Percentage Of Reticulated Platelets Research Articles

Evaluation of darbepoetin therapy on platelet count and function in healthy cats and cats with surgically induced chronic kidney disease.

To evaluate the effects of darbepoetin on platelet population and reactivity in healthy cats (HCs) and azotemic cats with remnant kidney (RK) model-induced chronic kidney disease. 12 purpose-bred domestic shorthair cats (n = 6 HCs and n = 6 RK). In this pilot study, all cats received darbepoetin (1 µg/kg, SC) on days 0, 7, and 14. Blood was sampled at baseline and on days 3, 10, 15, 17, 20, and 21. At each time point, a CBC was performed, platelet aggregometry was assessed by impedance and optical methods, and platelet P-selectin (CD62P) was quantified before and after thrombin stimulation. Additionally, reticulated platelets were quantified using both thiazole orange staining and proprietary analysis by the CBC analyzer. For RK cats, systemic blood pressure (BP) was serially measured. No adverse effects of darbepoetin were seen. There was no statistically significant change in platelet count between or within groups at any time point. Hematocrit increased significantly over time in the RK but not the HC group. RBC reticulocyte numbers in both groups increased over time. Reticulated platelet percentage did not increase in either group. Differences in platelet reactivity within or between groups were not seen in the aggregometry or flow cytometric assessments. In RK cats, indirect BP did not significantly change during the study. This preliminary investigation did not find evidence that darbepoetin administration impacted platelet number, reactivity, nor reticulated platelet count. Anemic RK cats experienced increased hematocrit and RBC reticulocytes as expected with darbepoetin therapy.

Autoimmune-mediated thrombocytopenia after allogeneic hematopoietic stem cell transplantation: significance of detecting reticulated platelets and glycoprotein-specific platelet autoantibodies

Autoimmune hematological disorders are rare complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Diagnosis of immune thrombocytopenia (ITP) is challenging, especially after allo-HSCT, because various complications such as graft-versus-host disease, disease relapse, viral infection, thrombotic microangiopathy, and drug side effects can also cause thrombocytopenia. Assessment of reticulated platelets (RP) and plasma thrombopoietin (TPO) levels may be useful to distinguish between ITP and hypoplastic thrombocytopenia. ITP is generally characterized by an increased percentage of RP, and a normal or slightly increased plasma TPO level. We now report three cases of thrombocytopenia after allo-HSCT. RP% was elevated in these patients, as it is in primary ITP. However, in contrast to primary ITP, plasma TPO levels were high in two of three patients. Anti-αIIbβ3 and anti-GPIb/IX-specific direct IgG antibodies were detected as well, suggesting occurrence of immune-mediated platelet destruction in addition to bone marrow suppression in two patients. All three patients were successfully treated with corticosteroids and/or thrombopoietin receptor agonists (TPO-RAs). These results suggest that increased RP% and detection of glycoprotein-specific platelet autoantibodies are useful for the diagnosis of ITP after HSCT.

Platelet reticulated forms, size indexes and functional activity. Interactions in healthy volunteers.

Reticulated platelets (RP) are young, functionally active platelet forms which are detected by RNA staining. Their content in the circulation reflects the intensity of bone marrow thrombocytopoesis. The aim of this study was to assess in healthy volunteers the relationship between RP percentage and platelet size and activity. RP were quantitated by thiazole orange staining using flow cytometry. Platelet size indexes included mean platelet volume (MPV), platelet large cell ratio (P-LCR) measured in a Coulter type hematological analyzer and forward scattering (FSC) measured in a flow cytometer. Platelet functional activity was evaluated by expression of activated glycoprotein (GP) IIb-IIIa (PAC-1 antibody binding) and P-selectin with the use of flow cytometry. Platelets were activated by thrombin receptor activating peptide (TRAP) (10 and 1 µM) and ADP (20 and 2.5 µM). The percentage of RP in healthy volunteers varied from 2.9% to 23.8% (mean ± SD ‒ 11.7 ± 4.7%, n = 99) and correlated with all platelet size indexes: MPV, P-LCR and FCS (r from 0.452 to 0.529, p < .001, n = 87–99). On average, RP were distributed at a ratio of 9:1 between 50% subpopulations of large and small platelets according to their FSC index. Expression of GP IIb-IIIa activated form correlated with RP percentage and platelet size indexes when platelets were activated by TRAP and ADP at both applied concentrations (r from 0.309 to 0.560, p from 0.014 to < 0.001, n = 50–62). P-selectin expression correlated with RP percentage and platelet size indexes when platelets were activated by 10 µM TRAP inducing maximum expression of this activation marker (r from 0.332 to 0.556, p from 0.008 to < 0.001, n = 65), but not by weaker agonists: 1 µM TRAP, 20 and 2.5 µM ADP (r < 0.3, n = 54–66). Thus, high RP content in healthy volunteers is associated with increased platelet size and activity in the whole platelet population.

