Bone marrow and peripheral blood findings in patients with extreme thrombocytosis. A report of 63 cases

Sixty-three bone marrow (BM) and peripheral blood specimens from patients with platelet counts of 1000 x 10(9)/L or greater were examined in an attempt to determine if any BM or peripheral blood findings could be used reliably to distinguish primary thrombocythemia from other myeloproliferative disorders and extreme examples of reactive thrombocytosis. Our results indicated that the BM findings in primary thrombocythemia were quite similar to those in polycythemia vera and chronic granulocytic leukemia with associated extreme thrombocytosis. However, statistically significant differences between the BM findings in myeloproliferative disorders and extreme reactive thrombocytosis were found in the numbers of megakaryocytes, presence or absence of megakaryocyte clusters, stainable iron, cellularity, and reticulin content. We concluded that BM examination is a useful procedure as an aid in determining the cause of extreme thrombocytosis.     

Thrombopoietin-induced CXC chemokines,NAP-2 and PF4, suppress polyploidization and proplatelet formation during megakaryocyte maturation

BACKGROUND: We previously reported that the expressions of two CXC chemokines, neutrophil activating peptide-2 (NAP-2) and platelet factor-4 (PF-4), were induced by megakaryocyte-specific cytokine thrombopoietin (TPO) in mouse bone marrow megakaryocytes. The roles of these chemokines on megakaryocyte maturation/differentiation processes, including polyploidization and proplatelet formation (PPF) remain unresolved. RESULTS: NAP-2 and PF-4 suppressed the PPF of mature megakaryocytes freshly prepared from mouse bone marrow as well as that of the megakaryocyte progenitors, c-Kit+CD41+ cells, isolated from mouse bone marrow and cultured with TPO. NAP-2 and PF-4 inhibited polyploidization of c-Kit+CD41+ cells in the presence of TPO, and also inhibited the proliferation of c-Kit+CD41+ cells. CONCLUSIONS: NAP-2 and PF-4 produced by TPO stimulation in megakaryocytes suppress megakaryocyte maturation and proliferation as a feedback control.     

Haematological changes in HIV infection

HIV infection is associated with a wide range of hematological abnormalities. The peripheral blood findings and the morphological abnormalities in the bone marrow can simulate myelodysplastic syndrome, myeloproliferative disorders, and T cell lymphoma. We studied the peripheral blood smear and bone marrow findings of 42 patients with HIV infection over a 3-year period with the aim of recognising the morphological findings sufficiently characteristic of HV infection. The salient peripheral blood smear findilngs were anemia, bicytopenia and pancytopenia. The bone marrow revealed trilineage dysplasia, plasma cells and eosinophils, increased megakaryocytes, increased iron and reticulin fibrosis. In two cases the bone marrow revealed granulomata.     

The platelet volume-number relationship in patients infected with the human immunodeficiency virus

The pathophysiology of thrombocytopenia in the acquired immune deficiency syndrome has not been elucidated completely. Many findings in these patients are identical to those with immune thrombocytopenic purpura. However, recent findings in acquired immune deficiency syndrome patients including the effect of zidovudine on platelet count and the demonstration of ultrastructural changes and viral RNA in megakaryocytes, have suggested that the human immunodeficiency virus may directly infect megakaryocytes, and play a role in acquired immune deficiency syndrome-related thrombocytopenia. To investigate further the mechanism of decreased platelet counts in human immunodeficiency virus-infected patients, the platelet volume-number relationship and corresponding bone marrow findings in 34 patients infected with human immunodeficiency virus were studied. Parameters evaluated included platelet count and mean platelet volume; bone marrow cellularity, megakaryocyte number, and number and percentage of denuded megakaryocyte nuclei. Two thirds of the platelet counts were low, and of these 92% had an inappropriately low mean platelet volume. These individuals had a platelet-volume number relationship that is very similar to that seen in myelosuppressive disorders. In addition, more than 90% of the bone marrows from thrombocytopenic patients had either normal or decreased numbers of megakaryocytes. These observations provide additional evidence to support the hypothesis that the pathophysiology of human immunodeficiency virus-associated thrombocytopenia may be due, at least in part, to a direct effect on the megakaryocytes.     

Analysis of loss of heterozygosity and X chromosome inactivation in spleens with myeloproliferative disorders and acute myeloid leukemia.

