The Independent Effect of Platelet Count On Mortality in a Large Inner City Elderly Outpatient Population

Abstract Abstract 4645 Introduction: Platelets play a critical role in hemostasis and are also important in the development of pathologic processes including atherosclerosis and arterial thrombosis. Both thrombocytopenia and thrombophilia are common findings in several illnesses including liver diseases, infections, autoimmune disorders and malignancies. However, to date no study has directly investigated the effect of thrombocytopenia and thrombocytosis on the overall survival of the general population. The present study was thus undertaken to elucidate the independent association of abnormal platelet counts with subsequent mortality among a large inner city outpatient elderly cohort comprised of different ethnicities and comorbid conditions. Methods: All 36,262 non-acutely ill individuals >65 years of age who were seen at an outpatient clinic within the Montefiore medical system from January 1st 1997 to May 1st 2008 and who underwent a complete blood count within 3 months of the visit were included in our cohort. Thrombocytopenia and thrombocytosis were defined as a platelet count of <150,000 per microliter or >450,000/microliter respectively. Data on 19 co-morbidities that could affect mortality and confound the analysis were extracted from the electronic medical records of each patient. These included the presence of anemia, neutropenia, congestive of heart failure, baseline anemia and/or neutropenia, prior myocardial infarction, peripheral vascular disorders, cerebrovascular disease, dementia, chronic pulmonary disease, rheumatologic disease, peptic ulcer disease, liver disease, diabetes, renal disease, malignancy, neurologic deficits and HIV infection. A Cox proportional hazard model was constructed to assess the independent association of abnormal platelet counts with subsequent mortality after controlling for all extracted co-morbidities, age, gender and race. Results: As shown in table 1, caucasians had higher thrombocytopenia rates compared to blacks, Hispanics and other ethnicities (p<0.001 for all comparisons). Ethnicities other than Caucasians, Blacks or Hispanics had significantly lower rates of thrombocytosis compared to Caucasians or Blacks (p values <0.01). The median platelet count was 237,000/microliter. The median follow-up period was 3.3 years with a total of 134,132 person-years of observation. Fully adjusted hazard ratios (HR) using normal platelet counts (≥150,000 and ≤450,000) as the reference group showed a significant association of thrombocytopenia (HR=1.43, 95% CI 1.34–1.53; p<0.001) and thrombocytosis (HR=1.73, 95% CI 1.54–1.94; p<0.001) with shorter overall survival. A statistically significant interaction term was also identified between race and thrombocytopenia (Table 1). More specifically, we found that Hispanics with thrombocytopenia are at a higher risk compared to Caucasians with thrombocytopenia (HR=1.34, 95% CI 1.13–1.59; p=0.001). Conclusions: Thrombocytopenia and thrombocytosis are independently associated with increased mortality in our study population. The effect of thrombocytopenia on overall survival is more prominent on Hispanics. These findings suggest that abnormal platelet count may be a mediator of increased mortality risk in older patients and this effect may differ by ethnicity. Disclosures: No relevant conflicts of interest to declare.

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A Case of Clopidogrel Induced TTP Recurring After Four Years

Blood ◽

10.1182/blood.v116.21.4678.4678 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4678-4678

Author(s):

Nanda K. Methuku ◽

Abhinav B. Chandra ◽

Anuradha Belur ◽

Lech Dabrowski

Keyword(s):

Platelet Count ◽

Renal Insufficiency ◽

Peripheral Blood ◽

Blood Smear ◽

Reticulocyte Count ◽

Conflicts Of Interest ◽

Peripheral Blood Smear ◽

Severe Thrombocytopenia ◽

Platelet Counts ◽

Multiple Organs

Abstract Abstract 4678 Case description - A 61 year old woman was started on clopidogrel after having PTCA with stent placement in February 2006. Four weeks after starting clopidogrel she developed thrombocytopenia with platelet nadir of 17,000. Her LDH was 700 IU/L and she was anemic with hemoglobin of 7.4 gm/dl with elevated reticulocyte count. Peripheral blood smear showed schistocytes and diagnosis of TTP secondary to clopidogrel was made. She did not have renal insufficiency. Clopidogrel was discontinued and patient was started on plasmapheresis with recovery of platelet counts. Early attempts in weaning plasmapheresis resulted in drop in platelet count and Rituximab was given to the patient weekly for four weeks. Subsequently, patient was weaned off plasmapheresis. For four years patient was followed periodically with CBC showing platelet counts greater than 250,000. In May 2010, four years after initial event patient was admitted to hospital for abdominal pain and found to have splenic infarcts. Subsequently, she also developed bilateral cerebral infarcts. Platelet count had decreased to less than 100,000. Her LDH was elevated at 419 IU/L. Reticulocyte count was 2.3%. Peripheral blood smear revealed significant number of schistocytes. There was no renal insufficiency or fever. Trans-esophageal echocardiogram (TEE) was done that did not reveal any vegetations. Patient was diagnosed as having recurrence of TTP and started on plasmapheresis with recovery in platelet counts. Pt was also treated with Rituximab. Discussion- We describe a case of TTP initially occurring within weeks of starting clopidogrel. Patient was treated with plasmapheresis and Rituximab and clopidogrel was discontinued. Patient had recurrence after four years as manifested by infarcts in multiple organs, with mild thrombocytopenia, elevated LDH and significant number of schistocytes on peripheral blood smear. It is very uncommon for clopidogrel associated TTP to recur after such a prolonged period of 4 years. Most cases of clopidogrel associated TTP have mild thrombocytopenia. This patient had severe thrombocytopenia on first presentation of TTP but had mild thrombocytopenia on recurrence. This case illustrates the importance of extended follow up and high index of suspicion for TTP as delays in initiation of plasmapheresis has a poor clinical outcome. Disclosures: No relevant conflicts of interest to declare.