Correlation between Percentage of Reticulated Platelets and Heart Score in Patients with Suspected Non-ST Elevation Acute Coronary Syndromes

Thrombus formation in non-ST Elevation Acute Coronary Syndrome (NSTE-ACS) causes increased plateletconsumption, leading to a 20-fold increase of Reticulated Platelets (RP) release. Reticulated platelets have more granulesand proteins that make them quickly forming thrombus than mature platelets, potent to form bigger thrombus, andincrease the risk of Major Adverse Cardiac Events (MACE). HEART score is a risk stratification for possible NSTE-ACS, whichcan predict MACE. The study aimed to analyze the correlation between the percentage of reticulated platelets and HEARTscore. This research was a correlation observational cross-sectional study performed in Dr. Hasan Sadikin Hospital,Bandung, from August 2018 to May 2019. The subjects were patients suspected with NSTE-ACS by clinicians in theEmergency Department of Dr. Hasan Sadikin Hospital. These subjects were assessed for the HEART score andRP percentage. This study involved 52 subjects consisting of a higher number of males (76.9%) aged 45-64 years old (69.2%).HEART score stratification in this study was mostly high risk (69.2%), but none was low risk. Mean of platelet count, absolute3 3 RP, and RP percentage were 271±73 x103/mm , 9.3±4.3 x 103/mm , and 3.6±1.7%, respectively. The correlation testbetween RP percentage and HEART score with a 95% confidence interval using Spearman's correlation test showed asignificant positive correlation with moderate strength (p &lt; 0.001 and r=0.475). The percentage of RP in this study was in thenormal range. However, there was a significant positive correlation with moderate strength between the percentage of RPand HEART scores in patients with suspected non-ST elevation acute coronary syndrome.

Patients with Essential Thrombocythemia may be Poor Responders to Enteric-Coated Aspirin, but not to Plain Aspirin.

Essential thrombocythemia (ET) patients are treated with aspirin (acetylsalicylic acid [ASA]) to prevent thrombosis. Previous studies showed that serum thromboxane (Tx) B2 was high 24 hours after enteric-coated (EC)-ASA in ET patients, due to increased number of noninhibited reticulated platelets (RPs), consequent to high platelet turnover, and that ASA should be given twice a day to ET patients. We studied ET patients (n = 17) and healthy subjects (n = 10) on 100 mg EC-ASA once daily; experiments were repeated after 14-day treatment with 100 mg plain-ASA once daily. Serum TxB2, plasma ASA, and salicylic acid (SA) were measured before the morning dose and up to 8 hours thereafter. Blood activity of ASA-deacethylating esterases, in vitro inhibition of collagen-induced TxB2 production by ASA (10-1,000 µM), and number of RP were measured. TxB2 inhibition by ASA in vitro and esterases activities were normal in all subjects. EC-ASA elicited highly variable responses; 6 ET patients were poor responders, as their serum TxB2 was high after EC-ASA; their plasma levels of ASA and SA were low/undetectable. In contrast to EC-ASA, plain ASA decreased serum TxB2 and increased plasma ASA and SA in all subjects. Serum TxB2 was high in ET patients at 24 hours and significantly correlated with RP count (but not RP percentage) and platelet count. Plain ASA should be used in ET patients to inhibit platelets efficiently. The identification of ET patients who might benefit from twice a day ASA could simply be based on their platelet count: since their platelet turnover is not increased, ET patients with normalized platelet count should not need twice a day ASA treatment.

Immature Platelets As a Predictor of Disease Severity and Mortality in Sepsis and Septic Shock: A Systematic Review.

