Prediction of clear cell renal cell carcinoma by integrity of cell-free DNA in serum.

OBJECTIVES: To hypothesize that serum cell-free DNA integrity may be clinically useful for prediction of clear cell renal cell carcinoma (cRCC). The integrity of cell-free DNA released from cancer cells was different from that released from apoptotic cells. METHODS: We collected peripheral blood samples from 78 patients before surgery; among these patients, 22 with tumor, both pre- and postoperation, and 42 controls without tumor. After the column extraction of DNA, we performed conventional polymerase chain reaction using different sizes of primers (size of products: 109, 193, 397, and 456 bp) of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase for measurement of serum cf-DNA integrity. RESULTS: Age and gender were not associated with cf-DNA integrity in controls (P = .136 and P = .345). Among the cRCC patients, we observed no significant association between cf-DNA integrity and gender, age, tumor grade (P = .510, P = .618 and P = .052), except tumor stage and size (P = .001 and P = .001). All specimens contained 109 bp products. The 193 bp product was detected in 75 of 78 cRCCs and 37 of 42 controls (P = .091), 21 of 22 patients preoperation and 19 of 22 postoperation (P = .294). The 397 bp product was detected in 71 of 78 cRCCs and 0 of 42 of controls (P = .001), 18 of 22 patients preoperation- and 7 of 22 postoperation (P = .003). The 456 bp product was detected in 69 of 78 of cRCCs and 0 of 42 of controls (P = .001), 18 of 22 preoperation and 3 of 22 postoperation (P = .002). CONCLUSIONS: Serum cf-DNA integrity may be a potential tool for the detection of clear cRCC.

Enumeration of peripheral lymphocyte subsets using 6 vs. 4 color staining: a clinical evaluation of a new flowcytometer.

UNLABELLED: Technological advances in instruments allow the evaluation of many lymphocyte subsets in one step. The aim of this study was to evaluate the new FACSCanto flowcytometer in routine conditions, using a 6 color combination, single platform, whole blood, lysis, no wash protocol. METHODS: Two systems were simultaneously compared on 67 blood samples and external quality controls, using CD3,CD4, CD8, CD19, CD16/56, and CD45 in one tube TRUCOUNT beads (BD Biosciences) or two tubes (TetraChrome and Flowcount, Beckman-Coulter and DakoCytomation). RESULTS: The day-to-day instrument detection but automatic compensations were stable. Manual compensation settings were satisfactory using available facilities. Commercial and UK NEQAS quality control results were acceptable. The intra-experiment reproducibility was good (coefficient of variation (CV)<3%) but highly operator-dependent (CD4+ T cell count CVs from 1.2 to 9.7, six operators). Storage of samples was acceptable, but storage of stained samples altered absolute count reliability. Serial dilutions show a good count accuracy. The FACScanto T subsets and B cell data were highly correlated with our reference values (r2>0.87) and absolutes values were very close (slopes>0.89). The gating strategy, fluorochrome choice, and compensation setting are discussed. A few improvements are expected (sample loader, data management, auto-gating, acquisition parameters, sample mixing, absolute values calculation, etc). In conclusions, despite its complexity, 6 color staining is a reliable, stable, and highly informative technique for lymphocyte subset monitoring but remains to be optimized.

Significance of unconventional peripheral CD4+CD8dim T cell subsets.

Routine T cells phenotyping occasionally reveals a CD4+CD8dim T cell subset with an apparently homogeneous dot plot. The aim of this study was to elucidate their immunological significance from analysis of 31 healthy donors, 21 elderly and 220 immune deficient patients. CD4+CD8dim T cells expressed reduced levels of CD8 (11-17,000 compared to 96-128,000 mol/cell on CD8+ T Cells). CD4 was expressed at the same level as on CD4+ T cells. The occurrence of raised CD4+CD8dim T cells (> 20 cells/muL) was similar in kidney transplant recipients (28.4%) and healthy donors (26%). It was somewhat lower in HIV+ patients (19.7%) possibly due to virally induced CD4+ T lymphopenia. However, an age effect is possible because the occurrence was raised (33.3%) in 70 volunteers (chi2 test NS). On the other hand, the size of the CD4+CD8dim subset was not correlated with age. CD4+CD8dim T cells did not express the activation markers CD69 (n = 220) or CD25 (n = 10) and expressed the homodimeric (alphaalpha) isoform of CD8, suggesting they are related to mucosal immunity (MALT). We selected 29 patients with unambiguous dot plots. In 26 of them one predominant TCR Vbeta clonotype was expressed on 18 to 94% of CD4+CD8dim T cells and never on more than 10% of conventional T cells. The predominant clonotypes were Vbeta8 (n = 5), Vbeta2 (n = 4), Vbeta13.1 and Vbeta 21 (n = 3 each). Whether this reveals a chronic stimulation or an emerging lymphoproliferative disorder must be elucidated. We propose to name this entity: "Oligoclonal Clonopathy of Undetermined Significance (OCUS)."