A flow cytometric assay for the study of dense granule storage and release in human platelets

The clinical manifestations of platelet dense (δ) granule defects are easy bruising, as well as epistaxis and bleeding after delivery, tooth extractions and surgical procedures. The observed symptoms may be explained either by a decreased number of granules or by a defect in the uptake/release of granule contents. We have developed a method to study platelet dense granule storage and release. The uptake of the fluorescent marker, mepacrine, into the platelet dense granule was measured using flow cytometry. The platelet population was identified by the size and binding of a phycoerythrin-conjugated antibody against GPIb. Cells within the discrimination frame were analysed for green (mepacrine) fluorescence. Both resting platelets and platelets previously stimulated with collagen and the thrombin receptor agonist peptide SFLLRN was analysed for mepacrine uptake. By subtracting the value for mepacrine uptake after stimulation from the value for uptake without stimulation for each individual, the platelet dense granule release capacity could be estimated. Whole blood samples from 22 healthy individuals were analysed. Mepacrine incubation without previous stimulation gave mean fluorescence intensity (MFI) values of 83±6 (mean ± 1 SD, range 69–91). The difference in MFI between resting and stimulated platelets was 28±7 (range 17–40). Six members of a family, of whom one had a known δ-storage pool disease, were analysed. The two members (mother and son) who had prolonged bleeding times also had MFI values disparate from the normal population in this analysis. The values of one daughter with mild bleeding problems but a normal bleeding time were in the lower part of the reference interval.     

A flow cytometric assay using mepacrine for study of uptake and release of platelet dense granule contents

Summary. Diagnosis of platelet dense granule storage pool disease and release defects at present requires a combination of studies including lumiaggregometry, conventional platelet aggregation, radioactive serotonin uptake and release, and electron microscopy. Flow cytometric methods have been developed to study platelet activation, aggregation, and α-granule protein release. Here, we have investigated the use of flow cytometry for analysis of platelet dense granule content uptake and release using mepacrine as a fluorescent marker. Mepacrine (quinacrine) is rapidly taken up and localized in dense granules of platelets. For the assay, as little as 20 μl of blood from a fingerstick collected without anticoagulant or venous blood collected in 3.8% sodium citrate were diluted 1:40 with 2 ml Hanks balanced salt solution (BSS). 300 μl of this cell suspension were incubated with mepacrinc alone, or simultaneously with a mouse monoclonal antibody to human platelet glycoprotein IIb (Tab), used as a platelet-specific marker. The bound monoclonal antibody was then indirectly labelled with the fluorochrome, RED670. 100 μl of the sample were further diluted with Hanks BSS for one- or two-colour flow cytometric analysis. To verify that mepacrine uptake was related to platelet dense granule content, platelets of beige mice, a strain with dense granule deficiency, were examined. Their mepacrine uptake was substantially decreased compared to that of normal mice. Decreased mepacrine uptake also was demonstrated in platelets of a patient with Hermansky-Pudlak syndrome in which a deficiency of platelet dense granules is characteristic. In both human and mouse platelets, mepacrine uptake was proportional to platelet size. Thrombin induced mepacrine release in a dose-dependent manner from 0.003 to 0.4 U/ml. Therefore both platelet uptake and release of mepacrine can be readily detected by flow cytometry. Flow cytometry provides an attractive alternative to aggregation and radioactive serotonin as methods to study defects in platelet dense granule function.     

Thrombin and ionophore A23187-induced dense granule secretion in storage pool deficient platelets: evidence for impaired nucleotide storage as the primary dense granule defect

