increased when marrow fractions from an albumin density gradient were cultured. PBMC from Patient 3 suppressed control marrow CFU-E and BFU-E, but serum had no effect. Patient 4 had normal CFU-E and BFU-E that increased with autologous serum and remained unchanged with autologous PBMC. We conclude that the red cell aplasia in Patients 1, 2, and 3 was due to suppressed erythropoiesis via IgG, IgM, and cell-mediated inhibition, respectively. In contrast, in Patient 4 no immune mechanism was demonstrated. Whereas transient red cell aplasia has a uniform clinical presentation, there are at least four pathogenetic mechanisms that can be detected in vitro.     

Fenbufen induced pure red cell aplasia in rheumatoid arthritis

Pure red cell aplasia occurred after fenbufen administration in a patient with severe rheumatoid arthritis. In vitro studies were performed to determine the pathogenesis of the selective red cell aplasia. No cellular or humoral inhibitory mechanisms were demonstrated on growth of erythroid and multipotent bone marrow progenitors. Also, no direct effect of fenbufen alone or in combination with IgG and/or patient serum was found. It is possible that a metabolite of the drug formed from its metabolism was responsible for the aplasia and that the target marrow cell precursor affected is later than both erythroid bone marrow progenitors (BFU-E and CFU-E) and therefore not apparent in our studies. Recovery upon cessation of fenbufen suggests its implication in the pure red cell aplasia.     

Congenital dyserythropoietic anaemia type II (CDA-II): chromosomal banding studies and adherent cell effects on erythroid colony (CFU-E) and burst (BFU-E) formation

Bone marrow CFU-E and BFU-E from a patient with CDA-II formed erythroid colonies and bursts which contained multinucleated erythroblasts in vitro . Adherent cell depletion of the patient's marrow increased CFU-E derived colonies six-fold (98 ± 17 v. 640 ± 15 per 10⁵ marrow cells plated) and co-culture of CDA-II marrow adherent cells with CDA-II adherent cell depleted marrow significantly suppressed erythroid colony formation. Similar adherent cell suppression of the patient's BFU-E also occurred. Adherent cell depletion of normal marrow did not increase CFU-E derived colony formation (488 ± 63 v. 495 ± 108) and decreased BFU-E derived burst formation. Addition of normal adherent cells to normal marrow increased erythroid colony and burst formation. Karyotype and chromosomal banding studies of the patient's multinucleated cells did not show chromosomal inversions, deletions or translocations.     

Demonstration of three distinct immunological disorders on erythropoiesis in a patient with pure red cell aplasia and autoimmune haemolytic anaemia associated with thymoma

A patient with pure red cell aplasia (PRCA) and autoimmune haemolytic anaemia (AIHA), associated with a thymoma which had already been removed, was studied in order to investigate the pathogenesis of PRCA and AIHA. The autoantibody eluted from the surface of the patient's red blood cells (RBC) reacted with the large E antigen of the Rh complex. Immunoglobulin-G (IgG) purified from the patient's serum suppressed CFU-E and BFU-E but not CFU-GM colony formation in the presence of complement. This antibody was not adsorbed with large E antigen. T-lymphocytes in the bone marrow suppressing autologous CFU-E and BFU-E colonies were demonstrated. Thus, three distinct immunological disorders on erythropoiesis were present in this patient with PRCA and AIHA associated with thymoma in a thymectomized state.     

Aplastic anemia associated with in vitro inhibition of erythropoiesis by bone marrow-adherent cells

A patient with aplastic anemia that evolved following pure red cell aplasia is described. Cultures of the patient's marrow cells revealed greatly reduced numbers of primitive (BFU-E) and relatively mature (CFU-E) erythroid progenitors, but normal numbers of multipotential (CFU-GEMM) precursors. The BFU-E/CFU-GEMM and CFU-E/BFU-E ratios in the patient's marrow cell cultures were also reduced. T cell- or antibody-mediated inhibition of in vitro erythropoiesis could not be demonstrated in this patient. However, the patient's marrow-adherent cells suppressed the growth of autologous and allogeneic BFU-E and CFU-E, without influencing the growth of CFU-GEMM. Medium conditioned by the patient's adherent cells failed to inhibit the growth of normal erythroid precursors. Our findings suggest a role for marrow-adherent cells in the pathogenesis of aplastic anemia in this patient.     

