Regulation of thrombopoiesis: effects of the degree of thrombocytopenia on megakaryocyte ploidy and platelet volume

Abstract We have established a murine model and techniques with which to serially study thrombocytopoiesis after induction of experimental immune thrombocytopenia of variable severity and duration. Bone marrow megakaryocyte ploidy distribution was determined by using unfractionated bone marrow, a polyclonal megakaryocyte-specific probe, and two-color, fluorescence-activated flow cytometry. With these techniques, the modal megakaryocyte ploidy class in normal murine bone marrow was 16N. Serial studies of bone marrow megakaryocyte ploidy after the induction of acute, severe thrombocytopenia (platelet count, less than 0.05 X 10(6) microL) demonstrated no detectable change in the ploidy distribution at 12, 24, and 36 hours after the onset of thrombocytopenia. At 48 hours, the modal ploidy class shifted from 16N to 32N, and the 64N class increased significantly (P less than .001). The ploidy distribution returned to normal 120 hours after the onset of thrombocytopenia. A lesser degree of thrombocytopenia (platelet count reduction to 0.100 to 0.200 X 10(6)/microL) delayed the modal ploidy class shift from 16N to 32N until 72 hours after the onset of thrombocytopenia. Chronic, severe thrombocytopenia (platelet count, less than 0.05 X 10(6)/microL for seven days) resulted in a modal ploidy class shift from 16N to 32N during the thrombocytopenic phase and an enhanced increase in the 64N megakaryocyte class during the recovery phase. Mean platelet volume (MPV) was simultaneously measured on isolated total platelet populations after induction of thrombocytopenia. MPV was significantly increased (P less than .001) as early as eight hours after the onset of acute, severe thrombocytopenia, 40 hours before a shift in the ploidy distribution. Mild thrombocytopenia (platelet count reduction to 0.400 X 10(6)/microL) was not associated with a ploidy shift but did result in a significantly increased MPV (P less than .001). These studies demonstrate that the temporal relationship and magnitude of the effects of thrombocytopenia upon megakaryocyte ploidy distribution are dependent upon the degree and the duration of the thrombocytopenic stimulus and that the effects of experimental thrombocytopenia on platelet volume and megakaryocyte ploidy are dissociated.

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Regulation of thrombopoiesis: effects of the degree of thrombocytopenia on megakaryocyte ploidy and platelet volume

Blood ◽

10.1182/blood.v70.1.177.bloodjournal701177 ◽

1987 ◽

Vol 70 (1) ◽

pp. 177-185 ◽

Cited By ~ 1

Author(s):

L Corash ◽

HY Chen ◽

J Levin ◽

G Baker ◽

H Lu ◽

...

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Immune Thrombocytopenia ◽

Temporal Relationship ◽

Recovery Phase ◽

Severe Thrombocytopenia ◽

Detectable Change ◽

Platelet Volume ◽

Murine Bone Marrow ◽

Ploidy Class

We have established a murine model and techniques with which to serially study thrombocytopoiesis after induction of experimental immune thrombocytopenia of variable severity and duration. Bone marrow megakaryocyte ploidy distribution was determined by using unfractionated bone marrow, a polyclonal megakaryocyte-specific probe, and two-color, fluorescence-activated flow cytometry. With these techniques, the modal megakaryocyte ploidy class in normal murine bone marrow was 16N. Serial studies of bone marrow megakaryocyte ploidy after the induction of acute, severe thrombocytopenia (platelet count, less than 0.05 X 10(6) microL) demonstrated no detectable change in the ploidy distribution at 12, 24, and 36 hours after the onset of thrombocytopenia. At 48 hours, the modal ploidy class shifted from 16N to 32N, and the 64N class increased significantly (P less than .001). The ploidy distribution returned to normal 120 hours after the onset of thrombocytopenia. A lesser degree of thrombocytopenia (platelet count reduction to 0.100 to 0.200 X 10(6)/microL) delayed the modal ploidy class shift from 16N to 32N until 72 hours after the onset of thrombocytopenia. Chronic, severe thrombocytopenia (platelet count, less than 0.05 X 10(6)/microL for seven days) resulted in a modal ploidy class shift from 16N to 32N during the thrombocytopenic phase and an enhanced increase in the 64N megakaryocyte class during the recovery phase. Mean platelet volume (MPV) was simultaneously measured on isolated total platelet populations after induction of thrombocytopenia. MPV was significantly increased (P less than .001) as early as eight hours after the onset of acute, severe thrombocytopenia, 40 hours before a shift in the ploidy distribution. Mild thrombocytopenia (platelet count reduction to 0.400 X 10(6)/microL) was not associated with a ploidy shift but did result in a significantly increased MPV (P less than .001). These studies demonstrate that the temporal relationship and magnitude of the effects of thrombocytopenia upon megakaryocyte ploidy distribution are dependent upon the degree and the duration of the thrombocytopenic stimulus and that the effects of experimental thrombocytopenia on platelet volume and megakaryocyte ploidy are dissociated.

