Interference of blast cell fragments with automated platelet counting.

INTRODUCTION: Automated haematology analysers may inaccurately determine platelet counts in several circumstances. Spuriously elevated automated platelet counts have been reported in some acute leukaemia (AL) cases because of fragmentation of circulating blast cells (pseudoplatelets). Haemorrhagic diathesis is a common manifestation of AL, which is often caused by severe thrombocytopenia. Therefore, overestimation of the actual platelet count in patients with AL can affect its clinical management. We aimed to detect the frequency of pseudoplatelets in patients with AL. METHODS: Complete blood cell counts were performed on 86 AL patients with three automated analysers (ADVIA 2120, Coulter LH 750 and Sysmex XE-2100D). Platelet counts were also performed by quantitative flow cytometry (QFC). The platelet counts of the automatic analysers were compared to the platelet counts by QFC. Blood smears were checked for the presence of pseudoplatelets. RESULTS: The automated analysers overestimated the platelet count due to the presence of pseudoplatelets in patients with AL. Pseudoplatelets were observed in the blood smears of 11 patients (13%). Three of these patients were near the prophylactic platelet transfusion threshold. CONCLUSION: Spurious increases in automated platelet counts by blast cell fragments are little known but frequent artefacts that should be ruled out by careful examination of peripheral blood smears.

Combination of CD160 and CD200 as a useful tool for differential diagnosis between chronic lymphocytic leukemia and other mature B-cell neoplasms.

INTRODUCTION: Chronic lymphocytic leukemia is usually diagnosed through the characteristic morphology/immunophenotype of the lymphocytes, but some CLL cases remain atypical resulting in diagnostic uncertainty. METHODS: Using flow cytometry analysis, we investigated the expression of CDs160/200 on B cells from 124 patients (82 CLL, 42 other B-cell neoplasms) and nine controls. CDs160/200 measurements were determined as a ratio of the mean fluorescence intensities of leukemic cells/controls and were considered positive when the ratios were ≥2 and 20, respectively. RESULTS: Sixty and 83% CLL expressed CDs160/200 as compared to 5% and 10% of other B-cell neoplasms, respectively. None of the controls showed CDs160/200 expressions. Combination of both markers was observed in 55% of CLL but only in 2% of other B-cell neoplasms, and absence of both markers occurred in 12% of CLL but in 86% of other B-cell neoplasms. CONCLUSION: CDs160/200 were associated with markers of the gold standard 'Matutes score' and could be useful markers to differentiate atypical CLL from other B-cell neoplasms in the absence of available biopsies or cytogenetics and molecular studies.

Mean platelet volume: comparison of three analysers towards standardization of platelet morphological phenotype.

INTRODUCTION: The aim of this study was to show variability in the measurement of the mean platelet volume (MPV) depending on the instrument used. METHODS: This prospective analysis was carried out to measure MPV with three instruments, in 30 healthy controls and 113 hospital patients. RESULTS: Firstly, for values in the normal range, the values obtained with the Siemens Advia(®) 2120 are lower than those given by the Beckman Coulter LH750(®) (-0.89), which are in turn lower than those obtained with the Sysmex XE-2100D(®) (-1.11), which represents a 20-25% variation in the measurement. These results emphasize the lack of universal external calibration for MPV analysis and thus make any intercentre comparison of MPV impossible unless the automated haematology analyser used is indicated. Secondly, we stress the differences in behaviour of the instruments in the presence of abnormally large platelets, i.e. an underestimate of the platelet count and the MPV may be provided because instruments using impedance technology may fail to take into account these platelets, but they rightly flag them. CONCLUSION: To harmonize our procedures, we propose definitions of platelet size (normal size, macroplatelets and giant platelets) based on the coordinated interpretation of the MPV, the distribution of platelet volume and the morphological appearance.

Chimerism analysis following nonmyeloablative stem cell transplantation using a new cell subset separation method: Robosep.

