Characterization of efficacy and safety of pathogen inactivated and quarantine plasma in routine use for treatment of acquired immune thrombotic thrombocytopenic purpura.

BACKGROUND: Auto-immune thrombotic thrombocytopenic purpura (TTP) is a morbid multi-organ disorder. Cardiac involvement not recognized in initial disease descriptions is a major cause of morbidity. Therapeutic plasma exchange (TPE) requires exposure to multiple plasma donors with risk of transfusion-transmitted infection (TTI). Pathogen inactivation (PI) with amotosalen-UVA, the INTERCEPT Blood System for Plasma (IBSP) is licensed to reduce TTI risk. METHODS: An open-label, retrospective study evaluated the efficacy of quarantine plasma (QP) and IBSP in TTP and defined treatment emergent cardiac abnormalities. Medical record review of sequential patient cohorts treated with QP and IBSP characterized efficacy by remission at 30 and 60 days (d) of treatment, time to remission, and volume (L/kg) of plasma required. Safety outcomes focused on cardiac adverse events (AE), relapse rates, and mortality. RESULTS: Thirty-one patients (18 IBSP and 13 QP) met study criteria for auto-immune TTP. The proportions (%) of patients in remission at 30 d (IBSP = 61·1, QP = 46·2, P = 0·570) and 60 d (IBSP = 77·8, QP = 76·9, P = 1·00) were not different. Median days to remission were less for IBSP (15·0 vs. 24·0, P = 0·003). Relapse rates (%) 60 d after remission were not different between cohorts (IBSP = 7·1, QP = 40·0, P = 0·150). ECG abnormalities before and during TPE were frequent; however, cardiac AE and mortality were not different between treatment cohorts. CONCLUSIONS: Cardiac and a spectrum of ECG findings are common in TTP. In this study, IBSP and QP had similar therapeutic profiles for TPE.

Patient outcomes and amotosalen/UVA-treated platelet utilization in massively transfused patients.

BACKGROUND: Amotosalen/UVA-treated platelet concentrates (PCs) have demonstrated efficacy for treating and preventing bleeding in clinical trials and in routine use; however, most studies were performed in haematology/oncology patients. We investigated efficacy during massive transfusion (MT) in general hospitalized patients. METHODS: Universal amotosalen/UVA treatment (INTERCEPT Blood System) of platelets was introduced at a large Austrian medical centre. We performed a retrospective cohort analysis comparing component use, in-hospital mortality and length of stay after MT that included platelet transfusion, for two periods (21 months each) before and after implementation. RESULTS: A total of 306 patients had MT. Patients were mostly male (74%) and ≥18 years old (99%), including 93 liver transplant, 97 cardiac or vascular surgery and 51 trauma patients. There were no differences in demographics between the periods. Component use on the day and within 7 days of the MT event was unchanged post-IBS implementation, except trauma patients received fewer RBCs on the day. The mean ratio of RBC:platelets:plasma on the day of the MT was close to 1:1:1 in both periods, except for liver transplants with MT who received more plasma components. Overall, in-hospital mortality (preimplementation = 27·6% vs. postimplementation = 24·0%; P = 0·51) and median time to discharge (preimplementation = 27 vs. postimplementation = 23 days; P = 0·37) did not change, except for cardiac and vascular surgery patients who were discharged earlier. CONCLUSION: The introduction of amotosalen/UVA-treated, pathogen-reduced PC did not adversely affect clinical outcomes in massively transfused patients in terms of blood product usage, in-hospital mortality and length of stay for a range of clinical indications for platelet transfusion support.

Red blood cell concentrates treated with the amustaline (S-303) pathogen reduction system and stored for 35 days retain post-transfusion viability: results of a two-centre study.

