Evaluation of platelet parameters on the ADVIA 120 as the quality indicator for stored platelets

In order to investigate the possible use of platelet parameters on the ADVIA 120 hematologic analyzer as the routine quality control indicator for preparation and storage of platelets, platelet parameters, pH and CD62P expression were determined in stored platelet concentrates. Platelet component distribution width (PCDW) was decreased progressively on days 1 and 3 of storage for 5 days when compared with 0 day. PCDW correlated with CD62P expression on unstimulated platelets and the difference in CD62P expression following agonistic stimulation (a measure of functional reserve). Mean platelet component (MPC) was decreased on day 1 of storage. It did not however, show a progressive decrease over storage time and did not correlate with CD62P, although MPC has been known to be a useful screening test for platelet activation. Therefore, PCDW is considered to be a simple, convenient and cost-effective quality indicator for determining the viability and storage lesion of platelets for transfusion.     

The effects of coronary artery disease severity on time-dependent changes in platelet activation indices in stored whole blood

Background Platelets play a pivotal role in the pathogenesis of the thrombotic complications in cardiovascular disease (CVD). Abnormal platelet activation indices are evolving as potentially useful markers in CVD risk stratification. Whilst there has been some investigation into the effects of storage time on several of these indices, the effects of underlying disease severity on these temporal changes have not been previously studied. Methods Using the ADVIA^TM120 haematology analyser, we assessed the effects of time-dependent storage of whole blood in EDTA, on a number of platelet activation indices: mean platelet volume (MPV), mean platelet component (MPC, measure of platelet density) and platelet component distribution width (PCDW, a marker of platelet shape change. We studied three age- and sex-matched patient groups: (i) healthy controls (n = 10), (ii) stable patients with coronary artery disease (CAD, n = 9); and (iii) patients with acute myocardial infarction (n = 8). Whole blood samples were processed at exactly 5 min following venesection and at 15, 30, 60 and 120 min later in storage in EDTA tubes at room temperature. Results There was a significant and stepwise increase in MPV (P = 0.01) and decrease in PCDW (P = 0.03), with a non-significant trend to increasing MPM and decreasing MPC with increasing underlying disease (that is healthy, ‘stable’ and ‘acute’ artery disease). There was a significant time-dependent increase in MPV and decrease in MPC and PCDW (all P < 0.05), which were all significant on ‘post-hoc’ analyses by 30 min. There were no significant changes in platelet count or MPM with time. There was no interaction of underlying disease with whole-blood storage time for any of the platelet indices reported (P = NS). Conclusion There is a temporal increase in MPV and decrease in MPC and PCDW in venous blood stored over 2 h in EDTA. These changes are not influenced by the underlying CVD disease severity.     

Flow cytometric analysis of platelet activation under calcium ion‐chelating conditions

Platelet activation and aggregation results in factitious counting and sizing in routine haematology testing. In this study, the possibility of platelet activation in anticoagulated solutions was examined. Whole blood was examined using an automated counter and a flow cytometer before and after strong vortex agitation. Blood treated with ethylenediaminetetraacetic acid (EDTA) exhibited platelet activation both pre‐ and postagitation but activated platelets did not cause platelet aggregation. With sodium citrate, platelets were only minimally activated both pre‐ and postagitation. Heparin‐treated blood exhibited minimal platelet activation preagitation, but agitation resulted in strong platelet activation and aggregation. Platelet size was increased by agitation in blood with EDTA and with sodium citrate, in association with significant increases in mean platelet volume (MPV) and platelet distribution width (PDW), but MPV and PDW were significantly higher in EDTA solution than in sodium citrate solution. Change in platelet size was observed even in the presence of EDTA, indicating that careful sampling and processing are needed in the collection of specimens. Specimens obtained from patients with EDTA‐dependent pseudothrombocytopenia exhibited the same level of activation as controls, although platelets exhibited aggregation in such specimens. In conclusion, platelet activation involving platelet size change can occur in the absence of calcium ions in blood treated with EDTA.     

