Ultrastructure of the bone marrow in HIV infection: evidence of dyshaemopoiesis and stromal cell damage

The ultrastructure of bone marrow cells was studied in nine patients infected with the human immunodeficiency virus (HIV). Two of these (cases 1 and 3) were thrombocytopenic, had never suffered from opportunistic infections and had not received any drugs prior to the time of study. A number of ultrastructural abnormalities were found in a variable proportion of the affected cell types in all nine patients. These were: (a) an increased prevalence of multivesicular bodies within several cell types and of abnormalities of the nuclear membrane in neutrophil granulocytes, (b) an increase in the size of the Golgi apparatus and in the quantity of endoplasmic reticulum in neutrophil granulocytes, (c) dysplastic features, including multiple long intranuclear clefts and large cytoplasmic vacuoles in some erythroblasts and (d) vacuolation of the plasma cells. Other abnormalities seen in a proportion of the patients were: (a) cylindrical confronting cisternae (CCC) in some of the lymphocytes, macrophages (phagocytic reticular cells), non-phagocytic reticular cells (including adventitial cells) and endothelial cells of marrow sinusoids, (b) tubuloreticular structures (TRS) in some lymphocytes, plasma cells, monocytes and endothelial cells and (c) precipitates of protein within occasional erythroblasts and marrow reticulocytes. There was also a striking and hitherto undescribed abnormality of the structure of the nucleus in intersinusoidal and perisinusoidal non-phagocytic reticular cells. This was seen in six patients, including case 3, and was characterized by the extensive detachment of masscs of abnormally electrondense heterochromatin from the nuclear membrane, the presence of a uniformly thin layer of electron-dense material at the inner surface of the areas of nuclear membrane denuded of heterochromatin masses and an abnormal electron lucency of areas containing euchromatin. The CCC and TRS were found in the six patients with the lowest number of circulating CD4-positive T cells. The precipitation of protein within erythroid cells may have been caused by the oxidant effect of dapsone or high doses of co-trimoxazole. The abnormalities in the stromal cells and in particular the nuclear changes seen in the non-phagocytic reticular cells support the possibility that one of the mechanisms underlying the cytopenia in patients infected with HIV may be a disturbance of the microenvironmental regulation of haemopoiesis.     

Ultrastructural abnormalities and arrest of protein biosynthesis in some erythroblasts from homozygotes for haemoglobin C and double heterozygotes for haemoglobin C and beta-thalassaemia

Various ultrastructural abnormalities were found in the erythroblasts of three homozygotes for haemoglobin C (HbC), one patient with HbC/beta(+)-thalassaemia and one patient with HbC/beta (0) thalassaemia. These included a coarsely granular or reticular appearance and altered electron-density of the heterochromatin, loss of parts of the nuclear membrane, and oozing of nuclear material into the cytoplasm. In addition, the two patients with HbC/beta-thalassaemia, but not the others, showed precipitated intracytoplasmic alpha-chains in a few profiles of polychromatic erythroblasts and marrow reticulocytes. Electron microscope autoradiographic studies of bone marrow cells from two of the patients with HbC disease and the patient with HbC/beta (0)-thalassaemia showed a marked depression or failure of incorporation of 3H-leucine into protein in some of the ultrastructurally abnormal erythroblasts. This impairment of protein synthesis may lead to alterations in the erythroblast membrane that are involved in the recognition and phagocytosis of the abnormal erythroblasts by macrophages.     

Ultrastructural abnormalities and arrest of protein biosynthesis in some erythroblasts from homozygotes for haemoglobin C and double heterozygotes for haemoglobin C and β-thalassaemia

Summary. Various ultrastructural abnormalities were found in the erythroblasts of three homozygotes for haemoglobin C (HbC), one patient with HbC/β⁺-thalassaemia and one patient with HbC/β thalassaemia. These included a coarsely granular or reticular appearance and altered electron-density of the heterochromatin, loss of parts of the nuclear membrane, and oozing of nuclear material into the cytoplasm. In addition, the two patients with HbC/β-thalassaemia, but not the others, showed precipitated intracytoplasmic α-chains in a few profiles of polychromatic erythroblasts and marrow reticulocytes. Electron microscope autoradiographic studies of bone marrow cells from two of the patients with HbC disease and the patient with HbC/β thalassaemia showed a marked depression or failure of incorporation of ³H-leucine into protein in some of the ultrastructurally abnormal erythroblasts. This impairment of protein synthesis may lead to alterations in the erythroblast membrane that are involved in the recognition and phagocytosis of the abnormal erythroblasts by macrophages.     

