Megakaryocyte Abnormalities Occur In The Absence Of Cell-Mediated Immunity In a Murine Model Of Passive Immune Thrombocytopenia (ITP)

Abstract Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by increased peripheral immune platelet destruction and megakaryocyte defects in the bone marrow. Although ITP was originally thought to be primarily due to humoral mediated autoimmunity it is now evident that T cells can also play a contributing role to the thrombocytopenia. In fact, the exact interplay between platelet destruction, megakaryocyte dysfunction, and the elements of both the humoral and cell mediated immune systems still remain incompletely defined. In murine passive models of ITP, the direct administration of anti-platelet antibodies can result in severe thrombocytopenia which is evident within 24 hours of injection. While most studies have focused on immune platelet destruction in the spleen, an additional possibility is that the anti-platelet antibody also has an effect on megakaryocytes. To unequivocally determine if antiplatelet antibodies have an effect on megakaryocytes in an in vivo model, BALB/c mice were intravenously administered 2 ug of an anti-GPIIbIIIa antibody (MReg30) or 50 uL of a high tittered anti-GPIIIa (anti-β3) serum from BALB/c GPIIIa (CD61) knockout mice immunized with wild type platelets. Platelet counts were assessed over time and the bone marrow and spleens were harvested for histological examination of megakaryocytes. Both preparations of antiplatelet antibodies significantly reduced platelet numbers within 1 day of antibody or serum administration. This thrombocytopenia could be rescued by administration of 2 g/kg of IVIg ip. Compared with naïve control mice, histological (H&E staining) examination of the bone marrow and spleens revealed that megakaryocytes were significantly increased in number and all exhibited abnormalities consistent with apoptosis e.g. pyknotic nuclei. IVIg administration completely prevented these megakaryocyte abnormalities. These results show that passively administered anti-platelet antibodies not only affect platelet counts but also significantly affect megakaryocyte physiology in the absence of cell mediated immunity. Disclosures: No relevant conflicts of interest to declare.

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Growth Factor Independence 1b (Gfi1b) Is An Essential Regulator of Late Stage Megakaryocyte Maturation and Platelet Production

Blood ◽

10.1182/blood.v118.21.2358.2358 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2358-2358

Author(s):

Lothar Vassen ◽

Tarik Moroy

Keyword(s):

