异基因造血干细胞移植后网织血小板的临床观察

目的：观察网织血小板(RP)在异基因造血干细胞移植后的变化规律，初步探讨其预测骨髓造血重建的价值。方法：应用荧光染料TO和流式细胞仪动态检测17例异基因造血干细胞移植(allo-HSCT)患者移植后外周血RP％o，同时监测其白细胞数、血小板数的变化。结果：移植后RP％随血小板下降而下降，并最早开始回升，较血小板回升提前9d；再随血小板数的升高，由峰值迅速回落至正常水平。结论：RP％可能更早反映allo-HSCT后造血重建，并不受血小板输注影响。     

辐照与去白细胞过滤单采血小板对血小板诸参数与部分细胞因子含量影响

目的：了解应用去白细胞过滤器制备去白细胞单采血小板以及经γ射线辐照单采血小板血小板参数与血浆部分细胞因子含量变化情况。方法：应用SysmexF-800血液分析仪检测血小板参数与应用ELISA法检测血浆IL-2、IL-6、INF-γ与TNF-α细胞因子。结果：去白细胞单采血小板与经γ射线辐照单采血小板悬液血小板包括：血小板数、血小板平均体积、血小板平均宽度、血小板压积以及血浆IL-2、IL-6、INF-γ与TNF-α含量与对照组相互比较差异均无统计学意义（P〉0．05）。结论：单采血小板经去白细胞或经γ射线辐照后，不会影响血小板参数与血浆部分细胞因子含量。     

脐带血造血干细胞向巨核细胞诱导分化研究进展

正大剂量的放、化疗或进行造血干细胞移植会导致患者血小板急剧减少而致出血,严重时危及生命。输注他人捐献的血小板成为目前主要的治疗手段。但血小板储存时间短、易于激活、储存条件苛刻的原因导致血小板供应紧张[1],且反复输注容     

免疫抑制剂联合脐血治疗重型再生障碍性贫血的机制及临床研究

目的:本研究通过监测细胞因子在治疗前后的变化及脐带血干细胞的植入,探讨免疫抑制剂联合脐血输注(IS+CBI)治疗重型再生障碍性贫血(SAA)的作用机制。方法:选择SAA患者30例(初诊组),均采用IS+CBI治疗。其中23例患者有效(有效组),7例患者无效(无效组)。以20名健康体检者作为对照组。采用ELISA法检测各组外周血中白细胞介素(IL)-22、IL-17、IL-21、干扰素(IFN)-γ、肿瘤坏死因子(TNF)-α的表达水平。使用DNA可变短串联重复序列多态性分析(STR-PCR)检测脐血干细胞的植入情况。结果:1SAA初诊组IL-22、IL-17、IL-21、IFN-γ、TNF-α的表达水平均明显高于对照组(均P0.05);有效组治疗3个月后细胞因子水平较治疗前无明显变化,治疗6、12个月后细胞因子水平明显低于治疗前(P0.05);无效组在治疗前后各细胞因子水平无明显差异。2STR-PCR检测发现治疗后有效组1、3个月脐带血干细胞有嵌合性植入,治疗6个月后均排斥;治疗无效组未观察到有效植入。3与外周血造血干细胞移植(PBSCT)相比,IS+CBI的白细胞、血小板和血红蛋白恢复时间均明显延长(均P0.05),但总有效率为76.67%,与PBSCT的77.08%相比差异无统计学意义。4供患者的人类白细胞抗原(HLA)配型相合性在有效组和无效组间差异无统计学意义。结论:1SAA患者体内IL-22、IL-17水平升高,可能与SAA的发生发展呈正相关;2在治疗过程中存在早期脐带血造血干细胞植入和中晚期免疫抑制剂调节的桥接机制,治疗后前3个月依靠脐血干细胞植入来促进造血恢复,治疗3个月后依靠免疫抑制剂来维持正常造血;3IS+CBI是无合适HLA配型SAA患者有效的治疗方案,供患者的HLA配型相合性与疗效之间无相关性。     

