Platelet Storage Pool Deficiency Research Articles

Multicolor flow cytometry in clinical samples for platelet signaling assessment

BackgroundAvailability of multichannel cytometers and specific commercial antibodies makes flow cytometry a new option to simultaneously assess multiple intracellular platelet signaling pathways for clinical purposes, in small volume of blood or low platelet count. ObjectivesTo describe a multicolor flow cytometry with fluorescent barcoding technique for screening signaling pathways downstream membrane receptors of major platelet agonists (adenosine diphosphate, thrombin, thromboxane, and collagen). MethodsBy comparison with immunoblotting, we first selected the target phosphoproteins, AKT, P38MAPK, LIMK, and SPL76; the times of stimulation; and phosphoflow barcoding conditions. We then performed a clinical study on whole blood of patients without evidence of blood platelet disorder on standard biological screening, consulting for trivial or occasionally provoked bleeds without familial antecedent (bleeding of unknown origin, n = 23) or type-1 von Willebrand disease (n = 9). In addition, we included a small group of patients with definite platelet disorders (Glanzmann thrombasthenia, δ-storage pool deficiency, and immune glycoprotein VI–related disease with granule secretion defect). ResultsThe range, kinetics, and distribution of fluorescence intensity were established for each agonist-target protein combination. Principal component analysis indicates a correlation in response to a target phosphoprotein (AKT and P38MAPK) to different agonists but no correlation in the response of different target phosphoproteins to the same agonist. The heterogeneity of individual responses in the whole population displayed was analyzed using clustering algorithm. Patients with platelet storage pool deficiency were positioned as lowest responders on the heatmap. ConclusionIn complement of functional tests, this study introduces a new approach for rapid platelet signaling profiling in clinical practice.

Experience With Pre-procedural Hemostatic Medications versus Platelet Transfusion in Patients With Platelet Storage Pool Deficiency

Background Storage pool deficiency (SPD) is a rare bleeding disorder characterized by reduction in the number of delta granules within platelets, interfering with hemostasis. Current literature lacks well-designed studies from which to draw concrete conclusions regarding pre-procedural management of bleeding complications. Objective: The purpose of this study is to describe bleeding and safety outcomes of SPD patients receiving either pre-procedural platelet transfusions or platelet-sparing regimens. Methods: An exploratory retrospective cohort study was conducted among SPD patients, comparing major bleeding events between those who received platelet transfusion and those who received desmopressin, tranexamic acid, and/or aminocaproic acid within 24 hours prior to procedure. Results: Rates of major bleeding were not found to be higher among patients who received a platelet-sparing regimen [platelet-sparing: 2/25 (8%); platelet transfusion: 2/29 (6.9%); P = .99]. Incidence of non-major bleeding was higher in the platelet transfusion group, but this was not statistically significant [platelet-sparing: 0/25 (0%); platelet transfusion: 3/29 (10.3%); P = .24]. Treatment-related adverse effects were observed following 8 of 54 procedures (14.8%). Conclusion: Use of a platelet-sparing regimen was not associated with a significantly higher incidence of major or non-major bleeding events. Future prospective trials are recommended to compare outcomes between therapies.

Hermansky-Pudlak syndrome type 2: A rare cause of severe periodontitis in adolescents-A case study.

Background and aimsHermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by oculocutaneous albinism (OCA) and platelet storage pool deficiency. The HPS-2 subtype is distinguished by neutropenia, and little is known about its periodontal phenotype in adolescents. AP3B1 is the causative gene for HPS-2. A 13-year-old Chinese girl presented to our department suffering from gingival bleeding and tooth mobility. Her dental history was otherwise unremarkable. Suspecting some systemic diseases as the underlying cause, the patient was referred for medical consultation, a series of blood tests, and genetic tests. In this case study, periodontal status and mutation screening of one HPS-2 case are presented.MethodsBlood analysis including a complete blood count (CBC) and glycated hemoglobin levels were measured. Platelet transmission electron microscopy (PTEM) was performed to observe the dense granules in platelets. Whole-exome sequencing (WES) and Sanger sequencing were performed to confirm the pathogenic variants.ResultsA medical diagnosis of HPS-2 was assigned to the patient. Following the medical diagnosis, a periodontal diagnosis of “periodontitis as a manifestation of systemic disease” was assigned to the patient. We identified novel compound heterozygous variants in AP3B1 (NM_003664.4: exon7: c.763C>T: p.Q255*) and (NM_003664.4: exon1: c.53_56dup: p.E19Dfs*21) in this Chinese pedigree with HPS-2.ConclusionThis case study indicates the importance of periodontitis as a possible indicator of underlying systemic disease. Systemic disease screening is needed when a young patient presents with unusual, severe periodontitis, as the oral condition may be the first of a systemic abnormality. Our work also expands the spectrum of AP3B1 mutations and further provides additional genetic testing information for other HPS-2 patients.