Bone Marrow ◽

Late Stage ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Pcr Analysis ◽

Severe Thrombocytopenia ◽

Facs Analysis ◽

Platelet Counts ◽

Platelet Release ◽

Deficient Mice

Abstract Abstract 2358 Absence of Gfi1b in mice is embryonically lethal and causes failure to produce functional megakaryocytes and platelets. Thrombopoiesis, the production of platelets by megakaryocytes, is an essential process in hemostasis that needs to be well controlled. Too many platelets can cause thrombosis, too few cause excessive bleeding. How terminal megakaryocyte maturation and platelet release works is incompletely understood but requires many factors such as Fli1, Gata1, MyH9, p45-NFE2, or c-Myc. Expression array analysis of hematopoietic cells from conditionally Gfi1b deficient mice (Gfi1bfl/fl Mx-Cre) revealed an up-regulation of many factors important for megakaryocyte function like Itga2b, Itgb3, CD9, CD41, CD61, PF4 and Ppbp. Gfi1b ablation in adult Gfi1bfl/fl Mx-Cre mice leads to a severe drop in platelet counts to less than 20% of wt mice with an increase in mean platelet volume (MPV) by 40%. However, megakaryocyte numbers rise up to 100 fold over normal levels when Gfi1b is absent. FACS analysis of bone marrow cells of Gfi1b deficient mice showed a higher number of MEPs, a higher proportion of smaller megakaryocytes and an aberrant population of cKithiCD41hiCD9veryhi cells, which are not present in wt animals. Gfi1b−/− megakaryocytes can reach wt size and normal ploidy as shown by FACS analysis and immunofluorescence microscopy. Transmission electron microscopy (TEM) of pIpC induced Gfi1bfl/fl Mx-Cre megakaryocytes revealed an excess number and larger size of so called “demarcation membranes” in unusual parallel layers and a strongly reduced number of dense granula. Strikingly, both intact and fragmented megakaryocytes were frequently found within bone marrow blood vessels in Gfi1b−/− mice. In addition, a high percentage of megakaryocytes were found in a state of disintegration, without signs of proper platelet release. These features are rarely seen in wt mice. It is known that Gfi1b is required for erythropoiesis and Gfi1bfl/fl Mx-Cre mice show signs of anemia and stress erythropoiesis, which might explain high MEP numbers, which could explain the high numbers of megakaryocytes. To better define the function of Gfi1b in late stage megakaryocyte development, we decided to abrogate Gfi1b expression more specifically by using mice that express Cre recombinase under the megakaryocyte specific promoter of the PF4 gene (PF4-Cre). We observed that Gfi1bfl/fl PF4-Cre mice develop a very severe thrombocytopenia reaching only 2% of wt platelet counts in peripheral blood accompanied by an increase of MPV by 80% over wt levels. Most of Gfi1bfl/fl PF4-Cre mice died at 6–8 weeks of age from severe internal bleedings. Megakaryocyte numbers increase in these mice by 5 to 10 fold and they also reach high ploidy, but their morphology is highly disturbed. Gfi1bfl/fl PF4-Cre megakaryocytes contain less cytoplasm, few dense granula organized in a small patch and a lobulated, ring-shaped nucleus localized close to the cell membrane giving the cells an almost “inside-out” appearance and indicating a disturbed cytoskeleton organization. Gfi1bfl/flPF4-Cre mice show a stress induced splenic erythropoiesis and an increase in MEP numbers, probably a consequence of their substantial hemorrhaging owing to the low platelet counts. The high MEPs number might explain the increase in megakaryocytes in these mice compared to wt controls. Immunofluorescence analysis of Gfi1bfl/fl PF4-Cre megakaryocytes compared to wt counterparts showed less expression of van Willebrand factor (vWF), an important regulator of thrombopoiesis. Q-PCR analysis on mRNA from sorted Gfi1bfl/fl PF4-Cre wt megakaryocytes revealed a lower expression of vWF, but higher PF4, very high Mpl and high CCNE1 expression. Our data show that Gfi1b controls the production of platelets from megakaryocytes, but does not affect the maturation of megakaryocytes as such. However, Gfi1b is required to maintain the cellular organelle structure in megakaryocytes and, more specifically is required to control the formation of dense granula and demarcation membrane formation. Gfi1b ablation in megakaryocytes results in a phenotype with high similarities to Gata1-low mice and syndromes involving mutations in the Gata1 target Gpib, the receptor for vWF, causing Bernhard-Soulier Syndrome (BBS). It is thus possible that Gfi1b is another candidate gene involved in megakaryocyte related diseases. Disclosures: No relevant conflicts of interest to declare.

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Recognition of Megakaryocyte-Specific T-Antigen By Macrophages Negatively Regulates Platelet Production in Bone Marrow

Blood ◽

10.1182/blood.v126.23.420.420 ◽

2015 ◽

Vol 126 (23) ◽

pp. 420-420

Author(s):

Melissa M. Lee-Sundlov ◽

Renata Grozovsky ◽

Silvia Giannini ◽

Martina McGrath ◽

Haley Elizabeth Ramsey ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Flow Cytometric Analysis ◽

