Clonality assessment of cutaneous B-cell lymphoid proliferations: a comparison of flow cytometry immunophenotyping, molecular studies, and immunohistochemistry/in situ hybridization and review of the literature.

Cutaneous lymphoid infiltrates are diagnostically challenging. Although ancillary techniques to assess clonality can help distinguish between reactive lymphoid hyperplasia and lymphoma, one of the most widely used techniques in hematopathology, flow cytometry immunophenotyping (FCI), has not been routinely applied to skin specimens. We performed FCI on 73 skin specimens from 67 patients clinically suspected of having a cutaneous B-cell lymphoma (CBCL) and compared the results with those obtained from immunoglobulin heavy chain (IGH) gene molecular studies (58 cases, primarily by polymerase chain reaction) and either immunohistochemistry (IHC) or in situ hybridization to evaluate for light chain restriction (22 and 2 cases, respectively). Sufficient quantity of CD45 (leukocyte common antigen)-positive cells and staining quality were achieved in 88% of cases by FCI, and clonality was detected in 68% of CBCLs versus molecular studies showing sufficient DNA quality in 74% and only 39% clonality detection, and interpretable/contributory IHC results in 84% of cases with 55% clonality detection. Clonality was documented more frequently in secondary rather than primary CBCLs by all 3 techniques. Therefore, FCI is feasible and appears to be more reliable than molecular studies or IHC/in situ hybridization for detecting clonality in CBCLs and can provide additional prognostically and therapeutically relevant information. The exception is cases with plasmacytic differentiation such as marginal zone lymphoma for which IHC might be a superior tool. We have also shown that a large subset of primary cutaneous follicle center lymphomas express CD10 and/or BCL2 by FCI. Recent advances in FCI beg the question of applicability to cutaneous T-cell and NK-cell lymphomas.

Cytokine expression profiles of immune imbalance in post-mononucleosis chronic fatigue.

BACKGROUND: As Chronic Fatigue Syndrome (CFS) has been known to follow Epstein-Bar virus (EBV) and other systemic infections; our objective was to describe differences in immune activation in post-infective CFS (PI-CFS) patients and recovered controls. We studied 301 adolescents prospectively over 24 months following the diagnosis of monospot-positive infectious mononucleosis (IM). We found an incidence of CFS at 6, 12 and 24 months of 13%, 7% and 4% respectively. METHODS: Using chemiluminescent imaging we measured the concentrations of IL-1a, 1b, 2, 4, 5, 6, 8, 10, 12 (p70), 13, 15, 17 and 23, IFN-γ, TNF-α and TNF-ß in duplicate plasma samples available in bio-bank from 9 PI-CFS subjects and 12 recovered controls at 24 months post-infection. RESULTS: Standard comparative analysis indicated significant differences in IL-8 and 23 across subject groups. In constructing a linear classification model IL-6, 8 and 23 were selected by two different statistical approaches as discriminating features, with IL-1a, IL-2 and IFN-γ also selected in one model or the other. This supported an assignment accuracy of better than 80% at a confidence level of 0.95 into PI-CFS versus recovered controls. CONCLUSION: These results suggest that co-expression patterns in as few as 5 cytokines associated with Th17 function may hold promise as a tool for the diagnosis of post-infectious CFS.

Design and analytic validation of BCR-ABL1 quantitative reverse transcription polymerase chain reaction assay for monitoring minimal residual disease.

CONTEXT: Monitoring minimal residual disease by quantitative reverse transcription polymerase chain reaction has proven clinically useful, but as yet there are no Food and Drug Administration-approved tests. Guidelines have been published that provide important information on validation of such tests; however, no practical examples have previously been published. OBJECTIVE: To provide an example of the design and validation of a quantitative reverse transcription polymerase chain reaction test. DESIGN: To describe the approach used by an individual laboratory for development and validation of a laboratory-developed quantitative reverse transcription polymerase chain reaction test for BCR-ABL1 fusion transcripts. RESULTS: Elements of design and analytic validation of a laboratory-developed quantitative molecular test are discussed using quantitative detection of BCR-ABL1 fusion transcripts as an example. CONCLUSIONS: Validation of laboratory-developed quantitative molecular tests requires careful planning and execution to adequately address all required analytic performance parameters. How these are addressed depends on the potential for technical errors and confidence required for a given test result. We demonstrate how one laboratory validated and clinically implemented a quantitative BCR-ABL1 assay that can be used for the management of patients with chronic myelogenous leukemia.

