Platelet Electron Microscopy Research Articles

NBEAL2 gene mutations do not always lead to gray platelet syndrome: A case report.

Gray platelet syndrome (GPS) is a rare disease caused by homozygosity and compound heterozygosity for autosomal mutations on the NBEAL2 gene, which is characterized by a deficiency of platelet α-granules, bleeding symptoms. However, in this study, we report 2 NBEAL2 gene mutations in an easy bruising family without gray platelet and bleeding. A 33-year-old female nurse sought admission to our laboratory due to a tendency to bruise easily and unwell in daily life. However, there are no signs of petechiae or excessive bleeding in her daily life. Coagulation tests, routine blood tests and platelet staining of blood smears were all normal. The whole exome sequencing and Sanger sequencing were used to identify the causative variant of the patient. Furthermore, the morphology of platelets was examined using electron microscopy. Whole exome sequencing revealed the presence of 2 mutations in the NBEAL2 gene: p.Thr365fs and p.Ala310Thr. This prompted the consideration of a GPS diagnosis. However, platelet electron microscopy did not identify any abnormalities, leading to the exclusion of GPS. These 2 NBEAL2 gene mutations (p.Thr365fs and p.Ala310Thr mutations) do not affect the degranulation of platelets.

Platelet Electron Microscopy Does Not Correlate with Bleeding Scores in a Pediatric Population

Background:Platelet electron microscopy (PEM) is the diagnostic test for delta storage pool deficiency, a bleeding disorder due to qualitative or quantitative deficiency in dense granules leading to abnormal platelet function. Many people have non-syndromic forms which are challenging to diagnose. Aims: We aimed to describe the population for whom PEM is sent and correlate PEM results with bleeding phenotype and platelet function testing. Methods:We performed a single-center, retrospective study including patients who had PEM sent between 2/1/2018 and 8/30/2022 and had a hematology encounter. Demographics, PEM results, platelet aggregation studies, von Willebrand factor (VWF) testing, coagulation studies, genetic testing, and International Society on Thrombosis and Hemostasis Bleeding Assessment Tool (ISTH-BAT) scores were collected. ISTH-BAT was calculated based on the note closest in time to PEM testing. ISTH-BAT was either retrospectively calculated from clinical documentation or extracted as calculated by the clinician during the visit. ISTH-BAT normal range was defined as <4 for adult males, <6 for adult females, and <3 for children (<18 years) (Elbatarny et al., Hemophilia 2014). Descriptive statistics were used to summarize the results. Pearson's correlation coefficient and a Student's t-test were used for normally distributed data and Spearman rank correlation for non-parametric data. Categorical variables were compared with a Pearson chi-squared test. Statistical analysis included the first PEM result. Alpha<0.05 was considered significant. Results: There were 92 PEM tests sent from 77 subjects (Table 1). PEM testing was completed at either University of Toledo (N=79, normal range: 4-6 dense granules per platelet [dg/pl]) or Mayo Clinic (N=13, normal range: ≥1.2 dg/pl). Forty-six (60%) subjects had abnormal PEM results. Repeat testing was sent from 15 subjects, 13 of whom had an initial abnormal reported result with 12/13 repeat tests confirmed abnormal. Two initially indeterminant results were repeated with 1 abnormal and 1 normal on repeat. The mean dense granule per platelet for the cohort was 2.68 (standard deviation [SD] 1.4) dg/pl. The mean dense granules per platelet was 3.9 dg/pl (range 1.8 - 5.47) for subjects with normal PEM results and 1.97 dg/pl (range 0 - 3.39) for those with abnormal results, p<0.001. Most ISTH-BATs were retrospectively calculated from clinical documentation (71/77, 92%). The mean ISTH-BAT for the cohort was 3.6 (SD 2.2) and 50/77 (65%) had an elevated ISTH-BAT score for age and gender. There was no correlation between the ISTH-BAT score and dg/pl (Pearson's r=0.17, 95% CI: -0.60 - 0.379, p=0.15), (Figure 1). There was a weak correlation between dg/pl and ISTH-BAT for female gender (Pearson's r=0.39, 95% CI: 0.105 - 0.611, p=0.009) that is not present with male gender (Pearson's r= -0.057, 95% CI: -0.404 - 0.303, p=0.759). There was no difference between the mean dense granules per platelet for those with a normal ISTH-BAT (2.5 dg/pl, 95%CI: 1.79 - 3.22) and an abnormal ISTH-BAT (2.8 dg/pl 95% CI: 2.45 - 3.10), p=0.48. Similarly, there was no association between ISTH-BAT and PEM results as a binary variable, based on reference lab normal versus abnormal result (chi-squared p=0.22). Seventy-three platelet function tests were performed in 74% (57/77) of the cohort. The majority (43/57, 75%) had only one platelet function test. Most were whole blood impedance lumi-aggregometry (WBILA) (65/73, 89%); 8 were light transmission aggregometry. The first platelet function test was abnormal in 42% (24/57) of those tested. In the 49 subjects with WBILA as their first test, the most common abnormality was ADP secretion (N=20). ADP secretion values are not correlated with dg/pl (Spearman's r=0.28, p=0.05). At least one set of VWF tests were sent in 79% of the cohort (61/77). Two subjects were diagnosed with VWD, one of whom had a reported abnormal PEM result (2.96 dg/pl) and an ISTH-BAT of 7. The other had a normal EM result (4.38 dg/pl) and an ISTH-BAT of 4. Conclusions:There was no association between platelet EM results and bleeding phenotype by ISTH-BAT in the overall pediatric cohort, however there was a weak correlation between bleeding score and dg/pl in female gender. There was also no correlation between dg/pl and ADP secretion. Future work will focus on the utility of platelet EM testing in pediatrics and identifying subpopulations who may benefit from this test.