Evaluation of blood collection methods and anticoagulants for platelet function analyses on C57BL/6J laboratory mice

Abstract

The exploration of thrombotic mechanisms relies on the application of blood collection methods from laboratory mice with a minimal pre-activation of platelets and the clotting system. So far, very little is known on how the blood collection method and the anticoagulant used influence pre-activation of mouse platelets and coagulation. To determine the most suitable blood collection method, we systematically compared blood collection by heart puncture, Vena cava puncture, and puncture of the retro-orbital vein plexus and the use of citrate, heparin, and EDTA as frequently used anticoagulants with regard to platelet activation and whole blood clotting parameters. The activation of platelet-rich plasma diluted in Tyrode’s buffer was analyzed by flow cytometry, analyzing the exposure of P-selectin and activated integrin α_IIbβ₃. Clotting of whole blood was profiled by thrombelastometry. Puncture of the retro-orbital vein plexus by plastic capillaries is not superior in terms of blood volume and platelet pre-activation, whereas heart puncture and Vena cava puncture resulted in similarly high blood volumes. Cardiac puncture and Vena cava puncture did not result in pre-activated platelets with citrate as an anticoagulant, but the use of EDTA resulted in increased levels of integrin α_IIbβ₃ activation. Puncture of the retro-orbital vein plexus by plastic capillaries resulted in increased platelet integrin α_IIbβ₃ activation, which could be prevented by soaking with citrate or coating with heparin. Further, activation of coagulation in citrated whole blood by puncture of the retro-orbital vein plexus using a blunt plastic capillary was observed by thromboelastometry. The use of citrate is the optimal anticoagulant in mouse platelet assays. Blood collections from the heart or Vena cava represent reliable alternatives to retro-orbital puncture of the vein plexus to avoid pre-activation of platelets and coagulation.     

Apheresis platelet concentrates contain platelet‐derived and endothelial cell‐derived microparticles

Background and Objectives Microparticles (MP) are membrane vesicles with thrombogenic and immunomodulatory properties. We determined MP subgroups from resting platelets, activated platelets and endothelial cells in donors and apheresis platelet concentrates (PC). Material and Methods MP were double stained with annexin V and CD61 (platelet‐derived MP; PMP), P‐selectin or CD63 (MP from activated platelets) and CD144 plus E‐selectin (endothelial cell‐derived MP; EMP) and detected by flow cytometry in platelet donors (n = 36) and apheresis PC (n = 11; Trima?). Results PC contained MP, mainly from resting platelets [93% (90–95)], and minor fractions of PMP from activated platelets [P‐selectin⁺ or CD63⁺; 4·8% (3·2–7·7) and 2·6% (2·0–4·0)]. Compared to donors, levels of annexin V+ MP, PMP, P‐selectin⁺ and CD63⁺ MP were 1·7‐, 2·3‐, 8·6‐ and 3·1‐fold higher in PC (all P < 0·05). During storage (1–5 days), levels of annexin V+ MP and PMP did not increase, although small increases in the fraction of P‐selectin⁺ or CD63⁺ MP occurred (both P < 0·05). PC also contained EMP, which were 2·6‐ to 3·7‐fold enriched in PC compared to donors (P < 0·05). Conclusions Transfusion of apheresis PC also results in transfusion of HLA‐carrying PMP and EMP. This might counteract the aim of reducing transfused HLA load by leucodepletion. The increases in PMP exposing P‐selectin or CD63 reflect mild platelet activation during storage. We conclude that in leucodepleted platelet apheresis using fluidized particle bed technology, MP are harvested mainly from the donor by apheresis. Improvement in apheresis technology might reduce MP load.     

Blood platelet and monocyte activations and relation to stages of liver cirrhosis

