Pathogenic heparin-induced thrombocytopenia and thrombosis (HIT) antibodies determined by rapid functional flow cytometry.

OBJECTIVES: Reliable diagnosis of heparin-induced thrombocytopenia and thrombosis (HIT) is mandatory for patient management, yet prompt determination of pathogenic antibodies remains an unmet clinical challenge. Common immunoassays carry inherent limitations and functional assays which detect antibody-mediated platelet activation are not usually readily available to routine laboratories, especially the serotonin release assay (SRA), being technically demanding, time consuming, and requires high level expertise. To overcome some of these limitations, we have developed a practical functional flow cytometric assay (FCA) for routine clinical use. METHODS: A simple FCA is described which avoids platelet manipulation, is highly specific and sensitive compared with SRA, and provides rapid results. RESULTS: Of the 650 consecutive samples, from HIT-suspected patients, 99 (15.3%) were positive by the PaGIA Heparin/PF4 immunoassay and 31 (4.8%) by FCA. Average platelet activation was 11-fold higher in PaGIA+/FCA+ vs PaGIA-/FCA- samples. Of 21 SRA-positive samples, 19 were FCA-positive (relative sensitivity 90.5%), and of 42 SRA-negative samples, 40 were FCA-negative (relative specificity 95.2%). The FCA showed significantly higher correlation with the clinical presentation of HIT (4Ts score) performed on 182 patients, compared with PaGIA Heparin/PF4 (ROC-plot analysis, AUC 0.93 vs 0.63, P < 0.001). At a 92% sensitivity, the assay specificity was 96%. CONCLUSIONS: The present FCA is practical for routine testing, providing prompt reliable results for initial diagnosis and confirmation, to effectively assist in HIT patient management.

DIC score in pregnant women--a population based modification of the International Society on Thrombosis and Hemostasis score.

OBJECTIVES: The objectives of this study were: 1) To determine the component needed to generate a validated DIC score during pregnancy. 2) To validate such scoring system in the identification of patients with clinical diagnosis of DIC. MATERIAL AND METHODS: This is a population based retrospective study, including all women who gave birth at the 'Soroka University Medical Center' during the study period, and have had blood coagulation tests including complete blood cell count, prothrombin time (PT)(seconds), partial thromboplastin time (aPTT), fibrinogen, and D-dimers. Nomograms for pregnancy were established, and DIC score was constructed based on ROC curve analyses. RESULTS: 1) maternal plasma fibrinogen concentrations increased during pregnancy; 2) maternal platelet count decreased gradually during gestation; 3) the PT and PTT values did not change with advancing gestation; 4) PT difference had an area under the curve (AUC) of 0.96 (p<0.001), and a PT difference ≥1.55 had an 87% sensitivity and 90% specificity for the diagnosis of DIC; 5) the platelet count had an AUC of 0.87 (p<0.001), an 86% sensitivity and 71% specificity for the diagnosis of DIC; 6) fibrinogen concentrations had an AUC of 0.95 (p<0.001) and a cutoff point ≤3.9 g/L had a sensitivity of 87% and a specificity of 92% for the development of DIC; and 7) The pregnancy adjusted DIC score had an AUC of 0.975 (p<0.001) and at a cutoff point of ≥26 had a sensitivity of 88%, a specificity of 96%, a LR(+) of 22 and a LR(-) of 0.125 for the diagnosis of DIC. CONCLUSION: We could establish a sensitive and specific pregnancy adjusted DIC score. The positive likelihood ratio of this score suggests that a patient with a score of ≥26 has a high probability to have DIC.

Advances in megakaryocytopoiesis and thrombopoiesis: from bench to bedside.