BACKGROUND AND OBJECTIVES: Pathogen reduction technology using amustaline (S-303) was developed to reduce the risk of transfusion-transmitted infection and adverse effects of residual leucocytes. In this study, the viability of red blood cells (RBCs) prepared with a second-generation process and stored for 35 days was evaluated in two different blood centres. MATERIALS AND METHODS: In a single-blind, randomized, controlled, two-period crossover study (n = 42 healthy subjects), amustaline-treated (Test) or Control RBCs were prepared in random sequence and stored for 35 days. On day 35, an aliquot of 51 Cr/99m Tc radiolabeled RBCs was transfused. In a subgroup of 26 evaluable subjects, 24-h RBC post-transfusion recovery, mean life span, median life span (T50 ) and life span area under the curve (AUC) were analysed. RESULTS: The mean 24-h post-transfusion recovery of Test and Control RBCs was comparable (83·2 ± 5·2 and 84·9 ± 5·9%, respectively; P = 0·06) and consistent with the US Food and Drug Administration (FDA) criteria for acceptable RBC viability. There were differences in the T50 between Test and Control RBCs (33·5 and 39·7 days, respectively; P < 0·001), however, these were within published reference ranges of 28-35 days. The AUC (per cent surviving × days) for Test and Control RBCs was similar (22·6 and 23·1 per cent surviving cells × days, respectively; P > 0·05). Following infusion of Test RBCs, there were no clinically relevant abnormal laboratory values or adverse events. CONCLUSION: RBCs prepared using amustaline pathogen reduction meet the FDA criteria for post-transfusion recovery and are metabolically and physiologically appropriate for transfusion following 35 days of storage.

A prospective, active haemovigilance study with combined cohort analysis of 19,175 transfusions of platelet components prepared with amotosalen-UVA photochemical treatment.

BACKGROUND AND OBJECTIVES: A photochemical treatment process (PCT) utilizing amotosalen and UVA light (INTERCEPT(™) Blood System) has been developed for inactivation of viruses, bacteria, parasites and leucocytes that can contaminate blood components intended for transfusion. The objective of this study was to further characterize the safety profile of INTERCEPT-treated platelet components (PCT-PLT) administered across a broad patient population. MATERIALS AND METHODS: This open-label, observational haemovigilance programme of PCT-PLT transfusions was conducted in 21 centres in 11 countries. All transfusions were monitored for adverse events within 24 h post-transfusion and for serious adverse events (SAEs) up to 7 days post-transfusion. All adverse events were assessed for severity (Grade 0-4), and causal relationship to PCT-PLT transfusion. RESULTS: Over the course of 7 years in the study centres, 4067 patients received 19,175 PCT-PLT transfusions. Adverse events were infrequent, and most were of Grade 1 severity. On a per-transfusion basis, 123 (0.6%) were classified an acute transfusion reaction (ATR) defined as an adverse event related to the transfusion. Among these ATRs, the most common were chills (77, 0.4%) and urticaria (41, 0.2%). Fourteen SAEs were reported, of which 2 were attributed to platelet transfusion (<0.1%). No case of transfusion-related acute lung injury, transfusion-associated graft-versus-host disease, transfusion-transmitted infection or death was attributed to the transfusion of PCT-PLT. CONCLUSION: This longitudinal haemovigilance safety programme to monitor PCT-PLT transfusions demonstrated a low rate of ATRs, and a safety profile consistent with that previously reported for conventional platelet components.

INTERCEPT plasma: comparability with conventional fresh-frozen plasma based on coagulation function--an in vitro analysis.

BACKGROUND: An effective pathogen inactivation (PI) technology for plasma must inactivate a broad range of pathogens with retention of haemostatic function suitable for therapeutic support. This study evaluated a broad panel of coagulation factors regarding functionality in plasma treated with the INTERCEPT Blood System (I-FFP). STUDY DESIGN AND METHODS: Apheresis plasma (600 ml) was treated with amotosalen and UVA. Aliquots of plasma were collected prior to and after photochemical treatment and frozen prior to analysis. Pro-coagulants, inhibitors and fibrinolytic proteins, contact pathway components, activation markers, the von Willebrand complex and complement proteins were analyzed. RESULTS: Retention of procoagulant factors in I-FFP ranged from 77 to 92% of pretreatment levels. Components of the von Willebrand complex, including multimers and von Willebrand cleavage protease activity (vWF:CP), remained within normal ranges after treatment. Endogenous inhibitors of coagulation were retained at 93 to 100% of baseline. Plasminogen and alpha-2 antiplasmin were retained at 94 and 78% respectively. Retention of contact factors was variable as some factors were below the reference range prior to PI treatment. With the exception of thrombin-antithrombin complexes (TAT) in one of six replicates all markers of coagulation activation were well within normal ranges. Anaphylatoxins were not increased and C1-esterase inhibitor was fully retained. CONCLUSION: Treatment of plasma with the INTERCEPT Blood System preserves proteins necessary for haemostasis without inappropriate activation of coagulation, fibrinolytic or complement pathways.