Antigen Expression ◽

Blood Platelet ◽

T Antigen ◽

Severe Thrombocytopenia ◽

Extrinsic Factors ◽

Platelet Production ◽

Platelet Counts

Abstract Glycosylation defects have been associated with low platelet counts. Six genes encoding sialyltransferases (ST), ST3gal1 to 6, that synthesize an α2,3 sialic acid (SA) linkage have been identified in the mammalian genome, and deletion of St3gal1 and St3gal4 genes has been associated with macrothrombocytopenia in mice. Despite the similarity in transferring SA in a α2,3-linkage to terminal galactose residues, St3gal1 and St3gal4 sialylate distinct glycans: St3gal1 is associated with core 1 O-glycan Galβ1,3GalNAcα1-Ser/Thr expression, also known as tumor-associated or Thomsen-Friedenreich antigen (T-antigen), whereas St3gal4 sialylates lactosaminyl Galβ1,4GlcNAc N-glycans. It has been previously shown that St3gal4-null platelets are cleared by the hepatic Ashwell-Morell receptor, causing severe thrombocytopenia in these mice. Herein, we generated St3gal1loxP/PF4+ mice specifically lacking ST3Gal1 in the megakaryocyte (MK) lineage to investigate the detailed mechanisms of macrothrombocytopenia associated with St3gal1 deficiency. Both St3gal1loxP/PF4+ circulating platelets and bone marrow (BM) MKs had increased T-antigen expression, compared to control, as evidenced by peanut agglutinin (PNA) binding. As expected, other blood cell lineages had no increase in T-antigen expression. Blood platelet counts were reduced by ~50% and platelets were enlarged in St3gal1loxP/PF4+ mice, compared to control, despite a virtually indistinguishable platelet clearance. BM MK numbers were normal despite the observed thrombocytopenia, BM MK colony forming units (CFUs) were reduced and in vitro proplatelet production was normal in St3gal1loxP/PF4+ mice, suggesting that extrinsic factors in the St3gal1loxP/PF4+ BM environment affected platelet production. We hypothesize that recognition of the T-antigen epitope on MKs mediate phagocytosis by macrophages. Macrophages in St3gal1loxP/PF4+ mice had increased expression of CD68 (macrosialin), indicative of an activated macrophage state. Flow cytometric analysis of BM derived macrophages of St3gal1loxP/PF4+ mice showed an increased population of resolving M2-type macrophages, which are normally involved in apoptotic cell clearance. Additionally, St3gal1loxP/PF4+ BM smears revealed increased hemophagocytosis, as evidenced by May-Grunwald/Giemsa, indicative of an unspecific increase in phagocytic macrophages. Macrophage ablation by in vivo injection of clodronate-encapsulated liposomes significantly reduced the numbers of activated macrophages in St3gal1loxP/PF4+ mice, thereby normalizing blood platelet counts and size. Taken together data show the contrasting effects of different SA loss on platelet homeostasis: Platelets lacking α2,3-linked SA on N-glycans have increased platelet clearance, whereas a lack of α2,3-linked on O-glycans do not affect platelet half-life, but cause defective thrombopoiesis in MKs. Disclosures No relevant conflicts of interest to declare.

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Calr Mutants Retroviral Mouse Models Lead to a Myeloproliferative Neoplasm Mimicking an Essential Thrombocythemia Progressing to a Myelofibrosis

Blood ◽

10.1182/blood.v124.21.157.157 ◽

2014 ◽

Vol 124 (21) ◽

pp. 157-157 ◽

Cited By ~ 4

Author(s):

Caroline Marty ◽

Nivarthi Harini ◽

Christian Pecquet ◽

Ilyas Chachoua ◽

Vitalina Gryshkova ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Myeloproliferative Neoplasm ◽

In Vivo Model ◽

Common Mechanism ◽

Loss Of Function ◽

Hematopoietic Stem ◽

Platelet Counts

Abstract Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocytemia (ET) and Primary Myelofibrosis (PMF). They are malignant homeopathies resulting from the transformation of a multipotent hematopoietic stem cell (HSC). The common mechanism of transformation is the constitutive activation of the cytokine receptor/JAK2 pathway that leads to the myeloproliferation. The acquired point mutation JAK2V617F is the most prevalent (95% of PV and 60% of ET or PMF). In addition, other mutations affecting the same signaling pathway have been described such as JAK2 exon 12 mutations, mutations of MPL affecting W515, and loss-of-function mutations of LNK and also mutations of c-Cbl in 3% of PMF. Recently, whole exome sequencing allowed identifying a new recurrent genetic abnormalities in the exon 9 of the calreticulin gene (CALR) in about 30% of ET and PMF patients. All CALR mutants induce a frameshift of the same alternative reading frame and generate a novel C-terminus tail. To address the role of these new mutants in the pathophysiology of MPN, the goal of this study was to investigate the effect of the CALR mutant (del52 and ins5) expression by a retroviral mouse modeling. For that purpose, we transduced bone marrow cells with retrovirus expressing either CALRdel52, CALRins5, CALRWT or CALRDexon9 and performed a transplantation in lethally irradiated recipient mice (10 mice / group), which were then followed over one year. CALRdel52 expressing mice showed a rapid and strong increased in platelet counts (over 5 x106/mL) without any other changes in blood parameters during 6 months. In contrast, CALRins5 expressing mice presented platelet counts much lower than CALRdel52 but significantly higher than CALRWT or CALRDexon9 expressing mice. After 6 months, CALRdel52 expressing mice showed a decreased in platelets count associated with anemia and development of splenomegaly suggesting the progression to a myelofibrosis. Importantly, the disease was transplantable to secondary recipient for both CALRdel52 and CALRins5 mutants. The bone marrow and spleen were also analyzed over time. We observed a progressive increased in immature progenitors (SLAM cells) as well as a hypersensitivity of the megakaryocytic progenitors (CFU-MK) to thrombopoietin. Altogether, these results demonstrate that CALR mutants are able and sufficient to induce a thrombocytosis progressing to myelofibrosis in retroviral mouse model, thus mimicking the natural history of MPN patients. It will offer a good in vivo model to investigate therapeutic approaches for CALR-positive MPN. Disclosures No relevant conflicts of interest to declare.