Serum erythropoietic inhibitors in patients with pure red cell aplasia

Summary Inhibitory mechanisms of erythropoiesis in 20 patients with pure red cell aplasia (PRCA) were investigated using the technique of in vitro hematopoiesis and an assay for human parvovirus. Complement-dependent serum inhibitors against late erythroid progenitors (CFU-E) were demonstrated in seven of 19 patients examined, and complement-dependent inhibitors against early erythroid progenitors (BFU-E) were demonstrated in three of these seven patients. Nonspecific and complement-independent inhibitors against CFU-E were thought to be associated with the etiology of PRCA in one patient. Human parvovirus-mediated erythropoietic suppression was demonstrated in a patient with complete remission of acute lymphoblastic leukemia complicated with marrow erythroid aplasia, whose serum showed a perfect inhibition against erythroid progenitor cells. T-cell-mediated erythroid suppression was not demonstrated in the patients examined. These findings reveal that erythroid aplasia is associated with complement-dependent serum erythropoietic inhibitor in some patients (36.8% in the present study) with PRCA, but it is difficult to identify the mechanism of erythroid aplasia in more than half of the patients with PRCA. In addition, our present study discovered the presence of parvovirus-mediated marrow pure red cell aplasia in one adult patient with acute lymphoblastic leukemia.     

Adult T-cell leukemia associated with pure red cell aplasia-like lesion

An adult T cell leukemia associated with pure red cell aplasia-like lesion was described in this paper. A 51 year-old woman was admitted because of headache and palpitation in October 1988. On admission, physical examination showed marked pallor but no detectable superficial lymphadenopathies. Hepatosplenomegaly was not observed. The blood examination revealed normocytic anemia with Hb of 6.6 g/dl and marked leukocytosis of 18,800/microliters with 43% ATL cells. The bone marrow aspirate showed moderate infiltration of ATL cells and a few erythroblasts. The bone marrow biopsy disclosed moderate infiltration of ATL cells, only a few erythroblasts with maturation arrest and marked fibrosis. The erythropoietin in serum was elevated (686 IU/microliters). To clarify the mechanism of development of the PRCA-like lesion, the peripheral blood lymphocytes (ATL cells) or serum of the patient was added to in vitro erythroid colony formation. The patient's serum increased BFU-E but either serum or lymphocytes didn't inhibit the growth of CFU-E compared with control.     

Mode of action of the IgG inhibitor of erythropoiesis in transient erythroblastopenia of children

Twelve cases of transient erythroblastopenia of childhood (TEC) have been studied to evaluate their marrow cell erythropoiesis in vitro and the effect on it of their serum or IgG. The number of colony-forming units-erythroid (CFU-E) and burst-forming units-erythroid (BFU-E) in the bone marrow of nine cases was extremely variable and did not allow any conclusion regarding the pathogenesis of this anemia. An IgG inhibitor of growth of erythroid colonies or bursts was detected in 8/12 cases. This IgG inhibitor had no effect on the growth of granulocyte-macrophage colonies. Further studies on its mode of action indicated that the IgG did not have antierythropoietin antibody properties and did not affect the mature erythroblasts, as shown by a lack of inhibition of their responses to erythropoietin and by the lack of a cytotoxic effect on 59Fe-labeled erythroblasts. In four cases, preincubation studies demonstrated a direct effect of the IgG on the CFU-E, which was complement-mediated in three cases and complement- independent in one case. In two other cases, the IgG suppressed the growth of normal BFU-E only without affecting the growth of CFU-E. The IgG inhibitor was no longer present after the erythroblastopenia had remitted. These studies demonstrate that in the majority of cases of TEC, an IgG suppressor of erythropoiesis in vitro is present. Its mode of action is heterogeneous regarding its requirement for complement. Its target cells are the earlier or later erythroid progenitors, BFU-E or CFU-E, but not the differentiated erythroblasts.     

Pure red cell aplasia: lymphocyte inhibition of erythropoiesis

The pathogenesis of pure red cell aplasia (PRCA) was studied in a patient who had no evidence of malignancy. In marrow culture, no erythroid colonies (from late erythroid progenitors [CFU-E]) but normal numbers of well-haemoglobinized erythroid bursts (from early erythroid progenitors [BFU-E]) were found, indicating that BFU-E existed in the patient but that their subsequent in vivo differentiation was inhibited. Autologous coculture studies suggested that inhibition was mediated by the patient's ER+ lymphocytes. After remission was induced with cyclophosphamide, autologous ER + cells no longer suppressed in vitro erythropoiesis. However, cryopreserved ER + cells, obtained with anaemia, suppressed BFU-E growth from remission marrow. An expanded population of large granular lymphocytes (LGL) with ER +, F_cγ+. T3 +, T8 +, HNK-1 +, Ia —, M1 — phenotype and no functional natural killer (NK) cell activity was noted during PRCA that reverted to normal with remission. For this patient, both in vivo and in vitro evidence demonstrates a cellular inhibition of erythropoiesis at the level of differentiation between BFU-E and CFU-E.     

