Circulating Cd34+ cell count differentiates primary myelofibrosis from other Philadelphia-negative myeloproliferative neoplasms: a pragmatic study.

A high number of circulating CD34+ cells has been advocated to distinguish primary myelofibrosis from other Philadelphia-negative myeloproliferative neoplasms. We re-evaluated the diagnostic interest of measuring circulating CD34+ cells in 26 healthy volunteers and 256 consecutive patients at diagnosis for whom a myeloproliferative neoplasm was suspected. The ROC curve analysis showed that a number of CD34+ <10/µl excludes the diagnosis of primary myelofibrosis with a sensitivity of 97 % and a specificity of 90 % (area under the curve: 0.93 [0.89-0.98]; p < 0.001). Patients with PMF harboring a CALR mutation had more circulating CD34+ cells than patients with either a JAK 2 or MPL mutation (p = 0.02 and p < 0.01, respectively). These results suggest that this fast, simple, non-invasive, and standardized test is of particular interest to exclude the diagnosis of primary myelofibrosis.

Plasma cell morphology in multiple myeloma and related disorders.

Normal and reactive plasma cells (PC) are easy to ascertain on human bone marrow films, due to their small mature-appearing nucleus and large cytoplasm, the latter usually deep blue after Giemsa staining. Cytoplasm is filled with long strands of rough endoplasmic reticulum and one large Golgi apparatus (paranuclear hof), demonstrating that PC are dedicated mainly to protein synthesis and excretion (immunoglobulin). Deregulation of the genome may induce clonal expansion of one PC that will lead to immunoglobulin overproduction and eventually to one among the so-called PC neoplasms. In multiple myeloma (MM), the number of PC is over 10% in most patients studied. Changes in the morphology of myeloma PC may be inconspicuous as compared to normal PC (30-50% patients). In other instances PC show one or several morphological changes. One is related to low amount of cytoplasm, defining lymphoplasmacytoid myeloma (10-15% patients). In other cases (40-50% patients), named immature myeloma cases, nuclear-cytoplasmic asynchrony is observed: presence of one nucleolus, finely dispersed chromatin and/or irregular nuclear contour contrast with a still large and blue (mature) cytoplasm. A peculiar morphological change, corresponding to the presence of very immature PC named plasmablasts, is observed in 10-15% cases. Several prognostic morphological classifications have been published, as mature myeloma is related to favorable outcome and immature myeloma, peculiarly plasmablastic myeloma, is related to dismal prognosis. However, such classifications are no longer included in current prognostic schemes. Changes related to the nucleus are very rare in monoclonal gammopathy of unknown significance (MGUS). In contrast, anomalies related to the cytoplasm of PC, including color (flaming cells), round inclusions (Mott cells, Russell bodies), Auer rod-like or crystalline inclusions, are reported in myeloma cases as well as in MGUS and at times in reactive disorders. They do not correspond to malignant changes of PC but are related to abnormal synthesis, trafficking, or excretion of the immunoglobulin that is stored in excess within the cytoplasm. Occurrence of crystalline inclusions within PC may be the first anomaly leading to the diagnosis of adult Fanconi syndrome. After a historical perspective, the authors report on the various morphological aspects of PC that may occur in multiple myeloma and related disorders, and discuss about their clinical and pathophysiological significance. Today, morphological identification and accurate determination of % PC within bone marrow remain ancillary criteria for the diagnosis of MM and help for the diagnosis of rare renal disorders.

[DIC and peripheral gangrene in a severe Plasmodium falciparum malaria: the coagulation-inflammation cycle with Plasmodium falciparum as a model]. / CIVD et gangrène périphérique dans un cas de paludisme sévère : le cycle coagulation-inflammation appliqué au Plasmodium falciparum.

Peripheral gangrene with disseminated intravascular coagulation (DIC) during severe Plasmodium falciparum malaria has already been described but is unfrequent. We report here the case of a 62-year-old man admitted in the intensive care unit of our hospital for severe Plasmodium falciparum malaria with disseminated intravascular coagulation (DIC) and peripheral gangrene of his toes that needed amputation. Pathophysiological mechanisms leading to DIC in malaria can be used as a model to explain the relation between coagulation and inflammation. Therapeutic targeting of coagulation, by acting on inflammation, could be useful to limit the coagulation-inflammation cycle.

