Reticulated Platelet Counts 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.

Jak2V617F clonal hematopoiesis promotes arterial thrombosis via platelet activation and cross talk

AbstractJAK2V617F (JAK2VF) clonal hematopoiesis (CH) has been associated with atherothrombotic cardiovascular disease (CVD). We assessed the impact of Jak2VF CH on arterial thrombosis and explored the underlying mechanisms. A meta-analysis of 3 large cohort studies confirmed the association of JAK2VF with CVD and with platelet counts and adjusted mean platelet volume (MPV). In mice, 20% or 1.5% Jak2VF CH accelerated arterial thrombosis and increased platelet activation. Megakaryocytes in Jak2VF CH showed elevated proplatelet formation and release, increasing prothrombogenic reticulated platelet counts. Gp1ba-Cre–mediated expression of Jak2VF in platelets (VFGp1ba) increased platelet counts to a similar level as in 20% Jak2VF CH mice while having no effect on leukocyte counts. Like Jak2VF CH mice, VFGp1ba mice showed enhanced platelet activation and accelerated arterial thrombosis. In Jak2VF CH, both Jak2VF and wild-type (WT) platelets showed increased activation, suggesting cross talk between mutant and WT platelets. Jak2VF platelets showed twofold to threefold upregulation of COX-1 and COX-2, particularly in young platelets, with elevated cPLA2 activation and thromboxane A2 production. Compared with controls, conditioned media from activated Jak2VF platelets induced greater activation of WT platelets that was reversed by a thromboxane receptor antagonist. Low-dose aspirin ameliorated carotid artery thrombosis in VFGp1ba and Jak2VF CH mice but not in WT control mice. This study shows accelerated arterial thrombosis and platelet activation in Jak2VF CH with a major role of increased reticulated Jak2VF platelets, which mediate thromboxane cross talk with WT platelets and suggests a potential beneficial effect of aspirin in JAK2VF CH.

Reticulated Platelet Count as a Diagnostic Tool in Immune Thrombocytopenia (ITP).

This study aimed to compare the reticulated platelet count between patients having thrombocytopenia secondary to autoimmune destruction (immune thrombocytopenia {ITP}), bone marrow failure, and healthy controls who presented to a tertiary care hospital in Karachi, Pakistan. A cross-sectional study wasconducted from February 2021 to October 2022 in the Department of Hematology, National Institute of Blood Disease (NIBD) Hospital in Karachi, Pakistan, that involved examining three groups: 30 patients with immune thrombocytopenia, 30 patients with thrombocytopenia secondary to reduced production from bone marrow, and 30 healthy controls. The study utilized the Sysmex XN-1000 (Hyogo, Japan: Sysmex Corporation) automated hematology analyzer to perform a complete blood count (CBC) test. Additionally, peripheral blood was stained with Leishman stain and examined under a microscope to eliminate pseudo thrombocytopenia and identify any abnormal cells or dysplasia. The immature platelet fraction (IPF) was then performed on Sysmex XN 1000 after ensuring adequate quality control. Finally, the data were analyzed using DATAtab (Graz, Austria: DATAtab) and SPSS version 25 (Armonk, NY: IBM Corp.). Of the ninety participants, the median age was 33 years with a range of 18-71 years. Patients with ITP had a significantly higher median IPF% (median=26.65, IQR=15-39.4) than thrombocytopenia due to bone marrow failure (median=9.25, IQR=4.55-14.30) and healthy controls (median=7, IQR=4.40-9.90), with a p-value of 0.001. The immune thrombocytopenia group demonstrated an increase in IPF% as platelet counts increased, indicating a significant moderate correlation between IPF% and platelets in these patients (r=0.438, p=0.016) and confirming that IPF% was an independent predictor for the detection of ITP. Reticulated platelet count may be a useful diagnostic tool to differentiate between ITP and thrombocytopenia caused by bone marrow failure. Because of its non-invasive nature, IPF is a valuable tool for expediting the management of thrombocytopenia associated with increased IPF.

2021 Chinese consensus on the diagnosis and management of primary immune thrombocytopenia in pregnancy.

2021 Chinese consensus on the diagnosis and management of primary immune thrombocytopenia in pregnancy.

Impact of platelet turnover on long-term adverse cardiovascular outcomes in patients undergoing percutaneous coronary intervention.

