Determination of GPI-anchored protein expression in bone marrows of normal individuals and patients with paroxysmal nocturnal hemoglobinuria (PNH)

阵发性睡眠性血红蛋白尿(PNH)是一种获得性造血干细胞疾病。PNH是磷脂酰肌醇多糖A类(PIG-A)基因体细胞突变的结果。这个基因的产物在糖基磷脂酰肌醇(GPI)锚的合成的初始阶段是至关重要的，GPI锚是一种将许多重要蛋白质附着到细胞膜上的结构。由于PIG-A基因突变起源于具有分化能力的多能造血干细胞，所有由突变的HSC衍生的祖细胞都存在GPI缺陷，并且细胞表面所有GPI锚定蛋白(GPI-APs)的表达全部或部分丧失。其中一些GPI锚定蛋白，如CD55和CD59，在补体调控中起着重要作用。已知活化的血清补体在溶血性贫血中起关键作用。在正常人中，红细胞不受补体介导的破坏，而那些缺乏锚定蛋白CD55和CD59的人发生溶血，主要是血管内溶血。PNH型克隆的扩大通常与特发性再生障碍性贫血(AA)和骨髓增生异常综合征(MDS)有关，这是免疫介导的正常造血失败的条件。研究表明，在再生障碍性贫血中，高达60%的患者拥有PNH型细胞。在MDS中，大约15%的患者拥有PNH类型的细胞。 目前对PNH患者的诊断和随访是通过基于流式细胞术(FC)的检测来完成的，包括评估外周血细胞中GPI-AP的表达缺陷。除了检测两个GPI-AP CD55和CD59在红细胞和中性粒细胞上的表达外，还出现了一种新的、更敏感的PNH检测方法，包括荧光标记的非活性蛋白溶血素变体(FLAER)。外周血红细胞和白细胞在PNH中得到了广泛的研究。对这些患者骨髓细胞群体的异常进行评估的工作较少。由于GPI-APs在造血细胞成熟的不同阶段有不同的表达，因此骨髓标本通常被认为不适合于外周血，因此，很少有人研究PNH评估。然而，包括骨髓衰竭综合征在内的原因不明的红细胞减少症患者的骨髓抽吸物经常被提交给实验室用于一般诊断目的。 本项目的具体目标：1.分析正常志愿者和外周血(PB)中可检测到PNH的骨髓衰竭患者(如再生障碍性贫血(AA)或骨髓增生异常综合征(MDS))与骨髓细胞群结合的荧光标记溶气素(FLAER)。2.相同细胞类型的骨髓和外周血中PNH克隆大小的比较。3.用临床实验室常规诊断骨髓疾病的抗体组合检测骨髓中的PNH。4.检测同一骨髓标本中PNH细胞和正常细胞的增殖和凋亡情况。 方法：用流式细胞术分析BM细胞中FLAER的结合，以及CD55/CD59与其他抗体组合的表达。流式细胞仪检测细胞增殖和凋亡。 结果：FLAER可与所有正常骨髓细胞结合，但与CD55和CD59结合的红细胞除外。除红细胞外，在各谱系中，FLAER结合随着细胞的成熟而增加，成熟成分的FLAER结合水平最高。在PNH中，淋巴细胞的缺陷(克隆大小)比其他BM细胞小。BM和PB中的PNH克隆大小非常相似，使得BM同样适用于PNH的检测和定量。结合常规诊断抗体(抗CD45、CD13、CD11b、CD64和锚定蛋白CD16、CD14)，也可在骨髓成熟中性粒细胞和单核细胞中检测到PNH克隆，具有较高的特异性和敏感性。目前，正在继续分析数据，以回答基于对先前值的克隆大小的纵向分析来预测克隆大小的变化(具有治疗意义)的问题。 演讲 1.Dulau Florea A，Young NS，Maric I，Braylan RC。人类病理学(海报摘要)。糖基磷脂酰肌醇(GPI)锚定蛋白(AP)在正常人和再生障碍性贫血伴阵发性睡眠性血红蛋白尿症(PNH)患者骨髓中的表达。人类病理学(海报摘要)。USCAP年会2016，西雅图，华盛顿州(2016年3月12-18日)。 2.Dulau Florea A、Young NS、Maric I、Jordan EK、酱C、Ahmad F、Braylan R免疫表型异常提示阵发性睡眠性血红蛋白尿(PNH)可用常规骨髓流式细胞术(BM)检测。人类病理学(海报摘要)。USCAP 2017年年会，德克萨斯州圣安东尼奥(2017年3月4日至10日)。 3.Dulau Florea A，Young NS，Jordan E，Maric I，Braylan R.154骨髓抽吸物与外周血相当，用于检测阵发性睡眠性血红蛋白尿(PNH)。Leuk Res.2017年4月，第55卷：S96(海报摘要)。第14届骨髓增生异常综合征国际研讨会(MDS 2017)，西班牙巴伦西亚(2017年5月3日至6日)。

PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH) is an acquired disorder of the hematopoietic stem cell. PNH is the result of a somatic mutations in the phosphatidylinositol glycan class A (PIG-A) gene. The product of this gene is crucial in the initial stage of synthesis of the glycosylphosphatidyl-inositol (GPI) anchor, a structure which attaches numerous important proteins to cell membranes. Because PIG-A gene mutation originates in a multipotent hematopoietic stem cell capable of differentiation, all progenitor cells deriving from the mutated HSC harbor the GPI defect, and have a complete or partial loss of expression of all GPI-anchored proteins (GPI-APs) on the cell surface. Some of these GPI-anchored proteins, such as CD55 and CD59, play important roles in the control of complement. Activated serum complement is known to play a crucial role in the hemolytic anemia. In normal individuals, red blood cells are protected from complement-mediated destruction, whereas those deficient in anchored proteins CD55 and CD59 undergo hemolysis, predominantly intravascular. Expansion of PNH type clones is commonly associated with idiopathic aplastic anemia (AA) and myelodysplastic syndromes (MDS), conditions of immune-mediated failure of normal hematopoiesis. In was shown that in AA, up to 60% of patients harbor PNH-type cells. In MDS, approximately 15% of patients harbor PNH-type cells. Diagnosis and follow-up of PNH patients is currently done by flow cytometry (FC)-based assays involving evaluation of deficient expression of GPI-APs in PB cells. Besides