Optimizing the management of analytical interferences affecting red blood cells on XN-10 (Sysmex®).

INTRODUCTION: Interferences on red blood cells (RBCs) measurement and the associated parameters in haematology analyzers are very common. Many sources of interferences are described but their management remains uncertain depending on the measurement system; we aimed at developing an optimized scheme allowing the accurate management of most interferences affecting RBCs, based on the alternative "optical" parameters from SYSMEX XN-10. METHODS: Samples from 12 groups of relevant interferences were analysed and compared with a control group allowing (1) the determination of deviation thresholds beyond which an interference is likely, and (2) the development of two flowcharts for their subsequent management. These flowcharts were then evaluated among a bank of retrospective typical cases of interferences and in the routine flow of the laboratory. RESULTS: After verifying the excellent agreement between standard and alternative parameters, the comparative study between analytical channels allowed to determine an acceptable deviation and then discriminate technical concerns caused by cold agglutinins, leukocytosis and plasma-related interferences. This led to the development of flowcharts ensuring the accurate management of these interferences, whether MCHC is <320 or >365 g/L. These proposed flowcharts allowed the correction of 63/65 historical confirmed interferences cases (97%). Furthermore, they corrected 18 results among 901 unselected prospective samples. CONCLUSION: The resulting flowcharts allow a relevant correction for most common interferences affecting RBCs and are now definitively included in the routine analytical management and will be directly incorporated in the middleware of the laboratory.

Hairy lymphoproliferations: a case report and review of the literature / Hémopathies à lymphocytes « chevelus ¼ : à propos d'un cas et revue de la littérature

Mature B-cell lymphoproliferation with hairy lymphocytes include Marginal Zone Splenic Lymphoma (SMZL), Hairy Cell Leukemia (HCL), Splenic Diffuse Red Pulp Lymphoma (SDRPL), and Variant Hairy Cell Leukemia (HCL-v), the two latter being provisional entities that appeared in the 2008 WHO classification. We report the case of a 75-year-old man who benefited from a diagnostic re-evaluation of his SMZL. The good clinical evolution, the flow cytometry investigation (HCL score < 3, SDRPL score > 3, strong CD180 and CD200/CD180 ratio < 0.5) and the histological assessment favored a SDRPL. This entity did not exist at the time of the diagnosis in 2006. The differential diagnosis between these diseases sometimes remains uneasy. Here are mentioned some practical clues to assess the diagnosis.

Les syndromes lymphoprolifératifs B matures avec des lymphocytes d'aspect « chevelus ¼ comprennent le lymphome splénique de la zone marginale splénique (SMZL), la leucémie à tricholeucocytes (HCL), le lymphome diffus de la pulpe rouge (SDRPL) et la leucémie à tricholeucocytes variante (HCL-v), ces deux dernières étant des entités provisoires apparues dans la classification OMS 2008. Nous rapportons le cas d'un homme de 75 ans qui a bénéficié d'une réévaluation diagnostique de son SMZL. En effet, la bonne évolution clinique, les données des explorations par cytométrie en flux (score HCL < score SDRPL > 3, CD180 fort et ratio CD200/CD180 < 0,5) et les données anatomopathologiques ont conclu à un SDRPL. Cette entité n'existait pas lors du diagnostic en 2006. Le diagnostic différentiel entre ces différentes pathologies n'étant pas toujours aisé, nous tenterons de donner quelques pistes pratiques pour conduire au diagnostic précis.

[Investigation of large granular lymphocytic leukemias: data from the laboratory of hematology at Nancy University Hospital, France]. / Investigation des leucémies à grands lymphocytes à grains : expérience d'un laboratoire d'hématologie.