Sepsis is associated with high morbidity and mortality, and short-term mortality remains above 30% despite relevant supportive and antibiotic treatments. The aim of this systematic review was to summarize and discuss the current evidence of the association of an increased number of circulating immature platelets with disease severity and mortality in patients with sepsis or septic shock. The review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement and was registered at the PROSPERO database (registration number: CRD42018104326). A systematic literature search was performed in PubMed, Web of Science, Scopus, and Embase on June 20, 2018, without time restrictions. The included studies were quality-assessed by the National Institutes of Health's Quality Assessment Tools. In total, 14 studies were included. The parameters used for the determination of platelet maturity were mean platelet volume, immature platelets fraction, reticulated platelet percentage, and absolute immature platelets count. Nine studies reported significantly increased immature platelet markers in nonsurvivors of septic shock compared with survivors, as well as in patients with severe sepsis or septic shock compared with patients without severe sepsis and septic shock. Six of these nine studies demonstrated that increased immature platelet markers were predictors of mortality and/or disease severity (area under the receiver operating curve: 0.599-0.886). This review suggests that an increased number of circulating immature platelets is associated with increased disease severity and mortality in patients with sepsis and septic shock. Larger studies are needed to confirm whether immature platelets should be routinely monitored to support the prediction of disease severity and mortality in septic patients.

Impact of Atrial Fibrillation and Sinus Rhythm Restoration on Reticulated Platelets

ObjectiveTo assess the impact of nonvalvular atrial fibrillation (NVAF) and sinus rhythm restoration on the distribution of reticulated platelets (RPs), which are known to be associated with thrombotic propensity and have a greater predilection for thrombus participation. Participants and MethodsThe RP content was assessed by flow cytometry (thiazole orange/CD61) in 110 consecutive patients with NVAF before and 3 to 4 months after catheter ablation of the pulmonary veins. Results were compared with those of 55 age- and sex-matched controls with normal sinus rhythm. ResultsThe mean ± SD percentage of RPs was higher in patients with NVAF compared with controls (28.5%±7.3% vs 6.4%±5.3%; P<.001). The RP content did not vary by CHA2DS2-VASc score. After catheter ablation of the pulmonary veins, 63 patients were available for follow-up assessment. A significant reduction of RPs was observed compared with preintervention values (29.85%±7.1% vs 20.79%±7.6%; P<.001). During follow-up, 19% of patients (12 of 63) had confirmed AF recurrence. The mean ± SD percentage of RPs was higher in this group than in those without a recurrence (24.7%±6.5% vs 18.9%±7.5%; P=.01). ConclusionNonvalvular atrial fibrillation affects the percentage of RPs, independent of the CHA2DS2-VASc score. After ablation, RP content dropped significantly. High RP content in patients with NVAF may explain the potential mechanism of thromboembolic complications and the lack of efficacy of currently available antiplatelet therapy for stroke prevention in this dysrhythmia.

Issue Highlight 2015.

Issue Highlight 2015.

Evaluation of reticulated platelets in dogs with breed-related thrombocytopenia.

The aim of this study was to evaluate the percentage of reticulated platelets in healthy dogs with breed-related thrombocytopenia. Seventy two dogs, clinically healthy, were enrolled in the study. Blood was collected from the patients and anticoagulated with tripotassium ethylenediaminetetraacetic acid (K3-EDTA) and sodium citrate. Platelet count was obtained by an impedance haematology analyser and platelet morphology was evaluated by examination of blood smears. Patients were allocated into two groups. Group 1 consisted of 30 dogs with normal platelet count, whereas group 2 was composed of 42 dogs with thrombocytopenia. Thrombocytopenia was present in both K3-EDTA and citrate blood samples. Patients with thrombocytopenia were divided into two subgroups: the first subgroup included dogs with platelet count in K3-EDTA anticoagulated blood from 100 to 200 x10(9)/L, patients in the second subgroup had a platelet count of less than 100 x10(9)/L. The percentage of young reticulated platelets (RPs) labelled with thiazole orange, and the percentage of platelets coated with platelet surface-associated IgG, were determined in platelet-rich plasma (PRP) by a flow cytometer. The mean percentage of RPs in K3-EDTA and citrate PRP was significantly higher in dogs with thrombocytopenia than in dogs with normal platelet count. The mean percentage of RPs was significantly higher in citrate PRP than in K3-EDTA PRP in all groups. The results suggest that idiopathic, asymptomatic thrombocytopenia is not caused by platelet surface-associated IgG. Dogs with breed-related thrombocytopenia have a competent bone marrow.

