Further studies on the relationship between platelet buoyant density and platelet age

The relationship between platelet buoyant density and platelet age was investigated in eight human subjects submitted to an autologous chromium labeled platelet survival study. Platelets were isolated after isopycnic centrifugation using either discontinuous isoosmotic stractan gradients (five subjects), or various continuous and linear isoosmolar gradients (three subjects). A paradoxical radioactivity enrichment of the dense platelets and a premature loss of radioactivity in the light platelets were observed. These results are explained by a shift of the radioactivity distribution curve toward higher densities during the 3–4 days after platelet injection, while the standard deviation of the distribution was conserved throughout the platelet life span. These results suggest that young platelets are heterogeneous and slightly less dense than the total platelet population.     

Dawning of the age of genomics for platelet granule disorders: improving insight,diagnosis and management

Inherited disorders of platelet granules are clinically heterogeneous and their prevalence is underestimated because most patients do not undergo a complete diagnostic work‐up. The lack of a genetic diagnosis limits the ability to tailor management, screen family members, aid with family planning, predict clinical progression and detect serious consequences, such as myelofibrosis, lung fibrosis and malignancy, in a timely manner. This is set to change with the introduction of high throughput sequencing (HTS ) as a routine clinical diagnostic test. HTS diagnostic tests are now available, affordable and allow parallel screening of DNA samples for variants in all of the 80 known bleeding, thrombotic and platelet genes. Increased genetic diagnosis and curation of variants is, in turn, improving our understanding of the pathobiology and clinical course of inherited platelet disorders. Our understanding of the genetic causes of platelet granule disorders and the regulation of granule biogenesis is a work in progress and has been significantly enhanced by recent genomic discoveries from high‐powered genome‐wide association studies and genome sequencing projects. In the era of whole genome and epigenome sequencing, new strategies are required to integrate multiple sources of big data in the search for elusive, novel genes underlying granule disorders.     

Testing agonist-induced platelet aggregation by the Impact-R [Cone and plate(let) analyzer (CPA)]

The Impact-R [Cone and plate(let) analyzer (CPA)] is useful to assess platelet adhesion in different diseases and to monitor antiplatelet therapy. The purpose of the present study was to adapt this system to test agonist-induced platelet aggregation. Blood samples were tested by light transmission platelet aggregometry (LTA), Impact-R regular test and Impact-R agonist-response test. In the latter, samples were pre-incubated for 1 min with an agonist leading to platelet activation, micro-aggregates formation and reduced adhesion. Impact-R regular test of ten healthy volunteers demonstrated platelet adhesion (surface coverage, SC) of 11.2 ± 2.6% while LTA induced by ADP, ristocetin, epinephrine, collagen and arachidonic acid (AA) yielded maximal aggregation (81% to 93%). In the Impact-R agonist-response test, SC was reduced to 2.2 ± 1.0%, 1.2 ± 0.9%, 2.3 ± 1.0%, 2.2 ± 0.8% and 2.4 ± 0.4%, respectively. Prostaglandin E₁ treatment weakened SC reduction in response to ADP and epinephrine (SC of 8.8 ± 1.8% and 9.5 ± 2.0%, respectively). Inhibition of P2Y₁₂ receptor with 2MeSAMP resulted in a dose-dependent decrease in maximal aggregation in the ADP-induced test, which inversely correlated to SC in the Impact-R ADP-response test. The Impact-R agonist-response tests detected aggregation defects in patients with storage pool disease, severe von Willebrand disease and epinephrine response deficiency, and may be useful to assess the effect of different agonists on platelet aggregation.     