[Peripheral blood Sézary cells and the diagnosis of Sézary syndrome]. / Recherche de cellules de Sézary dans le sang périphérique: morphologie ou immunophénotype?

Sézary syndrome (SS) is a rare and aggressive cutaneous lymphoma, and its diagnosis is based both on clinical, histological and biological features. None of these criteria taken alone is specific, and the two clinical observations reported here agree with this. Peripheral blood involvement, due to the presence of a variable number of Sézary cells, has been recently fully delineated by the ISCL/EORTC international organizations, and taken into account in the diagnosis and the prognosis of this syndrome. Identification and quantification of peripheral blood Sézary cells on the blood smear is an essential criterion for the diagnosis and is sufficient when associated with relevant clinical or/and histological grounds. Flow cytometry is another tool to demonstrate Sézary cells within the peripheral blood mononuclear cells. The authors discuss about the respective advantages and limits of morphology and flow cytometry in the identification and enumeration of circulating Sézary cells. Molecular biology is helpful in peculiar situations.

[Free and intracellular bacteria on peripheral blood smears: an uncommon situation related to an adverse prognosis]. / Présence de bactéries libres et intraleucocytaires sur l'étalement sanguin : une situation rare mais de pronostic vital.

Bacterial infections are responsible for several changes in the cell blood count, which are usually non specific, although some morphological changes of polymorphonuclear neutrophils may be indicative of sepsis. The presence of bacteria on peripheral blood smears is a rare but extreme situation, related in most instances to a fatal prognosis. The presence of both free and intracellular bacteria was observed in the peripheral blood smear of a critically ill patient with a pneumococcal septicaemia which led to a fatal outcome within the next following hours. If the finding of bacteria on the blood smear is a sign of severe sepsis, the literature review shows that less than 10% of septic patients demonstrate bacteria on the blood smear, and routine search for the diagnosis of sepsis is not recommended. Samples taken from infected central venous catheters are another situation of bacteraemia which must be known, but prognosis is usually not fatal if prompt medical care is performed. Some preanalytical conditions are also associated with the presence of bacteria on the peripheral blood smear, but unrelated to infection of the relevant patient.

[Bone marrow necrosis in two patients with neoplastic disorders]. / Nécrose médullaire chez deux patients atteints de maladies cancéreuses.

Bone marrow necrosis is defined by extensive necrosis of the myeloid tissue and bone marrow stroma. Diagnosis is done on characteristic cytological pattern of the bone marrow aspiration and/or biopsy. We report two observations. The first patient, aged 75, has been hospitalized for fever, asthenia and lower back pain. An haematological malignancy was suspected after observation of a few peripheral blood blast cells, but necrosis was found on the bone marrow aspiration and could not lead to further haematological diagnosis. Within next days, the white blood cell count and the number of blasts increased, leading to the diagnosis of acute monoblastic leukaemia. A chemotherapy was started but the patient died 20 days after admission. The second patient, aged 28, has been hospitalized for severe bleeding a few days after the diagnosis of a metastatic gastric tumour. The bone marrow aspiration, made for the evaluation of a thrombocytopenia, showed a massive necrosis. The patient deceased shortly after hospitalization. According to literature, bone marrow necrosis is in most instances secondary to either an haematological malignancy (60%) or to a solid tumour (30%), but only at times observed with a non-malignant disorder. Bone pain, fever, cytopenias and elevated serum lactic dehydrogenase and alkaline phosphatase are frequently reported, but are mostly non specific of the diagnosis in these malignant conditions. Examination of the bone marrow leads to the diagnosis: cells are pycnotic, scarcely recognizable in a background of amorphous extracellular eosinophilic proteinaceous material, and histology shows disappearance of fat spaces with preservation of the bone tissue. Tissue hypoxemia due to microcirculation failure may be the main mechanism leading to the necrosis, whatever the related disorder. Supportive care together with specific therapy of the causal disease must be started promptly. The prognosis depends on the underlying illness and is generally very poor when extensive necrosis is observed.

The effect of nitric oxide donors on nitric oxide synthase-expressing myenteric neurones in culture.