造血干细胞移植与输血

近半个世纪以来 ,造血干细胞移植 (HSCT)已经成为治疗恶性和难治性血液病、某些实体瘤、免疫性缺陷病、先天性和代谢性等疾病的重要手段[1] 。造血干细胞移植前的预处理会造成患者一定时段的“骨髓空虚” ,引起外周血细胞极度低下 ,甚至合并出血和严重感染等 ,输血就成为一个重要的支持治疗措施。随着血液成分分离技术的进步 ,成分输血在造血干细胞移植中已被普遍应用。移植中最常输注的成分血为血小板与红细胞 ,两者的用量约为 10∶1,偶尔也有输注新鲜冰冻血浆、粒细胞以及冷沉淀等的需要[2 ] 。1　血小板的输注不同类型的造血干细胞移植…     

血液病血小板无效输注的原因探讨

目的:探讨输注血小板无效的原因。方法:观察131例血液病患者的659例次单采血小板输注情况及效果并讨论其影响因素。结果:①急性白血病组(AL)、再生障碍性贫血组(AA)、特发性血小板减少性紫癜(ITP)组输注有效率分别为66.49%、71.19%、51.39%(P<0.05);②AL、AA发热组输注有效率低于未发热组(P<0.05);③AL脾脏肿大与无肿大组及弥散性血管内凝血(DIC)组与无DIC组输注有效率差异在统计学有显著差异(P<0.05);④外周造血干细胞移植组、骨髓移植组输注有效率分别为52.54%、38.22%(P<0.05),造血干细胞移植死亡组与未死亡组输注有效率分别为23.21%、52.91%(P<0.05)。结论:引起血小板输注无效的病因复杂。血小板输注应视患者一般状况、血小板计数及出血情况作出综合判断,避免或减少血小板输注无效,提高血小板输注的有效率。     

自体骨髓间充质干细胞联合造血干细胞共移植治疗恶性血液病五例疗效观察

目的 研究自体骨髓间充质干细胞(MSCs)与造血干细胞共移植治疗恶性血液病的安全性和可行性,及其对移植后造血重建的影响.方法 从无骨髓浸润的恶性血液病患者本人骨髓中分离、培养间充质干细胞,经放化疗等预处理后,与造血干细胞共移植治疗恶性血液病患者5例,其中恶性淋巴瘤4例,粒细胞肉瘤1例,并观察其对移植后造血重建的影响.结果 MSCs联合造血干细胞共移植治疗恶性血液病患者5例,MSCs输注过程顺利,未见明显不良反应.移植后造血恢复过程中,中性粒细胞≥0.5×109/L的中位时间为9.4(8～11)d、血小板≥20×109/L的中位时间为12.2(10～14)d.结论 MSCs联合造血干细胞共移植治疗恶性血液病安全性好,未见明显副作用.结果 提示输注MSCs可促进造血恢复,但其远期疗效仍有待于进一步观察.     

难治及复发性急性白血病的造血干细胞移植

造血干细胞移植 ( HSCT)是指用具有正常造血和免疫活性的造血干细胞去替代受者恶变的、缺陷的或缺乏的细胞 ,从而达到造血和免疫功能的重建。1　造血干细胞移植过程1.1　采集骨髓造血干细胞　骨髓造血干细胞采集应在手术室无菌操作 ,术前数天应分次采取 4 0 0～ 80 0 ml自体血备用。采髓前 2小时给予地塞米松 10 mg,可使骨髓有核细胞增多 ,有利于采髓。在髂骨后多点抽取骨髓 ,收集造血干细胞 ,CD34 细胞应 >2× 10 6 /kg,采集的细胞在输注前应行细菌培养。1.2　采集外周血造血干细胞　用肿瘤药物或硫酸葡聚糖或人造血细胞集落刺激因子作…     

用重组人粒-巨噬细胞集落刺激因子动员的血干细胞成功用于自身移植

自身血干细胞移植(ABSCT)可以代替骨髓造血干细胞移植,用于造血系统恶性肿瘤或实体瘤化疗和/或全身照射(TBI)后,以促进骨髓造血功能的恢复。在自身移植时,成功植入的干细胞数目是至关重要的。本文报道用重组人粒-巨噬细胞集落刺激因子(rhu GM-CSF)以增加外周血干细胞的结果     