AIM: Blood platelets (pIt) and monocytes are the cells that play a crucial role in the pathogenesis of liver damage and liver cirrhosis (LC). In this paper, the analysis of mutual relationship between platelets and monocytes activation in LC was conducted. METHODS: Immunofluorescent flow cytometry was used to measure the percentage of activated platelet populations (CD62P, CD63), the percentage of plt-monocyte aggregates (pma) (CD41/CD45), and activated monocytes (CD11b, CD14, CD16) in the blood of 20 volunteers and 40 patients with LC. Platelet activation markers: sP-selectin, platelet factor 4 (PF4), beta-thromboglobulin (PTG) and monocyte chemotactic peptide-1 (MCP-1) were measured and compared in different stages of LC. RESULTS: Platelet activation with the increase in both βTG serum concentration and elevation of pIt population (CD62P and CD63 as well as MIF CD62P and CD63) is elevated as LC develops and thrombocytopenia rises. There is a positive correlation between medial intensity of fluorescence (MIF) CD62P and MIF CD63 in LC. We did not show any relationship between monocyte activation and pma level. SP-selectin concentration correlates positively with pIt count and pma, and negatively with stage of pIt activation and MIF CD62P and MIF CD63. There was no correlation between MCP-1 concentration and pIt, monocyte activation as well as pma level in LC. CD16 monocytes and MIF CD16 populations are significantly higher in the end stage of LC. A positive correlation occurs between the value of CDllb monocyte population and MIF CD14 and MIF CD16 on monocytes in LC. CONCLUSION: Platelet and monocyte activation plays an important role in LC. Platelet activation stage does not influence monocyte activation and production of pIt aggregates with monocytes in LC. With LC development, thrombocytopenia may be the result of pIt consumption in platelet-monocyte aggregates.     

Prognostic value of platelet indices as determined by ADVIA 120 in patients suspected of having disseminated intravascular coagulation

Recent technological advances have made it possible to record a variety of platelet indices using automated hematology analyzers. Disseminated intravascular coagulation (DIC) is associated with the dramatic hemostasis activation, with evidence of fibrin formation and platelet consumption. We investigated the prognostic significance of platelet indices as measured by ADVIA in 222 patients suspected of having DIC. The presence of overt DIC was defined using the scoring system of the International Society of Thrombosis and Haemostasis Subcommittee. Twenty-eight day hospital mortality was used as a clinical prognosis parameter. Median platelet count and platelet-crit (PCT) levels markedly decreased in nonsurvivors, whereas mean platelet volume (MPV), platelet component distribution width (PCDW) and platelet dry mass distribution width (PMDW) were significantly increased in nonsurvivors. In terms of ROC analysis, which was conducted to predict 28-day mortality, areas under the receiver operating characteristic curve (AUC) were; 0.73 platelet count, 0.72 for PCT, 0.69 for PCDW, 0.65 for PMDW and 0.61 for MPV. The odds ratio of a reduced platelet count for the relative risk of 28-day mortality was 5.249 (95% CI: 2.399-11.486), and the odds ratio for PCDW was 3.240 and for PMDW 3.262. Among these indices, platelet count, PCDW and PMDW were found to be more predictive of 28-day hospital mortality. Our results suggest that these indices may provide prognostic information on hospital mortality in the patients suspected of having DIC.     

Formation of platelet-leukocyte aggregates in inflammatory bowel disease

OBJECTIVES: Formation of platelet-leukocyte aggregates (PLAs) is increased in several inflammatory and thrombotic conditions. This may result from and enhance platelet and neutrophil activation and could contribute to the inflammatory process in inflammatory bowel disease (IBD). We investigated platelet-leukocyte aggregation in patients with IBD and its relation to treatment, disease activity and platelet and neutrophil activation. METHODS: PLAs, platelet activation (P-selectin expression) and neutrophil activation (L-selectin expression) were assessed 30 and 180 minutes after drawing blood into EDTA/citrate-theophylline-adenosine and dipyridamole, a novel anticoagulant, using fluorescent antibodies to CD45 (for leukocytes), CD42a (for platelets), CD62P (P-selectin) and CD62L (L-selectin) and flow cytometry. Platelet activation was also measured using the ADVIA 120 hematology analyser. RESULTS: Samples from 67 patients with IBD measured within 30 minutes had a higher platelet count (P < 0.001), more platelets expressing P-selectin (P = 0.01), and more PLAs (P < 0.01) than from 20 healthy controls and more PLAs (P < 0.05) than from 9 controls with inflammatory arthropathies. IBD patients on thiopurines had fewer PLAs than those not taking them (P < 0.05); corticosteroids and aminosalicylates had no such effects. Incubation for 180 minutes increased the number of platelets expressing P-selectin (P < 0.0001), and the number of PLAs (P < 0.0001). The PLAs correlated with the number of platelets expressing P-selectin before (r=+0.40, P < 0.001) and after (r=+0.66, P < 0.0001) incubation. CONCLUSIONS: The number of PLAs is higher in patients with IBD than in healthy and inflammatory controls, but their numbers are lowered by thiopurines. Increased PLA formation may in part be due to increased platelet activation and could be pathogenic in IBD.     

