Use of flow cytometric light scattering to recognize the characteristic vacuolated marrow cells in VEXAS syndrome

VEXAS中的细胞质空泡主要存在于髓系和红系前体细胞中。通过流式细胞术测量的SSC强度反映了细胞的内部复杂性，并且可以预期空泡的存在增加了SSC光的强度。我们的结果证实了这一点。 我们的分析表明，中性粒细胞，单核细胞和红细胞前体细胞的SSC比显着较高。这种差异在中性粒细胞前体中最为显著，而在单核细胞前体中不太突出，这可能是由于在许多条件下在单核细胞中观察到的频繁胞质空泡化。相比之下，当使用成熟中性粒细胞而不是前体细胞进行测量时，SSC比率没有差异。基于整个VEXAS和对照患者队列的ROC曲线（AUC = 0.97），使用中性粒细胞前体的SSC比截止值11，检测VEXAS病例的阳性预测值（PPV）为91.7%，而阴性预测值（NPV）为96.3%。我们还使用成熟中性粒细胞作为内部对照计算了中性粒细胞前体的SSC强度。 该前体/成熟中性粒细胞SSC比率在VEXAS骨髓中也显示出显著更高的值。然而，成熟的中性粒细胞可能不是理想的对照，因为这些细胞中的低粒度可能导致SSC强度降低。 在VEXAS以外的疾病中，可发现髓系和红系前体细胞中的细胞质空泡。在这项研究中，我们发现，与含有细胞质空泡的前体细胞的非VEXAS骨髓相比，VEXAS病例中的中性粒细胞前体细胞的SSC比率也显著较高。VEXAS患者通常表现为特征性大红细胞性贫血，并可能发生进行性全血细胞减少和骨髓衰竭。当与再生障碍性贫血患者的骨髓相比时，VEXAS病例中骨髓前体中的SSC比率也显著较高，当VEXAS病例与其他条件的骨髓相比时也观察到相同的结果。 骨髓细胞过多伴异型增生常出现在VEXAS综合征中，这些结果通常与非VEXAS MDS中观察到的结果重叠。我们比较了VEXAS骨髓与非VEXAS（UBA 1-WT）患者的MDS骨髓。VEXAS患者骨髓中的中性粒细胞前体细胞显示出比MDS UBA 1-WT骨髓中显著更高的SSC比率。在伴有MDS UBA 1-WT的VEXAS患者的MDS病例中观察到类似结果。在VEXAS队列中，当非MDS与MDS病例相比时，中性粒细胞前体细胞的SSC比率无显著差异。在具有UBA 1-WT的MDS中的髓样细胞中可以发现骨髓化。然而，在3例伴有空泡化的MDS UBA 1-WT骨髓病例中，中性粒细胞前体中的SSC强度低于VEXAS病例，表明MDS（UBA 1-WT）病例中的空泡可能比VEXAS病例少或更小4。 值得注意的是，由于并发浆细胞肿瘤而移植并且骨髓细胞中未显示空泡的VEXAS患者中骨髓前体细胞的SSC强度远低于具有空泡化的VEXAS病例，并且与对照组相当，进一步表明VEXAS患者中中性粒细胞和红细胞前体细胞中较高的SSC确实是由于空泡化。 细胞质空泡不是VEXAS综合征的特异性，因为在其他疾病中也有报道。然而，大量明显空泡化的髓样前体细胞的存在与VEXAS综合征相关，具有良好的敏感性和特异性。 因此，在适当的临床和实验室背景下，检测到来自骨髓或红细胞前体的高SSC强度信号应高度怀疑VEXAS，这是一种必须通过适当的分子检测证实的诊断。 此外，量化细胞空泡化的能力可能有助于评估VEXAS患者的疾病负担。 此外，在VEXAS综合征中通过流式细胞术鉴定空泡化细胞也可以促进突变细胞的分选，这可能有助于这种疾病的功能和生化研究。

Cytoplasmic vacuoles in VEXAS are predominantly found in myeloid and erythroid precursors. SSC intensity measured by flow cytometry reflects the internal complexity of cells and it would be anticipated that the presence of vacuoles increases the intensity of the SSC light. This was confirmed by our results. Our analysis demonstrated a significantly higher SSC ratio for neutrophil, monocytic and erythroid precursors. The difference was most striking among neutrophil precursors and less prominent for monocyte precursors, probably due to the frequent cytoplasmic vacuolization observed in monocytes in many conditions. In contrast, there was no difference in SSC ratios when the measurements were made using mature neutrophils instead of precursors. Based on the ROC curve for the entire VEXAS and control patient cohort (AUC = 0.97), using a SSC ratio cutoff for neutrophil precursors of 11, the positive predictive value (PPV) for detecting VEXAS cases is 91.7% while the negative predictive value (NPV) is 96.3%. We additionally calculated the SSC intensity of neutrophil precursors using mature neutrophils as internal controls. This precursor/mature neutrophil SSC ratio also demonstrated a significantly higher value in VEXAS marrows. However, mature neutrophils may not be ideal controls, since hypogranularity in these cells may result in decreased SSC intensity. Cytoplasmic vacuoles in myeloid and erythroid precursors can be found in conditions other than VEXAS. In this study, we found significantly higher SSC ratios for neutrophil precursors in VEXAS cases also when compared to non VEXAS marrows containing precursor cells with cytoplasmic vacuoles. VEXAS patients usually present with characteristic macrocytic anemia and may develop progressive pancytopenia and bone marrow failure. When compared with marrows with aplastic anemia patients, the SSC ratio in marrow precursors was also significantly higher in VEXAS cases and the same was observed when VEXAS cases were compared to marrows with other conditions. Bone marrow hypercellularity with dysplasia is frequently present in VEXAS syndrome and these findings often overlap with those seen in non-VEXAS MDS. We compared the VEXAS marrows with those with MDS in non-VEXAS (UBA1-WT) patients. The neutrophil precursors in the marrows from VEXAS patients showed significantly higher SSC ratios than in marrows with MDS UBA1-WT. Similar findings were observed in MDS cases of VEXAS patients with MDS UBA1-WT. There was no significant difference in SSC ratio of neutrophil precursors when non-MDS were compared with MDS cases within the VEXAS cohort. Vacuolization may be found in myeloid cells in MDS with UBA1-WT. However, in the three cases of MDS UBA1-WT marrow with vacuolization, the SSC intensity in neutrophil precursors was lower than that in the VEXAS cases, suggesting that the vacuoles in the MDS (UBA1-WT) cases may be less numerous or smaller compared with VEXAS cases4. Of interest, the SSC intensity of marrow precursors in a VEXAS patient who was transplanted due to a concurrent plasma cell neoplasm and did not show vacuoles in the marrow cells was much lower than in VEXAS cases with vacuolization, and comparable to that of the control group, further suggesting that the higher SSC in neutrophil and erythroid precursors in VEXAS patients is indeed due to vacuolization. Cytoplasmic vacuoles are not specific of VEXAS syndrome since they have been reported in other disorders. However, the presence of a high number of significantly vacuolated myeloid precursors has been associated with VEXAS syndrome with an excellent sensitivity and specificity. Thus, in the appropriate clinical and laboratory context, the detection of high SSC intensity signals from myeloid or erythroid precursors should raise high suspicion for VEXAS, a diagnosis that would have to be confirmed by the appropriate molecular testing. Furthermore, the ability to quantify cellular vacuolization may potentially be helpful in assessing disease burden in VEXAS patients. Additionally, the identification of vacuolated cells by flow cytometry in VEXAS syndrome could also facilitate the sorting of mutated cells, which may be useful for functional and biochemical studies in this disease.