Intravascular and total body platelet equilibrium in healthy volunteers and in thrombocytopenic patients transfused with single donor platelets

Instrument platelet counts used in corrected count increment (CCI) and percent platelet recovery (PPR) formulas presume the transfused platelets are in equilibrium during the first hour after platelet transfusion. The timing of the pre-transfusion count affects CCI results, and we postulate that timing of CCI post transfusion affects CCI results. Platelet equilibrium using indium-111 platelet transfusions has not been reported. Platelet redistribution was studied in 16 healthy volunteers and 12 thrombocytopenic patients by generally infusing less than 72-hr stored single-donor platelets along with an aliquot of indium-111-labeled platelets by intravenous push. Counts were measured at 10, 15, 20, 60, and 120 min, and 24, 48, 72 hr along with continuous body scanning for 2 hr in healthy volunteers, and static organ scanning in patients and volunteers. Results indicated transfused platelets do not reach intravascular equilibrium for 60 min post-infusion and that the 10-min count cannot detect platelet refractoriness. However, total body equilibrium varies considerably between normal volunteers and thrombocytopenic patients. It is recommended to continue with the 1-hr post transfusion count. Am. J. Hematol. 58:165–176, 1998. © 1998 Wiley-Liss, Inc.     

Increase in density and accumulation of serotonin by human aging platelets

⁵¹Cr-labeled autologous platelets were infused into splenectomized subjects and the specific radioactivities of high-density (HD) and low-density (LD) platelet subpopulations were determined sequentially in postinfusion samples. A rapid decrease in the specific radioactivity of LD cohorts (T 1/2 = 2.5 days) was observed, but the specific radioactivity of HD platelets remained constant or increased slightly during the first 4 days and then gradually declined for the next 5 days. No experimental artifacts during the platelet-labeling steps that could account for these results were demonstrated. These findings confirm previous observations in eusplenic individuals and support the hypothesis that human LD platelets are, on the average, younger than HD platelets. LD platelets contain 33.8 ± 13.5 ng serotonin (5HT)/10⁸ platelets and HD platelets 76.8 ± 9.5 ng 5HT/10⁸ platelets (P < 0.001). Sequential measurements of 5HT in PRP platelets were performed during the recovery phase of thrombocytopenia following splenectomy in patients with idiopathic thrombocytopenic purpura (ITP), a condition associated with aging of platelets in circulation. Presplenectomy platelet 5HT was 17.7 ng/10⁸ platelets and on days 1, 6, and 12 after surgery it increased to 18.1, 37.8, and 61.0 ng/10⁸ platelets (n = 7). When three healthy volunteers were given aspirin (500 mg/day) for up to 15 days, no significant change in the 5HT content of circulating platelets was observed. If aspirin blocks, at least partially, the secretory process in vivo without interfering the 5HT uptake by the platelets, this finding stands against the possibility that a net depletion of 5HT occurs during the life-span of normal human platelets. The observation that human HD platelets, enriched with older cells, contain more 5HT than LD platelets taken together with the parallel increase in platelet 5HT and age during the recovery from thrombocytopenia in ITP patients and the lack of effect of aspirin on platelet 5HT content, provides initial evidence that human platelets accumulate 5HT during their life-span in circulation.     

Autologous platelet-rich plasma isolated using the Haemonetics Cell Saver 5 and Haemonetics MCS+ for the preparation of platelet gel.

BACKGROUND AND OBJECTIVES: We compared three methods of isolating platelet-rich plasma (PRP) using the Haemonetics Cell Saver 5 and one method of isolating PRP by plateletpheresis using the Haemonetics MCS+. PRP contains both platelets and fibrinogen, which are used in the preparation of haemostatic agents. MATERIALS AND METHODS: When the Haemonetics Cell Saver 5 was used, 500 ml of blood from each of 30 normal volunteer donors was collected into 70 ml of citrate-phosphate-dextrose (CPD) anticoagulant. In a further 14 normal volunteers, the Haemonetics MCS+ was used to isolate PRP by plateletpheresis using an acid citrate dextrose (ACD) to blood ratio of 1 : 9. In a separate study, CPD-anticoagulated whole blood from another 30 volunteers was used for measurement of fibrinogen levels in the plasma and cryoprecipitate. RESULTS: A larger volume of PRP can be collected using the Haemonetics Cell Saver 5 than by using the Haemonetics MCS+. The platelet concentration and the total number of platelets were higher in the PRP isolated using the Haemonetics MCS+ than in the PRP isolated by the three methods used with the Haemonetics Cell Saver 5, with differences in platelet concentration and PRP volume among the four methods. The mean fibrinogen level in the plasma was 253 mg % +/- 47 (SD) and in the cryoprecipitate was 1085 mg % +/- 304 (SD). CONCLUSIONS: The most appropriate method of PRP isolation for preparation of platelet gel is dependent upon the specific surgical procedure to be undertaken and the patient's needs.     