Automated assessment of the neutrophil and platelet activation status in patients with essential thrombocythemia

Neutrophil and platelet activation are consistently found in essential thrombocythemia (ET), but the techniques employed to demonstrate such abnormalities are complex. To ascertain whether the ADVIA 120 analyzer can be employed to assess neutrophil and platelet activation status in ET, 55 such patients and the same number of matched healthy individuals were studied and the results correlated with neutrophil CD11b and platelet P-selectin expressions measured by flow cytometry. Compared with controls, ET patients had significantly higher values of neutrophil myeloperoxidase index (MPXI), mean platelet volume (MPV), platelet distribution width (PDW), and platelet component distribution width, and significantly lower values of neutrophil lobularity index and mean platelet component (MPC). Patients with the JAK2 mutation had significantly lower values of MPC and higher values of MPV and PDW than those with wild-type allele. A positive correlation was observed between MPXI and neutrophil CD11b expression and a negative correlation between MPC and platelet P-selectin expression. The intensity of the agreement between the variables obtained by the two methods was moderate. These results support the possible value of MPC as surrogate parameter of platelet activation in ET.     

The influence of platelet additive solutions on cytokine levels and complement activation in platelet concentrates during storage

BACKGROUND AND OBJECTIVES: The accumulation of platelet-derived cytokines in platelet concentrates (PC) during storage may contribute towards non-haemolytic transfusion reactions (NHTR). We investigated the effect of platelet storage medium on platelet activation, complement activation and cytokine levels in leucocyte-reduced PC. MATERIALS AND METHODS: Hyperconcentrated platelets (3000-6000 x 109/l) were collected by apheresis and diluted in 100% plasma, 70% PASIII, or 70% or 80% PASIII supplemented with magnesium and potassium (PAS IIIM). RESULTS: Levels of transforming growth factor-beta (TGF-beta) and regulated on activation, normal, T-cell expressed, and secreted (RANTES) increased during storage, as did the expression of P-selectin (CD62P), and were highest in PC stored in PASIII. In PC stored in PASIIIM, however, levels of TGF-beta and RANTES were not significantly different from PC stored in plasma. Levels of CD62P expression, however, remained higher in PASIIIM PC than in those stored in plasma by day 5, but were lower than PC stored in PASIII. C3a des arg levels increased during storage in all media with the exception of PASIII and, on day 7, were higher in PC stored in plasma compared to PC stored in the other media. CONCLUSIONS: Our results indicate that replacing plasma in PC with unmodified PASIII for storage results in higher levels of platelet-derived cytokines in PC. Furthermore, it appears that the nature of the medium used for storage of PC has a significant impact on platelet activation and cytokine levels of the PC. These implications should be taken into account when considering replacement of plasma with PAS.     

射频消融对血管内皮及血小板功能的影响

目的　研究射频导管消融 (RFCA)术对血管内皮和血小板功能的影响。方法　应用放射性免疫法、酶联免疫法、单克隆抗体标记及流式细胞技术 ,观察 31例心动过速患者RFCA手术前、后血浆内皮素 (ET)、血管性假血友病因子 (vWF)水平及血小板α 颗粒膜蛋白 (CD62 P)、血小板溶酶体膜蛋白 (CD63 )表达的变化。结果　RFCA术前、后血浆ET、vWF水平无明显改变 ,但术后即刻血小板膜CD62 P、CD63 表达分别由术前的 (4 .75± 2 .32 ) %和 (9.6 2± 4 .0 8) %增高至 (7.6 4± 5 .2 5 ) % (t =3.0 5 ,P <0 .0 1)和 (12 .2 3± 5 .70 ) % (t=2 .10 ,P <0 .0 5 ) ,术后 4 7～ 115h(平均 6 5h)均降至基础水平。多元相关分析结果显示CD62 P表达变化与累积放电能量呈显著性正相关 (r =0 .30 ,P <0 .0 5 )。结论　RFCA术不引起明显的内皮损伤 ,但可导致血小板膜CD62 P、CD63 表达增加 ,促进血小板活化 ,其中手术累积放电能量是重要的影响因素。     

