Application Information Bulletin: Lymphocyte Subset Analysis

Transcription

1 Application Information Bulletin: Lymphocyte Subset Analysis Use of Precision Profiles to Estimate Precision of Lymphocyte Subset Analysis by Flow Cytometry. Robert Magari 1, Diana, B. Careaga 1, Karen, K. Lo 1, Xiangyang Dong 2, Elena Afonina 2, Liliana, M. Tejidor 1 1 Clinical Research, Diagnostics, Beckman Coulter, Inc., Miami, FL, United States 2 Clinical Applications, Life Sciences, Beckman Coulter, Inc., Miami, FL, United States

2 Use of Precision Profiles to Estimate Precision of Lymphocyte Subset Analysis by Flow Cytometry. INTRODUCTION A method to estimate precision based on profiles is presented in this paper. Because of the counting statistic and the performance of the flow cytometers, precision of measuring the lymphocyte subsets depends on the range of measurement. In addition, CD4 marker has several medical decisions levels, making it necessary to characterize precision throughout the range of measurements. MATERIALS AND METHODS Normal and abnormal donors covering the measuring range and tested in duplicates on a Navios* Flow Cytometer System were used in the study. Precision profiles were based on the relationship between the mean for each sample and the coefficient of variation (CV%). Mean represented the measuring range while CV% represented the precision performance. Mean and standard deviation were calculated for each sample. The relationship between mean and CV% throughout the range of measurement was modeled with a power functions as: CV =α Mean β + ε Where α and β were the parameters of the model and ε was the random error. Gauss-Newton method provided in PROC NLIN of SAS 9.3 was used to obtain the non linear least squares estimates of the parameters and their standard errors. Profiles were estimated for markers separately and all markers combined. * In U.S., Navios is intended for use as an in vitro diagnostic device for immunophenotyping with Navios tetra software and CYTO-STAT tetrachrome reagents. All other uses are for research use only. CE marked for 10 color in-vitro diagnostic use within the EU. LYMPHOCYTE SUBSET ANALYSIS BECKMAN COULTER 2

3 Repeatability performance (CV%) was calculated from the profile at the 25-th, 50-th, and 75-th percentile of the measuring range of each marker and for different medical decision levels for CD4 as: CV = a M b Where CV was the estimated repeatability coefficient of variation, M was the percentile/ medical decision level, while a and b were the estimates of the parameters of the model (α, β). The Taylor s series approximation was used to calculate the standard error of CV based on the variances and covariance of a and b. The upper limit of CV was calculated as: UL = CV + t α,df se (CV) Where t was the statistic from the student distribution, α = 0.05 (corresponding to 95% confidence), df were degrees of freedom, and se (CV) was the standard error. LYMPHOCYTE SUBSET ANALYSIS BECKMAN COULTER 3

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6 CONCLUSION Precision profiles are a very efficient tool for characterizing precision throughout the measuring range and for point evaluations of precision at different medical decision levels. CV% and their upper confidence limit at each medical decision level can be compared to the acceptance criteria for making decisions on precision performance of a medical device. The examples shown demonstrate excellent precision for CD4 + measurements at important medical decision points using the Navios flow cytometer with CYTO-STAT tetrachrome system. For in vitro diagnostic use. Beckman Coulter and the stylized logo are registered trademarks of Beckman Coulter, Inc and are registered in the USPTO. LYMPHOCYTE SUBSET ANALYSIS BECKMAN COULTER 6 BR-18548A B