Treatment of whole blood (WB) and red blood cells (RBC) with S-303 inactivates pathogens and retains in vitro quality of stored RBC.

A pathogen inactivation (PI) process has been developed using the frangible anchor linker effector (FRALE) compound S-303. A series of experiments were performed in whole blood (WB) to measure the level of viral and bacterial inactivation. The results showed that 0.2mM S-303 and 2mM glutathione (GSH) inactivated >6.5 logs of HIV, >5.7 logs of Bluetongue virus, >7.0 logs of Yersinia enterocolitica, 4.2 logs of Serratia marcescens, and 7.5 logs of Staphylococcus epidermidis. Recent development for S-303 is focused on optimization of the PI process for red blood cell concentrates (RBC). A series of studies in RBC showed that 0.2mM S-303 and 20mM GSH inactivated approximately 5 logs or greater of Y. enterocolitica, E. coli, S. marcescens, S. aureus, HIV, bovine viral diarrhoea virus, bluetongue virus and human adenovirus 5. In both applications of the S-303 process, in vitro parameters of RBC function and physiology were retained compared to conventional RBC. Results from these studies indicate that S-303 can be applicable for PI of RBC and WB.

Blood lead concentrations in a remote Himalayan population.

The lead content in the air at the foothills of the Himalayas in Nepal was found to be negligible. The concentration of lead in the blood of 103 children and adults living in this region was found to average 3.4 micrograms per deciliter, a level substantially lower than that found in industrialized populations.

An active haemovigilance programme characterizing the safety profile of 7437 platelet transfusions prepared with amotosalen photochemical treatment.

BACKGROUND: An active haemovigilance programme was implemented to survey adverse events (AE) associated with transfusion of platelets photochemically treated with amotosalen and ultraviolet A (PCT-PLT). The results of 5106 transfusions have already been reported. Here we report the results of an additional 7437 PCT-PLT transfusions. METHODS: The focus of this ongoing haemovigilance programme is to document all AEs associated with PCT-PLT transfusion. Data collected for AEs include: time of event after starting transfusion, clinical descriptions, vital signs, results from radiographs and bacterial cultures, event severity (Grade 0-4) and causal relationship to PCT-PLT transfusion. RESULTS: One thousand four hundred patients (mean 60 years, range 1-96) received PCT-PLT transfusions. The majority of the patients (53.4%) had haematology-oncology diseases and required conventional chemotherapy (44.8%) or stem cell transplantation (8.6%). Sixty-eight PCT-PLT transfusions were associated with AE. Acute transfusion reactions (ATR), classified as an AE possibly related, probably related, or related to PCT-PLT transfusions were infrequent (n = 55, 55/7437 = 0.7%) and most were of Grade 1 severity. Thirty-nine patients (39/1400 = 2.8%) experienced one or more ATRs. The most frequently reported signs/symptoms were chills, fever, urticaria, dyspnoea, nausea and vomiting. Five AEs were considered severe (> or = Grade 2); however, no causal relationship to PCT-PLT transfusion was found. Repeated exposure to PCT-PLT did not increase the likelihood of an ATR. No cases of transfusion-related acute lung injury and no deaths due to PCT-PLT transfusions were reported. CONCLUSIONS: Routine transfusion of PCT-PLT is well-tolerated in a wide range of patients. ATRs related to PCT-PLT transfusion were infrequent and most were of mild severity.

In vivo lability of glucose-6-phosphate dehydrogenase in GdA- and GdMediterranean deficiency.