Previously, we demonstrated that intestinal inflammation leads to a postinflammatory loss of nitric oxide synthase (NOS)-expressing myenteric neurones and motility disturbances. Here, we investigated whether high NO concentrations could be responsible for the decrease in NOS neurones. Myenteric neurone cultures, prepared from guinea-pig small intestine, were incubated with NO donors [sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1)]. After fixation, NOS neurones were identified by NADPH diaphorase staining and neurone-specific enolase (NSE)-positive neuronal content was assessed with an enzyme-linked immunosorbent assay (ELISA)-based method. Twenty-four hours incubation with SIN-1 (10(-3) mol L(-1)) or SNP (10(-4) mol L(-1) or higher) reduced the number of NADPH diaphorase-positive neurones. SNP incubation did not affect the NSE-positive neuronal content. Shorter incubations (SNP: 4 and 12 h) had no significant effect. The SNP-induced reduction was reversed by glutathione (GSH), but not by NO- or O-scavengers, whereas GSH depletion enhanced the decrease. The NO-dependent guanylate cyclase-blocker 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) did not affect the SNP effect. This reduction can be explained by either specific apoptosis of NOS neurones or downregulation of NOS activity. However, TdT-mediated X-dUTP nick end labelling (TUNEL stainings argue in favour of the latter. In conclusion, the NO donor SNP decreases the number of NOS-expressing myenteric neurones time and concentration dependently, without affecting the amount of neuronal material. Glutathione plays an important protective role.

Induction of apoptosis by all-trans retinoic acid in the human myeloma cell line RPMI 8226 and negative regulation of some of its typical morphological features by dexamethasone.

We investigated the effects of all-trans retinoic acid (RA) and dexamethasone (Dex) on the in vitro growth of the human myeloma cell line RPMI 8226. RA inhibited RPMI 8226 cell growth by both antiproliferative effect and induction of apoptosis. Typical morphological and biochemical characteristics of apoptosis including chromatin condensation, apoptotic bodies formation and internucleosomal DNA cleavage were detected after 4 days of treatment with 1 microM RA. In situ TUNEL assay demonstrated that DNA cleavage preceded chromatin condensation. The expression of tissue transglutaminase (tTG), an enzyme proposed to play a role in apoptosis was induced with RA, as shown by both enzymatic assay and in situ immunofluorescence detection. Dex, when used alone, had no effect on cell growth and apoptosis. When combined to RA, Dex did not interfere with the RA-dependent inhibition of cell proliferation, but unexpectedly inhibited both quantitatively and qualitatively several morphological and biochemical features of the apoptosis induced by RA. Dex did not affect RA-induced DNA breaks formation but impeded the progression of chromatin condensation and the formation of apoptotic bodies. Interestingly, Dex also inhibited the RA-dependent induction of tTG. RU486, a glucocorticoid antagonist, counteracted all Dex effects. Taken together these data demonstrate that key cytoplasmic and nuclear events occurring during apoptosis are differentially regulated by RA and Dex in myeloma cell line RPMI 8226.

Interphase fluorescence in situ hybridization (FISH) as a powerful tool for the detection of aneuploidy in multiple myeloma.

Conventional cytogenetic (CC) studies performed in multiple myeloma (MM) are difficult because of the low proliferation rate of plasma cells (PC). The purpose of this study was to compare results obtained by CC and by FISH for the detection of numeric chromosomal changes in patients with MM. PC DNA content, CC and interphase FISH analysis were performed on 29 consecutive patients with MM. Fifteen patients (control group) had known Cytogenetic abnormalities identified by CC. The other 14 patients (study group) had a normal karyotype but an abnormal DNA content. Bone marrow material prepared for CC or cytospin slides were probed with classical satellite III or alpha satellite DNA sequences for chromosomes 3, 7, 8, 9, 11 and 15 (chromosomes 3, 7, 9, 11, 15 probes for hyperdiploid patients and the chromosome 8 probe for hypodiploid patients). In the control group, an unexplained discrepancy between CC and FISH occurred for only one chromosome in one patient. Also in this group, four patients had only one abnormal cell by CC and the numeric changes in these patients were always confirmed by FISH analysis. In the study group, FISH analysis showed an abnormal result in all but one patient. From these data, we conclude that FISH improves the detection of cytogenetic abnormalities in multiple myeloma. Using commercially available DNA probes for the most frequent numeric changes and slides for CC or cytospin slides, we demonstrated abnormal cytogenetics by FISH in 28/29 patients. In further studies, use of FISH could permit a more accurate description of numeric changes and their prognostic value in MM as well as an approach to clonal evolution. It would also be of interest in the study of monoclonal gammopathies of undetermined significance.