Neoplastic myeloid proliferations are seen in the spleens of some patients with acute and chronic myeloproliferative disorders. Both acute myeloid leukemia (AML) and chronic myeloproliferative disorders have a variety of underlying cytogenetic defects that can be evaluated by loss of heterozygosity (LOH) studies. LOH studies have advantages over conventional cytogenetics by allowing the use of archival tissues. We evaluated the spleens in AML and chronic myeloproliferative disorders with neoplastic myeloid proliferations for the presence of LOH at several chromosome loci, and X-chromosome inactivation. A total of 17 spleens were evaluated (chronic myelogenous leukemia = 6; chronic idiopathic myelofibrosis = 6; essential thrombocythemia = 1; AML arising from previous chronic myeloproliferative disorders = 4). We examined LOH loci 7q (D7S2554), 8q (D8S263), 9p (D9S157, D9S161), 13q (D13S319), common sites of genetic abnormality in chronic myeloproliferative disorders, and TP53. In six cases, spleen LOH findings were compared to those of concurrent or preceding bone marrow biopsies. Five spleens of female patients were evaluated for the presence of clonality using X-chromosome inactivation. Of the 16 cases analyzed, 14 (88%) had at least one abnormal LOH locus, with 6/16 with two abnormal loci. The abnormalities were distributed as follows: D9S161-7/15 (47%), TP53-6/16 (38%), D7S2554-5/16 (31%), D9S157-5/15 (33%), D8S263-3/14 (21%), and D13S319-2/14 (14%). Of the six bone marrows, 4/6 showed concordance in bone marrow and spleen specimens, with additional LOH abnormalities being identified in the spleen specimens of all four cases. X-chromosome inactivation studies were showed nonrandom (clonal) patterns in two cases. Our results show that allelic losses were common in the neoplastic extramedullary hematopoiesis found in spleens of chronic myeloproliferative disorders and AML. Comparison of spleen and bone marrow specimens by LOH demonstrated additional abnormalities in the spleen compared to the marrow.     

Umbilical cord blood produces small megakaryocytes after transplantation.

Delayed platelet engraftment is a major complication of umbilical cord blood (CB) transplantation. Megakaryocytes derived from CB in vitro are smaller than megakaryocytes derived from bone marrow (BM) or mobilized peripheral blood from adults. Small megakaryocyte size may contribute to delayed platelet engraftment. To test whether small size persists after transplantation, we measured megakaryocyte size, concentration, and maturational stage in BM biopsy specimens obtained after transplantation in archived BM samples from patients receiving CB (CB group, n = 10) versus mobilized peripheral blood or BM transplantation (BM group, n = 9). Megakaryocytes in the postengraftment BM samples were significantly smaller in the CB group than in the BM group (median diameter, 16.7 vs 22.0 microm). There were no significant differences in megakaryocyte concentration or maturational stage between the CB and BM groups. For the first time, we demonstrate that the attainment of adult size in CB-derived megakaryocytes is delayed after human CB transplantation.     

DNA content and nuclear size of megakaryocytes in thrombocythaemia

Total nuclear DNA content and nuclear size of megakaryocytes were studied in biopsies of the iliac bone marrow of individuals with normal or increased platelet counts. The DNA content was determined using Feulgen cytophotometry of bone marrow smears and the nuclear area by morphometric analysis of megakaryocytes of bone marrow sections. The mean DNA content and the mean nuclear area were both significantly larger in megakaryocytes of patients with thrombocytosis as a result of myeloproliferative disease than in patients with secondary thrombocytosis as well as in two control groups of individuals with normal platelets counts, one comprising healthy volunteers, the other with various non-haematological disorders. There was a statistically significant correlation between the DNA content and nuclear area of the megakaryocytes (r = 0.92) in the entire group of bone marrows studied.     

Prominent gelatinous bone marrow transformation presenting prior to myelodysplastic syndrome: a case report with review of the literature