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Platelet-Associated IgG May Be Associated with Thrombocytopenia In Patients with Myelodysplastic Syndromes

Blood ◽

10.1182/blood.v116.21.2943.2943 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2943-2943

Author(s):

Simone Gilli ◽

Samuel de Souza Medina ◽

Vagner Castro ◽

Sara T. Olalla-Saad

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Myelodysplastic Syndromes ◽

Screening Test ◽

Immune Thrombocytopenia ◽

Severe Thrombocytopenia ◽

Platelet Indices ◽

Wilcoxon Rank Sum Test ◽

Highly Sensitive ◽

Spearman Test

Abstract Abstract 2943 Background. Myelodysplastic Syndromes (MDS) are a heterogeneous group of bone marrow disorders characterized by ineffective and dysplastic hematopoiesis, progressive bone marrow failure, cytopenias and a high risk of transformation into acute leukemia. Thrombocytopenia is detected in up to two thirds of patients with MDS and severe thrombocytopenia is present in approximately 10%. Besides ineffective thrombopoiesis, immune destruction of platelets could be an additional factor in the genesis of thrombocytopenia, since immunological abnormalities are also frequent in patients with MDS. The detection of platelet associated IgG (PAIgG) by immunofluorescence (platelet immunofluorescence test or PIFT) is a highly sensitive assay. In addition, some morphological platelet indices (PDW and MPV) are correlated with the occurrence of immune thrombocytopenia. We prospectively analysed platelet-bound IgG and platelet indices (PDW and MPV) in 35 patients with MDS. Methods: Thirty-five patients with MDS (mean age ± SD: 63 ± 19 yo; range 21–89 yo; 15female/20male) were evaluated. According to FAB, 27 patients were classified as RA, 5 as RARS and 3 as RAEB. Clinical manifestations of immunological disorder were not present in this population. Blood samples were analyzed by PIFT, in order to detect platelet associated IgG and results were expressed as a ratio of patient fluorescence/negative control fluorescence (R). Cell-dyn Sapphire blood cell analyzer (Abbott, Illinois, USA) was used to measure platelet count, mean platelet volume (MPV) and platelet size deviation width (PDW). Thrombocytopenia was defined as a platelet count <100 × 109/L. All samples were analyzed on the day of collection. Results. Platelet counts of the entire population ranged from 6.7 to 708 ×109/L, with median of 95.4 × 109/L. Eighteen patients (51.43%) had platelet count <100 × 109/L. A strong association between thrombocytopenia and PAIgG measured by PIFT (R) was demonstrated taking into account three analysis: an inverse correlation between the number of platelets and the fluorescence ratio (p=0.01, r=-0.39, Spearman test), the higher positivity of PIFT in patients with platelet count <100 × 109/L (p=0.007, Wilcoxon rank sum test) and a lower platelet count in patients with positive PIFT (p=0.059, Wilcoxon rank sum test). MPV was significantly higher in patients with platelet count <100 × 109/L (median, min-max: 9.38, 6.9–23.1 vs 8.46, 4.88–12.83; p<0.001, Wilcoxon rank sum test). PDW showed no statistical difference between these groups. Discussion: Immune thrombocytopenia is a relatively frequent hematological disorder of unknown origin and until today depends mainly upon clinical diagnosis. Assays for the detection of glycoprotein-specific antibodies such as MAIPA (monoclonal antibody-specific immobilization of platelet antigens) are highly specific but less sensitive than PIFT. MAIPA is laborious and require a certain amount of platelets, which is not always available in thrombocytopenia Thus, PIFT could be recommended as a screening test to discriminate patients with MDS in whom the hyperdestructive component of thrombocytopenia is important. Our results also showed a higher MPV in patients with platelet count <100 × 109/L, suggesting that this index may be of interest for detection of immune-mediated hyperdestructive thrombocytopenia in MDS. Conclusion: Thus, we propose that a combination of a simple index as MPV and a highly sensitive and easy to perform screening test for PAIgG as PIFT could be applied to select a subset of MDS patients in which we would be able to prevent the overuse of unnecessary platelet transfusions and who could be candidates for an immunosuppressive therapeutic approach. Supported by INCTS, FAPESP, CNPq. Disclosures: No relevant conflicts of interest to declare.

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Can Immature Platelet Fraction (IPF) be Used to Assess Bleeding Risk in Pediatric Immune Thrombocytopenia (ITP) and to Differentiate ITP from Bone Marrow Failure/Aplastic Anemia? A Retrospective Analysis

Blood ◽

10.1182/blood.v126.23.3474.3474 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3474-3474 ◽

Cited By ~ 1

Author(s):