Flow cytometric evaluation of platelet activation in blood collected into EDTA vs. Diatube-H,a sodium citrate solution supplemented with theophylline,adenosine, and dipyridamole

With platelet activation, there is modulation of platelet surface molecule expression. In flow cytometric analyses of in vivo platelet activation, results are often confounded by activation induced in vitro by the preparative procedures. It is particularly important therefore to prevent or retard platelet activation as soon as possible after withdrawal of the blood sample. Taking blood into paraformaldehyde, or fixing the cells with paraformaldehyde as soon as possible after withdrawal, has been employed to prevent platelet activation in vitro, but paraformaldehyde-fixed platelets cannot be further used in functional studies. We investigated the efficacy of Diatube-H, a commercially available combination of platelet antagonists (theophylline, adenosine, and dipyridamole), in preventing or retarding platelet activation in vitro, along with its effects on modulation of platelet membrane glycoproteins (GP) and adhesion molecules. In contrast to blood taken into EDTA, blood taken into Diatube-H vacutainer tubes could be stored at room temperature for up to 4 hr prior to paraformaldehyde fixation without significant in vitro platelet activation, as measured by CD62P, CD63 and modulation of GPIb and GPIIbIIIa surface expression. Hence, paraformaldehyde fixation could be deferred for several hours, permitting transport of samples from distant sites. Studies of thrombin-induced platelet activation indicated that platelets taken into Diatube-H remained functional i.e. were able to be activated. Expression of the CD29, CD49b and CD31 adhesion molecules on the platelet surface was unaffected by storage in Diatube-H. The results suggest that Diatube-H may be a useful reagent for flow cytometric studies of platelets when the samples cannot be processed immediately.     

Plasma thrombopoietin level and platelet indices in hemodialysis patients receiving recombinant human erythropoietin

We focused on thrombocytopenia in hemodialysis patients (HD) receiving recombinant human erythropoietin (rHuEPO) and investigated thrombopoietin (TPO) level and platelet indices. We analyzed platelet parameters including mean platelet volume (MPV), platelet-crit (PCT), mean platelet component (MPC) concentration and platelet count (PLT) using ADVIA 2120 in 375 HD patients. This study included 25 HD patients undergoing treatment with rHuEPO at 9000 IU/week. These patients were divided into two groups by reference PLT of 130 × 10⁹/l [eight patients with low PLT (L-PLT group) and 17 patients with normal PLT (N-PLT group)], and TPO level and platelet indices in each group were compared with those in nine HD patients not receiving rHuEPO. In HD patients, the mean value of MPV was slightly higher and the mean values of PLT, PCT, and MPC were significantly lower than those in healthy controls. TPO levels were significantly higher in patients receiving rHuEPO than in patients not receiving rHuEPO. However, no significant difference was found between TPO levels in patients in the L-PLT group and patients in the N-PLT group. TPO levels were not correlated with PLT in these patients and that MPC levels decreased remarkably regardless of PLT.     

Blood platelet activation evaluated by flow cytometry: optimised methods for clinical studies

A variety of flow cytometry techniques are in use to evaluate in vivo blood platelet activation. We have in this study further developed and optimised these methods to be suitable for use in clinical studies. By preloading the Monovette EDTA vacuum blood sampling tubes with 1/8 vol 4% (w/v) paraformaldehyde (PFA), we were able to assess platelet CD62P (P-selectin) expression in whole blood with less than 0.2% activated platelets. No washing or neutralising steps were required to remove excess fixative. Both basal and agonist-stimulated CD62P expression were stable for at least 48 h after sampling. The standard curve was linear from 1.9 (basal) to 8.1 x 10(3) (TRAP-stimulated) molecules of equivalent soluble fluorochrome units (MESF) in phycoerythrein-conjugated anti-CD62P labelled whole blood samples. These assay conditions were also well suited for assessment of platelet expression of CD41, CD42a, CD61 and CD63. The preanalytic storage period was extended from 10 min to at least 2 h for platelet PAC-1 and fibrinogen binding analysis by preloading Monovette citrate tubes with 8/10 vol buffer. With PFA preloading, blood sampled into citrated tubes could be analysed for fractions of microparticles and platelet-platelet aggregates as well as for aggregate size.     