Circumcision bleeding rates in patients diagnosed with delta-storage pool deficiency later in life.

Male circumcision is a common procedure, generally performed during the newborn period. Bleeding is an uncommon but feared complication of circumcision. Few reports have evaluated circumcision bleeding rates in patients with bleeding disorders. To study circumcision bleeding rates in male subjects who were diagnosed later in life with delta-storage pool deficiency (δ-SPD). We retrospectively reviewed the medical records of male subjects (≤18years of age) who were circumcised without hemostatic prophylaxis prior to δ-SPD diagnosis from 2000 to 2020. Bleeding rates and severity were the main outcomes evaluated. We collected demographic data, bleeding scores using a validated assessment tool, laboratory data, and platelet electron microscopy results. A descriptive analysis was performed. Twenty-five male subjects were included. The median bleeding score at presentation was 3.5 (range: 2-9). The diagnosis was confirmed using platelet electron microscopy. A value of <2 dense granules/platelet was considered abnormal. Circumcision was performed at a median age of 2days (range: 1day to 4months). None of the subjects had intraoperative or postoperative bleeding. With a bleeding rate of zero, we suggest that newborn males with a family history of δ-SPD may be safely circumcised without hemostatic prophylaxis.

Platelet Function Analyzer-100 Shows Poor Predictive Value for Children with Low VWF and Mild Platelet Function Defects

Background: The Platelet Function Analyser (PFA)-100 (Siemens) has proven to be a useful screening tool for primary hemostasis. Studies have demonstrated a pooled sensitivity around 90% for all individuals with Von Willebrand Disease (VWD), however the sensitivity for Type 1 is notably poorer than types 2 and 3. Our institution utilizes the PFA-100 as part of a panel of laboratory studies called the "bleeding screen"; this panel is often used as first line screening for inherited bleeding disorders across the hospital system. As the relative frequency of Type 1 VWD and low VWF (defined as Von Willebrand factor (VWF) levels 30-50%) greatly exceed the frequency of individuals with Type 2 and Type 3, when used as a general screen, the PFA-100 likely has reduced sensitivity than documented in the literature. Methods: To evaluate the utility of the PFA-100 as a screening test for VWD and platelet function defects, we retrospectively examined the ordering practices of PFA-100 in relation to the Von Willebrand panel (VWF:antigen, VWF:ristocetin cofactor, Factor VIII), platelet aggregation studies and platelet electron microscopy. We compared results of the testing with diagnostic codes to characterize the test characteristics of the PFA-100 for the diagnosis of VWD or platelet function defects in a pediatric population. We included all patients tested with the PFA-100 from 2017 to 2018 at any Children's Healthcare of Atlanta facility. Exclusion criteria include age under 30 months (reference range established for those over 30 months), hematocrit &amp;lt;35% or platelets &amp;lt;150,000/uL at time of testing due to impact on PFA-100 results. Demographics, laboratory results, bleeding symptoms and diagnoses were collected via chart review. Bleeding disorders were determined by ICD-9 and ICD-10 codes and confirmed via chart review. Descriptive statistics were calculated with frequency and median (interquartile range); comparative statistics calculated with t-tests, chi-square, Fisher's Exact, Mann-Whitney and Spearman rank tests. Test characteristics for PFA-100 and VWD were determined by correlation between PFA-100 and VWD studies sent concurrently; a ristocetin co-factor &amp;lt;50% was considered positive for VWD in the comparative analysis. Test characteristics determined for platelet function testing were dependent on results of platelet aggregation studies and/or platelet electron microscopy. Results: There were 609 children who met inclusion criteria. Median age was 12.2 years (7.3-15.6). The majority were female (62.4%), white (58.5%) and non-Hispanic (77.0%). The most common reason for testing was epistaxis (28.1%), followed by heavy menstrual bleeding (24.8%) and bruising (18.4%). Almost 30% had no bleeding symptoms documented. The majority of individuals also had a VWD profile sent (91.5%), with 418 having the PFA-100 and VWD Profile sent concurrently. The majority of PFA-100 tests were normal (70.6%). Only 91 (14.9%) had additional platelet evaluation with platelet aggregation studies or electron microscopy. Overall, 170 individuals (27.9%) were found to have a bleeding disorder. VWD was diagnosed in 146 individuals (24.0%), a platelet function defect 14 (2.3%) and 11 individuals had other diagnoses including Factor VII deficiency, Hemophilia A and Hemophilia B. The sensitivity of the PFA-100 for VWD was 60.4% (95%CI 46.9-73.6%) and the sensitivity was 60.0% (26.2-87.4%) for platelet function defects. For those with an abnormal PFA, the results of a VWD profile did not determine the likelihood of a provider sending additional testing with platelet aggregation studies or platelet microscopy. Of the 104 individuals with an abnormal PFA and normal VWD studies, only 31 had platelet aggregation testing. There were no differences in demographics or reported symptoms between the two groups. With normal VWD studies, individuals with an abnormal PFA were about 3 times more likely to have platelet aggregation studies sent (p&amp;lt;0.0001). Conclusion: The PFA-100 has poor sensitivity for VWD and mild platelet function defects in pediatric patients. Most individuals screened with a PFA-100 also had a complete VWD panel sent concurrently indicating that this is used as a screen for platelet function defects in our hospital. Given the poor sensitivity for platelet function defects, advanced platelet function testing should be sent based on clinical concern regardless of PFA-100 results when VWD testing is normal. Disclosures Brown: National Hemophilia Foundation, Takeda Clinical Fellowship: Research Funding. Woods:Takeda: Membership on an entity's Board of Directors or advisory committees.