骨髓移植和胎肝输注治疗急性白血病

随着化疗药物的不断更新,急性白血病的治疗有了显著的进展。但大剂量化疗可导致患者骨髓严重抑制和全身衰竭,同时因多种药物所产生的耐药性,急性白血病化疗后的复发率仍然很高,患者长期存活者也不多。因而使其应用受到了限制。近年来,由于组织相溶性配型技术和造血干细胞研究的进展,造血组织的移植取得了可喜的成果。现已知,造血干细胞是造血组织移植中起主要作用的细胞类型。除外周血有少量造血干细胞外,主要存在于骨髓和胎肝中。骨髓移植或胎肝输注的本质,就是造血干细胞的移植。由于供体或未受肿瘤细胞侵犯的骨髓中含有造血干细胞,可在患者骨髓造血微环境中增殖,故可重建体内正常造血功能,并有效地防止严重     

Use of total leukocyte and platelet counts to guide stem cell apheresis in healthy allogeneic donors treated with G-CSF

Healthy stem cell donors start leukapheresis 4-5 days after starting G-CSF based on the peripheral blood CD34+ cell count (PBCD34). Data from 137 harvests (68 donors) were analyzed to determine correlation between pre-apheresis leukocytes (11.0-94.8x10(9)/l; median 38.8) and platelets (49-374x10(9)/l; median 180), and PBCD34 (3-276/microl; median 40). PBCD34 correlated positively with leukocytes (r=0.48; P<0.0001) and platelets (r=0.40; P<0.0001). When pre-apheresis leukocytes were >or=25 and platelets were >or=100, PBCD34 and CD34+ collection were 5-276/microl (median 57) and 0.5-27.6x10(6)/kg (median 4.7), respectively; significantly higher than PBCD34 of 3-74/microl (median 17) and CD34+ collection of 0.2-8.9 x 10(6)/kg (median 2.2) when leukocytes were <25 and/or platelets were <100. With leukocytes >or=25 and platelets >or=100, PBCD34 was low (<20/microl) 8% of the time, compared to 57% of the time with leukocytes <25 and/or platelets <100 (P<0.0001). Our data suggest that it is not always necessary to measure PBCD34 to guide leukapheresis in healthy donors because pre-apheresis leukocytes and platelets >or=25 and >or=100, respectively, are associated with excellent mobilization. When blood counts do not meet these criteria, PBCD34 should be determined prior to initiation of apheresis.     

Alleviation of myocardial stunning by leukocyte and platelet depletion

Neutrophils accumulate in myocardium rendered ischemic and reperfused. Activated neutrophils release mediators such as metabolites of oxygen that can compromise myocellular integrity and provoke cardiac dysfunction. Although it is established that leukopenia reduces infarct size, the role of leukocytes and the source of free radicals in postischemic contractile dysfunction is unresolved. A carotid left anterior descending coronary-artery extracorporeal circuit without (n = 8) or with a Leukopak filter (n = 6) to deplete the leukocytes and platelets from blood entering the left anterior descending artery was established in the anesthetized, open-chest dog 30 minutes before ischemia. Subendocardial segmental function was monitored by sonomicrometry, and ischemia was produced by stopping flow for 15 minutes followed by 3 hours of reperfusion. Depleting leukocytes by 90 +/- 3.2% and platelets by 100% improved segmental function (from 30.5 +/- 7% to 74.1 +/- 12.7% for control versus leukocyte-depleted dogs, respectively) at 15 minutes of reperfusion. In the leukopenic group, however, there was a progressive decline in contractility to 32.5 +/- 13.8% by 3 hours of reperfusion that was associated with a return of leukocytes and, to a lesser extent, a return of platelets in the extracorporeal blood to 70.2 +/- 21.9% and 15.5 +/- 4.3% of systemic values, respectively. Removal of leukocytes and platelets from blood perfusing the coronary vascular bed only at reperfusion improved contractile function to 67.7 +/- 6.9% at 15 minutes and 54.7 +/- 12.1% at 3 hours (n = 6). Scanning electron microscopy revealed adherent leukocytes in the epicardial coronary arteries of control animals after 3 hours of reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased platelet cytosolic free calcium concentration in essential hypertension