Myelodysplastic syndromes and acute myeloid leukemia with 17p deletion. An entity characterized by specific dysgranulopoïesis and a high incidence of P53 mutations.

We looked for correlations between cytogenetic rearrangements leading to 17p deletion and presence of dysgranulopoïesis and p53 mutations in MDS and AML. Forty-nine (4.3%) of the MDS and AML studied cytogenetically at our institution over a period of 11 years had detectable 17p deletion, through monosomy 17 (14 cases) or rearrangements of chromosome 17 (generally unbalanced translocations between 17p and another chromosome) (35 cases). Most of the patients had additional complex cytogenetic findings, and 10 cases were therapy related. In 70% of the patients with 17p deletion, a particular type of dysgranulopoïesis, combining pseudo-Pelger-Huët anomaly and small vacuolated neutrophils was seen in > 5% marrow neutrophils, whereas 69% of the patients had a p53 mutation, generally in a missense mutation involving exons 5 to 8 of the p53 gene. FISH analysis, performed in eight cases, confirmed loss of one P53 allele in all of them. No DNA fragmentation suggesting increased apoptosis was found in marrow samples. Response to chemotherapy was almost uniformly poor and median survival was only 3 months. Analysis of dysgranulopoïesis and p53 mutations were also made in 'control' groups of MDS and AML without 17p deletion. 'Typical' dysgranulopoïesis, combining pseudo-Pelger-Huët anomaly and small vacuolated neutrophils in > 5% marrow neutrophils, was not seen in any of the 47 MDS and AML without 17p deletion analyzed and without p53 mutation (P = 10(-4) with patients having 17p deletion), and was seen in one of five patients without 17p deletion but with a p53 mutation. Only 3.1% of 256 MDS and AML without 17p deletion had a p53 mutation (P = 10(-4) with patients having 17p deletion). These findings suggest that 17p deletion, in MDS and AML, is strongly correlated to the presence of a particular type of dysgranulopoïesis and to a high incidence of p53 mutations, and that MDS and AML with 17p deletion could constitute a new morphological-cytogenetic-molecular entity in myeloid disorders.

Is t(14;18)(q32;q21) a constant finding in follicular lymphoma? An interphase FISH study on 63 patients.

The translocation (14;18)(q32;q21) is the hallmark of follicular lymphoma (FL). However, conventional cytogenetics and PCR techniques fail to detect it in at least 10% of cases. In order to evaluate the true incidence of this translocation in FL, we analyzed 63 patients with FL, and 17 patients with diffuse large cell lymphoma (DLCL) corresponding to suspected FL transformations using interphase fluorescence in situ hybridization (FISH). Colocalized signals related to the translocation were observed in 19-92% of cells (median = 51%), corresponding to positivity over the threshold in all (63/63) cases. Similarly, 16/17 possibly secondary DLCL displayed the translocation. Although some cytogenetic changes might be missed by this FISH assay (such as rare insertion, or translocations with other chromosomal partners), our results stress t(14;18)(q32;q21) as an almost constant finding in FL. Our sensitive interphase FISH assay should be of great value to define FL more accurately, namely in patients included into therapeutic trials. Furthermore, this approach could be of interest in (re)defining some types of FL, especially the grade 3 FL which frequently lack t(14;18).

Philadelphia negative, BCR-ABL positive adult acute lymphoblastic leukemia (ALL) in 2 of 39 patients with combined cytogenetic and molecular analysis.

