The implication of follicular lymphoma patients receiving allogeneic stem cell transplantation from donors carrying t(14;18)-positive cells.

We performed real-time quantitative polymerase chain reaction (RQ-PCR) in peripheral blood (PB) and/or bone marrow (BM) samples collected pre- and post transplant from 23 recipient-donor pairs receiving allogeneic stem cell transplantation (allo-SCT) for follicular lymphoma (FL). Of 23 donors, 11 had a PB and/or BM sample positive for t(14;18) (BCL2/IGH fusion) at low levels (

Pharmacogenomics as molecular autopsy for postmortem forensic toxicology: genotyping cytochrome P450 2D6 for oxycodone cases.

Pharmacogenomics, the study of the impact of heritable traits on pharmacology and toxicology, may serve as an adjunct for certifying opioid fatalities. Oxycodone, frequently prescribed for the relief of moderate to severe pain, is metabolized by cytochrome P450 (CYP) 2D6, encoded by a polymorphic gene with three mutations (*3, *4, and *5) with a combined 95% allelic frequency and about 10% prevalence. Individuals with variant alleles are more susceptible to oxycodone toxicity. By assessing the prevalence of CYP2D6 polymorphisms and covariables, we hypothesized that oxycodone fatality may be partially due to poor drug metabolism caused by CYP2D6 variant alleles. From the Milwaukee County Medical Examiner's Office (MCMEO), a retrospective analysis of 15 oxycodone cases was followed by genotyping blood samples for the variant alleles by conventional and real-time PCRs. Institutional Review Board approval was obtained. Oxycodone, extracted from blood and/or urine, was quantitated by GC-MS. The results show two homozygous for 2D6*4 and four heterozygous for 2D6*4. The MCMEO was not significantly different from those in the control group (n = 26) (p > 0.05, Fisher's Exact Test). However, genotyping CYP2D6 provided a more definitive interpretation of the oxycodone toxicity in four cases. Therefore, pharmacogenomics may serve as an adjunct in the determination of the cause and manner of death in forensic toxicology and a pharmacogenomic algorithm for genotyping has been proposed.

A novel multiparametric approach for analysis of cytoplasmic immunoglobulin light chains by flow cytometry.

We describe a novel flow cytometric approach using a two-step acquisition technique to determine the cytoplasmic immunoglobulin light chains (LC) expression. Samples were prepared by a lysed-whole-blood technique and incubated with CD38-PE (phycoerythrin) and CD45-FITC (fluorescein isothiocyanate). The cells were fixed and acquired on an FACSCalibur flow cytometer (first acquisition). The cells were then permeabilized, incubated with either kappa-FITC or lambda-FITC and reacquired (second acquisition). Analysis of the data was performed by gating on the differing intensities of CD38 and evaluating them for the presence of a shifting FITC-positive population from the first acquisition to the second acquisition. The FITC fluorescence intensity of the second acquisition was equal to the sum of surface CD45 expression obtained during the first acquisition and the cytoplasmic LC expression obtained during the second acquisition. Thus, the shifting (increase) of FITC fluorescence intensity during the second acquisition is specifically due to the cytoplasmic expression of either the kappa or lambda LC. We studied 15 multiple myeloma (MM) patients and 10 controls (samples from patients without plasma cell dyscrasias). None of the controls showed evidence of any clonal populations. Thirteen of 15 MM patients exhibited clonal plasma cells (CD38 bright), ranging from 0.01% to 34% of total events collected. In addition, we identified another minute clonal population of lymphocytes (CD38 dim, CD45 bright, low forward and side scatter) in 12 of 13 MM patients with clonal plasma cells. This population, ranging from 0.01% to 0.6% of total events collected, had the same LC restriction as the clonal plasma cells. Patients with a ratio of minor clonal population to clonal plasma cells less than 0.07 tended to remain in partial or complete remission than those with a ratio > or =0.07 (4/5 versus 1/4, P <.1, chi(2)). We conclude that this method is highly sensitive and permits us to identify the minute clonal population of lymphocytes in MM patients. Our preliminary observations with a small cohort of patients imply that this minute clonal population may have important prognostic significance. The prognostic significance should be confirmed by further studies.

Genotyping of cytochrome P450 2D6*3 and *4 mutations using conventional PCR.

Cytochrome P450 (CYP450) mixed-function mono-oxygenases, consisting of more than 30 enzymes, are responsible for the metabolism of a large number of drugs and metabolites. With the rapid advances in the human genome project, the role of genetic polymorphism in drug metabolism may become an important adjunct for rational drug therapy, and for the explanation of drug toxicity and interactions. This preliminary study modified a previously described procedure for genotyping CYP2D6*3 and *4. An additional step included uracil-DNA glycosylase for the prevention of "carry-over" contamination. DNA was extracted from peripheral blood using PureGene DNA Isolation kit. CYP2D6*3 and *4 sequences were amplified by PCR, followed by digestion with restriction endonuclease Msp1 and Mva1, respectively. Resulting fragments were analyzed by electrophoresis and visualized by ethidium bromide staining. Poor metabolizers of *3 mutation showed 168-, 82- and 20-bp bands, while those of *4 showed a single 355-bp band. Using these protocols, 22 individuals were genotyped, showing the following prevalence for *3 and *4: 0 and 3, respectively-comparable to those of the general population. This method provides a reliable means of genotyping CYP2D6*3 and *4.

Predicting onset and chronicity of women's problem drinking: a five-year longitudinal analysis.

BACKGROUND: Longitudinal studies of adult drinking have typically excluded or sampled only small numbers of problem drinking women, and have measured a limited range of influences on women's drinking behavior. METHODS: To study the development of women's problem drinking over time, five-year follow-up interviews were conducted with two groups of respondents from a 1981 national survey of women's drinking: 143 problem drinkers and 157 nonproblem drinkers. Regression analyses examined effects of 1981 predictors on six measures of 1986 problem drinking, for problem drinkers and nonproblem drinkers separately. RESULTS: Among 1981 nonproblem drinkers, predictors of onset of problem drinking indicators by 1986 included younger age, cohabiting, and lifetime use of drugs other than alcohol. The most consistent predictor of persistent (chronic) problem drinking was sexual dysfunction; other predictor included being employed part-time or never married, and experiencing recent depression. Divorce or separation predicted lower levels of subsequent alcohol dependance among problem drinkers. CONCLUSIONS: Findings suggest that different personal and social factors predict the onset of problem drinking as compared with its continuation, and point to nontraditional life-style, sexual dysfunction, and role deprivation as potentially important variables.