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Post-Transplant Platelet Recovery Kinetics and Its Association with Overall Survival in Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplant (HSCT)

Blood ◽

10.1182/blood.v118.21.2049.2049 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2049-2049

Author(s):

Jie Li ◽

Jillian Alyse Deppa ◽

Zahir Ali ◽

Michael Graiser ◽

Amelia Langston ◽

...

Keyword(s):

Overall Survival ◽

Platelet Count ◽

Early Death ◽

Disease Status ◽

P Value ◽

Term Survival ◽

Allogeneic Hsct ◽

Post Transplant ◽

Platelet Counts

Abstract Abstract 2049 Background: Post-transplant thrombocytopenia is universal among recipients of hematopoietic stem cell transplantation (HSCT). We have previously reported a secondary post-transplant thrombocytopenia following autologous HSCT which is associated with poor survival (Ninan MJ, et al., BBMT 2007), however the clinical significance of a fall post-engraftment in platelet counts among recipients of allogeneic HSCT has not been studied. Methods: A total of 929 consecutive pts who underwent allogeneic HSCT between 1993 and 2009 were studied in an IRB-approved retrospective analysis. 55% of pts were male and 45% were female with a median age of 43 ± 12.6 years. Diagnoses included: acute leukemia (423, 46%), chronic leukemia (197, 21%), non-Hodgkin's lymphoma (110, 12%),myelodysplastic syndrome (93, 10%), multiple myeloma (26, 3%), and other less common malignancies (80, 8%). Disease status was classified into five different categories: complete remission (287, 31%), partial remission (297, 34%), refractory (180, 19%), untreated (28, 3%) and incompletely classified (137, 15%). Grafts were obtained from related donors in 595 pts (64%), and unrelated donors in 334 pts (36%), with 55% peripheral blood stem cell (PBSC), 42% bone marrow (BM), and 3% cord blood units or multiple sources. Blood platelet counts and platelet transfusions were collected from 15 days pre-transplant until 100 days post-transplant. Platelet engraftment was defined as a platelet count ≥ 50 x10E3/mcL without a platelet transfusion in the previous 7 days. Pts (n=816) who achieved platelet engraftment and survived at least 30 days were selected for further analysis. Results: The 816 evaluable pts were divided into cohorts based upon their post-transplant survival: 146(18%) who died within 100 days post-transplant (early death); 267 (33%) that survived 100 days −2 years post-transplant (late death), 319 (39%) who survived > 2 years (long-term survival), and 84 (10%) were lost of follow-up within the first 2 years. Transfusion-independent platelet engraftment was achieved at median of 15 days post-transplant with no significant differences seen in the kinetics of initial engraftment among the different pt cohorts. Median platelet counts at different time points post-transplant were plotted for each pt cohort (Figure 1). Pts in the early-death cohort had a continuous decline in median platelet counts from engraftment values of > 50 x10E3/mcL to a median values of ∼20 x10E3/mcL. Univariate analyses indicated that higher platelet counts at day −15 (prior to conditioning) or at day 100 post-transplant were significantly associated with improved overall survival (HR of 0.63 and 0.39 respectively, P < 0.01). Cox-regression analysis was performed to evaluate significance of pre- and post-transplant platelet counts with clinical covariates that have been previously associated with survival including age, diagnosis, disease status and the source of the grafts. The multivariate model confirmed the significant association of the following factors with overall survival: higher platelet counts on day 15 pre-transplant (HR:0.81; 95%Cl:0.70∼0.93; P-value <0.01), the platelet count on day 100 post-transplant (HR: 0.62; 95%Cl:0.55∼0.70; P-value:<0.01 ), a diagnosis of acute leukemia (HR:1.64; 95%Cl:1.13∼2.39; P-value <0.01), a diagnosis of multiple myeloma (HR: 2.12; 95%Cl:1.05∼4.23; P-value= 0.04), a disease status of complete remission (CR) versus not in CR (HR: 0.66; 95%Cl:0.44∼0.97; P-value = 0.04), and age (HR: 1.01; 95%Cl:1.00∼1.02; P-value= 0.08). Kaplan-Meier estimates for survival were performed based upon stratification of pt groups on the platelet count at day-15 pre-transplant or the day +100 post-transplant platelet count (Figure 2). Pts with a platelet count > 80 × 10E3/mcL on day +100 had 5 year survival of more than 50% compared with 30% survival in the pt cohort with platelet counts < 50 x10E3/mcL on day +100. Conclusion: Pts with continuously low platelet count after initial platelet engraftment are at high risk for early death. Higher pre-transplant platelet may be a surrogate for disease status and extent of prior therapy and are associated with long-term survival among pts undergoing allogeneic HSCT. Post-transplant thrombopoiesis at day 100 is highly correlated with long-term survival after allogeneic HSCT, identifying a high-risk group of transplant pts for whom additional treatment strategies are needed. Disclosures: Gleason: Celgene, Merck, Millenium: Consultancy.