Gelatinous bone marrow transformation (GMT) is a rare disorder characterized by the presence of fat cell atrophy, loss of hematopoietic cells, and deposition of extracellular gelatinous materials. GMT is not a specific disease, but is strongly associated with malnutrition and drugs. Albeit extremely rare, GMT has been reported in patients with myeloproliferative disorders. Herein, we report the second documented case of hypoplastic myelodysplastic syndrome (MDS) accompanying GMT. A 73-year-old Japanese male with excellent nutrition status and no history of alcohol or drug intake was detected with pancytopenia. The initial bone marrow aspirate specimen reveled hypocellular marrow without dysplastic signs in the myeloid cells. Bone marrow biopsy demonstrated hypocellular bone marrow with prominent GMT. He received blood transfusions, however, pancytopenia continued to progress. The second bone marrow aspirate specimen showed dysplastic changes, such as pseudo-Pelger-Huët cells, hypogranular or agranular granulocytes, and megakaryocytes with multiple small nuclei. Cytogenetic study demonstrated deletion of chromosome 7. Therefore, an ultimate diagnosis of hypoplastic MDS accompanying GMT was made. Only a limited number of cases of myeloproliferative disorders with GMT have been reported. Our analysis of these cases revealed that chromosome 7 abnormality is frequently observed in this condition. Moreover, findings from the current case suggested that myeloproliferative disorders including MDS must be included in the differential diagnostic considerations of GMT patients, who have no history of malnutrition or drugs, and careful examination of the bone marrow smear specimen and cytogenetic analysis are necessary for early detection of underlying myeloproliferative disorders.     

Immunoreactivity of MIC2 (CD99) in acute myelogenous leukemia and related diseases.

MIC2 is characteristically expressed in lymphoblastic lesions and Ewing's/primitive neuroectodermal tumor sarcomas. Although MIC2 has recently been reported in chloroma and rare terminal deoxynucleotidyl transferase-positive acute myelogenous leukemia (AML), the incidence and the significance of MIC2 (CD99) immunoreactivity in myeloid lesions is not clear. In this study, we evaluated MIC2 positivity in a variety of myeloid diseases and normal marrow to determine its incidence and distribution in myeloid diseases; its correlation with flow cytometric and cytogenetic data in AML; and its association with leukemic transformation, relapse, and chloroma formation. Paraffin sections of 11 chloromas and 94 bone marrow core biopsies from 66 patients were stained with CD99 monoclonal antibody 12E7. Of 94 bone marrow core biopsies, there were 30 AML (fragment antigen binding M0 to M6), 23 remissions, 5 relapses, 12 myeloproliferative disorders, 13 myelodysplastic syndromes, and 11 normal marrows from patients who did not have leukemia. CD99 immunoreactivity was evaluated with light microscopy. MIC2 expression was seen in leukemic blasts in 6 of 11 chloromas (55%) and 13 of 30 AML (43%) but rarely in myeloproliferative disorders, myelodysplastic syndromes, remission, and normal marrow. CD99 tended to be positive in M1-, M3-, and HLA-Dr-negative AML and negative in AML with relapse. MIC2 expression did not correlate with the karyotype independent of French-American-British Cooperative Group classification and the disease remission or occurrence of chloroma in AML. We concluded that MIC2 is commonly expressed in leukemic blasts of AML and is not predictive of leukemic transformation from myeloproliferative disorders and myelodysplastic syndromes or chloroma formation. Caution should be taken when using MIC2 as a marker for Ewing's sarcoma/ primitive neuroectodermal tumor or lymphoblastic lymphoma on paraffin sections of either soft tissue or bone marrow specimens.     

Histopathologic diagnosis of bone marrow in leukemia and related disorders]

Histopathologic diagnosis of the bone marrow in leukemia is usually a supplementary method to the cytological in acute and chronic leukemia. However, for patients with MDS and MPD and with dry tap bone marrow biopsy is very important. Important morphological findings and useful immunohistochemical methods for differentiation and characterization of leukemia are reported and the usefulness of sequential examination of bone marrow in leukemia during and after chemotherapy is emphasized. In addition to leukemia, histological features and differential points of myelodysplastic syndrome (MDS) and myeloproliferative disorders (MPD) are mentioned. The proliferating megakaryocytes differed in size and shape between MDS and MPD. The difference in proliferating rate of the cells examined by PCNA was also useful to differentiate the two disorders histologically.     

