A panel of antibodies for the immunostaining of Bouin's fixed bone marrow trephine biopsies.

AIMS: To assess a panel of antibodies on Bouin's fixed bone marrow trephine (BMT) biopsies. These biopsies are widely used in routine diagnosis of various haematological malignancies and may be the sole material available in many centres; however, information regarding the immunostaining of this material is lacking. METHODS: Biopsies were taken from 72 patients presenting with various haematological malignancies (leukaemia, 38; lymphoma, 14; multiple myeloma, 20). A panel of antibodies was assessed on Bouin's fixed BMT biopsies by the alkaline phosphatase-antialkaline phosphatase method. RESULTS: Three B (MB2, LN-2, Ki-B5) and two T cell lineage antibodies (UCHL-1, CD3-r) reliably identified lymphoid cells, while MPO-r, Leu-M1/CD15, and KP-1/CD68 recognised cells from the myeloid or histiocytic/macrophage series. Reed-Sternberg cells were stained by LN-2, Leu-M1, and CD30. Antibodies specific for plasma cells (VS38) and hairy cells (DBA.44) gave a variable pattern of staining. Among the proliferation markers, proliferative cell nuclear antigen but not Ki-67 related antibodies were effective. CONCLUSION: This study presents a panel of antibodies with reactivity not restricted to common fixatives that are also suitable for Bouin's fixed BMT biopsies.

P-glycoprotein positive, drug resistant invasive lymphoepithelial thymoma: treatment response to chemotherapy with cyclosporin and quinine.

A case of invasive drug resistant thymoma, expressing P-glycoprotein, which showed noticeable clinical response to chemotherapy and the multidrug resistance modulating agents cyclosporin and quinine is reported. A 46 year old man presented with severe left shoulder pain and a diagnosis of invasive lymphoepithelial thymoma was made following chest x ray and a computed tomography scan. The patient underwent extensive chemotherapy without resolution of the tumour. More than 90% of the malignant epithelial cells were strongly positive for P-glycoprotein and based on this observation, cyclosporin and quinine were added to the chemotherapy regimen. The mediastinal mass completely resolved and the size of the pleural metastasis decreased substantially. The patient, however, died of an intercurrent infection. This case report highlights the feasibility and efficacy of using cyclosporin and quinine in combination with VAD chemotherapy in the treatment of invasive thymoma.

Haematological changes and infectious complications in anorexia nervosa: a case-control study.

To determine the prevalence of haematological abnormalities in patients with anorexia nervosa (AN), and assess the relationships between these changes, the severity of AN and the propensity to infections, we retrospectively studied 67 patients who met the DSM-III-R diagnostic criteria for AN. We recorded physical findings and routine haematological data on admission, and infectious events during hospitalization. The patients were compared with 67 normal controls matched for age and sex. Mean haemoglobin (Hb) was normal but lower in AN patients than in controls (131 +/- 19 vs. 137 +/- 11 g/l, p = 0.03) and the prevalence of anaemia (Hb < 120 g/l) was higher in the AN group (27% vs. 1.5%, p < 0.0001). Patients had a lower leucocyte count (4.94 +/- 1.9 vs. 6.78 +/- 2.4 x 10(9)/l, p < 0.0001), and increased prevalence of leucopenia (< 4 x 10(9) cells/l)(36% vs. 1.5%, p < 0.0001), neutropenia (< 1500 x 10(6) cells/l)(17% vs. 0%, p = 0.0015) and thrombocytopenia (< 150 x 10(9)/l) (10% vs. 0%, p = 0.03). Only 2 patients (3%) had pancytopenia, but 9/17 patients with anaemia (53%) also had leucopenia. There was a slight but significant correlation between body-mass index (BMI) and total leucocyte, neutrophil and red blood cell counts. Severe infectious complications occurred in 9% of AN patients vs. 0% in controls (p = 0.01); they were more frequent with neutropenia (relative risk, 15.1: 95% CI, 10-20.2) or low (< 12) BMI (relative risk, 11.6: 95% CI, 6.6-16.6) on admission. Compared with controls, AN patients thus had an increased prevalence of anaemia, leucopenia and thrombocytopenia.(ABSTRACT TRUNCATED AT 250 WORDS)

Philadelphia-negative (Ph-) chronic myeloid leukemia (CML): comparison with Ph+ CML and chronic myelomonocytic leukemia. The Groupe Français de Cytogénétique Hématologique.

