BackgroundCirculating CD34+ endothelial progenitor cells (EPCs) are capable of differentiating into mature endothelial cells to assist in angiogenesis and vasculogenesis. We sought to quantify the numbers of apoptotic progenitors in patients with congestive heart failure.Methods and ResultsPeripheral blood mononuclear cells were isolated by Ficoll density-gradient from 58 patients with various degrees of heart failure and 23 matched controls. Apoptosis in progenitor CD34+ cells was assessed using the Annexin V-PE/PI detection kit, and FACS analysis was performed with triple staining for CD34, annexin-V and propidium iodide. The percentage of early and late apoptotic progenitor cells was determined in the subject groups and was correlated with clinical characteristics. While there was no significant difference in total CD34 positive cells or early apoptotic progenitors between control subjects and CHF patients (p = 0.42) or between severe and mild/moderate CHF groups (p = 0.544), there was an elevated number of late apoptotic progenitors in the severe CHF group compared with the mild/moderate CHF group (p =  0.03). Late apoptotic progenitors were significantly increased in CHF patients as compared to matched controls. There was also an inverse correlation between late apoptotic progenitors and ejection fraction (r = −0.252, p = 0.028) as well as a positive association with NYHA class (r = 0.223, p = 0.046).ConclusionSevere heart failure patients exhibited higher numbers of late apoptotic progenitors, and this was positively associated with NYHA class and negatively correlated with ejection fraction. This finding may shed light on the numerous factors governing the pathophysiology of CHF.

The acute phase response is accompanied by the appearance of aggregated red blood cells in the peripheral blood. The Westergren erythrocyte sedimentation rate (ESR) is an indirect measurement of this enhanced aggregability. We adopted a simple slide test and image analysis to reveal the adhesiveness/aggregation of red blood cells. A significant correlation was found between the erythrocyte adhesiveness/aggregation test (EAAT) and the ESR. A predictive model for ESR based on EAAT and the age of the patients was created. This new approach will enable us to obtain within a few minutes a good estimate of whether a given individual has a mild moderate or significant acute phase response. With further development, we will be able to use a bedside small cartridge that will deliver the extrapolated ESR at low costs and within a couple of minutes.

The results of this study could explain, at least in part, the differential adhesive behavior of the WBC in the peripheral blood in the two populations.

BackgroundEthyl-chloride (EC) spray was recently shown to be an effective antipruritic agent, when given 15 min after histamine skin-prick test (SPT), without changing the wheal and flare reaction. We aimed to investigate the antipruritic effect of EC on SPT, when given prior to SPT.MethodsA double-blind placebo-controlled prospective study. Overall, 44 volunteers underwent histamine SPT on both arms to trigger local pruritus. Prior to test, they were randomly treated with EC spray on one arm and saline spray (placebo) on the other. Subjects as well as researchers were blinded to the type of applied sprays. The wheal and flare reaction was measured after the SPT and subjects reported the intensity of pruritus following EC/placebo using a validated pruritus questionnaire (indexes 1–3) and a visual analog scale (VAS).ResultsSignificant improvement in pruritus was reported following treatment with EC compared with placebo for all four studied parameters. Index 1 in EC 3.7 ± 2.3 versus 5 ± 3.5 (p = 0.007) in placebo, index 2 in EC 2.6 ± 2.1 versus 3.8 ± 2.8 (p = 0.002) in placebo, index 3 of EC 6.3 ± 3.8 versus 8.8 ± 5.8 (p = 0.03) and VAS in EC 3.7 ± 1.9 versus 4.4 ± 2.3 (p = 0.003). There were no significant differences between EC and placebo in terms of the wheal and flare indurations area.ConclusionsEthyl-chloride has an effective antipruritic agent, when given before histamine SPT. Its use did not change the wheal and flare reaction, making it ideal for prevention of pruritus, secondary to allergy skin test, without masking the results.

IgVH mutational status and molecular cytogenetics have dramatically improved the ability to predict the prognosis of CLL patients. These tests, however, are highly sophisticated, complex and costly for routine use. ZAP-70, a syk family tyrosine kinase normally expressed in T cells, is a newly described marker which correlates with clinical progression and shorter survival in CLL. A flow cytometry assay to detect ZAP-70 described by Crespo et al (1), appears to be the simplest approach for routine clinical stratification in B-CLL. It is highly informative, and has a strong correlation between the expression of ZAP-70 in CLL cells and clinical outcome. However, in this analysis there are some technical aspects that should be improved to enable it to be standardized as a routine flow cytometry assay. ZAP-70 expression in B-CLL cells is not quantitative but assessed relative to its expression in the T- and NK cells (CD3+, CD56+). This approach can be problematic at times, as ZAP-70 levels in T cells vary in CLL patients as well as in normal controls, probably due to its up regulation following activation. An additional quandary in this assay is that all results are recorded relative to the subjectively delineated T-cell gate. Accordingly, small changes in expression in the T cells can significantly alter the results obtained in some B-CLL samples. In this study we aimed to improve the resolution of the assay by performing a quantitative analysis of ZAP-70 expression within the B-CLL cell population which is uncoupled from T cells. Blood samples were stained by the method described by Crespo et al (1) and ZAP 70 levels in B cell populations in CLL patients (CD19+CD5+) and in healthy volunteers (CD19+) were determined using a standard curve generated by an absolute fluorescent standard of FITC high levels beads with a range of 50–2000 x103 molecules of equivalent soluble fluorochrome (MESF) units per microsphere. Quantitation of expression levels were generated using Quick Cal V2.2 via www.bangslab.com. (Bangs laboratories). Using this analysis system the mean expression levels of ZAP 70 were calculated in healthy B cells (n=11) to be 11,177±1812 MESF units while in CLL (n=36) the mean value was >143,000 MESF units. To determine the reliability of this new method and its clinical relevance we compared our results to data generated using the analysis method of Crespo et al (1). We found a significant correlation between the two methods (r2 = 0.7558). Using ROC curve analysis with maximum sensitivity and specificity, our minimum positive value was found to be 46,700 MESF, with >95% sensitivity at 27,000 MESF and >92% specificity at 67,000 MESF and a Pearson correlation of 0.877 (P<0.0005). We conclude that this assay can provide a more reproducible and reliable analysis of Zap-70 expression in B-CLL, which is easily standardized. This analysis is highly specific as it is quantitative, not subjective and uncoupled from T cell activation in the sample.

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