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Successful Treatment of Acquired Amegakaryocytic Thrombocytopenia with Rituximab: A Case Report.

Blood ◽

10.1182/blood.v114.22.4223.4223 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4223-4223

Author(s):

Laura R. Goldberg ◽

Lewis Glasser ◽

Tony Wu ◽

Ikue Shimizu ◽

Peter J. Quesenberry ◽

...

Keyword(s):

Bone Marrow ◽

Case Report ◽

Bone Marrow Biopsy ◽

Conflicts Of Interest ◽

Immunosuppressive Agents ◽

Peripheral Blood Smear ◽

Severe Thrombocytopenia ◽

Literature Report ◽

Platelet Counts ◽

Acquired Amegakaryocytic Thrombocytopenia

Abstract Abstract 4223 Acquired amegakaryocytic thrombocytopenia is a rare disorder in which there is a marked decrease in bone marrow megakaryocytes leading to severe thrombocytopenia with preserved hematopoeisis in the remaining lineages. The clinical course is variable and no standardized treatment exists. Multiple cases in the literature report treatment using immunosuppressive agents including cyclosporine and antithymocyte globulin. In this case report, we describe the first successful use of rituximab in a patient with amegakaryocytic thrombocytopenia in the absence of any underlying autoimmune disorders. Our patient, an 86- year-old woman, presented with epistaxis, ecchymoses, and blood-tinged sputum. She had thrombocytopenia (platelets 6 × 109/L, MPV 8.3) with a normal hemoglobin and white blood cell count. Peripheral blood smear showed no obvious cause for thrombocytopenia. Bone marrow biopsy revealed focal lymphocytic infiltrates and severe megakaryocytic hypoplasia with nearly absent megakaryocytes. Cytogenetic and molecular analyses were normal, revealing no evidence of a clonal myelo- or lymphoproliferative disorder. The diagnosis of amegakaryocytic thrombocytopenia was made. She was started on prednisone 50mg per day. There was no significant improvement in her platelet count and she was then given IVIG without response. Repeat bone marrow biopsy showed persistent severe bone marrow hypoplasia with near absence of megakaryocytes. After 6 weeks of platelet transfusion dependence and steroids, she was tapered off her steroids and was given rituximab 375mg/m2 weekly for 4 doses. Three weeks after starting rituximab, her platelet counts began to recover and by day 37, her platelet counts normalized and remained within normal limits 12 months after treatment. This case demonstrates the possible utility of rituximab in treating patients with isolated acquired amegakaryocytic thrombocytopenia. Disclosures: No relevant conflicts of interest to declare.

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The Day 1 Postoperative Platelet Count Predicts Splenectomy Response in Patients with Immune Thrombocytopenia (ITP)

Blood ◽

10.1182/blood.v124.21.1448.1448 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1448-1448

Author(s):

Shylaja Mani ◽

Hashim Abbas ◽

Akhil Parashar ◽

Keith R. McCrae

Keyword(s):