To better understand the Philadelphia-negative (Ph-) chronic myeloid leukemia (CML) and its relationships with Philadelphia-positive (Ph+) CML and chronic myelomonocytic leukemia (CMML), a study was undertaken by the Groupe Français de Cytogénétique Hématologique. Thirty-five Ph- CML patients were investigated and compared with 55 chronic phase Ph+ CML and 100 CMML patients. There were 12 M-BCR positive (M-BCR+) and 23 M-BCR negative (MBCR+) patients. No clinical or biologic differences were found between Ph+ and Ph-, M-BCR+ patients. In the Ph- group, M-BCR+ and M-BCR- patients differed significantly in age (47.7 +/- 6.6 v 67.0 +/- 6.1 years, respectively; P = .001), leukocytosis (153.4 +/- 135.1 v 58.5 +/- 37.7 10(9)/L, P = .002), relative monocytosis (1.8% +/- 1.2% v 5.6% +/- 1.4%, P = .048), absolute basophilia (8.5 +/- 9.7 v 0.9 +/- 1.5 10(9)/L, P = .001), percentage of immature myeloid precursors (IMP) in peripheral blood (29.0% +/- 9.5% v 15.3% +/- 8.1%, P = .001), and percentage of erythroblasts in bone marrow (BM) (6.5% +/- 3.5% v 14.6% +/- 3.6%, P = .001). Karyotypic abnormalities other than the Ph chromosome occurred in 0 of 12 M-BCR- at diagnosis and 7 of 23 M-BCR- Ph- CML (P = .033). None of the 13 investigated BCR- patients had detectable BCR/ABL transcripts using polymerase chain reaction (PCR) and none had an N-RAS mutation. Cytologic findings showed a marked morphologic difference between M-BCR+ and M-BCR- patients, especially in the monocytic lineage. Dysmyelopoietic features in CMML and M-BCR- patients were very similar, and the differences were of quantitative order only. Using four criteria (monocytosis, percentage of IMP, basophilia, and percentage of erythroblasts in BM), patients could be divided into typical and atypical CML and this classification correlated well with molecular findings. We conclude that, while Ph-, M-BCR+, and Ph+ CML are identical diseases, Ph-, M-BCR- CML, and CMML have many similarities and might be only different aspects of a same entity.

A disseminated alveolar rhabdomyosarcoma in a 9-year-old boy disclosed by chromosomal translocation (2;13) (q35;q14)

A 9-year-old boy presented with a small subcutaneous tumor of the trunk and diffuse bone marrow involvement. The first histological diagnosis given was undifferentiated malignancy possibly of neural crest origin and chemotherapy was started immediately using vincristine, cyclophosphamide, cisplatin, and teniposide (OPEC). Complete response was achieved after four courses of chemotherapy. Histological slides were then reviewed and the final diagnosis of alveolar rhabdomyosarcoma (RMS) was retained. Moreover, chromosome analysis of malignant cells in the bone marrow revealed a translocation involving chromosomes 2 and 13:t(2;13) (q35;q14). This specific karyotype finding has been recently reported in a few cases and could be specific for alveolar RMS. The patient had a relapse 7 months after diagnosis and died 4 months later.

Breakpoints of 11q25 in three similar cases of B non Hodgkin's lymphoma.