Increased platelet turnover and high platelet reactivity are associated with short-term major adverse cardiovascular events (MACE) after percutaneous coronary intervention (PCI) for acute coronary syndrome (ACS) or stable coronary artery disease (SCAD). We investigated the impact of platelet turnover on long-term MACE. Consecutive patients presenting with ACS or SCAD undergoing PCI between 2009 and 2010 were included. All patients received clopidogrel and aspirin as dual antithrombotic therapy regimen. Multivariable Cox proportional hazard models were applied to assess the prognostic impact of platelet turnover (reticulated platelet count [RPC], mean platelet volume [MPV]) and function on long-term MACE, a composite of cardiovascular death, nonfatal myocardial infarction and nonfatal stroke. In total, 477 patients were eligible. Mean age was 64.3±12.7years, 68.8% were male. Median follow-up was 5.8 (IQR 4.2-6.5) years. Median RPC was 7.6 (IQR 5.6-10.4) g/L and median MPV was 10.7 (IQR 10.1-11.3) fL. In univariable analysis, RPC was associated with MACE, both as continuous (HR 1.064 [95%CI 1.021-1.111]; P=.006) and dichotomized (HR 1.693 [95%CI 1.156-2.481]; P=.006) variable. After adjustment, continuous RPC (HR 1.055 [95%CI 1.012-1.099]; P=.010) and dichotomized RPC (HR 1.716 [95%CI 1.152-2.559]; P=.007) remained significantly associated with MACE. Neither MPV nor platelet function testing was associated with long-term adverse outcome. Increased platelet turnover is associated with long-term adverse outcome in patients with coronary artery disease undergoing PCI. Platelet turnover represents a new marker of atherothrombotic risk and might help to guide composition or duration of antiplatelet therapy.

A Model of Platelets in the Aging Organism Reveals Increased Numbers Due to Reduced Clearance and Enhanced Activatability of Gp2b/3a and P-Selectin, Resulting in a Larger Stroke Burden

Background and AimsAge isakey risk factor for cardiovascular disease (CVD). The prevalence of people aged > 60 y old is projected to rise to 25 % by the year 2050, associated with a 10 % increase in CVD.Platelets play a major role in the progression and the final pathway of CVD. Yet, little is known on the changes in platelet function due to aging per se.Thus, we aimed to further analyze platelets in the aging organism and their putative mechanistic role in ischemia/reperfusion (I/R) brain injury.To dissect the potentially specific aging effects from confounding factors, such as interfering medications (eg. antiplatelet drugs) and comorbidities (eg. hypertension, diabetes), we chose an animal model of young and very old mice to tackle this apparent hen and egg problem.Methods and Results:C57BL/6 wildtype littermates, not spontaneously developing atherosclerosis, were studied at the ages of 12 weeks (young Cohort, yoC) and > 20 months respectively (old Cohort, oldC; n=7/7).Platelet mass was increased in the oldC in number (yoC: 791.42 x 103/µl vs. oldC: 1719.14 x 103/µl, p = 0.013) and in size (6.35 fl vs. 7.2 fl, p=0.0037). Numerically higher reticulated platelet counts were found in the young (11.31 % vs. 4.3 %, p=0.14), as determined by thiazole orange staining and flow cytometric analysis, suggesting a decreased clearance in the old as an underlying mechanism. The red blood cell count in the yoC was significantly higher than in the old (12.34 x 106/mm3 vs. 9.97 x 106/mm3, p=0.0028), whereas white blood cell counts did not differ.Cryosections from liver and spleen stained with a rat-anti-mouse CD41 antibody revealed larger areas marked in the yoC, thus supporting the hypothesis of reduced clearance with aging (hepatic area covered 0.83 % vs. 0.27 %, p=0.014; splenic area covered 2.04 % vs. 1.04 %, p=0.012). The glycocalicin (GC) concentration as determined by ELISA was increased in the oldC (43.26 µg/ml vs. 71.4 µg/ml, p=0.0018) but after correction for the platelet count, the GC index was similar between the groups, suggesting a steady state of platelet survival at a higher count.For functional studies of platelet receptors and activation, washed platelets were activated with thrombin (0.1 U/ml) or collagen type I (5 µg/ml), incubated with specific anti-P-selectin and anti-activated integrin aIIbb3 (JON/A) antibodiesand analyzed by flow cytometry. While the baseline receptor expression between groups was similar, we observed an increased GP IIb/IIIa (fold increase 2.41 vs. 8.26, p=0.04) and numerically also P-selectin (fold increase 7.4 vs. 18.1, p=0.08) exposure after activation with thrombin in the oldC (Fig.1).The baseline surface expression of GPVI and GPIb was similar between groups before and upon activation.To study, whether the observed changes could alter stroke size and functional outcome after I/R brain injury, we studied the model of transient middle cerebral artery occlusion (tMCAO), in which a silicone-covered thread is introduced into the ICA and blocks cerebral blood flow for 30 minutes followed by 48 h of reperfusion. In a preliminary set of experiments (n=3/4), stroke size was analyzed using triphenyltetrazolium chloride (TTC) staining and revealed a doubling of the stroke volumes (21.15 mm3 vs. 43.32 mm3, p=0.20) in the old mice. This finding related to a significantly poorer neurological outcome after 48 h, as demonstrated by the RotaRod test, measuring the latency to fall from a rotating rod (55.17 s vs. 9.83 s, p = 0.0015) and by the Bederson test, a global assessment of neurological function using a scale from 0-4 (1 vs. 1.83, p=0.0036) (Fig.2) .Summary & Conclusions:Platelet studies in our aging model reveal 1) higher platelet numbers and larger platelet size not related to reticulated platelets, 2) a reduced hepatic and splenic clearance and 3) an increased procoagulant potential (activatability of GPIIb/IIIa), as well as a strong tendency to a pro-inflammatory response (P-selectin expression) upon thrombin exposure. These obviously acquired changes in platelet function and number may in part be responsible for 4) larger stroke sizes and poorer functional outcome observed in old mice, overall pointing towards a genuine, primarily age dependent procoagulant platelet phenotype. The model will help in delineating the specific role of platelets in age-related CVD and allow to test specific molecular or pharmacological interventions, targeting the platelet changes with aging. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

