Jesús Villarrubia

Immunophenotypic characterization of human bone marrow mast cells. A flow cytometric study of normal and pathological bone marrow samples

The goal of the present paper was to define the immunophenotype of bone marrow mast cells (BMMC) from healthy controls and patients with hematologic malignancies (HM) based on the use of multiple stainings with monoclonal antibodies analyzed by flow cytometry. Our results show that BMMC from both groups of individuals display a similar but heterogenous immunophenotype. The overall numbers of BMMC are higher in the HM group of individuals (p = 0.08). Three patterns of antigen expression were detected: (1) markers constantly positive in all cases analyzed (CD9, CD29, CD33, CD43, CD44, CD49d, CD49e, CD51, CD71, CD117, and FcεRI), (2) antigens that were constantly negative (CD1a, CD2, CD3, CD5, CD6, CD11a, CD14, CD15, CD16, CD19, CD20, CD21, CD23, CD25, CD30, CD34, CD38, CD41a, CD42b, CD65, CD66b, HLA-DR, and CD138), and (3) markers that were positive in a variable proportion of cases – CD11b (50%), CD11c (77%), CD13 (40%), CD18 (20%), CD22 (68%), CD35 (27%), CD40 (67%), CD54 (88%) and CD61 (40%). In addition, BMMC from all cases explored were CD45+, and this antigen was expressed at an intensity similar to that of mature granulocytes. In summary, our results show that BMMC from both healthy controls and HM patients display a relatively heterogenous immunophenotype. Interestingly, we have observed clear differences between the immunophenotype of BMMC and MC from other tissues. This could be due either to the heterogeneity of human MC according to their tissue localization or to the sensitivity of the method used for antigen detection.

Expression of Bcl-2 by human bone marrow Mast Cells and its overexpression in Mast cell leukemia

Bcl‐2 protein plays a major role in the prevention of programmed cell death of differentiating cells. In the present study, the expression of cytoplasmic bcl‐2 by human Bone Marrow Mast Cells (BMMC) from both normal and pathological bone marrow samples was examined. A total of 35 subjects corresponding to 9 healthy volunteers, 8 cases of adult indolent systemic mast cell disease (SMCD), 4 cases of pediatric mastocytosis (PM), 11 cases of hematological malignancies (HM), 2 cases of reactive bone marrow, and 1 case of mast cell leukemia (MCL) were analyzed. The expression of bcl‐2 was studied using quantitative three‐color flow cytometry. We also studied the molecular configuration of the bcl‐2 gene and other relatives by Southern blot and polymerase chain reaction (PCR) in the MCL case. Bcl‐2 expression was detected in BMMC from all samples analyzed. No significant differences on the expression of bcl‐2 were detected between BMMC from healthy subjects and patients with SMCD, PM, HM, and reactive bone marrow. By contrast, bcl‐2 protein was overexpressed in BMMC from MCL patient without gene rearrangement. Our results show that bcl‐2 protein was constitutively expressed by BMMC. BMMC from MCL display overexpression of bcl‐2, which could not be related to molecular rearrangements involving the bcl‐2 gene. The expression of this protein by mature MC may play a role in the prevention of MC apoptosis and thus help to explain the long survival of these cells. The overexpression of bcl‐2 by BMMC in MCL may help to explain their resistance to chemotherapy‐induced apoptosis. Am. J. Hematol. 60:191–195, 1999.

Expression of lymphoid-associated antigens in mast cells: report of a case of systemic mast cell disease

Summary. In this study the expression of‘classically’considered lymphoid‐associated antigens (CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD19, CD20, and CD22) was explored both in peripheral blood (PB) and bone marrow (BM) mast cells (MC) in a case of systemic mast cell disease (SMCD) by means of using multiple stainings and a direct immunofluorescence technique. CD2 and CD22 were expressed in both PB and BM MC, all the remaining lymphoid‐associated markers were negative. Our results suggest that the reactivity for both CD2 and CD22 in PB and BM MC would be aberrant.

The CD69 early activation molecule is overexpressed in human bone marrow mast cells from adults with indolent systemic mast cell disease.

We have analysed the quantitative expression of surface CD69 antigen on human mast cells (MC), from both normal and pathological bone marrow (BM) samples, using flow cytometry. Our major aim was to analyse whether CD69 is constitutively expressed by normal BMMC and to explore the possible differences between CD69 expression by BMMC from normal controls and patients suffering from different pathological conditions.

Amelanotic bone marrow infiltration secondary to pigmented malignant melanoma.

Amelanotic melanomas account for aproximately 2 to 3% of malignant melanomas. Some authors suggest an increased aggressiveness and a higher rate of metastases for these clinical variants (l). In autopsy series, bone marrow involvement is observed in 45% of patients with malignant melanoma (MM) (2). However, in vivo staging procedures disclose neoplastic infiltration only in 7% of the cases (2). As far as we can determine, bone marrow infiltration by amelanotic cells of malignant melanoma has not been previously described. We report the case of a woman with a personal history of inelanotic MM who developed involvement of bone marrow by amelanotic cells presenting as primary fibrinolysis.

δβ-Thalassemia trait: how can we discriminate it from β-thalassemia trait and iron deficiency anemia?