The secretion of the dense granule constituents ATP, ADP, calcium, pyrophosphate (PPi), and orthophosphate (Pi), and the release of magnesium induced by thrombin and the divalent cation ionophore A23187 have been quantitated directly in gel-filtered platelets from patients with storage pool deficiency (SPD). Both the contents and the maximal amounts of the dense granule constituents secretable by thrombin were decreased in all the patients studied, while the nonsecretable, retained amounts of these substances were identical in SPD and normal platelets. In response to both thrombin and A23187, the amounts of secretable ATP and ADP were strongly correlated in the platelets of individual patients; in contrast, secretable calcium showed no correlation with the nucleotides, and significant amounts of calcium were secreted in the total absence of nucleotide secretion in the platelets of several patients. The contents of magnesium were normal in all patients, and approximately 12% of platelet magnesium was liberated by thrombin in both SPD and normal platelets. A23187 induced the release of up to 70% of the magnesium content of normal platelets, but released significantly less (46%) magnesium from SPD platelets. Platelet aggregation induced by A23187 in platelet-rich plasma was also markedly decreased in SPD platelets. The correlations among secretable dense granule constituents suggest the presence in SPD platelets of abnormal dense granule structures that sequester calcium and other constituents but little or no adenine nucleotides, and are thus consistent with a hypothesis that impaired nucleotide transport and/or storage may be the primary dense granule defect in this disorder. In addition, these results demonstrate that certain responses to A23187 are impaired in SPD platelets.     

Quantification of a novel dense granule protein (granulophysin) in platelets of patients with dense granule storage pool deficiency.

An antigen-capture sandwich enzyme-linked immunosorbent assay (ELISA) was developed for a novel protein granulophysin, a constituent of the platelet dense granule (DG) membrane and used to characterize patients with dense granule storage pool deficiency (delta-SPD). The assay uses two monoclonal antibodies against the protein, one of which is conjugated to peroxidase. Purified DGs, an enriched source of the protein, were used for the standard curve. Granulophysin levels were only low in forms of delta-SPD associated with albinism. Granulophysin levels in platelet homogenates of 30 patients with the Hermansky-Pudlak syndrome form of delta-SPD were 1/4 to 1/5 of levels in controls or obligate heterozygotes. Two patients with the Chediak-Higashi form of delta-SPD syndrome also had markedly reduced levels of granulophysin. Patients with other forms of delta-SPD had normal levels of granulophysin. Two sisters with delta-SPD in one family had normal granulophysin present in empty dense granule membrane vesicles. Three members of another family with delta-SPD had low DG counts but normal granulophysin levels, indicating that in this group the level of granulophysin was maintained despite the reduction in granule formation. Thus, granulophysin quantitation facilitates characterization of delta-SPD patients and may provide clues to the nature of defective granules in delta-SPD subtypes.     

A sensitive and specific functional flow cytometric assay for the diagnosis of heparin-induced thrombocytopenia

A functional flow cytometric assay (FCA) for the immediate diagnosis of heparin-induced thrombocytopenia (HIT), with simultaneous compatibility testing for alternative anticoagulant therapies, has been developed to provide rapid and reliable results which effectively support patient management. The assay provides results within 1–2 h, uses readily available non-radioactive reagents, and employs standard equipment. Using the highly sensitive annexin V protein probe, the method detects activated platelets induced by heparin immune-complexes, with 300-fold increased binding to activated platelets. Twenty-five samples from patients clinically-suspected of having HIT (131 tests) and 10 normal control (NC) samples (36 tests) were simultaneously tested with unfractionated heparin (UH) and low-molecular-weight heparin (LMWH), and by the radioactive serotonin-release assay (SRA) (62 and 16 tests respectively). The FCA highly correlated with the SRA, showing 100% specificity and 95% sensitivity. Moreover, the FCA exhibited higher resolution between positive and negative samples (an average value of 8.6-fold the NC versus 4.0-fold the NC by SRA). The LMWH showed concordant results with UH ( r =0.95). We conclude that the functional FCA for HIT is practical, specific and sensitive, thereby permitting the rapid diagnosis of HIT and the suitability of alternative therapies.     