Large granular lymphocytic leukemia (LGLL) is a rare clonal lymphoproliferative disorder from T or NK origin. PURPOSE: to report on the diagnostic and therapeutic management of LGLL investigated in the university hospital at Nancy, France. METHODS: retrospective (7 years) collection of clinical and biological data and patients' cohort analysis. RESULTS: Eight out of fifteen patients presented with neutropenia, including five profound neutropenia (neutrophils < 500 × 109/L). Four patients had an infection. Two patients have rheumatoid arthritis and an associated Felty's syndrome, one a Sweet syndrome. Two also suffered from chronic Lymphocytic Leukemia, and one from a diffuse large B-cell lymphoma. Twelve patients had LGLL-T and 3 had a chronic LGLL-NK. Eleven out of twelve patients had a clonal LGLL-T when polymerase chain reaction assessed. No KIR clonality was sought among the 3 LGL-NK patients. Five patients out of fifteen received immunosuppressive treatment. CONCLUSION: Although using simple and robust investigations, our series demonstrates a high heterogeneity in LGLL detection and assessment.

[Use of the multiparametric panel CD3/CD4/CD8/CD7/CD26/CD158k in the detection and use of flow cytometry of Sezary cells]. / Apport du panel multiparamétrique CD3 CD4 CD8 CD7 CD26 CD158k dans la détection et le suivi par cytométrie en flux des cellules de Sézary.

The Sezary syndrome has been defined by a triad combining erythrodermia, generalized lymphadenopathy, and the presence of circulating Sezary cells > 1 × 109/L characterized by a CD4+/CD8- phenotype with loss of one or more T antigens (mainly CD7 and/or CD26). We retrospectively reviewed the immunophenotypic profiles of 10 SS patients followed in our institution (University Hospital at Nancy, France). The application of the WHO criteria resulted in a diagnostic confirmation for 9 out of 10 cases. Since 2008, new diagnostic and staging criteria have been proposed, including the CD158k/KIR3DL2 receptor detection. The application of these new criteria to our cohort led us to notice a phenotypic heterogeneity of our cases but allowed to achieve a relevant diagnosis of Sezary syndrome in all cases, especially for patients with lymphopenia. The use of such a panel of monoclonal antibodies also optimized the follow-up of the patients.

Combined use of multiparametric flow cytometry and cytomorphology to enhance detection of neuroblastoma metastatic cells in bone marrow.

INTRODUCTION: In the context of neuroblastoma (NB), the screening for bone marrow (BM) metastasis is a recurrent issue for hematology laboratory routine practice. Detection of low tumor burden using light microscopy is often difficult. In this regard, our objective was to evaluate the performance of multiparametric flow cytometry (FC) for detecting NB metastatic cells in BM. METHODS: We applied a new FC multiparametric panel allowing the analysis of the co-expression of 5 surface markers: GD2 (disialoganglioside 2), CD9, CD56, CD81, and CD90, on CD45-negative BM cell populations, and compared results with BM biopsy immunohistochemistry, which is the reference method. RESULTS: In spike-in tests, the multiparametric FC successfully detected NB cells mixed in peripheral blood mononuclear cells to a level of 0.01%. FC analysis was performed on 45 sets of BM aspirates sampled from 21 children, either at diagnosis or during follow-up. Combining multiparametric FC with light microscopy improved NB metastasis detection, with a higher sensitivity (76.9% vs 61.5%) and a higher specificity (94.4% vs 77.8%) as compared to light microscopy alone. At the time of diagnosis, multiparametric FC detected NB metastatic cells in all cases. CONCLUSION: These results illustrate the performance of multiparametric FC analysis to detect metastatic BM infiltration of NB. This is of particular interest in an emergency context, since when combined with light microscopy, it enhances the detection of metastatic invasion within a short timeframe, allowing an adapted and rapid clinical management.

Diagnostic and prognostic value of plasma volume status at emergency department admission in dyspneic patients: results from the PARADISE cohort.