ACTN1-related thrombocytopenia: identification of novel families for phenotypic characterization

AbstractInherited thrombocytopenias (ITs) are a heterogeneous group of syndromic and nonsyndromic diseases caused by mutations affecting different genes. Alterations of ACTN1, the gene encoding for α-actinin 1, have recently been identified in a few families as being responsible for a mild form of IT (ACTN1-related thrombocytopenia; ACTN1-RT). To better characterize this disease, we screened ACTN1 in 128 probands and found 10 (8 novel) missense heterozygous variants in 11 families. Combining bioinformatics, segregation, and functional studies, we demonstrated that all but 1 amino acid substitution had deleterious effects. The clinical and laboratory findings of 31 affected individuals confirmed that ACTN1-RT is a mild macrothrombocytopenia with low risk for bleeding. Low reticulated platelet counts and only slightly increased serum thrombopoietin levels indicated that the latest phases of megakaryopoiesis were affected. Given its relatively high frequency in our cohort (4.2%), ACTN1-RT has to be taken into consideration in the differential diagnosis of ITs.

Clinical significance of serum cytokine levels and thrombopoietic markers in childhood idiopathic thrombocytopenic purpura.

Biological markers useful for defining children with newly diagnosed immune thrombocytopenic purpura (ITP) who are likely to develop the chronic form of the disease are partially lacking. The purpose of this study was to assess the clinical role of both immunological and thrombopoietic markers in children with ITP and correlate their levels with different disease stages. We enrolled 28 children with ITP at the onset of their disease, who were followed-up for a whole year and divided according to whether their disease resolved within the 12 months (n=13) or became chronic (n=15), 11 subjects with chronic ITP off therapy for at least 1 month at the time of enrolment, and 30 healthy matched controls. Serum levels of T helper type 1 and 2 and T regulatory-associated cytokines, such as interferon γ, tumour necrosis factor α, interleukin (IL) 2, IL6, IL10, and thrombopoietin were measured in all children using quantitative immunoenzymatic assays, while reticulated platelets were evaluated by flow cytometric analysis. Serum IL10 levels were significantly higher in patients with an acute evolution of ITP than in either healthy controls (p<0.001) or patients with chronic progression of ITP (p<0.05). Reticulated platelet count and thrombopoietin levels were significantly higher in ITP patients at the onset of their disease, whether with acute resolution or chronic progression, than in healthy subjects (p<0.01; p<0.001), but did not differ between the groups of patients. IL-10 seems to predict the clinical course of ITP, as it is significantly higher at the onset of disease in patients who obtain disease remission in less than 1 year.

Measurement of thrombopoietic activity through the quantification of megakaryocytes in bone marrow cytology and reticulated platelets

Reticulated platelets are considered as marker for bone marrow thrombopoiesis. The aim of the study was evaluate the role of reticulated platelets as markers of thrombopoiesis in dogs. Reticulated platelets analysis by flow cytometry and megakaryocyte quantification by bone marrow cytology were determined in 29 healthy adult dogs (control group), 14 dogs with thrombocytopenia without megakaryocytic hypoplasia (group A) and 14 dogs with thrombocytopenia which presented megakaryocytic hypoplasia (group B), detected by bone marrow aspiration cytology. Blood samples were collected and the platelet rich plasma was obtained for reticulated platelets quantification in flow cytometry. Megakaryocytes were quantified in aspiration cytology by two techniques in marrow particles, and correlated to reticulated platelets counts. There are no differences between megakaryocyte quantification. Although there is no correlation between reticulated platelet values and megakaryocyte in bone marrow cytology, the interpretation of reticulated platelet values can be based both on absolute or relative corrected values.

Fluctuations in thrombopoietin, immature platelet fraction, and glycocalicin levels in a patient with cyclic thrombocytopenia