Platelet Storage Pool Deficiency and Elevated Inflammatory Biomarkers Are Prevalent in Postural Orthostatic Tachycardia Syndrome.

A significant number of postural orthostatic tachycardia syndrome (POTS) patients have platelet delta granule storage pool deficiency (δ-SPD). The etiology of POTS is unknown but a number of laboratories, including ours, have reported elevations of G-protein-coupled adrenergic receptor and muscarinic acetylcholine receptor autoantibodies in POTS patients, detected by a variety of techniques, suggesting that the disorder is an autoimmune condition. Thus, it could also be considered an inflammatory disease. In a pilot study, we investigated a limited number of platelet-related cytokines and chemokines and discovered many that were elevated. This case–control study validates our pilot study results that POTS patients have an activated innate immune system. Plasma of 35 POTS patients and 35 patients with unexplained bleeding symptoms and categorized as “non-POTS” subjects was analyzed by multiplex flow cytometry to quantify 16 different innate immune system cytokines and chemokines. Electron microscopy was used to quantify platelet dense granules. Ten of 16 biomarkers of inflammation were elevated in plasma from POTS patients compared to non-POTS subjects, with most of the differences extremely significant, with p values < 0.0001. Of particular interest were elevations of IL-1β and IL-18 and decreased or normal levels of type 1 interferons in POTS patients, suggesting that the etiology of POTS might be autoinflammatory. All POTS patients had δ-SPD. With a growing body of evidence that POTS is an autoimmune disease and having elevations of the innate immune system, our results suggest a potential T-cell-mediated autoimmunity in POTS characteristic of a mixed-pattern inflammatory disease similar to rheumatoid arthritis.

Inflammatory Biomarkers in Postural Orthostatic Tachycardia Syndrome with Elevated G-Protein-Coupled Receptor Autoantibodies.

A growing body of evidence suggests that postural orthostatic tachycardia syndrome (POTS) may be an autoimmune disorder. We have reported in a previous manuscript that 89% of POTS patients (n = 55) had elevations in G-protein-coupled adrenergic A1 receptor autoantibodies and 53% had elevations in muscarinic acetylcholine M4 receptor autoantibodies, as assessed by ELISA. Patients with autoimmune disorders have been reported with a variety of elevated cytokines and cytokines (such as rheumatoid arthritis); thus, we evaluated a limited number of cytokines/chemokines in POTS patients with elevated adrenergic and muscarinic receptor autoantibodies. We utilized the plasma of 34 patients from a previous study; all of the patients (100%) had autoantibodies against the A1 adrenergic receptor and 55.9% (19/34) had autoantibodies against the M4 muscarinic acetylcholine receptor. In particular, the plasma cytokine/chemokine levels were measured as biomarkers of inflammation by Quantibody® technology (Raybiotech, Peachtree Corners, GA, USA). We also evaluated the platelet dense granule numbers, as these patients frequently complain of symptoms related to platelet dysfunction. Patients were predominantly young females who displayed a multitude of co-morbidities but generally reported viral-like symptoms preceding episodes of syncope. Eighty five percent (29/34) had platelet storage pool deficiency. Patients had elevations in five of ten cytokine/chemokines biomarkers (IL1β, IL21, TNFα, INFγ, and CD30), whereas two biomarkers had decreased levels (CD40L and RANTES). Our observations demonstrate that POTS patients known to have autoantibodies against the G-protein-coupled adrenergic A1 receptor have abnormal plasma concentrations of inflammatory cytokines.