We report three cases of small B cell non Hodgkins lymphoma, two with plasmacytoid differentiation, presenting with a similar clinical picture and identical immunophenotype, who cytogenetically had breakpoints involving band 11q25. We suggest that this breakpoint may define a group of lymphomas closely related to the diffuse small cleaved cell lymphomas with t(11;14)(q13;q32).

Multipotent stem cell involvement in megakaryoblastic leukemia: cytologic and cytogenetic evidence in 15 patients.

Cytologic and cytogenetic results obtained from patients fulfilling the FAB criteria for the diagnosis of acute nonlymphocytic leukemia (ANLL) of megakaryocytic lineage (ANLL-M7) are reported. Eleven cases were de novo ANLL-M7, of whom three presented with acute myelofibrosis. Four cases were megakaryoblastic transformations of chronic myelogenous leukemia (two cases), refractory anemia with excess of blasts (one case), and polycythemia vera (one case). Four patients showed a minority of granular blasts, with occasional Auer rods in one. Positive myeloperoxidase and/or sudan black-B stainings and CD13 positivity in these cases were consistent with the presence of a myeloid involvement. Morphologic evidence of associated myelodysplastic features was detected in all evaluable patients with de novo ANLL-M7. These cytologic findings indicate that ANLL-M7 may frequently represent a multilineage proliferation. Cytogenetic studies revealed -7/7q- and +8, alone or in combination with additional aberrations, in three cases each. Rearrangements involving bands 3q21 or 3q26 were seen in two patients and +21, as an additional aberration, in one. Other structural rearrangements all observed in a single patient were inv(16)(p13q22) at megakaryoblastic relapse with bone marrow eosinophilia, t(13;20)(q13 or 14;q11), del(20)(q11), and der(7)t(7;17)(p14;q22). Most breakpoints of these aberrations are located at bands frequently rearranged in malignant myeloid stem cell disorders. A review of 31 cases of the literature showed a frequent occurrence of -7/7q- and -5/5q- in ANLL-M7. Many of the chromosome aberrations so far described in ANLL-M7 appear to be shared by a spectrum of myeloid neoplasias and may be related to mechanisms conferring proliferative advantage to undifferentiated stem cells.

Translocation t(6;9) occurring in acute myelofibrosis, myelodysplastic syndrome, and acute nonlymphocytic leukemia suggests multipotent stem cell involvement.

The cytological and cytogenetic features of six patients with myeloid neoplasia and t(6;9)(p23;q34) including a case of acute myelofibrosis (AMF), a refractory anemia with excess of blasts (RAEB), and four cases of acute nonlymphocytic leukemia (ANLL) are described. Two patients in this series, both affected by ANLL type M2, presented an increase of bone marrow basophils, suggesting that this cytological-cytogenetic association is not absolute and that it may be more frequently observed in ANLL with maturation. All patients with de novo ANLL showed associated myelodysplastic features, and one patient presented a dysmyelopoietic syndrome, later evolving into ANLL. The presence of the t(6;9) in a range of myeloid neoplasias, with either concurrent myelodysplastic features or a preleukemic phase in cases of ANLL, provide evidence that this chromosome aberration may always involve a multipotent myeloid stem cell. Data on toxic exposure of the patients suggests that myeloproliferative disorders with the t(6;9) may frequently represent environmentally induced neoplasias.

Immunodetection of small cell lung cancer metastases in bone marrow using three monoclonal antibodies.

Detection of bone marrow metastases by indirect immunofluorescence methods was investigated using three monoclonal antibodies (MoAbs) raised against small cell lung cancer (SCLC). These antibodies, designated anti-LCA1, -LCA2 and -LCA3, recognize three different antigens on the surface of SCLC cells. Eighty-four bone marrow samples from 74 different patients were studied. Whereas tumor cells were found in 32 (38%) by MoAb staining, only 10 (12%) were positively identified using conventional morphological methods. Nine out of the morphologically positive specimens showed reactivity with at least two monoclonal antibodies. Among the 32 samples proven positive by immunofluorescence, an important antigenic variability was noted. Anti-LCA1 recognized tumor cells in 62%, anti-LCA2 and anti-LCA3 in 53%. Due to the recognition of bone marrow involvement by fluorescence methods in 26% of the 34 patients classified as limited disease, a new subgroup of limited disease patients was defined whose prognosis remains undetermined. Our results confirm the utility of immunodetection in the diagnosis of SCLC bone marrow metastases and emphasize the advantage of using a panel of MoAbs with different antigenic specificities. Further study is needed to determine the prognostic significance of bone marrow involvement established by immunodetection.