Cyclic thrombocytopenia (CT) is an uncommon disorder characterized by periodic fluctuations in platelet count, resulting in alternating episodes of thrombocytopenia and normal platelet counts. Its pathogenesis remains unclear. Here, we report the case of a 65-year-old man with CT who was initially diagnosed with idiopathic thrombocytopenic purpura (ITP) concomitant with chronic thyroiditis. ITP was diagnosed 12 years ago in the absence of periodic fluctuations in platelet counts. He had been treated with corticosteroids; underwent splenectomy; and subsequently been treated with azathioprine, vincristine, high doses of dexamethasone, and herbal medicine. He had not received antibiotic eradication therapy for Helicobacter pylori because there was no evidence of infection with this organism. Since his symptoms did not improve, the treatments were discontinued, and he was placed under observation. He complained of mild bleeding symptoms, such as petechiae and purpura, which occurred intermittently for several months before he presented to us to undergo detailed investigations. His platelet count varied between 3 9 10/ll and 30 9 10/ll approximately every 3–4 weeks. His white blood cell count and hemoglobin concentration remained unchanged. To elucidate the mechanism underlying this disease, we evaluated the serum thrombopoietin (TPO) level, immature platelet fraction (IPF), plasma glycocalicin index normalized for the individual platelet count (GCI), and T helper (Th)1/Th2 ratio. The serum TPO level was measured with a human TPO ELISA kit (R&D Systems Inc., Minneapolis, MN, USA). The serum levels of TPO, a principal regulator of megakaryogenesis and thrombopoiesis, are thought to be regulated via receptor-mediated clearance by platelets and megakaryocytes. The median serum TPO concentration in healthy individuals is 51 pg/ml. The plasma glycocalicin level was measured with a Glycocalicin EIA kit (Takara Bio Inc., Ohtsu, Japan). The GCI was calculated as proposed by Steinberg et al. [1]: GCI = glycocalicin level 9 250 9 10/individual platelet count (/ll). Glycocalicin is a proteolytic fragment of the a-chain of glycoprotein Ib, a platelet membrane. The GCI reflects the rate of platelet destruction [2]. The mean GCI in healthy individuals is 1.27 (range 0.87–1.77). The IPF was determined to reliably quantify reticulated platelet counts by using a fully automated hematology analyzer XE-2100 equipped with special software IPF Master (Sysmex, Kobe, Japan). Reticulated platelets are RNA-rich immature platelets and are thought to reflect the ability of the bone marrow to produce platelets. Therefore, the IPF, as a surrogate marker of the reticulated platelet count, reflects the rate of thrombopoiesis [3]. The mean IPF in healthy individuals is 2.0% (range 0.5–5.7%). The Th1/Th2 ratio was determined by using flow cytometry analysis to measure the ratio of CD4? cells containing intracellular interferon-c to those containing interleukin-4. The fluctuations in the TPO levels, percentage of IPF, and GCI were synchronous and inversely proportional to those in the platelet counts (Fig. 1). In the thrombocytopenic phase, elevated GCI indicated platelet destruction in this patient. Thrombopoiesis was simultaneously accelerated in response to TPO. Consequently, the IPF, which is as an indicator of thrombopoiesis, increased. In the present case, CT seemed to be closely associated with cyclic increases in platelet destruction. However, the mechanism of cyclic platelet destruction remains to be elucidated. Th1/Th2 balance is T. Yujiri (&) Y. Tanaka M. Tanaka Y. Tanizawa Third Department of Internal Medicine, Yamaguchi University School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan e-mail: yujirit@yamaguchi-u.ac.jp

Reticulated platelets and thrombopoietin in schistosomiasis patients

Schistosomiasis mansoni is a non-cirrhotic liver disease. In cirrhosis patients with portal hypertension, a decreased number of reticulated platelets associated with increased thrombopoietin serum levels were reported. We previously reported a 120/nl platelet cutoff level as a marker of clinically significant portal hypertension in schistosomiasis patients. To evaluate reticulated platelet counts and thrombopoietin serum levels (TPO) in schistosomiasis patients and correlate them with portal hypertension markers. Thirty-three schistosomiasis patients without co-morbidities were endoscopically classified as those with (n = 19) or without (n = 14) clinically significant portal hypertension. Flow cytometric determination of reticulated platelets was performed using CD41 antibody and thiazol orange. Ultrasonographic examinations were performed according to the Niamey protocol. TPO and hyaluronic acid serum levels were determined in duplicate using ELISA methods. The platelet number of 120/nl discriminates the two groups with 100% accuracy and 100% positive and negative predictive values, and correlates with spleen length and portal and splenic vein diameters. Differences in reticulated platelets and hyaluronic acid serum levels between both groups were significant (P = 0.025 and 0.012, respectively), but thrombopoietin serum levels were not (P = 0.769). Schistosomiasis patients with portal hypertension have increased reticulated platelets associated with normal TPO serum levels.