Cytosolic free calcium concentration [Ca2+] was studied in platelets of hypertensive patients with the use of the fluorescent indicator Quin 2/AM. Cytosolic free Ca2+ was significantly higher in platelets of hypertensive patients than in those of normotensive subjects (241 +/- 9 versus 192 +/- 7 nmol/l, n = 58 and 57, respectively P less than 0.001). When all 115 subjects were included, there was a significant correlation between cytosolic free Ca2+ and systolic or diastolic blood pressure (r = 0.262, P less than 0.0025 and r = 0.251, P less than 0.0025, respectively). Intracellular Quin 2 concentration was measured to evaluate the formaldehyde production (a product of Quin 2/AM hydrolysis which has been described as reducing the adenosine triphosphate (ATP) production). The Quin 2 concentrations in platelets of the two groups of subjects were observed to be similar (0.41 +/- 0.03 versus 0.38 +/- 0.03 mmol/l, n = 8 and 7 for hypertensives and normotensives, respectively). The effects of prostaglandin E1 (PGE1), an adenylate cyclase stimulator, on cytosolic free Ca2+ were studied. The presence of 10(-7) mol/l PGE1 lowered the Ca2+ in platelets of hypertensive patients only, suppressing the difference between the two groups.     

Studies in Red Blood Cell Preservation 1. Effect of the Other Formed Elements

The purpose of this study was to determine if the removal of most of the leukocytes and platelets would affect the in vitro characteristics of stored red blood cells (RBC). Fresh RBC concentrates prepared by removing platelet-rich plasma and filtration through Imugard IG 500 filters were compared with unmanipulated units after storage for up to 56 days. The filtered units were significantly better after 56 days storage for supernatant K+ (p = 0.001), hemolysis (p = 0.05), total vesicle membrane protein shed (p = 0.03), and RBC morphology score (p = 0.04). These differences occurred even though ATP levels were well maintained in both groups. The measurements that did not differ significantly were pH, hematocrit, ATP, 2,3-DPG, glucose and supernatant Na+. It is suggested that enzymes, leukotrienes, catecholamines and eicosanoids released by degenerating leukocytes and platelets may be inimical to RBC. Some may act as agonists on alpha-adrenergic and cholinergic muscarinic receptors present on RBC membranes.     

Greater inhibitory effects of bivalirudin compared with unfractionated heparin plus eptifibitide on thrombin-induced platelet activation

OBJECTIVES: The effects of antithrombotic agents on the activation of platelets by thrombin were determined in blood from patients (n=12) with symptomatic coronary artery disease. METHODS: Blood obtained immediately before cardiac catheterization was incubated in vitro with therapeutic concentrations of unfractionated heparin (1.2 and 2.0 U/ml) alone and in combination with eptifibatide (unfractionated heparin 1.2 U/ml+eptifibatide, 1.7 microg/ml) or bivalirudin (8 and 14 microg/ml). An activated clotting time was determined. Platelet activation was induced with thrombin (0, 5, 15, and 40 U/ml) and assessed with the use of flow cytometry. Fibrin polymerization was prevented by the peptide Gly-Pro-Arg-Pro. The activation of glycoprotein IIb-IIIa and surface expression of P-selectin were identified with antibodies (PAC-1 and anti-CD62). RESULTS: The activated clotting time was 258+/-13 s with 1.2 U/ml unfractionated heparin alone, 396+/-8 s with unfractionated heparin+eptifibatide, and 348+/-9 s with 8 microg/ml bivalirudin. The binding of PAC-1 (reflecting the potential to aggregate) was decreased most effectively by bivalirudin (percentage of platelets binding PAC-1 in response to 15 U/ml thrombin-unfractionated heparin=54+/-7%, unfractionated heparin+eptifibatide=12+/-4%, bivalirudin=1+/-0.3% of platelets, P<0.05 for bivalirudin compared with unfractionated heparin or unfractionated heparin+eptifibatide and unfractionated heparin+eptifibatide compared with unfractionated heparin). Bivalirudin prevented thrombin-induced surface expression of P-selectin more effectively than unfractionated heparin alone or unfractionated heparin+eptifibatide (percentage of platelets expressing P-selectin in response to 15 U/ml thrombin-unfractionated heparin alone=74+/-5%, unfractionated heparin+eptifibatide=53+/-7%, bivalirudin=1+/-0.3%, P<0.05 for bivalirudin compared with unfractionated heparin or unfractionated heparin+eptifibatide). CONCLUSION: Comparison between pharmacologic concentrations shown to be therapeutic demonstrated that bivalirudin inhibited thrombin-induced activation of platelets to a greater extent than unfractionated heparin alone or in combination with eptifibatide.     