LAT (linker for activation of T cells): a useful marker for megakaryocyte evaluation on bone marrow biopsies

Detection of atypical megakaryocytes in bone marrow biopsies, especially in cases of myelodysplastic syndromes (MDS), chronic myeloproliferative disorders (CMPD) and acute leukemias, is facilitated by staining for markers such as Ulex europaeus agglutinin (UEA)-J, CD31, CD61 and von Willebrand factor (VWF), the latter being considered the most sensitive. Recently, LAT (linker for activation of T cells), a molecule involved in T-cell activation and platelet aggregation, was found to be expressed by megakaryocytes and platelets in tissue sections. We compared VWF and LAT immunoreactivity on megakaryocytes in 64 bone marrow biopsies from 12 normal controls (NC), and from patients with MDS (n=18), CMPD (n=21) and acute megakaryocytic leukemia (AML-M7, n=13). Immunostaining was performed on paraffin sections with polyclonal antibodies against VWF and LAT. Immunoreactivity was evaluated by counting positive megakaryocytes in 10 high-power fields, and values were compared using Student's t test for paired data. Both VWF and LAT predominantly stained the cytoplasm of megakaryocytes, although LAT was also recognizable on the cell membrane. In most biopsies, the immunoreactivity of the two antibodies was quite similar. No significant differences were noticed between the mean values of VWF+ and LAT+ megakaryocytes. However, in 22 cases (5 NC; 5 MDS; 6 CMPD; 6 AML-M7), the number of LAT+ megakaryocytes was at least 30% higher than VWF+cells, while in 3 cases opposite findings were found. In 3 AML-M7 cases, anti-LAT antibodies stained numerous megakaryocytes, but anti-VWF staining was practically negative; in another 5 AML-M7 cases, anti-LAT labeling was much stronger than anti-VWF staining. LAT represents a useful immunohistochemical marker for megakaryocytes in normal and pathological conditions. It seems to be expressed by megakaryocytes more than VWF in most cases and, particularly, in conditions associated with poorly differentiated megakaryocytes, such as acute megakaryocytic leukemias. The use of LAT staining should be recommended in association with other megakaryocyte markers in the study of bone marrow biopsies in cases of hematopoietic disorders.     

Antiplatelet antibody‐induced thrombocytopenia does not correlate with megakaryocyte abnormalities in murine immune thrombocytopenia

Immune thrombocytopenia (ITP ) is an autoimmune bleeding disorder characterized by increased peripheral immune platelet destruction and megakaryocyte defects in the bone marrow. Although ITP was originally thought to be primarily due to antibody‐mediated autoimmunity, it is now clear that T cells also play a significant role in the disease. However, the exact interplay between platelet destruction, megakaryocyte dysfunction and the elements of both humoral and cell‐mediated immunity in ITP remains incompletely defined. While most studies have focused on immune platelet destruction in the spleen, an additional possibility is that the antiplatelet antibodies can also destroy bone marrow megakaryocytes. To address this, we negated the effects of T cells by utilizing an in vivo passive ITP model where BALB /c mice were administered various anti‐αII b, anti‐β3 or anti‐GPI b antibodies or antisera and platelet counts and bone marrow megakaryocytes were enumerated. Our results show that after 24 hours, all the different antiplatelet antibodies/sera induced variable degrees of thrombocytopenia in recipient mice. Compared with naïve control mice, however, histological examination of the bone marrow revealed that only 2 antibody preparations (mouse‐anti‐mouse β3 sera and an anti‐ αII b monoclonal antibody (MWR eg30) could affect bone marrow megakaryocyte counts. Our study shows that while most antiplatelet antibodies induce acute thrombocytopenia, the majority of them do not affect the number of megakaryocytes in the bone marrow. This suggests that other mechanisms may be responsible for megakaryocyte abnormalities seen during immune thrombocytopenia.     

Nucleolar organizer regions of megakaryocytes in chronic myeloproliferative disorders

Summary To study megakaryocyte activation, the argyrophilic staining method of nucleolar organizer regions (AgNOR) has been applied to decalcified bone marrow biopsies of 16 individuals with no haematopoietic disorders and 59 patients with chronic myeloproliferative disease. Of the 59 patients, 18 had chronic myeloid leukaemia (CML), 21 chronic megakaryocytic granulocytic myelosis (CMGM), 13 polycythaemia vera (PV) and 7 essential thrombocythaemia (ET). The AgNOR number of megakaryocytes in CML was significantly lower, and in CMGM, PV and ET significantly higher than in healthy individuals. The high number and the clusters of fine-grained AgNORs of megakaryocytes in CMGM, PV and ET are suggestive of active, proliferating cells. The AgNOR number of megakaryocytes and the platelet counts of the patients did not show a convincing correlation. In CMGM, PV and ET the pyknotic, heterochromatinized megakaryocytes with narrow rims of cytoplasm called bare (nude) nuclei, possessed few, large AgNOR granules. The AgNOR staining of bare nuclei and the roughly identical number of granules found in CMGM, PV and ET indicate a common, active mechanism of apoptosis.     

