王兆钺2010广州出血性疾病的诊断

1、出血性疾病的诊断 苏州大学附属第一医院 江苏省血液研究所 王兆钺,Initiation of coagulation, new model.,Amplification of coagulation,Propagation of coagulation,实验室检查的方法与步骤,一般先做过筛试验区别初期止血异常和凝血障碍。 1 初期止血检查：血小板计数。血小板数量明显减少时往往需做骨髓检查。如血小板数量基本正常，应检查出血时间。血管异常和血小板疾病都可导致出血时间延长。在怀疑有血小板功能异常时需使用多种诱导剂做血小板聚集试验。如血小板不聚集或聚集明显减低，则可能为血小板无力症。如聚集正常，需进一

2、步观察瑞斯托霉素诱导的血小板凝聚。如该试验异常，应怀疑有血管性血友病的可能。,实验室检查的方法与步骤,2 凝血系统的过筛试验：APTT。血友病A与血友病B患者分别缺乏因子与因子，APTT常明显延长。接触系统的缺陷也使APTT延长，但一般无出血倾向。PT。因子缺乏的患者常有PT延长。如果APTT与PT均有延长，提示有内、外途径和/或共同途径的多因子缺乏，或者有共同途径中某一因子缺乏。如PT和/或APTT异常可被等量的正常人血浆纠正，提示为某个（些）凝血因子缺乏；如不能被纠正，则可能有抗凝物质存在。TT。TT延长见于纤维蛋白原减少、异常纤维蛋白原血症、纤维蛋白（原）降解产物的增加或抗凝物质的存在。

3、,实验室检查的方法与步骤,出血过筛试验可大体估计止血障碍的部位和机理。但这些试验的敏感性与特异性较差，在有关因子减少至30%以下时才有可能出现阳性反应。另一方面，出血过筛试验异常并不等于就是止血系统本身的疾病。在严重的肝功能损伤或口服抗凝药时，PT和APTT常有延长。在尿毒症、多发性骨髓瘤、骨髓增生异常综合症以及服用非甾体抗炎药或其它某些药物时，也可发生出血时间延长与血小板聚集减低。,实验室检查的方法与步骤,在出血过筛试验异常并且临床上怀疑有出血性疾病时，应进一步选择更精确的实验检查以确定诊断。例如，测定血小板膜糖蛋白GPb-与GPb-a对巨大血小板综合症与血小板无力症有确诊的意义。血小板颗粒

4、与致密颗粒释放产物的测定有助于贮存池病的诊断。血栓烷与前列腺素测定是诊断先天性花生四烯酸代谢异常的必要条件。细胞内钙流的测定有助于判断血小板活化过程中信息传导的障碍。,实验室检查的方法与步骤,对怀疑有某个或某些凝血因子缺乏的病人应做有关因子定量与活性分析以明确诊断。例如，在PT与APTT延长怀疑有凝血共同途径异常时，可先测定血浆纤维蛋白原含量，如纤维蛋白原量正常再考虑因子、因子以及凝血酶原的测定。,出血性疾病的基因诊断,临床上实用的方法是进行基因多态性检测。主要的方法有限制性片断长度多态性（RFLPs）与可变数目串联重复序列（VNTR）分析。对因子基因内含子13、22多态性与内含子18双态性的

5、连锁分析可确定90%血友病家系中的患者和携带者。von Willebrand因子基因多态性位点也已发现了40种。基因多态性分析不能直接诊断血友病或其它出血性疾病，但可检出携带者并可用于产前诊断和遗传咨询。,出血性疾病的基因诊断,近年来新出现的DNA芯片技术结合了电子计算机与分子生物学的最新成果，可同时检测上万个DNA片断。DNA芯片将可能为遗传性出血性疾病的基因诊断带来革命性突破。,病例I（HA）男，14岁，右膝关节出血。 出血时间7分，凝血时间47分。 PT9.6秒，TT12秒，APTT118.7秒。 血小板聚集：ADP79.9%，瑞斯托霉素79.1% S-TGT60秒，加血清54.8秒，加

6、吸附血浆10.2秒。 因子VIII:C 1.8%,病例8（AVIII）女，22岁。在14岁以后皮炎，右臂与右腿反复发生血肿。用激素治疗有效，已自行停药一月余。入院前4日又出现右腿肿胀疼痛。无家族史。 凝血时间3小时。APTT98.5秒，PT15秒，TT13秒。纤维蛋白原3g/L。正常血浆与稀释至1/512的患者血浆温育时凝固时间9分20秒（对照3分18秒）。因子活性为2.9%，v WF:Ag125.7%。补做的实验：患者父亲的KPTT和F:C均正常；患者抗F抗体滴度16 Bethesda单位；患者F:Ag1%，而其父为114%。 患者在用激素治疗并输注血浆、因子与丙种球蛋白后出血停止。,病例2

