Macropolycytes: Severe stress time for neutrophils.

Macropolycytes are giant neutrophils found in a variety of benign and neoplastic conditions. Since megaloblastic anaemia is one of the recognised causes of macropolycytes, other blood film features of megaloblastic anaemia should be sought when they harbor hypersegmented nuclei. When they are hypolobulated and hypogranular, the occurrence of a myelodysplastic syndrome must be investigated. Finding macropolycytes in the context of a nonspecific reactive granulopoiesis is more questionable but is often associated with stressed myelopoiesis and/or granulocyte colony-stimulating factor therapy.

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.

Schistocytes in disseminated intravascular coagulation.

INTRODUCTION: The presence of schistocytes on the peripheral blood film during disseminated intravascular coagulation (DIC) remains controversial. METHODS: We examined schistocytes count on blood films from 35 DIC patients and checked morphological anomalies of all RBCs. RESULTS: Thirty of 35 patients presented with schistocytes and 22 with acanthocytes, which was the commonest shape anomaly. Mean percentage ± standard deviation was 0.33 ± 0.38%, median value was 0.1%, and range was 0-1.4%. The patients with schistocytes ≥ 1% had circumstances frequently associated with increased schistocytes count (promyelocytic leukaemia, pregnancy, severe infection). DISCUSSION: Schistocytes were thus frequently observed in DIC patients, usually with low percentage, within or close to the reference range (<0.5%). Schistocytes measurement is not a clue test for the initial diagnosis of DIC, but might be of clinical value to suggest an associated or underlying thrombotic microangiopathy if ≥ 1%.

Autologous transplantation in CLL patients with B and C Binet stages: final results of the prospective randomized GOELAMS LLC 98 trial.

The relevance of high-dose chemotherapy followed by auto-SCT in CLL remains to be defined. The aim of the prospective, randomized, GOELAMS LLC 98 trial was to compare two strategies in previously untreated CLL patients aged <60 years. Conventional chemotherapy (Arm A) consisted of six monthly courses of CHOP followed by six CHOP courses in every 3 months in those achieving a complete or PR. Arm A was compared with high-dose therapy with auto-SCT (Arm B), used as consolidation after three CHOP courses in case of CR or very good PR. A total of 86 patients were enrolled, of which 39 and 43 patients were evaluable in arm A and arm B, respectively. The primary endpoint was PFS. On an intent-to-treat basis and with a median follow-up time of 77.1 (range 1-135.5) months, the median PFS was 22 months in Arm A and 53 months in Arm B (P<0.0001). Median survival time was 104.7 months in arm A and 107.4 months in arm B. This trial demonstrates that frontline high-dose therapy with auto-SCT prolongs PFS but does not translate into a survival advantage in advanced CLL patients in the pre-rituximab era.

Evaluation of schistocyte monitoring after haematopoietic stem cell transplantation.

INTRODUCTION: Observation of schistocytes on the peripheral blood following haematopoietic stem cell transplantation (SCT) is a common finding. As their presence is not specific to the onset of SCT-related thrombotic microangiopathy, we evaluated the interest of schistocyte measurement twice a week during the entire follow-up of 195 patients undergoing SCT, particularly focussing on the 125 allogeneic SCT. METHODS: Schistocytes were strickly defined as triangular-, crescent- or helmet-shaped red blood cells according to consensus standards and were checked blindly under the microscope and with computer image analysis. RESULTS: Mean schistocyte percentage was 0.7% (±0.5%, reference value ≤0.5). High schistocyte percentage was observed after allografts (0.79%) when compared to autologous SCT (0.47, P < 0.001). All but one patients undergoing allogenic SCT had schistocytes ≥0.6%. Conversely, significant schistocytosis was observed in 20% of the autologous SCT. Initial diagnosis [chronic myelocytic leukaemia, acute lymphoblastic leukaemia (ALL)], high-risk status, unrelated transplant and conditioning regimen including total body irradiation influenced higher schistocyte percentage (≈0.9%). Significant rise in the schistocyte percentage was observed during acute/chronic graft-versus-host disease, veno-occlusive disease (VOD), cholestatic hepatitis, haemorrhagic cystitis (HC) and pulmonary complications. Multivariate analysis showed a significant association between thrombotic microangiopathy (TM), renal impairment and delayed thrombopaenia after day 50, and schistocyte >1.2%. SCT-TM grade ≥2 occurred in nine patients. A marked rise in schistocyte >4.5% was observed, which was not reached during the other SCT-related complications. Children with ALL, undergoing unrelated allogeneic SCT, with early acute graft-versus-host disease refractory to steroids were prone to present SCT-TM, associated with VOD, interstitial pneumopathy and HC, resulting in a high mortality rate (six of seven patients). Our data confirmed that schistocytosis was common after SCT. Mild percentages were likely concomitant with extensive endothelial damage but higher percentage should have prompted to a close monitoring with SCT-TM investigation. CONCLUSION: In our experience, systematic schistocyte count after HSCT proved to be useful: the occurrence of an increased percentage was a surrogate marker for complications even if unspecific for TM.

