L. Omar Henderson

Proficiency Testing of Human Leukocyte Antigen-DR and Human Leukocyte Antigen-DQ Genetic Risk Assessment for Type 1 Diabetes Using Dried Blood Spots

Background: The plurality of genetic risk for developing type 1 diabetes mellitus (T1DM) lies within the genes that code for the human leukocyte antigens (HLAs). Many T1DM studies use HLA genetic risk assessment to identify higher risk individuals, and they often conduct these tests on dried blood spots (DBSs) like those used for newborn bloodspot screening. One such study is The Environmental Determinants of Diabetes in the Young (TEDDY), a long-term prospective study of environmental risk factors. To provide quality assurance for T1DM studies that employ HLA genetic risk assessment, the Centers for Disease Control and Prevention (CDC) conducts both a voluntary quarterly Proficiency testing (VQPT) program available to any laboratory and a mandatory annual Proficiency testing (PT) challenge for TEDDY laboratories. Methods: Whole blood and DBS samples with a wide range of validated HLA-DR and HLA-DQ genotypes were sent to the participating laboratories. Results were evaluated on the basis of both the reported haplotypes and the HLA genetic risk assessment. Results: Of the reported results from 24 panels sent out over six years in the VQPT, 94.7% (857/905) were correctly identified with respect to the relevant HLA-DR or HLA-DQ alleles, and 96.4% (241/250) were correctly categorized for risk assessment significant improvement was seen over the duration of this program, usually reaching 100% correct categorization during the last three years. Of 1154 reported results in four TEDDY PT challenges, 1153 (99.9%) were correctly identified for TEDDY eligibility. Conclusions: The different analytical methods used by T1DM research centers all provided accurate (>99%) results for genetic risk assessment. The two CDC PT programs documented the validity of the various approaches to screening and contributed to overall quality assurance.

Titration of a CD45‐FITC conjugate to determine the linearity and dynamic range of fluorescence intensity measurements on lymphocytes

To produce biologic calibrators for relative fluorescence intensity (RFI) measurements, we stained leukocytes with serial dilutions of CD45-FITC conjugate and processed them using our regular whole blood lysis procedure. Cells were stained with conjugate concentrations ranging from twice recommended to a million-fold lower. At the highest concentrations of conjugate, the RFI reached a plateau near the top of the third decade, indicating saturation of CD45 binding sites. As the concentration decreased, the RFI declined in a highly linear relationship between the dilution factor and the histogram channel number. For channel numbers corresponding to the lowest percentiles of the RFI distribution, linearity persisted down to the first half decade. The slope of this relationship revealed a true dynamic range of 4.5 decades, which was comparable to the value obtained with microbead standards calibrated in molecules of equivalent soluble fluorochrome (MESF). Our results suggest that the lower limit of linearity for fluorescence intensity from fluorescein isothiocyanate (FITC)-stained lymphocytes is below 500 MESF and that cellular autofluorescence is the major limiting factor in detecting and quantifying FITC-specific staining. This procedure provides an adroit way of characterizing the linearity and dynamic range of measurements for quantitative fluorescence cytometry using exactly the same matrix, stains, and preparation methods as those used for cellular analytes.

Preparation of lyophilized human serum based reference materials with graded levels of apolipoproteins A-I and B

Surveys of national and international laboratories indicate that among-laboratory and between-laboratory sources of variance account for the majority of variability in laboratories measuring apolipoproteins A-I and B. These sources of variance are amenable to correction through the use of common quality control and reference materials. We utilized proven techniques employing ethyl alcohol and acetate buffer to precipitate either apolipoprotein A-I rich or apolipoprotein B rich fractions that were blended with whole or delipidated serum producing five pilot-sized pools containing graded levels of apolipoproteins. After lyophilization the pools were tested and each pool contained levels of analytes similar to frozen serum and contained varied amounts of apolipoproteins A-I and B. Temporal and accelerated thermal stability testing demonstrated stability of the analytes in the pools with time (three years) and temperature (up to 56 degrees C). This technology provides a preparative procedure for apolipoprotein reference materials over the extended range needed in clinical applications.