Report on the Tenth International Platelet Genotyping and Serology Workshop on behalf of the International Society of Blood Transfusion

BACKGROUND AND OBJECTIVES: The aims of the 10th International Platelet Serology and Genotyping Workshop were to evaluate the proficiency of platelet immunology determinations. MATERIALS AND METHODS: There were 40 participants from 25 countries of four continents. Thirty-eight institutions reported results for genotyping, and 38 institutions reported their serological results. For genotyping, EDTA-anticoagulated whole-blood samples were provided (to allow the inclusion of DNA-separation methodology in the analysis) as well as separated DNA of a and b alleles for human platelet antigen (HPA)-1 to -6. For serological evaluations, sera contained allo- and autoantibodies, and for sensitivity testing a standard freeze-dried sample of HPA-5 antibody. RESULTS: All participants reported HPA-1, -2, -3 and -5 genotyping results; HPA-4 was determined in 29 laboratories and HPA-6 in 21. Results from 16 laboratories were concordant with the majority vote for all allotypes, eight institutions reported one deviation, five laboratories two, and nine laboratories three or more deviations. Twelve institutions had no deviation from the majority vote for HPA antibodies, nine had one, three had two, and 14 had three or more deviations. Most laboratories reported a reactivity of the standard anti-HPA-5b sample with HPA-5b platelets at a dilution of 1:4-1:8. Four laboratories detected anti-Gova in one sample. Seventeen laboratories reported no deviation from the majority vote for pan-reactive platelet antibodies, 12 had one deviation, two had two, and seven had three or more deviations. In addition, seven participants reported antibodies against glycoprotein IV (GPIV), three against glycoprotein V (GPV) and three against CD 109. These results were discussed at a meeting organized jointly with the International Society of Blood Transfusion (ISBT) 2000 Congress. CONCLUSION: The results for pan-reactive antibodies were heterogeneous with most discrepancies from the majority vote. The provision of sufficient samples for many participants is difficult. Based on the results and discussion it is clear that frequent workshops are needed in the future. Therefore, workshops shall be organized regionally, and each region shall participate with one institution in international workshops. The latter are needed to assure international exchange of experience and quality.     

The effect of human platelet alloantigen polymorphisms on the in vitro responsiveness to adrenaline and collagen

A number of clinical studies have suggested that carriage of the low frequency allele (b) of the human platelet antigen 1 (HPA-1) system is a risk factor for coronary thrombosis. We have examined the effect of a series of HPA biallelic polymorphisms (systems -1, -2, -3 and -5) on the in vitro platelet aggregation in response to adrenaline and collagen in 30 healthy volunteers. There was a significantly higher prevalence (10 out of 18) of carriers of the HPA-1b polymorphism among subjects showing a > 50% aggregation response to adrenaline ('responders') than the prevalence (1/12) in 'non-responders' (P < 0.05). Platelets heterozygous for the HPA-1b polymorphism showed a significantly higher rate (slope) and greater extent (%) of adrenaline-induced aggregation than platelets not carrying the HPA-1b allele (P < 0.05). A greater extent of collagen-induced aggregation was also demonstrated in HPA-1ab platelets (P < 0.05). Inhibition of adrenaline-induced aggregation following incubation with aspirin was greater (P < 0.01) in HPA-1ab than in HPA-1aa platelets. Collagen-induced aggregation was slower in carriers of the HPA-5b allele than in HPA-5aa subjects (P < 0.05). Polymorphisms of the HPA-2 and HPA-3 systems were not associated with different aggregation responses to either adrenaline or collagen. These results support the clinical observation that polymorphism HPA-1b may predispose to increased platelet thrombogenicity and suggest that the presence of polymorphism HPA-5b might render the platelet less reactive to collagen.     