OBJECTIVES To analyze the differences not only in classic hematologic parameters but also in RBC subpopulations among δβ-thalassemia trait (δβ-TT), β-thalassemia trait (β-TT), and iron deficiency anemia (IDA) and to evaluate the role of fetal hemoglobin (HbF) in elevated RBC distribution width (RDW). METHODS Samples from 553 patients with microcytosis (74 δβ-TT, 272 β-TT, and 207 IDA) were run on an Advia 2120i analyzer (Siemens Medical Solutions Diagnostics, Tarrytown, NY). Classic hematologic parameters and RBC subpopulations were assessed. The correlation between HbF and RDW in patients with thalassemia (both β and δβ) was evaluated. An independent sample t test was used to compare classic hematologic parameters and RBC subpopulations among β-TT, IDA, and δβ-TT and receiver operating characteristic curves performed in the significant comparisons. RESULTS RDW was significantly higher in δβ-TT compared with β-TT (18.79% vs 16.04%, P < .001), as was mean corpuscular volume (66.39 vs 64.82 fL, P < .001), mean corpuscular hemoglobin (20.73 vs 20.04 pg, P < .001), and mean corpuscular hemoglobin concentration (31.16 vs 30.66 g/dL, P = .03). Pearson coefficient showed a good correlation between HbF and RDW. The values obtained for all the parameters were significantly different (P < .001) between patients with thalassemia (β and δβ) and IDA. CONCLUSIONS RDW is the best parameter to discriminate δβ-TT from β-TT. The degree of anisocytosis in patients with β-TT and δβ-TT is strongly correlated with HbF.

Metastatic malignant melanoma detected on bone marrow aspiration

A 75-year-old man presented with asthenia and epistaxis of 3 weeks duration. He had been diagnosed with malignant melanoma 4 months previously, and had received intensive chemotherapy. Physical examination showed pallor, petechiae of both legs and splenomegaly. A blood count showed a haemoglobin concentration of 98 g/l, white cell count of 1 7 9 10/l and platelet count of 18 9 10/l. A blood film showed a leucoerythroblastic reaction; metastasis was suspected and a bone marrow aspiration and trephine biopsy were therefore performed. A May-Gr€ unwaldGiemsa stain revealed numerous clusters of non-haemopoietic cells containing black pigment (top left and top right), some with giant granules (bottom). Immunohistochemical staining of the biopsy specimen showed positivity for S-100 protein, thus confirming metastatic melanoma. Liver and spleen metastases were also identified on a computed tomography scan. The patient was treated palliatively and died 2 months later. Although metastasis of malignant melanoma to the bone marrow is infrequent, this possibility should be considered in the presence of pancytopenia or leucoerythroblastosis.

Leprosy diagnosed by bone marrow aspiration

A 26-year-old previously healthy Paraguayan woman consulted because of persistent fever and painful cutaneous lesions of the arms and legs of 2 weeks duration. Physical examination revealed: numerous two cm diameter brown subcutaneous nodules, and ulcers of both feet (left); supraclavicular, axillary and inguinal lymphadenopathy; hepatosplenomegaly; and thermal hypoesthesia and decreased pain sensitivity in legs and arms. A blood film showed atypical lymphocytes, so a bone marrow aspiration and trephine biopsy were performed. A ZiehlNeelsen stain revealed numerous acid-fast bacilli (right). A week later, a biopsy of a cutaneous lesion showed panniculitis with mycobacteria and an infiltration by microvacuolated histiocytes, confirming the diagnosis of lepromatous leprosy. The patient was discharged home after clinical improvement with rifampicin, dapsone and clofazimine. Although the bone marrow is usually affected in lepromatous leprosy, the diagnosis is commonly made by skin biopsy. This case illustrates the importance of bone marrow aspiration in the differential diagnosis of fever of unknown origin.

Juvenile Chronic Lymphocytic Leukemia with Unusual Intracisternal Inclusions: An Ultrastructural Study

The electron microscopic analysis of intracisternal inclusions in lymphocytes of the bone marrow and peripheral blood in a case of juvenile chronic lymphocytic leukemia is described. These inclusions consist of well-ordered microtubules attached to a central axis. The contribution of electron microscopic analysis in establishing the substructural pattern of these inclusions is discussed.

Skin lesions and cytological features in HTLV-1 associated adult T-cell leukaemia/lymphoma

A 58-year-old woman born in South America was hospitalised for investigation of lymphocytosis and constitutional symptoms. She reported involuntary loss of weight of 5 kg in the previous 2 months, almost daily fever and intense asthenia. During the previous month, she had developed erythematous and itchy plaques, which had started in skin folds and spread to the limbs (image). A full blood count showed a haemoglobin concentration of 119 g/l, platelet count 278 9 10/l, total white cell count of 31 6 9 10/l and 12 6 9 10/l total lymphocytes. She had no renal or hepatic impairment and serum calcium level was normal, but lactate dehydrogenase was 1086 iu/l (normal 140–240). A total-body computed tomography scan showed splenomegaly and multiple enlarged lymph nodes at both sides of the diaphragm. A peripheral blood film revealed lymphocytes with basophilic cytoplasm and hyperlobulated, flower-shaped nuclei (image, centre top). Other lymphoid cells had immature nuclei with fine chromatin and a visible nucleolus that gave them a blastic appearance. Many cells showed intense vacuolization (image), which was positive on periodic acid–Schiff staining. Peripheral blood flow cytometric immunophenotyping showed that lymphocytes were positive for CD3, CD4, CD2, CD5, CD25 and CCR4 and negative for CD8 and CD7. The bone marrow and the cerebrospinal fluid were both infiltrated. Serology for human T-cell lymphotropic virus 1 (HTLV-1) was positive and this result was confirmed by Western blot. A diagnosis of HTLV-1-associated adult T-cell leukaemia/lymphoma (ATLL) was made and the patient was started on treatment with zidovudine plus alpha interferon and combination chemotherapy. HTLV-1-associated ATLL is an uncommon disease in Western countries. HTLV-1 is endemic mainly in Japan, the Middle East, the Caribbean region and South America. The clinical course of ATLL is very heterogeneous including indolent and aggressive forms. There is no standard treatment; both chemotherapy and antivirals should be used.