A decreased level of glucose-6-phosphate dehydrogenase might result from decreased rate of synthesis, synthesis of an enzyme of lower catalytic efficiency, increased lability, or a combined mechanism. To test the hypothesis of increased lability, the rate of decline of the enzyme in vivo was measured in three groups of individuals, controls, Gd(-),A-males, and Gd(-), Mediterranean males, by the slope of decline of activity in fractions containing erythrocytes of progressively increasing mean age. These fractions were obtained by ultracentrifugation on a discontinuous density gradient of erythrocyte suspensions free of contaminating platelets and leukocytes. The rate of in vivo decline of pyruvate kinase (another age-dependent enzyme) was also measured and found very similar in the three groups. The in vivo decline of glucose-6-phosphate dehydrogenase was found to follow an exponential rate, with a half-life of 62 days for controls and 13 days for Gd(-),A- erythrocytes. The activity in normal reticulocytes was estimated at 9.7 U and in Gd(-),A- reticulocytes at 8.8 U. These estimates were confirmed by direct measurements in reticulocytes isolated from patients with extreme reticulocytosis. In Gd(-),Mediterranean erythrocytes activity could be demonstrated only in reticulocytes, which were estimated to average 1.4 U. The rate of decline is so extreme that no activity could be detected in mature erythrocytes. These data suggest that the glucose-6-phosphate dehydrogenase deficiency of both the Gd(A-) and the GdMediterranean variant results from different degrees of in vivo instability of the abnormal enzyme.

Evidence that a 210,000-molecular-weight glycoprotein (GP 210) serves as a platelet Fc receptor.

We previously identified a 210,000-mol-wt platelet glycoprotein (GP 210) that is missing from Bernard-Soulier platelets, and found that an antibody against GP 210 inhibits ristocetin-induced platelet agglutination. We now show by immunoblotting that GP 210 binds heat-aggregated rabbit and human IgG, as well as keyhole limpet hemocyanin (KLH)-anti-KLH and ovalbumin (OA)-anti-OA immune complexes. Immune complex binding to GP 210 was preserved on chymotrypsin-treated platelets that lacked glycoprotein Ib (GP Ib). In contrast, ristocetin-induced platelet agglutination resulted in disappearance of immunologically detectable GP 210 and loss of immune complex binding, even though GP Ib remained intact. Purified Fc fragments inhibited binding of anti-GP 210 antibody to intact platelets and to GP 210 on immunoblots. The Fc fragments also blocked immune complex binding to GP 210. Conversely, anti-GP 210 antiserum and F(ab)2 fragments inhibited binding of fluorescein-labeled Fc fragments to intact platelets. We conclude that GP 210 functions as a platelet Fc receptor.

Acquired Bernard-Soulier syndrome. Evidence for the role of a 210,000-molecular weight protein in the interaction of platelets with von Willebrand factor.

A patient with a lymphoproliferative disorder developed bleeding associated with a prolonged bleeding time and a selective defect of platelet aggregation in response to ristocetin. The patient's purified IgG was shown to inhibit aggregation of washed normal platelets by ristocetin and von Willebrand factor (F VIII:vWF). By Western blotting, it was shown that antibody bound specifically to an antigen of Mr 210,000 present on normal platelets but missing on platelets from patients with congenital Bernard-Soulier syndrome (BSS). Binding was effected by the F(ab)2 portion of the IgG, indicating the presence of an autoantibody rather than an immune complex. These results suggest that the 210,000-Mr protein is involved in the interaction of F VIII:vWF with platelets. Furthermore, we have demonstrated the apparent absence of an additional protein on congenital BSS platelets. Heat-aggregated IgG was also shown to bind to the 210,000-Mr protein, suggesting that this protein may function as an Fc receptor on platelets. The relationship of the 210,000-Mr protein to glycoprotein Ib and the precise role of this protein in the interaction of platelets with F VIII:vWF need to be characterized.

Biochemical heterogeneity in glutathione synthetase deficiency.

Two different clinical syndromes are associated with glutathione synthetase deficiency, one presenting with hemolytic anemia and 5-oxoprolinuria, the other with isolated hemolysis. We have differentiated these disorders on an enzymatic basis. In 5-oxoprolinuria, all cell types examined have grossly deficient enzyme activity and glutathione content. In contrast, in the nonoxoprolinuric variant, erythrocytes have decreased enzyme activity and glutathione content, whereas nucleated cells maintain substantial levels of both. The enzyme in this disorder is unstable in vitro and has shortened survival in intact erythrocytes. Nucleated cells appear able to maintain sufficient enzyme activity and concentrations of glutathione to suppress overproduction of 5-oxoproline.

Intraoperative hemolysis. The initial manifestation of glucose-6-phosphate dehydrogenase deficiency.