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Differential Diagnosis of Hereditary and Acquired Thrombocytopenia By the Immature Platelet Fraction and Thrombopoietin Levels

Blood ◽

10.1182/blood.v126.23.1049.1049 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1049-1049 ◽

Cited By ~ 1

Author(s):

Fabio Luiz Bandeira Ferreira ◽

Marina Pereira Colella ◽

Samuel de Souza Medina ◽

Maiara Marx Luz Fiusa ◽

Loredana Nilkenes Gomes da Costa ◽

...

Keyword(s):

Bone Marrow ◽

Differential Diagnosis ◽

Platelet Count ◽

Healthy Volunteers ◽

Complete Blood Count ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Healthy Individuals ◽

Technical Limitation ◽

Immature Platelet Fraction

Abstract Introduction: The differential diagnosis of hereditary and acquired thrombocytopenias can be challenging, especially when between immune thrombocytopenia (ITP) and less well characterized hereditary thrombocytopenias (HT) such as MYH9-related disorders. Fundamental differences in the management of these two conditions add clinical relevance to the search for novel parameters that differentiate these conditions. The immature platelet count (IPF) represents the fraction of platelets with higher RNA content, and in analogy to the reticulocyte count for erythropoiesis is a biomarker of thrombopoietic activity. In a recent report (Miyazaki et al, 2015), IPF values that were more than 5-fold higher than those observed in ITP patients were reported in a population of 15 patients with HT. However, whether this increased values represented a real increase in thrombopoietic activity, or reflected a technical limitation of IPF determination in large platelets could not be clarified. Here, we aimed to evaluate the role of IPF determination in the differential diagnosis between HT and several forms of acquired thrombocytopenia in a larger and more diverse population of patients. We also evaluated thrombopoietin (TPO) levels in HT compared to ITP, to further investigate the mechanisms by which extremely large IPF values are observed in HT. Methods: IPF and mean platelet volume (MPV) were prospectively determined using a Sysmex XE5000 hematologic analyzer (as part of the complete blood count) in a cohort of patients with post-chemotherapy thrombocytopenia (n=56), bone marrow failure (myelodysplastic syndromes and aplastic anemia; n=22), ITP (ITP; n=105) and inherited thrombocytopenias (n=27). The latter population consists of a well-defined cohort of individuals with HT thrombocytopenia characterized by clinical, familial, laboratory and molecular data. TPO levels were determined by ELISA (R&D Systems) in 21 HT patients and 22 ITP patients matched for platelet count and age. A group of 178 healthy volunteers were used to determine normal IPF and MPV values. Results: Median platelet counts were similar in post-chemotherapy patients (CTx) (32.0*109/L), bone marrow failure (BMF) (33.5*109/L), ITP (52.0*109/L) and HT (52.0*109/L) (P=0.15). Similar IPF levels were observed in CTx and BMF patients (5.6%; IQR 3.4-8.8% and 6.5%; IQR 3.5-13.7%. Compared to these two groups, higher IPF values were observed in both ITP (12.3%; 7.0-21.0%) and HT patients (29.8%; 17.5-56.4%) (both P values < 0.05). In addition, IPF were significantly higher in HT compared to ITP (Kruskall-Wallis test and Dunn's post test,P=0.001). MPV values were different between HT and CTx/BMF groups, but could not differentiate ITP from HT. TPO levels. The accuracy of IPF to discriminate HT from all other causes of thrombocytopenia estimated by ROC analysis was 0.88 (CI95%0.8-0.96, p<0.0001). Similar TPO levels were observed in platelet count-matched ITP, HT patients and healthy volunteers without thrombocytopenia. Interestingly, TPO presented marked correlations with both platelet count (Rs = - 0.61, P=0.002) and IPF (Rs= 0.59, P=0.003), even with TPO levels in the same range of healthy individuals. In contrast, no significant correlation could be observed between TPO and IPF or platelet count in HT patients. Conclusions: IPF represents an informative biomarker for the differential diagnosis of hereditary and acquired thrombocytopenias, and accurately differentiates ITP from the most common HT. As expected, TPO levels in patients with ITP were not higher than in individuals with normal platelets counts. The inverse correlation between TPO and platelet count in these patients confirm a blunted TPO response to thrombocytopenia in these patients. Similarly, patients with HT did not present increased TPO levels compared to healthy individuals. Accordingly, increased IPF levels in these patients cannot be attributed to higher TPO levels. Disclosures No relevant conflicts of interest to declare.

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Safety of Lumbar Punctures in Adult Oncology Patients with Thrombocytopenia

Blood ◽

10.1182/blood.v126.23.1141.1141 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1141-1141 ◽

Cited By ~ 1

Author(s):

Shuoyan Ning ◽

Brent Kerbel ◽

Jeannie Callum ◽

Yulia Lin

Keyword(s):