A sensitive flow cytometric assay for circulating platelet–leucocyte aggregates

A whole blood flow cytometric method for studies of platelet–leucocyte aggregates (PLAs) in vivo , involving neither fixation nor centrifugation, is described. With this method, PLAs in the total leucocyte population (PLA/L) were 15.3 ± 8.5% in 36 healthy volunteers. Blocking antibodies had little effect on PLAs in the absence of in vitro stimulation, suggesting that the aggregates were preformed in vivo . Fixation with formaldehyde or paraformaldehyde increased PLA/L significantly. Similarly, prefixation of the blood or red blood cell lysis, with repeated washing and centrifugation, caused artefactual 3–5-fold increases in PLAs ( P  < 0.05). ADP, thrombin, platelet activating factor (PAF) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) each increased PLA formation in unfixed whole blood dose-dependently; additive effects were found when they were combined. Experiments with blocking MAbs suggested that different ligand–receptor systems mediate PLA formation by different agonists. PLA formation by the platelet agonist ADP was inhibited by P-selectin blockade, but enhanced by GPIIb/IIIa blockade (which inhibits platelet–platelet interactions). PLA formation by the leucocyte agonist fMLP was inhibited by GPIIb/IIIa blockade, suggesting linking via fibrinogen. Platelet–leucocyte aggregate analysis by this whole blood method appears to reflect in vivo conditions, and enables investigations of the mechanisms involved in their formation.     

Activation during preparation of therapeutic platelets affects deterioration during storage: a comparative flow cytometric study of different production methods

Three different separation methods, all using centrifugation, are routinely used to prepare therapeutic platelet concentrates from human donor blood. Platelet concentrates derived from platelet-rich plasma (PRP-PC), buffy coat (BC-PC) and apheresis (AP-PC) were investigated at the end of production, and over an 8 d storage period. Change in platelet surface markers were measured by flow cytometry, using fluorescein-conjugated antibodies to fibrinogen, P-selectin (CD62P), GPIIb–IIIa (CD41), GPIbα (CD42b) and GPV (CD42d), and fluorescein-conjugated Annexin V was used to measure expression of anionic phospholipid.
All concentrates showed some changes during preparation but PRP-PC underwent the greatest changes with significantly higher levels of P-selectin ( P  < 0.001) and bound Annexin V ( P  = 0.001) than AP-PC or BC-PC, and lower levels of GPIbα ( P  = 0.002) and GPV ( P  < 0.001). These changes were attributable to component separation rather than venesection. These markers all continued to change on storage with a strong positive correlation between the changes seen during production and those after 5 d storage. PRP-PC continued to show the greatest changes whereas BC-PC showed the least. Fibrinogen was bound to 40–50% of platelets in all preparations and this did not alter significantly on storage whereas total expression of GPIIb–IIIa remained unchanged throughout.
There was no evidence that the platelet surface changes were thrombin-mediated and leucocyte depletion of BP-PC by filtration had no effect on the changes. It is proposed that the deterioration of platelet concentrates during storage may be related to activation occurring during preparation. 'Whole blood' flow cytometry using a panel of fluorescein-labelled reagents provides an informative method for evaluating platelet concentrates.     

Electron dense chains and clusters in human platelets

Human platelets contain dense organelles that serve as storage sites for adenine nucleotides and serotonin. They also contain calcium and phosphate responsible for the opacity of dense bodies in the electron microscope. The present study has shown that there are also bead-like structures and clusters of beads that are electron opaque when viewed by the whole mount technique. Their origin and function are not known. However, their presence might interfere with the accuracy of imaging techniques currently being used to measure the number and volume of dense bodies in patients with platelet storage pool deficiency.     

Electron dense chains and clusters in human platelets

Human platelets contain dense organelles that serve as storage sites for adenine nucleotides and serotonin. They also contain calcium and phosphate responsible for the opacity of dense bodies in the electron microscope. The present study has shown that there are also bead-like structures and clusters of beads that are electron opaque when viewed by the whole mount technique. Their origin and function are not known. However, their presence might interfere with the accuracy of imaging techniques currently being used to measure the number and volume of dense bodies in patients with platelet storage pool deficiency.     