A zinc transporter, transmembrane protein 163, is critical for the biogenesis of platelet dense granules

Lysosome-related organelles (LROs) are a category of secretory organelles enriched with ions such as calcium, which are maintained by ion transporters or channels. Homeostasis of these ions is important for LRO biogenesis and secretion. Hermansky-Pudlak syndrome (HPS) is a recessive disorder with defects in multiple LROs, typically platelet dense granules (DGs) and melanosomes. However, the underlying mechanism of DG deficiency is largely unknown. Using quantitative proteomics, we identified a previously unreported platelet zinc transporter, transmembrane protein 163 (TMEM163), which was significantly reduced in BLOC-1 (Dtnbp1sdy and Pldnpa)–, BLOC-2 (Hps6ru)–, or AP-3 (Ap3b1pe)–deficient mice and HPS patients (HPS2, HPS3, HPS5, HPS6, or HPS9). We observed similar platelet DG defects and higher intracellular zinc accumulation in platelets of mice deficient in either TMEM163 or dysbindin (a BLOC-1 subunit). In addition, we discovered that BLOC-1 was required for the trafficking of TMEM163 to perinuclear DG and late endosome marker–positive compartments (likely DG precursors) in MEG-01 cells. Our results suggest that TMEM163 is critical for DG biogenesis and that BLOC-1 is required for the trafficking of TMEM163 to putative DG precursors. These new findings suggest that loss of TMEM163 function results in disruption of intracellular zinc homeostasis and provide insights into the pathogenesis of HPS or platelet storage pool deficiency.

Planning for Birth for a Patient With Hermansky-Pudlak Syndrome

Hermansky-Pudlak Syndrome (HPS) is an autosomal recessive disorder that affects 1 in 500,000–1,000,000 people worldwide. There are three elements of the disorder, including oculocutaneous albinism, disorder of ceroid metabolism, and platelet storage pool deficiency. Due to the platelet storage pool deficiency, risk of bleeding during labor and birth are significantly increased. A 25-year-old gravida 2, para 0101 pregnant woman presented at the maternal-fetal medicine office for a consult related to her diagnosis of HPS. The patient had confirmed HPS Type 1 by genetic testing. She also has ocular albinism and a history of blood transfusions for severe menorrhagia and anemia. The patient’s previous pregnancy was complicated by preterm labor, which ended in a cesarean for breech presentation, requiring a T incision. The patient received regional anesthesia with the previous delivery without adverse outcome and did not experience a postpartum hemorrhage. Collaboration among anesthesia, obstetrics, and hematology resulted in a birth plan that consisted of repeating coagulation studies on the day of surgery to determine type of anesthesia and need for desmopressin (DDAVP) prophylaxis or platelet transfusion before delivery. On the scheduled day of birth, hematology was consulted; platelets, activated partial thromboplastin time (APTT), fibrinogen, collagen epinephrine (EPI), platelet function interpretation, and Von Willebrand factor activity were drawn. Although coagulations studies were normal, hematology recommended general anesthesia for the cesarean due to risk of epidural hematoma and lack of definitive information on the safety of regional anesthesia in HPS. The patient gave birth via cesarean at 36 weeks and did not require prophylactic treatment with DDAVP or platelets. Patients with HPS need thoughtful birth planning and consultation with anesthesia and hematology to obtain the best possible outcome based on the limited evidence available. The delivery mode; anesthesia type; predelivery treatment, including DDAVP or platelet transfusion; and postpartum pain management should be thoroughly reviewed, discussed, and documented in the patient’s medical record to limit adverse events. Understanding HPS and the risk factors associated with it can help nurses better assess and care for patients affected with this syndrome.

McCune Albright Syndrome: Gastrointestinal Polyps and Platelet Dysfunction over 12 Years

Background and Objective: Gastrointestinal (GI) polyps with unknown malignant potential and a platelet storage pool deficiency that increases the risk of severe intraoperative and other types of bleeding have been identified in McCune-Albright syndrome (MAS). The natural course of these disorders has not been well characterized. The aim of this study was to report the follow-up of GI polyps and platelet dysfunction (PD) in a cohort of 28 patients with MAS. Methods: Twenty-eight patients with MAS (15 females) were included. Endoscopic screening for GI polyps was undertaken in 14 subjects and 19 were tested for PD. Results: Six subjects (5 males) were diagnosed with GI polyps at a median age of 23 (range 15–43) years, and were monitored for a median period of 8 (range 4.5–11.5) years. At endoscopic follow-up, the 4 patients with hamartomatous polyps at first endoscopy had either normal findings (n = 2), or duodenal gastric metaplasia (n = 2). Two patients with caecal polyps were identified. Of 8 subjects with a platelet storage pool deficiency, 5 required transfusions during surgery, and subsequent platelet cover in 2 markedly reduced intraoperative blood loss. Conclusions: New polyps with uncertain malignant potential are diagnosed after long term follow-up in MAS. Platelet cover reduces the need for red blood cell transfusion during orthopaedic surgery and may be useful to reduce non-operative bleeding events. We recommend regular upper and lower endoscopy and screening for PD in all MAS patients.