Factors Differentially Associated with Low PFA-100 Closure Times in Patients on Chronic Aspirin Therapy

Background: The PFA-100 device tests platelet function in an in vitro simulated high-shear environment. Low closure times (CTs) on the collagen/ADP (C/ADP) cartridge indicate platelet hyper-reactivity; shortened CTs on the collagen/epinephrine (C/EPI) cartridge suggest residual platelet function after aspirin administration. The impact of patient-specific and pre-analytic variables such as citrate concentration on C/ADP and C/EPI CTs has not been well characterized for a single patient population.Methods: PFA-100 CTs were measured in 289 patients on chronic aspirin therapy who had undergone cardiac surgery 6 months previous and in whom there was complete inhibition of arachidonic acid (AA)-induce platelet aggregation. CTs were measured using C/ADP and C/EPI agonist cartridges in whole blood collected in both 3.2% and 3.8% citrate. Simultaneous measurements of hematologic parameters (complete blood count, reticulated platelet count by flow cytometry, ABO blood group,) as well as markers of inflammation (CRP, fibrinogen and vWF levels), oxidative stress (urine 8-iso-PGF2α) and aspirin responsiveness (urine 11-dehydro-TXB2, arachidonic acid platelet aggregometry) were also performed. Demographic data were recorded, as were medications that could potentially affect assay results. Univariate logistic regression was performed to predict odds of low closure time despite aspirin treatment: <= 71 sec for C/ADP and <=193 sec for C/EPI. Predictors were log transformed or treated categorically if necessitated by distribution of the data. Variables with P <= 0.20 on unadjusted analysis were included in the four initial multivariate models to predict CT. Thereafter, model fit was optimized using the Akaike Information Criteria, by testing stepwise removal of each variable.Results: Multivariate analysis identified several factors that differentially correlated with odds of low CT depending on whether the C/ADP or C/EPI agonist cartridges were used and whether the blood was collected in 3.2% vs. 3.8% citrate. Predictors of low CT are shown below in descending order of strength of evidence of association as assessed by t-statistic and importance to model fit:C/ADP3.2% Citrate3.8% CitrateOdds RatioP ValueOdds RatioP ValuevWF Antigen (>150 % vs. <=150%)2.990.0012.560.016White (vs. non-White)3.690.019NSNSWarfarin Use6.110.028NSNSHematocrit (%)1.110.029NSNSUrine 8-iso-Prostaglandin F2a (>= 1072 vs. <1072 pg/mg creatinine)1.790.062NSNSPlatelet Count (>= 150 vs. < 150 × 103/mm3)2.030.065NSNSC/EPI3.2% Citrate3.8% CitrateOdds RatioP ValueOdds RatioP ValuevWF Antigen (>150 % vs. <= 150 %)NSNS4.140.001Blood Group (O vs. non-O)0.530.030NSNSHematocrit (%)1.100.036NSNSImmature Platelet Fraction (per unit increase in natural-log-transformed %)1.680.064NSNSUrine 11-dehydro-Thromboxane B2(>= 400 vs. < 400 pg/mg creatinine)1.660.093NSNSConclusion: The significance of patient-specific, hematologic, inflammatory and oxidative indices on the performance of the PFA-100 varies differentially between cartridges and citrate concentrations. vWF antigen levels > 150% were the strongest predictor of low CT for three of four cartridge-citrate combinations. Odds of low CT are associated with a number of factors potentially extrinsic to platelet function. The collection of blood in 3.8% rather than 3.2% citrate may limit the influence of extrinsic variables on PFA-100 results. These results may have implications for how PFA-100 tests are analyzed and interpreted.

Effects of Sepsis on Neonatal Thrombopoiesis

We serially evaluated the effects of sepsis and/or necrotizing enterocolitis (NEC) on neonatal thrombopoiesis, using a panel of tests that included platelet counts, thrombopoietin concentrations (Tpo), circulating megakaryocyte progenitor concentrations (CMPs), and reticulated platelets (RPs). Variables analyzed included sepsis type, time after onset of sepsis, platelet counts, and gestational (GA) and postconceptional ages (PCA). Twenty neonates were enrolled. Ten had Gram-negative, six had Gram-positive, and four had presumed sepsis. Four neonates had NEC stage II or higher, and six developed thrombocytopenia. Overall, septic neonates had significantly elevated Tpo concentrations and circulating megakaryocyte progenitors. The highest Tpo levels were associated with Gram-negative or presumed sepsis. RP percentages were increased only in neonates with low platelet counts, while RP counts (RP% x platelet count) were elevated in neonates with high platelet counts. Our findings suggest that septic neonates up-regulate Tpo production, leading to increased megakaryocytopoiesis and platelet release, although the degree of upregulation is moderate. The changes in RP% and RP count most likely reflect increased thrombopoiesis with variable degrees of platelet consumption. In addition, our findings suggest that different factors, likely including level of illness and/or specific platelet or bacterial products, can down-regulate the magnitude of the thrombopoietic response.