Platelet Electron Microscopy: Utilizing LEAN Methodology to Optimize Laboratory Workflow.

Platelet electron microscopy (PEM) is the gold standard methodology for diagnosing storage pool disorder, defined as a paucity of delta granules, alpha granules, or both. PEM literature is limited with few published resources and without well-developed interlaboratory standardization for the preparation and examination of platelet samples. Whole mount (WM) dense body (DB) counts for 300 pediatric cases were reviewed to determine whether counting fewer platelets could yield the same results. For 6 cases, DB average was determined on the day of WM preparation and on 2 consecutive days. Both WM and thin section (TS) preparations were examined for all cases. Employing LEAN methodology, an algorithm was developed to markedly decrease the number of platelets counted and still ensure accurate results. WMs decay with time; a statistically significant difference in DB counts was noted between day 0 and day 1 (p < .1). Twelve of 300 cases required both WM and TS preparations for a complete diagnosis. It is possible to maintain accuracy and decrease 100 platelet DB counts by >75%. WMs must be counted on the day they are prepared to avoid false paucity of DB secondary to sample decay. An accurate evaluation of platelet morphology requires both the WM and TS techniques.

Identification of novel variants in ten patients with Hermansky-Pudlak syndrome by high-throughput sequencing

Background Hermansky-Pudlak syndrome (HPS) is a rare inherited platelet disorder characterized by bleeding diathesis, oculocutaneous albinism (OCA) and a myriad of often-serious clinical complications. Methods We established the clinical and laboratory phenotype and genotype of six unrelated pedigrees comprising ten patients with clinical suspicion of HPS; including platelet aggregation, flow cytometry, platelet dense granule content, electron microscopy and high-throughput sequencing (HTS). Results The clinical presentation showed significant heterogeneity and no clear phenotype-genotype correlations. HTS revealed two known and three novel disease-causing variants. The Spanish patients carried a homozygous p.Pro685Leufs17* deletion (n = 2) in HPS4, or the novel p.Arg822* homozygous variant (n = 1) in HPS3. In the case of two Turkish sisters, a novel missense homozygous HPS4 variant (p.Leu91Pro) was found. In two Portuguese families, genetic studies confirmed a previously reported nonsense variant (p.Gln103*) in DTNBP1 in three patients and a novel duplication (p.Leu22Argfs*33) in HPS6 in two unrelated patients. Conclusions Our findings expand the mutational spectrum of HPS, which may help in investigating phenotype-genotype relationships and assist genetic counselling for affected individuals. This approach is a proof of principle that HTS can be considered and used in the first-line diagnosis of patients with biological and clinical manifestations suggestive of HPS. Key messages We established the relationships between the clinical and laboratory phenotype and genotype of six unrelated pedigrees comprising ten patients with clinical suspicion of HPS. Molecular analysis is useful in confirming the diagnosis and may offer some prognostic information that will aid in optimizing monitoring and surveillance for early detection of end-organ damage. This approach is a proof of principle that HTS can be considered and used in the first-line diagnosis of patients with biological and clinical manifestations suggestive of HPS.

A Retrospective Study of Clinical and Laboratory Characteristics in Patients Diagnosed with Platelet Storage Pool Deficiency

Introduction:Platelet storage pool deficiency (PSPD) is a disorder of platelet dysfunction with a varying degree of bleeding severity that can range from no symptoms to severe bleeding with hemostatic challenges. The true incidence of the disorder is unknown; however, some believe that it could be as common as von Willebrand disease (vWD). The current standard for diagnosis of PSPD is platelet electron microscopy (EM). However, there is a lack of standardization of techniques and reference ranges. In addition, some experts debate the clinical significance of the diagnosis based on the abnormal platelet EM finding alone. Available literature on adult patients with PSPD is scarce. Therefore, we conducted this study with the aim to describe the clinical and laboratory characteristics of patients diagnosed with PSPD based on specified laboratory criteria. We also intended to evaluate if platelet EM testing can be used to discern patients under evaluation for bleeding symptoms.Methods:We performed a retrospective cohort study of all adult patients referred for hemostatic workup who had platelet EM testing performed at The Ohio State Medical Center from 1/1/2004 to 10/26/2016. Patients with confirmed hemophilia or vWD were excluded. We collected data including patient demographics, symptoms, laboratory data, management plans, and outcomes. The reference range in our laboratory is 3.68-6.24 delta granules per platelet. Patients with less than 3.68 granules per platelet were diagnosed with PSPD (cases), while patients with at least 3.68 granules per platelet were designated as the comparison group. Characteristics in the two groups were compared by Fisher's exact test for categorical variables and the non-parametric Wilcoxon rank sum test for continuous variables since some of the continuous variables had highly skewed distributions.Results:A total of 291 patients were included in the study, 207 patients with PSPD based on EM results (cases) and 84 in the comparison group. Of the 207 cases, median age was 39 (range 12-84) years old and 81.2% were female. Among the cases, 38.7% had <2 delta granules per platelet, 42.0% had 2-2.99, and 19.3% had 3-3.68. The most common presenting symptom was cutaneous bruising (42.5% of cases and 38.1 % in the comparison group). Other common presenting symptoms included menorrhagia (29.9% of cases and 26.2 % of comparison), post-op bleeding (23.7% of cases and 38.1% of comparison), and epistaxis (14.0% of cases and 10.7% of comparison). At presentation, 4.8% of cases were asymptomatic. With the exception of higher incidence of post-op bleeding as a presenting symptom in the comparison group (p=0.015), there was no significant difference in the incidence of other presenting symptoms between the case and the comparison groups.There were no significant differences in the standard hemostatic workup between the two groups, including platelet count, PT, PTT, platelet function assay (PFA-100), platelet aggregation, and von Willebrand panel (Table 1). In both case and comparison groups, 20% of patients had abnormal PFA-100 and 50% had abnormal platelet aggregation with at least one agonist. The only significant difference in the laboratory findings was platelet EM results used to categorize cases (Figure 1 and Table 1).Conclusions:This study showed that in adult patients referred for hemostatic workup, abnormal platelet EM does not correlate with bleeding symptoms or the results of other laboratory tests such as PFA-100 or platelet aggregation. This calls into question the relevance of abnormal platelet EM alone and the appropriate “reference range” in the adult population. Patients with delta granules below the reference range were commonly diagnosed with PSPD, and interventions such as desmopressin and/or antifibrinolytic agents were subsequently recommended. Over-diagnosis of PSPD is a concern, but under-diagnosis in patients with normal EM but persistent bleeding symptoms is also problematic. Future research is needed to characterize correlation of platelet EM values with severity of clinical presentation and to establish appropriate reference range. Furthermore, better diagnostic tools are needed to evaluate patients with bleeding symptoms. [Display omitted] DisclosuresWang:Daiichi Sankyo: Consultancy, Other: Travel.