Chimerism analysis has become an important tool to manage patients in the peri-transplant period of allogenic stem cell transplantation. During this period, cells of donor and host origin can coexist and increasing proportion of cells of host origin is considered as a recurrence of the underlying disease. We currently performed chimerism analysis on separate peripheral blood cell subsets, lymphocytes and granulocytes. To improve our isolation method, a new automated device from Stem Cell Technology Roboseptrade mark was tested and compared to our manual separation technique. The results obtained on T cell purification showed an improvement of the purity (98.42% with Robosep vs. 92.42% with the manual technique Rosettesep) and of the recovery (63.43% with Robosep and 38% with Rosettesep). The results were significantly improved on patient samples with less than 10% CD3 positive cells (purity: 90% vs. 44.44%; recovery: 73.79% vs. 43.98%). Granulocytes separation was based on CD15 expression. The results showed an improvement of the purity with Robosep (96.90% vs. 86.20% with the manual technique Polymorphprep) but the recovery was impaired (35.2% vs. 52.30%). Using a myeloid (CD66/CD33) cocktail, recovery was improved with the Robosep device (64.04% with the myeloid cocktail vs. 22.4% with the CD15 cocktail). Our data demonstrated that Robosep allowed a performant cell purification in the early period post-transplantation even for populations representing less than 10% of the peripheral blood cells.

[Persistent thrombocytopenia in a child: morphological examination of blood platelets established the diagnosis of Wiskott-Aldrich syndrome]. / Thrombopénie persistante chez un enfant: l'examen morphologique des plaquettes a conduit au diagnostic de syndrome de Wiskott-Aldrich.

Thrombocytopenia frequently occurs in laboratory practice. The present work illustrates, through the presentation of a case report of Wiskott-Aldrich syndrome, the difficulties encountered to identify and characterize thrombocytopenia. The clinicobiological validation of a low platelet count involves both the biologist, who must assume the validation of numeration while mentioning the morphological characteristics of the platelets and other blood cells, as well as the physician who has to interpret these data according to the clinical context.

Unusual direct phycoerythrin labeling of B-cells from a splenic marginal zone lymphoma.

Flow cytometry is the most widely used method for lymphocyte subset characterization. Two types of antibodies, directly labeled with fluorochrome, are currently used for immunological diagnosis of B-cell lymphoproliferation: monoclonal antibodies against leukocyte differentiation antigens and polyclonal antibodies against immunoglobulins and light chains. In this study is described the case of a patient with an uncommon immunophenotyping of a B-cell lymphoproliferative disorder. B-cells from peripheral blood and from bone marrow reacted positively with all the tested phycoerythrin (PE)-conjugated antibodies, including the isotypic control. So we thought about a B-cell proliferation carrying a surface receptor recognizing PE: these B-cells were directly labeled with streptavidin-PE, indeed. Moreover, the immunodots from the patient were able to fix the streptavidin-PE. Finally, this unusual immunophenotyping was solved by using antibodies labeled with other fluorochromes than PE.

[A marginal zone-B cell lymphoma revealed by platelet satellitism and lympho-agglutination phenomenon around atypical lymphocytes]. / Satellitisme plaquettaire et lympho-agglutination exclusivement aux lymphocytes atypiques révélant un lymphome B de la zone marginale.

A 48-year-old man, with persistent pyrexia, presented with thrombocytopenia and lymphocytosis. The peripheral blood smears showed atypical lymphocytes and a platelet satellitism phenomenon around atypical lymphocytes associated to lympho-agglutination. Platelet satellitism was exclusively observed with atypical lymphocytes in EDTA-treated blood and at room temperature. This phenomenon was not observed when adding normal plasma and could be reproduced several times. Flow cytometry analysis of the peripheral blood, cytological and histological studies revealed a marginal zone-B cell lymphoma. The mechanism underlying platelet satellitism is not fully understood, but is likely to involve an immunologic binding of EDTA-dependent antiplatelet autoantibodies directed against the platelets glycoprotein IIb/IIIa complex. The association between platelet satellitism and lymphoma could also involve a monoclonal Ig secreted by lymphoma cells.

[Incubated osmotic fragility test does not exclude red blood cell membrane disorders! About a case of hereditary elliptocytosis]. / Une résistance globulaire osmotique normale n'exclut pas une membranopathie érythrocytaire ! A propos d'un cas d'elliptocytose héréditaire.