Megakaryocytopoiesis involves the commitment of haematopoietic stem cells, proliferation and terminal differentiation of megakaryocytic progenitors (MK-p) and maturation of megakaryocytes (MKs) to produce functional platelets. This complex process occurs in specialized niches in the bone marrow where MKs align adjacent to vascular endothelial cells, form proplatelet projections and release platelets into the circulation. Thrombopoietin (THPO, TPO) is the primary growth factor for the MK lineage and necessary at all stages of development. THPO is constitutively produced in the liver, and binds to MPL (c-Mpl) receptor on platelets and MKs. This activates a cascade of signalling molecules, which induce transcription factors to drive MK development and thrombopoiesis. Decreased turnover rate and platelet number result in increased levels of free THPO, which induces a concentration-dependent compensatory response of marrow-MKs to enhance platelet production. Newly developed thrombopoietic agents operating via MPL receptor facilitate platelet production in thrombocytopenic states, primarily immune thrombocytopenia. Other drugs are available for attenuating malignant thrombocytosis. Herein, we review the regulation of megakaryocytopoiesis and platelet production in normal and disease states, and the innovative drugs and therapeutic modalities to stimulate or decrease thrombopoiesis.

[Megakaryocyte development and platelet production in normal and disease states].

Megakaryocytopoiesis involves the commitment of hematopoietic stem cell, the proliferation and terminal differentiation of the megakaryocytic progenitors and maturation to platelet producing megakaryocytes (MK). MKs align adjacent to bone marrow vascular endothelial cells, and form proplatelets. Platelets are released, or sheared from the MK directly into the circulation from the tips of proplatelets which protrude into the vascular lumen. The regulation of megakaryocytopoiesis is mediated through multiple hematopoietic growth factors, chemokines and cellular interactions via signal transduction pathways and integrated transcription factors. The primary physiological growth factor that regulates megakaryocytopoiesis and platelet production is thrombopoietin. Circulating Levels of thrombopoietin (TPO) induce concentration-dependent proliferation and maturation of MK progenitors by binding to the c-Mpl receptor and signaling induction. Increased concentration of free TPO resulting from decreased platelet turnover rates enables the compensatory response of marrow MKs to drive amplified platelet production. C-MpL signaling is orchestrated by a complex cascade of signaling molecules inducing the action of specific transcription factors to drive MK proliferation and maturation. Newly developed thrombopoietic agents operating via c-Mpl receptor have now been proven useful in supporting platelet production in thrombocytopenic states. In this article, the authors review the regulation of megakaryocytopoiesis and platelet production in normal and disease states, and new approaches of thrombopoietic therapy.

Platelet counts and platelet activation markers in obese subjects.

OBJECTIVE: In this work we studied the correlation between platelet count, platelet activation, and systemic inflammation in overweight, obese, and morbidly obese individuals. METHODS AND SUBJECTS: A total of 6319 individuals participated in the study. Complete blood counts, high sensitivity C-reactive protein (hs-CRP) serum levels, and body mass index (BMI) were measured during routine checkups. Platelet activation markers were studied among 30 obese (BMI = 41 +/- 8 kg/m(2)) and 35 nonobese (BMI = 24 +/- 3 kg/m(2)) individuals. Platelet activation status was evaluated by flow cytometry using specific antibodies against the activated platelet membrane glycoprotein IIb/IIIa, p-selectin (CD-62 p), and binding of Annexin-V to platelet anionic phospholipids. RESULTS: Overweight, obese, and morbidly obese females had significantly elevated platelet counts ( P < .0001) compared with normal-weight females. No significant elevation of platelet counts was observed in the male subgroups. A significant age adjusted correlation between BMI and platelet counts ( P < .0001) was found among females. This correlation was attenuated (P = .001) after adjustment for hs-CRP concentrations. The flow cytometry analysis of platelets showed no significant differences in activation marker expression between nonobese and obese individuals. DISCUSSION: Obesity may be associated with elevated platelet counts in females with chronic inflammation. Obesity is not associated with increased platelet activation.

Autoimmune thrombocytopenia: determination of platelet-specific autoantibodies by flow cytometry.

Autoimmune thrombocytopenia is a disorder characterized by antibody-mediated accelerated platelet destruction. Despite its clinical importance, the diagnosis of which is one of exclusion, thus inevitably associated with potential difficulties. Current methods used to determine antigen-specific antibodies including MAIPA and the radioactive immunobead assay, are not routinely used due to methodological and practical limitations. To facilitate diagnosis, flow cytometric methods have been developed, suitable for testing a single or multiple samples. The feasible flow cytometric methods with their high sensitivity and specificity should facilitate the routine use of diagnostic methods for autoimmune thrombocytopenia and permit follow-up to determine immune remission.