Effects of the synthetic ether phospholipid KO-286011 on cells and components of rabbit blood

The ether phospholipid KO-286011, which is related to platelet-activating factor (PAF), was examined for PAF-antagonistic action on rabbit platelets both in vitro and ex vivo. It inhibited the aggregation of washed platelets induced by PAF (5 nmol/l) concentration dependently (IC50 = 6.5 X 10(-7) mol/l). After injecting 0.5 mg/kg to rabbits, the ex vivo PAF-mediated aggregation in heparinized platelet-rich plasma was influenced selectively. The inhibition was short and maximal after 5 min. In contrast, hematocrit, counts of peripheral platelets and leukocytes, hemoglobin content, partial thromboplastin time, prothrombin time, and fibrinolytic activity in plasma were not affected. However, the therapeutic range of KO-286011 is limited because of membranolytic activity at higher dosages, leading to hemolysis and drop of circulating platelet counts.     

Elevation of endothelial microparticles, platelets, and leukocyte activation in patients with venous thromboembolism

OBJECTIVES: The purpose of this research was to determine the levels of platelet, leukocyte, and endothelial activation and markers of cellular interactions in patients with venous thromboembolism (VTE). BACKGROUND: The details of interactions between endothelium, platelets, and leukocytes in VTE are not well understood. METHODS: We studied 25 patients with VTE and compared 25 healthy controls. We used flow cytometry to measure: 1) endothelial microparticles (EMP) identified by CD31+/CD42b- (EMP(31)) or E-selectin (EMP(62E)); 2) platelet microparticles (CD31+/CD42b+); 3) surface expression of P-selectin in platelets and CD11b in leukocytes; 4) EMP-monocyte conjugates (percentage of monocytes positive for E-selectin); and 5) platelet-leukocyte conjugates (PLC) expressed as percentage of leukocytes positive for CD41. RESULTS: Patients with VTE had marked elevations of EMP(31) (2,193 vs. 383 counts/microl; p = 0.003), EMP(62E) (368 vs. 223 counts/microl; p = 0.001), and EMP-monocyte conjugates (3.3% vs. 2.5%; p = 0.002), as well as increased activation of platelets (35.2 vs. 5.0 fluorescence intensity units for P-selectin; p < 0.0001) and leukocytes (13.9 vs. 7.7 U for CD11b; p = 0.004). Also elevated in VTE were PLC (61.7% vs. 39.6%; p = 0.01). Expression of CD11b in leukocytes strongly correlated with PLC (r = 0.74; p < 0.0001). CONCLUSIONS: Marked activation of endothelium, platelets, and leukocytes occurs in VTE, and VTE, or the accompanying inflammatory process, involves the release of EMP and formation of EMP-monocyte conjugates and PLC. These findings support prior studies suggesting that release of EMP and their binding to monocytes are key events in thrombogenesis. Our findings also support the concept that the formation of PLC regulates leukocyte activation and participates in linking thrombosis with inflammation.     