We report a case of hereditary elliptocytosis in an infant diagnosed a few months after the birth, in a context of regenerative normocytic normochromic anaemia. The investigations, including incubated osmotic fragility, erythrocytic enzymes study and haemoglobin electrophoresis, were not contributive. Only the persistence of elongated (or cigar-shaped) erythrocytes on blood smears was noted. Hereditary elliptocytosis was confirmed by specialized investigations (rheological study and erythrocytic membrane proteins electrophoresis). Investigations in the mother were realized and led to the discovery of a similar biological pattern. Hereditary elliptocytosis is a red blood cell membrane disorder due to the defect in cytoskeleton proteins (spectrin or 4.1), leading to the loss of deformability properties of erythrocytes. This disorder is considered as rare; however, its incidence is probably underestimated because most cases are pauci- or asymptomatic and the discovery is often fortuitous. The absence of detection of this defect by incubated osmotic fragility should not discard the hypothesis of erythrocytes membrane disorders. The persistent observation of elongated erythrocytes on blood smear must encourage the biologist to evocate a hereditary elliptocytosis.

Human umbilical vein endothelial cells increase ex vivo expansion of human CD34(+) PBPC through IL-6 secretion.

BACKGROUND: Ex vivo expansion of hematopoietic stem cells (HSC) can help reduce cytopenia following transplantation, especially in NHL patients whose BM is deficient because of extensive chemotherapy. We have previously reported that human umbilical vein endothelial cells (HUVEC) can contribute to improved PBPC expansion when used in co-culture with CD34(+) cells. METHODS: We evaluated the roles of direct HUVEC CD34(+) contact and HUVEC-produced soluble factors. We cultured CD34(+) PBPC harvested from NHL patients in four different conditions: (1) liquid culture without HUVEC; (2) co-culture in contact with HUVEC; (3) co-culture with HUVEC but without direct contact; (4) liquid culture with HUVEC-conditioned medium (CM). Thrombopoietin (Tpo), Flk2Flt3 ligand (FL) and c-kit ligand (KL) with or without rhIL-6 were added to these four culture conditions. RESULTS AND DISCUSSION: Our results showed that HUVEC co-culture or addition of HUVEC-CM to Tpo, FL and KL (TFK) improved CD34(+) PBPC expansion compared with liquid culture, as determined by total viable nucleated cells (TNC), colony-forming cell assay (CFC) and week-6 cobblestone area-forming cells (Wk-6 CAFC) expansions. Non-contact culture led to similar PBPC expansion as contact co-culture; moreover, HUVEC-CM improved PBPC expansion. However, when rhIL-6 was added to HUVEC-CM with TFK, no significant difference was observed. Finally, high quantities of IL-6 were detected in HUVEC-CM and addition of anti-IL-6 Ab inhibited the positive effect of HUVEC on PBPC expansion. Our results thus suggest that HUVEC may improve PBPC expansion, at least through IL-6 secretion.

[A case of de novo acute basophilic leukaemia: diagnostic criteria and review of the literature]. / A propos d'un cas de leucémie à basophiles de novo: critères diagnostiques et revue de la littérature.

We report a case of a de novo acute basophilic leukaemia, revealed by an infectious pneumopathy in a 73 year old man. The full blood count revealed an hyperleucocytosis associated with an unregenerative normocytic normochrom anaemia and a thrombocytopenia. The blood and bone marrow smears showed a mixture of undifferentiated blast cells and basophiloblasts (high nucleo-cytoplasmic ratio, coarse basophilic cytoplasmic granules), along with basophilic precursors and basophilic polymorphonuclears. All the blasts were MPO negative but positive for the toluidine blue metachromatic coloration, which is considered as consistent with basophilic lineage. Immunophenotypic studies showed myeloid blasts, without maturity marker, CD 117 negative and CD203 cytoplasmic positive, the latter known to be highly representative of the basophilic lineage. This very clear-cut phenotype, associated with the morphology of cells, were arguments to ascertain the basophilic lineage of the blasts without the need of electron microscopic study. Cytogenetic and RNA analysis revealed the presence of a Philadelphia chromosome and of a BCR-ABL transcript with the unusual junction e6a2. Thus, imatinib was added to the conventional chimiotherapy and the patient is currently in complete remission. This clinical prompted allows us to review the literature on acute basophilic leukaemia and to state on the different diagnostic criteria of this rare disorder.