Megakaryocyte development and platelet production.

Megakaryocytopoiesis involves the commitment of haematopoietic stem cells, and the proliferation, maturation and terminal differentiation of the megakaryocytic progenitors. Circulating levels of thrombopoietin (TPO), the primary growth-factor for the megakaryocyte (MK) lineage, induce concentration-dependent proliferation and maturation of MK progenitors by binding to the c-Mpl receptor and signalling induction. Decreased platelet turnover rates results in increased concentration of free TPO, enabling the compensatory response of marrow MKs to increased platelet production. C-Mpl activity is orchestrated by a complex cascade of signalling molecules that induces the action of specific transcription factors to drive MK proliferation and maturation. Mature MKs form proplatelet projections that are fragmented into circulating particles. Newly developed thrombopoietic agents operating via c-Mpl receptor may prove useful in supporting platelet production in thrombocytopenic state. Herein, we review the regulation of megakaryocytopoiesis and platelet production in normal and disease state, and the new approaches to thrombopoietic therapy.

Bivalirudin for anticoagulation in mechanical aortic valve replacement and heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a potentially serious syndrome. Since there are some alternatives to treatment with heparin in patients who develop HIT, the decision as to which to use should be based on renal and hepatic function, drug availability and the available monitoring resources. We report a patient who received heparin for mechanical aortic valve replacement. Her clinical course was complicated by HIT, which was treated initially by danaparoid. The syndrome progressed with new thrombotic complications, and eventually was treated successfully by bivalirudin (Angiomax; Medison Pharma Ltd, Petach Tikva, Israel) for 9 days. We propose that treatment with bivalirudin for several days is a safe and effective alternative to heparin therapy in patients who develop HIT.

Flow cytometry for the diagnosis of autoimmune thrombocytopenia.

Autoimmune thrombocytopenia is a disorder characterized by antibody-mediated accelerated platelet destruction. Despite its clinical importance, the diagnosis of autoimmune thrombocytopenia is one of exclusion, thus inevitably associated with potential difficulties. Current clinically applicable methods used to determine antigen-specific antibodies, primarily directed to GPIIb/IIIa (CD41a) and GPIb (CD42b), include the monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay and the radioactive immunobead assay. Neither of these assays is commonly used by clinical laboratories, however, because of methodologic and practical limitations. As a result, diagnoses are generally based on clinical impression despite patient presentations that are sometimes complex. To overcome some of these difficulties, flow cytometric techniques have been developed, employing standard methods and equipment suitable for testing a single sample or multiple samples, as may occur in cases of autoimmune thrombocytopenia. The availability of a feasible technique such as flow cytometry, with improved sensitivity and specificity, should facilitate the routine use of a diagnostic method in the evaluation of thrombo-cytopenic patients suspected of having an autoimmune disorder and permit follow-up to determine immune remission.

Human marrow megakaryocyte differentiation: multiparameter correlative analysis identifies von Willebrand factor as a sensitive and distinctive marker for early (2N and 4N) megakaryocytes.

Human megakaryocyte differentiation and maturation were studied in fresh marrow aspirates by using multiparameter flow cytometric correlative analysis. The expression of glycoprotein (GP)IIb/IIIa, GPIIIa, GPIb, and CD36 correlated directly with cell size and ploidy (r > 0.97); however, GPIb acquisition was relatively slow. von Willebrand factor (VWF) is robustly expressed by early (2N and 4N) megakaryocytes, enabling their complete resolution from the other marrow cells at a level superior to that achieved with GPIIb/IIIa. Expression of myeloid CD45 and immunoglobulin G (IgG)-FcgammaRII receptor (CDw32) increased with megakaryocyte maturation and contrasted with the declining expression of HLA-DR (negative in platelets). Interleukin-6 receptor expression in megakaryocytes was higher than in other marrow cells. By using the time-of-flight technique, the diameter of the megakaryocyte population was 37 +/- 4 microm (mean +/- 1 SD) compared with 14 +/- 2 microm for the total marrow cells, ranging from 21 +/- 4 microm for 2N cells to 56 +/- 8 microm for 64N cells. Cell size directly correlated with cell DNA (r = 0.98). Receptor density of GPIIb/IIIa and GPIb decreased with the transition from 2N to 4N cells, then reached maximum at 32N cells. In conclusion, the present methods are useful for studying in vivo human megakaryocytopoiesis in normal and altered states. The expression of VWF is a sensitive and distinctive marker for the identification of young marrow megakaryocytes.