Unexpected intraoperative hemolysis suggestive of a hemolytic transfusion reaction was the initial clinical manifestation of Mediterranean type glucose-6-phosphate dehydrogenase (G-6-PD) deficiency in a 50-year-old Indian man. There was no evidence for immune mediated hemolysis since no unexpected RBC antibodies were found, and results of the direct antiglobulin test remained negative. Analysis of preoperative blood specimens demonstrated complete absence of G-6-PD activity. No etiologic agent clearly associated with G-6-PD hemolysis could be identified. Testing for G-6-PD deficiency should be considered when unexpected hemolysis occurs intraoperatively.

The relationship between megakaryocyte ploidy and platelet volume in normal and thrombocytopenic C3H mice.

Previous studies have suggested that platelet volume may be primarily regulated by the ploidy distribution of mature bone marrow megakaryocytes. However, earlier investigations from this laboratory using C57/BL mice have shown that in response to acute, severe, or moderate thrombocytopenia, platelet volume is regulated independently of megakaryocyte ploidy. Murine strains, including C57/BL, usually have a modal bone marrow megakaryocyte ploidy class of 16N. In contrast, the C3H mouse has a 32N modal bone marrow megakaryocyte ploidy class. We have examined the platelet count, platelet volume distribution, and bone marrow megakaryocyte ploidy distribution of C3H mice during steady-state thrombopoiesis and after depletion of platelets by antiplatelet serum. The platelet count and volume of normal C3H mice were not substantially different from those of C57/BL mice, but megakaryocyte frequency was marginally greater (p less than 0.05), and the ploidy distribution exhibited a a marked reduction in the proportion of 16N cells (p less than 0.001) and increased relative frequencies of 32N (p less than 0.001) and 64N (p less than 0.01) megakaryocytes. In response to acute severe thrombocytopenia, C3H mice demonstrated an increase in platelet volume equivalent to that previously reported for C57/BL mice, without a subsequent shift in the modal megakaryocyte ploidy class. The relative frequencies of 64N and 128N megakaryocytes increased significantly (p less than 0.005) compared to normal C3H mice, without a change in the frequency of 32N megakaryocytes. These studies indicate that during steady-state thrombopoiesis, a greater proportion of higher ploidy megakaryocytes (32N plus 64N) does not necessarily alter peripheral platelet count or platelet volume. Therefore, it appears that neither platelet volume nor count are primarily regulated by bone marrow megakaryocyte ploidy and that the magnitude of upward regulation of megakaryocyte ploidy is limited.

Sustained thrombocytopenia in mice: serial studies of megakaryocytes and platelets.

We studied thrombopoiesis in mice after the experimental induction of sustained, immune thrombocytopenia with platelet antiserum (PAS). Utilizing light and electron microscopy and a digital image analyzer to determine platelet sectional areas, we examined platelets and megakaryocytes (MK) after 120 h of sustained, severe thrombocytopenia (120CT) and during recovery from thrombocytopenia at 48 h (48R), 72 h (72R), and 120 h (120R) after cessation of administration of PAS. Mean platelet volume (MPV), determined by electrical impedance, also was measured at each time point. Platelets at 120CT (platelet count less than 50,000/microliter), 48R (platelet count 100-200,000/microliter), and 72R (platelet count approximately 1 x 10(6)/microliter) were significantly larger in sectional area than control platelets and contained increased profiles of endoplasmic reticulum and Golgi cisternae, a lower concentration of surface-connected canalicular system, and occasional membrane complexes. The largest median platelet sectional area was detected at 48R and was the largest median value observed in response to either chronic or acute thrombocytopenia. At 120R, most platelets were normal in size and cytoplasmic appearance, although some large cells remained present in the circulation. MPV paralleled the morphometric changes in platelet sectional area. MK were increased in number at 120CT, 48R, 72R, and 120R. In addition, at least half of the MK examined at 48R contained small areas of cytoplasm, devoid of organelles, that were interspersed between larger areas of organelle-filled, undemarcated cytoplasm. The modal bone marrow megakaryocyte ploidy class, determined using two-color fluorescence-activated flow cytometry, shifted from 16N to 32N in response to sustained thrombocytopenia. In contrast, during recovery and development of rebound thrombocytosis, the relative frequency of 8N megakaryocytes was significantly increased. Because there was no consistent correlation between megakaryocyte cytoplasmic characteristics and platelet morphology, these data support the hypothesis that platelet formation is not determined by compartmentalization of MK cytoplasm into platelet areas as MK mature in the bone marrow, but involves a rearrangement of MK cytoplasm immediately prior to platelet release.