Logistic Regression ◽

Platelet Count ◽

Medical Therapy ◽

Immune Thrombocytopenia ◽

Conflicts Of Interest ◽

Cleveland Clinic ◽

Complete Response ◽

Stepwise Logistic Regression ◽

Severe Thrombocytopenia ◽

Platelet Counts

Abstract Background: Immune thrombocytopenia (ITP) in adults is generally a chronic disorder that may lead to severe thrombocytopenia and bleeding. Though several medical modalities such as thrombopoietin receptor agonists have become available for the management of ITP withinin the last decade, splenectomy remains a valuable option for management of refractory ITP, with approximately 2/3 of treated patients remaining in complete remission 10 years afterwards. However, there are no consistent and reliable predictors of splenectomy response for an individual patient with ITP. Since patients with ITP who fail to respond to splenectomy can develop significant bleeding in the postoperative period it is important to identify those individuals early after their surgical procedure so that aggressive medical intervention may be employed. Despite this concern, there is little information available on the value of postoperative platelet counts obtained soon after splenectomy in predicting the ultimate outcome of surgery. Objectives: The goal of this study was to define the value of platelet counts determined soon after splenectomy on the ultimate success of splenectomy in inducing remission of ITP. Methods: We reviewed the medical records of 66 patients who underwent splenectomy for ITP at the Cleveland Clinic from 2000-2013. A complete response was defined as a stable platelet count >100 x109/L two months after splenectomy without medical therapy. Stepwise logistic regression with backward selection was used to identify significant predictors of complete response. Results: The 66 patients had a median age of 41(IQR 21-56) with a male:female ratio of 1:2. The median platelet count at the time of diagnosis was 12 x 109/L and 43% of the patients had severe ITP (defined per IWG guidelines as bleeding that mandates treatment). Ninety percent of patients were steroid dependent, and 39%, 15% and 5% had been treated with rituximab, eltrombopag or romiplostim respectively. The median time to splenectomy from diagnosis of ITP was 22 months (IQR 6-44 months). At a median follow up of 35 months after splenectomy, 39 patients (59%) achieved a complete response. The median platelet count prior to and 24 hours after splenectomy in responders and non-responders is shown in Table 1. Logistic regression analyses identified a post-op day 1 platelet count greater than the median platelet count of 112 x 109/L (OR- 3.72, CI- 1.14-12.16, p<0.03) and post-operative day 3 platelet count greater than median platelet count of 175x 109/L (OR- 4.87, CI- 1.37-17.2, p<0.01) as a significant predictor of splenectomy response. The probability of response based on the post-operative day 1 platelet count is depicted in Figure 1. The difference between the pre-splenectomy and post-operative day 1 platelet count was also a significant predictor of response (OR 1.01 (1.0001-1.02), p=0.04), (figure 2). The log of the time from the diagnosis of ITP to splenectomy (OR- 0.61, CI 0.40-0.94, p<0.02) was also a weak, but significant predictor. Increased numbers of prior treatments for ITP prior to splenectomy correlated with a decreased response, although this relationship was not statistically significant. Conclusion: The platelet count on postoperative day 1 is a significant predictor of long term response to splenectomy, with almost 4-fold increased probability of achieving remission if this value is >112 x 109/L .This is among the first studies to examine the prognostic value of the platelet count obtained this early after splenectomy, and suggests that in patients with severe ITP and a persistently low postoperative platelet count on day 1, medical therapy should be considered to prevent bleeding. Our data also suggests that responses to splenectomy may be less frequent in patients with a longer interval between ITP diagnosis and splenectomy. Disclosures No relevant conflicts of interest to declare.

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Understanding the Mechanisms Underlying Thrombocytopenia in Visceral Leishmaniasis

Blood ◽

10.1182/blood-2019-128133 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2378-2378 ◽

Cited By ~ 1

Author(s):