Suppression of in vitro megakaryocyte production by antiplatelet autoantibodies from adult patients with chronic ITP.

Chronic immune thrombocytopenic purpura (ITP) is manifested by autoantibody-induced platelet destruction. Platelet turnover studies suggest that autoantibody may also affect platelet production. To evaluate this, we studied the effect of plasma from adult patients with chronic ITP on in vitro megakaryocyte production. CD34(+) cells, obtained from healthy donors, were cultured in medium containing PEG-rHuMGDF and 10% plasma from either ITP patients or healthy subjects. Cultures containing plasma from 12 of 18 ITP patients showed a significant decrease (26%-95%) in megakaryocyte production when compared with control cultures. Positive ITP plasmas not only reduced the total number of megakaryocytes produced during the culture period but also inhibited megakaryocyte maturation, resulting in fewer 4N, 8N, and 16N cells. The role of antibody in this suppression is supported by 2 factors: (1) immunoglobulin G (IgG) from ITP patients inhibited megakaryocyte production when compared with control IgG; and (2) adsorption of autoantibody, using immobilized antigen, resulted in significantly less inhibition of megakaryocyte production when compared with unadsorbed plasma. These results show that plasma autoantibody from some adult patients with ITP inhibits in vitro megakaryocyte production, suggesting that a similar effect may occur in vivo.

Enhanced erythrocyte adhesiveness/aggregation in obesity corresponds to low-grade inflammation.

OBJECTIVE: Previous studies have suggested that obesity enhances the inflammatory response, producing macromolecules involved in the induction and/or maintenance of increased erythrocyte aggregation. The objectives of this study were to evaluate the correlation between inflammation markers, erythrocyte adhesiveness/aggregation, and the degree of obesity and to assess phosphatidylserine expression on erythrocyte surface membrane of obese vs. nonobese individuals. RESEARCH METHODS AND PROCEDURES: Erythrocyte adhesiveness/aggregation in the peripheral venous blood was evaluated by using a new biomarker, phosphatidylserine expression was assessed by means of flow cytometry, and markers of inflammation were measured in 65 subjects: 30 obese [body mass index (BMI) = 41 +/- 7.7 kg/m(2)] and 35 nonobese (BMI = 24 +/- 2.7 kg/m(2)) individuals. Pearson correlations and Student's t test were performed. RESULTS: A highly significant difference was noted in the degree of erythrocyte adhesiveness/aggregation and markers of inflammation between the study groups. BMI correlated with erythrocyte adhesiveness/aggregation (r = 0.42, p = 0.001), erythrocyte sedimentation rate (r = 0.42, p = 0.001), high-sensitive C-reactive protein (r = 0.55, p < 10(-4)), fibrinogen (r = 0.37, p = 0.004), and white blood cell count (r = 0.45, p < 10(-4)). The degree of erythrocyte adhesiveness/aggregation correlated with erythrocyte sedimentation rate (r = 0.5, p < 10(-4)), high-sensitive C-reactive protein (r = 0.56, p < 10(-4)), fibrinogen (r = 0.54, p < 10(-4)), and white blood cell count (r = 0.32, p = 0.01). DISCUSSION: Our results suggest that obesity-related erythrocyte adhesiveness/aggregation is probably mediated through increased concentrations of adhesive macromolecules in the circulation and not necessarily through hyperlipidemia or phosphatidylserine exposure on erythrocyte's membrane.

Antiphospholipid antibody syndrome: rapid, sensitive, and specific flow cytometric assay for determination of anti-platelet phospholipid autoantibodies.