The bone marrow in human cerebral malaria: parasite sequestration within sinusoids

Bone marrow aspirates from patients with cerebral malaria were studied with the light and electron microscopes. Various abnormalities were found including: (1) an increase in plasma cells and macrophages, sometimes to a marked degree; (2) phagocytosis of parasitized red cells by macrophages and of merozoites by neutrophil metamyelocytes, neutrophil granulocytes and macrophages; (3) an increase in the proportion of eosinophil granulocytes and their precursors; (4) the presence of giant metamyelocytes; and (5) morphological abnormalities of erythroblasts, particularly irregularly-shaped nuclei and karyorrhexis. A high percentage of the red cells within marrow sinusoids were parasitized and the parasitized cells were attached to the endothelium. Some marrow sinusoids were packed with and completely obstructed by parasitized cells. Strands of electron-dense material were sometimes found connecting the knobs of parasitized red cells to endothelial cells or to the knobs of adjacent parasitized red cells. A striking finding was a complex interdigitation between cytoplasmic processes developed by some of the parasitized red cells and those developed by the endothelial cells to which they were attached. Occasionally, cytoplasmic processes arising from marginated parasitized red cells completely penetrated the endothelial cell and emerged extravascularly. Several parasitized red cells were also found extravascularly between haemopoietic cells. Sequestration of parasitized red cells within small blood vessels may play a part in the pathogenesis not only of the encephalopathy of cerebral malaria but also of the bone marrow dysfunction in severe malaria.     

Observations on the ultrastructure of sinusoids and reticular cells in human bone marrow

Summary The ultrastructural characteristics of sinusoids in human bone marrow were generally similar to those previously reported in the rat. However, human sinusoids differed from rat sinusoids in displaying frequent tight junctions between adjacent overlapping or interdigitating endothelial cells. In both species, large areas of the sinusoidal walls were devoid of much subendothelial connective tissue and of an outer adventitial cell layer. The marrow sinusoids of humans resembled those of rabbits and rats in that processes of macrophage cytoplasm protruded through endothelial cells into the sinusoidal lumen; thin veils derived from such processes were apposed over the inner surfaces of some endothelial cells. Features observed in pathological human marrow not noted in rodent marrow are the phagocytosis of extruded erythroblast nuclei and of abnormal erythroblasts by perisinusoidal adventitial cells (reticular cells) and the presence of large secondary lysosomes and siderosomes in sinusoidal endothelial cells. When compared with mouse bone marrow, human bone marrow contained very few non-phagocytic reticular cells that were unassociated with sinusoids and other blood vessels. There were no absolute ultrastructural differences between perisinusoidal adventitial cells, intersinusoidal non-phagocytic reticular cells and macrophages lacking phagosomes or containing only a few small phagosomes. Consequently, on some occasions, these cell types could not be reliably identified from the study of a single thin section. Extracellular reticulin fibres were found adjacent both to non-phagocytic reticular cells and to macrophages. Mitosis was observed in macrophages, albeit very rarely.     

Observations on the ultrastructure of sinusoids and reticular cells in human bone marrow.

The ultrastructural characteristics of sinusoids in human bone marrow were generally similar to those previously reported in the rat. However, human sinusoids differed from rat sinusoids in displaying frequent tight junctions between adjacent overlapping or interdigitating endothelial cells. In both species, large areas of the sinusoidal walls were devoid of much subendothelial connective tissue and of an outer adventitial cell layer. The marrow sinusoids of humans resembled those of rabbits and rats in that processes of macrophage cytoplasm protruded through endothelial cells into the sinusoidal lumen; thin veils derived from such processes were apposed over the inner surfaces of some endothelial cells. Features observed in pathological human marrow not noted in rodent marrow are the phagocytosis of extruded erythroblast nuclei and of abnormal erythroblasts by perisinusoidal adventitial cells (reticular cells) and the presence of large secondary lysosomes and siderosomes in sinusoidal endothelial cells. When compared with mouse bone marrow, human bone marrow contained very few non-phagocytic reticular cells that were unassociated with sinusoids and other blood vessels. There were no absolute ultrastructural differences between perisinusoidal adventitial cells, intersinusoidal non-phagocytic reticular cells and macrophages lacking phagosomes or containing only a few small phagosomes. Consequently, on some occasions, these cell types could not be reliably identified from the study of a single thin section. Extracellular reticulin fibres were found adjacent both to non-phagocytic reticular cells and to macrophages. Mitosis was observed in macrophages, albeit very rarely.     