Karen Lee Bride ◽

Derick Lim ◽

Michele Paessler ◽

Michele P Lambert

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Immune Thrombocytopenia ◽

Bone Marrow Failure ◽

Bleeding Risk ◽

Medical Laboratory ◽

Bleeding Complications ◽

Severe Thrombocytopenia ◽

Immature Platelet Fraction ◽

Time Of Presentation

Abstract Immune Thrombocytopenia (ITP) usually presents with isolated, severe thrombocytopenia with very low platelet count (generally less than <30 x 109/L) in the absence of other hematologic abnormalities. However, ITP is a diagnosis of exclusion without any definite diagnostic test that can confirm the diagnosis at the time of presentation and clinicians occasionally worry at the time of presentation about other bone marrow processes that may present with thrombocytopenia, which would require considerably different therapy. In light of current guidelines suggesting that observation is likely to be safe in pediatric patients with low platelet counts without significant bleeding, identifying patients at risk for severe hemorrhage is even more important to help guide therapy. In addition, appropriately differentiating ITP from other diagnoses may also prevent inappropriate administration of ineffective therapies. The immature platelet fraction (IPF) is a measure of platelet turnover measuring RNA containing, large platelets by fluorescently labeling the platelets and utilizing flow cytometric gates programmed into the Sysmex XN-3000 hematology analyzer. We examined the medical laboratory records of 134 patients who had an IPF performed over the past 4 months for correlation between IPF and bleeding manifestations. In ITP patients who presented with significant bleeding symptoms (defined as epistaxis which was more than brief, oral bleeding more than palatal petechiae or GI or intracranial hemorrhage), the IPF was significantly lower than in those who presented with no bleeding or cutaneous bleeding only (bruising and petechiae): IPF=4.3%±1.6 SEM in bleeding patients versus 21.8%±1.8 SEM in not bleeding patients; p<0.0001. In two patients with life threatening hemorrhage and ITP (GI bleeding with drop in hemoglobin requiring both PRBC transfusion and treatment to raise the platelet count; ICH resulting in mortality), the IPF was low at the time of initial hemorrhage, but increased after ITP therapy (GI Bleed: plt 1K, IPF 5.3% increased to 20.3% after IVIG; ICH plt 6K, IPF 1.8% increased to 12.8% after IVIG and prednisone). We also examined first platelet count and IPF in 127 patients with ITP and 21 patients with BMF/AA who presented to our institution since October 2013. In this cohort of patients, the IPF in patients with ITP was significantly higher than in the BMF/AA patients and an IPF of >5.3 was associated with a negative predictive value of 80% for BMF/AA (IPF 16.6%±1.2 SEM in ITP vs. 2.9%±1.4 SEM in BMF/AA). In summary, we demonstrate that the IPF is a useful and simple adjunct in diagnosis of ITP which can help differentiate the patients most likely to have ITP from those who may need further diagnostic evaluation and require treatment to prevent bleeding complications. Further studies will focus on the ability of the IPF to prospectively predict the bleeding risk of patients and categorize patients. Disclosures Lambert: GSK: Consultancy; NovoNordisk: Honoraria; Hardin Kundla McKeon & Poletto: Consultancy.

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A Case of Acquired Amegakaryocytic Thrombocytopenia with Anti-c-mpl Autoantibody: Comparison with Idiopathic Thrombocytopenic Purpura

Acta Haematologica ◽

10.1159/000499523 ◽

2019 ◽

Vol 142 (4) ◽

pp. 239-243

Author(s):

Bora Son ◽

Hee sue Park ◽

Hye Sook Han ◽

Hee Kyung Kim ◽

Seung Woo Baek ◽

...

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Rare Disease ◽

Idiopathic Thrombocytopenic Purpura ◽

Immunosuppressive Treatment ◽

Severe Bleeding ◽

Severe Thrombocytopenia ◽

Thrombocytopenic Purpura ◽

Acquired Amegakaryocytic Thrombocytopenia

Acquired amegakaryocytic thrombocytopenia (AAMT) is a rare disease that causes severe bleeding. The pathogenesis and treatment of AAMT have not yet been defined. We report the case of a 60-year-old woman diagnosed with AAMT, who presented with severe thrombocytopenia, gastrointestinal bleeding, and significantly reduced bone marrow megakaryocytes. The patient was treated with methylprednisolone, cyclosporin, and intravenous immunoglobulin. After 2 weeks of treatment, her platelet count started to increase, and her bone marrow megakaryocyte count had normalized 3 months after diagnosis. At the time of diagnosis, the patient was seropositive for anti-c-mpl antibody but was seen to be seronegative once the platelet count recovered. In contrast, anti-c-mpl antibodies were not detected in the serum of 3 patients with idiopathic thrombocytopenic purpura. This case study suggests that anti-c-mpl antibody plays an important role in the development of AAMT, and that intensive immunosuppressive treatment is required for autoantibody clearance and recovery of megakaryocyte count.

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Association of Interleukin-18 Gene Polymorphism with Severity of Chronic Immune Thrombocytopenia (ITP).

Blood ◽

10.1182/blood.v116.21.3693.3693 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3693-3693

Author(s):

Takayuki Saitoh ◽

Norihiko Moriyama ◽

Tomonori Takani ◽

Takeki Mitsui ◽

Takumi Hoshino ◽

...

Keyword(s):