Are physicians ethically obligated to address hospice as an alternative to "usual" treatment of advancing end-stage disease?

Hospice care is ideally suited to meet the psychosocial and spiritual needs of dying patients, providing the opportunity to settle financial, property, and inheritance issues; to mend lacerations in important lifetime relationships, including forgiving and asking forgiveness; and to assure a degree of autonomous control over the environment and the social and spiritual processes that attend one's death. Physicians are not only imprecise in prognosticating a patient's time to die, they tend to be over-optimistic in their predictions. A "no" answer to the question, "Would I be surprised if this patient died in the next year?" is a reasonable starting-point for discussing hospice care as a potential treatment plan, now or in the future. Physicians have a duty to present palliative care in hospice as an alternative to the recurrent hospital interventions that are typical in the last six to 12 months of life tor patients who are failing and have declining prospects for one-year survival.

Validation of multiplex ligation-dependent probe amplification for confirmation of array comparative genomic hybridization.

The American College of Medical Genetics recommends that each laboratory should confirm abnormal or ambiguous results detected by array comparative genomic hybridization (aCGH). At present, the gold standard method for aCGH confirmation is fluorescent in situ hybridization (FISH). However, FISH is not well suited for small tandem duplications or very small deletions that are detectable by oligonucleotide arrays. Therefore, we developed and validated multiplex ligation-dependent probe amplification (MLPA) for aCGH confirmation. The method performance validation showed linearity through the expected analytical measurement range (0.05 to 2 genome equivalents). The interassay normalized coefficient of variation averaged 3.7% across 12 control and target probes. This low imprecision allowed detection of 20% mosaicism with exceptional confidence (P<0.006). Comparison with a combined gold standard of phenotype, aCGH, karyotype, and/ or FISH showed 100% concordance for 218 samples using an X/Y chromosome-specific probe set (95% confidence interval, 98.3%-100.0%). Patient-specific probe sets also showed 100% concordance to the gold standard for 18 genomic targets. In conclusion, we have developed and validated an MLPA assay using a novel approach to accommodate the fact that positive controls would not be available at the time of testing. We initially validated the MLPA method using X/Y chromosome-specific probes and well-characterized samples and then validated new probe sets by comparision with reference populations. We have successfully incorporated aCGH confirmation using custom-designed MLPA into our normal workflow, and used it for confirmation of all abnormal or ambiguous results.

Methylated arginine derivatives in children and adolescents with chronic kidney disease.

Asymmetric dimethylarginine (ADMA), a methylated L: -arginine (Arg) derivative is associated with endothelial dysfunction, vasoconstriction, and hypertension in animals and humans. We examined the relationship between these derivatives, estimated glomerular filtration rate (eGFR), and awake (AW) and asleep (AS) blood pressure (BP) load in children and adolescents (n = 28) with stage 2-3 chronic kidney disease (CKD) and in matched intra-familial controls (n = 10). Plasma L: -Arg, ADMA, and symmetric dimethylarginine (SDMA) levels were measured by high-performance liquid chromatography-tandem mass spectrometry. Subjects wore a 24-hr ambulatory BP monitor with BP load >95th percentile. ADMA, SDMA/ADMA ratio and SDMA were 38-200% higher in CKD patients while L: -Arg/ADMA and L: -Arg/SDMA ratios and the L: -Arg level were 11-64% lower. The eGFR explained 42-60% of L: -Arg/SDMA, SDMA/ADMA, and SDMA variability (n = 38). Using linear regression, SDMA and SDMA/ADMA separately explained 15-38% of AW and AS systolic (S) BP and diastolic (D) BP load variability (p < 0.001-0.022). Using multivariate stepwise regression with eGFR held constant, SDMA/ADMA was a significant independent variable for AW DBP load (p = 0.03). In conclusion, BP load and a disproportionate elevation of SDMA are seen in children and adolescents with stage 2-3 (mild-moderate) CKD. SDMA is a strong marker for reduced eGFR and serves as a moderate but significant indicator of 24-hr BP load variability.

