网织血小板测定对血小板减少疾病诊断价值的探讨

目的　探讨外周血网织血小板 (RP)数值的变化对各型血小板减少疾病的诊断价值 ;外周血RP与骨髓中巨核细胞 (MK)增生程度的关系。方法　以噻唑橙作为RNA的荧光染料 ,利用流式细胞仪测定外周血中含有RNA的RP ,并计算RP(% )及RP绝对值。结果　 (1)正常对照组 :RP(8 4±2 5 ) % ,RP绝对值为 (16 8± 6 8)× 10 9/L。 (2 )原发性血小板减少性紫癜 (ITP)和脾功能亢进症患者RP(% )显著高于正常对照组 ,而RP绝对值显著低于正常对照组 (P <0 0 1) ,在不同MK增生级别的ITP患者中 ,RP(% )、RP绝对值在各组之间均无差异 ;再生障碍性贫血患者RP(% )及绝对值均低于正常对照组 (P <0 0 5 ,P <0 0 1) ;而急性白血病、骨髓增生异常综合征患者RP(% )与正常对照组无明显差异 (P >0 0 5 ) ,但其RP绝对值明显低于正常对照组 (P <0 0 1)。 (3)上述疾病患者 ,经治疗有效者RP(% )恢复正常 ,而无效或疗效欠佳者 ,则RP(% )几无变化。结论　外周血RP测定 ,有助于血小板减少疾病的病因学诊断 ,是血小板减少疾病一有价值的诊断依据 ,也是疗效判断的监控指标。外周血RP与骨髓中MK增生的程度无相关性     

网织血小板检测在血小板减少症中的临床意义

目的 :探讨网织血小板 (RPs)检测在血小板减少症中的临床意义。方法 :采用流式细胞仪技术测定36例血小板减少性疾病患者 [其中特发性血小板减少性紫癜 (ITP) 2 2例 ,慢性再生障碍性贫血 (CAA) 9例 ,脾功能亢进 5例 ]全血中 RPs的比例和绝对值 ,观察 RPs在血小板减少性疾病中的变化 ,并与 2 3例健康对照组进行比较。结果 :健康对照组 RP s百分率和 RPs绝对值为 (2 .5 2± 1.12 ) %和 (3.32± 1.6 0 )× 10 9/ L;ITP患者的 RPs百分率为 (10 .77± 8.42 ) % ,明显高于健康对照组 ,但 RPs绝对值 [(1.74± 3.19)× 10 9/ L]却明显低于对照组 (P<0 .0 1;P <0 .0 5 ) ;CAA及脾功能亢进者的 RPs百分率接近健康对照组水平 ,分别为 (2 .92± 2 .19) %和 (1.82±0 .97) % ,但 RPs绝对值明显低于对照组 [(0 .5 8± 0 .86 )× 10 9/ L ;(0 .5 9± 0 .11)× 10 9/ L ]。结论 :采用本方法测定RPs可靠易行 ,在血小板破坏增多或血小板生成不足所致的血小板减少性疾病中 ,RPs的比例和绝对值均有相应的显著变化。在临床上可作为 ITP诊断的辅助条件 ,并可与其他血小板生成不良性疾病相鉴别     

网织血小板在血小板减少性疾病中的临床意义

网织血小板;血小板减少症特发性血小板减少性紫癜（rrP）是因免疫机制使血小板破坏增多的临床综合征。血小板减少原因归为两类：血小板生成减少和血小板消耗或破坏过多。网织血小板（RP）是新近从骨髓中释放入外周血中的血小板,RP数目反映了人体血小板的更新速度、血小板生成能力。2002年11月-2004年6月我们检测50例健康人、48例ITP病人和其中30例获完全缓解ITP及35例急性白血病（AL）、25例慢性再生障碍性贫血（CAA）外周血的RP％和绝对计数,旨在初步探讨RP在血小板减少性疾病中的临床意义。     

网织血小板检测在特发性血小板减少性紫癜中的临床价值

特发性血小板减少性紫癜(ITP)是一种因为产生抗自身血小板抗体促使血小板减少而使机体器官受损的特异性自身免疫性疾病。血小板减少引起的出血是ITP较常见的表现之一。血小板减少的主要原因大致为两类：血小板生成减少和血小板破坏加速。网织血小板(RP)是新释放入血液循环的血小板，是富含RNA类似于网织红细胞(Ret)的最年轻的血小板。RP的数目反映了人体血小板的更新速度、血小板增生情况。2002年11月～2003年4月我们检测了50例健康人、48例ITP患者、35例急性白血病、25例再生障碍性贫血外周血的RP，旨在探讨RP在特发性血小板减少性紫癜诊断和治疗中的临床价值，以便更好为临床上诊治ITP提供一种新的手段。     

