Affordable, Reliable Dual-Platform Approach to Quantitating Phosphatidylserine-Exposing Platelets in Platelet Components

Objective To compare the number of phosphatidylserine (PS)–exposing platelets obtained using the dual-platform approach and bead-based flow cytometry. Methods Platelets were enumerated using the ADVIA 2010i instrument (Siemens AG). The numbers and percentages of PS-exposing platelets in 175 platelet products were determined using a FACSCalibur flow cytometer (Becton, Dickinson and Company) and counting beads. Results Our results showed good correlation (r2 = 0.96; P <.001) between the PS-exposing platelets obtained using counting beads and the dual-platform approach. The results of Bland-Altman analysis showed a bias of +46,449 cells per µL and a limit of agreement (LOA) from −197,863 to 290,762 cells per µL. Also, 8 measurements (5.0%) revealed a number of PS-exposing platelets outside the LOA ranges. Further, 21 measurements (12.0%) revealed greater than 2-fold changes in the number of PS-exposing platelets. Conclusions The results suggest that the dual-platform approach is affordable and reliable for quantitating PS-exposing platelets as part of monitoring the quality of platelet products.

Evaluation of a Low-Cost Strategy for Enumerating CD4 Lymphocyte Absolute Count and Percentage Using the FACSCalibur Flow Cytometer in HIV-Infected Patients from a Resource-Limited Setting

Enumeration of CD4 lymphocytes is essential for the clinical management of HIV-infected patients, but it can be difficult to afford in developing countries. In this study we evaluated a reagent reduction strategy for reducing the cost of enumerating CD4 cell absolute count and percentage using the FACSCalibur flow cytometer (Becton Dickinson). We compared the protocol recommended by the manufacturer with a protocol that used half of the usual amount of CD3/CD4/CD45 monoclonal antibody reagent in 100 samples from HIV-infected patients in a rural hospital in India. The concordance correlation coefficient between the two protocols was 0.976 for CD4 cell count and 0.984 for CD4 cell percentage. We did not find significant bias when performing Deming regression or Bland-Altman analysis. Sensitivity and specificity were 97% and 98.5% for identifying patients with less than 200 CD4 cells/μL, 98.1% and 93.8% for identifying patients with less than 350 CD4 cells/μL, and 100% and 94.7% for identifying patients with less than 25% CD4 cells, respectively. This reagent reduction strategy can be used for reducing the cost of enumerating CD4 lymphocytes in high-volume laboratories from resource-limited settings.

Prognostic Significance of CD33, CD34 and CD117 Expression on Bone Marrow Blasts of Patients with Myelodysplastic Syndromes.

Introduction: Although blast-rich specimens immunophenotype studies in myelodysplastc syndromes (MDS) could associate bone marrow (BM) blast expression of CD7 and/or CD117 antigens with poor outcome (Ogata et al., Blood 2002), the prognostic role of markers of myeloid cell immaturity and committment in not enriched BM samples is largely unexplored. Patients and Methods: The expression of CD33, CD34 and CD117 antigens in not enriched BM samples of 50 newly diagnosed MDS was compared with both BM blast WHO category and IPSS score. Immunophenotyping was carried out by using the panel of quadruple monoclonal antibodies CD34/CD117/CD45/CD33, conjugated with the fluorochromes FITC, PE, PerCP, APC, respectively. Acquisition of information on 1x105 stained cells corresponding to the whole BM cellularity was assessed on a dual-laser FACSCalibur flow cytometer using the CellQUEST software (Becton Dickinson, San José CA USA). Multiple group comparisons were made using non parametric ANOVA for BM blasts; general linear model with Wald’s test and Kruskal-Wallis (KW) test to confirm significance was used for IPSS. Results: According to IPSS, 5 (10%) low risk, 27 (54%) intermediate risk-1, 14 (28%) intermediate risk −2 and 4 (8%) high risk pts were identified, respectively. The expression of CD33, CD34 and CD117 significantly correlated with both blast WHO category and IPSS, as shown in the Table 1. Interestingly, by analyzing the subset of 30 pts with BM blasts <5%, a correlation was found between IPSS and the expression of both CD33 (p=0.0097) and CD34 (p=0.0299); CD117 expression increased in pts with higher IPSS, without, however, reaching statistical significance. Conclusions: Both markers of myeloid cell committment (i.e., CD33 and CD34) and markers of myeloid cell immaturity (i.e., CD117) correlate with BM blast WHO category and IPSS score. Expression of CD33 and CD34 may have prognostic significance independently from blast percentage. The evaluation of CD33, CD34 and CD117 expression on not enriched BM samples may represent a simple tool for the prognostic assessment of MDS patients, in addition to previously established methods. Tab. 1: Correlation of CD33, CD34, CD117 expression with IPSS and percentage of BM blast in 50 MDS pts at diagnosis

Cell adherence-promoted activity of Plesiomonas shigelloides GroEL

Previously, it has been demonstrated that the invasion of Caco-2 cells by Plesiomonas shigelloides induces apoptotic cell death. Therefore, the attachment to and colonization of eukaryotic intestinal host cells by P. shigelloides are important steps in causing pathogenicity. In this study, the participation of P. shigelloides GroEL in the attachment of P. shigelloides was examined. The groESL operon of P. shigelloides was isolated by PCR. The nucleotide sequence of the groESL operon of P. shigelloides revealed two ORFs of 294 nucleotides for groES and 1647 nucleotides for groEL. Cell fractionation and immunostaining experiments suggested that the GroEL of P. shigelloides was associated with the bacterial cell surface. The expression of the groEL gene was upregulated during the attachment and apoptosis-induction stages, and the expression of the protein was also induced during the attachment stage. Furthermore, GroEL efficiently promoted the attachment of P. shigelloides to Caco-2 cells, as measured by a FACSCalibur flow cytometer. These results demonstrated that GroEL has a positive influence on the attachment of P. shigelloides to Caco-2 cells.