A MitoTracker Green-based flow cytometric assay for natural killer cell activity: variability, the influence of platelets and a comparison of analytical approaches

OBJECTIVE: A number of flow cytometric assays for natural killer (NK) cell cytotoxicity have been described, however, the relative merits of analytical approaches and the influence of platelets on measured responses have not been systematically evaluated. Information on the time-dependent variability in measured responses is also limited. MATERIALS AND METHODS: Human peripheral blood mononuclear cells were obtained using Nycoprep 1.077, or Nycoprep 1.077 followed by Nycoprep 1.068 (to remove platelets), and incubated for 3 hours with MitoTracker Green (MTG)-labeled K562 cells. Cells were stained with propidium iodide (PI) and the proportions of viable and nonviable target cells (MTG(+)PI(-), MTG(+)PI(+)) were determined by flow cytometry using quadrant and polygonal region analysis. RESULTS: Platelets inhibited NK cell cytotoxicity and the response was underestimated when the nonviable target cell population was not entirely enclosed within the nonviable target cell (upper right) flow cytometric quadrant. The variability in measured NK cell cytotoxic responses in samples obtained from five individuals on three occasions over a 3-week period was 28%, 24%, 26%, and 37%, and 19%, 23%, 27%, and 32% for the quadrant and region analyses (mean coefficient of variation at effector-to-target cell ratios of 100:1, 50:1, 25:1, and 12.5:1, respectively), and 24% and 20% when data were calculated as the area under the cytotoxic curve (AUCC). CONCLUSION: Polygonal regions and the calculation of data as the AUCC appear to be the best approach. This study will be of value to investigators that are wishing to incorporate an NK cell cytotoxicity assay into their portfolio of experimental techniques.     

Rapid flow cytometric quantitation of reticulated platelets in whole blood

Young or reticulated platelets contain some residual mRNA, which is rapidly degraded after platelet release into the circulation. In order to minimize platelet activation and possible loss of large platelets during sample handling a whole blood method has been developed utilising the RNA fluorochrome thiazole orange (TO) in combination with an antibody to anti-glycoprotein Ib (GpIb) directly conjugated to phycoerythrin (PE), to specifically stain reticulated platelets via flow cytometric analysis. In this study whole blood analysis of platelet mRNA was undertaken in healthy normal subjects and a variety of patients with haematological abnormalities. The percentage of Gp Ib positive platelets containing mRNA in normals (n = 22) was 11.61% with a two SD range of 3.19-20.01%. The percentage of reticulated platelets was significantly increased (mean mRNA content ± one SD) in sickle cell disorders (n = 22) 38.12% ± 18.42 (P < 0.001); thalassaemia (major, intermedia and trait) (n = 24) 29.76 ± 19.15 (P < 0.001); ITP (N = 20) 23.53% ± 13.04 (P < 0.02) and essentialthrombocythemia (N = 32) 37.12 ± 19.84 (P < 0.001). Platelets from patients with reactive thrombocytosis (N = 15) were only 12.23% positive (±6.95) and not significantly different from the normal range (P = 0.95). This method offers a rapid and simple procedure for assessment of reticulated platelets in whole blood and suggests that there may be an increased platelet turnover in certain haemoglobinopathies.     

A rapid flow cytometric technique for the detection of platelet-monocyte complexes, activated platelets and platelet-derived microparticles

Platelet activation occurs in a variety of clinical situations in which it directly contributes to the pathology. This study reports a simple flow cytometric assay for platelet activation which measures platelet-derived microparticles, activated platelets and platelet-monocyte complexes. Pre- and post analytical conditions were investigated and optimized and a normal range established on 20 healthy controls. Twenty patients pre- and post percutaneous coronary intervention (PCI) were tested with the technique. Soluble activation markers sCD40 ligand and sP-selectin and plasma phospholipid levels were measured in both groups. There was a significant increase in activated platelets and platelet-monocyte complexes between normal and pre-PCI (P = 0.005 and 0.0275, respectively) suggesting an activated state. There was a significant fall in activated platelets post-PCI (P = 0.0027) which was mirrored by a fall in soluble CD40 ligand, soluble P-selectin and plasma phospholipid levels (P = 0.0066, <0.0001 and 0.0032, respectively) consistent with antiplatelet therapy administered during the process. This is a reliable and rapid method for the assessment of ex vivo platelet activation which may be an aid in diagnosis and help guide therapy for patients with thrombotic disease.     