New insights into thrombopoiesis in neonatal sepsis

Thrombocytopenia is one of the most frequent hematologic abnormalities in the neonatal period, affecting about 18–35% of all patients admitted to the Neonatal Intensive Care Unit (NICU), with sepsis being among the most common causes of severe neonatal thrombocytopenia. It is unclear whether decreased platelet production or increased platelet consumption contributes to thrombocytopenia of septic neonates. To answer this question, we evaluated the effects of sepsis on neonatal thrombopoiesis using a panel of tests. This prospective case-control study was conducted on 50 neonates with culture-proven sepsis admitted to NICU at the Pediatrics Department, Ain Shams University Hospitals. Thirty healthy newborns were included as controls. The enrolled neonates were subjected to detailed history taking, thorough clinical examination, and laboratory investigations including complete blood count, C-reactive protein, blood cultures, and tests of thrombopoiesis; namely serum thrombopoietin (TPO) assay, flow cytometric analysis of reticulated platelet percentage (RP%), and calculation of absolute RP counts. Septic neonates comprised 24 males and 26 females with a mean gestational age of 36.0 ± 3.1 weeks. Twenty-eight (56%) of the septic neonates were thrombocytopenic (platelets < 150 000/µl). While platelet and RP counts were decreased, TPO and RP% were increased in septic neonates compared to healthy controls. Neonates with Gram-negative sepsis had the lowest platelet and RP counts and the highest TPO and RP% followed by those with fungal septicemia. Platelet counts showed inverse correlations with TPO and RP% and direct correlation with RP count. Our findings suggest that neonates respond to sepsis by up-regulating thrombopoiesis, where thrombocytopenia ensues when the rate of platelet consumption exceeds the rate of platelet production. Simultaneous measurements of serum TPO levels and RP% are helpful in discriminating hyperdestructive from hypoplastic thrombocytopenia among septic neonates.

Reticulated Platelets and Platelet Reactivity in Renal Transplant Recipients Receiving Antiplatelet Therapy

Introduction Renal transplant recipients are at increased risk of cardiovascular morbidity and mortality. We assessed platelet reactivity and reticulated platelets (RPs) in 90 recipients, 51 (56.6%) of whom were not receiving acetylsalicylic acid (ASA) therapy (group A) and 39 (43.3%) who were receiving ASA therapy, 100 mg (group B), and in 60 healthy controls (group C). Methods Reticulated platelets were measured using a hematology automated analyzer (XE-2100; Sysmex Corp, Kobe, Japan) and were expressed as the percentage of RPs in the total optical platelet count (immature platelet fraction [IPF]), as the percentage of highly fluorescent RPs, and as the absolute number of RPs (IPF#). Platelet function was assessed using optical aggregometry (platelet aggregation) induced using 1 mmol/L of arachidonic acid, 2 or 10 μmol/L of adenosine diphosphate, or 2 μg/mL of collagen. Results Group A demonstrated significantly higher values of RP compared with group B or group C. Group B demonstrated a substantially higher percentage of RPs compared with group C, which was significant only for the IPF parameter. Multiple regression analysis demonstrated that IPF and IPF# were significantly and positively related to collagen-induced platelet aggregation. Conclusion We documented the presence of higher concentrations of RPs in transplant recipients compared with a control population, and a significant association between RPs and platelet function.