Gulab Fatima Rani ◽

Olivier Preham ◽

Ian Hitchcock ◽

Paul Kaye

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Visceral Leishmaniasis ◽

Conflicts Of Interest ◽

Experimental Models ◽

Severe Thrombocytopenia ◽

Hematopoietic Stem ◽

Platelet Production ◽

Platelet Counts ◽

Splenic Macrophages

Visceral leishmaniasis (VL) is a neglected tropical parasitic disease caused by Leishmania parasites and only second to malaria in terms of worldwide morbidity and mortality. According to recent WHO report, there are 500,000 cases of VL worldwide leading to ~30,000 deaths per year. VL is endemic in 98 countries but the major disease burden is contributed by Brazil, India and Sudan. Disease manifestations include fever, weight loss, hepatosplenomegaly, immune dysregulations and extensive hematological complications. We have shown previously using experimental models of infection that the infiltration of CD4+ T cells results in disruption to the bone marrow environment, resulting in dysfunctional hematopoietic stem and progenitor cells self-renewal (Pinto et al, PLOS Pathogens, 2017) and aberrant medullary erythropoiesis causing pathological anemia (Preham et al, Frontiers in Immunology, 2018). Thrombocytopenia is also dominant hematological feature seen in both human and experimental models that may reflect either reduced platelet production or enhanced clearance. However, the mechanisms of VL-driven thrombocytopenia remain poorly understood. The aim of this study is to explore the possible underlying mechanisms from platelet production to phagocytic cells dependent clearance. Using a murine experimental model of VL, we demonstrate a steady decrease in the platelet count from d14 onwards in infected mice culminating in severe thrombocytopenia on d28 of infection (infected: 225.9 ±35.7 vs naïve: 1005 ±90.6, x 106/µl). Critically, thrombocytopenia is completely reversible after a single dose of liposomal amphotericin B (Ambisome @ 8mg/kg bodyweight, IV) which clears parasites by delivering the drug directly to parasite harbouring tissue macrophages, thereby improving parasite clearance and reducing toxicity. Despite significant thrombocytopenia, the number and gross morphology of bone marrow megakaryocytes (MKs) were not altered, but MK ultrastructure studies using transmission electron microscopy identified significantly reduced demarcation membranes in infected mice compared to naïve. Levels of plasma thrombopoietin (TPO), the key regulator of MK differentiation and platelet production, were decreased in infected vs naïve mice (1254 ± 95.49 vs 3249 ± 125.1 pg/ml) and administration of exogenous TPO resulted in complete recovery of platelet counts. Given that the majority of TPO is produced by the liver, reduction in the levels of circulating TPO during infection is likely due to destruction of liver architecture by parasite loaded hepatic granulomas. Together, these data suggest that despite some changes in MK cytoplasmic maturation, the bone marrow microenvironment remains supportive of MK differentiation capacity during VL. As platelet production is not significantly altered by VL, we next determined effects on platelet clearance. Large number of highly active splenic macrophages are common in VL and are known for their phagocytic properties. Experiments conducted on VL-infected splenectomised mice demonstrated a reduction in thrombocytopenia compared to sham-operated infected mice (685 ±32 vs 297± 16, x 106/µl) and showed a great response to exogenous TPO, implying splenic clearance may be involved in thrombocytopenia. Partial depletion of splenic macrophages in infected mice using clodronate liposomes did not alter platelet count, whereas neutrophil deletion (anti-Gr1 mAb @ 250ug/g IP) in infected mice resulted in a near 2-fold increase in platelet counts. Furthermore, circulating platelets in VL infected mice were IgG coated compared to naive which is likely to further enhance autoimmune platelet clearance. Severe thrombocytopenia and bleeding are important clinical manifestations of VL. Our findings clearly demonstrate that the mechanisms of thrombocytopenia in VL are multifactorial but do not cause permanent long term damage to the BM microenvironment. Critically, these changes could be reversed rapidly by clearing parasitemia, using TPO agonists to increase numbers of circulating platelets and/or by reducing platelet clearance. This highlights the possibility of re-evaluating the current treatment regimen in VL endemic countries by including therapeutic interventions aimed at reversing severe thrombocytopenia. Disclosures No relevant conflicts of interest to declare.

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Haemodialysis-associated thrombocytopenia: interactions among the immune system, membranes and sterilisation methods

BMJ Case Reports ◽

10.1136/bcr-2019-229594 ◽

2019 ◽

Vol 12 (9) ◽

pp. e229594

Author(s):

Felipe Batalini ◽

Gabriel Francisco Aleixo ◽

Asaf Maoz ◽

Shayna Sarosiek

Keyword(s):

Bone Marrow ◽

Differential Diagnosis ◽

Electron Beam ◽

Immune System ◽

Severe Thrombocytopenia ◽

Platelet Destruction ◽

Platelet Counts ◽

Immune Mediated ◽

Relationship Of

We present a case of a 47-year-old man with severe thrombocytopenia. The differential diagnosis for thrombocytopenia is wide. The assessment includes an evaluation for falsely low platelet counts (pseudothrombocytopenia), immune-mediated platelet destruction, bone marrow dysfunction, or increased consumption and sequestration. After extensive and systematic workup, we found a relationship of his thrombocytopenia with haemodialysis. Although not widely recognised by clinicians, partly due to an incomplete understanding of its pathophysiology, haemodialysis is also a potential cause of thrombocytopenia. His platelet counts completely normalised after the substitution of his haemodialysis membrane. We concluded that our patient had haemodialysis-induced thrombocytopenia, most likely secondary to electron-beam sterilisation.

