Antenatal platelet antibody testing by flow cytometry—results of a pilot study

BACKGROUND: Maternal platelet antibodies can cause fetomaternal alloimmune thrombocytopenia (FMAT), which has significant mortality and morbidity even in a first pregnancy. Prenatal diagnosis of FMAT has not previously been possible in the first affected pregnancy. STUDY DESIGN AND METHODS: Using flow cytometry, a sensitive, inexpensive test for the detection of platelet antibodies has been developed. It was adapted for use as a possible antenatal screening test, and 600 pregnant women were tested in a pilot study. RESULTS: In the study group, two women tested positive for platelet-specific IgG antibodies, one for anti-HPA- 1a and the other for anti-HPA-1a with anti-HLA. In each case, the fetus was found to be affected in utero, and treatment was initiated before successful delivery. Another woman was shown to have a platelet- reactive autoantibody without IgG specificity, and her infant was unaffected. A total of 95 (15.8%) of the women tested had HLA antibodies alone, and the majority demonstrated IgG specificity. On follow-up of 62 infants born to these women, none had thrombocytopenia; thus HLA antibodies were not shown to lead to FMAT in this study. CONCLUSION: The flow cytometry-based test for platelet antibodies can detect clinically significant maternal antibodies, and it may be that early diagnosis and treatment in utero can enhance outcome in FMAT. A population screening program is planned to determine the predictive power of this test, in addition to its sensitivity, specificity, and efficiency.     

流式细胞术测定血小板的实验评价

目的探讨流式细胞术(FCM)计数血小板的临床应用价值.方法用FCM的红细胞与血小板比值法(RBC/PLT-R法)计数血小板并与参考方法(手工法)及电阻法进行比较.结果 3种方法计数血小板均有相关性(P<0.001),血小板计数在(1～805)×109/L范围时相关系数>0.97;血小板计数在(1～89)×109/L范围时相关系数>0.84.与参考方法比较,血小板计数>89×109/L时2种方法的相关系数分别为>0.99、>0.95;血小板<89×109/L时,相关系数为>0.97、>0.84.但是对部分病例标本,FCM与电阻法存在较大的误差.结论 FCM法计数血小板优于电阻法,特别对血小板减少症的患者.     

流式细胞术测定血小板的实验评价

目的探讨流式细胞术(FCM)计数血小板的临床应用价值。方法用FCM的红细胞与血小板比值法(RBC/PLT-R法)计数血小板并与参考方法(手工法)及电阻法进行比较。结果3种方法计数血小板均有相关性(P<0.001),血小板计数在(1~805)×109/L范围时相关系数>0.97;血小板计数在(1~89)×109/L范围时相关系数>0.84。与参考方法比较,血小板计数>89×109/L时2种方法的相关系数分别为>0.99、>0.95;血小板<89×109/L时,相关系数为>0.97、>0.84。但是对部分病例标本,FCM与电阻法存在较大的误差。结论FCM法计数血小板优于电阻法,特别对血小板减少症的患者。     

Establishment of a flow cytometric assay for determination of human platelet glycoprotein VI based on a mouse polyclonal antibody

Glycoprotein VI (GPVI) is the major signaling receptor for collagen on platelets. Recent studies suggest that the surface density of GPVI is related to the activation of platelets by collagen. To measure the level of GPVI on platelets, a mouse polyclonal antibody BJ010 was prepared using an amplified fragment of extracellular domain in GPVI. The specific reactivity of BJ010 was identified by anti-GPVI specific monoclonal antibody 11a12 using immunoprecipitation, sandwich enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The antibody BJ010 recognized both native and denatured human GPVI so that it was used to set up a flow cytometric assay to detect the level of GPVI in normal subjects. The relative level of GPVI on platelets was detected in 101 healthydonors. The median geometric mean fluorescence intensity (GMFI) of platelet GPVI level was 57.7. The variation between minimum and maximum values of platelet GPVI and integrin alpha2beta1 were found to be 3.5- and 4.1-fold, respectively, in the normal subjects. There was a week correlation between the amount of GPVI and integrin alpha2beta1 on platelet surfaces. For the method, the intraassay and interassay coefficient of variation was 6.3% and 8.8%, respectively. The flow cytometric assay described here provides a simple, reliable, reproducible, and readily available means of quantitation of collagen receptor GPVI density on the platelet surface in a larger number of blood samples.     