High platelet turnover and reactivity in renal transplant recipients patients

Renal transplant recipients (RTRs) patients are at increased risk of cardiovascular morbidity and mortality. We aimed this study to assess reticulated platelets (RP), platelet reactivity and von Willebrand factor (vWF) levels in RTRs patients. In 150 RTRs patients [84 (56%) not on acetylsalicylic acid (ASA) treatment, group A; 66 (44%) on ASA 100 mg treatment, group B] and in 60 healthy control subjects, RP were measured by a Sysmex XE-2100 and were expressed as the percentage of RP of the total optical platelet count (immature platelet fraction; IPF), as the percentage of RP highly fluorescent (H-IPF) and as the absolute number of RP (IPF#). Platelet function was assessed by optical aggregometry (PA) induced by 1 mmol arachidonic acid (AA-PA), 2 and 10 μM ADP (ADP2-PA and ADP10-PA) and 2 μg/ml collagen (Coll-PA). vWF levels were measured by using a miniVidas analyser. Group A and group B showed significant higher values of RP than controls. At a multiple linear regression analysis IPF and IPF# were significantly and positively related to collagen-PA. By analysing group B according to residual platelet reactivity (RPR), we observed a significant higher number of RP among patients with RPR by collagen. Moreover at a multiple logistic regression analysis, IPF# significantly affected the risk of having a RPR by collagen. With regard to vWF, RTRs patients showed higher levels than control subjects. We documented a higher platelet turn-over in both groups of RTRs patients and increased platelet reactivity in RTRs patients not on ASA therapy than controls.

Reticulated platelet monitoring after autologous peripheral haematopoietic progenitor cell transplantation

Reticulated platelets are the youngest marker of megakaryopoiesis. We monitored 13 haematological patients undergoing autologous peripheral haematopoietic progenitor cell transplantation. We used a flow cytometric method based on thiazole orange platelet staining. Patients showed a fall in reticulated platelet percentage at a mean of 5 days before a nadir of the platelet count. In all cases, platelet recovery is preceded by a rise of reticulated platelets; in 10 cases patients received platelet prophylactic transfusions after RP rise. The rise in reticulated platelets is an early sign of engraftment and may represent an easy parameter for transfusion management of transplanted patients.

Platelet turnover, coagulation factors, and soluble markers of platelet and endothelial activation in essential thrombocythemia: Relationship with thrombosis occurrence and JAK2 V617F allele burden

Patients with essential thrombocythemia (ET) have an increased frequency of thrombosis, but the relationship of both thrombosis and JAK2 V617F allele burden with platelet turnover, acquired activated protein C resistance (aAPCR), and levels of coagulation factors and soluble markers of platelet, and endothelial activation is not well known. In 53 ET patients (26 with a history of thrombosis), reticulated platelets (RP) percentage, aAPCR, platelet tissue factor (TF) expression, and plasma levels of TF, coagulation factors, soluble P-selectin (sP-selectin), soluble CD40 ligand (sCD40L), von Willebrand factor antigen (VWF:Ag), soluble thrombomodulin (sTM), D-dimer and prothrombin fragment 1 + 2 were compared with those in matched healthy individuals and correlated with thrombosis occurrence and JAK2 mutational load. ET patients with thrombosis had significantly higher values for RP percentage, aAPCR, and levels of factors V and VIII, VWF:Ag, sP-selectin, and sCD40L than patients without thrombosis and controls. At multivariate study, RP percentage, factor V levels, and aAPCR were independently associated with an increased risk of thrombosis. Patients with JAK2 mutation had significantly lower levels of free protein S (PS) and higher levels of TF, sP-selectin, sCD40L, VWF:Ag, and sTM than those with wild-type allele. A mutant allele dosage effect (>or= 12%) was observed for TF, sP-selectin, sCD40L, VWF:Ag, and PS levels. These results support a role for platelet turnover, factor V, and aAPCR in the thrombosis of ET as well as the association between JAK2 V617F allele burden and either decreased free PS or increased TF and soluble markers of platelet and endothelial activation.

Reticulated platelets as a marker of platelet recovery after allogeneic stem cell transplantation

Reticulated platelets (RP) are the youngest forms of platelets in blood and reflect the rate of bone marrow platelet production. In the present study, we used flow cytometric analysis to determine the percentage of RPs in patients undergoing allogeneic stem cell transplantation. We investigated 10 patients after transplantation from HLA identical siblings: five with acute myeloid leukemia (AML), four with chronic myeloid leukemia (CML), and one patient with myelodysplastic syndrome (MDS). Of the patients examined, four patients underwent allogeneic bone marrow transplantation and six patients underwent peripheral blood stem cell transplantation. It was observed that the initially reduced percentage of RPs (2.9 +/- 1.7%; mean +/- SD) was significantly higher (P = 0.0109) in all patients (13.6 +/- 6.4%) in the following 10-26 days. The RP percentage peak preceded the recovery of peripheral platelet count up to 45.6 x 10(9)/l on average by 3 days. We found no difference in RP% between the AML and CML patients but we did observe that in CML patients the RP percentage increased on average 7 days earlier than in AML patients. The elevated RP percentage reflects increased bone marrow regeneration and can be considered an additional marker of thrombopoietic recovery in the patients undergoing allogeneic stem cell transplantation.