[Thrombocytopenia: clinicobiologic validation and classification]. / Validation et classification clinicobiologique d'une thrombopénie.

Thrombocytopenia occurs frequently. We will illustrate, through the presentation of a clinical case, the difficulties encountered to identify and characterize thrombocytopenia. The clinicobiological validation of a low platelet count implies, at the same time, the biologist, who must assume the validation of numeration while mentioning the morphological characteristics of the platelets and other blood cells, as well as the clinician who must interpret these data according to the clinical context. Firstly, we will detail the basic rules to correctly ensure this validation. Secondly, we will see which are the arguments which that make it possible to direct the diagnosis towards an acquired or inherited thrombocytopenia. Lastly, we will approach the classification of inherited thrombocytopenias.

Osteopetrotic mouse stroma with thrombopoietin, c-kit ligand, and flk-2 ligand supports long-term mobilized CD34+ hematopoiesis in vitro.

OP-9 cells are stromal cells derived from macrophage colony-stimulating factor (M-CSF)-deficient osteopetrotic mice. To evaluate the OP-9 capability to sustain long-term hematopoiesis, we reported the expansion of granulocyte colony-stimulating factor (G-CSF)-mobilized human peripheral blood (PB) CD34(+) cells in co-culture with murine OP-9 and MS-5 stromal cells, either transfected with various combinations of adenovectors (Ad) expressing c-kit ligand (KL) (either soluble or transmembrane form), thrombopoietin (TPO), flt-3/flk2 ligand (FL), and granulocyte-macrophage (GM)-CSF or with weekly addition of these cytokines. Expression of TPO as well as association of TPO, FL, and KL increased progenitor cell and week-6 cobblestone area forming cell (CAFC) production in all stromal co-cultures. Similar progenitor expansion was obtained by weekly addition of soluble cytokine. Five weeks of co-culture with OP9 and TPO, FL + KL resulted in the greatest expansion of progenitor cells and week-6 CAFC as measured by secondary assay on MS-5. In contrast to MS-5 and TPO or TPO + FL + KL cultures where hematopoiesis declined by week 4, progenitor as well as week-6 CAFC expansion continued for over 3 months in TPO + FL + KL OP9 cocultures. This was associated with decrease of CD14(+) macrophage production. The addition of human macrophage (M)-CSF or CD14(+) cells to the co-culture decrease progenitor and stem cell expansion; however, murine M-CSF to OP-9 co-cultures did not decrease progenitor expansion. High levels of stromal-derived factor-1 (SDF-1) production by MS-5 and low or absent production by OP-9 may account for stem cell adhesion and CAFC formation in the former cultures and the predominance of stem and progenitor cells in the nonadherent fraction in the latter cultures.

Pseudo-storage pool disease due to platelet degranulation in EDTA-collected peripheral blood: a rare artifact.

We report a patient who was referred for acute myocardial infarction and presented a pseudo-storage pool disease. The platelets from the blood collected with ethylene diamine tetra-acetic acid (EDTA) were moderately underestimated by the automated analyser, stained poorly on the blood smear and appeared agranular under the microscope. This artifactual anomaly does not occur in samples anticoagulated with citrate, heparin or the mixture citrate theophyline adenine dipyridamole. Reports of EDTA-induced pseudo-storage pool disease are scarce, possibly because underestimation of this poorly explained and difficult to diagnose phenomenon.

[Labour epidural analgesia for a woman with hereditary macrothrombocytopenia]. / Analgésie péridurale obstétricale chez une patiente présentant une macrothrombocytopénie familiale.

Epidural analgesia is often considered as risk of epidural haematoma in a patient with thrombocytopenia. In this observation, uncomplicated epidural analgesia was performed in a pregnant woman with hereditary macrothrombocytopenia. She received continuous epidural labour analgesia for a vaginal delivery with a platelet count at 63x10(9)/l but platelets with high mean platelet volume (20fL) and normal function. No neurological sequelae or excessive bleeding occurred.

[Childhood essential polycythemia: an unusual disorder]. / Polyglobulie primitive de l'enfant: un diagnostic rare.