Successful treatment of chronic refractory pure red cell aplasia with antithymocyte globulin: correlation with in vitro erythroid culture studies

Two contrasting cases of chronic refractory pure red cell aplasia (PRCA) responsive to a commercial preparation of horse antihuman thymocyte globulin (ATG) are reported. Both cases were refractory to trials of cyclophosphamide, corticosteroids, and plasmapheresis. One patient developed a reticulocytosis after a single intravenous infusion of ATG; the other patient responded after administration of 14.7 g of ATG over a 28-day course. At presentation, erythroid progenitors (CFU-E and BFU-E) in one patient were normal; in the second patient, the number of erythroid progenitors was severely reduced. Neither patient had a serum IgG inhibitor to progenitor cells as judged by in vitro erythroid colony studies. Both patients had increased numbers of marrow T-cells and co-culture studies in one case were consistent with T-cell-mediated suppression of erythropoiesis. These studies confirm that ATG is a useful agent in the treatment of refractory PRCA. However, ATG may not act by removal of T suppressor cells in all cases.     

Case report: isoimmune inhibition of erythropoiesis following ABO-incompatible bone marrow transplantation.

A 26-year-old ABO-O positive patient with aplastic anemia received a bone marrow transplant from his genotypically HLA identical, but ABO-A positive, brother. Engraftment of myeloid and megakaryocytic lineages occurred within 4 weeks but pure red cell aplasia and transfusion dependent anemia persisted for 160 days. The authors postulated that the failure of erythropoiesis was due to a high titer of anti-A isohemagglutinins. They tested this hypothesis with clonal cell cultures and flow cytometric analysis of ABO antigen expression by colony forming cells in vitro. During the period of prolonged red cell aplasia, the patient had normal numbers (85 +/- 12 per 10(6) cells) of circulating donor derived, burst forming units-erythroid (BFU-E). Immunophenotypic analysis of erythroid burst colonies derived from culture of the patient's bone marrow cells showed that 91 +/- 5% of 274 nucleated red cells were A-antigen positive, confirming full donor engraftment. Autologous plasma and complement added on day 1 of culture did not affect the colony growth (82.5 +/- 15 per 10(6) cells). However, when the addition of complement was delayed until day 7 of culture, there was 90% inhibition of BFU-E (7.5 +/- 5 per 10(6) cells) compared to controls (p less than 0.0004). Based on this, the authors propose a model for expression of ABO antigens during erythropoiesis, in which BFU-E do not express ABO antigens but their progeny do. The data support the hypothesis that the mechanism of prolonged pure red cell aplasia after ABO-incompatible bone marrow transplantation is complement mediated immune destruction of erythroid progenitors past the stage of BFU-E in differentiation.     

Suppression of normal human erythropoiesis by human recombinant DNA-produced alpha-2-interferon in vitro

Interferons (IFN) have been shown to suppress the proliferation of human erythroid progenitors (BFU-E, CFU-E) in vitro. We have previously demonstrated that the inhibition of erythroid colony formation by gamma-IFN in vitro is mediated, in part, through the activation of monocytes and T-lymphocytes. In order to examine the mechanism(s) underlying the inhibitory action of one type of recombinant alpha-IFN (alpha-2-IFN) on erythropoiesis, the effect of different doses (80-10,000 U) of alpha-2-IFN on erythroid colony formation by normal human bone marrow cells in the presence or absence of monocytes and/or T cells was studied. The addition of alpha-2-IFN to whole marrow caused the suppression of BFU-E (10%-68%) and CFU-E (5%-75%) in a dose-dependent fashion. This inhibition occurred with the direct addition of alpha-2-IFN to culture plates but not with brief preincubation of marrow cells with alpha-2-IFN followed by washing of the cells. By contrast, brief exposure of marrow cells to gamma-IFN resulted in significant suppression of erythroid colony formation. The inhibitory action of alpha-2-IFN was not influenced by erythropoietin. Removal of monocytes and/or T cells prior to the addition of alpha-2-IFN failed to significantly reduce the suppressive effects of this molecule (BFU-E: 21%-66%; CFU-E: 20%-83%). Coculture of purified monocytes or T-lymphocytes preexposed to alpha-2-IFN with autologous bone marrow cells did not cause suppression of erythropoiesis; monocytes or T cells similarly treated with gamma-IFN, however, inhibited autologous BFU-E and CFU-E in vitro. These results demonstrate that, unlike gamma-IFN, the inhibitory effect of alpha-2-IFN on erythroid colony formation in vitro is not mediated to any significant degree through monocytes and T-lymphocytes. The suppressive effect of alpha-2-IFN occurs either directly at the erythroid progenitor(s) level and/or through accessory cell(s) other than monocytes and T cells.     