流式细胞仪法用于血小板相关抗体检测和血小板交叉试验

目的 应用流式细胞仪(FCM)进行血小板相关抗体检测和血小板交叉试验。方法 利用血小板抗体阳性、阴性血清探寻FCM法实验条件,血小板经血清致敏,洗涤后,加入荧光标记鼠抗人IgG反应,由FCM获取和分析。52人次正常人血清经FCM法检测后,统计界定正常值范围。1份阳性血清经9个稀释度倍比稀释,分别用FCM法和固相ELISA抗体筛选法检测。31名临床血小板输注无效患者分别用FCM法和抗原捕获酶免分析法(MACE)进行102人次血小板交叉配合试验。结果 FCM法检测正常值为荧光强度比值R<1.156,9个稀释度的阳性血清检测表明,FCM法最高可检稀释度为86.67,固相ELISA法最高可检稀释度为56.34(抗-HPA)和67.25(抗-HLA)。102人次血小板交叉结果显示,FCM法和MACE法无显著性差异(P>0.05)。结论 FCM法可用于血小板相关抗体检测和血小板交叉配血,是一种快速、灵敏的新方法。     

Validation of flow cytometric detection of platelet microparticles and liposomes by atomic force microscopy

Summary. Background: Platelet microparticles (PMPs) are a promising prognostic marker for thrombotic disorders because of their release during platelet activation. The use of flow cytometry for the enumeration of PMPs in plasma has generated controversy due to their size, which is below the stated detection limits of conventional flow cytometry instruments. The potential impact of this is an underestimation of PMP counts. Objectives/Methods: To address this possibility, we used a combination of fluorescence‐activated cell sorting (FACS) and atomic force microscopy (AFM) to determine the size distribution of PMPs present in plasma from acute myocardial infarction (AMI) patients and normal volunteers, and PMPs generated by expired platelet concentrates and washed platelets treated with agonists such as thrombin and calcium ionophore (A23187). Results: According to AFM image analysis, there was no statistically significant difference in height or volume distributions in PMPs from thrombin‐activated, calcium ionophore‐activated, expired platelet concentrates and plasma from healthy volunteers and AMI patients. Based on volume, expired platelets released the greatest proportion of exosomes (< 1.0 × 10^?22 L³ in volume) in relation to the entire PMP population (29.7%) and the smallest proportion of exosomes was observed in AMI patient plasma (1.8%). Moreover, AFM imaging revealed that PMPs from expired platelets exhibited smooth surfaces compared with other PMP types but this was not statistically significant. Conclusions: We confirm that flow cytometry is capable of analyzing PMPs from plasma by using AFM to perform nanoscale measurements of individual PMP events isolated by FACS. This method also provided the first quantitative nanoscale images of PMP ultrastructure.     

Validation of flow cytometric analysis of platelet function in patients with a suspected platelet function defect

Essentials

• The diagnosis of mild platelet function disorders (PFDs) is challenging.
• Validation of flow cytometric testing in patients with suspected PFDs is required.
• Flow cytometry has added value to light transmission aggregometry (LTA) in diagnosis of PFDs.
• There is fair agreement in diagnosing PFDs between LTA and flow cytometry.

Summary

Background

Light transmission aggregometry (LTA) is the most commonly used test for the diagnosis of platelet function disorders (PFDs), but has moderate sensitivity for mild PFDs. Flow cytometry has been recommended for additional diagnostics of PFDs but is not yet standardized as a diagnostic test. We developed a standardized protocol for flow cytometric analysis of platelet function that measures fibrinogen binding and P‐selectin expression as platelet activation markers in response to agonist stimulation.

Objectives

To determine the additional value of flow cytometric platelet function testing to standard LTA screening in a cross‐sectional cohort of patients with a suspected PFD.

Methods

Platelet function was assessed with flow cytometry and LTA in 107 patients suspected of a PFD in whom von Willebrand disease and coagulation factor deficiencies were excluded. Both tests were compared in terms of agreement and discriminative ability for diagnosing patients with PFDs.