BACKGROUND: Systemic congestion, evaluated by estimated plasma volume status (ePVS), is associated with in-hospital mortality in acute heart failure (AHF). However, the diagnostic and prognostic value of ePVS in patients with acute dyspnea has been insufficiently studied. OBJECTIVES: To assess the association between the first ePVS calculated from blood samples on admission in the emergency department (ED) and discharge diagnosis of AHF and in-hospital mortality in patients admitted for acute dyspnea. METHODS: The study included 1369 patients admitted for dyspnea in the ED in 2015. ePVS was calculated from hematocrit and hemoglobin values at admission. Comparisons of baseline characteristics according to ePVS tertiles were carried out and then associations between ePVS and the two outcomes "AHF diagnosis" and "intra-hospital mortality" were assessed using a logistic regression model. RESULTS: 36.6% had a BNP > 400 pg/mL and median ePVS was 4.58 dL/g [3.96-5.55]. Overall in-hospital mortality was 11.1% (n = 149). In multivariable analysis, the third ePVS tertile (> 5.12 dL/g) had a significantly increased risk of having AHF (OR = 1.64 [1.16-2.33], p = 0.005). In-hospital mortality rose across ePVS tertiles (8.4-13.8% p < 0.01). ePVS greater than the first or second tertile threshold (respectively, 4.17 dL/g and 5.12 dL/g) were both significantly associated with a higher risk of in-hospital mortality (OR for 2nd/3rd tertile = 2.06 [1.25-3.38], p = 0.004 and OR for 3rd tertile = 1.54 [1.01-2.36], p = 0.04). CONCLUSION: Higher ePVS values determined from first blood sample at admission are associated with a higher probability of AHF and in-hospital mortality in patients admitted in the ED for acute dyspnea.

Hyponatraemia, hyperglycaemia and worsening renal function at first blood sample on emergency department admission as predictors of in-hospital death in patients with dyspnoea with suspected acute heart failure: retrospective observational analysis of the PARADISE cohort.

OBJECTIVES: To assess the prognostic value of hyponatraemia, hyperglycaemia and impaired estimated glomerular filtration rate (eGFR) in predicting in-hospital death in patients with acute heart failure (AHF) admitted for acute dyspnoea in the emergency department. DESIGN: Retrospective observational study. SETTING: Emergency Department of the University Hospital of Nancy. Data were collected from August 2013 to October 2015. PARTICIPANTS: The analysis included 405 patients with AHF admitted for acute dyspnoea in an emergency department. RESULTS: The population was elderly (mean age 82 years), 20.1% had hyponatraemia, 45.1% had hyperglycaemia and 48.6% had eGFR <50 mL/min/1.73 m2. Sixty-one patients (15.1%) died in hospital, mostly due to cardiac aetiology (58.3%). In multivariable analysis adjusted for key potential confounders, adjusted hyponatraemia (OR=2.40, (1.16 to 4.98), p=0.02), hyperglycaemia (OR=2.00, 1.06 to 3.76, p=0.03) and eGFR <50 mL/min/1.73 m2 (OR=1.97 (1.00 to 3.80), p=0.04*) were all identified as significant independent predictors of in-hospital death. CONCLUSIONS: Results of basic routine laboratory tests (hyponatraemia, hyperglycaemia and impaired eGFR) performed on admission in the emergency department are independently associated with in-hospital death. These inexpensive tests, performed as early as patient admission in the emergency department, could allow the early identification of patients admitted for AHF who are at high risk of in-hospital death. TRIAL REGISTRATION NUMBER: NCT02800122.

[New complete blood count parameter to evaluate iron status of haemodialysis patients]. / Apport des nouveaux paramètres de l'hémogramme dans l'evaluation du statut martial chez le patient dialysé chronique.