A rapid, sensitive and specific flow cytometric assay has been developed for the determination of autoantibodies directed against platelet anionic phospholipids in antiphospholipid antibody syndrome (APLAS). The method is based on demonstrable competition between the placental anticoagulant protein I, annexin V, and the patients' autoantibodies on the platelet anionic phospholipids (the binding site for the prothrombinase complex; prothrombin and factors Va and Xa). The method is practical and rapid, uses readily available reagents, and involves standard equipment. The assay is inexpensive and cost-effective for both single and multiple samples. Results are provided within 2 hours from obtaining blood samples, thereby supporting clinical decision-making and management. Ten serum samples from patients with the clinical diagnosis of APLAS (48 tests), 10 from normal individuals (35 tests), and 10 from patients with immune thrombocytopenia (33 tests) were tested. Platelet preparations preincubated with normal sera showed high binding of fluorescein-labeled annexin V with an average fluorescence of 202.9 +/- 22.0 (arbitrary units). Patients with immune thrombocytopenia exhibited similar results, with an average fluorescence of 192.5 +/- 32.1 (P >.05). In contrast, incubation with sera from patients with APLAS resulted in a marked decline in the binding of annexin V to an average fluorescence of only 14.6 +/- 7.4 (P <.001). Preincubation with annexin V followed by the addition of patients' sera showed displacement of annexin V to a similar degree. Because annexin V attenuates procoagulant activity by competing with factors Va and Xa on the platelet anionic phospholipids, its displacement by patients' antibodies may result in the acceleration of procoagulant activity, thereby promoting thrombogenesis in APLAS.

Effects of anagrelide on in vivo megakaryocyte proliferation and maturation in essential thrombocythemia.

To define the mechanism by which anagrelide normalizes the platelet count in essential thrombocythemia, we studied in vivo megakaryocytopoiesis in 10 newly diagnosed patients prior to and while on anagrelide therapy. Using flow cytometric analysis of aspirated marrow, megakaryocytopoiesis was quantified and correlated with the autologous platelet production rate. Megakaryocytes were identified by CD41a expression and enumerated in relation to the nucleated marrow erythroid precursors. Megakaryocyte diameters were directly measured by time-of-flight technique, and cell ploidy was measured by DNA staining. Two to 3 thousand megakaryocytes were analyzed in each sample. In the 10 patients, the platelet count was 1063 +/- 419 x 10(9) platelets/L (mean +/- 1 SD) with markedly increased production (237 +/- 74 x 10(9) platelets/L per day versus 43.1 +/- 8.4 x 10(9) platelets/L per day in healthy individuals). The platelet survival was 8.2 +/- 1.1 days versus 9.0 +/- 0.5 days in healthy controls (P >.05). Megakaryocyte diameter was increased to 46 microm (versus 37 microm in controls; range, 21 microm for 2N to 56 microm for 64N cells). The volume increased to 48 x 10(3) microm(3) versus 26 x 10(3) microm(3) in controls, and the number increased to 14 x 10(6)/kg (versus 7 x 10(6)/kg in controls), resulting in 3.7-fold increase in megakaryocyte mass (66 x 10(10) microm(3)/kg versus 18 x 10(10) microm(3)/kg). Cell ploidy was enhanced showing a modal ploidy of 32N (versus 16N in healthy controls) with marked increase in 64N and 128N cells (P <.05). Anagrelide therapy reduced the platelet counts to 361 +/- 53 x 10(9) platelets/L and the turnover rate to 81 x 10(9) platelets/L per day. The platelet survival was unchanged. Following therapy, megakaryocyte number decreased to 8 x 10(6)/kg, diameter to 40 microm, and volume to 34 x 10(3) microm(3) with a normalized modal ploidy of 16N, resulting in a megakaryocyte mass reduced by 60% (28 x 10(10) microm(3)/kg; P <.05). This reduction in cell mass closely correlated with the reduction in platelet count and production rate by 66% (r = 0.96). The present data indicate that in essential thrombocythemia anagrelide therapy decreases circulating platelets by reducing both megakaryocyte hyperproliferation and differentiation.