Japanese Society for Laboratory Hematology flow cytometric reference method of determining the differential leukocyte count: external quality assurance using fresh blood samples

<jats:title>Summary</jats:title><jats:sec><jats:title>Introduction</jats:title><jats:p>To provide target values for the manufacturers’ survey of the Japanese Society for Laboratory Hematology (<jats:styled-content style="fixed-case">JSLH</jats:styled-content>), accurate standard data from healthy volunteers were needed for the five‐part differential leukocyte count. To obtain such data, <jats:styled-content style="fixed-case">JSLH</jats:styled-content> required an antibody panel that achieved high specificity (particularly for mononuclear cells) using simple gating procedures. We developed a flow cytometric method for determining the differential leukocyte count (<jats:styled-content style="fixed-case">JSLH</jats:styled-content>‐Diff) and validated it by comparison with the flow cytometric differential leukocyte count of the International Council for Standardization in Haematology (<jats:styled-content style="fixed-case">ICSH</jats:styled-content>‐Diff) and the manual differential count obtained by microscopy (Manual‐Diff).</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>First, the reference laboratory performed an imprecision study of <jats:styled-content style="fixed-case">JSLH</jats:styled-content>‐Diff and <jats:styled-content style="fixed-case">ICSH</jats:styled-content>‐Diff, as well as performing comparison among <jats:styled-content style="fixed-case">JSLH</jats:styled-content>‐Diff, Manual‐Diff, and <jats:styled-content style="fixed-case">ICSH</jats:styled-content>‐Diff. Then two reference laboratories and seven participating laboratories performed imprecision and accuracy studies of <jats:styled-content style="fixed-case">JSLH</jats:styled-content>‐Diff, Manual‐Diff, and <jats:styled-content style="fixed-case">ICSH</jats:styled-content>‐Diff. Simultaneously, six manufacturers’ laboratories provided their own representative values by using automated hematology analyzers.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>The precision of both <jats:styled-content style="fixed-case">JSLH</jats:styled-content>‐Diff and <jats:styled-content style="fixed-case">ICSH</jats:styled-content>‐Diff methods was adequate. Comparison by the reference laboratory showed that all correlation coefficients, slopes and intercepts obtained by the <jats:styled-content style="fixed-case">JSLH</jats:styled-content>‐Diff, <jats:styled-content style="fixed-case">ICSH</jats:styled-content>‐Diff, and Manual‐Diff methods conformed to the criteria. When the imprecision and accuracy of <jats:styled-content style="fixed-case">JSLH</jats:styled-content>‐Diff were assessed at seven laboratories, the <jats:styled-content style="fixed-case">CV</jats:styled-content>% for lymphocytes, neutrophils, monocytes, eosinophils, and basophils was 0.5~0.9%, 0.3~0.7%, 1.7~2.6%, 3.0~7.9%, and 3.8~10.4%, respectively. More than 99% of <jats:styled-content style="fixed-case">CD</jats:styled-content>45 positive leukocytes were identified as normal leukocytes by <jats:styled-content style="fixed-case">JSLH</jats:styled-content>‐Diff.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>When <jats:styled-content style="fixed-case">JSLH</jats:styled-content>‐Diff method were validated by comparison with Manual‐Diff and <jats:styled-content style="fixed-case">ICSH</jats:styled-content>‐Diff, <jats:styled-content style="fixed-case">JSLH</jats:styled-content>‐Diff showed good performance as a reference method.</jats:p></jats:sec>