The effect of oscillatory flow on the release reaction and aggregation of human platelets

The effect of variable shear rate on the thrombin-induced release of [¹⁴C]serotonin from human platelets was investigated by subjecting washed suspensions containing 5 × 10⁵ cells/μl to laminar oscillatory flow at 37°C in 1-meter lengths of 1.57-mm polyethylene tubing. The suspensions were prepared at 37°C in Tyrodes-albumin containing apyrase to avoid desensitization of the platelets by released ADP. After flow, the sheared sample and a control, incubated at rest at 37°C, were stirred with thrombin at concentrations of 0.04 units/ml (threshold at which aggregation just occurred) and 0.02 units/ml, and the ¹⁴C-activity in the supernatant measured after centrifugation.At time-averaged wall shear rates $\text{G}\text{(R}{}_0\text{) = 2350}\text{sec}{}^{\mathrm{?1}}$ and 20 min shear, there was no significant release of [¹⁴C]serotonin due to flow alone. However, in 53 experimental runs the mean release at 0.02 and 0.04 units/ml exceeded that in the paired controls by 6.1%(±1.4%, SE) and 10.1%(±1.3%) of the total activity in the suspension. The increase in serotonin release was not significant at $\text{G}\text{(R}{}_0\text{) < 1300}\text{sec}{}^{\mathrm{?1}}$.The synergistic effects of flow were also noted when [Ca²⁺] was increased from 4 to 12 mEq, and in the presence of ASA (1 mg/ml), release being less inhibited in the sheared samples. When adrenaline (2.5 μM) was added, the serotonin release, which increased by 26.4%(±3.8%) in 17 controls at the lower thrombin level, was further enhanced by 10.9%(±2.6%) in the paired sheared samples. However, when adrenaline was added after oscillatory flow, the reverse occurred, the sheared samples releasing 8.5% less than the controls.Studies in the aggregometer showed that the rate and extent of aggregation in the sheared samples was a little lower than that in the controls.     

A flow cytometric assay of CD34-positive cell populations in the bone marrow

Summary CD34⁺ BM cells form a heterogenous population of primitive stem cells and more mature progenitors committed to different lineages of differentiation. By combining CD45 expression with SSC, it is possible to separate immature cells from more diferentiated BM cells, and, by three-colour flow cytometry, analyse the antigens expressed in various subsets of cells. In this paper we show that in the normal BM at least four distinct CD34⁺ cell populations can be identified by their different patterns of CD45 expression and SSC. The most immature CD34⁺ cell population (0.1% of the BM cellularity) lacked all signs of lineage commitment and was CD45RA negative and only weakly CD45 positive. With increasing expression of the CD45 antigens, a second CD34⁺ population (0.2% of the BM cellularity) was formed expressing mainly primitive lymphatic antigens. However. 30% of the cells co-expressed B-cell line antigens and myeloid antigens. Cells committed to the myeloid cell line lost B-cell line antigens, gained CD45 antigen expression and SSC and formed two CD34⁺ cell population (0.2% and 0.1% of the BM cellularity. respectively) differing only with respect to the pattern of myeloid antigen expression and SSC characteristics. Similarly, differentiation along the lymphatic pathway implicated down-regulation of myeloid antigens, loss of the stem cell antigen and immature lymphatic antigens and gain of CD45 expression and mature lymphatic antigens.     

Immature dense granules in platelets from mice with platelet storage pool disease

Mepacrine uptake into platelets and bone marrow megakaryocytes was analyzed to further characterize the dense granule defects in a group of seven mouse pigment mutants that have characteristics of platelet storage pool disease (SPD). In contrast to our previous studies using electron microscopy, this method revealed that all mutants had normal numbers of dense granules. However, total mepacrine uptake in all mutant platelets was significantly diminished to less than 50% of normal uptake. Also, the flashing phenomenon observed when normal dense granules are irradiated with ultraviolet light was either greatly diminished or absent when platelets of individual mutants were similarly irradiated. Therefore the principal defect in the mutant platelets is an inability to accumulate dense granule contents rather than an absence of the granules. Mepacrine uptake into megakaryocytes was indistinguishable in normal and mutant mice. This indicates the mutant dense granule defects appear either very late in megakaryocyte development or early in platelet formation in correlation with development of the mature dense granule. By standard transmission electron microscopy we have not been able to detect gross structural or subcellular abnormalities in either platelets or megakaryocytes of mutant mice. It appears all seven mutants produce immature or functionally abnormal dense granules.     

