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

阵发性夜间血红蛋白尿（PNH）是一种获得性造血干细胞疾病。PNH是磷脂酰肌醇聚糖A类（PIG-A）基因体细胞突变的结果。 该基因的产物在合成糖基磷脂酰肌醇（GPI）锚的初始阶段至关重要，GPI锚是将许多重要蛋白质附着到细胞膜上的结构。 因为PIG-A基因突变起源于能够分化的多能造血干细胞，所以从突变的HSC衍生的所有祖细胞都具有GPI缺陷，并且具有细胞表面上的所有GPI锚定蛋白（GPI-AP）的表达的完全或部分丧失。这些GPI锚定蛋白中的一些，如CD 55和CD 59，在补体控制中起重要作用。已知活化的血清补体在溶血性贫血中起关键作用。在正常个体中，红细胞被保护免受补体介导的破坏，而锚定蛋白CD 55和CD 59缺陷的红细胞发生溶血，主要是血管内的溶血。PNH型克隆的扩增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）相关，这是免疫介导的正常造血功能衰竭的病症。 研究表明，在AA中，高达60%的患者携带PNH型细胞。在MDS中，大约15%的患者携带PNH型细胞。 PNH患者的诊断和随访目前通过基于流式细胞术（FC）的测定来进行，所述测定涉及评估PB细胞中GPI-AP的表达缺陷。除了测试红细胞和中性粒细胞上两种GPI-AP CD 55和CD 59的表达外，还出现了一种更新且更敏感的PNH测试方法，涉及荧光标记的蛋白气溶素（FLAER）的无活性变体。PB红细胞和白色细胞在PNH中已被广泛研究。对这些患者的BM群异常进行评估的工作较少。 由于在造血细胞成熟的各个阶段GPI-AP的表达不同，BM标本通常被认为不如PB合适，因此很少研究用于PNH评价。 然而，来自不明原因血细胞减少症（包括骨髓衰竭综合征）患者的BM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。在正常志愿者和具有骨髓（BM）衰竭病症（例如再生障碍性贫血（AA）或骨髓增生异常综合征（MDS），其在外周血（PB）中具有可检测的PNH）的患者中，分析荧光标记气单胞菌溶素（FLAER）与骨髓（BM）细胞群的结合。2.在相同细胞类型内BM和PB中PNH克隆大小的比较。3.通过临床实验室中常规用于诊断BM疾病的抗体组合检测BM中的PNH。4.在相同的BM样品中评估PNH细胞和正常细胞的增殖和凋亡。 方法：流式细胞术分析FLAER结合，以及CD 55/CD 59与其他抗体组合在BM细胞中的表达。通过流式细胞术的增殖和凋亡测定。 结果：FLAER与所有正常骨髓细胞结合，红细胞除外，红细胞与CD 55和CD 59结合。在除RBC外的每个谱系内，FLAER结合随着细胞成熟而增加，并在成熟元件上达到最高水平。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检测和定量。 与常规用于一般诊断的抗体（抗CD 45、CD 13、CD 11b、CD 64和锚定蛋白CD 16和CD 14）组合，也可以在BM成熟中性粒细胞和单核细胞中以高特异性和灵敏度检测PNH克隆。 目前，数据继续进行分析，以回答预测克隆大小的变化（具有治疗意义）的基础上，在以前的值克隆大小的纵向分析的问题。 专题介绍 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）锚蛋白（AP）在正常受试者和阵发性睡眠性血红蛋白尿症（PNH）的再生障碍性贫血患者骨髓中的表达。人类病理学（海报摘要）。USCAP 2016年年会，西雅图，华盛顿州（2016年3月12-18日）。 2. Dulau Florea A，Young NS，Maric I，Jordan EK，Jiang C，Ahmad F，Braylan R.免疫表型异常高度提示阵发性睡眠性血红蛋白尿症（PNH），可通过骨髓（BM）常规流式细胞术分析检测。人类病理学（海报摘要）。USCAP年会2017，圣安东尼奥，德克萨斯州（2017年3月4日至10日）。 3. [10]杨文，杨文. 154例骨髓穿刺标本与外周血检测阵发性睡眠性血红蛋白尿症（PNH）的效果相当。勒克研究，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），瓦伦西亚，西班牙（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).