Bleeding Severity and Health Care Utilization in Patients with Platelet Function Disorders

Introduction: Disorders affecting any platelet pathway mediating adhesion, activation, aggregation and procoagulant surface exposure can result in a bleeding diathesis. Platelet function disorders (PFDs) are classified based on abnormalities in cytoskeleton, membrane phospholipids adhesive protein receptors, soluble agonist receptors, platelet granules or signal transduction pathways. Due to variability in platelet dysfunction, the clinical presentation and hence the treatment of bleeding manifestations in these patients vary. Use of a bleeding assessment tool can help in the assessment of bleeding severity, predict recurrent bleeding events in patients and help in predicting rates of hospitalization in these patients. Severe PFDs likely have higher BAT scores, and higher rates of hospitalization; but has not been reported widely. Resource utilization in PFDs is largely undescribed or quantified, yet a very important factor in the delivery of health care. Our study aims to assess bleeding severity at diagnosis and assess health care utilization (number of ER visits, hospitalizations, procedures and medications used) in patients with platelet dysfunction, to understand the burden of illness in these patients.Methods: This is an IRB-approved, two-institution retrospective analysis of 26 patients with PFDs younger than 21 years of age, from 2012 - 2018. Patients included were diagnosed with a PFD during the study period at Rainbow Babies and Children's Hospital and Children's Healthcare of Atlanta. The International Society on Thrombosis and Hemostasis - Bleeding Assessment Tool (ISTH - BAT) was used to assess bleeding severity at diagnosis. Demographic characteristics, diagnostic labs, details of hospitalizations, ER visits, outpatient procedures, details on medications, transfusions, and length of stay at these visits were collected via medical record abstraction. Descriptive statistics were used to summarize results.Results: 26 patients were included, of which 10 were male and 16 were female. The age of presentation with symptoms ranged from 2 months to 13 years of age. The mean age of platelet function diagnosis was 5 years, except for the most severe PFDs, who presented prior to 1 years of age. The 2 most common symptoms at presentation were easy bruising (n =11, 42%) and epistaxis (n = 8, 30%). Of the 26 patients, we had 2 patients (7.7%) with Bernard Soulier syndrome, 5 patients (19.2%) with Glanzmann Thrombasthenia, 7 patients (27%) with dense granule storage pool defect and 12 patients (46.1%) with one or more agonist aggregation defects. Light transmission aggregometry was used for diagnosis in 26 (100%), release assay in 5 (19.2%), and platelet electron microscopy in 7 (26.9%) patients. ISTH BAT scores were highest for patients with more severe platelet dysfunction, such as Bernard Soulier syndrome and Glanzmann Thrombasthesia (mean score = 9) and lower for milder PFDs (mean score = 2). The BAT scores among patients with 1, 2 or 3 agonist defects were similarly low at an average score of 2. Rates of ER visits, outpatient procedures and hospitalizations were higher in patients with higher BAT scores, i.e. more severe platelet dysfunction as seen in Table 1. Of the 5 patients with Glanzmann's Thrombasthenia, 3 were started on Novo7 prophylaxis, indicating severity of symptoms in these patients. Platelet transfusions were only used in severe disorders, while amicar was the preferred modality in milder platelet dysfunction, minor procedures and minor bleeds.Conclusions: Based on our retrospective analysis, we conclude that patients with milder platelet dysfunction have lower BAT scores at presentation, lower bleeding manifestations and therefore less health care utilization. In our study, higher BAT scores in patients signified severe platelet dysfunction and recurrent bleeding episodes, requiring frequent ER visits and hospitalizations. We plan to continue this study on a larger patient database to estimate resource utilization from a national perspective, and to identify independent predictors of increased resource use for these patients and understand the economic impact of platelet function defects. DisclosuresSidonio:Kedrion: Research Funding; Novo Nordisk: Other: Advisory Board; Shire: Other: Advisory Board, Research Funding; CSL Behring: Other: Advisory Board; Genentech: Other: Advisory Board, Research Funding; Grifols: Other: Advisory Board, Research Funding; Biomarin: Other: Advisory Board; Octapharma: Other: Advisory Board; Bioverativ: Other: Advisory Board, Research Funding; Uniqure: Other: Advisory Board. Ahuja:Bayer: Honoraria; Shire: Honoraria, Speakers Bureau; Bioverativ: Honoraria, Speakers Bureau.