The distribution of PIG-A gene abnormalities in paroxysmal nocturnal hemoglobinuria granulocytes and cultured erythroblasts

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia that is characterized by a deficiency of glycosylphosphatidylinositol-anchored membrane proteins due to phosphatidylinositol glycan-class A (PIG-A) gene abnormalities in various lineages of peripheral blood cells and hematopoietic precursors. The purpose of our study was to clarify the distribution of PIG-A gene abnormalities among various cell lineages during differentiation and maturation in PNH patients.The expression of CD16b or CD59 in peripheral blood granulocytes or cultured erythroblasts from three Japanese PNH patients was analyzed using flow cytometry. PIG-A gene abnormalities in both cell types, including glycophorin A(+) bone marrow erythroblasts, were examined using nucleotide sequence analysis. The expression study of PIG-A genes from each patient was also performed using JY-5 cells.Flow cytometry revealed that the erythroblasts consisted of negative, intermediate, and positive populations in Cases 1 and 3 and negative and intermediate populations in Case 2. The granulocytes consisted of negative and positive populations in all three cases. DNA sequence analysis indicated that all the PNH cases had two or three types of PIG-A gene abnormalities, and that a predominant clone with an abnormal PIG-A gene was different in granulocytes and erythroblasts from Cases 2 and 3. Expression studies showed that all the mutations from the patients were responsible for the null phenotype.PIG-A gene abnormalities result in deficiencies of glycosylphosphatidylinositol-anchored proteins in PNH erythroblasts and granulocytes. The distribution of predominant PNH clones with PIG-A gene abnormalities is often heterogeneous between the cell types, suggesting that a clonal selection of PIG-A gene abnormalities occurs independently among various cell lineages during differentiation and maturation.     

Granulocytic nuclear differentiation of lamin B receptor-deficient mouse EPRO cells

OBJECTIVE: Lamin B receptor (LBR) is an integral protein of the inner nuclear membrane. Recent studies have demonstrated that genetic deficiency of LBR during granulopoiesis results in hypolobulation of the mature neutrophil nucleus, as observed in human Pelger-Hu?t anomaly and mouse ichthyosis (ic). In this study, we utilized differentiated early promyelocytes (EPRO cells) that were derived from the bone marrow of homozygous and heterozygous ichthyosis mice to examine changes to the expression of nuclear envelope proteins and heterochromatin structure that result from deficient LBR expression. MATERIALS AND METHODS: Wild-type (+/+), heterozygous (+/ic), and homozygous (ic/ic) granulocytic forms of EPRO cells were analyzed for the expression of multiple lamins and inner nuclear envelope proteins by immunostaining and immunoblotting techniques. The heterochromatin architecture was also examined by immunostaining for histone lysine methylation. RESULTS: Wild-type (+/+) and heterozygous (+/ic) granulocytic forms revealed ring-shaped nuclei and contained LBR within the nuclear envelope; ic/ic granulocytes exhibited smaller ovoid nuclei devoid of LBR. The pericentric heterochromatin of undifferentiated and granulocytic ic/ic cells was condensed into larger spots and shifted away from the nuclear envelope, compared to +/+ and +/ic cell forms. Lamin A/C, which is normally not present in mature granulocytes, was significantly elevated in LBR-deficient EPRO cells. CONCLUSIONS: Our observations suggest roles for LBR during granulopoiesis, which can involve augmenting nuclear membrane growth, facilitating compartmentalization of heterochromatin, and promoting downregulation of lamin A/C expression.     

Dyserythropoiesis in homozygous haemoglobin C disease

Summary. Electron microscope studies of the bone marrow of three patients with homozygous haemoglobin C (HbC) disease have shown marked ultrastructural abnormalities in several of the polychromatic erythroblasts and marrow reticulocytes and the presence of phagocytosed erythroblasts within the macrophages. Such abnormalities were not found in the bone marrow of three patients with sickle cell anaemia indicating that the abnormalities represented a feature of HbC disease rather than a disturbance secondary to peripheral haemolysis. The characteristic ultrastructural finding in the polychromatic erythroblasts in HbC disease was the presence of grossly-disorganized nuclei showing multiple intranuclear clefts, the loss of parts of the nuclear membrane, oozing of nuclear material into the cytoplasm and an alteration of the structure and stainability of the nuclear chromatin. It is proposed that both the dyserythropoiesis and ineffective erythropoiesis in HbC disease may have resulted from the formation in vivo of very small aggregates of HbC within erythropoietic cells.     