Report on the Eleventh International Society of Blood Transfusion Platelet Genotyping and Serology Workshop

BACKGROUND AND OBJECTIVES: The aims of the Eleventh International Workshop were to evaluate proficiency in platelet genotyping and antibody detection, to equip laboratories to perform Gov antigen system genotyping and antibody detection, and to evaluate the laboratory and clinical approach to cases of neonatal alloimmune thrombocytopenia (NAIT). MATERIALS AND METHODS: There were 34 participating laboratories from 22 countries on five continents. Participating laboratories were provided with 10 DNA samples, 15 unknown sera, and three monoclonal antibodies for titration, as well as primer pairs and a protocol for Gov genotyping and Gov antibody screening. They were also provided with a questionnaire on investigation and clinical management of patients with NAIT. RESULTS: Thirty-three participants reported human platelet antigen (HPA)-1, -2, -3 and -5 genotyping results, 25 reported HPA-4 typing results, 17 reported HPA-6 typing results and 24 reported Gov typing results. For HPA-1-6 genotyping, 23 laboratories were concordant with a majority vote for all allotypes tested, five laboratories reported one deviation, three laboratories reported two deviations and one laboratory reported three deviations. For Gov genotyping, six deviations occurred in three of the 24 laboratories reporting results. Antibody detection was 90% concordant for anti-HPA-1a, anti-HPA-5a and anti-HPA-5b detection. Anti-HPA-2b and anti-Gova were detected by 20 and 14 out of 33 laboratories, respectively. Approaches to the clinical management of NAIT vary widely, especially for mothers with a history of a previous infant with mild NAIT. CONCLUSIONS: The overall error rate for HPA-1-6 genotyping decreased from 2.7% in the tenth workshop to 0.8% in the eleventh workshop. The majority of laboratories were able to perform Gov genotyping, although the error rate was 7.5%. Detection of common clinically significant antibodies was good, although detection of the much rarer HPA-2b was problematic. There was considerable progress in the detection of anti-Gova. The lack of consensus over treatment of NAIT demonstrates uncertainty over optimal management of these patients.     

Platelet aggregability and platelet volume in the postoperative course: problems in the platelet aggregation test derived from the measurement of platelet volume

Platelet count, aggregability and volume in the postoperative course of 20 patients were examined. Platelet count was decreased on the 1st postoperative d, and increased on the 7th and 14th d compared with the preoperative value. The maximal aggregation rate of platelets induced by ADP was decreased on the 3rd postoperative d, and then recovered to the preoperative level. In contrast, platelet volume was only slightly increased on the 3rd postoperative d. In this study, there was no correlation between platelet aggregability and platelet volume in PRP. We have proposed one parameter, 'platelet concentration ratio' (platelet concentration in PRP/platelet concentration in whole blood). In the postoperative course, this concentration ratio changed depending on platelet volume, and possibly on other conditions of blood such as hematocrit, viscosity and specific gravity. The concentration ratio influenced the subpopulations of platelets in PRP. Platelet aggregation tests may be performed using PRP in which platelet subpopulations differ from those in whole blood, especially in the postoperative state.     

Organ distribution and fate of human platelets: studies of asplenic and splenomegalic patients

Little is known about the organ distribution and fate of human platelets. We investigated the kinetics, organ distribution, and fate of autologous 111In-oxine-labeled platelets in 12 normal volunteers, four asplenic subjects, and four patients with splenomegaly. The initial recovery of infused 111In-platelets from the circulation was 97.8 +/- 9.8% (means +/- SD) for asplenic subjects and 26.3 +/- 5.9% for splenomegalic patients as compared to 59.2 +/- 9.3% for normal controls. The mean platelet survival times as derived from the multiple-hit model were 9.2 +/- 1.0 days for asplenics and 6.2 +/- 0.6 days for splenomegalic subjects (8.4 +/- 0.8 days for normals). At 30 min postinfusion, 79.4 +/- 19.2% of the infused 111In-platelets pooled in the spleen of splenomegalic subjects and 42.7 +/- 12.2% in normal controls. There was 7.1 +/- 2.0, 12.6 +/- 3.7, and 29.3 +/- 8.4% pooling in the liver of splenomegalic, normal, and asplenic subjects, respectively. At 10 days postinfusion, 37 and 24% of the 111In-platelets were sequestered in the spleen and liver of normal control subjects, respectively. Similar figures for splenomegalic subjects were 71 and 14%, respectively. In asplenic subjects, 89% was sequestered in the liver. We conclude that spleen and liver are the primary sites of platelet destruction, accounting for 61% of infused 111In-platelets in normal volunteers and 85% in splenomegalics, while the liver is the primary site of platelet destruction, accounting for 89% in asplenic subjects.     