Platelet Count ◽

Immune Thrombocytopenia ◽

Initial Diagnosis ◽

Severe Thrombocytopenia ◽

Interleukin 18 ◽

Single Nucleotide ◽

Chronic Itp ◽

Significant Difference ◽

Haplotype Frequencies ◽

And Control

Abstract Abstract 3693 Introduction: Immune thrombocytopenia (ITP) is a chronic acquired organ-specific autoimmune disorder characterized by the production of antibodies against antigens on the membranes of platelets. Several cytokine studies have shown Th1 polarization in ITP patients. Interleukin-18 (IL-18) plays an important role in Th1 and Th2 immune response. Recent studies showed that single-nucleotide promoter polymorphisms influence the transcriptions of IL-18 mRNA. IL-18 polymorphism has been implicated in autoimmunity, including Crohn's disease, rheumatoid arthritis, and asthma. We examined the single nucleotide polymorphisms (SNPs) in the promoter regions of the IL-18 genes in patients with ITP, and analyzed the relationship between IL-18 SNPs and clinical features. Patients and Methods: One hundred patients (male/female; 22/78, median age; 54.5) diagnosed as chronic ITP and 151 healthy controls were included. Chronic ITP was defined as thrombocytopenia (platelet count < 100×109/L) persisting greater than 12 months, normal or increased marrow megakaryocytes, and no secondary immune or non-immune abnormality that could account for the thrombocytopenic state. ITP with severe thrombocytopenia was defined as thrombocytopenia (platelet count < 10×109/L) at presentation of ITP. The response criteria of the ITP International Working Group was used. A complete response (CR) is defined as any platelet count of at least 100×109/L, and a response (R) was defined as any platelet count between 30 and 100×109/L and at least doubling of the baseline count. Allparticipants gave written informed consent about the study. Genomic DNA was isolated from peripheral blood using the DNA Kit (QIAGEN, Hilden, Germany). An allele-specific polymerase chain reaction was used to analyze polymorphism in IL-18 –607A/C and -137G/C. Genotype and allele frequencies were compared between the study groups using Χ2-test. The characteristics and laboratory features of the ITP patients with each IL-10 promoter polymorphism were compared using X2-tests and student t-tests. Probability values <0.05 were considered statistically significant. Results: The platelet count was at an initial diagnosis ranged from 1×109/L to 98 ×109/L, with a median of platelet count of 15×109/L. Thirty-five patients (35%) had severe thrombocytopenia. Steroid treatment was given to 68 patients (68%), while splenectomy was used in 11 patients (11%).The frequencies of the genotypes were as follows: AA (34%), AC (57%), and CC (9%) for -607; GG (77%), GC (21%), and CC (2%) for -137 loci. The frequencies of each haplotype were as follows: C-G/C-G haplotype (9%), A-G/C-G haplotype (47%), A-C/C-G haplotype (10%), A-G/A-G haplotype (21%), A-G/A-C haplotype (11%) and A-C/A-C haplotype (2%). No significant differences in the genotype or haplotype frequencies demonstrated between chronic ITP patients and control group. However, patients with -137CC genotypes showed severe thrombocytopenia at initial diagnosis compared to those with -137GG/GC genotypes (5×109/L vs. 22×109/L, p=0.002). Furthermore, patients with A-C/A-C haplotype showed severe thrombocytopenic state (5×109/L vs. 22×109/L, p=0.002) compared to those without A-C/A-C haplotype. No significant difference of treatment response was observed according to IL-18 polymorphism. Conclusion: No significant differences in the genotype or haplotype frequencies demonstrated between chronic ITP patients and control. However, -137CC genotypes or AA/CC haplotype was associated with severity of chronic ITP. Our data suggest that the group with low IL-18 inducibility (i.e. -137CC genotype, A-C/A-C haplotype) may have more severe thrombocytopenia. Disclosures: No relevant conflicts of interest to declare.

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The Cytokine Polymorphisms Affect the Severity of Thrombocytopenia at Diagnosis in Chronic Immune Thrombocytopenia.

Blood ◽

10.1182/blood.v120.21.2194.2194 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2194-2194

Author(s):

Takayuki Saitoh ◽

Chiaki Ushie ◽

Atsushi Iwasaki ◽

Norihiko Moriyama ◽

Tomonori Takani ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Platelet Count ◽