Alpha interferon in the treatment of symptomatic myelofibrosis with myeloid metaplasia.

2 patients with myelofibrosis and myeloid metaplasia had symptomatic splenomegaly and were treated with interferon alpha-2c (IFN alpha-2c). The splenic pain and pressure symptoms disappeared, accompanied by a decrease in the size of the spleen. However, the peripheral blood count worsened and no improvement in the bone marrow fibrosis could be observed.

Assessment of bone marrow blood flow using positron emission tomography: no relationship with bone marrow cellularity.

Bone marrow blood flow has been assessed using positron emission tomography and the 15O-labelled carbon dioxide steady-state technique. The measurements were performed at the site of the posterior iliac crest. The bone marrow blood flow was 10.0 ml/min/100 cm3 +/- 3.0 (SD) in normal volunteers. It was markedly increased in patients with polycythaemia vera (26.9 +/- 4.6), chronic granulocytic leukaemia (25.2 +/- 3.9) and myelofibrosis (35.1 +/- 7.3). However, bone marrow blood flow did not differ from normal in patients with aplastic anaemia, chronic haemolysis or chronic lymphocytic leukaemia. There was no relationship between bone marrow cellularity and bone marrow blood flow. The data show that bone marrow blood flow is markedly elevated in polycythaemia vera, myelofibrosis and chronic granulocytic leukaemia and suggest that bone marrow cellularity is not a major factor in regulating bone marrow blood flow.

A variant of the congenital dyserythropoietic anaemia type II with structural abnormalities in the granulocytic series.

Typical features of congenital dyserythropoietic anaemia (CDA) were found in a 13-year-old girl admitted for chronic recurrent multifocal osteomyelitis. The findings on light microscopy were in agreement with those described in CDA type II, whereas on electron microscopy, the ultrastructure findings were compatible with both types I and II. The acidified serum lysis test (Ham test) performed with eight normal sera was negative. The patient's red blood cells showed an increased agglutinability with anti-i antibodies. Morphological changes were also shown in the mature neutrophilic granulocyte suggesting that the primary disorder exists already in the multipotent stem cell.

Quantitative assessment of erythropoiesis in bone marrow expansion areas using 52Fe.

Quantitative 52Fe scans were performed in 180 patients. Expansion of bone marrow was observed in 70. This bone marrow expansion was a nearly constant feature in haemolytic anaemia and in sideroblastic anaemia. It occurred in a third of the patients with myelofibrosis. In patients with polycythaemia rubra vera, expansion was noticed in only two out of seven. Erythropoiesis in expansion areas occurred despite persistence of fat in the iliac crest bone marrow biopsy. It could exist with a slight increase in erythropoiesis and might develop only after a long period of erythropoietic stimulation. Increased marrow activity can take place without erythropoietic expansion in long bones. The fraction of iron uptake in expansion areas did not exceed a third of total marrow iron uptake. With increasing erythropoiesis, the increase in iron uptake in expansion areas was less marked than the increase in the central areas. Erythropoiesis in expansion areas was usually not of major quantitative importance but could nevertheless reach the erythropoiesis of a normal adult.

Effect of lithium on diffusion chamber granulopoiesis.

