Impact of blood storage and sample handling on quality of high dimensional flow cytometric data in multicenter clinical research

Obtaining reliable and reproducible high quality data in multicenter clinical research settings requires design of optimal standard operating procedures. While the need for standardization in sample processing and data analysis is well-recognized, the impact of sample handling in the pre-analytical phase remains underestimated. We evaluated the impact of sample storage time (approximate to transport time) and temperature, type of anticoagulant, and limited blood volume on reproducibility of flow cytometric studies. EDTA and Na-Heparin samples processed with the EuroFlow bulk lysis protocol, stained and stored at 4 degrees C showed fairly stable expression of cell surface markers and distribution of the major leukocyte populations for up to 72 h. Additional sample fixation (1% PFA, Fix & Perm) did not have any beneficial effects. Blood samples stored for < 24 h at room temperature before processing and staining seemed suitable for reliable immunophenotyping, although losses in absolute cell numbers were observed. The major losses were observed in myeloid cells and monocytes, while lymphocytes seemed less affected. Expression of cell surface markers and population distribution were more stable in Na-Heparin blood than in EDTA blood. However, storage of Na-Heparin samples was associated with faster decrease in leukocyte counts over time. Whole blood fixation strategies (Cyto-Chex, TransFix) improved long-term population distribution, but were detrimental for expression of cellular markers. The main conclusions from this study on healthy donor blood samples were successfully confirmed in EDTA clinical (patient) blood samples with different time delays until processing. Finally, we recognized the need for adjustments in bulk lysis in case of insufficient blood volumes. Despite clear overall conclusions, individual markers and cell populations had different preferred conditions. Therefore, specific guidelines for sample handling should always be adjusted to the clinical application and the main target leukocyte population

Improved standardization of flow cytometry diagnostic screening of primary immunodeficiency by software-based automated gating

Background Multiparameter flow cytometry (FC) is essential in the diagnostic work-up and classification of primary immunodeficiency (PIDs). The EuroFlow PID Orientation tube (PIDOT) allows identification of all main lymphocyte subpopulations in blood. To standardize data analysis, tools for Automated Gating and Identification (AG&I) of the informative cell populations, were developed by EuroFlow. Here, we evaluated the contribution of these innovative AG&I tools to the standardization of FC in the diagnostic work-up of PID, by comparing AG&I against expert-based (EuroFlow-standardized) Manual Gating (MG) strategy, and its impact on the reproducibility and clinical interpretation of results. Methods FC data files from 44 patients (13 CVID, 12 PID, 19 non-PID) and 26 healthy donor (HD) blood samples stained with PIDOT were analyzed in parallel by MG and AG&I, using Infinicyt (TM) software (Cytognos). For comparison, percentage differences in absolute cell counts/mu L were calculated for each lymphocyte subpopulation. Data files showing differences >20% were checked for their potential clinical relevance, based on age-matched percentile (p5-p95) reference ranges. In parallel, intra- and inter-observer reproducibility of MG vs AG&I were evaluated in a subset of 12 samples. Results The AG&I approach was able to identify the vast majority of lymphoid events (>99%), associated with a significantly higher intra- and inter-observer reproducibility compared to MG. For most HD (83%) and patient (68%) samples, a high degree of agreement (<20% numerical differences in absolute cell counts/mu L) was obtained between MG and the AG&I module. This translated into a minimal impact (<5% of observations) on the final clinical interpretation. In all except three samples, extended expert revision of the AG&I approach revealed no error. In the three remaining samples aberrant maturation and/or abnormal marker expression profiles were seen leading in all three cases to numerical alarms by AG&I. Conclusion Altogether, our results indicate that replacement of MG by the AG&I module would be associated with a greater reproducibility and robustness of results in the diagnostic work-up of patients suspected of PID. However, expert revision of the results of AG&I of PIDOT data still remains necessary in samples with numerical alterations and aberrant B- and T-cell maturation and/or marker expression profiles

CSF cells in tuberculous meningitis. Humoral and cellular immune response

In 10 patients with tuberculous meningitis both T and B lymphocyte activation was observed in the CSF from clinical onset of disease. During the course of the illness, the humoral response remained most marked, disappearing by 2 months, while the cellular immune response continued for about another 3 months. This contrasts with acute viral meningitis, in which both responses cease within 1-2 weeks of disease.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Burkitt's lymphoma - A correlated light and electron microscopic study on the malignancy in the CSF

SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Fanconi's anaemia. Simultaneous onset in 2 siblings and unusual cytological findings

SCOPUS: ar.jinfo:eu-repo/semantics/publishe

CSF cytology of acute viral meningitis and meningoencephalitis

SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Platelet survival in myelodysplastic syndromes

SCOPUS: ar.jinfo:eu-repo/semantics/publishe

A case of L1 acute lymphoblastic leukaemia relapsing in L3 (Burkitt's type) with non-T, non-B cells

A case of acute lymphoblastic leukaemia with L1 morphology relapsed in L3 (Burkitt's type). These L3 cells disclosed neither T nor B markers and were CALLA positive. This morphology change occurred simultaneously with a rising antibody titer to the Epstein-Barr virus.SCOPUS: ar.jinfo:eu-repo/semantics/publishe