Ultrastructure and cell cycle distribution of bone marrow cells in protein-energy malnutrition

Bone marrow aspirates from four children with kwashiorkor and three with marasmus were studied using the techniques of electron microscopy and combined Feulgen microspectrophotometry and 3H-thymidine autoradiography. The majority of the erythroblasts were ultrastructurally normal, the distribution of the early polychromatic erythroblasts between the various stages of the cell cycle was normal or almost normal, and the macrophages did not contain ingested erythroblasts. Since erythropoietin production has been shown to be normal in protein-energy malnutrition, these findings suggest that at least in some cases of PEM the impairment of erythropoiesis results primarily from an abnormality in the erythroid progenitor cell pool rather than from dyserythropoiesis and ineffective erythropoiesis. In one afebrile and apparently uninfected patient with marasmus, a substantial proportion of the neutrophil granulocytes and their more mature precursors contained electron-dense, myelin-containing intracytoplasmic structures which were presumed to be abnormal primary granules. In four of the patients, the 3H-thymidine labelling index of the neutrophil promyelocyte-myelocyte pool was increased. In addition, in all of the cases, neutrophils at various stages of degradation were readily found within the cytoplasm of some of the macrophages. Thus, whereas the techniques employed did not reveal a major disturbance in the morphologically recognizable precursor cells of the erythroid series in PEM, they demonstrated some abnormalities in such cells of the neutrophil series.     

Observations on the Ultrastructure of Erythropoietic Cells and Reticulum Cells in the Bone Marrow of Patients with Homozygous β-Thalassaemia

An electron microscopic study of marrow fragments from patients with homozygous beta-thalassaemia has shown that 3% of early polychromatic erythroblast profiles and 20% of late polychromatic erythroblast profiles contain intracytoplasmic alpha-chain precipitates. Various nuclear abnormalities were found including the loss of parts of the nuclear membrane and the presence of intranuclear alpha-chain precipitates, and these abnormalities were virtually confined to the non-dividing, late polychromatic erythroblasts. As most profiles of the proliferating early polychromatic erythroblasts did not contain intracytoplasmic or intranuclear alpha-chain precipitates, it is suggested that the arrest of many of these cells in the G1 phase of the cell cycle may be related to the presence of an excess of free alpha-chains rather than to the presence of alpha-chain precipitates within them. The cytoplasm of the bone marrow reticulum cells contained early and late polychromatic erythroblasts at various stages of degradation, providing direct evidence of ineffective erythropoiesis.     

Some Aspects of the Biology of Multinucleate and Giant Mononucleate Erythroblasts in a Patient with CDA, Type III

Marrow cells of a patient with CDA, type III, were (1) incubated with 3H-thymidine for 0.5 h and studied using a combination of Feulgen microspectrophotometry and light microscope autoradiography and (2) incubated with 3H-thymidine, 3H-uridine or 3H-leucine for 1 h and studied using the technique of electron microscope autoradiography. The data revealed multiple abnormalities in the proliferation of erythroblasts but no abnormality in the distribution of neutrophil promyelocytes and myelocytes in the different stages of interphase. Some mononucleate erythroblasts had DNA contents of 4-20c and the multinucleate erythroblasts had total DNA contents of 2-40c. In about 40% of the multinucleate erythroblasts, only some of the nuclei within the same cell incorporated 3H-thymidine and the electron microscope autoradiographic studies revealed that the nuclei which failed to synthesize DNA virtually always showed abnormalities in the electron-density of the heterochromatin or euchromatin or a Swiss-cheese appearance of the heterochromatin. Furthermore, in some multinucleate cells but not in others, the ultrastructurally abnormal nuclei showed a marked depression of RNA synthesis when compared with the normal-looking nuclei within the same cell. An occasional binucleate, multinucleate and giant mononucleate erythroblast showed ultrastructural changes suggestive of advanced degeneration and such cells, which showed a marked depression of RNA and protein synthesis, appeared to be phagocytosed by macrophages.     