Rapid platelet turnover in WASP(−) mice correlates with increased ex vivo phagocytosis of opsonized WASP(−) platelets

Our objective was to determine a mechanism for the thrombocytopenia of murine Wiskott-Aldrich syndrome (WAS). Consumption rates of WAS protein (WASP)(-) and wild-type (WT) platelets were measured by injection of 5-chloromethylfluorescein diacetate (CMFDA)-labeled platelets into WT or WASP(-) recipients, and by in vivo biotinylation. Platelet and reticulated platelet counts were performed using quantitative flow cytometry. Bone marrow megakaryocyte number and ploidy was assessed by flow cytometry. Phagocytosis of CMFDA-labeled, opsonized platelets was assessed using bone marrow-derived macrophages. Serum antiplatelet antibodies were assayed via their binding to WT platelets. CMFDA-labeled WASP(-) platelets are consumed more rapidly than WT platelets in either WT or WASP(-) recipients. In vivo biotinylation studies corroborate these findings and show a normal consumption rate for WASP(-) reticulated platelets. The number of reticulated platelets is reduced in WASP(-) mice, but a significant number of the mice show an increased proportion of reticulated platelets and more severe thrombocytopenia. Sera from some of the latter group contain antiplatelet antibodies. Compared to WT platelets, WASP(-) platelets opsonized with anti-CD61 or 6A6 antibody are taken up more rapidly by bone marrow-derived macrophages. In vivo consumption rates of WASP(-) platelets are more accelerated by opsonization than are those of WT platelets. Both rapid clearance and impaired production contribute to the thrombocytopenia of murine WAS. Increased susceptibility of opsonized WASP(-) platelets to phagocytosis leads to increased in vivo clearance. This correlates with a higher incidence of individuals with an elevated fraction of reticulated platelets, a more severe thrombocytopenia, and antiplatelet antibodies.

Rapid In Vivo Consumption and Ex Vivo Phagocytosis of WASP(−) Platelets.

Patients with the Wiskott-Aldrich Syndrome (WAS) have thrombocytopenia and increased platelet consumption rates, and may have reduced platelet production rates. WASP(−) mice have been reported to have only a mild thrombocytopenia. We find that when fully crossed onto the C57Bl/6J background, WASP(−) mice have a >50% reduction in platelet counts. Ex vivo labeled WASP(−)platelets are consumed 2 to 4-fold faster than are WT platelets in WT mice, and with exponential rather than linear kinetics. Clearance rates of WASP(−) platelets in WT mice, and vice versa, indicate that rapid consumption is due to factors both intrinsic and extrinsic to platelets. In vivo biotinylation demonstrates consumption rates comparable to those seen with ex vivo labeling, and shows a normal rate of consumption of WASP(−) reticulated (immature) platelets. Reticulated platelet counts are reduced, indicating that their production rate is reduced. Megakaryocytes are increased in spleen and bone marrow, and in the latter their ploidy distribution is normal, suggesting that impaired platelet production occurs at the level of thrombopoiesis. The absolute turnover rates of mature and reticulated platelets, however, indicate that maturation of the latter can account for only a fraction of the former's turnover in either WT or WASP(−) mice. A subset of WASP(−) mice show an increased fraction of reticulated platelets and more severe thrombocytopenia, and some members of this subset also express serum anti-platelet antibodies. CMFDA-labeled WASP(−) platelets opsonized with anti-CD61(IgG1) antibody are more susceptible to ex vivo phagocytosis by bone marrow derived macrophages (BMDM) than WT platelets, and as susceptible as CD47(−/−) platelets. After opsonization with 6A6(IgG2b) antibody, WASP(−) platelets are also taken up more rapidly than WT platelets by BMDM, but less rapidly than are CD47(−/−) platelets. The in vivo consumption rate of WASP(−) platelets in WT recipients is more accelerated by opsonization with anti-CD61 antibody than is that of WT platelets. Increased phagocytosis is not due to (A) altered levels of the targeted antigens or of CD47 (B) increased exposure of phosphatidyl serine, or (C) antibody-induced activation as assayed by CD62P expression. Increased phagocytosis is not due to selective sensitivity to IgG1 vs IgG2b antibodies, as opsonization with an engineered 6A6(IgG1) antibody leads to reduced phagocytosis for both CD47(−/−) and WASP(−) platelets. Anti-CD61 opsonized platelets deficient in both WASP and CD47 show markedly increased ex vivo phagocytosis compared to platelets deficient in either protein, suggesting that platelet WASP does not function to amplify signals from platelet CD47 through macrophage SIRP-alpha. These results raise the possibility that the binding of low affinity or low titer antibodies that might have no effect on WT platelets could cause thrombocytopenia when the platelets lack WASP. Alternatively, rapid phagocytosis of opsonized WASP(−) platelets could promote a self-reinforcing cycle of increased host antigen presentation and increased immune responses to host antigens.

In Vivo Effects of Eltrombopag on Human Platelet Function.

