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单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,CD157,鉴定粒,/PNH,克隆,唐古生 副主任医师、副教授,、,1,概 要,PNH,概念、发病机制和临床表现,PNH,流式诊断指标变迁及现状,流式,PNH,诊断的临床应用建议,CD157,对,PNH,临床诊断应用的评价,2,精品资料,3,你怎么称呼老师？,如果老师最后没有总结一节课的重点的难点，你是否会认为老师的教学方法需要改进？,你所经历的课堂，是讲座式还是讨论式？,教师的教鞭,“不怕太阳晒，也不怕那风雨狂，只怕先生骂我笨，没有学问无颜见爹娘 ”,“太阳当空照，花儿对我笑，小鸟说早早早”,4,What is,PNH,An acquired clonal disorder resulting,from a somatic mutation of the,X-linked phosphatidylinositol glycan complementation Class A,(,PIGA,) in,a hematopoietic stem cell,This gene encodes an enzyme that catalyzes,the first step in the biosynthetic pathway of,the,glycosylphosphatidylinositol,(,GPI,) anchor,Expansion of this clone is responsible for the,clinical manifestations of the disease,5,6,European Journal of Haematology, 2015,Eculizumab,7,Consequences of Hemolysis in PNH,Defective Red Cells Lack,Complement Defense Proteins,Attack by,Complement,Anemia,Intravascular Hemolysis and,Release of Cell Contents,Hemoglobinemia,Iron Loss,Hemoglobinuria,Nephrotoxicity,Nitric oxide,sequestration,Increased Lactate,Dehydrogenase,Muscle spasm and abdominal pain,Vascular smooth muscle effect Pulmonary hypertension,Platelet activation and thrombosis,8,Pathogenesis of PNH,Deficiency of complement regulatory proteins,is responsible for nearly all the important,pathologic findings in PNH patients, Hemolytic anemia, Thrombosis, Pain, Renal failure,Bone marrow failure,appears to occur by a,different mechanism,9,Classification scheme for PNH,Classical PNH, which includes hemolytic and thrombotic patients;,PNH in the context of other primary disorders, such as aplastica nemia or myelodysplastic syndrome;,Subclinical PNH, in which patients have small PNH clones but no clinical or laboratory evidence of hemolysis or thrombosis.,Blood 2005;106:36993709,International PNH Interest Group (I-PIG),10,Diagnosis of PNH,Ham test, sucrose hemolysis test and the complement lysis sensitivity test,Antibodies against,CD55,and,CD59,on RBCs were the most common flow method used to diagnose PNH until recently,Flow cytometry now universally accepted as the best method of choice of,diagnosing PNH,11,The Problem With CD55/CD59,Sent out 18 specimens to 92 laboratories using stabilized whole blood material,10 had PNH clones with expected values ranging from about 10% to 90%, 8 normal specimens,12,Results of initial EQA study,Labs using only CD55 and CD59 for granulocyte analysis performed poorly,False negative rate of 19% and false positive rate of 17.6%,Ten labs falsely detected clones in 50% of normal specimens,Thus, CD55 and CD59 are not desirable reagents for detecting PNH,13,14,15,Detecting 1% clone sizes,Adequate for patients with large clones associated with hemolytic/thrombotic PNH,Testing only red cells may miss patients,Possible to test both monocytes and granulocytes as one serves as a check for the other,Lymphocytes not a suitable target,Routine Testing,16,High sensitivity assays,Detecting clones as small as 0.01% (or even less),Up to 40% patients with aplastic anemia have identifiable PNH populations,Identification is important because about 20-30% of patients with aplastic anemia progress to PNH,High sensitivity flow cytometry is needed to detect these small clones,17,Prevalence,PNH clones in AA, MDS and other BMF, except clinical PNH.,PNH clones 1% were detected in 199 of all 5398 patients (3.7%);,18.5% in AA; 1.1% MDS ; 2.3% in other BMF,.,PNH clones 0.01% in 167 of 1746 patients from all groups (9.6%) ;,1.8% in MDS; 39.5% in AA, and 7.8% in other BMF patients,.,Up to 35% of PNH patients die within 5 years of diagnosis, and up to 50% of patients die within 1015 years of diagnosis.,A Prospective Multicenter Study of Paroxysmal Nocturnal Hemoglobinuria Cells in Patients with Bone Marrow Failure,18,High sensitivity assays,Sensitivity is determined by a number of factors,Number of events collected,Background rate (frequency of abnormal cells in normal populations),Difference between normal and abnormal population,Need to combine two GPI-linked WBC markers to maximize sensitivity and specificity,19,How many cells to collect,Collecting,250,000,cells of interest (i.e. granulocytes) and requiring 25 events to identify a population would allow a sensitivity of 0.01% with a precision of about 25%; 50 events out of,500,000,gives a precision of 14%,However, it is possible to screen fewer cells,If there are 0 cells with loss of GPI anchors out of 50,000 events, 99+% probability that true frequency is 0.01% (Poisson distribution);,0 out of 30,000 events has 95% probability 10,万，单核细胞计数,2,万。,43,结果,纳入骨髓或外周血标本合计,131,，其中,15,份阳性标本，来自,9,例阳性患者,（,PNH,克隆大小：,0.19%-93.5%,）,Flaer/CD157,组合检出全部,PNH,阳性患者，敏感性、特异性、准确度均达到,100%,。,CD157/Flaer,组合检出的,PNH,克隆与其他组合检出的克隆大小一致。,本研究中常见（,69.8%,，,88/126,）单核细胞,CD14,弱表达而,Flaer,表达正常，外周血与骨髓标本类似。,44,PNH,阴性标本,45,PNH,阳性微小克隆,46,PNH,阳性克隆,47,CD157,异常证实可疑,PNH,克隆,1,例,MDS,患者存在单核细胞和红细胞,PNH,克隆，粒细胞未见,Flaer/CD24,低表达克隆，该患者,CD157,在粒、单核细胞表面均不表达。,48,7,例标本粒,/,单核细胞表面,CD157,表达同时缺失,巨幼贫患者治疗前后,49,讨论,本研究首次在同一标本管内标化各种混杂因素，证实,Flaer/CD157,组合用于识别,PNH,克隆其诊断性能与,Flaer/CD24/CD14,组合无差异，提示其可用于临床替代,CD14,和,CD24,，提高成本效益。,本研究也首次证实部分患者存在,CD157,在粒单核细胞上同时缺失，但不伴有,Flaer,表达下调，临床应用中应注意以免误判。,CD14,在单核细胞上表达弥散，应注意避免影响,PNH,诊断,50,
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