testing the expression of the two GPI-APs CD55 and CD59 on both red cells and neutrophils, a newer and more sensitive approach for PNH testing emerged, involving fluorescently labeled inactive variant of the protein aerolysin (FLAER). PB red and white cells have been extensively studied in PNH. Less has been done to assess the abnormalities of BM populations in these patients. BM specimens are generally considered less suitable than PB owing to variable expression of GPI-APs during the various stages of hematopoietic cell maturation and for that reason, are rarely studied for PNH evaluation. However, BM aspirates from patients with unexplained cytopenias, including bone marrow failure syndromes, are frequently submitted to laboratories for general diagnostic purposes. Specific goals of this project: 1. Analysis of fluorescently labeled aerolysin (FLAER) binding to bone marrow (BM) cell populations in normal volunteers and patients with BM failure conditions such as aplastic anemia (AA) or myelodysplastic syndrome (MDS) who have detectable PNH in the peripheral blood (PB). 2. Comparison of PNH clone size in BM and PB within the same cell types. 3. Detection of PNH in BM by combinations of antibodies routinely used for diagnosis of BM disorders in clinical laboratories. 4. Assessment of proliferation and apoptosis of PNH cells and normal cells in the same BM samples. Methods: Flow cytometry analysis of FLAER binding, and expression of CD55/CD59 in conjunction with combinations of other antibodies in BM cells. Proliferation and apoptosis assays by flow cytometry. Results: FLAER binds to all normal BM cells, except for erythrocytes, which bind CD55 and CD59. Within each lineage except for RBCs, FLAER binding increases with cell maturation and reaches the highest level on mature elements. In PNH, the defect (clone size) is smaller in lymphocytes than in other BM cells. PNH clone sizes in BM and PB are very similar, making BM equally suitable for PNH detection and quantitation. With combination of antibodies routinely used for general diagnosis (anti CD45, CD13, CD11b, CD64 and the anchored proteins CD16 and CD14) PNH clones can also be detected in BM mature neutrophils and monocytes with high specificity and sensitivity. Currently, data continue to be analyzed to answer the question of predicting variation in clone size (which has therapeutic implications)based on longitudinal analysis of clone sizes at prior values. PRESENTATIONS 1. Dulau Florea A, Young NS, Maric I, Braylan RC. Human Pathology (Poster Abstract). Expression of Glycosylphosphatidylinositol (GPI) Anchor Protein (AP) in Bone Marrows of Normal Subjects and Aplastic Anemia Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH).Human Pathology (Poster abstract).The USCAP Annual Meeting 2016, Seattle, WA (March 12-18, 2016). 2. Dulau Florea A, Young NS, Maric I, Jordan EK, Jiang C, Ahmad F, Braylan R. Immunophenotypic Abnormalities Highly Suggestive of Paroxysmal Nocturnal Hemoglobinuria (PNH) can be Detected with Routine Flow Cytometric Analysis of Bone Marrow (BM). Human Pathology (Poster abstract).The USCAP Annual Meeting 2017, San Antonio, TX (March 4-10, 2017). 3. Dulau Florea A, Young NS, Jordan E, Maric I, Braylan R. 154 Bone Marrow Aspirate Samples are Equal to Peripheral Blood for the Detection of Paroxysmal Nocturnal Hemoglobinuria (PNH). Leuk Res. April 2017, Vol.55: S96 (Poster abstract). The 14th International Symposium on Myelodysplastic Syndromes (MDS 2017), Valencia, Spain (May 3-6, 2017).