Clinical Features ◽

Immune Thrombocytopenia ◽

Steroid Treatment ◽

Severe Thrombocytopenia ◽

Bleeding Tendency ◽

Chain Reaction ◽

Chronic Itp ◽

Polymerase Chain

Abstract Abstract 2194 Introduction: The severity of immune thrombocytopenia (ITP) depends on the degree of the thrombocytopenia and the extent of bleeding. Some investigators have reported the association between the thrombocytopenia and cytokine dysregulation in ITP. We investigated the association between the severity of thrombocytopenia at diagnosis in ITP patients and several cytokine polymorphisms, including IL-10-1082A/G, -819T/C, -592A/C, IL-17F-7488T/C and IL-18-607A/C, −137G/C. Patients and methods: We examined 102 patients (male/female, 24/78; median age, 42) diagnosed with chronic ITP. The definition, response criteria, including complete response (CR)and response (R), loss of CR,and “corticosteroid-dependence” were assessed according to the criteria of the ITP International Working Group. ITP with severe thrombocytopenia (ST group)was defined as thrombocytopenia (platelet count < 10×109/L) at the initial diagnosis of ITP. Genotyping of IL-10 (rs1800870 − 1082 A/G, rs1800871 − 819 T/C, and rs1800872 − 592 A/C) and IL-17F (rs763780, 7488 T/C) polymorphisms were determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and the genotyping of the IL-18 polymorphism (rs187238 −137G/C and rs1946518−607 A/C) was determined by the allelic specific polymerase chain reaction technique. To confirm the accuracy of the assay, amplification products of several individuals were sequenced using an ABI Prism Genetic Analyzer. Genotype and allele frequencies were compared between the study groups using χ2-test. The characteristics and laboratory features of ITP patients with each polymorphisms were compared using χ2-tests and student t-tests. Odds ratios (OR) and 95% confidence intervals (CIs) were estimated for each study. All patients were provided written information about the study. This study was approved by the Institutional Research Board of Gunma University Hospital. Results: Clinical features of chronic ITP: The platelet count ranged from 1×109/L to 98×109/L with a mean of platelet count of 32×109/L at the initial diagnosis. Fifty seven patients (49%) had bleeding tendency. Steroid treatment was given to 68 patients (66.7%) and eradication of Helicobacter pylori (H. pylori) was performed in 32 patients (31.4%), while splenectomy was performed in only 11 patients (10.8%). Clinical features of ST group vs. non-ST group in chronic ITP: Of these 102 patients, 17 (16.7%) had severe thrombocytopenia (platelet count < 10×109/L) (ST group). ST group were significantly older (ST group: median 59 years vs. non-ST group: 41 years, p<0.01) and had more severe bleeding tendency (ST group: 100% vs. non-ST group: 54%, p<0.0001). Steroid treatment was frequently given to ST group than to non-ST group (ST group: 100% vs. non-ST group: 59.5%, p<0.001). Though the response to corticosteroids treatment was not significantly different between ST group and non-ST group (CR rate, ST group: 50% vs. non-ST group: 51.0%, p=0.94), corticosteroid-dependent patients in ST group was significantly higher than in non-ST group (76.9% vs. 25.3%, p<0.005). Polymorphism study of ST group vs. non-ST group in chronic ITP: The frequencies of genotypes of cytokines in patients with chronic ITP according to the definition of criteria of ST were as follows: AA (93.3% vs. 97.1%) and AG (6.7% vs. 2.9%, p=0.48) for IL-10–1082; TT (46.7% vs. 33.3%), TC (33.3% vs.55 %) and CC (20% vs. 11.7%) for IL-10–819; AA (46.7% vs. 33.3%), AC (33.3% vs.55 %) and CC (12.2% vs. 11.5%) for IL-10–592; TT (100% vs. 81%) and TC (0% vs. 19%) for IL-17F; GG (82.4% vs. 74.4%), GC (17.6% vs. 23.2%) and CC (0% vs. 2.4%) for IL-18–137; AA (35.3% vs. 34.1%), AC (58.8% vs. 53.7%) and CC (5.9% vs 12.2%) for IL-18–607 loci (ST group vs. non-ST group, respectively). No significant difference was observed between ST group and non-ST group according to IL-10–1082A/G, −819T/C, −592A/C, and IL-18–607A/C, −137G/C polymorphism. However, the numbers of IL-17F 7488TT genotype (higher function type) in ST group were significantly higher than in non-ST group (ST group: 100% vs. non-ST group: 81% p<0.05). Conclusion: These findings suggest that severe thrombocytopenia at diagnosis have an impact of bleeding tendency and corticosteroid-dependency of chronic ITP. Furthermore, IL-17F polymorphism may affect the severity of thrombocytopenia of chronic ITP. Disclosures: No relevant conflicts of interest to declare.

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Dynamic Changes in Platelet Responsiveness to Thrombin and Coated Platelet Potential May Inform Risk of Hemorrhage and/or Thrombosis in a Novel Canine Mode of Immune Thrombocytopenia.

Blood ◽

10.1182/blood.v120.21.2193.2193 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2193-2193

Author(s):

Marshall A. Mazepa ◽

Dana N LeVine ◽

Adam J Birkenheuer ◽

Marjory B Brooks ◽

Shila K Nordone ◽

...

Keyword(s):