特发性血小板减少性紫癜患者血小板相关抗体及网织血小板检测的临床意义

目的探讨血小板相关抗体(PAIgG)和网织血小板(RP)检测在特发性血小板减少性紫癜(ITP)治疗中的临床意义。方法收集常州市第一人民医院2003-06～2004-10住院的ITP患者48例,根据疗效分为两组,其中临床有效组40例,临床无效组8例,以50例健康人为正常对照。应用流式细胞仪(FCM)测定其治疗前后血浆和(或)血清PAIgG及RP%,并常规血小板计数。结果ITP患者临床有效组治疗后较治疗前血小板计数明显升高、PAIgG和RP%显著降低(P<0.01)。临床无效组治疗前后血小板计数、PAIgG和RP均无明显变化(P>0.05)。两组治疗前3项指标与对照组比较差异有显著性意义(P<0.01)。结论PAIgG和RP动态检测将是ITP患者血小板治疗效果的预测指标之一。     

老年血小板减少患者网织血小板测定及意义

血小板减少症为血液系统常见疾病 ,多见于儿童及中青年 ,老年人相对较少。我们应用流式细胞仪测定了 41例老年血小板减少患者及 50例正常老年人外周血网织血小板 (RP) ,研究其对老年人血小板减少性疾病的诊断价值和意义。1　材料和方法1.1　对象　 41例老年血小板减少患者均来自本院血液科门诊及住院病人 ,根据张之南主编的《血液病诊断及疗效标准》[1],其中特发性血小板减少性紫癜(ITP) 2 6例 ,男 10例 ,女 16例 ,年龄 60～76岁 ,平均 (68 7± 4 7)岁 ;再生障碍性贫血 (AA) 6例 ,男 4例 ,女 2例 ,年龄 61～ 72岁 ,平均 (66 5± 2 6)岁 …     

网织血小板检测及临床意义

巨核细胞数和形态的观察一直作为判断骨髓血小板生成情况的手段应用于临床 ,但骨髓穿刺和骨髓活检不仅给患者带来痛苦 ,而且容易造成人为误差。 1 969年 Ingram和 Coopersmith应用新亚甲蓝染色发现放血后的 Beagle犬外周血液中含有一种清晰的浓染网状结构的血小板 ,被命名为网织血小板 ( Reticulated platelet,简称 RP) ,放血前 RP为0 .8%～ 3.0 % ,放血后 5～ 1 0天增加至 6%以上 ,认为这种 RP与网织红细胞类似 ,能反映骨髓血小板的生成状态〔1,2〕。由于检测技术的进步 ,RP的检测已应用于临床。现就其检测方法及其临床意义作综述。1 …     

特发性血小板减少性紫癜患者网织血小板的测定

目的 :观察慢性血小板减少性紫癜 (c ITP)患者网织血小板百分率 (RP% )和绝对值水平及其在治疗过程中的变化。方法 :采用噻唑橙染色 ,流式细胞仪检测 6 4例正常人和 47例 c ITP患者的 RP%和 RPs绝对值。结果 :c ITP组 RP%〔(2 9.31± 16 .30 ) %〕明显高于对照组〔(5 .83± 1.81) %〕,RPs绝对值明显低于对照组 ,分别为 (6 .33± 4.5 0 )× 10 9/ L 和 (8.70± 2 .6 0 )× 10 9/ L,分别与对照组比较 ,差异有显著性意义 (P <0 .0 5 ) ;患者在接受治疗后 ,RP%和成熟巨核细胞明显降低 ,产板巨核细胞明显增高 ,分别与治疗前比较 ,差异有显著性意义 (P <0 .0 5 ) ;还观察到 ,在血小板升高前 ,RP%升高达峰值 ,此后随血小板升高 ,RP%逐渐下降。结论 :RP%和 RPs绝对值能反映 c ITP患者骨髓血小板生成活性 ,且 RP%能作为血小板恢复的预测指标     

网织血小板的检测及其临床应用

网织血小板是新生成的释放入血液循环中的最年轻的血小板。网络血小板的检测有画于判断血小板减小的原因，也可应用于某些疾病的疗效观察以及预后判断。本文对这方面的研究进行综述。     

山莨菪碱联合血浆置换对老年血栓性血小板减少性紫癜患者网织红细胞计数的影响

<正>据流行病学调查显示血栓性血小板减少性紫癜(TTP)患病率约为3.7/100万〔1〕,TTP以微血管病性溶血性贫血主要特征,常伴有神经精神异常、肾脏损害、发热、出血等症状和体征的血栓性微血管病。老年TTP患者因红细胞变形能力差,红细胞容易破碎,微血管溶血性贫血尤其突出。本文观察山莨菪碱联合血浆置换(PE)对TTP患者细胞百分数(Ret%)的影响。     