Risk Factors ◽

Platelet Count ◽

Platelet Transfusion ◽

Conflicts Of Interest ◽

Tertiary Care ◽

Bleeding Risk ◽

Von Willebrand Disease ◽

Oncology Patients ◽

Platelet Counts ◽

Hemorrhagic Complications

Abstract Introduction: Lumbar puncture (LP) is a frequently performed diagnostic and therapeutic intervention in adult oncology patients. While thrombocytopenia is common in this patient population, the minimum "safe" platelet count required for LPs is unknown. Recent guidelines from the AABB (American Association of Blood Banks) recommend a pre-procedure platelet count of 50 x 109/L. However this recommendation is largely based on expert opinion, and there remains a paucity of studies in the adult oncology literature to address this important question. Methods: We retrospectively reviewed all oncology patients ≥18 years who underwent 1 or more LPs over a 2 year period at a single tertiary care institution to determine 1) the range of platelet counts at which LPs are performed; 2) the rate of traumatic taps; and 3) the rate of hemorrhagic complications. Laboratory, clinical, and transfusion information were extracted through the Laboratory Information System, chart review, and blood bank database, respectively. Thrombocytopenia was defined as a platelet count of < 150 x 109/L. Pre-LP platelet counts were those collected ≤24 hours from, and closest to the time of the LP. The following bleeding risk factors were documented: end stage renal disease; platelet dysfunction; von Willebrand disease; hemophilia. Anticoagulation, anti-platelet, and non-steroidal inflammatory use was also recorded, with accuracy limited by the study's retrospective nature. All patients with coagulopathy were excluded (INR ≥ 1.5, aPTT ≥ 40, fibrinogen ≤ 1.0). Traumatic tap was defined as 500 or more red blood cells per high-power field in the cerebrospinal fluid. A follow up of 1 week after LP was used to capture any hemorrhagic complications. Results: From January 2013 to December 2014, 135 oncology patients underwent 369 LPs; 64 (47.4%) patients were female, and the mean age was 59 years (range 20-87). 119 (88.1%) patients had a primary hematological diagnosis. 113 (30.6%) LPs were performed in thrombocytopenic patients. 28 (7.6%) procedures had a pre-procedure platelet count of ≤ 50 x 109/L, with 18 receiving a single platelet transfusion on the day of the LP. Of these 18 transfusions, only 1 had a post-transfusion platelet count available prior to LP with no improvement in platelet count (33 x 109/L). 15 transfusions had post-LP platelet counts within 24 hours of the transfusion (8 below 50 x 109/L with lowest 14 x 109/L), 1 had post-LP platelet count within 24-48 hours (54 x 109/L) and 1 did not have a post-transfusion platelet count. Traumatic taps occurred in 17 (15.0%) LPs in patient with thrombocytopenia, compared to 26 (11.0%) LPs in patients with a normal platelet count (fisher's exact test P=0.39). There was 1 traumatic tap in a patient with a pre-LP platelet count of ≤ 50 x 109/L; however, this patient received a pre-LP platelet transfusion for a platelet count of 42 x 109/L and had a post-LP platelet count of 66 x 109/L. Presence of bleeding risk factors did not increase the risk of a traumatic tap (present in 48.8% of traumatic taps vs. 88.3% of non-traumatic taps). There were no hemorrhagic complications. Conclusion: Among this cohort of adult oncology patients undergoing diagnostic and therapeutic LPs, there were no hemorrhagic complications. There was no significant increase in traumatic taps in patients with thrombocytopenia or bleeding risk factors. While platelet transfusions were frequently administered for patients with a platelet count of ≤ 50 x 109/L, post-transfusion platelet counts were infrequently assessed prior to the procedure. Our findings question whether a platelet transfusion threshold of 50 x 109/L is necessary for lumbar puncture.Table 1.Platelet Count Pre-LP(x109/L)Number of LPsNumber of Traumatic TapsNumber of Hemorrhagic Complications0-90N/AN/A10-2030021-5070051-1003380101-1495270> 150242270Unknown1400< 50 x 109/L and received platelet transfusion on day of LP181*0Total369430*There was one traumatic tap in a patient with a platelet count of 42 x 109/L who received a platelet transfusion pre-LP. The post transfusion platelet count was 66 x 109/L. Disclosures No relevant conflicts of interest to declare.

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Severe Thrombocytopenia Does Not Increase Bleeding Complications in Patients Undergoing Bronchoscopy

Blood ◽

10.1182/blood.v124.21.4293.4293 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4293-4293

Author(s):

Lakshminarayanan Nandagopal ◽

Muthu Veeraputhiran ◽

Tania Jain ◽

Ayman Soubani ◽

Charles A. Schiffer

Keyword(s):