1946 Recurrent Lower Gastrointestinal Bleeding Secondary to Hermansky-Pudlak Syndrome

INTRODUCTION: Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder with a prevalence of 1 in 1,800 in Puerto Rico. It is characterized by oculocutaneous albinism with nystagmus, variable skin hypopigmentation, loss of visual acuity, and platelet storage pool deficiency resulting in bleeding diathesis. Hematochezia in HPS primarily occurs due to inflammatory bowel disease (IBD) presenting in first to third decades. However, lower gastrointestinal bleeding can occur even without evidence of endoscopic or histological findings of colitis. CASE DESCRIPTION/METHODS: A 20-year-old Puerto Rican male with history of HPS presented with 3 to 5 episodes of bright red blood per rectum for the past one week. The patient has been experiencing intermittent hematochezia since the age of 7 years. Previous work-up with double balloon enteroscopy 4 years ago showed small bowel erosions. Colonoscopy performed during that time showed normal findings, however biopsy was not performed. Meckel’s scan did not show any evidence of Meckel’s diverticulum. He did not experience any bleeding for 2 years before this presentation. His vital signs on presentation were stable. Laboratory findings showed hemoglobin of 13 gm/dl, platelet count of 330,000/ul and white blood cell count of 8,900/µL. International normalized ratio (INR) was 1.1, prothrombin time (PT) 14.6 seconds and partial thromboplastin time (PTT) 32.3 seconds. The patient was started on intravenous fluids, pantoprazole infusion and was given one dose of aminocaproic acid. Following the dose of amino-caproic acid, the patient’s hematochezia resolved and he did not have any recurrent bleeding episodes. He was discharged after 3 days, and followed up one month later, when he did not report of recurrent bleeding episodes. DISCUSSION: Previous studies have shown that mucosal inflammation can occur in absence of endoscopic abnormalities in symptomatic patients. The likely cause of hematochezia in our patient was mucosal inflammation along with platelet dysfunction. Patients with mild intermittent episodes of hematochezia can be managed conservatively. For acute bleeding episodes, desmopressin acetate (DDAVP) or aminocaproic acid can be used and in case of severe bleeding, platelet transfusion should be performed.

Infantile-onset inflammatory bowel disease in a patient with Hermansky-Pudlak syndrome: a case report

BackgroundHermansky-Pudlak syndrome (HPS) is a rare, genetically heterogeneous disorder that manifests oculocutaneous albinism together with bleeding diatheses that reflect a platelet storage pool deficiency. Ten genetic subtypes of this autosomal recessive condition have been described to date. Some patients with Hermansky-Pudlak syndrome type 1, 4, or 6 develop Crohn’s-like inflammatory bowel disease at any age including early childhood, but most often in adolescence or young adulthood. Here we report infantile-onset of inflammatory bowel disease in a patient with Hermansky-Pudlak syndrome type 1 who responded to infliximab.Case presentationA Japanese boy, the second child of non-consanguineous healthy parents, was born with chalky white skin, silvery-white hair, and gray eyes, representing oculocutaneous albinism. He developed frequent diarrhea and fever accompanied by weight loss at 6 months, and was diagnosed with Crohn’s-like inflammatory bowel disease based on the endoscopic finding of longitudinal ulcerations in the colon and the histopathologic finding of nonspecific chronic inflammation without granulomas at the age of 11 months. Treatment with an elemental diet, salazosulfapyridine, and corticosteroids failed to improve clinical or laboratory abnormalities, and the diarrhea became bloody. At 13 months he began treatment with infliximab, which produced marked improvement followed by clinical remission. Endoscopy at 20 months demonstrated healing of the colonic mucosa. At 22 months he is in sustained clinical remission receiving only infliximab. Because albinism with inflammatory bowel disease suggested Hermansky-Pudlak syndrome, we performed genetic screening using next-generation sequencing in a targeted gene panel analysis for primary immunodeficiency disease and/or inflammatory bowel disease. The patient proved to have a compound heterozygous mutation of the HPS1 gene resulting in Hermansky-Pudlak syndrome type 1.ConclusionsWe consider this report to be the first account of type 1 Hermansky-Pudlak syndrome with infantile-onset of inflammatory bowel disease. Early administration of infliximab was effective. We recommend next-generation sequencing for patients with very early-onset inflammatory bowel disease suspected to be monogenic.