Platelet-leukocyte interaction: selective binding of thrombin- stimulated platelets to human monocytes, polymorphonuclear leukocytes, and related cell lines

The association of platelets with leukocytes was investigated, using gel-filtered platelets stimulated with thrombin and then fixed with formaldehyde. Evidence is presented that stimulation of gel-filtered platelets with low concentrations of thrombin (0.01 to 0.1 U/mL) induces the expression of surface determinants interacting strongly with monocytes and polymorphonuclear leukocytes (PMNs) but only weakly with lymphocytes. Both monocyte-platelet binding and PMN-platelet binding occurred at 37 degrees C and more intensively at 0 degrees C; it was Ca2+-dependent and was unaffected by the addition of sodium azide. The binding also occurred with the monocytoid cell lines HL 60 and U 937 in exponential growth and was much less two days after induction of terminal differentiation by phorbol myristate acetate (PMA). No other tested cell lines (B cells, T cells, and myeloid cells) bound thrombin-stimulated platelets. Monocyte-derived macrophages kept in culture for one week also exhibited reduced binding of thrombin- stimulated platelets. IgG and fibronectin could be ruled out as ligands that mediate binding.     

Endothelin-1 induced proinflammatory markers in the myocardium and leukocytes of guinea-pigs: role of glycoprotein IIB/IIIA receptors

AIM: To assess whether endothelin-1 (ET-1) induces the in vivo expression of inflammatory-related proteins, namely cyclooxygenase-2 (COX-2) and tissue factor, in the myocardium and circulating leukocytes of guinea-pigs. The involvement of platelets was also analyzed. METHODS: ET-1 (0.013 microg/min) was infused to male guinea-pigs for 45 min in the presence and absence of tirofiban, a nonpeptidic blocker of the glycoprotein IIb/IIIa receptor (GPIIb/IIIa). Tissue factor and COX-2 expression were determined by Western blot. RESULTS: No changes in mean arterial pressure and heart rate were detected. ET-1-infused guinea-pigs showed a marked increase in the number of platelets expressing activated GPIIb/IIIa receptors (0.8+/-0.03% vs. 6.5+/-0.2%; P<0.05). Tirofiban (10 microg/Kg bw/min) blunted ex vivo platelet aggregation in response to ADP, although only partially reduced COX-2 and tissue factor expression in both the myocardium and leukocytes of ET-1-infused guinea-pigs. The myocardium of platelet-depleted guinea-pigs also showed a reduced COX-2 expression after ET-1 infusion (57+/-3% reduction; P<0.05). In vitro studies demonstrated that platelets (10(7) and 10(9) platelets/well) enhanced ET-1 (10(-7) mol/l)-induced COX-2 expression in heart slices. CONCLUSION: ET-1 stimulated in vivo the expression of the pro-inflammatory proteins COX-2 and tissue factor in the myocardium and in leukocytes by a mechanism GPIIb/IIIa platelet receptors.     

Quality of Red Cell Concentrates in Relation to the Volume of the Buffy Coat Removed by Automated Processing in a Top and Bottom System

The effect of automated removal of increasing volumes of buffy coat in a 'top and bottom' system on the composition of red cell concentrates (RCC) was investigated. The volume of the buffy coat was adjusted to group 1:50 ml (n = 31), group 2: 70 ml (n = 31) and group 3: 100 ml (n = 31), respectively. The numbers of platelets and leukocytes in the buffy coats were comparable between the groups, whereas the red cell volumes in the buffy coats showed a significant difference (17 +/- 3.6 ml group 1, versus 22 +/- 4.1 ml group 2 and 26 +/- 3.88 ml group 3; p less than 0.001). The volumes, hematocrits and cell counts of the RCC were not significantly different. The plasma volumes were inversely correlated with the volume of buffy coat removed, i.e. 268 +/- 19 ml group 1, versus 257 +/- 15 ml group 2 and 233 +/- 20 ml group 3 (p less than 0.001). We conclude that in the 'top and bottom' system an increase of the volume of the buffy coat from 50 to 100 ml did not improve the quality of the RCC regarding contamination with leukocytes and platelets.