Measurement of reticulated platelets by simple flow cytometry: An indirect thrombocytopoietic marker

BACKGROUND: The aim of this study was to determine whether measurement of reticulated platelets (RP) by flow cytometry directly from whole blood, with no fixation or manipulation, is as useful a thrombocytopoietic marker as other more complex techniques. METHODS: RP percentage was prospectively assessed in thrombocytopenic patients (platelets <100x10(9)/L) and non-thrombocytopenic controls using a direct, whole-blood, dual-labelling flow cytometric method. Direct, whole-blood double coverage was achieved using a monoclonal antiglycoprotein (GP)-III antibody (CD61-PerCP(R)) for platelet identification and thiazol orange (Retic-count(R)) as platelet mARN stain. After establishing thrombocytopenia etiology, patients were grouped according to whether their rate of thrombopoiesis was increased or decreased. RESULTS: RP were measured in 53 thrombocytopenic patients with several etiologies and in 53 non-thrombocytopenic controls. The mean RP in 14 thrombocytopenic patients with no increased thrombopoietic activity was 4.8% (95% CI: 3.2-6.4) and the RP absolute number was 1.98x10(9)/L (95% CI: 1.3-2.6). The mean RP in 17 thrombocytopenic patients with increased thrombopoietic activity was 29.4% (95% CI: 24.7-34.1) and the RP absolute number was 7.24x10(9)/L (95% CI: 4.9-9.5). CONCLUSIONS: RP measurement by flow cytometry, directly from whole blood without manipulation, is a useful screening test to differentiate thrombocytopenia with high or low thrombopoietic activity.     

Evaluation of platelet function in thrombocytopenia

Whole blood aggregometry is a functional assay for determination of platelet function. Until now, whole blood aggregometry has not been considered feasible at low platelet counts. Hence, the objectives of the present study were to explore platelet function in thrombocytopenia using a novel index of impedance aggregometry adjusted for platelet count and evaluate the association to platelet function assessed by flow cytometry. Hirudin anticoagulated blood was collected from 20 healthy volunteers, 20 patients with primary immune thrombocytopenia (ITP), and 17 hematological cancer patients. Platelet function was analyzed by impedance aggregometry and by flow cytometry. Collagen, adenosine diphosphate, thrombin receptor agonist peptide-6, and ristocetin were used as agonists for both analyses. Thrombocytopenia in healthy whole blood was induced in vitro employing a recently published method. Platelet aggregation of thrombocytopenic patients was evaluated relative to the aggregation of healthy volunteers at the same platelet count. In flow cytometry, platelet function was described as expression of the platelet surface glycoproteins: bound fibrinogen, CD63, and P-selectin. Similar platelet counts were obtained in the patient groups (p = 0.69) (range: 13–129 × 10⁹/l). Aggregation adjusted for platelet count was significantly increased in ITP patients compared to healthy platelets across all agonists. The platelet aggregation was high in the 95% prediction interval, with 18 ITP patients above the prediction interval in at least two agonists. In contrast, the platelet aggregation was low in the prediction interval in cancer patients, and three cancer patients with platelet aggregation below the prediction interval in at least one agonist. ITP patients displayed increased expression of bound fibrinogen and CD63 following activation, compared with particularly cancer patients, but also compared with healthy platelets. This study demonstrated the feasibility of a novel approach to perform platelet function analyses in thrombocytopenia using impedance aggregometry adjusted for platelet count.     

Immature platelet fraction related parameters in the differential diagnosis of thrombocytopenia

Abstract

The pathogenesis of thrombocytopenia can be divided into increased destruction (ID) of platelets in the peripheral blood and decreased production (DP) of platelets in the bone marrow. This study aimed to analyze the efficacy of immature platelet fraction (IPF) related parameters, including the IPF count (IPF#), IPF percentage (IPF%) and highly fluorescence IPF percentage (H-IPF%), measured by XN-9000, in the differential diagnosis of thrombocytopenia. One hundred and twenty healthy volunteers were enrolled in the healthy control (HC) group, and 180 thrombocytopenia patients were grouped into either the increased destruction (ID) group or the decreased production (DP) group according to their final diagnosis. IPF# was significantly lower in the DP group than in the ID and HC groups (P < .01). Among the three groups, the ID group had the highest IPF% and H-IPF%, and the HC group had the lowest IPF% and H-IPF%. The differences between the three groups were all statistically significant (P < .01). In differentiating the ID patients from the DP patients, the areas under the operating characteristics curve of IPF#, IPF% and H-IPF% were 0.859, 0.944 and 0.930, respectively. False positive rates were below 0.04 when IPF#, IPF% and H-IPF% were above 2.65, 7.55 and 2.35, respectively. IPF related parameters showed high efficacy in the differential diagnosis of thrombocytopenia. However, due to the small numerical values of the IPF related parameters in some thrombocytopenia patients, the fluctuations of IPF% and H-IPF% should also be taken into consideration. Though H-IPF% is a new parameter, its effectiveness in the differential diagnosis of thrombocytopenia is not better than IPF%’s.     