We studied the effect of lithium on diffusion chamber (DC) granulopoiesis. When DC loaded with bone marrow cells were implanted into the peritoneal cavity of mice previously injected with lithium carbonate, more proliferative and nonproliferative granulocytes were produced as compared to DC implanted into control hosts. The number of DC CFU-c was increased significantly in the lithium-treated group, but there was no difference in the number of DC CFU-s. Levels of DC fluid CSF showed no evident correlation with DC myelopoiesis. These data suggest that a humoral factor other than CSF mediates the action of lithium in DC granulopoiesis, and that lithium's influence on DC hematopoietic stem cell proliferation occurs mainly at the CFU-c level.

Assessment of bone marrow and splenic erythropoiesis in myelofibrosis.

The iron uptake in bone marrow and spleen was measured in 29 patients with myelofibrosis using 52Fe and quantitative scanning. In 10 patients, no iron uptake in the marrow could be observed and active erythropoiesis was extramedullary only. In the bone marrow of patients with myelofibrosis, the iron uptake per nucleated red cell was less than that observed in conditions without myelofibrosis or extramedullary erythropoiesis. Increasing splenic iron uptake was likely to be associated with a decreasing bone marrow iron uptake and was related to the size of the spleen. The data suggest that in myelofibrosis, the spleen dominates iron uptake through intense erythropoiesis and a high splenic blood flow, thus restraining iron supply to the bone marrow.

Myeloid stem cell kinetics in children hypertransfused during remission induction of acute lymphoblastic leukemia.

Experimental studies in animals and recent preliminary clinical evidence raised the possibility that hypertransfusion might be capable of producing a beneficial effect on granulopoiesis recovery following irradiation or chemotherapy. This prompted us to design a study to determine the effect of hypertransfusion on the blood and marrow CFU-c of leukemic children during remission induction. Nineteen children with acute lymphoblastic leukemia have been randomized in pairs to normotransfused (Hb: 12-14 g/dl) and hypertransfused (Hb: 16-18 g/dl) groups. Anti-leukemic chemotherapy (vincristine and adriamycin weekly during 4 weeks and prednisone daily) was identical in all children. As expected, suppression of erythropoiesis was observed in the hypertransfused group. During the first three courses of chemotherapy, the number of marrow CFU-c remained very low in both groups. One week after the third course of chemotherapy the number of bone marrow CFU-c began to increase in both groups. One week after course four the CFU-c value was significantly larger in the hypertransfused group. We also observed that circulating CFU-c were almost absent before induction chemotherapy, whereas their number increased after course three and was higher in the hypertransfused group and remained higher after course four. These results show the kinetics of bone marrow recovery after chemotherapy and suggest that hypertransfusion increases the rate of recovery of granulopoiesis.

Selective hypoplasia of pelvic bone marrow.

6 patients with thrombocytopenia and anaemia had fatty marrow replacement at the site of an iliac crest. 52Fe scans showed marked abnormalities of bone marrow distribution. Particularly, the uptake of radioiron into the pelvis was almost absent. The sternal marrow was cellular. Studies of the bone marrow which included cytology, histology and electron microscopy failed to reveal why the pelvic marrow was aplastic. Occasional vascular lesions could not seen in the iliac crest bone marrow sections. The cellular morphology showed slight maturation abnormalities. Ferrokinetics were consistent with erythroid marrow hypoplasia. The pelvic and sternal marrow cellularity in patients with pancytopenia may not be representative of the cellularity of the whole marrow, and the pelvic marrow especially may be prone to aplasia.

Murine yolk sac hematopoiesis studied with the diffusion chamber technique.

The proliferation and differentiation of visceral and of parietal yolk sac cells from 9-day-old mouse embryos were studied in diffusion chambers (DC). After culture, the visceral yolk sac yielded predominantly macrophages whereas the parietal yolk sac displayed mainly plasmocyte-like cells. It is shown the latter do not synthesize immunoglobulin but rather elaborate Reichert's membrane. Neither visceral nor parietal yolk sac contained detectable pluripotent stem cells (CFUs) prior to or after DC culture, a finding which is discussed in the light of the current literature.