We performed cytogenetic and molecular analysis of the BCR-ABL rearrangement by polymerase chain reaction (PCR) in 39 consecutive cases of adult acute lymphoblastic leukemia (ALL). Eleven patients had a Philadelphia (Ph) chromosome. Thirteen patients had a BCR-ABL rearrangement, involving minor breakpoint cluster region (m-bcr, situated in intron 1 of the BCR gene) in 11 cases, and major breakpoint cluster region (M-bcr, 'specific' of chronic myeloid leukemia) in the remaining two cases. All of the 12 BCR-ABL cases studied immunologically were of early B, CALLA-positive immunophenotype. The 13 BCR-ABL positive cases included the 11 Ph-positive cases, and two patients with normal karyotype at diagnosis. In the two Ph-negative BCR-positive cases, seven (patient 1) and 18 (patient 2) mitoses had been examined at diagnosis. In patient 1, Ph negativity at diagnosis could certainly be explained by the small number of mitoses analyzed, as a Ph chromosome was found at relapse. This was less probable in patient 2, who raised the issue of whether authentic Ph-negative BCR-ABL-positive ALL exists (as in the chronic myeloid leukemia model) or not. Whatever the explanation, our results suggest that molecular detection of BCR-ABL should be more widely used in B-lineage ALL.

Involvement of peripheral blood cells in multiple myeloma: chromosome changes are the rule within circulating plasma cells but not within B lymphocytes.

The mononuclear cells in the peripheral blood are implicated in the myeloma process especially with the presence of peripheral blood plasma cells (PBPC) and clonal B lymphocytes found using phenotypic or gene rearrangement techniques. The purpose of this study was to look for aneuploidy in the two main B cell components of the peripheral blood: PBPC and CD20-positive B lymphocytes. Conventional cytogenetics (CC) or DNA content analysis and fluorescence in situ hybridization (FISH) with centromeric probes were performed on bone marrow plasma cells (BMPC) of 21 patients with multiple myeloma and peripheral blood cells were studied as follows: immunostaining to look for PBPC and to assess their number, image analysis cytometry for the determination of their DNA content, and FISH chromosomes analysis. FISH was performed using probes against the chromsomes that were lost or gained in BMPC and was coupled with immunostaining of the relevant light chain or CD20 antigen to study PBPC or B lymphocytes, respectively. Monotypic PBPC were found in 16 patients. Their DNA content was the same or nearly the same as for BMPC and they exhibited the same monosomies or trisomies as those found within their BM counterpart. By contrast, DNA content of mononuclear cells other than PBPC was within normal ranges, and in 13 of 15 patients CD20-positive B lymphocytes failed to show chromosomal changes by FISH analysis. In two patients however, a few CD20+ cells with lymphoid morphology exhibited chromosome changes, hypothesizing that a few cytogenetically abnormal B cells without plasmocytic morphology may circulate. From these data, we conclude that PBPC share the same genetic abnormalities as BMPC and thus belong to the malignant clone, whereas most peripheral blood B lymphocytes are unrelated to the tumor clone.

POEMS syndrome: report on six patients with unusual clinical signs, elevated levels of cytokines, macrophage involvement and chromosomal aberrations of bone marrow plasma cells.

POEMS syndrome is a multisystemic disorder characterized by the association of polyneuropathy, organomegaly, endocrinopathy, M protein, skin changes and various other systemic clinical signs. The pathophysiology of this syndrome remains largely unknown. In order to gain insight into its pathophysiology, we studied the clinical characteristics and performed serum analysis (auto-antibodies, cytokine levels) and phenotypic and cytogenetic studies of bone marrow plasma cells (BMPC) in six patients with unequivocal POEMS syndrome. Two unusual clinical signs were present in these patients: pulmonary hypertension (two patients) and diffuse cutaneous necrosis (one patient). No auto-antibodies against peripheral nerve (PN) antigens (SGPG and SGLPG glycolipids, GM1, GD1a, GD1b and GT1b gangliosides) were found. Sequential evaluations of serum cytokines (IL-1-beta, IL-6 and TNF-alpha) showed a moderate to marked elevations of IL-6 and TNF-alpha in all patients (up to six-fold for TNF-alpha and 16-fold for IL-6). Using in situ hybridization of these cytokines mRNAs on lymph node specimens of two patients who had an angiofollicular lymph node hyperplasia, a strong positivity was found with the IL-1-beta antisense probe in lymph node macrophages. On skin biopsy a high number of cells expressing TNF-alpha mRNA was observed in the dermis. The biological features of BMPC: phenotype (expression of CD19 and CD56 antigens), kinetics (Ki-67 index), karyotype, DNA content and chromosomal in situ hybridization remained those of BMPC found in monoclonal gammopathy of undetermined significance. We conclude that POEMS syndrome is a hypercytokinemic syndrome in which BMPC are not of malignant type. Macrophages are involved in this syndrome and their role has to be further investigated as well as treatments which act through an anti-cytokine mechanism.

