Westers Theresia M, Alhan Canan, Visser-Wisselaar Heleen A, Chitu Dana A, van de Loosdrecht Arjan A
Department of Hematology, Amsterdam University Medical Centers, Location VU University Medical Center, Cancer Center Amsterdam, Amsterdam, The Netherlands.
Department of Hematology, HOVON Data Center, Erasmus MC Cancer Institute, Rotterdam, The Netherlands.
Cytometry B Clin Cytom. 2023 Mar;104(2):162-172. doi: 10.1002/cyto.b.22038. Epub 2021 Nov 22.
Flow cytometry is increasingly applied in cytopenic patients suspected for myelodysplastic syndromes (MDS). Analysis includes evaluation of antigen expression patterns in granulocytes of which, for example, partial lack of CD16 may indicate dysplasia, but presence of paroxysmal nocturnal hemoglobinuria (PNH)-type cells should be considered. However, diagnostic bone marrow (BM) samples hamper PNH analysis because immature stages in the granulo-/monocytic compartment lack expression of certain glycophosphatidyl-inositol-anchored proteins. In this prospective study, we evaluated the presence of PNH-type cells in BM next to aberrancies from routine MDS immunophenotyping.
We combined antibodies defining maturation trajectories with FLAER. Validation of the designed method against routine PNH analysis and parallel analysis of BM and blood samples revealed similar results (granulocytes: Wilcoxon p = 0.25 and p = 0.82, respectively). We analyzed BM samples from 134 MDS, 17 chronic myelomonocytic leukemia, 15 aplastic anemia (AA), 1 PNH, 51 non-clonal cytopenic controls, and 12 normal controls.
Most AA/PNH-BM samples showed clear PNH clones: median 1.1% (0%-35%); CD16 loss on mature neutrophils paralleled PNH-clone sizes. In MDS-BM, only 3.7% of cases showed ≥0.1% PNH-type cells, whereas partial CD16 loss was more frequent and abundant.
Our findings confirm that dysplastic features in MDS-BM may point to presence of PNH-type cells, though only few cases displayed FLAER-negative cells. We showed that identification of these cells in the granulocyte compartment of BM specimen is feasible, but-according to international guidelines-results need to be confirmed in peripheral blood.
流式细胞术越来越多地应用于疑似骨髓增生异常综合征（MDS）的血细胞减少患者。分析包括评估粒细胞中的抗原表达模式，例如，CD16部分缺乏可能表明发育异常，但应考虑阵发性睡眠性血红蛋白尿（PNH）型细胞的存在。然而，诊断性骨髓（BM）样本妨碍了PNH分析，因为粒/单核细胞区室中的未成熟阶段缺乏某些糖磷脂酰肌醇锚定蛋白的表达。在这项前瞻性研究中，我们评估了BM中PNH型细胞的存在情况以及常规MDS免疫表型异常情况。
我们将定义成熟轨迹的抗体与FLAER相结合。针对常规PNH分析对设计方法进行验证，并对BM和血液样本进行平行分析，结果相似（粒细胞：Wilcoxon检验p值分别为0.25和0.82）。我们分析了134例MDS、17例慢性粒单核细胞白血病、15例再生障碍性贫血（AA）、1例PNH、51例非克隆性血细胞减少对照和12例正常对照的BM样本。
大多数AA/PNH-BM样本显示出明显的PNH克隆：中位数为1.1%（0%-35%）；成熟中性粒细胞上CD16的缺失与PNH克隆大小平行。在MDS-BM中，只有3.7%的病例显示≥0.1%的PNH型细胞，而部分CD16缺失更频繁且更明显。
我们的研究结果证实，MDS-BM中的发育异常特征可能表明存在PNH型细胞，尽管只有少数病例显示FLAER阴性细胞。我们表明，在BM标本的粒细胞区室中识别这些细胞是可行的，但根据国际指南，结果需要在外周血中得到证实。