A flow cytometric assay for the determination of cell proliferation with a monoclonal antibody directed against DNA-methyltransferase

The enzyme DNA-methyltransferase is responsible for the methylation of a newly synthesized DNA-strand. A monoclonal antibody directed against DNA-methyltransferase was used to determine cell proliferation by means of flow cytometry. The reactivity of DNA-methyltransferase antibody was compared with the known proliferation markers transferrin-receptor and Ki67. All three methods showed comparable reactivity with the erythroblastic cell line K562 (86%, 81%, 76% respectively). In a second set of experiments peripheral blood mononuclear cells were stimulated with phytohaemagglutinin; in three experiments, a mean of 63% of the cells reacted with DNA-methyltransferase antibody after 72 h of culture as compared to a mean of 6% in the case of unstimulated control cells. HL60 cells were incubated with DMSO and harvested on day 5 of culture. The results obtained show that in differentiated cells the fraction positive with DNA-methyltransferase antibody decreased to levels below 10%. It is concluded that the technique described is a fast and easy method for the flow cytometric determination of cellular proliferation.     

A whole-blood flow cytometric assay for leukocyte CD11b expression using fluorescence signal triggering

A flow cytometric assay for measurements of leukocyte CD11b expression in whole blood has been developed and evaluated. The method is based on triggering of the flow cytometer by a fluorescent pan leukocyte marker, RPE-CD45. This enabled flow cytometric analysis in whole blood, and avoidance of in vitro artefacts related to cell purification and hemolysis. Our methodological evaluation suggested the following routine procedure: sampling with sodium citrate as the anticoagulant, sample incubation at 22 degrees C, and mild sample fixation with 0.5% formaldehyde saline. The latter provided good sample stability during 24 h. Moreover, the assay provided good assay reproducibility, low labelling antibody consumption, and minimal sample manipulation (< 30 min) and acquisition time demands. The assay seems to reflect the CD11b expression of circulating leukocytes, and is also suitable for studies of agonist stimulated CD11b expression in leukocyte subpopulations in vitro. When full CD11b responsiveness to agonist stimulation is desired, samples should be incubated at 37 degrees C, but this also elevated CD11b expression in unstimulated samples. The present whole-blood technique is thus suitable for analyses of CD11b expression for both research and clinical routine laboratory use. The assay can easily be modified for measurements of other leukocyte antigens by use of other specific fluorescent antibodies.     

四色流式分析冠心病患者外周血单核细胞亚群及单核细胞-血小板聚集体的变化

目的:建立基于四色的流式细胞术检测外周血单核细胞亚群及各亚群单核细胞-血小板聚集体(MPA)的方法,并观察冠心病患者外周血单核细胞亚群及各亚群MPA的变化特点。方法:本研究纳入经冠状动脉造影证实的冠心病患者和健康对照者各25例,记录所有研究对象的一般临床资料,并采集外周静脉血,依据单核细胞和血小板的标志性分子CD86、CD14、CD16和CD41对单核细胞亚群和MPA进行四色的流式细胞术(FCM)分析。利用抗CD86-PE-Cy5圈出总的单核细胞(总Mon)群,利用抗CD14-FITC和抗CD16-PE将总Mon分为:经典型(CD14++CD16-,Mon1)、中间型(CD14++CD16+,Mon2)和非经典型(CD14+CD16++,Mon3)3个亚群,最后应用CD41-PE-Cy7分析总Mon和各个亚群的MPA,并应用Flow-count TM荧光微球对各个亚群及MPA的绝对量进行计数,比较两组研究对象外周血单核细胞各亚群及各亚群MPA的差异。结果:冠心病组患者外周血Mon2和Mon3所占百分比和绝对计数均显著高于对照组(P<0.05),而Mon1和总Mon两组间比较差异无统计学意义;与对照组相比,冠心病患者总MPA和Mon2相关MPA所占百分比和绝对计数亦显著增高(P<0.05),而Mon1相关MPA和Mon3相关MPA的百分比及绝对计数两组间比较差异无统计学意义。结论:基于CD14、CD16、CD86和CD41的四色流式分析能够有效定量分析外周血单核细胞各亚群及各亚群MPA。中间型单核细胞及其形成的MPA水平显著升高,为临床上冠心病患者的诊治策略提供了新思路。     

