Time and temperature stability of T-cell subsets evaluated by a dual-platform method.

INTRODUCTION: T-cell subset enumeration in HIV patients is routinely performed for monitoring infection stage and response to antiretroviral therapy. Studies have examined the effect of specimen refrigeration and age for single-platform (SP) methods, but there is limited data for time and temperature requirements of dual-platform (DP) methods. METHODS: Using a DP method, we analyzed peripheral blood (PB) from 52 HIV patients at room temperature (RT) at 24, 72, and 96 hours. PBs from 34 HIV patients had baseline RT analysis within 24 hours, and then were refrigerated and analyzed at 24, 48, and 72 hours. The coefficient of variation (CV) and residuals (changes in lymphocyte subsets) were recorded at each time point and compared to assess the precision and bias under the various conditions. Testing performance under different conditions was compared by linear regression. RESULTS: Mean CV was ≤7.3% and median residuals were <30/µl for absolute CD4 and CD8 determinations. There was good correlation between baseline analysis data at RT and at various time points, both at RT and 4°C. CONCLUSIONS: Our results are similar to those published for SP methods for aging or refrigerated specimens. The high level of agreement between measurements supports the robustness of this DP methodology.

Expression of natural killer receptors in T- and NK-cells: comparison of healthy individuals, patients with prior stem cell transplant, and patients undergoing chemotherapy.

We studied the expression of natural killer receptors (NKRs) on peripheral blood cytotoxic T lymphocytes and NK cells in patients who underwent an allogeneic stem cell transplant (SCT), and compared these findings with results from healthy individuals (CTRL) and patients undergoing chemotherapy (CHEMO), respectively. Peripheral blood mononuclear cells were analyzed by flow cytometry with antibodies against the NKRs CD158a, CD158b, CD158e (known as killer immunoglobulin-like receptors, KIRs), and CD94. Expression of NKRs was evaluated separately in CD56+, CD57+, and CD56/CD57 (double +) subsets of T and NK cells. We found mainly differences in CD158a and CD94 expression between the three cohorts, with the SCT and CHEMO groups usually showing similar changes, when compared to the CTRL population. None of the patients with SCT or CHEMO demonstrated patterns of restricted NKR expression. Our results provide a comprehensive overview of KIR and CD94 expression in T and NK cells following SCT and chemotherapy.

Validation of a CYP2D6 genotyping panel on the NanoChip Molecular Biology Workstation.

BACKGROUND: CYP2D6 is a highly polymorphic phase I enzyme that metabolizes 20%-25% of clinically used drugs. The objective of this study was to validate a CYP2D6 genotyping assay with the NanoChip Molecular Biology Workstation. METHODS: We genotyped 200 anonymized human DNA samples with the Pyrosequencing platform at the Medical College of Wisconsin and with the NanoChip platform at Dartmouth Medical School. We compared CYP2D6 genotypes and resolved samples with genotypic discrepancies with the Jurilab CYP2D6 duplication/deletion assay or with traditional DNA sequencing. The Jurilab assay is a long-range PCR assay used to evaluate sequence structures 3' of the CYP2D7 and CYP2D6 coding regions. For the NanoChip platform, we performed multipad addressing and duplicate runs to test the intra- and intercartridge precision, within- and between-run precision, and reproducibility of the defined genotypes. RESULTS: We used both platforms to genotype all 200 DNA samples for CYP2D6*3, *4, *5, *6, *7, *8, and gene duplication. The 2 methods showed 99.4% concordance in the genotyping results; we found only 8 discrepant genotypes among 1400 DNA analyses. Confirmatory molecular analysis of the discrepant genotypes revealed that the NanoChip assay showed better agreement. The imprecision of the NanoChip method (CV) was 8.9%-17.7%. CONCLUSIONS: This validation study of the NanoChip electronic microarray-based CYP2D6 genotyping assay revealed a CV <20% and good concordance with the Pyrosequencing method and a confirmatory sequencing method.

Negative urine opioid screening caused by rifampin-mediated induction of oxycodone hepatic metabolism.

INTRODUCTION: Oxycodone has become widely used in the clinic for the treatment of chronic pain. This reflects its favorable pharmacokinetics and side effect profiles. CASE REPORT: We report a 60-y-old man who had a clinically significant drug interaction between rifampin and oxycodone, resulting in 3 consecutive negative urine oxycodone screens in a 2-month period, suggesting non-adherence. A combination of urine opioid metabolite quantification by GC/MS and CYP genotyping confirmed that he was compliant with his oxycodone therapy. Determination of the complete oxycodone metabolite profile and the CYP3A4/5 and 2D6 genotype allowed the physician to be confident that the patient was compliant with the medication (and not diverting it) and to increase his oxycodone dose to optimize his pain control. CONCLUSION: This case demonstrates how the combination of analytical toxicology and pharmacogenetic analyses enhances a physician's ability to personalize drug therapy in patients with chronic pain syndromes.