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.

Use of Sysmex XE-2100 and XE-5000 hematology analyzers for the diagnosis of malaria in a nonendemic country (France).

INTRODUCTION: Most studies dealing with automated hematology analyzers (HAs) and malaria diagnosis are conducted in endemic countries. METHODS: We retrospectively studied cell blood counts (CBCs) performed with Sysmex XE-2100 and XE-5000 HAs in our center (Angers, France) regarding 67 patients returning from endemic areas and infected with various Plasmodium species. RESULTS: In 83% of infected samples with Plasmodium vivax (Pv), ovale (Po), or malariae (Pm), extra clouds of dots were present in neutrophil and/or eosinophil area(s) on routine differential (DIFF) scattergrams. In contrast, samples infected with Plasmodium falciparum (Pf) failed to show such DIFF scattergrams, or any other suggesting malaria infection (0/ 49 pts). Abnormal areas from DIFF scattergrams were related to the presence of mature schizonts and gametocytes, undestroyed by lysis agent, the latter not observed in Pf-infected patients from our series. The internal parameter WBC[DIFF] - WBC[BASO] raised in parallel to parasitemia in Pv, Po, and Pm samples but could not be used as a surrogate for parasitemia. In Pf infection, reticulocyte/ immature reticulocyte fraction (IRF) ratio showed a significant correlation with parasitemia (P < 0.05). A diagnostic model developed for Pf in endemic countries showed sensitivity of 77%. CONCLUSION: Using SYSMEX analyzers, Pv, Po, and Pm infections are easy to ascertain as DIFF scattergrams are almost specific (specificity = 99.9%). Pf infection diagnosis by CBC may be a more promising tool.

[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.

Characterization of myenteric neuropathy in the jejunum of spontaneously diabetic BB-rats.

Decreased gastric expression and function of neuronal nitric oxide synthase (nNOS) has been proposed as a potential mechanism underlying diabetic gastroparesis. As gastric nNOS expression is vagally controlled, these changes might occur secondarily to vagal neuropathy. In addition, it is unclear whether other inhibitory neurotransmitters are also involved. We used the type 1 diabetic BioBreeding (BB)-rat model to study jejunal motor control and nNOS expression, which is independent of the vagus. Jejunal segments were used for in vitro contractility studies, and measurement of nNOS expression after 8 or 16 weeks of diabetes compared with age- and sex-matched controls. Unlike electrical field stimulation and acetylcholine (ACh)-induced contractions, non-adrenergic non-cholinergic (NANC) relaxations were significantly reduced in diabetic rats. In contrast to control rats, NANC relaxations in diabetic rats were N(omega)-nitro-L-arginine methyl ester (L-NAME) insensitive. Jejunal nNOS expression was significantly decreased in diabetic rats. Both in diabetic and in control animals, L-NAME resistant relaxations were sensitive to P(2)-receptor antagonists. In the jejunum of spontaneously diabetic rats, decreased nitric oxide responsiveness and decreased nNOS protein expression occur while purinergic transmission is unaffected. These findings indicate that nitrergic enteric neuropathy may be a primary dysfunction in diabetes, independent from vagal dysfunction.

[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.

[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.

Spurious counts and spurious results on haematology analysers: a review. Part I: platelets.

The widespread use of haematology analysers (HA) has led to a major improvement of cellular haematology, because of quick and accurate results found in most instances. However, in several situations, spurious results are observed. Inadequate blood samples, situations induced by the anticoagulant(s) used, peculiar changes related to the pathology in the patient, and technical considerations about performances of the various HA must be considered. Spurious thrombocytopenia occurs in several circumstances related to the presence of ethylenediamine tetra-acetic acid (EDTA) used as the anticoagulant. Mechanism of EDTA-dependent platelet (PLT) agglutination is related to circulating (auto)antibodies directed against normally hidden epitope(s) in the glycoprotein alpha IIb/beta IIIa complex from PLT membrane exposed only in the presence of EDTA. Other spuriously low PLT counts may be related to EDTA, including PLT rosetting around white blood cells (WBC; satellitism) and PLT-WBC aggregates, but mechanisms responsible for those latter phenomena are less well known. Spurious increase of PLT count may be related to several situations, including fragmented red blood cells, cytoplasmic fragments of nucleated cells, cryoglobulins, bacteria or fungi, and lipids. Flags generated in several of these situations alert the operator on possible abnormal findings and may identify the problem. Analysing only PLT parameters is not sufficient: in many situations the WBC differential scattergram is of crucial help for flagging. Flags generated depend on the software version on the HA used, the performance in detecting the same anomalies may differ according to which analyser is used, even those from the same manufacturer. Operators must be aware of the characteristics of their analyser and be able to recognize and circumvent anomalous results.