Heparin-induced thrombocytopenia: ELISA optical density value and 4T score in correlation with panel donor platelets activation in functional flow cytometric assay

BackgroundSerological assays for the diagnosis of heparin-induced thrombocytopenia (HIT) detect both platelet-activating and platelet non-activating anti-heparin/platelet factor 4 (PF4) antibodies and have therefore a limited positive predictive value. Functional assays confirm the presence of platelet-activating antibodies but require platelets from healthy donors, whose response to patient serum can differ. Our aim was to investigate the correlation between the level of anti-heparin/PF4 antibodies, 4T score, and the extent of panel donor platelet activation in the functional assay.Materials and methodsIn total, 38 sera from enzyme immunoassays (ELISA) positive patients were tested against panel platelets obtained from 10 healthy, randomly selected donors, using our routine flow cytometry functional test for CD62P expression. Levels of anti-heparin/PF4 antibodies from medical and surgical patients and 4T pretest probability scores (where available) were correlated with the number of activated panel platelets.ResultsSera with low ELISA optical density (OD) values (0.4–1) activated on average 5.6, sera with intermediate ELISA OD values (>1–2.5) activated on average 7.3, and sera with high ELISA OD values (>2.5) activated on average 8.6 out of 10 panel platelets. One serum with low 4T score did not activate donor platelets, 12 sera with intermediate 4T score activated on average 6.3 donors, 8 sera with high 4T score activated on average 8.5 panel platelets.DiscussionSera with higher ELISA OD values activated platelets from a higher number of platelet donors, independently of patient type (medical or surgical). The average number of activated panel platelets increased with rising 4T score. Results indicate that both donor platelet reactivity and quantity of anti-heparin/PF4 antibodies affect the result of the functional assay, meaning special attention is needed in platelet donor selection when testing sera with low levels of antibodies.     

Heterogeneous abnormalities of platelet dense granule ultrastructure in 20 patients with congenital storage pool deficiency

Studies on platelet dense granule structure were carried out in 20 patients with various types of congenital storage pool deficiency (SPD), including 15 with specific deficiencies of dense granules and dense granule substances (δ-SPD), and five with combined deficiencies of dense and α-granules (αδ-SPD). Dense granules were identified by their high affinity for uranyl ions (uranaffin reaction), by their ability to accumulate the fluorescent dye mepacrine, and by their inherent electron opacity on unfixed, unstained whole mount preparations. By all these methods, dense granules were markedly decreased in seven albino patients with the Hermansky-Pudlak syndrome (HPS) variant of δ-SPD. These findings suggest that the basic defect in these patients is a specific abnormality in organelle development which prevents the formation of an intact granule structure, a quantitative abnormality which may differ from that in animals with related pigment disorders. In contrast, eight non-albino patients with δ-SPD had, on average, only a slightly reduced number of uranaffin-positive and mepacrine-positive granules, but a shift in uranaffin-granule distribution towards those lacking a dense core (‘empty granules’), suggesting a more qualitative type of dense granule defect. These results are consistent with previous evidence suggesting a decreased uptake of ATP across the granule membrane in δ-SPD. In addition, on whole mounts, these patients’platelets contained substantial numbers of electron dense chains and clusters which contained P and Ca, but with a P/Ca ratio less than that of typical dense granules, and which were retained, along with a larger amount of ATP, after thrombin treatment of the platelets. The various findings in these patients raise the possibility that these structures may represent microvesicles, derived from the Golgi apparatus, which provide a transport mechanism for concentrating adenine nucleotides and calcium in dense granules and which is impaired in some patients with SPD. Additional defects may account for the more extensive granule abnormalities observed in αδ-SPD.