CD157在PNH诊断中的应用

1、CD157鉴定粒/单核细胞PNH克隆12020/4/9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22022/8/2What is PNHAn acquired clonal disorder resulting from a somatic mutation of the X-linked phosphatidylinositol glycan complementation Class A (PIGA) in a hematopoietic stem cellThis gene encodes an enz

2、yme that catalyzes the first step in the biosynthetic pathway of the glycosylphosphatidylinositol (GPI) anchorExpansion of this clone is responsible for the clinical manifestations of the disease32022/8/242022/8/2European Journal of Haematology, 2015Eculizumab52022/8/2Consequences of Hemolysis in PN

3、HDefective Red Cells LackComplement Defense ProteinsAttack byComplementAnemiaIntravascular Hemolysis andRelease of Cell ContentsHemoglobinemiaIron LossHemoglobinuriaNephrotoxicityNitric oxidesequestrationIncreased LactateDehydrogenaseMuscle spasm and abdominal pain Vascular smooth muscle effect Pulm

4、onary hypertension Platelet activation and thrombosis62022/8/2Pathogenesis of PNHDeficiency 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 diff

5、erent mechanism72022/8/2Classification scheme for PNHClassical 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 labora

6、tory evidence of hemolysis or thrombosis.Blood 2005;106:36993709International PNH Interest Group (I-PIG)82022/8/2Diagnosis of PNHHam test, sucrose hemolysis test and the complement lysis sensitivity testAntibodies against CD55 and CD59 on RBCs were the most common flow method used to diagnose PNH un

7、til recentlyFlow cytometry now universally accepted as the best method of choice of diagnosing PNH92022/8/2The Problem With CD55/CD59Sent 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

8、102022/8/2Results of initial EQA studyLabs using only CD55 and CD59 for granulocyte analysis performed poorlyFalse negative rate of 19% and false positive rate of 17.6%Ten labs falsely detected clones in 50% of normal specimensThus, CD55 and CD59 are not desirable reagents for detecting PNH112022/8/

9、2122022/8/2132022/8/2Detecting 1% clone sizesAdequate for patients with large clones associated with hemolytic/thrombotic PNHTesting only red cells may miss patientsPossible to test both monocytes and granulocytes as one serves as a check for the otherLymphocytes not a suitable targetRoutine Testing

10、142022/8/2High sensitivity assaysDetecting clones as small as 0.01% (or even less)Up to 40% patients with aplastic anemia have identifiable PNH populationsIdentification is important because about 20-30% of patients with aplastic anemia progress to PNH High sensitivity flow cytometry is needed to de

11、tect these small clones152022/8/2PrevalencePNH 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

12、 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 Failure162022/8/2High sensitivity assaysSensit

13、ivity is determined by a number of factorsNumber of events collectedBackground rate (frequency of abnormal cells in normal populations) Difference between normal and abnormal populationNeed to combine two GPI-linked WBC markers to maximize sensitivity and specificity172022/8/2How many cells to colle

14、ctCollecting 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 cellsIf there are 0 cells with loss o

15、f 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。412022/8/2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。422022/8/2PNH阴性标本432022/8/2PNH阳性微小克隆442022/8/2PNH阳性克隆452022/8/2CD157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。462022/8/27例标本