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Thrombopoietin Can Induce Tolerogenic Responses Via the Modulation of Thymic and Splenic T Lymphocyte Populations in a Murine Model of Immune Thrombocytopenia (ITP)

Blood ◽

10.1182/blood.v120.21.1088.1088 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1088-1088

Author(s):

John W. Semple ◽

Xu Zhang ◽

Yu Hu ◽

Li Guo ◽

Edwin R. Speck ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Murine Model ◽

Immune Thrombocytopenia ◽

Conflicts Of Interest ◽

T Regulatory Cell ◽

Platelet Counts ◽

Platelet Antibodies ◽

Lymphocyte Populations ◽

Platelet Antibody

Abstract Abstract 1088 Clinical trials using thrombopoietin (TPO) mimetics have clearly shown their benefit in raising platelet counts in patients with immune thrombocytopenia (ITP), however, little is known whether TPO may affect immunologic responses. It has been demonstrated that, like other therapies, TPO treatment can rescue the peripheral CD4+CD25+FoxP3+ T regulatory cell (Tregs) deficiency that is commonly observed in patients with ITP (Bao W et al Blood 116;4639, 2010). Of interest, an earlier murine study also suggested that TPO could affect the production of T cells, particularly CD4+ T cells (Zhao JZ et al Haematologica 83:572, 1998). Using a murine model of ITP that demonstrates both antibody and T cell mediated thrombocytopenia, we studied T cell responses in ITP mice treated with TPO. ITP was induced in severe combined immunodeficient (SCID) mice by administration of 2×104 splenocytes from CD61 (GPIIIa) knockout mice immunized against CD61 and the mice where either treated or not with weekly subcutaneous injections of 1ug of murine TPO. Mice were bled weekly to count platelets and obtain sera for anti-platelet antibody production and at 3 weeks after the splenocyte transfer, they were sacrificed and their spleens and thymi were harvested. Splenocytes and thymocytes were isolated and examined by flow cytometry to quantify CD4+CD25+FoxP3+ (Tregs) and CD8+CD25+FoxP3+ (Tc-regs). We observed that TPO treatment caused an expected significant increase in platelet counts, however, the treatment also caused a significant reduction in the titre of serum anti-platelet antibodies. This was associated with significant changes in the proportions of both CD4+ Tregs and CD8+ Tc-regs within the thymus and spleen. TPO treatment rescued the peripheral splenic Treg and Tc-reg deficiencies and the cells ability to functionally suppress the proliferation of conventional CD4+CD25- T cells. The data suggests that in addition to its megakaryocyte-stimulating properties, TPO treatment has additional tolerance-inducing effects associated with anti-platelet antibody reduction and CD4+ Treg and CD8+ Tc-reg stimulation. Disclosures: No relevant conflicts of interest to declare.

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Immature platelet values indicate impaired megakaryopoietic activity in neonatal early-onset thrombocytopenia

Thrombosis and Haemostasis ◽

10.1160/th09-03-0148 ◽

2010 ◽

Vol 103 (05) ◽

pp. 1016-1021 ◽

Cited By ~ 27

Author(s):