We report the case of an 2-year-old boy presenting an essential polycythemia since birth, with details of the diagnostic procedures used and clinical course. Pediatric cases are very rare, and a secondary acquired polycythemia should be first investigated. Most causes of primary childhood polycythemia remains unknown. Erythropoietin (EPO) level may help to separate diseases with high EPO (Chuvash, or yet unclassified), or with normal/low EPO (congenital with truncation of the EPO receptor, polycythemia vera-Vaquez disease-, or currently with unknown mechanism).

A standardized procedure for quantitation of CD11b on polymorphonuclear neutrophil by flow cytometry: potential application in infectious diseases.

An up-regulation of the surface marker CD11b has been demonstrated during polymorphonuclear (PMN) cell activation. CD11b over-expression is often associated with inflammation and is considered as an early marker of infection. However, the absence of standardized assay and the variability of preanalytical settings leading to PMN artifactual activation have compromised the interest of this marker. In the present study a standardized quantitative flow cytometry assay directly performed in whole blood has been used to determine CD11b expression on PMN cells. The results indicate that quantitative flow cytometry can provide consistent CD11b density values between laboratories provided that a calibration system is used including specific calibrators, reagents and protocols. This method allowed us to evidence an up-regulation of CD11b expression for infected patients. This quantitation is a standardized and potentially useful method in clinical situations implying quantitative CD11b expression variations.

Complete recovery from Cryptosporidium parvum infection with gastroenteritis and sclerosing cholangitis after successful bone marrow transplantation in two brothers with X-linked hyper-IgM syndrome.

We describe two brothers who suffered from hyper-IgM syndrome (HIGM1) with similar clinical features: recurrent infections, especially cryptosporidium gastroenteritis with cholangitis. Their activated T cells did not express CD40L. Nucleotide sequencing revealed a mutation in both boys with respect to intron 4 and exon 5 boundaries of the CD40L gene in Xq26. They underwent successful bone marrow transplantation (BMT) from HLA-geno-identical siblings. The Cryptosporidium infection and cholangitis resolved thereafter. At 6 months after BMT, expression of CD40L on activated T lymphocytes was normal. After 1 year, both boys are well, and immune reconstitution has improved. Based on these two successful experiences, BMT with a genoidentical sibling seems a reasonable therapeutic approach for HIGM1, if Cryptosporidium infection occurs.

CD34+ cells homing: quantitative expression of adhesion molecules and adhesion of CD34+ cells to endothelial cells exposed to shear stress.

To investigate the function of the main adhesion receptors (CD62L, CD49d, CD49e, CD11b and CD18) on CD34+ cells during homing, their expression was quantified by flow cytometry using calibration beads. CD34+ cells were isolated from bone-marrow (BM), cord blood (CB) or peripheral blood (PB) from patients with myeloma. As this process might mimic the mature leukocyte migration, we also observed the effect of exposing endothelial cells to shear stress (7 dyn/cm(2)) on the adhesion of CB CD34+ cells. The proportion of CD34+/CD62L+ cells was greater in PB than in BM (p<0.05). Likewise, we found a significantly greater expression of CD62L receptor on PB cells compared to BM cells (p<0.05) and on BM cells compared to CB cells (p<0.05). The proportions of CD34+/CD49d+ cells and CD34+/CD49e+ cells were significantly higher in the BM and CB than in PB. However, no significant difference in CD49d or CD49e antigen densities was observed. The beta_2 integrins (CD11b and CD18) receptors are also implicated in CD34+ cells homing to BM. No significant variation in CD34+/CD11b+ and CD34+/CD18+ cells frequency was noted. However quantitative analysis revealed that CD18 was more strongly expressed on BM cells than on PB and CB cells. The adhesion assay showed that fluid flow may favour a firm adhesion of CB CD34+ cells to endothelial cells whereas static conditions just allowed CD34+ cells sedimentation. In conclusion, quantitative expression of the main receptors on CD34+ cells indicates that the three main sources of CD34+ cells currently used for transplantation have neither the same phenotype nor the same number of antigenic sites for a receptor. So, we hypothesize that migrational capacity of these cells might be different. Moreover, it seems that shear stress could favor adhesion of CD34+ cells to endothelial cells.