Blood platelet activation evaluated by flow cytometry: optimised methods for clinical studies

A variety of flow cytometry techniques are in use to evaluate in vivo blood platelet activation. We have in this study further developed and optimised these methods to be suitable for use in clinical studies. By preloading the Monovette® EDTA vacuum blood sampling tubes with 1/8 vol 4% (w/v) paraformaldehyde (PFA), we were able to assess platelet CD62P (P-selectin) expression in whole blood with less than 0.2% activated platelets. No washing or neutralising steps were required to remove excess fixative. Both basal and agonist-stimulated CD62P expression were stable for at least 48 h after sampling. The standard curve was linear from 1.9 (basal) to 8.1·10 3 (TRAP-stimulated) molecules of equivalent soluble fluorochrome units (MESF) in phycoerythrein-conjugated anti-CD62P labelled whole blood samples. These assay conditions were also well suited for assessment of platelet expression of CD41, CD42a, CD61 and CD63. The preanalytic storage period was extended from 10 min to at least 2 h for platelet PAC–1 and fibrinogen binding analysis by preloading Monovette® citrate tubes with 8/10 vol buffer. With PFA preloading, blood sampled into citrated tubes could be analysed for fractions of microparticles and platelet–platelet aggregates as well as for aggregate size.     

Concurrent and separate inside‐out transition of platelet apoptosis and activation markers to the platelet surface

The cell plasma membrane is tightly coupled with the vital processes of apoptosis and activation. In the current study, we investigated exposure of the apoptosis marker phosphatidylserine (PS) and activation marker P‐selectin (CD62) on the plasma membrane of anucleate platelets. We found that, depending on triggering stimuli, the plasma membrane of human platelets may exist in four states with predominant exposure of (i) PS but not CD62 (75·9 ± 2·8% of total cells), (ii) CD62 but not PS (86·2 ± 1·3%), (iii) both PS and CD62 (89·6 ± 1·0%) or (iv) neither PS nor CD62 (87·9–97·5%), when platelets were treated at optimal conditions with pro‐apoptotic BH3 mimetic ABT‐737, thrombin, calcium ionophore A23187 or control diluents, respectively. The dynamics of PS exposure induced by ABT‐737 is a slow temperature‐dependent process requiring 90 min treatment at 37°C rather than at room temperature for obtaining high levels of PS exposure. In contrast, thrombin‐induced CD62 exposure and A23187‐induced PS and CD62 exposure showed fast temperature‐independent dynamics. This model of selective and concurrent stimulation of PS and/or CD62 transition to the platelet surface provides an experimental horizon for elucidating the roles of plasma membrane markers of platelet apoptosis and activation in platelet clearance.     

肺栓塞与血小板活化标志物

目的探讨肺栓塞与血小板活化的关系,以发现更加简便、可靠的检测血小板活化的方法。方法用酶联免疫吸附法测定血浆中可溶性P-选择素（PS）;流式细胞术检测血小板膜上活化标记物P-选择素（CD62p）。结果PTE患者血小板膜上P-选择素及血浆中可溶性P-选择素均明显高于高危人群及正常对照组（P〈0．01）;高危人群血小板膜上P-选择素及血浆中可溶性P-选择素高于正常对照组;栓塞面积越大血小板膜上P-选择素及血浆中可溶性P-选择素越高,血小板的活化程度越高;说明血小板活化在肺栓塞的发病中起重要作用。结论PTE时,血浆P-选择素水平明显增高,参与了PTE的发生、发展。通过早期监测高危人群血浆P-选择素,对于预防肺栓塞有一定帮助。P-选择素可以作血栓性疾病的导向诊断和治疗提供崭新的途径。P-选择素简单、易测,上述两种方法均可作为常规检查的指标。     

Correlation between the extent of platelet activation in platelet concentrates and in vitro and in vivo parameters