Current status and future prospects for platelet function testing in the diagnosis of inherited bleeding disorders

Platelets play a crucial role in haemostasis by preventing bleeding at the site of vascular injury. Several defects in platelet morphology and function have been identified and described over the years. Although a range of methodologies is available to assess platelet function, a significant proportion of subjects with bleeding symptoms and normal coagulation parameters still appear to have normal results on platelet function testing. This might suggest that the reason for bleeding is multifactorial and is due to a combination of several minor defects in platelet function and/or other parts of the haemostatic system or might indicate that the currently available platelet function tests do not provide optimal diagnostic power. This review will summarize the established platelet function tests used for diagnosing inherited platelet abnormalities in adults and children, and discuss the newly developed methodologies as well as unmet challenges and potential areas for further improvement in this field.     

A review of inherited platelet disorders with guidelines for their management on behalf of the UKHCDO

The inherited platelet disorders are an uncommon cause of symptomatic bleeding. They may be difficult to diagnose (and are likely to be under-diagnosed) and pose problems in management. This review discusses the inherited platelet disorders summarising the current state of the art with respect to investigation and diagnosis and suggests how to manage bleeding manifestations with particular attention to surgical interventions and the management of pregnancy.     

血小板相关指标与心力衰竭关系的研究进展

血小板相关指标，包括血小板计数、平均血小板体积、血小板分布宽度，被多项研究证实与心力衰竭具有一定相关性，甚至部分指标与预后相关。部分治疗心衰的药物对血小板相关指标也有影响。本人将对上述指标与心力衰竭的相关研究做一综述。     

浓缩血小板中白细胞表型和功能变化的研究

目的检测浓缩血小板和冰冻浓缩血小板中白细胞的表型和功能变化。方法用N ageotte法计数新鲜浓缩血小板和冰冻浓缩血小板中的白细胞数量,台酚蓝拒染法计数白细胞存活率,流式细胞仪对白细胞表面标记进行测定,用双向混合淋巴细胞培养法对同种免疫功能进行分析。结果新鲜浓缩血小板中的白细胞数约为2.4×106/U,经3个月的冻存有85%的白细胞存活,浓缩血小板中白细胞表型为CD3+CD8+、CD3+CD4+、CD3+CD4-和CD3+CD8-等,并存在较强的同种免疫活性。结论新鲜浓缩血小板和冰冻浓缩血小板中的白细胞均有免疫原性和免疫反应性,此为输注过程中产生不良反应的主要原因;为预防输注浓缩血小板引起非溶血性输血发热反应、血小板输注无效、输血相关的移植物抗宿主病(TA-GVHD)等,建议应用滤除白细胞输血器(PL型滤器)。     

Influence of the Pure Synthetic PAF (Platelet Aggrregating Factor) on Clot Retraction and Platelet Aggregation

Pure synthetic platelet aggregating factor (PAF) (1-O-Hexadecyl-2-acetyl-sn-glycero-3-phosphorylcholine) induces a dose-dependent platelet aggregation in platelet-rich plasma (PRP) and in gel-filtered platelets. Irreversible platelet aggregation was observed at final concentrations of PAF higher than 2 times 10^-7 mol/l, while reversible or two-wave aggregation was obtained with lower final concentrations. The second wave was inhibited by acetylsalicylic acid, indomethacin, dipyridamole, EDTA, EGTA, theophylline, caffeine, PGE₁ and verapamil. PAF does not induce reptilase clot retraction (RCR); however, it does not inhibit RCR induced by ADP or thrombin. Since all substances known to activate platelets also induce RCR, the lack of this activity by PAF would support the existence of a third pathway in platelets.