Idiopathic Purpura With Gray Platelets: an Acquired Form of Gray Platelet Syndrome.

An acquired, transient bleeding disorder that predominantly affects children in Southeast Asia has been reported for the last 4 decades. The condition has been named idiopathic purpura with gray platelets (IPGP) or acquired platelet dysfunction with eosinophilia. In a retrospective review from a private pediatric clinic over an 8-year period, 10 consecutive children were diagnosed as IPGP with a mean age of 8.4 (3.7 to 16.2) years. Eosinophilia (>0.5×10/L) was absent in 1, while gray platelets were consistently found in all cases with a mean proportion of 64.5% (40% to 80%). Platelet aggregation tests were performed in 9 patients with abnormal responses consistent with platelet storage pool defect. All children recovered completely and spontaneously from 1 to 4 months after diagnosis without specific therapy. In an otherwise well child who presents abruptly with easy bruising and a platelet count >100×10/L, IPGP can be readily recognized as an acquired form of gray platelet syndrome. Eosinophilia is common but not mandatory for diagnosis.

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.

P467Rare electrics and rare blood: properties of a left posteroseptal accessory pathway with decremental conduction and successful ablation in a patient with platelet storage pool disorder

P467Rare electrics and rare blood: properties of a left posteroseptal accessory pathway with decremental conduction and successful ablation in a patient with platelet storage pool disorder

Combined alpha-delta platelet storage pool deficiency is associated with mutations in GFI1B.

Combined alpha-delta platelet storage pool deficiency is characterized by the absence or reduction in the number of both alpha granules and dense bodies. This disorder can have variable severity as well as a variable inheritance pattern. We describe two patients from unrelated families with combined alpha-delta storage pool deficiency due to mutations in GFI1B, a zinc finger protein known to act as a transcriptional repressor of various genes. We demonstrate that this disease is associated with either a heterozygous mutation (de novo or familial) abrogating the binding of the zinc fingers with the promoter of its target genes, or by hypomorphic biallelic mutations in GFI1B leading to autosomal recessive inheritance.

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.

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.

Identification of a novel mutation in HPS6 in a patient with hemophilia B and oculocutaneous albinism.

Hemophilia B, an X-linked disease, manifests with recurrent soft tissue bleeding episodes. Hermansky-Pudlak syndrome, a rare autosomal recessive disorder, is characterized by oculocutaneous albinism and an increased tendency to bleed due to a platelet storage pool defect. We report a novel mutation in HPS6 in a Caucasian man with hemophilia B and oculocutaneous albinism. The patient was diagnosed with hemophilia B at age 4months due to recurrent soft tissue bleeding episodes, and he was also diagnosed with Hermansky-Pudlak syndrome at 32years of age due to unexplained oculocutaneous albinism. His factor IX level was markedly reduced at 13%; whole exome and Sanger sequencing showed the Durham mutation in F9 (NM_000133.3). The diagnosis of Hermansky-Pudlak syndrome subtype 6 was established by demonstrating absence of platelet delta granules on whole mount electron microscopy, an abnormal secondary wave in platelet aggregation studies, and a novel homozygous c.1114 C>T (p.Arg372*) mutation in HPS6 (NM_024747.5) on exome analysis and Sanger sequencing. Clinical phenotyping revealed no evidence of recurrent or unusual infections, interstitial lung disease or pulmonary fibrosis, or neurological disorders. The patient was treated with fresh frozen plasma, recombinant factor IX, and aminocaproic acid. Treatment with desmopressin was added to his regimen after he was diagnosed with Hermansky-Pudlak syndrome. Treatment of bleeding episodes results in effective hemostasis, and the patient has not required platelet or blood product transfusions. This report highlights the need to consider Hermansky-Pudlak syndrome as an etiology of oculocutaneous albinism even in patients with known hematologic disorders associated with bleeding. Identification of a novel mutation in HPS6 in an individual with hemophilia B shows that, although quite rare, patients may be diagnosed with two independent inherited bleeding disorders. No evidence of lung disease was found in this adult patient with Hermansky-Pudlak syndrome subtype 6.

Postural orthostatic tachycardia syndrome is associated with platelet storage pool deficiency.

