Qualification of the differential leukocyte count and immunophenotyping in cryopreserved ex vivo whole blood assay.

We developed a flow cytometry-based assay, termed Differential Leukocyte Counting and Immunophenotyping in Cryopreserved Ex vivo whole blood (DLC-ICE), that allows quantification of absolute counts and frequencies of leukocyte subsets and measures expression of activation, phenotypic and functional markers. We evaluated the performance of the DLC-ICE assay by determining inter-operator variability for processing fresh whole blood (WB) from healthy donors collected at multiple clinical sites. In addition, we assessed inter-operator variability for staining of fixed cells and robustness across different anticoagulants. Accuracy was evaluated by comparing DLC-ICE measurements to real-time cell enumeration using an accredited hematology analyzer. Finally, we developed and tested the performance of a 27-colour immunophenotyping panel on cryopreserved fixed WB and compared results to matched fresh WB. Overall, we observed <20% variability in absolute counts and frequencies of granulocytes, monocytes and lymphocytes (T, B and NK cells) when fresh WB was collected in different anti-coagulant tubes, processed or stained by independent operators. Absolute cell counts measured across operators and anti-coagulants using the DLC-ICE method exhibited excellent correlation with the reference method, complete blood count (CBC) with differential, measured using a hematology analyzer (r2 > 0.9 for majority of measurements). A comparison of leukocyte immunophenotyping on fresh WB versus DLC-ICE processed blood yielded equivalent and linear results over a wide dynamic range (r2 = 0.94 over 10-104 cells/µL). These results demonstrate low variability across trained operators, high robustness, linearity and accuracy, supporting utility of the DLC-ICE assay for large cohort studies involving multiple clinical research sites.

Optimization and Interpretation of Serial QuantiFERON Testing to Measure Acquisition of Mycobacterium tuberculosis Infection.

RATIONALE: Conversion from a negative to positive QuantiFERON-TB test is indicative of Mycobacterium tuberculosis (Mtb) infection, which predisposes individuals to tuberculosis disease. Interpretation of serial tests is confounded by immunological and technical variability. OBJECTIVES: To improve the consistency of serial QuantiFERON-TB testing algorithms and provide a data-driven definition of conversion. METHODS: Sources of QuantiFERON-TB variability were assessed, and optimal procedures were identified. Distributions of IFN-γ response levels were analyzed in healthy adolescents, Mtb-unexposed control subjects, and patients with pulmonary tuberculosis. MEASUREMENTS AND MAIN RESULTS: Individuals with no known Mtb exposure had IFN-γ values less than 0.2 IU/ml. Among individuals with IFN-γ values less than 0.2 IU/ml, 0.2-0.34 IU/ml, 0.35-0.7 IU/ml, and greater than 0.7 IU/ml, tuberculin skin test positivity results were 15%, 53%, 66%, and 91% (P < 0.005), respectively. Together, these findings suggest that values less than 0.2 IU/ml were true negatives. In short-term serial testing, "uncertain" conversions, with at least one value within the uncertainty zone (0.2-0.7 IU/ml), were partly explained by technical assay variability. Individuals who had a change in QuantiFERON-TB IFN-γ values from less than 0.2 to greater than 0.7 IU/ml had 10-fold higher tuberculosis incidence rates than those who maintained values less than 0.2 IU/ml over 2 years (P = 0.0003). By contrast, "uncertain" converters were not at higher risk than nonconverters (P = 0.229). Eighty-seven percent of patients with active tuberculosis had IFN-γ values greater than 0.7 IU/ml, suggesting that these values are consistent with established Mtb infection. CONCLUSIONS: Implementation of optimized procedures and a more rigorous QuantiFERON-TB conversion definition (an increase from IFN-γ <0.2 to >0.7 IU/ml) would allow more definitive detection of recent Mtb infection and potentially improve identification of those more likely to develop disease.