Peripheral blood candidosis infection leading to spurious platelet and white blood cell counts.

We report a patient with thrombocytopenia secondary to disseminated stomach adenocarcinoma and sepsis whose platelet and white blood cells were falsely enumerated by two automated haematology analyzers. The cause of the spurious counts became obvious when numerous yeast forms were observed on the peripheral blood smear. Artefactual automated analyzer results are detailed.

[Acquired alpha-thalassemia as early sign for myelodysplastic syndrome (refractory anaemia) with secondary haemochromatosis]. / Alpha-thalassémie acquise révélatrice d'un syndrome myélodysplasique de type anémie réfractaire avec hémochromatose secondaire.

We report the case of a 59 year old man presenting a regenerative microcytic hypochromic anaemia. The investigations revealed the presence of haemoglobin H, suggesting abnormalities in the alpha-globin chains synthesis. Alpha-thalassemia was thus suspected. The patient had no personal or familial history. The association with aniso-poïkilocytosis and a marked iron overload (ferritinemia > 1,500 microg/L) suggested a myelodysplastic syndrome, which was confirmed with a bone marrow aspiration. The pattern was consistent with the Acquired alpha-Thalassemia-Myelodysplastic Syndrome (ATMDS). About a hundred cases are listed worldwidely and collected in an international registry. The causes of ATMDS are ignored, but recent reports indicate that the ATRX gene may be implicated in the pathogenesis. ATRX is a chromatin-associated protein, involved in the transcription of several genes. The alpha globin genes could be one of the targets of the ATRX protein.

EDTA-dependent lymphoagglutination in a patient with non-Hodgkin lymphoma.

Lymphoagglutination is a rare EDTA-dependent phenomenon far less frequent than neutrophil agglutination. It usually involves normal lymphocytes, but has also been reported in lymphoproliferative disorders. We report the case of a woman who developed artifactual agglutination of both normal and lymphoma cells in the context of an unusual primary cutaneous leg type B-cell non-Hodgkin's malignancy, raising the potential of misdiagnosis.

[Evaluation of a telemedicine system for the transmission of morpho/immunological data at the inclusion of patients in a therapeutic trial (Goelams LLC 98)]. / Validation du diagnostic morpho-immunologique par télétransmission en vue de l'inclusion dans un protocole thérapeutique (Goelams LLC 98).

The performances of the images digitalization and teletransmission systems make them more and more used. Applied to cellular haematology, they contribute to confrontations of diagnosis mostly within the framework of therapeutic trials. We present one of the first approaches of the use of telehematology for the inclusion of patients in the Goelams Chronic Lymphocytic Leukaemia 98 trial. The advantages were the constitution of a protected data bank, conveniently consultable; expertise on identical documents; facility of the exchanges between experts. We were able to set new standards of images sampling for CLL, solve the semantic divergences, to point out the inter-observer variability for the morphology. The limiting factors were the personal investment of the experts, but mainly the implication of first line morphologists which should benefit from adequate tools to apprehend this system of second reading like a quality control.

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.

[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).

Automated measurement of schistocytes after bone marrow transplantation.

Bone marrow transplantation-related thrombotic microangiopathy (BMT-TMA) is a severe complication partly suspected on the evidence of a microangiopathic haemolysis. Microscopic schistocyte observation confirms the mechanical origin of the haemolysis, but remains a tedious procedure that lacks standardization. Direct measurement of abnormal red blood cell (RBC) fragments is now available on some automated haematology systems. We compared in 131 patients (69 BMT with five BMT-TMA, 38 thrombotic thrombocytopenic syndromes, 11 macroangiopathies, 13 dyserythropoiesis) percentages of microscopic schistocytes and automated RBC fragments (Bayer ADVIA 120) to evaluate the clinical relevance of the automated measurements for BMT-TMA detection. The analyser correlated well with the microscope (intraclass correlation coefficient: 0.82) and quantified RBC fragments with a moderate overestimation (+0.4%) as compared to microscopic counts. BMT patients had higher RBC fragments when they had TMA (1.1 vs 0.4% without TMA). Automated counting was useful to flag BMT-related TMA, particularly when RBC fragments were above 1%. As RBC fragments were frequently detected in BMT patients even without TMA, a threshold of less 1% that ruled out TMA was determined with a 98% negative predictive value. The new RBC fragment automated parameter proved its clinical value to assess BMT-TMA, which might be useful for day-to-day monitoring of the post BMT period.