Mechanisms have been postulated to explain postural orthostatic tachycardia syndrome (POTS), however, the etiology of this often debilitating disorder remains unknown. We conducted a retrospective case–control study of 181 POTS patients who exhibited/reported bleeding symptoms for a specific platelet (PL) dysfunction disorder, delta granule storage pool deficiency (δ-SPD).Patients were included only if results of blood tests for δ-SPD were available. Electron microscopy was utilized to diagnose δ-SPD. An ELISA assay was used to determine serotonin (5HT) concentration in PLs and medical record review was employed to collect patients’ clinical symptoms.The most common bleeding symptom was easy bruising (71%) but frequent nose bleeds, heavy menstrual bleeding, and a family history of bleeding were also commonly reported. Of the patients studied, 81% were diagnosed with δ-SPD. Our investigation of 5HT concentration extracted from PLs revealed significantly lower levels of 5HT in POTS patients when compared to that of control subjects. Our data suggest that patients with POTS have significant comorbidities including bleeding symptoms and/or family bleeding histories, and have diminished PL 5HT levels supporting the hypothesis that POTS is a low 5HT level disorder. While we describe a significant relationship with POTS and δ-SPD, this finding does not constitute an etiology for POTS.Our results establish an additional comorbidity frequently seen in POTS that could explain a number of disparate symptoms often affecting the severity of POTS.

Assessment and validation of a defined fluid restriction protocol in the use of subcutaneous desmopressin for children with inherited bleeding disorders.

Despite the availability of subcutaneous desmopressin (1-deamino-8-d-arginine vasopressin, SC-DDAVP) as a haemostatic agent for children with mild bleeding disorders, few publications specifically address the safety or efficacy of this mode of administration. Our aim was to assess whether a defined fluid restriction protocol was effective in preventing hyponatremia in children receiving perioperative SC-DDAVP, and to document adequate biological and clinical response in this setting. We retrospectively analysed a cohort of children with mild bleeding disorders prescribed SC-DDAVP over a 5-year period following institution of a 'two-thirds maintenance' fluid restriction protocol. Sixty-nine patients received SC-DDAVP following this protocol, including 15 with mild haemophilia A, 49 with von Willebrand disease (VWD) and five with platelet storage pool disorder. In patients who underwent formal preoperative assessment a complete or partial response was observed in 28/29 with type 1 VWD and 14/15 with mild haemophilia A. Perioperative SC-DDAVP provided excellent haemostasis in all patients, with no requirement for factor concentrate or blood products. Mild asymptomatic hyponatremia was detected in seven children who received multiple doses of DDAVP (lowest sodium 129 mmol L(-1) ); however, adherence to the prescribed fluid restriction protocol was questionable in six of these cases. Symptomatic hyponatremia was not observed. Subcutaneous desmopressin was well-tolerated, with no serious side-effects observed, and good biological responses in preoperative trials. A two-thirds maintenance fluid regimen was effective at preventing symptomatic hyponatremia in our cohort, and is now the standard protocol for fluid restriction post-DDAVP administration in our centre.

Mutations in AP3D1 associated with immunodeficiency and seizures define a new type of Hermansky-Pudlak syndrome

AbstractGenetic disorders affecting biogenesis and transport of lysosome-related organelles are heterogeneous diseases frequently associated with albinism. We studied a patient with albinism, neutropenia, immunodeficiency, neurodevelopmental delay, generalized seizures, and impaired hearing but with no mutation in genes so far associated with albinism and immunodeficiency. Whole exome sequencing identified a homozygous mutation in AP3D1 that leads to destabilization of the adaptor protein 3 (AP3) complex. AP3 complex formation and the degranulation defect in patient T cells were restored by retroviral reconstitution. A previously described hypopigmented mouse mutant with an Ap3d1 null mutation (mocha strain) shares the neurologic phenotype with our patient and shows a platelet storage pool deficiency characteristic of Hermansky-Pudlak syndrome (HPS) that was not studied in our patient because of a lack of bleeding. HPS2 caused by mutations in AP3B1A leads to a highly overlapping phenotype without the neurologic symptoms. The AP3 complex exists in a ubiquitous and a neuronal form. AP3D1 codes for the AP3δ subunit of the complex, which is essential for both forms. In contrast, the AP3β3A subunit, affected in HPS2 patients, is substituted by AP3β3B in the neuron-specific heterotetramer. AP3δ deficiency thus causes a severe neurologic disorder with immunodeficiency and albinism that we propose to classify as HPS10.