Weak correlation of bleeding scores to platelet electron microscopy: A retrospective chart review of pediatric patients with delta-storage pool disorder.

Delta granule storage pool deficiency (δ-SPD) is a rare platelet disorder in which a deficiency of platelet granules leads to poor aggregation, resulting in varying clinical bleeding phenotypes. Children with δ-SPD have variable laboratory results, making the proper diagnosis and evaluation controversial. To describe the demographic and laboratory trends of this population and to assess the value of electron microscopy in diagnostic evaluation and its correlation to bleeding symptoms. We performed a retrospective review of 109 pediatric patients diagnosed with δ-SPD. We collected demographic information and bleeding scores using a validated bleeding assessment tool. A descriptive and exploratory analysis was performed. The majority of patients were female, with an average age at diagnosis of 11.61 years. Females were diagnosed at a significantly older age presenting most often with menorrhagia, while males presented most commonly with epistaxis. The majority showed normal lumiaggregometry, the mean platelet electron microscopy (PEM) value was 2.37, and the mean bleeding score was 6. Bleeding assessment tool and PEM had a significantly weak correlation. Patients with more dense granules per platelet had higher bleeding scores than those with fewer dense granules per platelet. The current body of evidence does not favor the use of PEM in routine clinical practice, and results are difficult to interpret. In patients with severe mucocutaneous bleeding symptoms and normal platelet aggregation studies, consideration should be given to an alternative diagnosis and further evaluation is warranted.

GFI1B Mutation Associated Alpha-Delta Platelet Storage-Pool Deficiency: A Case Report and Its Potential Important Implication

Background: In addition to NBEAL2, a single dominant-negative mutation in the GFIB1 gene has been associated with grey platelet syndrome but no association has been mentioned with dense granule deficiency. Here we describe a child with thrombocytopenia, alpha-delta granule deficiency, and a homozygous missense mutation in GFIB1.Case Report:The patient is an 8y/o Hispanic male born to non-consanguineous parents. Prenatal and birth history were unremarkable. No family history of blood disorders or pediatric malignancies. The boy has 2 healthy older siblings, no dysmorphic features, and normal skin pigmentation and eye findings. Renal function and hearing are normal. At 3mo he was noted to have multiple spontaneous petechiae along with an isolated thrombocytopenia of 46K/uL. At 10 months, a bone marrow evaluation showed increased megakaryocytes suggestive of ITP. By 3years of age, he received three treatments of IVIG without an adequate response. His platelet counts have generally ranged between 30-50K/uL. With acute illnesses, they drop to 15-20K/uL. His bleeding symptoms have primarily been spontaneous bruising and petechiae as well as prolonged epistaxis. His symptoms have been generally controlled with anti-fibrinolytic agents alone. Platelet transfusions have been reserved for surgical procedures or significant bleeding symptoms.Further evaluations over the past 5 years have included peripheral smears, showing atypical large hypo-granular platelets, and two additional bone marrow aspirates, showing megakaryocytic hyperplasia with numerous osteoclast-like forms and occasional small mono-lobated megakaryocytes and evident emperipolesis. Anti-nuclear antibody testing and platelet direct and indirect antibody testing were negative. Platelet electron microscopy showed that platelets virtually contained no dense granules (0.05 dense granules/platelet, 200 platelets) and about 30-40% of the platelets had markedly decreased alpha granules. Some platelets had complex canalicular networks and membrane vacuoles. These features are consistent with an alpha-delta platelet storage-pool deficiency. A myeloid malignancy mutation panel, performed using next generation sequencing, detected no variants of known significance. Gene sequencing for MHY9-related disorders as well as DNA breakage analysis for Fanconi’s anemia were negative. However, DNA sequencing of the patient’s sample revealed a homozygous missense mutation (c923 T>C; p.Leu308Pro) in the GFIB1gene; each parent carried one copy of this change.Conclusion:GFIB1, mapped to chromosome 9q34.13, encodes a transcriptional repressor that is important for megakaryopoiesis. It has been reported in patients with gray platelet syndrome; an inherited platelet disorder associated with thrombocytopenia and decreased alpha granules. This case points to an association of biallelic GFIB1 mutations with alpha-delta granule deficiency. This case underscores the importance of platelet esoteric testing and molecular analysis in the diagnosis of hereditary platelet disorders. As a targetable mutation in the future, this could revolutionize the early diagnosis and treatment of this rare platelet disorder. DisclosuresNo relevant conflicts of interest to declare.

Practice patterns in the diagnosis of inherited platelet disorders within a single institution.

: The diagnosis of inherited platelet disorders (IPDs) is challenging with variable diagnostic practices existing between institutions. To determine patterns and utility of diagnostic testing practices for IPDs within a single institution, a retrospective cohort study was performed. Records of 50 patients (50% women), median age 32 years (1 day to 81 years) were analyzed. In total, 28 (53%) had a positive International Society of Thrombosis and Hemostasis Bleeding Assessment Tool score. Test-ordering patterns were highly variable. All patients had platelet morphology analysis by light microscopy. In total, 42 (84%) underwent light transmission aggregometry, 43 (86%) platelet function analyzer, 37 (74%) platelet electron microscopy, 25 (50%) flow cytometry, and 15 (30%) genetic testing. Platelet function analyzer and light transmission aggregometry were always used as first-order tests, followed by platelet transmission electron microscopy and flow cytometry (81 and 84%, respectively). Genetic testing was obtained up front in five cases (33% of orders), mostly in patients with syndromic thrombocytopenia or in the setting of a known genetic disorder. Test-ordering practices did not adhere to published algorithms. Even within a single institution, great heterogeneity exists in the testing approach to IPDs. Although, a large proportion of cases were studied with platelet transmission electron microscopy and flow cytometry, standard platelet assays established the diagnosis in a great majority. Standardization of testing practices, first beginning at the institutional level is a much needed step forward.