Megakaryocytes carry the fused bcr-abl gene in chronic myeloid leukaemia: a fluorescence in situ hybridization analysis from bone marrow biopsies

Histological examination of bone marrow biopsies shows that about one-third of chronic myeloid leukaemia (CML) patients exhibit an increase of megakaryocytes. The megakaryocytic predominance may be so striking that differentiation from other chronic myeloproliferative disorders (CMPD) may be difficult in some CML patients. Megakaryocytes in CML are clonal as demonstrated by loss of glucose-6-phosphate dehydrogenase isoenzymes. The Ph translocation, fusing the abl and bcr genes on chromosomes 9 and 22, however, obviously occurs as a second step in tumour development. So far, the Ph translocation has not been assigned explicitly to megakaryocytes. The question is whether the megakaryocytic cell lineage could harbour the bcr/abl fusion in those CML cases with striking proliferation of megakaryocytes but lack this genetic defect in cases with normal or decreased megakaryocyte counts. We therefore performed triple-colour fluorescence in situ hybridization (FISH) for portions of the bcr and abl genes flanking the breakpoint in CML in paraffin sections of CML cases with normal and with increased numbers of megakaryocytes. This method allows identification of the bcr/abl fusion in single, morphologically intact cells, whereas conventional cytogenetics requires lysis and thus destruction of the cell. Among the 21 CML patients examined by FISH, 10 were informative for bcr and abl genes and displayed distinct hybridization signals within nuclei of bone marrow cells. Besides the granulopoietic cells, megakaryocytes of all those patients (4 without and 6 with varying grades of megakaryocytic increase) displayed bcr/abl fusion signals indiciative of a Ph translocation. The lack of hybridization signals in the remaining 11 cases indicates that this technique is not of value diagnostically and should be reserved for scientific questions. Positive controls consisted of conventional chromosome preparations from bone marrow aspirates demonstrating the Ph chromosome in all patients examined, and negative controls of paraffin sections of bone marrow biopsies from non-CML patients. These showed no fusion signals in bone marrow cells, including megakaryocytes, using FISH. Our results demonstrate clearly that not only the transforming event but also the Ph translocation leading to the bcr/abl fusion happens prior to the differentiation of the pluripotent stem cell into different myeloid lineages. The megakaryocytic proliferation evident in some CML cases is probably a consequence of the disease progress.     

The relationship between ploidy and morphological classification of human megakaryocytes]

The relation between polyploidy and morphological classification of human megakaryocytes was studied in bone marrow aspirates from five normal individuals. On a Wright-Giemsa stained smear, megakaryocytes were morphologically classified into four groups according to a modification of Feinendegen's classification which is considered to reflect megakaryocyte maturation. The DNA of the morphologically classified cell is measured by microcytofluorometry using DAPI (4',6-diamidino-2-phenylindole) staining after removing the Wright-Giemsa stain. Most of the normal megakaryocytes were classified into type III (mature megakaryocytes) and the maximum peak in population of the megakaryocyte ploidy was observed at 16N. In each individual, the ploidy showed a similar pattern regardless of the classification. These findings suggest that the development of ploidy depends on a factor different from the one that determined the megakaryocyte maturation of cytoplasm and the ploidy is determined at the level of a megakaryocyte precursor or the most juvenile megakaryocyte.     

The physiology of platelet production

The production of platelets from the bone marrow megakaryocytes is a well-regulated process. Nearly 100 years ago, James Homer Wright described how platelets formed from megakaryocytes and entered the circulation. Subsequent clinical and animal studies have enumerated a number of principles of platelet physiology: the platelet count is constant in any one individual but varies greatly between individuals; an inverse relationship exists between the platelet count and platelet size; the body conserves the mass, not the number, of platelets; and megakaryocyte number, size and ploidy vary in response to changing demands for platelets. With the discovery of thrombopoietin (TPO), a number of additional physiological principles have emerged: TPO takes 24 h to rise maximally and has a maximal half-life of 45 min; TPO levels are inversely and exponentially proportional to the platelet mass; platelets bind and clear TPO from the circulation; and hepatic TPO product on is not altered by changes in the platelet mass. Using these principles, a model for the regulation of platelet production by TPO has been proposed in which the constitutive hepatic TPO produced is removed from the circulation by the platelet mass. Changes in the platelet mass or its ability to clear TPO produce changes in TPO levels resulting in an altered platelet production rate. Using this model, a number of pathological disorders of platelet production, such as essential thrombocythemia and idiopathic thrombocytopenic purpura, are analyzed.     