Platelet Count ◽

Renal Dysfunction ◽

Platelet Transfusion ◽

Conflicts Of Interest ◽

Bleeding Complications ◽

British Thoracic Society ◽

Severe Thrombocytopenia ◽

Platelet Counts ◽

Number Of Patients ◽

Platelet Transfusions

Abstract Introduction Prophylactic platelet transfusions are often performed prior to bronchoscopy or broncho-alveolar lavage (BAL) to prevent bleeding in thrombocytopenic patients. There is a paucity of data to validate this approach, with a platelet transfusion threshold of <50,000/mm3 largely based on expert opinion. We conducted a retrospective study on the incidence of bleeding complications in thrombocytopenic patients undergoing bronchoscopy. Methods We identified 150 consecutive patients with platelet counts <100,000/mm3 who underwent bronchoscopy and/or BAL from January 2009 to May 2014 at our institution. Bronchoscopies performed in patients with frank hemoptysis and trans-bronchial lung biopsy procedures were excluded. Patient characteristics, underlying diagnosis, platelet count prior to bronchoscopy, administration of platelet transfusions and bronchoscopy details were recorded. Factors affecting bleeding risk including presence of renal dysfunction (defined as BUN >30 and/or Cr>2.0) and coagulation studies (PT, PTT, INR) were identified. The British Thoracic Society guidelines1 were used to categorize bleeding as a result of bronchoscopy. Data were analyzed using descriptive statistics. Results The median age was 59 years (range 27-90), with two-thirds of patients (63%) being male. One hundred and seventeen (78%) patients had underlying malignancy and 55 (37%) had thrombocytopenia related to malignancy. Fellows and residents under the supervision of a bronchoscopy certified attending performed all but 4 of the bronchoscopies. Infection (40%) was the primary indication for bronchoscopy with BAL performed in 127 (85%) patients. Fifty-eight of 89 (65%) patients with baseline platelet counts <50,000/mm3 received prophylactic transfusions compared to 8% of those with platelet counts >50,000/mm3. The platelet count did not rise to >50,000//mm3 in many transfused patients. Seventy patients (47%) had counts <50,000/mm3 and eighty patients (53%) had counts >50,000/mm3 at the time of bronchoscopy. 49% were receiving immunosuppressive medications, 45% had renal dysfunction and 8% had INR >1.5. Bloody lavage that resolved spontaneously without continuous suctioning (Grade 0) was observed in 9 (6%) patients. Bleeding that required continuous suctioning but then resolved spontaneously (Grade 1) was noted in 1 patient with a platelet count of 61,000/mm3. Of 10 total bleeding events, 7 occurred in patients who were intubated. Two additional patients with platelet counts of 30,000/mm3 and 53,000/mm3 had diffuse alveolar hemorrhage, which was present before bronchoscopy. “Old” blood and blood clots were observed in 6 patients. Discussion The low incidence of bleeding complications from bronchoscopy +/- BAL even in patients with platelet counts <30,000/mm3 (3 episodes in 31 patients, all grade 0) demonstrates that bronchoscopy can be safely done in severely thrombocytopenic patients. Adopting a lower threshold for prophylactic transfusions could save a considerable number of platelet units and translate into significant cost savings and decreased risk of transfusion-related complications. Table 1 Platelet count, transfusion history and bleeding complications during bronchoscopy Platelet count at the time of bronchoscopy Number (n) and percentage (%) of patients who underwent bronchoscopy Number of patients who received prior platelet transfusion Bleeding during bronchoscopy n % 0-15,000/mm3 9 6% (9/150) 5 Grade 0=1 pt 16-29 22 15% 16 Grade 0=2 pts 30-39 17 11% 9 Grade 0=1 pt 40-49 22 15% 9 Grade 0=3 pts 50-75 44 29% 14 Grade 1=1 pt 76-100 36 24% 10 Grade 0=2 pts Total 150 63 Grade 0=9 pts, Grade 1=1 pt. 1.Du Rand IA, Blaikley J, Booton R, et al. British Thoracic Society guideline for diagnostic flexible bronchoscopy in adults: accredited by NICE. Thorax. 2013:68 Suppl 1:i1-i44 Disclosures No relevant conflicts of interest to declare.

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Mice with LMAN1 Deficiency Exhibit Thrombocytopenia and Reduced Serum Thrombopoietin Level

Blood ◽

10.1182/blood.v128.22.412.412 ◽

2016 ◽

Vol 128 (22) ◽

pp. 412-412

Author(s):

Rami Khoriaty ◽

Lesley Everett ◽

Jennifer Chase ◽

Guojing Zhu ◽

Bin Zhang ◽

...

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Conflicts Of Interest ◽

Critical Role ◽

Coagulation Factors ◽

Hematopoietic Stem ◽

Platelet Counts ◽

Normal Platelet ◽

Transmission Electron ◽

Wide Range

Abstract LMAN1 and MCFD2 encode the components of a mammalian cargo-receptor that facilitates the ER-to-Golgi transport of coagulation factors V (FV) and VIII (FVIII) for secretion to the plasma. Mutations in LMAN1 or MCFD2 result in a rare bleeding disorder known as combined deficiency of coagulation factors V and VIII (F5F8D), characterized by FV and FVIII levels that are ~10% of normal. No other clinical phenotypes are known in human patients. Lman1 null mice have ~50% of normal FV and FVIII levels and exhibit a partially penetrant, perinatal lethality, suggesting a critical role for yet unknown LMAN1 secretory cargo(s). To further characterize the function of the LMAN1/MCFD2 complex and identify new cargos, we generated several murine models of F5F8D, including ubiquitous null Lman1 (Lman1-/-) and Mcfd2 (Mcfd2-/-) mice maintained on a C57BL/6J genetic background. Adult Lman1-/- mice were mildly thrombocytopenic, exhibiting a 25% decrease in platelet count relative to wild-type (WT) mice (9.3 x 105 vs. 12.3 x 105 cells/uL, p < 0.004), but no other CBC abnormalities. In contrast, Lman1 heterozygous and Mcfd2-/- mice exhibited normal platelet counts. To further explore the role of LMAN1 in megakaryocyte/platelet development or survival, bone marrow (BM) histology and platelet transmission electron microscopy were performed. Lman1-/-mice had no platelet morphologic abnormalities by light or transmission electron microscopy, as well as normal number and morphology of BM megakaryocytes. Hematopoietic stem cells and megakaryocyte progenitors were indistinguishable between WT and Lman1-/- mice by flow cytometry. In order to determine whether the thrombocytopenia phenotype results from LMAN1 deficiency specifically in the hematopoietic compartment, mice with tissue-specific knockout of Lman1 in hematopoietic and endothelial cells (Tie2-Cre) were generated. Platelet counts of mice with LMAN1 deficiency restricted to the hematopoietic compartment were indistinguishable from those in WT controls. In contrast, hepatocyte-specific (Alb-Cre) Lman1 deficiency, resulted in significant thrombocytopenia relative to WT controls (p < 0.017), with platelet counts comparable to those observed in ubiquitous Lman1 null mice. Since thrombopoietin (TPO) is a major hepatocyte-derived regulator of platelet synthesis, plasma TPO levels were measured by ELISA in ubiquitous Lman1 and Mcfd2 null mice. Plasma TPO levels in Lman1-/- mice were ~56% lower than those in WT levels (128.7 x 103 vs. 229.5 x 103 pg/mL, p < 0.0025). However, TPO levels were not reduced in Mcfd2-/- mice (p > 0.17). TPO mRNA expression in the liver of Lman1-/-mice was indistinguishable from livers of WT littermate controls. In conclusion, global LMAN1-deficient and hepatocyte-specific LMAN1 deficient mice exhibit thrombocytopenia, a phenotype not previously reported in F5F8D patients. Lman1-/- mice, but not Mcfd2-/- mice, exhibit low plasma TPO levels, suggesting that TPO may be a novel LMAN1-dependent secretory cargo. These results raise the possibility that F5F8D patients with LMAN1 mutations may have mild thrombocytopenia, previously unappreciated as a result of the small number of F5F8D patients and the wide range of clinically normal platelet counts. Disclosures No relevant conflicts of interest to declare.