Myelokathexis, a congenital disorder of severe neutropenia characterized by accelerated apoptosis and defective expression of bcl-x in neutrophil precursors

Myelokathexis is a congenital disorder that causes severe chronic leukopenia and neutropenia. Characteristic findings include degenerative changes and hypersegmentation of mature neutrophils and hyperplasia of bone marrow myeloid cells. The associated neutropenia can be partially corrected by treatment with granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). These features led us to propose that accelerated apoptosis of neutrophil precursors might account for the neutropenic phenotype. Blood and bone marrow aspirates were obtained from 4 patients (2 unrelated families) with myelokathexis before G-CSF therapy and from 2 of the affected persons after G-CSF therapy (1 microg/kg per day subcutaneously for 3 weeks). Bone marrow was fractionated using immunomagnetic bead cell sorting into CD34(+), CD33(+)/CD34(-), and CD15(+)/CD34(-)/CD33(- )cell populations. Examination of these cells by flow cytometry and electron microscopy revealed abundant apoptosis in the CD15(+) neutrophil precursor population, characterized by enhanced annexin-V binding, extensive membrane blebbing, condensation of heterochromatin, and cell fragmentation. Colony-forming assays demonstrated significant reduction in a proportion of bone marrow myeloid-committed progenitor cells. Immunohistochemical analysis revealed a selective decrease in bcl-x, but not bcl-2, expression in the CD15(+)/CD34(-)/CD33(-)cell population compared with similar subpopulations of control bone marrow-derived myeloid precursors. After G-CSF therapy, apoptotic features of patients' bone marrow cells were substantially reduced, and the absolute neutrophil counts (ANC) and expression of bcl-x in CD15(+)/CD34(-)/CD33(-)cells increased. The authors concluded that myelokathexis is a disease characterized by the accelerated apoptosis of granulocytes and the depressed expression of bcl-x in bone marrow-derived granulocyte precursor cells. These abnormalities are partially corrected by the in vivo administration of G-CSF. (Blood. 2000;95:320-327)     

Correlation between bone marrow karyotype and the occurrence of erythroblast micronuclei and nuclear budding in patients with myelodysplastic syndromes

147 patients with myelodysplastic syndromes were investigated for the presence of micronuclei and nuclear budding in bone marrow erythroblasts. The patients were divided into subgroups on the basis of bone marrow karyotype, 31 healthy bone marrow donors constituted a control group. Patients with monosomy 7 or 7q- and patients with major karyotypic abnormalities (MAKA) had significantly more erythroblasts with micronuclei and nuclear budding than the control group. Patients with a 5q- chromosome as the sole karyotypic aberration had more micronuclei than the controls. For other patients with MDS the differences were statistically nonsignificant.     

An Electron Microscopic Study of the Nuclear Abnormalities in Erythroblasts in Beta-Thalassaemia Major

S ummary . Erythroblasts in the peripheral blood of 20 splenectomized patients with β-thalassaemia were examined by electron microscopy. A number of alterations in the structure and integrity of the nucleus and nuclear membrane was noted and these included apparent widening of the nuclear pores, partial absence and areas of reduplication of the nuclear membrane. Intranuclear inclusions were found, some identical to the electron-dense inclusions (Heinz bodies) present in the cytoplasm, while others contained aggregates of finer particles, probably iron micelles. Some erythroblast nuclei appeared to contain portions of cytoplasm with similar inclusions. Nuclear alterations were most prominent when inclusion bodies were abundant in the cytoplasm and when they were in close approximation to or in contact with nuclear material. These nuclear abnormalities may be related to the recognized disturbance in proliferation of thalassaemic erythroblasts and the ineffective erythro-poiesis encountered in this disease.     

Nuclear bridging of erythroblasts in acquired dyserythropoiesis: an early and transient preleukemic marker

The clinical, hematologic, and histological characteristics of two patients who progressed from refractory anemia to acute leukemia are described. When first studied, nuclear bridging of erythroblasts, similar to that seen in congenital dyserythropoietic anemia type I and megakaryocytic dysplasia, were the only abnormalities. Within 6 years, both patients died, the first of acute nonlymphocytic leukemia, the second of erythroleukemia. Nuclear bridging of erythroblasts in the marrow of these patients was an early and transient phenomenon and was not observed during the terminal phase of leukemia.     