Thrombopoietin receptor (Mpl) expression by megakaryocytes in myeloproliferative disorders

The thrombopoietin receptor (Mpl) is involved in the pathogenesis of chronic myeloproliferative disorders (CMPD). In this study, we determined Mpl expression by bone marrow cells and megakaryocytes in CMPD by applying laser microdissection, real-time RT-PCR, and immunohistochemistry. Mpl mRNA expression was significantly increased up to 9-fold in total bone marrow cells (p < 0.001) and up to 4-fold in megakaryocytes in chronic myeloproliferative disorders (n = 73) compared to normal controls (n = 26, p = 0.01). Immunohistochemistry revealed heterogeneous Mpl expression by megakaryocytes in CMPD with a stronger accentuation in idiopathic myelofibrosis (IMF) in comparison to polycythaemia vera (PV) and essential thrombocythemia (ET). In addition to megakaryocytes, the erythropoietic lineage was prominently labelled by Mpl antiserum, with considerably stronger staining in polycythaemia vera. We conclude that, in CMPD, megakaryocytes and erythroid cells exhibit increased Mpl expression levels which may contribute to the sustained proliferation of both cell lineages in CMPD.     

Use of monoclonal anti-actin as a megakaryocyte marker in paraffin wax embedded bone marrow biopsy specimens.

Monoclonal anti-actin was used as a marker of megakaryocytes in Zenker's fixed, paraffin wax embedded bone marrow tissue, using an immunoperoxidase staining method. Twenty bone marrow samples were studied, including controls, and different myeloproliferative and myelodysplastic syndromes. The results were compared with those obtained using factor VIII related antigen (F VIII RAg) immunolabelling. Anti-actin is as good a marker for megakaryocytes as anti-FVIIIRAg and is potentially clinically useful when morphological identification is difficult, when bone marrow aspiration is unsuccessful, or when quantitative evaluation of tissue sections is required.     

Ovarian granulocytic sarcoma as the primary manifestation of acute myelogenous leukemia

Granulocytic sarcoma (GS) usually occurs concomitantly with or after the onset of acute myeloid leukemia (AML) or other myeloproliferative disorders, however, GS of the ovary as the primary manifestation of AML is exceedingly rare. To the best of our knowledge, eight cases of ovarian GS as the first sign of AML have been reported in the literature. Here, we report the ninth case: a 27-year-old female who presented with an ovarian mass without any underlying hematologic disorder. A high index of suspicion aided by immunohistochemistry established the correct diagnosis of undifferentiated GS that involved the ovary. Simultaneously, laboratory findings indicated that the blood counts continually increased after surgery. Five days after the surgery, bone marrow biopsy confirmed the presence of AML. After establishing the diagnosis, the patient was sent to the hematology department to receive cytosine arabinoside and idarubicin chemotherapy. This report outlines an exceedingly rare case of AML that initially manifested as an ovarian GS. Awareness of this entity will enable earlier diagnosis and appropriate treatment.     

Detection of minimal residual disease in peripheral blood stem cells from two acute myeloid leukemia patients with trisomy 8 predicts early relapse after autologous bone marrow transplantation

We report two cases of acute myeloid leukemia (AML) French-American-British M4 classification with trisomy 8 at diagnosis as the sole chromosome abnormality. Both patients were treated with the GIMEMA AML-10 protocol and underwent autologous bone marrow transplantation (ABMT) in hematologic remission. Peripheral blood stem cells (PBSC), and bone marrow in one patient, were collected after consolidation therapy and tested by fluorescence in situ hybridization (FISH) analysis with an alpha-satellite probe for chromosome 8. It revealed that all samples were positive for minimal residual disease (MRD) as the value of trisomic cells exceeded the mean +3 standard deviations of the controls. ABMT was done following a myeloablative regimen (busulphan/cyclophosphamide) and PBSC were reinfused. Both patients relapsed, 4 and 2 months, respectively, after autotransplant. Although more data are needed, these results suggest that the persistence of MRD, as detected by FISH, in stem cell collections, is associated with a poor outcome in AML patients with trisomy 8 undergoing ABMT.