New Monoclonal Antibodies Cocktail for the Flow Cytometry Leukocyte Differential Method

Abstract 4726

Automated blood cell counters provide a leucocyte count and five-part (some 6-part) leucocyte differential, however haematology instrument differentials provide only limited information on cell morphology using abnormal cell flags and are often unable to reliably classify abnormal and immature cells. There are also limitations with the standard microscopic differential, identification of cells is subjective and there is significant inter and intra observer variation (Koepke et al. 1985). It is also subject to significant statistical variance (Rumke 1985).

There have recently been reports of using monoclonal antibody cocktails for an extended leucocyte differential by flow cytometry. The International Council for Standardization in Haematology has set up a group to prepare an international reference method for an extended flow differential; this is intended to replace the current reference manual microscopic 2 × 200 cell count (CLSI H20-A2). Currently, there are several different protocols in use for leucocyte differential using different monoclonal antibodies and gating strategies. The aim of this study was to compare the differential results from a protocol from Singapore (SGP) with published flow cytometric protocols for the leucocyte differential from France (Faucher et al. 2007, Roussel et al. 2010) and to the automated count from the Beckman Coulter LH750 analyser and the current reference microscopic method. The French flow cytometric method uses 6 antibodies and allows detection of all white blood cells, mature neutrophils, total lymphocytes, total monocytes, eosinophils, basophils, immature granulocytes, B lymphocytes, non-cytotoxic T-lymphocytes, cytotoxic T/NK lymphocytes, CD16 positive and CD16 negative monocytes, and blasts cells with lineage orientation. The SGP method uses 8 antibodies (CD3, CD34, CD117, CD45, CD13, CD20, CD16, CD56), premixed in single tube. It detects mature neutrophils, total lymphocytes, total monocytes, eosinophils, basophils, CD16 positive and CD16 negative monocytes, T-lymphocytes, B-Lymphocytes, NK-cells, immature granulocytes and blasts. A 5-colour flow cytometer, the Beckman Coulter FC500, was used in this study.

EDTA blood was analysed on 27 normal and 148 abnormal samples, either with complete blood count values outside the reference range or which demonstrated abnormal cell flags on the LH750. These samples included blast cells, immature granulocytes and abnormal lymphocytes.

Results for most cell populations measured by the SGP flow differential compared well with the LH750, the manual reference method and French protocol. Comparative results using Pearson correlation are presented in Table 1.

For the SGP protocol, correlation with the LH750 and with the manual differential was good for neutrophils, lymphocytes, monocytes and eosinophils. Excellent correlation was observed for all cells apart from basophils when the two flow methods were compared to each other. There was no correlation for basophils between the SGP flow method and the manual method. Similarly, there was no correlation between SGP flow method and LH750 nor between both flow methods. This is not surprising as basophils are usually present in very low numbers. Hence, without a positive marker for basophils in the flow cytometric panel correlation may depend on the type of samples used for the evaluation.

Very good correlation of blast cells, r=0.99 and immature granulocytes, r=0.88 was seen between the manual and the SGP method. When comparing the flow methods to each other correlation for blast cells shows an r value of 0.96 and immature granulocytes 0.97.

Our study shows that this flow cytometric method performs well with both normal and abnormal patient samples. A differential using monoclonal antibodies for immunological recognition of cells provides more information than either the manual or automated differential. In addition to the detection of the common cell populations, blast cells, immature granulocytes, subpopulations of lymphocytes and inflammatory monocytes are enumerated.