Measurement of arginine derivatives in pediatric patients with chronic kidney disease using high-performance liquid chromatography-tandem mass spectrometry.

BACKGROUND: The arginine derivatives asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) interfere with endothelial nitric oxide synthesis. Plasma ADMA and SDMA have been shown to be risk factors for cardiovascular disease and/or kidney function deterioration in a variety of patient populations. METHODS: We developed a method to quantitatively measure arginine, ADMA, and SDMA using HPLC-tandem mass spectrometry. 13C6-L-Arginine was used as the internal standard, while the derivatives were separated on a silica column in less than 14 min. Plasma levels of ADMA, SDMA, and arginine were measured in children with stage II or III chronic kidney disease (CKD) and age- and gender-matched siblings. RESULTS: The chromatography exhibited no observable ion suppression in the patient specimens tested. There was no apparent carryover for any of the analytes. The assay was linear over 0.32-2.29, 0.23-4.43, and 1.00-303.89 micromol/L for ADMA, SDMA, and arginine, respectively. Plasma ADMA, SDMA, and arginine (mean+/-SD) were 1.10+/-0.35, 2.06+/-1.11, and 57.93+/-22.10 mumol/L for children with CKD, and 0.78+/-0.16, 0.71+/-0.23, and 65.29+/-21.30 micromol/L for the healthy siblings. CONCLUSIONS: The method exhibited no observable ion suppression in the patient specimens tested and has an acceptably short analytical cycle time. Children with CKD had higher levels of ADMA and SDMA than the healthy siblings.

Measurement of serum testosterone using high-performance liquid chromatography/tandem mass spectrometry.

Low levels of serum testosterone typically found in women and children cannot be reliably measured by immunoassay. We developed a simple and sensitive method using high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS). Sample preparation involved protein precipitation of serum (1.0 mL) with acetonitrile containing the internal standard (testosterone-d3) followed by liquid extraction with methylene chloride. The chromatographic cycle per specimen was 10 min. The performance was evaluated according to the CLSI EP10-A2 protocol. Within- and between-run imprecision was 20.9%, 2.29% and 1.80%, and 1.81%, 3.58% and 2.97% at mean concentrations of 0.17, 14.1 and 28.8 nM, respectively, with no apparent carryover. The method was linear from 0.21 to 53.1 nM and the analytical recovery was 100.5-106.2% across the concentrations tested. There was no interference observed from other steroids that were tested. Correlation using de-identified patient specimens with a commercial HPLC-MS/MS method showed a slope of 0.991, an intercept of -0.017 and a correlation coefficient (R(2)) of 0.998 by linear regression over concentrations ranging from 0.21 to 16.7 nM. In conclusion, we report here an HPLC-MS/MS method suitable for clinical measurement of serum testosterone.

Evaluation of an immunoassay of whole blood sirolimus in pediatric transplant patients in comparison with high-performance liquid chromatography/tandem mass spectrometry.

BACKGROUND: Sirolimus is widely used as an immunosuppressant, along with calcineurin inhibitors. Because of its variable pharmacokinetics and narrow therapeutic range, therapeutic drug monitoring of sirolimus is critical to optimize its therapeutic effect and to minimize toxicity. Although liquid chromatography/tandem mass spectrometry is considered the method of choice, the technical and financial challenges of this method are obstacles to its use. A microparticle enzyme immunoassay on the Abbott IMx has recently been reintroduced to the clinical diagnostic market. METHODS: We evaluated this immunoassay using high-performance liquid chromatography/tandem mass spectrometry as the reference method. Precision and carryover were evaluated using an expanded CLSI EP10-A2 protocol. Linearity was studied by serial dilution of high-level whole blood samples, and clinical utility was demonstrated by correlation with the reference method using 56 de-identified pediatric patient samples. RESULTS: The total imprecision was less than 12% across the concentrations tested. The method was linear from 2.6 to 31 nM. The immunoassay showed a mean positive bias of 11.5% in patient specimens relative to high-performance liquid chromatography/mass spectrometry (p<0.001), with a correlation coefficient (R) of 0.953. CONCLUSION: We conclude that the reintroduced immunoassay is useful for therapeutic drug monitoring of sirolimus.