Reticulated platelets as a screening test to identify thrombocytopenia aetiology

Thrombocytopenia is a common haematological abnormality and no simple diagnostic test is available to diagnose thrombocytopenia pathogenesis. To evaluate sensitivity and specificity of reticulated platelets (RP) as a diagnostic test for thrombocytopenia with increased thrombopoietic activity. Prospective observational study in thrombocytopenic patients. A direct, whole-blood, dual-labelling flow cytometric method was used. Direct, whole-blood double coverage was achieved using a monoclonal anti-glycoprotein (GP)-III antibody (CD61 PerCP) for platelet identification and thiazole orange (Retic-count) as platelet mARN stain. RP were measured in 101 thrombocytopenic patients and 104 non-thrombocytopenic controls. The mean RP percentage in 60 thrombocytopenic patients with no increased thrombopoietic activity was 7.5% (CI for 95%: 5.2-9.7) and RP absolute number was 3.2 x 10(9)/l (CI for 95%: 2.1-4.3). The mean RP percentage in 41 thrombocytopenic patients with increased thrombopoietic activity was 30.3% (CI for 95%: 25.1-35.5) and RP absolute number was 6.2 (CI for 95%: 4.8-7.7). The RP percentage cut-off for a diagnosis of thrombocytopenia with increased thrombopoietic activity was 11% [sensitivity 93%, specificity 85%, positive predictive value (PPV) 83%, negative predictive value (NPV) 95%]. RP measurement by flow cytometry, directly from whole-blood, is a useful screening test to differentiate between thrombocytopenia with high or low thrombopoietic activity. A RP percentage in excess of 11%, has a high sensitivity and good specificity for a diagnosis of thrombocytopenia with increased thrombopoietic activity.

Relationship between high platelet turnover and platelet function in high-risk patients with coronary artery disease on dual antiplatelet therapy

A high platelet turnover rate produce a population of immature reticulated platelets (RP) that could confer, despite of antiplatelet drugs, a residual platelet reactivity (RPR) in coronary artery disease (CAD) patients. To assess the influence of RP on platelet reactivity in CAD patients on dual antiplatelet therapy we measured RP in 372 patients by using the Sysmex XE-2100 haematology analyzer and platelet function by optical platelet aggregometry (PA) on platelet-rich-plasma induced by 1 mmol arachidonic acid (AA-PA) and 10 microM ADP (ADP-PA). RPR was defined as either AA-PA>20% or ADP-PA>70%. RP were expressed as a percentage of RP of the total optical platelet count (immature platelet fraction; IPF) and as the percentage of RP highly fluorescent (highly fluorescent immature platelet fraction; H-IPF). Moderate but significant positive correlations between PA, IPF, H-IPF, and mean platelet volume (MPV) were found. According to tertiles of IPF, H-IPF and MPV, a significant trend for an increase of platelet aggregation by AA and ADP was evidenced. Furthermore, a significant difference for IPF, H-IPF and MPV between patients with and without RPR was observed. A linear regression analysis showed that IPF, H-IPF and MPV significantly affected PA measured by AA and ADP. At multivariate linear regression analysis these associations were confirmed. Moreover, a logistic regression analysis demonstrated that IPF, H-IPF and MPV significantly influenced the risk of RPR, and in the multivariate model these results remained significant. This study indicates that a high rate of platelet turnover is a new mechanism associated with platelet reactivity in high risk CAD patients on dual antiplatelet therapy.