Light Transmission Aggregometry Does Not Correlate With the Severity of δ-Granule Platelet Storage Pool Deficiency.

Delta-granule platelet storage pool deficiency (δ-PSPD) is a poorly studied bleeding diathesis resulting from either decreased granule content or decreased average number of platelet δ-granules. Light transmission aggregometry (LTA) is commonly used to evaluate for δ-PSPD and platelet electron microscopy (EM) is used to confirm the diagnosis. Currently, little data exist examining the relationship between the likelihood of abnormal platelet aggregation findings, severity of δ-granule deficiency on platelet EM, and severity of bleeding symptoms in patients with δ-PSPD. Patients diagnosed with δ-PSPD by platelet EM who also underwent LTA testing were identified at a single institution for correlation between severity of bleeding, average number of platelet δ-granules, and number of agonist abnormalities on LTA. No statistically significant association was identified between the average number of δ-granules per platelet and likelihood of an abnormal LTA. LTA abnormalities were quite varied and only 50% diagnosed with δ-PSPD on EM had abnormal aggregation testing. Also, no correlation was seen between the number of clinical bleeding symptoms, number of average δ-granules per platelet, and the number of LTA agonist abnormalities. Our findings highlight the difficulties inherent in the laboratory evaluation of platelet function.

Platelet Function Disorders in Adolescents with Heavy Menstrual Bleeding: Clinical Presentations, Laboratory Testing and Treatment Options

Background: Platelet Function Disorders (PFD) is receiving increasing attention as an important etiology of Heavy Menstrual Bleeding (HMB) in adolescents. An understanding of presentation profiles, diagnostic limitations and effective treatment regimens for these disorders are important for effective management of these disorders. Methods: This study reviews the literature on the diagnosis and treatment of platelet function disorders in adolescents with heavy menstrual bleeding. Results: Heavy menstrual bleeding affects the quality of life of many adolescents, and PFDs appearing to be an important underlying etiology of this disorder. The storage pool subtype is the most prevalent of the platelet functional defects in these patients. Standard platelet functions screening has a number of limitations in diagnosing many of the PFD subtypes, and platelet aggregation studies and electron microscopy are important diagnostic adjuncts. Hormonal and non-hormonal treatment regimens are available and treatment recommendations will depend on the severity of bleeding. Conclusion: Clinicians treating adolescent with HMB should be aware of PFDs and the diagnostic limitations in detecting many of the subtypes. Management depends on the severity of bleeding.

Familial Myeloid Neoplasms: Clinical Spectrum and Associated Abnormalities

BackgroundFamilial myeloid neoplasms are rare causes of myeloid malignancies. Over the last few years germline mutations involving RUNX1, JAK2, GATA1, GATA2 and CEBPA genes have been associated with myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN) and acute myeloid leukemia (AML). We carried out this study to describe the spectrum of familial myeloid neoplasms seen at our institution. MethodsAfter due IRB approval, the myeloid neoplasms data base (1990-2012) at the Mayo Clinic in Minnesota was queried for patients presenting with, or having a strong family history of myeloid neoplasms. Selective sequencing of germline DNA was done in appropriate patients. Germline mutations that were screened for included; RUNX1, JAK2, GATA1, GATA2 and CEBPA. In one family with none of the aforementioned gene mutations, exome sequencing was performed. Families with chromosomal breakage and primary telomere disorders were excluded. All patients had bone marrow and cytogenetic studies at diagnosis. ResultsWe identified six families with familial myeloid neoplasms (table one). There were two with germline GATA2 mutations (no.1 and 2); one with a novel c1339A>C, pS447R, non-zinc finger gene abnormality. In this family nine members have developed MDS/AML. All members had profound B/NK cell deficiency, viral warts and one member had primary lymphedema (Embergers syndrome). Three of the 6 seen at the clinic have successfully undergone allo- SCT. The second GATA2 mutated family had a less extensive history of myeloid neoplasms.There was one patient (no.5) who presented with long standing thrombocytopenia, clinically diagnosed as ITP. She eventually developed MDS with trisomy 8. She had three first degree relatives with myeloid neoplasms. Germline sequencing identified a RUNX1 mutation. Platelet electron microscopy identified characteristic ultrastructural changes.There was one family (no.4) with a germline JAK2V617F mutation. The propositus was diagnosed to have primary myelofibrosis and then demonstrated clonal evolution to a concomitant BCR-ABL1 p190 driven chronic myeloid leukemia. His sister and mother have essential thrombocytosis (ET) and his brother has a detectable JAK2 mutation (no evidence for disease). The patient has successfully undergone an allo-SCT.The patient with a GATA1 mutation (no.3) presented with congenital anemia and had craniofacial anomaly, atrial septal defect and developmental delays. Red cell and platelet ultrastructural abnormalities lead to the diagnosis with an additional family member having chronic anemia and suspected MDS.One family (no.6) presented with seven first degree relatives affected by MDS/AML. Germline sequencing for the aforementioned genes was negative. Exome sequencing has been performed and results should be available at the time of the meeting. The prepositus has successfully undergone allo-SCT. ConclusionFamilial myeloid neoplasms are uncommon but warrant early recognition. Clues to diagnosis include strong family history, associated primary immunodeficiency states, morphological/skeletal abnormalities and platelet ultrastructural defects (RUNX1, GATA2, and GATA1). Exome and whole genome sequencing is an exciting prospect for further discoveries.Table 1Familial Myeloid Neoplasms.Germline mutationAgeSexHematologic-al diagnosisCytogeneticsAssociated featuresFamily members affectedAllo-SCTStatus1.GATA2 (c1339A>C)37MAML46,XY, t(1;21)[9] +8[4]46,XY[7]NK/B cell defViral wartsHydroceleMDS-3AML-3Neutropenia-1YesAlive9MChronic NeutropeniaNoneNK/B cell defViral wartsEmbergers syndromePulmonary stenosisNoAlive17FMDSNoneNK/B cell defViral wartsYesAlive31FMDSNoneNK/B cell defViral wartsInfertilityYesAlive57FNoneUnknownNK/B cell defViral wartsNoAlive2. GATA21FNone46,XX,del(3) (q13.2q22.2)HypotoniaDysmorphic featuresDevelopmental delayASDMPN-1MDS-1NoAlive3. GATA18MCongenital AnemiaNoneDevelopmental delay.ASDSplenomegalyCongenitalAnemia- 1NoAlive4. JAK 2 V617F59MPMF/CML46,XY,del(13) (q12q22)[5]/ 46,XY[15]BCR/ABLp190MelanomaHiatal HerniaMPN- 2YesAlive5. RUNX165FMDS47,XX,+8[20]Chronic ThrombocytopeniaMDS-2AML-1NoAlive6. Exome sequencing performed61MAML46, XY [20]Pilonidal cystMDS-4AML-3MM-1DLBCL-1YesAlive Disclosures:No relevant conflicts of interest to declare.

