“6 markers/5 colors” extended white blood cell differential by flow cytometry

Abstract

Electronic white blood cell (WBC) differential by standard cytology (hematology analyzer and visual inspection of blood smears) is limited to five types and identification of abnormal cells is only qualitative, often problematic, poorly reproducible, and labour costing. We present our results on WBC differential by flow cytometry (FCM) with a 6 markers, 5 colors CD36-FITC/CD2-PE1CRTH2-PE/CD19-ECD/CD16-Cy5/CD45-Cy7 combination, on 379 subjects, with detection of 12 different circulating cell types, among them 11 were quantified. Detection of quantitative abnormalities of whole leucocytes, neutrophils, eosinophils, basophils, monocytes, or lymphocytes was comparable by FCM and by standard cytology in terms of sensitivity and specificity. FCM was better than standard cytology in detection and quantification of circulating blast cells or immature granulocytes, with a first lineage orientation in the former case. All cases of lymphocytosis, with lineage assignment, were detected by FCM. FCM identified a group of patients with excess of CD16pos monocytes as those having an inflammatory syndrome. WBC differential by FCM is at least as reliable as by standard cytology. FCM superiority consists in identification and systematic quantification of parameters that cannot be assessed by standard cytology such as lineage orientation of blast cells or lymphocytes, and expression of markers of interest such as CD16 on inflammatory monocytes. ' International Society for Analytical CytologyKey terms white blood cell differential; flow cytometry; screening for hematological malignancies SCREENING for hematological disorders is routinely performed by counting circulating cells with hematology analyzers. But identification of circulating white blood cells (WBCs) by electronic counters is limited to only five cell-types: lymphocytes, monocytes, neutrophils, eosinophils, and basophils. Moreover, although most cell hematology analyzers are very good in detection of quantitative abnormalities, qualitative recognition of abnormal WBCs is poor, and microscopic examination of blood smears is needed for most cases to ascertain the presence of abnormal circulating cells. Therefore, both electronic WBC count and microscopic inspection of blood smears are needed to establish a reliable WBC differential. This traditional scheme, referred to as traditional or standard cytology, was set up in the 70s.Standard cytology is based on the expertise of cytologists and technicians, which is noticeably variable. Blood smear reviewing is time consuming and difficult to standardize. A recent study shows that, in a median institution among 263 hospitals and independent laboratories, manual review of peripheral blood smears were performed on 26.7% of specimens. The authors raised clearly the question of how to reduce the rate of manual peripheral blood smear review and to improve the efficiency of generating blood cell count results (1). In our institutions (JF, FL), <5% of reviewed blood smears will lead to further investigations. Last but not leas...

Refining the white blood cell differential: The first flow cytometry routine application

“…All the threshold counts that were used were established on leucocyte flagged samples and normal controls. The reference values established in this study were close to those previously published (i) B-, T-, and NK-lymphocytes, (ii) neutrophils and monocytes, and (iii) blasts and immature granulocytes (5,(19)(20)(21). Of note the choice of thresholds will dramatically impact the amount of failed analyses by flow, although they should be adapted to the specificity and the sample population of each laboratory.…”

“…However, all these studies were based on single cell type identification per antibody combination and were not suitable for a full differential count. Recently, efforts have been made to develop relevant combinations of antibodies for a five part differential (5,6). This study sought to investigate the use of flow cytometry as an integral part of the diagnostic process and, importantly, use it for the automated validation of all samples with suspect cells.…”

“…At the time of our study, only one antibody combination has been previously published based on a specific gating strategy, the detection of 16 subsets of cells (5). Although the gating strategy has been improved with the exclusion of RBCs and platelet aggregates, several limitations exist including the fact that cytotoxic T cells (Tc) and NK cells (CD2 (14,15).…”

“…The aim of our gating strategy, adapted from Faucher et al (5), was to sort all populations and subpopulations of interest as described in Table 3. As shown in Figure 1, the master group of plots was proposed to: (i) eliminate platelet clumps, RBCs, and cell lysates (dot plot CD36/CD45); (ii) target the WBC (dot plot SSC/CD45); (iii) define the neutrophils and to orient the algorithm tree as described below (dot plot CD16/ SSC).…”

“…Recent studies have demonstrated the feasibility of the flow cytometry approach using combinations of monoclonal antibodies allowing the detection and quantification of up to 16 cell subsets (5,6).…”