Measurement of reticulated platelets by simple flow cytometry: An indirect thrombocytopoietic marker

Background The aim of this study was to determine whether measurement of reticulated platelets (RP) by flow cytometry directly from whole blood, with no fixation or manipulation, is as useful a thrombocytopoietic marker as other more complex techniques. Methods RP percentage was prospectively assessed in thrombocytopenic patients (platelets < 100 × 10 9/L) and non-thrombocytopenic controls using a direct, whole-blood, dual-labelling flow cytometric method. Direct, whole-blood double coverage was achieved using a monoclonal antiglycoprotein (GP)-III antibody (CD61-PerCP®) for platelet identification and thiazol orange (Retic-count®) as platelet mARN stain. After establishing thrombocytopenia etiology, patients were grouped according to whether their rate of thrombopoiesis was increased or decreased. Results RP were measured in 53 thrombocytopenic patients with several etiologies and in 53 non-thrombocytopenic controls. The mean RP in 14 thrombocytopenic patients with no increased thrombopoietic activity was 4.8% (95% CI: 3.2–6.4) and the RP absolute number was 1.98 × 10 9/L (95% CI: 1.3–2.6). The mean RP in 17 thrombocytopenic patients with increased thrombopoietic activity was 29.4% (95% CI: 24.7–34.1) and the RP absolute number was 7.24 × 10 9/L (95% CI: 4.9–9.5). Conclusions RP measurement by flow cytometry, directly from whole blood without manipulation, is a useful screening test to differentiate thrombocytopenia with high or low thrombopoietic activity.

Evaluation of platelet turnover by flow cytometry

The number of circulating newly produced platelets depends on the thrombopoietic capacity of bone marrow as well as platelet removal from the bloodstream. Flow cytometric analysis with thiazole orange (TO), a fluorescent dye that crosses platelet membranes and binds intracellular RNA, has been used to measure circulating reticulated platelets (RPs) with high RNA content as an index of platelet turnover. We first assessed the specificity of TO flow cytometry and then applied this method in the diagnosis of thrombocytopenia caused by impaired platelet production or increased destruction. We also explored the utility of TO flow cytometry to predict thrombocytopoiesis after chemotherapy-induced bone marrow aplasia. Venous blood, anticoagulated with K2EDTA, was incubated with 0.6 µg/ml TO plus an anti-GPIIIa monoclonal antibody. The mean percentage of RPs in control subjects (n = 23) was 6.13 ± 3.09%. RPs were 10.41 ± 9.02% in patients (n = 10) with hematological malignancies during aplasia induced by chemotherapy and a significant increase in RPs (35.45 ± 6.11%) was seen in the recovery phase. In 10 patients with idiopathic thrombocytopenic purpura, the percentage of TO positive platelets was 67.81 ± 18.79 (P < 0.001 vs. controls). In patients with thrombocytopenia associated with hepatic cirrhosis (n = 21; 21.04 ± 16.21%, P < 0.001 vs. controls) or systemic lupus erythematosus (n = 6, 29.08 ± 15.57%; P < 0.001 vs. controls) increases in TO-stained platelets were also observed. Measurement of TO positive platelets may be a reliable tool for the laboratory identification of platelet disorders, with a higher sensitivity than measurement of platelet volume. Measurement of RPs may also prove useful to recognize the underlying pathogenetic mechanisms in thrombocytopenia.

Reticulated platelets: a reliable measure to reduce prophylactic platelet transfusions after intensive chemotherapy

Reticulated platelets (RPs) are the youngest circulating platelets (PLTs). The aim of our study was to predict PLT recovery with RP percentage (RP%) and therefore to identify PLT transfusions that could be avoided after autologous peripheral blood progenitor cell (PBPC) transplantation. With a whole-blood dual-labeling flow cytometric method, RP% was prospectively assessed in 47 patients who received myeloablative chemotherapy followed by autologous PBPC transplantation. Retrospective analysis of RP evolution identified three time points: nadir of the RP% (NRP), imminent PLT recovery (IPR) corresponding to an RP% of greater than 7 percent, and PLT transfusion autonomy (PTA). Median occurrences of NRP, IPR, and PTA were on Days +5, +8, and +12 after transplantation, respectively. The RP% value at NRP (4%) was significantly lower compared to the IPR (15%) and PTA (14%). Thirty patients (64%) achieved PTA within 4 days after IPR. On Day +8, if RP% was greater than 7 percent, positive and negative predictive values for PTA within 4 days, specificity, and sensitivity were 79, 63, 66, and 76 percent, respectively. Fever between IPR and PTA was the only factor found to negatively influence PLT recovery (p = 0.02). All patients required at least one PLT transfusion. Among patients with rapid PLT recovery (IPR-PTA interval < 4 days; n = 30), half of them received one PLT transfusion after RP increase, which could be avoided. These encouraging results may allow us to reduce the prophylactic PLT transfusion according to patients RP% increase.