Background: Diagnosing the cause of thrombocytopenia often requires a bone marrow aspiration or biopsy, an invasive procedure. Reticulated platelets (RP) are immature RNA containing platelets, accurate RP enumeration has yet to be achieved, partially due to the lack of a robust reference method. Goal: To refine previous work and gating strategies distinguishing RP from mature platelets while incorporating accurate platelet enumeration into the analysis. After reviewing previously published studies on Thiazole Orange (TO) staining of RP, we systematically evaluated CD41/CD61 in combination with a commercial source of TO (BDBiosciences). Previous RP methods have not taken advantage of platelet enumeration therefore our goal was to incorporate the ICSH platelet enumeration protocol into our method.Methods: TO concentration, incubation, and fixation method were determined to be 10% of stock concentration, 30 min, and 1% formaldehyde respectively. Gating strategy to determine RP fraction used an unstained control tube to set the limit of TO staining.Results: Normal range (n = 51) was 9.9 6 3.1%. Analysis of 40 patients with immunethrombocytopenia-purpura (ITP) showed a RP range from 4.3% to 81.2%. Platelet enumeration was consistent with our previous studies in this area. INTRODUCTIONReticulated platelets (RP), first recognized in 1969 (1), are young, RNA-containing platelets, freshly released from the bone-marrow into the peripheral circulation: RP are analogous to the reticulocytes of erythropoiesis. Subsequent studies showed RP were readily stained with nucleic acid dyes such as Thiazole Orange (TO) (2-4) and that RP have obvious potential as a sensitive early predictor of bone marrow engraftment (5,6). However difficulties with consistency of analysis have hampered wide-spread adoption. The nucleic acid dye based flow methods are problematic to control and these difficulties with specificity and reproducibility have prevented routine adoption of RP counts in the clinical setting. Though automated methods have been available on some Sysmex and Abbott hematology analyzers for some time, there are distinct methodological differences between them, which has further contributed to slow adoption of the assay by clinicians (7-10). The lack of consensus over the selection of dye (11-13), the temperature and duration of incubations (13-15) and the use of whole blood (16) versus platelet rich plasma (14) has further contributed to the slow adoption of this promising measurement. Previous studies determining RP have used various methods for gating this immature fraction of platelets. Work by Ault et al. (3) and Kienast and Schmitz (2) evaluated TO as a stain for RP, as the ability to enter cells without pretreatment and large increase in fluorescent emission upon binding made it ideal. Studies in murine and human samples demonstrated that this was predominately due to RNA content. However, TO fluorescence in platelets is not entirely due to binding of dye to mRNA: Robinson et al. (17) demonstrated a proportion of fluores...

T lymphocytes may play a central role in MS. The search for more targeted immunosuppression than is currently available has led to recent clinical trials of novel therapeutics. We studied 29 patients in a double-blind placebo-controlled trial of the chimeric monoclonal anti-CD4 antibody, cM-T412 (Centocor, Leiden, Holland) over a period of 18 months. Total and differential WBC counts; T, B, and natural killer lymphocytes; CD4+ and CD8+ T cells; CD4+ and CD4- naive cells; CD4+ and CD4- memory cells; interleukin-2 receptor- and major histocompatibility class II-positive T cells; serum tumor necrosis factor alpha (TNF-alpha); and PHA (phytohemagglutinin)/LPS (lipopolysaccharide)-stimulated whole blood TNF-alpha production were all examined serially in peripheral blood for the duration of the trial. In addition, for the first two treatment cycles, the above variables were tested 1 and 7 days after treatment. The results demonstrated significant long-term reductions, lasting up to 12 months after the last treatment cycle in all CD4+ subsets studied, but with a relative preservation of CD4+ memory cells as opposed to CD4+ naive cells. CD4- subsets also showed significant reductions after treatment but returned to baseline levels within 7 days. Monocyte counts were unaffected by cM-T412. Serum TNF-alpha and 2- and 18-hour PHA/LPS-stimulated TNF-alpha levels were also unchanged in the long term, although significant increases were observed in the 2- and 18-hour PHA/LPS-stimulated TNF-alpha levels the day immediately after treatment. There was no significant correlation between any of the immunologic markers studied and MRI measures of disease activity.

Background Identification of human T‐helper cell subsets is possible by measurement of intracellular cytokines after coincubation of lymphocytes with phorbol myristate acetate (PMA), calcium ionophore, and brefeldin A for up to 20 h. However, exposure to PMA leads to internalization of membrane CD4 and to loss of resolution of the CD4+ cells. Detection of CD3+CD8‐ cells or preselection of CD4+ cells prior to stimulation is more cumbersome than direct measurement of CD4+ cells. We report the use of the Leu3a/Leu3b multiclone for the accurate determination of CD4 cells after PMA stimulation. Methods Peripheral blood lymphocytes were isolated from healthy normal donors and the proportion of CD3+ / CD4+ T cells was determined by flow cytometry before and after incubation with PMA, calcium ionophore, and brefeldin A for 20 h using a variety of anti‐CD4 monoclonal antibodies. Results The Leu3a/3b multiclone reagent was the only anti‐CD4 monoclonal antibody capable of resolving more than 98% of the initial CD4+ events after incubation with PMA. Conclusions The higher signal‐to‐noise ratio associated with Leu3a3b reagent, compared with other CD4‐specific antibodies available, allows the direct and accurate identification of the CD4 subset even after PMA treatment of cells. Cytometry 44:148–152, 2001. © 2001 Wiley‐Liss, Inc.