Evaluation of minimal residual disease by interphase FISH in multiple myeloma: does complete remission exist?

As in other hematological malignancies, the achievement of a complete remission (CR) is important in multiple myeloma but is still based on common cytological and electrophoretic criteria. In this report, we studied 14 patients who achieved an apparent CR following high-dose therapy using fluorescence in situ hybridization (FISH) analysis. Although the results were difficult to interpret in two patients, 12 of 14 patients had unequivocal persistence of abnormal plasma cells in their bone marrow. Our results suggest that only a few patients, if any, are in true CR following one course of high-dose therapy and are in favor of post-transplantation treatments.

[Refractory anaemia with ringed sideroblasts (RARS) associated with marked thrombocytosis: a provisional entity in the WHO classification of haematological malignancies]. / Thrombocytose majeure et anémie réfractaire avec sidéroblastes en couronne: une entité provisoire dans la classification OMS des hémopathies.

The WHO classification describes a group of myelodysplastic/myeloproliferative diseases, including a provisional entity, refractory anaemia with ringed sideroblasts (RARS) associated with marked thrombocytosis, underlining that is a provisional entity without consensus of belonging to myelodysplastic rather than to myeloproliferative syndromes. The authors report two cases with features of refractory anaemia with excess of ringed sideroblasts and marked thrombocytosis. In the first case, RARS is concomitant with thrombocytosis and fits the WHO criteria for this temporary entity. The second case is a typical RARS, who developed a thrombocytosis after several years and emphasizes that a link, at least progressive, exists between RARS and myeloproliferative disorders. The authors summed up the various situations related to secondary or primary acquired sideroblastic anaemia, likewise to primitive and reactive thrombocytosis. The cases of RARS + marked thrombocytosis reported in the literature are few in number and do not allow to settle between a particular form of myelodysplastic syndrome and a myeloproliferative disorder, a fully justified reason to classify these patients in a temporary group. To date, there is no codified therapy for this disorders.

[Idiopathic hypereosinophilic syndrome: toward a new molecular-targeted therapy and a new cytomorphological and molecular definition]. / Hyperéosinophilie essentielle: nouvelle thérapeutique ciblée et nouvelle définition cytologique et moléculaire.

Idiopathic hypereosinophilic syndrome is characterised by chronic hypereosinophilia leading to tissue damage, and after exclusion of reactive eosinophilia. Until recently no specific or efficient therapeutic was available. In 2003, a recurrent interstitial deletion 4q12 leading to the fusion of the FIP1L1 and PDGFRA genes was detected in hypereosinophilic syndromes. The resulting protein has constitutive tyrosine kinase activity which explains clinical and cytological remission of hypereosinophilic syndrome after treatment by a specific tyrosine kinase inhibitor, imatinib mesylate or Glivec, usually used in chronic myeloid leukaemia. Here we report a patient with hypereosinophilic syndrome associated to peculiar morphology of neutrophilic series and the 4q12 deletion. He presented clinical and haematological remission since the introduction of imatinib mesylate therapy.

Mature plasma cells as indicator of better prognosis in multiple myeloma. New methodology for the assessment of plasma cell morphology.

The relationship between plasmablastic cells and outcome in multiple myeloma (MM) has been established for nearly 15 years. But the assessment of these cells is not easy to perform and it allows the identification of only a small proportion of patients. We investigated the plasma cell morphology using a progressive evaluation of consecutive criteria: nucleolus, chromatin and nuclear-cellular ratio (N/C). The combination of these three items produces a subclassification where four cellular subtypes identify 93% of the plasma cells, and these subtypes are related to the outcome. The interest of this methodology is to be based on the mature plasma cells that are easier to identify than the plasmablastic cells. These new cell subtypes introduce a new classification for patients: Group 1 includes patients with at least 66% mature plasma cells (P000). Both Group 2 and 3 have less than 66% P000 and are separated by their degree of maturation (Proplasma I > or = Proplasma II + plasmablastic). The distinction of these three groups of patients is highly related to the prognosis (P < 10(-4)). These results have been confirmed on a second group of patients coming from a different institution. In conclusion, we propose a new methodology for the plasma cell evaluation in MM, that is based on the morphological criteria and that has the advantage of identifying an intermediate (30%) subgroup of patients with a prognostic significance.