Results

Out of 107 patients, 51 patients had an elevated bleeding score; 62.7% of the patients had abnormal platelet function measured with flow cytometry and 54.2% of the patients were abnormal based on LTA. There was fair agreement between LTA and flow cytometry (κ = 0.32). The discriminative ability of flow cytometric analysis in patients with an elevated bleeding score was good (AUC 0.82, 0.74–0.90), but moderate for LTA (AUC 0.70, 0.60–0.80). Both tests combined had a better discriminative ability (AUC 0.87, 0.80–0.94).

Conclusion

Flow cytometric analysis of platelet function has added value in diagnostics of PFDs in patients with unexplained bleeding tendency.     

Residual red cell and platelet content in WBC-reduced plasma measured by a novel flow cytometric method.

BACKGROUND: The levels of residual red blood cells (RBC) and platelets (PLT) in WBC-reduced plasma are often below the lower detection limit of automated blood cell counters. This study established a novel flow cytometric method for the enumeration of residual RBC and PLT in plasma. Furthermore, their levels in WBC-reduced plasma prepared by using various filters were investigated. MATERIALS AND METHODS: WBC-reduced plasma was prepared from two sources: (i) filtration of buffy-coat reduced plasma using dock-on Baxter, Pall and Maco Pharma plasma filters; (ii) filtered whole blood using integral Asahi RZ2000, Maco Pharma LST1, NPBI, and Pall WBF2 whole blood filters. Residual RBC and PLT counts were assessed by using a TruCount tube (Becton Dickinson) containing a known number of lyophilized fluorescent beads. RBC and PLT were labelled with dual monoclonal antibodies, anti-CD41-R-phycoerythrin and anti-glycophorin A-fluorescein isothiocyanide, and analyzed by flow cytometer. RESULTS: The flow cytometric method used in this method can detect residual RBC, PLT as well as RBC-MV simultaneously. The sensitivity of the assay was 50 x 10(6) cells/l with the coefficient of variations < or = 10%. Baxter and Maco Pharma plasma filters consistently reduced both RBC, RBC-MV and PLT to below 50 x 10(6/)l. Plasma derived from day 1 RZ2000 filtered whole blood contained PLT below 50 x 10(6) cells/l, whereas day 0 NPBI filtered whole blood showed the highest level of residual PLT. CONCLUSION: A sensitive and accurate method for the detection of low levels RBC, RBC-MV, and PLT was established to measure their levels in WBC-reduced plasma. The procedure is simple and practical for routine quality monitoring of plasma, as well as for setting a new specification for WBC-reduced plasma.     

Quantitative detection of platelet GPIIb-IIIa receptor antagonist activity using a flow cytometric method

Platelet-membrane surface receptors are important targets for pharmacologic intervention in cardiovascular disease. Among these, glycoprotein (GP) IIb-IIIa is dominant and integrally involved in platelet aggregation and thrombus formation. When activated, GPIIb-IIIa binds soluble fibrinogen (Fb) in a key, early step of this process. New drugs are under development that block Fb binding to GPIIb-IIIa and inhibit platelet aggregation. A thorough understanding of the relationship between circulating drug levels and the extent of GPIIb-IIIa receptor occupancy in humans is crucial for safe and efficacious use of these agents. Described here is the development of a new technique for measurement of GPIIb-IIIa receptor occupancy. In this assay, activated human platelets are incubated with biotinylated fibrinogen (Fb-biotin) followed by antibiotin-FITC. The extent of Fb binding is determined using flow cytometric analysis. Our results indicate that Fb-biotin binds rapidly to activated platelets and its detection is dependent on incubation temperature. Platelets that were pre-incubated with the GPIIb-IIIa antagonist echistatin were inhibited from binding Fb-biotin in a concentration-dependent manner. The fluorescence of processed samples was stable for two weeks in the cold. The assay described here is simple, cost effective, and can be adapted for use in clinical evaluations of these new drugs. J. Clin. Lab. Anal. 12:191–196, 1998. © 1998 Wiley-Liss, Inc.     