7、（vWD1）男，34岁，龈血、鼻衄。 出血时间14分，凝血时间9.5分。 PT10秒，TT11秒，APTT57秒，纤维蛋白原 3.3 g/L。 血小板聚集：ADP65.3%，瑞斯托霉素6.7%。 因子VIII:C 40%，vWF 23%,患者BSS，女性，22岁，自幼皮肤易出现瘀斑、鼻与牙龈出血。13岁月经初潮后每次出血量大，持续达半月，可引起出血性贫血。患者父母非近亲婚配。父亲两年前死于恶性淋巴瘤。母亲与一兄弟无出血倾向。患者血小板计数为10109/L，血小板巨大，平均血小板体积为17 fl（正常范围为7-11 fl）。最大血小板直径可达8m。出血时间20 min（正常范围 4-8 min）

8、。凝血检查正常。 ADP 2M 诱导的血小板聚集率为12.4%，肾上腺素 4.5M 诱导的血小板聚集率为10%，但瑞斯托霉素12.5 mg/ml不能引起血小板的聚集反应。,图1 BSS患者血小板的光学显微镜与电子显微镜图象,左为患者外周血光学显微镜照片，显示血小板大小与红细胞相当；中为患者血小板电子显微镜照片（10 000倍），显示血小板巨大，颗粒增多；右为正常对照血小板电子显微镜照片（10 000倍）。,A为BSS患者，B为正常对照。括号内为阳性细胞百分数 图 2 流式细胞仪检测血小板膜糖蛋白,A,B,GP- (99.4%),GPb (99.5%）,GPa (94.9%）,阴性对照 （2.5

9、%）,阴性对照 （2.5%）,GPb (14.2%),GP- (21.7%),GPb (99.2%),GPb (98.8%),GPa (97.9%）,1为正常对照；2 为BSS患者。箭头示核苷酸突变部位。 图3 糖蛋白基因DNA测序,1,2,Marker Control Mother Brother Patient,259bp,192bp,67bp,The G to A transition in GP IX gene creates a unique site for restriction enzyme HpyCH4 III.,血小板糖蛋白IX基因在CHO细胞的表达,突变型,野生型,血小板

10、糖蛋白Ib基因在CHO细胞的表达,突变型,野生型,Immunostaining analysis of GP band GP in the cytoplasm of cotransfected cells. (A) Untransfected CHO cells; (B) GP in cells transfected with plasmids for GP b, GP band wild-type GP ; (C) GP in cells transfected with plasmids for GPb, GPb and mutant GP; (D) GPbin cells transf

11、ected with plasmids for GPb, GPband wild-type GP; (D) GPbin cells transfected with plasmids for GPb, GPband mutant GP.,Immunoblotting analysis of GPband GP in transfected CHO cells lysate. GPband GP are all detectable in the cells cotransfected with plasmids for GPb, GPb and wild-type or mutant GP.,

12、A number of GP Ib-IX comples gene mutations have been found. To date, only six mutations were described in GP, which are all located in the extracellular domain. Asp21（GAC）Gly（GGC）and Asn45（AAC） Ser（AGC）compound heterozygous missenseAsn45（AAC）Ser（AGC）homozygous missenseTrp126（TGG）stop（TGA）nonsense m

13、utationCys73（TGG）Tyr（TAT） homozygous missensePhe55（TTT）Ser（TCT） homozygous missenseCys 97 （TGT）Tyr（TAT） homozygous missense,HPLC elution pattern of plasma sterols in subjects with sitosterolemia. A, patient; B, heterozygote; C, control. Peak1, stigmasterol; Peak2, cholestanol; Peak3, sitosterol.,DNA

14、 sequence surrounding nucleotide 18802 of ABCG5 exon 1. (A) DNA sequence of three affected patients. The CT transition was present in the homozygous state. (B) DNA sequence of the patients parents. The CT transition was present in the heterozygous state. (C) DNA sequence of the normal control. ABCG5

15、基因外显子1中18802位CT突变,导致22位的谷氨基酸（Q）变为终止密码子。,郁女，30岁。2月前因妇科病查血发现血小板计数仅2 109/L，多次复查血小板计数为0-10109/L，但无任何出血表现，用大剂量激素冲击治疗与丙种球蛋白治疗无效。骨髓检查正常。PAIg正常，MAIPA示抗GPb 弱阳性，抗GPb与a 阴性。患者血小板计数在EDTA抗凝液中为0，在枸椽酸中为124109/L。,Case 3 was a 13 year-old female. Since the age of 6 months, a hemihypertrophy on the right side of her b