Flow Cytometry ◽

Platelet Count ◽

Exploratory Study ◽

Immune Thrombocytopenia ◽

Treated Group ◽

Canine Model ◽

Severe Thrombocytopenia ◽

Dynamic Changes ◽

Platelet Recovery ◽

Time Zero

Abstract Abstract 2193 In both canine and human patients with Immune Thrombocytopenia (ITP), bleeding risk is challenging to predict, and potentially leads to over-treatment of patients at low risk. Conversely, recent studies have highlighted the risk of thrombosis in ITP during platelet recovery. Given these clinical observations, we hypothesized that in ITP, changes in platelet response to agonists may occur in addition to changes in platelet numbers. In response to dual agonist activation (thrombin and convulxin), a subpopulation of platelets in both humans and dogs develops enhanced procoagulant activity. This subpopulation is termed coated platelets, and differences in individuals' potential to form coated platelets have been correlated with both hemorrhagic and thrombotic outcomes. In this exploratory study, we serially evaluated ex vivo platelet responsiveness to both thrombin and dual agonists (termed coated platelet potential) in a novel canine model of ITP. Dogs (n=4) were infused with a murine monoclonal anti-GPIIb antibody (2F9) in order to model ITP and generate predictable severe thrombocytopenia. Control dogs (n=3) were infused with a control antibody. Platelet count, thrombin responsiveness, and coated platelet potential were measured at baseline, time zero, 6 hours, 24 hours, and every 24hrs thereafter until the platelet count was ≥ baseline for at least two consecutive measures (recovery). Time zero was defined as the time when platelet count first fell to ≤ 30,000/μl following 2F9 infusion, or 1 hour following control antibody infusion. For platelet thrombin responsiveness, a monoclonal antibody to P-selectin was used to determine platelet P-selectin surface expression by flow cytometry after stimulation with graded doses of thrombin. The ED50 Thrombin was defined as the concentration of thrombin required for half-maximal P-selectin expression. Coated platelet potential was defined as the percent of platelets activated to the highly procoagulant state after dual stimulation with thrombin and convulxin, as determined by binding of biotinylated fibrinogen by platelets by flow cytometry. All dogs in the treated group developed severe thrombocytopenia (median=6×103, range=4–11×103 platelets/uL); no dogs in the control group developed thrombocytopenia. All treated dogs had platelet recovery by 240 hours (median=132 hours, range 120–240hours). Of interest, at 6 hours, ED50 Thrombin in the treated group increased nearly twofold (fig 1A) (ratio of median ED50 Thrombin treated/baseline=1.6, range 1.3–2.3), which correlated with a decline in coated platelet potential by nearly half of baseline (fig 1B) (median 52.4% of baseline, range 19.6–61.5%); minimal change from baseline was observed in controls. In both groups, ED50 Thrombin was lower at recovery than baseline (fig 1A) (treated median ED50 Thrombin=71.5% of baseline; control median ED50 Thrombin=67% of baseline). A trend of rising coated platelet potential was also noted as platelets recovered in the treated group. In conclusion, in this exploratory study of a canine model of ITP, we observed dynamic changes in platelet responsiveness. During severe thrombocytopenia, we observed a rise in ED50, indicating a decline in response to thrombin, which correlated with a fall in coated platelet potential. We speculate that this early fall in platelet thrombin response and coated platelet potential could contribute to hemorrhage risk in ITP. As a complement to this finding, in the treated group, there was a rise in coated platelet potential as platelets rebounded and coated platelet potential was slightly greater than baseline at recovery. This is consistent with others' observation that younger platelets are more likely to have coated platelet potential. We also observed a decline in ED50 Thrombin at recovery, not only in the treated dogs, but also control dogs. Thus, at recovery, the decline in ED50 Thrombin was independent of treatment group. However, this may be an artifact of our small sample size. Our observed increase in coated platelet potential during platelet recovery could potentially contribute to the thrombotic tendency of some ITP patients. Future studies are planned to explore the relationship of hemorrhagic and thrombotic risk with platelet thrombin responsiveness and coated platelet potential in this model of ITP and clinical studies of canine and human ITP. Disclosures: No relevant conflicts of interest to declare.

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Novel Management of Immune Thrombocytopenia in Gaucher Disease Patients

Blood ◽

10.1182/blood.v124.21.5006.5006 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5006-5006

Author(s):

Hanna Rosenbaum

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Avascular Necrosis ◽

Gaucher Disease ◽

Immune Thrombocytopenia ◽

High Dose ◽

Type I ◽

Platelet Counts ◽

Skeletal Complications

Abstract Type I Gaucher disease (GD) the is characterized by hepatosplenomegaly, pancytopenia and skeletal complications due to the accumulation of glucocerebroside in macrophages. Thrombocytopenia is usually related to hypersplenism and infiltration of bone marrow by lipid-laden macrophages namely Gaucher cells. Enzyme replacement therapy (ERT) restores the hemoglobin and platelet count in GD patients. In GD ERT treated patients, manifesting persistent low platelet counts, immune thrombocytopenia (ITP) should be considered.Treatment of GD with concomitant ITP is a challenge. Splenectomy may worsen bone manifestations in GD patients and is controversial. Steroids should be used with caution because of possible induction of osteopenia and joints avascular necrosis. Thrombopoietin receptor analogues (TPO-RA) are therapeutic option in GD patients with ITP. Beneficial use of TPO-RA is reported in 2 cases. Patient 1: 39 YO male with new onset of purpura and low platelet count failed treatment with 1 mg/kg of Prednisone. Bone marrow biopsy (BM) showed Gaucher cells infiltration, numerous atypical megakaryocytes, normal erythropoiesis and myelopoiesis with no fibrosis. Low level of ß-glucocerebrosidase activity with compound heterozygosity for 84GG /R495H mutations, established the diagnosis of Type I GD. Low C4 and detection of IgG platelet antibodies added to the diagnosis of concomitant immune thrombocytopenia. ERT with taliglucerase alfa (ElelysoTM) 60 Units/kg/month was given with Prednisone for six weeks. Occurrence of retinal bleeding and purpura, with decrease of platelet count necessitated addition of high-dose IVIG with no response regarding platelet counts. Splenectomy was not considered due to known bony complication risk in splenectomised GD patients. Rituximab was given to prevent wet purpura recurrence with short response regarding platelet count. Romiplostim was initiated raising platelet count from 29,000/µL to 60,000/µL after 3 wks. and to 90,000/µL after 8 wks. enabling corticosteroids withdrawal. Same dose Romiplostim is maintained for the last 30 months with platelet counts of 90,000 - 110,000/µL with no bleeding events. Repeated BMB showed no increase in collagen fibrosis. Patient 2: 63 YO female patient diagnosed with Gaucher at age 33 with a history of purpura, ecchymosis, and occasional vaginal bleeding episodes. At age 53 the platelet count dropped to < 20,000/µL with presence of Anti Platelets Ab (IgG). BMB revealed megakaryocytic hyperplasia with atypical forms, focal infiltration by Gaucher cells and no fibrosis. Combined therapy by ERT (Imiglucerase® followed by Velaglucerase Alfa®), Prednisone (1mg/kg/d for 2 months) and one course of IVIG yielded no increase in platelet count. The patient refused Rituximab®. Romiplostim was initiated increasing platelet count to100,000/µL maintained throughout a year of follow up. Repeated BMB showed slight increase of fibrosis and marked hyperplasia of atypical megakaryocytes. Discussion: Thrombocytopenia is often present in GD and may be severe in approximately 15% of the patients. Persistent cytopenias may be caused by other underlying pathologies such as autoimmune disorders and are important to be recognized and addressed. Before ERT era GD patients with hypersplenism and severe cytopenia were splenectomised. Risks of splenectomy include serious bacterial infection and vascular complications limiting its use in chronic refractory ITP. Splenectomy is avoided in Gaucher patients, due to risk of exacerbating skeletal complications (bone infarcts, avascular necrosis). Stable bone marrow results regarding fibrosis in our patients are consistent with data from a recent 2-year follow-up of 100 ITP patients receiving Romiplostim treatment with no evidence of BM fibrosis. Conclusion: In patients with type I Gaucher disease and concomitant ITP, adjunctive treatment with Romiplostim was successful in maintaining haemostatic platelet counts with no adverse effects. Traditional treatment regimens of corticosteroids and splenectomy should be used with caution or avoided in GD patients due to possible aggravation of Gaucher skeletal disease and the risk of osteopenia and avascular necrosis resulting in increased morbidity in this cohort of patients. Use of TPO-RA should be considered in GD patients with ITP. Disclosures Off Label Use: Romiplostim in gaucher patients.