Pure red cell aplasia and pseudothrombocytopenia associated with hepatitis A

We report a rare case who developed pure red cell aplasia (PRCA) and pseudothrombocytopenia associated with hepatitis. A 50-year-old woman was admitted to the hospital because of acute hepatitis A. On the 22nd hospital day, normocytic normochromic anemia without reticulocytosis was developed. A bone marrow aspirate revealed erythroid hypoplasia with a small percentage of proerythroblasts and basophilic erythroblasts, but almost complete absence of polychromatophilic and orthochromic erythroblasts. This case was diagnosed as PRCA characterized by the maturation arrest of erythropoiesis. Anemia was spontaneously recovered following marked reticulocytosis. Afterward, transient EDTA-dependent pseudothrombocytopenia developed for 3 months. The serum taken during the acute phase of clearly inhibited BFU-E colony formation. This data suggests that some humoral factor in the serum of this patient may be involved in the pathogenesis of PRCA.     

Pure red cell aplasia following autoimmune haemolytic anaemia. Cell-mediated suppression of erythropoiesis as a possible pathogenesis of pure red cell aplasia.

We report here a rare case of pure red cell aplasia (PRCA) following autoimmune haemolytic anaemia (AIHA). After 4 years of AIHA, the patient developed anaemia with severe erythroid hypoplasia and was diagnosed as having PRCA. At this time, Coombs' test was negative and parvovirus infection was not recognized. The patient received azathioprine, and PRCA improved. To determine the pathogenesis of PRCA, in vitro culture studies of erythropoietic and granulopoietic precursors were performed. The patient's serum or IgG did not suppress the colony formation of bone marrow colony-forming units-erythroid (CFU-E) of normal subjects and the patient. In contrast, mononuclear cells in the peripheral blood of the patient significantly suppressed CFU-E of normal subjects. Media conditioned by the patient's mononuclear cells did not significantly suppress CFU-E. The significant production of suppressive cytokine such as tumour necrosis factor or gamma-interferon by the patient's mononuclear cells was not recognized. These findings suggest that the cytotoxic mononuclear cells affected directly the erythropoietic precursors and caused PRCA in this patient. The pathogenesis of PRCA was, therefore, considered to be different from that of the preceding AIHA.     

Anti-K562 cell monoclonal antibody RTF8X recognizes tumor-associated antigen of erythroid lineage

A new anti-K562 cell monoclonal antibody, RTF8X, a cytotoxic IgM, recognized a surface antigen on erythroblasts from patients with erythroleukemia and polycythemia vera. RTF8X, which is highly specific to K562 cells, did not react with the other 14 hematopoietic cell lines and the seven nonhematopoietic cell lines. RTF8X antigen was not detected in normal peripheral blood, but was found in less than 1% of normal marrow cells. RTF8X did not inhibit in vitro colony formation of CFU-E and BFU-E in a complement-dependent cytotoxicity assay. Cell- sorting analysis showed that, morphologically, the RTF8X-positive marrow cells from the patients and normal volunteers contained more than 60% erythroblasts and that CFU-E and BFU-E were not demonstrated in cells with RTF8X antigen. Enzyme treatment suggested that RTF8X antigen was a sialoglycolipid. These results indicate that RTF8X may recognize the surface antigen found increasingly in association with tumors of erythroid lineage. RTF8X should be useful for studies of erythroid differentiation and proliferation in patients.     

Studies on hematopoietic stem cells: XI. Lack of erythroid burst-forming units (BFU-E) in patients with aplastic anemia.