Preanalytical requirements for flow cytometric evaluation of platelet activation: choice of anticoagulant

Accurate assessment of in vivo or in vitro platelet activation requires optimal preanalytical conditions to prevent artefactual in vitro activation of the platelets. The choice of anticoagulant is one of the critical preanalytical conditions as anticoagulants exert different effects on the activation of platelets ex vivo. We tested the effectiveness of Diatube-H (also known as CTAD; sodium citrate, theophylline, adenosine and dipyridamole) and citrate vacutainer tubes in preventing artefactual activation of platelets and preserving functional reserve. Platelet surface expression of the CD62P (reflecting alpha granule release), CD63 (reflecting lysosomal release) and modulation of normal platelet membrane glycoproteins CD41a and CD42b, were measured in whole blood and in isolated platelets immediately after collection and at 6, 24 and 48 h after venipuncture. Samples taken into Diatube-H showed less spontaneous platelet activation than did those taken into citrate. To measure in vitro platelet functional reserve, thrombin was added as agonist to blood stored for varying periods up to 48 h. Although Diatube-H suppressed in vitro platelet activation for up to 4 h, in samples kept for 6-24 h before thrombin addition, the inhibitory effect was lost and platelets responded fully to agonist activation. Hence, Diatube-H preserved platelets and allowed for measurement of in vivo platelet activation as well as thrombin-induced in vitro platelet activation after 6-24 h, in both whole blood and isolated platelets.     

Head and neck paragangliomas: a clinicopathologic study with DNA flow cytometric analysis

A total of 11 head and neck paragangliomas were the subject of pathologic study, including histologic, immunohistochemical, and DNA flow cytometric analyses. We cannot absolutely predict aggressive clinical behavior using histologic parameters alone, but we can use such parameters to segregate patients into low-risk and high-risk groups. Several trends were observed in the current study. Tumors with higher S-phase fractions, G2/M fractions, or aneuploid cell populations tended to behave "aggressively." The presence of sustentacular cells in the primary tumors cannot be used as an absolute indicator of tumor metastatic potential, as two metastatic paragangliomas in this study contained sustentacular cells in both the primary and metastatic lesions. DNA ploidy status cannot be used as an absolute prognostic parameter as the two metastatic tumors were composed of diploid primary and metastatic lesions. The three tumors with aneuploid cell populations showed "aggressive" histologic and clinical features, but the length of the follow-up period for these cases is too limited to draw any conclusions. Although no absolute criteria can be used at present to gauge aggressiveness, close follow-up of these patients is essential, especially if pathologic findings suggest an "aggressive" course (ie, "malignant" histology, higher S-phase fractions, G2/M fractions, aneuploid cell populations, or decreased sustentacular cell density).     

化学发光法与ELISA法检测梅毒抗体的一致性比较

目的研究化学发光法与酶联免疫吸附试验(ELISA)法检测梅毒螺旋体特异性抗体的一致性比较。方法分别用ELISA、梅毒螺旋体明胶颗粒凝集试验(TPPA)、微粒子化学发光法(CMIA)检测120例送检梅毒的血清样本。结果 ELISA和TPPA检测梅毒血清结果一致性较好(κ=0.966,P<0.05);CMIA和TPPA检测梅毒血清结果一致性好(κ=0.867,P<0.05);ELISA与TPPA一致性较化学发光法稍差。结论 ELISA和CMIA均与TPPA具有较好的相关性,CMIA敏感性最高,梅毒血清学试验检测可采用CMIA为筛查试验,TPPA作为确证试验,以减小梅毒的漏诊率和假阳性率。     

Monitoring temporary immunodepression by flow cytometric measurement of monocytic HLA-DR expression: a multicenter standardized study