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Four Cases of Lenalidomide-Associated Immune Thrombocytopenia

Blood ◽

10.1182/blood.v128.22.4927.4927 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4927-4927 ◽

Cited By ~ 1

Author(s):

Alessandra Pompa ◽

Francesca Guidotti ◽

Anna Ines Gregorini ◽

Maria Cecilia Goldaniga ◽

Francesca Gaia Rossi ◽

...

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Steroid Therapy ◽

Partial Remission ◽

Immune Thrombocytopenia ◽

Line Treatment ◽

The Third ◽

Bone Marrow Evaluation ◽

Grade 3 ◽

D 20

Abstract Introduction Hematologic neoplasms are associated with an increased incidence of autoimmune events, particularly evident in non‐Hodgkin lymphomas (NHL) and especially chronic lymphocytic leukemia (CLL); until now only few cases have been described in patients affected by multiple myeloma (MM) and other plasma cell disorders. The introduction of new drugs, in particular immunomodulatory drugs (IMiDs), could increase the risk of these complications. Herein we describe four cases of immune thrombocytopenia (ITP) occurred during Lenalidomide (LEN) therapy in 3 patients affected by MM and 1 with light‐chain amyloidosis. Case reports Case 1. A 66‐year old woman with relapsed IgGk MM started 2th line treatment with LEN/DEX 25 mg/d-20 mg/w. During the first 5 cycles she presented moderate hematologic toxicity which resolved after dose reduction (LEN 15 mg/d). A severe and isolated thrombocytopenia appeared after 5th cycle with patient in good partial remission (PR) for MM. A bone marrow evaluation showed absence of plasma cells and abundant megakaryocytes leading to a diagnosis of ITP. LEN was interrupted and steroid therapy (prednisone 1 mg/Kg) and IV Ig infusion administered, obtaining a transient good response (from 17 to 114 x 109/l). LEN was then restarted at lower dosage but a month after, while myeloma still was in good response, ITP relapsed complicated by intracranial hemorrhage and was successfully treated with IV Ig; Len was definitely interrupted. At the third relapse Rituximab (750 mg/w x 4 weeks) was administered with only minimal increase of platelet count (Fig 1). Afterwards, the patient died due to rapid MM progression. Case 2. A 76‐year old woman with relapsed IgGλ MM started 2th line treatment with LEN 15 mg. After the 3rd cycle, having obtained a very good partial remission (VGPR), she presented with grade 3 thrombocytopenia which did not ameliorate with LEN suspension and resolved only after introduction of steroid, thus supporting ITP diagnosis. She underwent a 4th cycle of LEN/DEX maintaining a normal platelet count but then LEN was interrupted due to rapid MM progression. (Fig 2). Case 3. A 78‐year old woman with renal amyloidosis in IgAλ gammopathy started a 2nd line treatment with LEN/DEX 15 mg/d-20 mg/w with a significant reduction in proteinuria. During the 6th cycle she presented with diffuse purpura and a platelet count of 18x109/L. She received platelet transfusion and bone marrow evaluation showed abundant megakaryocytes supporting the diagnosis of ITP. Furthermore anti‐platelet antibodies search resulted positive. Steroid therapy was started with partial response on platelet count but the patient remained steroid‐therapy dependent. Case 4. A 66‐year‐old woman affected by MM started a 2th‐line treatment with LEN‐DEX 25 mg/d-20 mg/w. After the third cycle, being the patient in PR, a isolated grade 3 thrombocytopenia developed, persisting despite discontinuation of LEN. Viral infections were excluded and antiplatelet antibodies search resulted positive. Therefore she started steroid therapy (prednisone 1 mg/Kg) with an progressive increase of platelet count from 44 to 80x109/L after a month of therapy, that is ongoing. Lenalidomide has not yet been restarted. Discussion We report four cases of ITP developing during LEN therapy with the characteristics of ITP. None of these patients had a history of previous autoimmune events, the decline of platelet count was rapid and other causes of thrombocytopenia were excluded or unlikely in all the cases. Furthermore diagnosis was supported by consistent bone marrow evaluation in two cases. Considering the temporal association in these four cases, the ITP mechanism may be related to the immunomodulation and T‐cell activation caused by LEN. Even if further other studies are needed, it seems reasonable to consider a possible association between LEN and autoimmune phenomena, in particular ITP. Disclosures No relevant conflicts of interest to declare.