Increased apoptosis in mononucleated cells but not in CD34+ cells in blastic forms of myelodysplastic syndromes.

INTRODUCTION: Myelodysplastic syndromes are characterized by peripheral refractory cytopenias together with normo or hyper cellular marrow. Increased apoptosis has been shown to be involved in the process leading to this paradox. MATERIALS AND METHODS: Early apoptosis detection, based on the modification of mitochondrial transmembrane potential (deltapsim), was performed on bone marrow cells from 42 MDS patients (21 RA, four RARS, 10 RAEB, two RAEB-t, three sAML, and two CMML) and seven normal healthy donors. Phosphatidylserine (PS) expression, a late/intermediate marker of the apoptotic cascade, was also quantified. Apoptosis was analysed by flow cytometry both on unsorted mononuclear cells and on progenitor cells after CD34+ magnetic cell sorting. RESULTS: A significant increase of apoptosis of MNC was observed in RA, RARS, RAEB and to a lower extent in RAEB-t and AML samples. In the progenitor compartment, RA and RARS samples presented a high level of apoptosis, whereas a switch to a low level of apoptosis was detected in the blastic forms RAEB, RAEB-t and sAML. Fas (CD95/APO-1), a member of the death domain receptor family, has been reported to be overexpressed on MDS CD34+ marrow cells. A functional assay of Fas cross-linking using the CH11 antibody on CD34+ marrow cells was performed on samples of 17 MDS patients; 8/17 were found to be sensitive to Fas-induced apoptosis. However, no correlation was observed with the level of in vivo spontaneous apoptosis. CONCLUSION: This study demonstrates increased apoptosis of MNC in all MDS subgroups as measured by deltapsim collapse. Moreover, while important apoptosis is still observed at the progenitor level in early MDS, blastic forms show a clear reduction of apoptosis. Study of Fas functionality modulates the implication of this receptor in the pathophysiology of the disease.

Comparison of four serum-free, cytokine-free media for analysis of endogenous erythroid colony growth in polycythemia vera and essential thrombocythemia.

INTRODUCTION: The assay of endogenous erythroid colony formation (EEC), a characteristic of polycythemia vera and essential thrombocythemia, is not standardized. In this multicentric study, we tested four semisolid, serum-free, cytokine-free media based on either methylcellulose (M1, M2) or collagen (C1, C2) commercialized for the EEC assay. MATERIALS AND METHODS: Bone marrow mononuclear cells (BMMC) from 73 individuals (62 patients with either polycythemia vera (26), essential thrombocythemia (19), secondary polyglobuly (17) or chronic myeloid leukemia (2) and 11 healthy donors) were grown in parallel in the four media without, or with 0.01 U/ml erythropoietin (EPo). RESULTS: In all four media EEC formation was specific, as it was not observed in cultures of patients with secondary polyglobuly or chronic myeloid leukemia, nor of healthy donors. Analysis of fresh or MGG-stained collagen gel cultures allowed detection of EEC formation significantly more frequently than methylcellulose-based media; addition of 0.01 U/ml of EPo had little or no effect on EEC formation. Collagen-based medium C1 gave better results than the other media tested: the 'C1' EEC assay was positive for 68.2% of polycythemia vera cultures with significantly higher median EEC numbers (6.5/10(5) BMMC for patients with one major criteria of polycythemia vera and 19 and 21/10(5) BMMC for patients with two or three major criteria, respectively). Medium C1 was also better for essential thrombocythemia cultures with 47.4% of positive results but with a low median EEC number (6.7/10(5) BMMC). When associated with the ELISA dosage of serum EPo, the 'C1' EEC assay allowed confirmation or elimination of the diagnosis of polycythemia vera for 91% (20/22) of polyglobulic patients. CONCLUSION: We propose that serum-free collagen-based culture systems be considered to standardize the EEC assay, now part of the new criteria of polycythemia vera.