Spurious counts and spurious results on haematology analysers: a review. Part II: white blood cells, red blood cells, haemoglobin, red cell indices and reticulocytes.

Haematology analysers provide quick and accurate results in most situations. However, spurious results, related either to platelets (part I of this report) or to other parameters from the cell blood count (CBC) may be observed in several instances. Spuriously low white blood cell (WBC) counts may be observed because of agglutination in the presence of ethylenediamine tetra-acetic acid (EDTA). Cryoglobulins, lipids, insufficiently lysed red blood cells (RBC), erythroblasts and platelet aggregates are common situations increasing WBC counts. In most of these instances flagging and/or an abnormal WBC differential scattergram will alert the operator. Several situations lead to abnormal haemoglobin measurement or to abnormal RBC count, including lipids, agglutinins, cryoglobulins and elevated WBC counts. Mean (red) cell volume (MCV) may be also subject to spurious determination, because of agglutinins, excess of glucose or salts and technological considerations. In turn, abnormality related to one measured parameter will lead to abnormal calculated RBC indices: mean cell haemoglobin content (MCHC) is certainly the most important RBC indices to consider, as it is as important as flags generated by the haematology analysers (HA) in alerting the user to a spurious result. In many circumstances, several of the measured parameters from CBC may be altered, and the discovery of a spurious change on one parameter frequently means that the validity of other parameters should be considered. Sensitive flags now allow the identification of several spurious counts, but only the most sophisticated HA have optimal flagging and more simple HA, especially those without a WBC differential scattergram, do not possess the same sensitivity for detecting anomalous results. Reticulocytes are integrated now into the CBC in many HA, and several situations may lead to abnormal counts.

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.

[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.

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).

Is there any significance for intracellular crystals in plasma cells from patients with monoclonal gammopathies?

Several plasma cells morphological changes have been described in monoclonal gammopathies, including intracytoplasmic crystals. We report one case of indolent kappa-chain multiple myeloma with renal insufficiency, featuring plasma cells with Auer-rod-like intracytoplasmic inclusions. The relationship between such aberrations and those found in multiple-myeloma-associated adult Fanconi syndrome is discussed. The significance of intracellular storage and crystallisation of immunoglobulin within plasma cells remains partially unknown.

[Chronic lymphoid hemopathies in adults: chronic lymphocytic leukaemia and the phase of dissemination of small cell lymphomas]. / Les héopathies lymphoïdes chroniques de l'adulte: la leucémie lymphoïde chronique et le phase de dissémination des lymphomes à petites cellules.

Chronic lymphocytic leukaemia is the most frequent haematological cancer in adult patients, and its incidence raises with aging. Diagnosis needs several clinical and biological data, but hemogram together with the morphological and immunophenotypic analysis of the lymphoid cells take the major place. If the diagnosis is performed easily in about 65% of the patients, various clinicobiological entities were reported in the past few years that must be identified, at least because some are of adverse prognosis. Moreover, the other chronic lymphoid neoplasms, corresponding to the various low and intermediate grade non-Hodgkin's lymphomas (mainly of follicular type, marginal zone, mantle cell zone), may disseminate within the blood and the bone marrow. Those circulating lymphoma cells must be identified at diagnosis in order to perform the accurate diagnosis and to avoid an erroneous diagnosis of atypical chronic lymphocytic leukaemia. Up to 90% of lymphoid malignancies are B cell disorders, contrasting with only a few cases of T cell origin: some of those latter cases cannot be neglected however, as they may be observed in Western countries. Most recent classifications (REAL and WHO) defined all hematological malignancies: each entity referred to clinical, morphological, immunological, cytogenetic, and molecular findings. The basis of these classifications is pathophysiological, trying in each disorder to define a normal counterpart to the pathological clone. Reviewing main steps of the immune response in the normal patient, corresponding to those involving B cells, it is possible indeed to localize and demonstrate a function for many of the cells that expand in lymphoid malignancies.

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.