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Cross-Strain Platelet Immunization in Selected Mouse Strains Recapitulates Clinical and Serologic Findings Typical of Post-Transfusion Purpura (PTP)

Blood ◽

10.1182/blood-2019-126815 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2349-2349

Author(s):

Daniel W. Bougie ◽

Jessica Sutton ◽

Richard H. Aster

Keyword(s):

Flow Cytometry ◽

Platelet Count ◽

Amino Acid ◽

Conflicts Of Interest ◽

Human Condition ◽

Autoimmune Response ◽

Single Amino Acid ◽

Mouse Strains ◽

Igg Antibody ◽

Platelet Counts

Post-transfusion purpura (PTP) is an uncommon but life-threatening condition characterized by profound thrombocytopenia (TP) occurring one week after transfusion of blood products. The hallmark of PTP is a potent IgG antibody specific for a transfused human platelet antigen (HPA), usually HPA-1a located on αIIb/β3 integrin (GPIIb/IIIa). It is widely thought that, in PTP, the alloantibody somehow causes destruction of the recipient's platelets even though they lack the antigen for which the alloantibody is specific. Several reports have suggested that the underlying cause of PTP is a platelet-specific autoantibody that can be difficult to detect because it is absorbed in the process of destroying autologous platelets and is overshadowed by the accompanying, very potent alloantibody but experimental support for this concept is minimal. Platelet alloantigens comparable to HPAs have not been defined in animals. Using a public database, we identified four mouse strains (C57BL/6J (C57), 129S1/Svlmj (129), PWK/PhJ (PWK), AND SPRET/EIJ) differing from each other at amino acid residues in extracellular domains of GPIIb/IIIa that could comprise potential alloantigens. Cross-strain platelet immunizations (intraperitoneal with adjuvant) were performed weekly for 5 weeks while monitoring platelet counts and platelet associated IgG (PAIgG) and saving plasma samples for serologic studies. After 2-4 immunizations, each of 39 cross-strain but none of 9 strain-identical immunizations induced "alloantibodies" that recognized donor but not recipient platelets (flow cytometry). Thrombocytopenia (<50% of maximum platelet count) developed in 28 of 39 mice (71%) given strain-disparate platelets but not in mice given strain-identical platelets; 12 of these mice (30%) developed profound TP (<15%). The most consistent and severe declines in platelet counts occurred in PWK mice immunized with 129 platelets and vice versa (N=13) in which the mean platelet count decline was 88% (range 59-96%, median 92%). Autoantibodies recognizing syngeneic platelets were identified in all animals that developed profound TP and their potency (measured by flow cytometry) correlated closely with the severity of TP (p<0.001) (Fig 1). Alloantibodies were shown by immunoprecipitation to be mainly specific for GPIIb/IIIa (N=13) and GPIb/IX (N=1) on donor platelets. Two monoclonal antibodies (mAbs MBC417.1 and MBC425.1) specific for a single polymorphic amino acid at positions 111(Gly) and 37(Val), respectively, on GPIIb of C57 and PWK mice were generated using spleen cells of two immunized mice. To our knowledge, these are the first alloantibodies in mice that are specific for single amino acid polymorphisms in a platelet membrane glycoprotein and are thus comparable to HPA antibodies found in humans. The findings define a model (platelet immunization between PWK and 129 mice) in which a routine alloantibody response recognizing GPIIb/IIIa on donor platelets regularly transitions to an autoimmune response capable of causing profound thrombocytopenia, thus mimicking the course of PTP in human patients and supporting the hypothesis that PTP is an autoimmune disorder. Successful development of the model could be related to use of the more recently developed, wild-caught PWK strain as one of the partners for immunization. The inherent utility of a mouse model is expected to facilitate further work to define the molecular basis for a transition from allo- to auto-immunity in the human condition, post-transfusion purpura. Disclosures No relevant conflicts of interest to declare.