BACKGROUND: Single-center trials have shown that monocytic HLA-DR is a good marker for monitoring the severity of temporary immunodepression after trauma, major surgery, or sepsis. A new test for measuring monocytic HLA-DR is now available. METHODS: We evaluated a new test reagent set for monocytic HLA-DR expression (BD Quantibritetrade mark HLA-DR/Monocyte reagent; Becton Dickinson) in single-laboratory and interlaboratory experiments, assessing preanalytical handling, lyse-no-wash (LNW) vs lyse-wash (LW) values, reference values, and the effect of use of different flow cytometers and different instrument settings on test variance. RESULTS: For preanalytical handling, EDTA anticoagulation, storage on ice as soon as possible, and staining within 4 h after blood collection gave results comparable to values obtained for samples analyzed immediately after collection (mean increase of approximately 4% in monocytic HLA-DR). Comparison of LNW and LW revealed slightly higher results for LNW ( approximately 18% higher for LNW compared with LW; r = 0.982). Comparison of different flow cytometers and instrument settings gave CVs <4%, demonstrating the independence of the test from these variables and suggesting that this method qualifies as a standardized test. CV values from the interlaboratory comparison ranged from 15% (blood sample unprocessed before transport) to 25% (stained and fixed before transport). CONCLUSIONS: For the BD Quantibrite HLA-DR/Monocyte test, preanalytical handling is standardized. Single-laboratory results demonstrated the independence of this test from flow cytometer and instrument settings. Interlaboratory results showed greater variance than single-laboratory values. This interlaboratory variance was partly attributable to the influence of transport and can be reduced by optimization of transport conditions.     

Antigen capture ELISA for platelet antibody detection: choice of conjugate influences assay result

The performance of an anti-IgG/A/M and two anti-IgG alkaline phosphatase (AP) conjugates in a commercial antigen capture ELISA (ACE) were compared for their ability to detect antibodies to human platelet antigens (HPAs) contained in 11 sera. Three anti-HPA-1a and one anti-HPA-3a were not detected by the anti-IgG/A/M conjugate, but both anti-IgG conjugates detected all HPA antibodies as a consequence of increased sensitivity in detecting specific antibody binding. This increase varied from 10% to 500%, depending on the serum being tested. The increased sensitivity in some instances was also accompanied by an apparent increase in nonspecific binding, as measured by activity against irrelevant glycoproteins that should not have been recognized by the relevant HPA antibodies in the sera in question. Hence, anti-IgG conjugates would appear to be preferable for detecting platelet-reactive antibodies in many clinical situations; however, the choice of anti-IgG conjugate should be made judiciously (and appropriately validated), in order to avoid false positive results arising from increased nonspecific binding, which may otherwise be erroneously attributed to the presence of auto-antibodies in the serum under investigation.     

三种流式细胞术计数血小板方法的比较研究

目的　探讨 3种流式细胞术计数血小板方法的临床应用价值。方法　用二维激光散射法 (ADVIA 12 0法 )、红细胞与血小板比值法 (RBC/PLT R法 )、荧光微球绝对计数法 (TruCOUNT法 ) 3种流式细胞术 (FCM)方法计数血小板 ,并与电阻抗法比较。结果　 3种FCM方法计数血小板具有高度相关性 (P <0 0 0 1) ,血小板计数在 ( 1～ 6 46 )× 10 9/L的较宽范围内时 ,相关系数 (r) >0 99;血小板计数在 ( 1～ 89)× 10 9/L的较低范围内时 ,相关系数 (r) >0 95。与TruCOUNT法血小板计数相比 ,ADVIA12 0法计数值偏高 ,RBC/PLT R法偏低。TruCOUNT法与ADVIA 12 0法的相关性最好 (P <0 0 0 1) ,在较低范围血小板计数时的相关系数 (r) >0 98。低血小板计数时 ,ADVIA 12 0法的变异系数 ( 3 46 % )最小 ,TruCOUNT法 ( 5 2 % )次之 ,RBC/PLT R法 ( 5 84% )稍高。电阻抗法与 3种FCM法均有相关性 (P <0 0 0 1) ,但血小板计数比 3种FCM法偏低 (P <0 0 1) ,而且对部分病例标本的血小板计数存在较大误差。结论　 3种FCM法计数血小板均优于电阻抗法。ADVIA 12 0全自动血细胞分析系统最适合于临床常规计数血小板 ,TruCOUNT法是较为低血小板计数和校准血细胞分析仪。对严重血小板减少症的患者 ,若临床需要采取预防性血小板输血时