The marrow concentration of erythropoietic precursors was examined in normal donors and patients with idiopathic aplastic anemia using a plasma clot culture system. On time course observations the heterogeneity of human erythroid precursors assayable in culture was demonstrated. To evaluate human erythropoiesis in vitro, the benzidine-positive colonies were divided into three groups: small colony, containing 8-50 cells; medium-sized colony, containing 50-500 cells; and large colony, containing more than 500 cells. The majority of the large colonies assumed the morphology of erythropietic bursts (BFU-E) consisted of several subcolonies. The small colonies were counted as CFU-E1, the medium-sized as CFU-E2, and the large as BFU-E to evaluate the erythroid precursor cell compartment in aplastic anemia. The marrow concentration of CFU-E1 and CFU-E2 was shown to be quantitatively diminished in aplastic anemia. In addition, there was no ability of the marrow cells from aplastic patieints to grow BFU-E in vitro even in the presence of a large dose of erythropoietin. This lack of BFU-E colony growth may play an important role in the mechanism of the erythropoietic deficiency in aplastic anemia.     

Erythropoietin receptor characteristics on primary human erythroid cells.

Erythropoietin (EP) exerts its effects on erythropoiesis by binding to a cell surface receptor. We examined EP receptor expression during normal human erythroid differentiation and maturation from the burst-forming unit-erythroid (BFU-E) to the reticulocyte level. In contrast to previous studies, we assessed EP receptor number and affinity in erythroid precursors immunologically purified from fresh bone marrow aspirates or fetal liver samples and in reticulocytes purified from peripheral blood. EP receptors were quantitated by equilibrium binding experiments with 125I EP. We found that purified primary erythroblasts from both adult and fetal sources exhibited a single high-affinity (kd 100 pmol/L) binding site for EP under our experimental conditions, and 135 or 250 receptors per cell, respectively. Reticulocytes were devoid of EP receptors. We compared these data to in vitro-derived BFU-E progeny at both early and late stages of maturation. Cultured BFU-E progeny also displayed a single class of receptors of slightly lower affinity (210 to 220 pmol/L). Preparations enriched in colony-forming units-erythroid (CFU-E) and proerythroblasts (day 9 BFU-E progeny) displayed approximately 1,100 receptors per cell, whereas populations containing mature erythroblasts (day 14 BFU-E progeny) exhibited approximately 300 receptors per cell. Furthermore, information from binding experiments was complemented by autoradiography in both enriched BFU-E preparations, cultured BFU-E progeny (days 9 and 14), and marrow mononuclear cells. These studies are consistent with a peak in EP receptor expression at the CFU-E/proerythroblast stage and a decrease with further maturation to undetectable levels at the reticulocyte stage. These data examining EP receptor characteristics on freshly isolated erythroid precursor cells complement previous data on EP receptor biology using culture-derived erythroblasts.     

Monoclonal antibodies detecting antigenic determinants with restricted expression on erythroid cells: from the erythroid committed progenitor level to the mature erythroblast

Two new cell surface antigens specific for the erythroid lineage were defined with cytotoxic IgM monoclonal antibodies (McAb) (EP-1; EP-2) that were produced using BFU-E-derived colonies as immunogens. These two antigens are expressed on in vivo and in vitro derived adult and fetal erythroblasts, but not on erythrocytes. They are not detectable on resting lymphocytes, concanavalin-A (Con-A) activated lymphoblasts, granulocytes, and monocytes or granulocytic cells or macrophages present in peripheral blood or harvested from CFU-GM cultures. Cell line and tissue distributions distinguish McAb EP-1 and EP-2 from all previously described monoclonal antibodies. McAb EP-1 (for erythropoietic antigen-1) inhibits the formation of BFU-E and CFU-E, but not CFU-GM, colonies in complement-dependent cytotoxicity assays. By cell sorting analysis, about 90% of erythroid progenitors (CFU-E, BFU-E) were recovered in the antigen-positive fraction. Seven percent of the cells in this fraction were progenitors (versus 0.1% in the negative fraction). The expression of EP-1 antigen is greatly enhanced in K562 cells, using inducers of hemoglobin synthesis. McAb EP-2 fails to inhibit BFU-E and CFU-E colony formation in complement-dependent cytotoxicity assays. EP-2 antigen is predominantly expressed on in vitro derived immature erythroblasts, and it is weakly expressed on mature erythroblasts. The findings with McAb EP-1 provide evidence that erythroid progenitors (BFU-E and CFU-E) express determinants that fail to be expressed on other progenitor cells and hence appear to be unique to the erythroid lineage. McAb EP-1 and EP-2 are potentially useful for studies of erythroid differentiation and progenitor cell isolation.