BACKGROUND AND OBJECTIVES: Platelet activation, which is necessary to stop bleeding, also occurs in vitro during the storage of platelet concentrates (PCs). However, it is unknown whether in vitro-activated platelets are able to reduce blood loss in the patient. We studied correlations between platelet activation in PCs and in vitro parameters (pH, platelet count, swirling effect, storage time). In addition, we studied the correlation between platelet activation and in vivo parameters [the volume of thorax drain fluid as a measure of blood loss, platelet count, international normalized ratio (INR), and activated partial thrombin time (APTT)] in a clinical pilot study. MATERIALS AND METHODS: White blood cell-reduced PCs prepared from five buffy coats and one plasma unit (n = 55; storage time: median, 5 days; range, 2-12 days) were sampled. Platelet activation (CD62p, CD63, CD42b), as measured by flow cytometry, pH, platelet count, swirling effect and storage time, was determined. For the in vivo pilot study, PCs (n = 21) stored for 2-7 days were also checked for the above parameters and transfused into patients (n = 21) immediately after coronary artery bypass graft surgery. The volume of thorax drain fluid was measured for up to 12 h after surgery, and the platelet count, INR and APTT were measured < 1 h and 16-24 h postsurgery. RESULTS: A good correlation (r2 > 0.5) was observed between CD62p and CD63, between CD62p and CD42b, between CD62p or CD63 and pH and between CD62p or CD63 and swirling effect. Also, a significant increase in platelet activation was observed for PCs stored for > or = 8 days (mean +/- standard deviation: CD62p, 41.6 +/- 30.7; CD63, 23.8 +/- 18.6), compared to PCs stored for 2-7 days (mean +/- standard deviation: CD62p, 12.3 +/- 4.8; CD63, 10.4 +/- 3.6). No correlation (r2 < or = 0.1) was observed between platelet activation and the in vivo parameters. CONCLUSIONS: Although a correlation between platelet activation and in vitro parameters was observed, no correlation was found between platelet activation and in vivo parameters. Possible explanations for this are a too low variance in platelet activation in transfused PCs, and too small a number of patients.     

Four cases of pseudothrombocytopenia due to platelet cold agglutinins

We report 4 cases of pseudothrombocytopenia due to platelet cold agglutinins. Case 1 was a 57 y.o. female whose platelet count was 97 x 10(3)/microl. Case 2 was a 37 y.o. male with a platelet count of 96 x 10(3)/microl. Case 3 was a 74 y.o. male with a platelet count of 28 x 10(3)/microl. Case 4 was a 62 y.o. female whose platelet count was 34 x 10(3)/microl. The platelet counts in these 4 cases were decreased and blood smears showed platelet clumping in blood drawn in a tube without anticoagulant just after withdrawal, as well as in blood drawn in a tube with anticoagulant. The platelets from these patients agglutinated at a temperature below 10 degrees C (case 1 and 4) and 24 degrees C (case 2). The immunoglobulin class of the platelet cold agglutinins in cases 1, 2 and 4 was IgM. Agglutinated platelets showed no activation marker, such as CD62P, CD63 or CD40L, on the surface of the platelets. The target antigen of cold agglutinins was GPIIb-IIIa in cases 1 and 2. We considered that the detection of platelet agglutination in blood without anticoagulant is important to diagnose pseudothrombocytopenia due to platelet cold agglutinins. Although this disease is considered to be very rare, we suspect that this disease may be misdiagnosed as pseudothrombocytopenia due to the presence of an anticoagulant, and overlooked.     

Pseudo grey platelet syndrome--grey platelets due to degranulation in blood collected into EDTA

We have studied a woman with a history of mild bruising and bleeding, with a normal platelet count and normal clotting factors, who had platelets that appeared grey when stained and viewed under the microscope. Unlike the grey platelet syndrome, the abnormality was only evident when blood had been collected into EDTA and not when citrate or heparin was used as anticoagulant. This 'pseudo grey platelet syndrome' was associated with platelet dense body and alpha granule secretion with no aggregation and occurred on removal of extracellular Ca2+. We discovered that a plasma factor was responsible which could be an immunoglobulin but which is clearly different from the EDTA-sensitive antibodies which cause platelet aggregation and agglutination. We were not able to demonstrate a relationship between the mild bleeding tendency and the in vitro abnormality.