16、ody became gradually apparent. In the age of 10 years, she had a problem of hip dislocation, and then was effectively treated by open reductiona. In the age of 12 years, she suffered from a severe hematochezia. Digital subtraction angiography revealed abnormality of vascular structure in her ascendi

17、ng colon. However, abdominal operation did not find any Meckel diverticulum or vascular tumour in her small intestine. In the age of 13 years, she was admitted to our hospital because of continuing gum bleeding.,患者，男，1970年生，自幼即有皮肤破损后出血不止，愈合缓慢。在肢体碰撞后出现血肿，经保守治疗后可逐渐消退，但无自发性皮肤粘膜出血。16岁时左大腿上部血肿压迫神经引起继发性肌萎

18、缩。两年前因右大腿陈旧性血肿引起肢体强直而手术，取出部分机化的血块。手术期间与术后渗血不止。,3 实验室检查结果凝血过筛试验正常(APTT 38.9s, PT 14.9s, TT 15.3s),纤维蛋白原3.19g/L, 血小板聚集试验正常(ADP 72%,肾上腺素60%, 胶原88%, 瑞斯托霉素2%),出血时间 7min,因子VIII活性72.9%,因子IX活性96.1%,vWF抗原95.3%,FXIII活性100,D-Dimer和FDP阴性,tPA活性0.33AU/ml(0.31AU/ml) tPA抗原20.4ng/ml(21.7/ml) 2AP活性127.7%(98.6%) 优球蛋白溶

19、解时间 70min(正常对照120min)血浆中加入PAI-1(50ng/ml)后延长至120min PAI-1活性0.04 AU/ml(0.41AU/ml) PAI-1抗原5.6ng/ml(44ng/ml),图,1,a PAI,-,1,外显子,2,PCR,产物直接测序（正向）,图,1,a PAI,-,1,外显子,2,PCR,产物直接测序（正向）,Case 3 was a 13 year-old female. Since the age of 6 months, a hemihypertrophy on the right side of her body became gradually

20、apparent. In the age of 10 years, she had a problem of hip dislocation, and then was effectively treated by open reductiona. In the age of 12 years, she suffered from a severe hematochezia. Digital subtraction angiography revealed abnormality of vascular structure in her ascending colon. However, ab

21、dominal operation did not find any Meckel diverticulum or vascular tumour in her small intestine. In the age of 13 years, she was admitted to our hospital because of continuing gum bleeding.,Alterations of DIC markers in two PS cases with giant hemangiomas Platelets APTT PT TT Fibrinogen AT D-dimer

22、(109/L) (s) (s) (s) (g/L) (%) (g/L) Case 1 Before splenectomy 71 61.7 20.1 22.8 0.6 50 19.1 After splenectomy 110 37.2 14.4 18.2 2.91 83 3.22 Case 2 Before splenectomy 81 44.9 14.7 24.0 0.34 70.4 18.0 After splenectomy 138 40.1 14.5 18.2 3.06 98.6 1.95 Normal control 100-300 28-40 10.8-13.5 14.0-21.

23、0 2.00-4.00 70-125 0.01-0.50,A scoring system for diagnozing Proteus syndrome Macrodactyly and/or hemihypertrophy 5 points Plantar or palmar cerebriform hyperplasia 4 points Lipomas/subcutaneous tumours 4 points Epidermal naevus 3 points Macrocephaly and/or skull exostosis 2.5 points miscellaneous o

24、ther minor abnormalities 1 point A score of 13 or greater confirms its diagnosis. Our two patients scored 15.5 and 13 points, respectively, and met the criteria of Proteus syndrome diagnosis.,A 10-year-old girl had recurrent episodes of prolonged bleeding and hematoma starting in her early childhood

25、, which could be stopped by transfusion of fresh frozen plasma or PCC. The coagulation tests revealed a markedly prolonged APTT (61.8-104 s) and TT (36-50.1 s), and a slightly prolonged PT (15.9-25 s). Fibrinogen, prothrombin and other coagulation factors as well as anticoagulant and fibrinolytic sy

26、stems were all normal. Treatment of the patients plasma in vitro with either protamine or heparinase could completely normalize the coagulation abnormalities, but not with normal plasma. The anticoagulant activity of her plasma corresponded to 0.2 heparin U/mL, and concentration 0.22 heparin U/mL. In ex vivo study, the abnormal coagulation tests could effectively be corrected when the patient was intravenously administed with protamine.,有出血性疾病的患者禁止使用影响止血与凝血的药物，如阿司匹林一类的解热止痛剂