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Extreme Thrombocytosis Under Azacitidine in Patients with Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v120.21.4961.4961 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4961-4961

Author(s):

Charikleia Kelaidi ◽

Dimitrios Kokkinidis ◽

Maria Protopappa ◽

Georgios Papaioannou ◽

Ioannis Batsis ◽

...

Keyword(s):

Platelet Count ◽

Myelodysplastic Syndrome ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasm ◽

Response To Treatment ◽

Jak2 Mutation ◽

Hematopoietic Stem ◽

Platelet Counts ◽

Hemorrhagic Event ◽

Count Response

Abstract Abstract 4961 Background: Platelet increase under azacitidine in patients with myelodysplastic syndrome (MDS) has been acknowledged as an early predictive factor of response to treatment. However, extreme thrombocytosis under azacitidine has not been reported. Methods: We studied consecutive patients with MDS or MDS/myeloproliferative neoplasm (MDS/MPN) who had platelet counts near or over 1, 000 G/L under azacitidine. Results: Four patients, sex ratio 1:1, with median age of 65 years, had extreme thrombocytosis under azacitidine. Baseline characteristics were: WHO classification RAEB-2/CMML-1/CMML-2 in 2/1/1 patients, median platelet count 248 G/L (<400 G/L in all), normal karyotype/+8, −9/−7 in 2/1/1 patients, IPSS low/int-2/high in 1/2/1 patients. None had reticulinic fibrosis or ring sideroblasts>15% at baseline. A median number of 8 cycles of azacitidine was administered. Individual platelet counts reached 2, 960 G/L, 800 G/L, 1, 188 G/L and 2, 740 G/L. Thrombocytosis occured early after treatment onset or resumption (Figure 1). Histologic findings under treatment were: Increased cellularity (N=4), micromegakaryocytes and other signs of megakaryocytic dysplasia (N=4), reticulinic fibrosis grade I and II in 1 and 2 patients, respectively. JAK2 V617F mutation was detected in 1 patient (with maximum platelet count of 2, 900 G/L) and was undetectable in the remaining patients. None had a thrombotic or hemorrhagic event. Two patients had a concomitant increase of WBC count. Response to azacitidine was CR, PR and stable disease in 1/1/2 patients. Three patients received hydroxyurea (HU) in addition to azacitidine and one patient underwent hematopoietic stem cell transplantation (HCT). AML transformation occurred in 1 patient 25 months after azacitidine onset. Median overall survival after azacitidine onset was 25 months. Conclusion: Extreme thrombocytosis of the range of essential thrombocytosis, with megakaryocytic dysplasia and hyperplasia, was noted under azacitidine in 4 patients with MDS-MDS/MPN and normal baseline platelet count. Hypothetically, azacitidine may induce the expression of critical genes of megakaryopoiesis or platelet release in patients with rare mutations. Notably, JAK2 mutation was detected in only one patient. Alternatively, demethylation could unmask an underlying unclassified MDS/MPN similar to RARS-T. Disclosures: No relevant conflicts of interest to declare.

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Short Term Treatment With Thrombopoietin-Receptor Agonists For Patients With Immune Thrombocytopenic Purpura (ITP) Before Splenectomy

Blood ◽

10.1182/blood.v122.21.1086.1086 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1086-1086 ◽

Cited By ~ 1

Author(s):

Yosef Kalish ◽

Galia Spectre ◽

David Varon

Keyword(s):

Platelet Count ◽

Immune Thrombocytopenic Purpura ◽

Complete Blood Count ◽

Conflicts Of Interest ◽

Short Term Treatment ◽

Thrombocytopenic Purpura ◽

Thrombopoietin Receptor Agonists ◽

Receptor Agonists ◽

Short Course ◽

Thrombopoietin Receptor

Abstract The thrombopoietin-receptor agonists (romiplostim and eltrombopag) were approved recently as treatments for patients with immune thrombocytopenic purpura (ITP). Splenectomy remains a common second line treatment for ITP with the highest remission rate compared with alternative therapies. It has been reported that splenectomy can be safely performed in patients with a platelet count of 40-50,000 per cubic millimeter but many patients do not reach these values. Intravenous immune globulins (IVIG) are often used before splenectomy in order to increase the platelet count before surgery. The aim of this study was to determine whether a short course of thrombopoietin-receptor agonists can be used as a reliable and safe treatment to increase the platelet count in patients with ITP before splenectomy. Between 2010 and 2012, fifteen patients with ITP, all refractory to steroids, were scheduled for splenectomy. Treatment with thrombopoietin-receptor agonists (romiplostim or eltrombopag) was started 3 weeks before splenectomy. Eight patients received eltrombopag at a dose of 50 mg/day orally until 3 days before splenectomy. For romiplostim, a subcutaneous injection of 3 mcg/kg was given weekly to 7 patients. The last injection was given one week before splenectomy. Complete blood count was repeated every week and the dose of romiplostim was adjusted (up to 10 mcg/kg or down to 1 mcg/kg) based on the platelet count increment. Response was defined as a platelet count of 50,000 or more per cubic millimeter. Mean platelet count before treatment was 11,000±8,000 cells per cubic millimeter. All patients, except one patient on romiplostim, responded to the treatment with a mean platelet count of 74± 25 cells per cubic millimeter on the day of splenectomy (p<0.01). Similar effect was noticed among responders of the two drugs. Four patients from the romiplostim group responded to a 3 mcg/kg dose. Two patients responded to increased doses of 7 and 10 and 10 mcg/kg. One patient did not respond to 10 mcg/kg of romiplostim but later responded to IVIG. The two drugs were well tolerated with no side effects except for mild liver function abnormalities in one patient in the eltrombopag group. No thromboembolic complications or excessive bleeding were reported for these patients. In summary, we report that a short course of thrombopoietin-receptors agonists can effectively and safely increase the platelet count in steroid resistant ITP patients before splenectomy. Disclosures: No relevant conflicts of interest to declare.

