Enumeration of CD34-positive stem cells: evaluation and comparison of three methods.

Accurate enumeration of CD34+ stem cells is important in assessing the need for continued mobilization and subsequent apheresis collections. We compared two new analysis systems, ProCOUNT (Becton Dickinson Immunocytometry Systems) and IMAGN 2000 STELLer (Biometric Imaging, Inc.) with our current (3-Color) flow cytometry-based method. The ProCOUNT system uses an absolute counting tube, which contains reference beads and a specific (multiple) gating strategy to determine an absolute count. The STELLer assay combines microvolume fluorimetry and automated analysis software to determine an absolute count. To evaluate linearity and reproducibility, peripheral blood was spiked with CD34+ cells (KG1a cell line). Three dilution series (measured at approximately equal to 0, 5, 10, 25, 50, and 100 CD34+ cells/microliter) were analyzed by each method. Analysis of predicted versus actual CD34+ concentration showed excellent correlation with all methods (r2 > or = 0.97, slope 0.98-1.04). To further assess precision, two PBSC samples, at approximately 200 and 800 CD34+ cells/microliter, respectively, were analyzed 10 times by each method. Coefficients of variation for the precision analysis of these samples were 5.1%-6.4% and 5.4%-12.3%, respectively. To assess overall performance, 75 patient specimens were analyzed. Excellent correlation (r2 values of 0.89-0.98) was observed among all three methods. We conclude that the three methods provide comparable linearity and reproducibility.

Post-transplantation lymphoproliferative disorder in the Epstein-Barr virus-naïve lung transplant recipient.

Post-transplantation lymphoproliferative disorder (PTLD) is a widely recognized and often catastrophic complication of organ transplantation. The incidence of PTLD after lung transplantation ranges from 6.2 to 9.4% and is two-fold higher than that seen after organ transplantation of other organs. Primary Epstein-Barr virus (EBV) infection is a major risk factor for PTLD, but the incidence of PTLD in EBV seronegative (EBV-) patients seems to vary with type of organ transplant. The goal of this study was to quantify the risk of PLTD based on pre-lung transplantation EBV serostatus in lung transplant patients. Pre- and post-lung transplant serostatus was defined in 80 patients, and our six cases of PTLD occurred in this group. Six of 94 lung transplant patients (6.4%) who survived > 1 mo developed PTLD. All cases of PTLD involved thoracic structures at presentation and occurred in the first post-operative year. Patients who were EBV- before lung transplant were much more likely to develop PTLD than those who were seropositive (EBV+) (five of 15 [33%] versus one of 60 [< 2%], p < 0.001). Consistent with the prevailing adult (donor) EBV+ rate (85%), two of our EBV-patients remained EBV-after lung transplant. Therefore, the rate of PTLD was 42% in those with primary EBV infection. As compared with EBV-patients that remained tumor-free, those who developed PLTD had similar levels of immunosuppressants and doses of anti-viral therapy. We conclude that PLTD occurs predominantly in EBV-naïve patients (risk approximately 1/3). EBV-patients should be monitored more closely after lung transplantation and, possibly, managed with lower immunosuppression. Our data also suggest that anti-viral therapy alone does not decrease the incidence of PTLD in high risk patients, PTLD can be successfully treated in most cases, and EBV-naïve patients should not be excluded from lung transplant because their risk of death from PTLD is < 15%.

Laboratory diagnosis of congenital syphilis by immunoglobulin M (IgM) and IgA immunoblotting.

We screened cord blood or serum samples from 101 infants at risk for congenital syphilis and serum samples from their mothers for immunoglobulin G (IgG), IgM, and IgA antibodies to Treponema pallidum by western blotting (immunoblotting). Clinical evaluation showed that six infants had signs and/or symptoms consistent with congenital syphilis. The sera from five of these infants were IgM blot positive, and four were IgA blot positive. Four asymptomatic infants had serologic evidence of congenital syphilis. The sera from three of these infants were IgM blot positive, and two were IgA blot positive. However, the IgM reactivity of the serum from one asymptomatic infant, which was also IgA positive, was abolished by protein G treatment. An IgM capture enzyme-linked immunosorbent assay corroborated the presence of IgM antibodies in six of seven IgM blot-reactive sera. Overall, for detection of symptomatic congenital syphilis, a sensitivity of 83% for IgM blotting and 67% for IgA blotting was obtained. The significance of positive IgM or IgA Western blots for asymptomatic infants requires further study to confirm infection in these infants.