Ineffective Erythropoiesis in Haemoglobin Eβ -thalassaemia: an Electron Microscope Study

S ummary. Electron microscope studies have been performed on the bone marrow cells of two non-splenectomized patients and the circulating erythroblasts and reticulocytes of three splenectomized patients with HbE/β-thalassaemia. Some intracellular precipitates (probably consisting of α-chains) and mild dyserythropoietic changes were found in the early polychromatic erythroblasts within the bone marrow. Larger quantities of precipitate and more marked dyserythropoietic changes were found in the late polychromatic erythroblasts and reticulocytes both within the marrow and within the circulation. The bone marrow macrophages contained phagocytosed erythroblasts within their cytoplasm. These data indicate that the anaemia in HbE/β-thalassaemia results largely from dyserythropoiesis and ineffective erythropoiesis. The ultrastructural abnormalities encountered in the cases of HbE/β-thalassaemia were qualitatively and quantitatively similar to those seen in homozygous β-thalassaemia.     

Electron Microscope Autoradiographic Studies of the Erythroblasts of a Case of Congenital Dyserythropoietic Anaemia,Type II

The bone marrow cells of a patient with congenital dyserythropoietic anaemia, type II, were incubated with ³H-thymidine, ³H-uridine or ³H-leucine for 1 h and studied using the technique of electron microscope autoradiography. Several of the erythroblasts which either displayed the characteristic subsurface double membranes or showed various non-specific abnormalities of the nuclear membrane were found to be actively engaged in DNA, RNA and protein synthesis. Both members of some pairs of erythroblasts which were joined together by a spindle bridge were found to be engaged in DNA synthesis, indicating that some spindle bridges persist for a period longer than the duration of the G₁ phase. A small proportion of mononucleate and binucleate late (non-dividing) erythroblasts showed a marked depression or arrest of protein synthesis and some or all of such cells were presumably destined to be phago-cytosed by the bone marrow macrophages.     

Bone marrow changes mimicking myelodysplasia in patients with hemophagocytic lymphohistiocytosis

In hemophagocytic lymphohistiocytosis (HLH), cytokine-induced pancytopenia is a common finding and is associated with hypoplastic and hypocellular bone marrow and abundant hemophagocytosis. To date, neutrophil nuclear segmentation abnormalities have not been clarified in HLH patients. We report a study of bone marrow from 6 cases of HLH that showed abnormal granulocytes, dyserythropoietic changes, and micromegakaryocytes mimicking the findings in myelodysplasia at the onset of disease. Pelger-Hu?t anomalies were particularly noted in all cases. The increased levels of cytokines in these cases may have caused cellular damage leading to the morphological changes in the bone marrow of these HLH patients. The impact of these findings on pathophysiology and prognosis in HLH patients remains to be determined.     

Poly(ADP-ribose) synthesis, a marker of granulocyte differentiation.

By using an indirect immunofluorescence technique, the distribution of poly(ADP-ribose) synthesis in human blood cells was investigated. The antibody used was reactive with poly(ADP-ribose) larger than trimers. The specific immunofluorescence of poly(ADP-ribose) synthesized in situ from NAD+ was observed in nuclei of lymphocytes and monocytes in normal peripheral blood. No immunofluorescence, however, was detected in granulocytes and erythrocytes. In agreement with this finding, no incorporation of radioactivity from [adenine-14C]NAD+ into acid-insoluble material was detectable in nuclei isolated from granulocytes. In normal bone marrow, the immunofluorescence of poly(ADP-ribose) was not observed in myelocytes or in their descendants but was observed in nuclei of lymphocytes and erythroblasts. Myelocytes and mature granulocytes in peripheral blood as well as in bone marrow of patients with chronic myelocytic leukemia were totally negative in the polymer-specific immunofluorescence. In marked contrast, prominent fluorescence was observed in nuclei of myeloblasts that appeared in peripheral blood as well as in bone marrow of patients with acute myeloblastic leukemia. Myeloblasts appearing in peripheral blood of patients in blastic crisis of chronic myelocytic leukemia also showed the nuclear immunofluorescence. These results suggest that the capacity for synthesizing poly(ADP-ribose) serves as a marker of differentiation of granulocytes, and its immunohistochemical analysis may be useful for differential diagnosis of leukemias, especially in blastic crisis.