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Mpl Expression on AML Blasts Predicts Cytopenia

Blood ◽

10.1182/blood.v126.23.1387.1387 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1387-1387

Author(s):

Philipp J Rauch ◽

Corinne Widmer ◽

Kristin Fritsch ◽

Jana M Ellegast ◽

Jeroen S Goede ◽

...

Keyword(s):

Platelet Count ◽

Negative Correlation ◽

Conflicts Of Interest ◽

Gene List ◽

Receptor Expression ◽

Severe Neutropenia ◽

Severe Thrombocytopenia ◽

Hematopoietic Stem ◽

Platelet Counts ◽

Blast Count

Abstract Acute myeloid leukemia (AML) induces profound impairment of healthy hematopoiesis. The production deficit in the bone marrow (BM) leads to development of peripheral anemia, thrombocytopenia and neutropenia, which is a major cause of AML-associated morbidity and mortality. Despite much progress in understanding of AML biology, the mechanisms by which AML blasts interact with elements of normal hematopoiesis to cause cytopenia are unclear. Conventional wisdom has it that blasts infiltrate the marrow and displace normal hematopoiesis. If this concept were to be true, there should be a strong correlation between BM blast count and peripheral cytopenia. Surprisingly, analysis of 223 patients with newly diagnosed AML at a tertiary referral center revealed lack of correlation between initial BM blast count [% of cellularity] and hemoglobin level (ρ=-0.11, P=0.12), platelet count (ρ=-0.00, P=0.53) and absolute neutrophil count (ρ=0.13, P=0.06). This indicates that mechanisms other than displacement of normal hematopoiesis dictate the severity of cytopenia in AML patients. Hematopoiesis is tightly regulated by cytokines. Among them, thrombopoietin (TPO) acts through its receptor c-Mpl as the master regulator of megakaryopoiesis, but also exerts upstream effects on hematopoietic stem and progenitor cells (HSPC). TPO levels are controlled by receptor-mediated scavenging by cells carrying c-Mpl on the surface, with platelets representing the lion's share in a healthy organism. This negative feedback loop results in strong negative correlation between serum TPO concentration and platelet count in the steady state. When we examined this relationship in our AML cohort, TPO levels did not follow the expected negative correlation with platelet counts (ρ=-0.10, P=0.59). Comparison with historic controls with thrombocytopenia induced by chemotherapy for non-hematopoietic malignancy revealed that the lack of correlation was driven by AML cases with severe thrombocytopenia that had lower than expected levels of TPO in the serum. As HSPC are known to express c-Mpl, we hypothesized that HSPC-derived AML blasts may also express the receptor and cause insufficiency of hematopoiesis by means of receptor-mediated TPO scavenging. To test this hypothesis, we compared c-Mpl expression on blasts in AML cases with severe thrombocytopenia and low TPO concentration (potential scavenger cases) to cases with TPO levels adequate for the degree of cytopenia. Both surface flow cytometry and qPCR demonstrated higher c-Mpl expression in potential scavenger cases (3.1-fold, P=0.02). To determine whether this difference in expression translates into increased serum TPO clearance, we incubated AML blasts with high (c-Mpl+) and low (c-Mpl-) receptor expression in serum containing recombinant human TPO at a concentration of 100 pg/mL. After 2h, TPO clearance reached 45 pg per 106 cells in wells with c-Mpl+ blasts, compared to only 4 pg per 106 cells in wells with c-Mpl- blasts (P=0.02). This confirms the hypothesis that AML blasts can lower TPO levels by virtue of their c-Mpl expression. Validation studies in an independent, multi-center Dutch-Belgian-Swiss cohort of 437 AML cases confirmed lack of correlation between initial BM blast count and cytopenia. Ranked gene list correlation analysis of whole genome microarray data proved significant enrichment of the MPL transcript in patients with severe thrombocytopenia when compared to patients with average platelet counts (rank 27/20'589, FDR<10-6). MPL enrichment could also be observed in patients with severe neutropenia (P<0.01), but there was no correlation between MPL transcript level and degree of anemia. Lastly, we asked if MPL expression was related to cytogenetic or molecular AML subtype: indeed, microarray analysis showed higher MPL expression in cases of AML with t(8;21) than in any other subtype (P<10-4). Concurrently, these patients displayed significantly lower platelet count (40 vs 83 x 109/L, P=0.02) when compared to all other AML cases. In summary, our study demonstrates that cytopenia in AML is independent of BM blast count, but strongly correlated with c-Mpl expression on blasts. We show that c-Mpl+ blasts clear TPO, causing insufficient TPO levels and contributing to development of thrombocytopenia and neutropenia. The work may have important ramifications for treatment of AML-induced cytopenia, especially in the relapsed or refractory setting. Disclosures No relevant conflicts of interest to declare.

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