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Association between Megakaryocyte Abnormalities on Bone Marrow Smear and Response to Thrombopoietin Receptor Agonists in Adult Patients with Primary Immune Thrombocytopenia

Blood ◽

10.1182/blood-2021-151435 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3160-3160

Author(s):

Ondine Walter ◽

Agnès Ribes ◽

Johanne Germain ◽

Jean-Baptiste Rieu ◽

Thibault Comont ◽

...

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Board Of Directors ◽

Research Funding ◽

Immune Thrombocytopenia ◽

Adult Patients ◽

Second Line ◽

Bone Marrow Smear ◽

Advisory Committees ◽

Thrombopoietin Receptor

Abstract Introduction: Immune thrombocytopenia (ITP) is an autoimmune disease due to peripheral destruction but also impaired central production of platelets. Autoimmune reaction directed against megakaryocytes (MKs) has been described, and may explain morphological abnormalities of MKs observed in some patients with primary ITP. Thrombopoietin receptor agonists (TPO-RAs) are indicated as second-line treatments for ITP, but no predictive factors of response used in clinical routine practice has been demonstrated. The utility of systematic bone marrow smears (BMS) at ITP diagnosis is discussed. Howerer, it is usually recommended before second-line treatments. Two studies have suggested an association between MK abnormalities and response to corticosteroids in primary ITP, but none have investigated this association for TPO-RAs. This study aimed to investigate the association between MK abnormalities and response to TPO-RAs in adult patients with primary ITP. Methods: The source of population was the CARMEN registry. The CARMEN (Cytopénies Auto-immunes: Registre Midi-PyréneEN) registry is aimed at the prospective follow-up of all incident ITP adults in the French Midi-Pyrénées region (South-West of France, 3 million inhabitants) since June 2013. Each investigator follows all adult patients (aged ≥18 years) with incident ITP in routine visit or hospital stay. ITP was defined by international definition (platelet count <100 x 10 9/L and exclusion of other causes of thrombocytopenia). The study population consisted in all patients included in the CARMEN registry between June 2013 and March 2018 with primary ITP, treated by TPO-RA and with a BMS before initiating TPO-RA. We excluded the patients with a number of MKs <10 MK on the BMS. Morphological abnormalities were established based on literature and defined by consensus among 3 expert cytologists (AR, JBR and VDM). All MKs present on each smear were analyzed. MKs were categorized by the presence of dysplasia (monolobed MK and/or separated nuclei and/or microMKs), and according to their stage of maturation (basophilic, granular and thrombocytogenic). All patients' medical charts were reviewed by two experts in ITP (OW and GM) to determine the response to TPO-RAs. Response was defined by a platelet count between 30 and 100 G/L with at least a doubling of basal platelet count according to the international definition. In case of subsequent exposure to both TPORAs in a single patient, response was defined by response to at least one TPO-RA in the main analysis. We performed a subgroup analysis by TPORAs. Results: During the study period, 451 patients with incident ITP were included in CARMEN-registry. Among them, 105 had been treated by TPO-RAs, including 65 with BMS before the exposure to TPORA. We then excluded 20 patients with secondary ITP and 7 with less than 10 MKs on the BMS. We finally included 38 patients in the analysis. Median age at diagnosis was 71 years (interquartile range - IQR: 31 - 94) and 34.2% were women. Thirty-three patients were treated with eltrombopag, 17 with romiplostim including 13 who were exposed to both TPORAs. Thirty-four (89.4%) achieved response. The median number of MKs analyzed per patient was 137 (IQR: 50 - 265). All results are presented in Table 1. In the main analysis, there was no significant difference in the median percentage of dysplastic MKs in responders (4.0%, 95% confidence interval - CI: 2.3 - 6.4) and non-responders (4.5%, 95% CI: 0.7 - 7.1). There was a trend for a higher proportion of granular MKs (4.5%, 95% CI: 3 - 6) and basophilic MKs (30.1%, 95% CI: 21.9 - 39.1) in non-responders comparing to responders (granular: 2.0%, 95% CI: 0 - 4.1; basophilic: 21.3%, 95% CI: 11.4 - 40.7). Results were similar in the subgroup of patients treated with eltrombopag (data not shown; the low number of patients treated with romiplostim precluded any analysis). Conclusion: In this study, neither MK abnormalities nor the pattern of MK maturation stages were significantly associated with response to TPO-RAs. These results do not support a systematic bone marrow smear in patients with primary ITP to look for morphological predictive factors of response to TPO-RA. Figure 1 Figure 1. Disclosures Comont: AstraZeneca: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Moulis: Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Grifols: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sobi: Membership on an entity's Board of Directors or advisory committees; Argenx: Membership on an entity's Board of Directors or advisory committees.

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