Correlation between immature platelet fraction and reticulated platelets. Usefulness in the etiology diagnosis of thrombocytopenia

Background: Reticulated platelets (RP) are a surrogate marker for megakaryocytic activity, but the limitation of this determination is the lack of standardization of methodology. The determination of the immature platelet fraction (IPF) is performed in a simple, automated, and reproducible way between laboratories. We analyzed the correlation between IPF and RP, and usefulness of IPF in patients with thrombocytopenia. Methods: RP were determined by flow cytometry using double staining with thiazole orange and CD61 PerCP^®. IPF was performed with Sysmex XE2100 analyzer. We used a control group with normal platelets, and thrombocytopenic patients were classified into three groups: Group 1. Central thrombocytopenia, Group 2. Thrombocytopenia as a result of enhanced peripheral platelet destruction, and Group 3. Peripheral non‐immune thrombocytopenia by abnormal distribution. Results: Fourteen controls and 66 patients were analyzed. Group 1: 25 patients, they had mean and confidence interval 95% (95% CI) for IPF 8.67% (6.49–10.46%) and RP 4.08% (2.86–5.30%). Group 2: 20 patients, they had mean and 95%CI for IPF 16.80% (12.20–21.39%) and RP 16.14% (9.89–22.40%). Group 3: 21 patients, they had mean and 95% CI for IPF 9.04% (6.95–11.14%) and RP 5.23% (3.41–7.05%). The overall Pearson linear correlation between IPF and RP was r: 0.65. There were statistically significant differences in values of IPF and RP between Group 2 and the other two groups (P < 0.01). Conclusion: There is a good correlation between IPF and RP mainly in thrombocytopenia by peripheral destruction. Determination of IPF is an easy technique in their implementation, standardized and reproducible, so it could be a useful screening technique in patients with thrombocytopenia.     

Investigation of platelet function and platelet disorders using flow cytometry

Patients with thrombocytopenia or platelet disorders are at risk of severe bleeding. We report the development and validation of flow cytometry assays to diagnose platelet disorders and to assess platelet function independently of platelet count. The assays were developed to measure glycoprotein levels (panel 1) and platelet function (panel 2) in sodium citrated blood. Twenty healthy volunteers and five patients diagnosed with different platelet disorders were included. Glycoprotein expression levels of the receptors Ia, Ib, IIb, IIIa and IX were measured and normalised with forward scatter (FS) as a measurement of platelet size. Platelet function was assessed by CD63, P-selectin and bound fibrinogen in response to arachidonic acid, adenosine diphosphate (ADP), collagen-related peptide, ristocetin and thrombin receptor-activation peptide-6. All patients except one with suspected δ-granule defect showed aberrant levels of glycoproteins in panel 1. Glanzmann's thrombasthenia and genetically verified Bernard–Soulier syndrome could be diagnosed using panel 1. All patients showed reduced platelet function according to at least one agonist. Using panel 2 it was possible to diagnose Bernard–Soulier syndrome, δ-granule defect and GPVI disorder. By combining the two assays, we were able to diagnose different platelet disorders and investigate platelet function independent of platelet count.     

Arachidonic acid causes lysis of blood cells and ADP-dependent platelet activation responses in platelet function tests

The use of arachidonic acid (AA) to stimulate platelets is considered as a specific approach to study aspirin treatment efficacy. However, very high concentrations of AA are used, and it has been previously reported that AA can induce cell lysis in other settings. Several clinical studies have reported decreased responses to AA in whole blood tests in the presence of clopidogrel.

Our aim was to investigate whether unspecific effects contribute to AA-induced aggregation and platelet activation in light transmission aggregometry (LTA) in platelet-rich plasma (PRP), and in assays using whole blood, multiple electrode aggregometry (MEA, Multiplate®), and flow cytometry.

We report that cell lysis, especially of red blood cells, does occur at concentrations of AA used in the clinical tests and that ADP is very important for the AA-induced platelet activation responses. In flow cytometry, very limited platelet activation was detected before reaching AA concentrations in the millimolar range, where cell lysis also occurred, making it problematic to develop a reliable flow cytometry assay using AA as reagent.

We conclude that cell lysis and ADP release contribute to AA-induced platelet responses, most markedly in whole blood assays. This finding could potentially explain some differences between studies comparing methods using whole blood and PRP and also how clopidogrel treatment could influence AA-induced aggregation results in previously published studies. Our findings highlight some issues with AA as reagent for platelet activation, which also have an impact on how platelet activation assays using AA should be interpreted.