Regulation of platelet heterogeneity: effects of thrombocytopenia on platelet volume and density.

We have examined the effects of variable degrees of acute thrombocytopenia on platelet levels, mean platelet volume (MPV), and buoyant density after induction of thrombocytopenia by platelet antiserum (PAS) in mice with or without spleens. Mice were studied serially 10-16, 36, 48, 60-64, 84, 108, 144, 180, 228, 276, 348-360, 372, and 516 h after PAS treatment. MPV and platelet count (PC) x 10(6)/microliters for normal intact mice (n = 136) were 4.7 +/- 0.3 fl (SD) and 1.69 +/- 0.52 (SD), respectively. Twelve hours after PAS-induced severe thrombocytopenia (PC less than 0.05 x 10(6)/microliters), MPV increased significantly (p less than 0.01) to 6.4 fl, was maximal at 36 h (8.2 fl), remained elevated until 144 h following PAS treatment, and then returned to normal. Platelet density decreased significantly (p less than 0.05) 64 h after PAS treatment and returned to normal at 144 h. Hematocrits of repeatedly bled intact control mice decreased from 45% to 30%, accompanied by thrombocytosis (maximal PC 2.24 x 10(6)/microliters) without significant changes in either MPV or platelet density. Moderate thrombocytopenia (PC 0.1-0.2 x 10(6)/microliters) in intact mice produced significantly (p less than 0.05) increased MPV, at 5.7 fl 12 h after PAS treatment, with a peak MPV of 7.6 fl (p less than 0.001) at 36 h; MPV returned to normal at 84 h. Platelet density decreased (p less than 0.001) 12 h after PAS treatment and returned to baseline at 228 h. Control splenectomized mice (n = 185) had an MPV of 5.0 fl +/- 0.7 fl and a PC of 2.14 +/- 0.6 x 10(6)/microliters. Comparably severe and moderate thrombocytopenia in splenectomized mice produced alterations in platelet count, MPV, and density similar to those in intact mice, although maximal MPV and the degree of rebound thrombocytosis after severe thrombocytopenia were more marked in splenectomized mice. In response to reduction of the platelet mass in both intact and splenectomized mice, MPV increased in proportion to the severity of thrombocytopenia, occurred as early as 4 h after induction, and persisted during early rebound thrombocytosis. Previous observations that megakaryocyte ploidy did not shift until 48 h after onset of thrombocytopenia confirm that both initial and maximal changes in MPV in response to this stimulus are regulated by processes other than alterations of megakaryocyte DNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Measurement of megakaryocyte frequency and ploidy distribution in unfractionated murine bone marrow.

Measurement of megakaryocyte frequency, ploidy distribution, and maturation stage have been complicated by the low frequency of megakaryocytes in either bone marrow or spleen. Due to harsh labeling conditions, previous studies utilizing flow cytometry have not quantified cell recovery or megakaryocyte frequency. We have modified our two-color fluorescence-activated cytometric technique in order to identify megakaryocytes in unfractionated murine bone marrow with a fluoresceinated cell surface immunologic probe (heterologous polyclonal platelet antiserum) and to selectively measure megakaryocyte DNA content with propidium iodide using isosmolar conditions. Under these conditions, total nucleated cell recovery from unfractionated normal murine bone marrow, after all preparative procedures, averaged 68.0% +/- 5.0% (SD) with 93.5% +/- 2.0% DNA staining efficiency. Mean megakaryocyte frequency (n = 30) was 0.14% +/- 0.04%. Using both our previously described gating technique with single parameter analysis and our modified dual parameter technique, the modal megakaryocyte ploidy class was 16N. Low ploidy megakaryocytes, 2N and 4N, constituted 9.8% and 10.8%, respectively, of the total megakaryocyte population. Analysis of the megakaryocyte population with respect to cell surface fluorescence intensity demonstrated that the dimly fluorescent population contained an increased proportion of lower ploidy class cells (2N + 4N + 8N) compared with brightly fluorescent cells, which contained an increased proportion of higher ploidy cells (16N + 32N). Our modifications of the two-color technique yielded improved recovery of total nucleated bone marrow cells from unfractionated bone marrow and provided more precise measurements of megakaryocyte frequency and ploidy than previously available.