The presence of functionnal iron deficiency during erythropoietin therapy especially in patients with chronic renal failure leads to a diagnostic problem due to the low sensitivity and specificity of the biological parameters usually used in the diagnosis of iron deficiency. However, this diagnostic appears to be essential in so far as its treatment consists in intravenous iron perfusion that allow an optimal efficiency of erythropoietin therapy. Hypochromic red blood cells and reticulocyte hemoglobin content (CHr) are two new parameters that are given by Bayer blood count analyser (H3 and ADVIA 120) that may be usefull in the diagnostic of functionnal iron deficiency. The measurement is based on optical red cell analysis by laser beam diffraction in two angles that permit to analyze content and volume. Reticulocytes are identified by dying of residual nucleic acid. Many papers have demonstrated the usefulness of these parameters in the initial take care and follow up of haemodialysis patient. It seems that these parameters are complementary and need to be analysed simultaneously; Hypochromic red blood cells having the most interesting sensibility specificity ratio but reticulocyte hemoglobin content being superior in the understanding of acute modification.

[How do we optimally evaluate iron stores in dialyzed patients treated with erythropoiesis stimulating agent?]. / Comment evaluer de façon optimale les réserves martiales d'un patient traité par agent stimulant l'erythropoïèse?

The treatment of renal anaemia with erythropoiesis stimulating agent is often associated with a functional iron deficiency characterized by normal or elevated iron stores but insufficient iron delivered for erythropoiesis. Biological markers of iron status depend on the compartment where it is located: stored, circulating or available for erythropoiesis. Ferritin is the protein of iron storage but also a protein of the acute phase of inflammation and serum ferritin increases in case of liver cytolysis. In the circulation iron is bound to transferrin (Tf). Tf dosage is necessary to calculate transferrin saturation coefficient (TSAT) which decreases below 20% in iron deficiency but also in inflammatory states. Another Limitation is the nycthemeral variations of serum iron. The best marker of functional iron deficiency is the percentage of hypo chromic red cells (> 6%) followed by reticulocyte Hb content (< 29 pg/cell). These 2 markers measure the body capacity to donate iron to erythroid precursors but necessitate specific laboratory equipment. In all cases evaluation of iron balance should be done at least eight days after the last iron infusion.

Comparative evaluation of schistocyte counting by an automated method and by microscopic determination.

Schistocytes are circulating RBC fragments. The morphologic identification of schistocytes is difficult because the shapes to which they correspond are still under discussion. Automated hematology systems permit the possibility of direct measurement of RBC fragments. We compared schistocyte counts performed by different biologists and technicians with the automated counts by the ADVIA 120 (Bayer Health Care, Tarrytown, NY). The agreement between the ADVIA 120 and the average of the observers gives a correlation coefficient of 0.7274 (95% confidence interval, 0.6285-0.8019). The ADVIA 120 has a tendency to overestimate the count (average, +0.445%). No false-negative case was recorded. The maximum sensitivity (detection of 100% of samples with schistocytes) of the analyzer was determined at a threshold value of 0.25%, but the specificity was low (20%). Therefore, a blood smear examination remains necessary to confirm schistocyte presence. However, the clinical features correlated particularly with negative automated RBC fragments, and the high negative predictive value of RBC fragments ruled out thrombotic events (macroangiopathies or microangiopathies).

Platelet counting.

Platelet counting is a daily basic hematological analysis of crucial interest in many clinical situations. Historical manual techniques (phase-contrast microscopy) have been replaced by automated techniques (impedance or optical analyzers) more rapid and precise. More recently, flow cytometry techniques using labeled monoclonal antibodies have been proposed as reference techniques. Nevertheless, pre-analytical and analytical variables should be respected to obtain reliable results and avoid validation pitfalls.

Platelet morphology analysis.

Platelets are very small blood cells (1.5-3 µm), which play a major role in primary haemostasis and in coagulation mechanisms. Platelet characterization requires their counting (see Chapter 15 ) associated with accurate morphology analysis. We describe the major steps in order to correctly obtain stained blood films, which can be analyzed by optical microscope. Platelet morphology abnormalities are found in acquired malignant hematological diseases such myeloproliferative or myelodysplastic syndromes and acute megakaryoblastic leukemia. A careful analysis of the platelet size and morphology, by detecting either normal platelets with or without excessive anisocytosis, microplatelets, or large/giant platelets, will contribute to inherited thrombocytopenia diagnosis and gather substantial data when looking for an acquired platelet disorders.