Combined accurate platelet enumeration and reticulated platelet determination by flow cytometry

Abstract

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...

Detection of platelet vesicles by flow cytometry

“…The use of fluorescence probes, in particular fluorescence-labelled antibodies, can provide information on the abundance of specific molecular components of cells and their functional physiology. Platelets represent the smallest “cellular” material that most flow cytometers were generally designed to detect, and multicolor flow cytometry is a useful tool for the characterization of platelets and their receptor repertoires (6). …”

Issue Highlights—May 2018 (94B3)

“…No adverse side effects were encountered. Platelet numbers in this patient rose from 36 × 10 9 /L to 85 × 10 9 /L, and fell within the normal range . Concordantly, T regs increased from 3.5% of CD4 T cells to 10.2% after 2 weeks of IL‐2 treatment and stabilized at 9% by the end of treatment.…”

Reticulated Platelets in Egyptian Patients with Chronic Hepatitis C Infection

“…Many studies showed that RP measurement is a non-invasive test that represents a surrogate marker for megakaryocytic activity [13][14][15][16][17][18][19][20][21] . Therefore, the current study aimed to evaluate the RP% in patients with chronic hepatitis C as an index of platelet production and to evaluate the relationship between the RP% and the platelet count in those patients.…”

“…Flow cytometric analysis had been considered the preferred method of measuring RP [14,15] . Thiazole orange (TO), a nucleic acid dye, [16][17][18] remains a good choice for RP staining [15] .…”

“…Thiazole orange (TO), a nucleic acid dye, [16][17][18] remains a good choice for RP staining [15] . However, there is an evident lack of analytical standardization as there is a significant variability in the various published protocols regarding the incubation time, the used substrate (whole blood or platelet-rich plasma), concentrations of the fluorescent dyes, and the use of a second antibody marker to distinguish platelets, which yielded much variation in the published reference intervals of reticulated platelets percent (RP%) [19] .…”