Eltrombopag, an orally-administered small-molecule agonist of the thrombopoietin receptor (c-Mpl), is under investigation as a treatment for immune thrombocytopenic purpura (ITP). Studies have indicated that eltrombopag does not ‘prime' platelets for activation in vitro, and eltrombopag administration to healthy volunteers does not increase platelet surface P-selectin or activated integrin αIIbβ3 (Jenkins J. Blood 2007). However, the effects of eltrombopag on platelet function in thrombocytopenic patients in vivo, either by direct binding to c-Mpl receptors on platelets or indirectly by altering the dynamics of platelet production and causing an influx of young, large platelets, is unknown. Whole blood flow cytometry, unlike other assays of platelet function, enables measurement of platelet function in the setting of marked thrombocytopenia (Michelson. Platelets, Elsevier, 2007). As a substudy of larger treatment studies, 17 adult patients with chronic ITP received eltrombopag at a starting dose of 50 mg daily, with the possibility of an increase to 75 mg daily after 3 weeks. Blood samples were drawn pre-treatment, and after 7 and 28 days of therapy. Platelet count, mean platelet volume (MPV), and the immature platelet fraction (IPF, or reticulated platelet count) were measured using a Sysmex XE-2100. Platelet surface P-selectin and activated integrin αIIbβ3 (reported by monoclonal antibody PAC1) were measured by whole blood flow cytometry in the presence and absence of 0.5 μM ADP, 20 μM ADP, 1.5 μM thrombin receptor activating peptide (TRAP), or 20 μM TRAP. Bleeding was quantified by a comprehensive scale that allocates grades of 0 (no), 1 (minor) or 2 (marked) bleeding at 10 anatomical sites according to physical examination and/or history (Page, L.K. Br J Haematol 2007). Eleven of the 17 patients responded to eltrombopag with a rise in platelet count of >30 x 109/L. The IPF increased in responders but not non-responders (table 1). Response to eltrombopag was not predicted by pretreatment MPV or IPF. The ITP bleeding score decreased in responders over the study period in parallel with the increases in platelet count (table 1). As determined by platelet surface P-selectin and activated integrin αIIbβ3, eltrombopag did not result in platelet activation or augment ADP- or TRAP-induced platelet activation (table 2). In summary, eltrombopag increases the platelet count and reduces bleeding in responding adult patients with chronic ITP through the release of new platelets into the circulation. While bleeding is reduced in responders, eltrombopag does not result in platelet activation or augmentation of platelet activation by ADP or TRAP. This suggests that the newer platelets released by eltrombopag stimulation are not hyper-functional (or are only transiently so prior to day 7).Table 1IPF (maximum absolute change, mean ± SEM x 109/L)Number in whom bleeding decreasedResponders57.0 ± 22.48/11Non-responders3.3 ± 1.51/6Table 2Study Day0728Activated αIIbβ3 MFINo agonist11.411.39.2Low ADP180.3159.498.4High ADP451.2348.2*251.8*Low TRAP158.1175.5143.9High TRAP385.2347.0299.6P-selectin MFINo agonist5.56.66.2Low ADP48.643.438.8High ADP144.5109.096.8Low TRAP113.8114.9107.8High TRAP457.3396.3330.9MFI, mean fluorescence intensity, *P <0.05 compared with day 0

The Immature Platelet Fraction in HIV Patients with Thrombocytopenia.

Thrombocytopenia is a common clinical feature of HIV infection. Given the number of possible etiologies of thrombocytopenia in a patient with known HIV, a peripheral blood test effective in determining the likely pathophysiologic basis of the thrombocytopenia would be a valuable clinical tool. Immature platelets are released early from the bone marrow in response to increased platelet turnover. These platelets contain residual megakaryocyte mRNA and have been termed reticulated platelets. A new assay, the Immature Platelet Fraction (IPF), measures the reticulated platelet count in peripheral blood. Patients with increased destruction of platelets from such conditions as ITP consistently have a higher IPF percent, while patients with decreased platelet production have a low or normal IPF percent. The goal of our study was to determine the performance characteristics of the IPF assay in HIV patients with thrombocytopenia and to see if the IPF percent could be used to help elucidate the etiology of low platelet counts in this group of patients. All adult patients admitted to the Johns Hopkins Hospital with a diagnosis of HIV and a platelet count less than 150,000 were eligible for enrollment. 62 patients were identified from February 2007 to June 2007. 34 control samples were obtained from inpatients with HIV who were not thrombocytopenic. In addition, 81 samples were available from non-HIV historical controls with normal platelet counts. The mean platelet count in the HIV thrombocytopenic group was 92,000 while the mean platelet count in the HIV control group was 254,000 (p value <.001). The mean platelet count in the non-HIV historical control group was 274 (p=.34 when compared to the HIV control group). The mean IPF percent in the HIV thrombocytopenic group was 10.2% as compared to 6.8% in the HIV control group (p=.001). The mean IPF in historical non-HIV controls was 3.1% (p<.001 for both the HIV thrombocytopenic and the HIV control group). Univariate analyses were conducted to identify potential individual predictors of a high IPF percent. Backward selection was then performed using multivariate linear models with a threshold Wald test p-value of 0.05. ITP, diabetes mellitus and cirrhosis were significantly associated with a higher IPF percent with a co-efficient (95% confidence interval) of 6.98 (3.05–10.91), 4.73 (1.39–8.06), and 14.18 (9.7–18.66), respectively. CD4 count, HIV viral load, hepatitis C and reticulocyte count were not correlated with IPF percent. Our results suggest that patients with HIV have increased platelet turnover as compared to patients without HIV. Thrombocytopenic patients with HIV have increased platelet turnover relative to both non-thrombocytopenic HIV patients and to historical non-HIV controls. History of ITP, diabetes mellitus, and cirrhosis are predictive of an elevated IPF percent. Reticulocyte count is not correlated to IPF percent, suggesting that a low reticulocyte count is not a reliable marker for decreased bone marrow production in HIV thrombocytopenia. It is unlikely that the IPF assay alone can be used to determine the pathophysiologic basis of thrombocytopenia in any single patient with HIV. Further work needs to be done to clarify the utility of the IPF assay in this group of patients.