Delta-storage pool disease as a mimic of abusive head trauma in a 7-month-old baby: A case report

A seven-month-old baby was admitted to a hospital emergency department after collapsing suddenly while staying with his nanny. The baby displayed classic symptoms of shaken baby syndrome, including subdural haemorrhage, cytotoxic cerebral oedema, and bilateral retinal hemorrhages. Child protection services were informed, but both the parents and the nanny denied any involvement. In the subsequent weeks, the baby developed three other episodes of new subdural bleeding and a medico-legal investigation was started into the origin of the repeated subdural bleeding. Eventually, platelet aggregation tests and electron microscopy diagnosed a delta-storage pool disease; that is, a haemostatic disorder involving dense granules of the platelets. Initial minor blunt trauma may have resulted in subdural bleeding, while subsequent retinal haemorrhage could have been facilitated by the underlying haemostatic disorder. Delta-storage pool disease should be considered as a possible mimic of abusive head trauma similar to other rare conditions such as Menkes disease and type 1 glutaric aciduria.

Thrombocytopenia and Platelet Ultra-Structural Abnormalities Associated with RUNX1 Haploinsufficiency in Monosomy 21 Mosaicism.

AbstractAbstract 2190RUNX1, also known as core binding factor A2 (CBFA2), is a transcription factor that regulates the expression of several hematopoietic specific genes through a highly conserved DNA-binding region called the RUNT homology domain. The significant role of RUNX1 on megakaryopoiesis and leukemogenesis is supported by the finding that germline, heterozygous mutations in the RUNX1 gene on 21q22.12 forms the genetic basis of the autosomal dominant, familial platelet disorder with predisposition to acute myeloid leukemia (FDP/AML). Patients with FDP/AML present with mild to moderate thrombocytopenia, qualitative platelet defects and a propensity to develop AML. While several platelet abnormalities have been reported in this cohort including abnormalities in platelet aggregation and secretion, Mpl receptors, activation of GPIIb-IIIa, phosphorylation of myosin light chain and deficiency of platelet factor 4 (PF4); to the best of our knowledge, platelet electron microscopy findings have never been described. Furthermore, data on phenotypic manifestations of partial monosomy 21 are sparse and anecdotal. Published reports from literature describe patients with growth restriction, developmental delay, heart defects and dysmorphic facies. Thrombocytopenia, bleeding and even fatal hemorrhage have been noted, though detailed hematological manifestations are not consistently described. It is plausible that deletion of RUNX1 may be responsible for some of the characteristics observed in this cohort.An 18 month old male with a history of unprovoked recurrent subdural hemorrhage was referred to our service for further investigation. Patient was born at 35 weeks of gestation with intra-uterine growth restriction (birth weight 1440 grams). Post-natal history was significant for dysmorphic facies, global developmental delay, seizure disorder, chronic thrombocytopenia (platelet count 70 – 140 × 109/L) and subaortic stenosis. Bleeding diathesis workup including INR, aPTT, von Willebrand antigen, ristocetin co-factor and multimer analysis were normal. PFA-100 showed consistently prolonged closure times with Col/Epi and Col/ADP cartridges and platelet aggregation showed dis-aggregation with ADP, but normal ATP release. Peripheral blood karyotype showed 46, XY, r(21) (p11.2q22.3)[22]/45, XY, −21[8]. Fluorescent-in-situ hybridization (FISH) analysis for the RUNX1 locus showed 2 copies of RUNX1 in 116/200 (58%) nuclei and one signal of RUNX1 (consistent with loss of the ring chromosome 21) in 84/200 (42%) nuclei analyzed. Transmission electron microscopy of patient platelets revealed frequent fused/large alpha granules, with approximately 20% of the platelet population characterized as agranular or having few granules. PF4 in patient total platelet lysates was not significantly decreased as assessed by immunoblotting.To our knowledge this is the first demonstration of ultra-structural platelet abnormalities in a patient with RUNX1 haploinsufficiency. The molecular data from our patient helps elaborate the role of RUNX1 on both thrombocytopenia and platelet developmental abnormalities which also affect platelet function. Further use of electron microscopy in RUNX1 haplodeficient cohort may help better understand and characterize this rare platelet disorder. Disclosures:No relevant conflicts of interest to declare.