Hannes Hammer ◽

Christoph Bührer ◽

Christof Dame ◽

Malte Cremer ◽

Andreas Weimann

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Intensive Care ◽

Early Onset ◽

Absolute Number ◽

Severe Thrombocytopenia ◽

Platelet Counts ◽

Immature Platelet Fraction ◽

Neonatal Thrombocytopenia ◽

Immature Platelets

SummaryNewly released platelets, referred to as immature platelets, can be reliably quantified based on their RNA content by flow cytometry in an automated blood analyser. The absolute number of immature platelets (IPF#) and the immature platelet fraction (IPF%) reflect megakaryopoietic activity. We aimed to analyse the implication of these parameters in analysing the pathomechanism of early-onset neonatal thrombocytopenia. Platelet counts and IPF were determined at day 1 to 3 (d1 to d3) in 857 neonates admitted to intensive care. In thrombocytopenic patients (platelet counts<150 x 109/l, n=129), IPF# was significantly lower (8.5 ± 2.7 x 109/l), than in non-thrombocytopenic neonates (9.5 ± 3.6 x 109/l, n=682, p<0.05). IPF% was significantly higher in thrombocytopenic (9.3 ± 7.9%) vs. non-thrombocytopenic neonates (4.1 ± 1.8%, p<0.001). While neonates with early-onset infection (n=134) had lower platelet counts (199 ± 75 x 109/l) compared to controls (230 ± 68 x 109/l, n=574, p<0.01), there were no differences in IPF# or IPF%. Likewise, “small for gestational age” infants (SGA, n=149) had lower platelet counts at d1 (199 ± 75 x 109/l, p<0.001) than controls, but no differences in IPF. A trend towards lower IPF# was detected if SGA infants with platelet counts <100 x 109/l (5.4 ± 3.9 x 109/l, n=11) and thrombocytopenic neonates with infection (9.9 ± 7.3 x 109/l, n=10, p=0.11) were compared. The evaluation of IPF# indicates that thrombocytopenia in neonates is likely due to a combination of increased platelet consumption and inadequate megakaryopoietic response by the neonatal bone marrow. Furthermore, SGA neonates with moderate and severe thrombocytopenia might have a pronounced suppression of megakaryopoiesis compared to neonates with infection.

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Impact of Platelet Count on Bleeding in the Setting of Anti-Platelet Therapy

Blood ◽

10.1182/blood-2020-142893 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 18-18

Author(s):

Robert Hugh Lee ◽

Wolfgang Bergmeier

Keyword(s):

Real Time ◽

Conflicts Of Interest ◽

Thrombotic Event ◽

Platelet Inhibition ◽

Intravital Imaging ◽

Severe Thrombocytopenia ◽

Platelet Counts ◽

Anti Platelet Therapy ◽

The Impact

Anti-platelet therapy (APT) is used for secondary prevention of thrombosis. The most commonly prescribed anti-platelet drugs are aspirin and P2Y12 inhibitors, including clopidogrel, prasugrel and ticagrelor. Dual anti-platelet therapy (DAPT) consisting of aspirin and a P2Y12 inhibitor is often used in the first 1-12 months after an initial thrombotic event and has a greater anti-thrombotic effect than single agents, but is also associated with a higher risk of bleeding. Due to this risk of hemorrhage, the appropriate use of DAPT in patients requiring percutaneous coronary intervention (PCI) with baseline or periprocedural thrombocytopenia remains unclear. To study the impact of thrombocytopenia on bleeding with APT, we used intravital imaging in a murine hemostasis model and adoptive platelet transfer to generate mice with specific platelet counts with or without platelet inhibition. To generate experimental mice, we used transgenic mice in which platelets express a chimeric GPIb receptor with the extracellular domain replaced with a domain of the human IL-4R (hIL-4R/GPIb-Tg). Endogenous platelets were depleted by injection of anti-hIL-4R antibody, and the recipient mice were then transfused with wild-type (WT) platelets from donor mice treated, or not, with single or dual APT (aspirin 20 mg/kg; clopidogrel 25 mg/kg) to achieve specific platelet counts ranging from 50,000 to 400,000 platelets/μL. We also compared these mice with WT mice (with normal platelet counts, ~1,200,000 platelets/μL) treated with APT. Platelet inhibition was confirmed prior to performing in vivo experiments. Hemostasis was determined by intravital imaging in our saphenous vein laser injury model, in which a 50 μm injury was induced by laser ablation. Real-time top-down epifluorescence imaging was used to determine time to initial hemostasis, rebleeding events, and platelet and fibrin accumulation. In each mouse, 3-5 injuries were induced at different sites and each injury was visualized for 10 minutes. Following real-time imaging, spinning disk confocal Z-stacks of platelet plugs were obtained for 3D reconstruction to compare platelet plug volume. In untreated WT mice, hemostasis was achieved in ~20 seconds. In WT mice treated with DAPT, initial hemostasis was often rapidly achieved but this was followed by significant rebleeding events. Paradoxically, platelet accumulation was increased in WT + DAPT mice due to extravascular accumulation of platelets which occurred during bleeding. However, in plugs that stabilized, plug volume was reduced in WT + DAPT mice. In hIL-4R/GPIb-Tg mice with reduced platelet counts, untreated platelets were able to form a stable hemostatic plug even at 50,000/μL, although time to hemostasis was slightly prolonged. However, as platelet counts decreased in mice with DAPT-treated platelets, initial hemostasis became more prolonged and many injuries never achieved initial hemostasis. These results suggest that DAPT may not be safe in the setting of severe thrombocytopenia. Disclosures No relevant conflicts of interest to declare.

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