Reticulated platelet counts correlate with treatment response in patients with idiopathic thrombocytopenic purpura and help identify the complex causes of thrombocytopenia in patients after allogeneic hematopoietic stem cell transplantation

In thrombocytopenic conditions of unknown origin, quantification of reticulated platelets (RP) in the peripheral blood by flow cytometry has been shown to differentiate increased platelet (Plt) turnover from insufficient Plt production. We used a whole blood flow cytometry method combining thiazole orange and anti-CD41a-staining to assess RP in 71 healthy subjects, six with thrombocytopenic myelodysplastic syndrome (MDS), nine with liver cirrhosis, 14 patients with idiopathic thrombocytopenic purpura (ITP), and 12 patients who had undergone hematopoietic stem cell transplantation (HSCT). Patients with MDS had normal, patients with liver cirrhosis had slightly elevated RP counts compared to healthy subjects. ITP patients had elevated RP counts, and RP >15% were associated with treatment response (P = 0.015). In 7/10 patients after HSCT, an increase of RP preceded Plt recovery, whereas in patients with secondary thrombocytopenia after normal regeneration, the assessment of RP allowed the differentiation between conditions with high Plt turnover, such as GvHD and microangiopathy, indicated by high RP counts, and graft failure, indicated by low RP counts. Our data provide the rationale for prospective studies on the diagnostic and prognostic value of RP counts in larger patient populations with ITP and after HSCT.

Evaluation of reticulated platelets in patients with sickle cell diseases

Background and purpose Reticulated platelet (RP) count provides an estimate of thrombopoiesis. The objective was to evaluate RP in patients in different stages of sickle cell disease (SCD) and to determine the relationship between interleukin-6 (IL-6), interleukin-3 (IL-3) and thrombopoietin (TPO) and RP count and degree of activation. Methods Eighty-nine adult patients with SCD were studied: 38 were in the steady state, 27 in hemolytic crisis (HC) and 24 in vaso-occlusive crisis (VOC). RPs and activated platelets were analyzed by flow cytometry. Soluble P-selectin, IL-6, IL-3 and thrombopoietin (TPO) levels were measured by ELISA tests. Results The patients in VOC had a higher absolute number of RPs and CD62P+ platelets than did the control group or patients in the steady state. A significant correlation was observed between the absolute number of CD62P+ platelets and RPs in patients in the steady state, HC and VOC. In the steady-state group of patients, the level of soluble P-selectin was found to be dependent on the RP values. IL-3 and TPO serum levels were higher in patients in the steady state, HC and VOC than in the control group. IL-6 serum levels were higher in HC and VOC patients than in the control group and higher in patients in the steady state than in the VOC group. Conclusion Our results suggest that PRs contribute to the vaso-occlusive process in sickle cell disease. Increased interleukin serum levels probably indicate that inflammatory process is involved in the vascular–occlusive phenomenon. However, it appears that these inflammatory mediators do not have an effect on thrombopoiesis in sickle-cell-disease patients.

Thrombocytopoesis in allergic asthma

Allergic asthma is characterized by bronchial inflammation and simultaneous repair processes that results in increased airway obstruction. Recent studies indicate that platelets could play an important role in this process. The aim of the present study was to evaluate the platelet count, the percentage of reticulated platelets, plasma thrombopoietin and interleukin 6 (IL-6) concentrations in patients with moderate bronchial asthma. The study was performed in 12 allergic chronic asthma patients and 12 nonallergic chronic asthma patients. Patients were treated according to the GINA 2004 recommendations for chronic moderate asthma. Seven healthy individuals served as negative controls. Blood was collected on CTAD from antecubital vein in the morning. The platelet count was statistically higher in allergic asthma patients compared with healthy controls. In nonallergic asthma patients the platelet count was also higher but the difference was not statistically significant. The percentage of the reticulated platelets in allergic asthma patients was statistically higher as compared to nonallergic and healthy subjects, who had similiar values. The concentration of IL-6 was significantly elevated in allergic asthma patients as compared to healthy controls. There was no difference in IL-6 levels between both asthma patient groups. In nonallergic asthma patients plasma thrombopoietin levels were slightly higher, but not statistically significant, in comparison with allergic asthma patients and controls. Our findings suggest that platelets may be involved in allergic inflammation and play a significant role in remodeling of the airways. In patients with allergic asthma we found increased reticulated platelet count suggesting enhanced megakaryocytopoiesis. Further studies on a role of platelets in the pathogenesis of asthma and their potential clinical implications are warranted and may contribute to the development of novel therapies.