Report of a Young Girl With MYH9 Mutation and Review of the Literature

MYH9 mutations cause the inherited macro-thrombocytopenic syndromes of May-Hegglin anomaly, Fechtner syndrome, Sebastian syndrome, and Epstein syndrome, collectively referred to as MYH9-related disease. We present the case of a girl with MYH9-related disease whose diagnosis was facilitated by platelet electron microscopy and MYH9 sequencing. We discuss our patient's clinical presentation, now with 12 years of follow-up. We also discuss management and her possible prognosis given her specific MYH9 mutation.

Platelet Function Disorders and Menorrhagia in Adolescents: A Review of Laboratory Diagnosis

This article provides an overview of the prevalence and pathophysiology of platelet function disorders (PFDs) in adolescents with menorrhagia. In addition, this article reviews the various testing modalities employed for diagnosing PFDs including platelet aggregometry, platelet function analyzer (PFA-100), platelet electron microscopy (EM), flow cytometry, and thromboelastography (TEG), discuss their utility and drawbacks, and allude to the recent recommendations and consensus statements about some of these modalities. Finally, the authors have sketched out a diagnostic algorithm for platelet function testing, which will guide treating physicians to a step-wise approach while evaluating adolescents with menorrhagia for PFDs.

The PFA‐100® does not predict delta‐granule platelet storage pool deficiencies

There are no published reports investigating the ability of the platelet function analyzer (PFA-100(®) ) to detect the presence of delta-granule platelet storage pool deficiencies (δ-PSPD), a common mild bleeding disorder. Prior studies of the PFA-100(®) and congenital platelet disorders have been limited by small numbers of patients with a variety of disorders. We examined PFA-100(®) results in a large paediatric patient population diagnosed specifically with δ-PSPD, and determined the relationship between PFA-100(®) and platelet electron microscopy (the gold standard for diagnosis). This study is a retrospective review of patients <19 years of age diagnosed with δ-PSPD at Nationwide Children's Hospital from 2008 to 2010. To examine the correlation between PFA-100(®) and average number of granules per platelet we used Spearman's Rho as a non-parametric measure of dependence. A total of 105 patients diagnosed with δ-PSPD were included, of which 99 patients underwent PFA-100(®) testing. Of those tested 46% had at least one abnormal closure time, whereas 16% had abnormal results for both cartridges. We found no statistical correlation between C-EPI closure time and average number of granules per platelet (ρ= -0.0095, P-value = 0.9328), nor between C-ADP closure time and the average number of granules (ρ = 0.0315, P-value = 0.7798). The PFA-100(®), a widely used screening test for suspected bleeding disorders, did not correlate with presence or severity of δ-PSPD as determined by platelet electron microscopy. When evaluating patients with suspected bleeding disorders, PFA-100(®) alone cannot be used to rule out the presence of a δ-PSPD.

The PFA-100 Does Not Predict Delta-Granule Platelet Storage Pool Deficiencies

AbstractAbstract 2518 Background:Although delta-granule platelet storage pool deficiencies (δ-PSPDs) are common disorders of platelet function, they are relatively poorly studied and described. One unknown is the relationship between δ-PSPDs and the PFA-100, a screening test originally developed for von Willebrand's disease but now widely used as a general screening test for coagulopathies. Previous studies have suggested that the PFA-100 is less effective in detecting mild platelet function disorders (which include δ-PSPDs) than more severe platelet function disorders. These studies, however, were limited by small numbers of patients with a variety of different platelet function defects. We examined PFA-100 results in a larger pediatric patient population diagnosed specifically with δ-PSPD, and determined the relationship between PFA-100 results and platelet electron microscopy (the standard for diagnosis of δ-PSPDs). Methods:This study is a retrospective medical record review of patients 0 to 18 years of age diagnosed with δ-PSPD at Nationwide Children's Hospital between January 1, 2008 and July 31, 2010. We defined δ-PSPD as patients with an average of fewer than 3.68 delta granules per platelet. We obtained demographic data including age, sex, and family history of bleeding. Lab data was also extracted, including PFA-100 and platelet electron microscopy. We determined the percentage of δ-PSPD patients with an abnormal PFA-100. To examine the correlation between the PFA-100 results and the average number of granules per platelet we used Spearman's Rho as our non-parametric measure of dependence. Results:A total of 88 patients diagnosed with δ-PSPD were included in this study. Of our patient population, 35% were male, and 61% had a first degree family history of easy bruising or bleeding, while only 15% had a family history that was positive specifically for platelet function disorders. The most common symptoms on presentation were easy bruising (56%), epistaxis (39%), oral cavity bleeding (35%) and menorrhagia (30%). Eighty-one of these patients underwent PFA-100 testing, of which 41% had an abnormal CEPI value, 17% had an abnormal CADP value, and 14% had abnormal results for both PFA cartridges. We found no statistical correlation between CEPI closure time and the average number of granules per platelet (rho = 0.0315, p value = 0.7798), nor between CADP closure time and the average number of granules (rho = -0.0095, p value = 0.9328)(Figure). Additionally, the number of bleeding symptoms in each patient was not statistically correlated with CEPI closure times, CADP closure times or platelet EM. Discussion: The PFA-100, which is widely used as a screening test for suspected bleeding disorders, was abnormal in fewer than half of pediatric patients diagnosed with δ-PSPD. We found that PFA-100 results did not correlate with presence or severity of delta-granule platelet storage pool deficiencies as determined by platelet electron microscopy. When evaluating patients with suspected bleeding disorders, PFA-100 testing alone cannot be used to rule out the presence of a δ-PSPD. [Display omitted] Disclosures:No relevant conflicts of interest to declare.