Strobel Sebastian, Hesse Naomi, Santhanakumaran Vidiyaah, Groeschel Samuel, Bruchelt Gernot, Krägeloh-Mann Ingeborg, Böhringer Judith
Department of Pediatric Neurology, University Children's Hospital Tübingen, Hoppe-Seyler-Straße 1, 72076 Tübingen, Germany.
Cells. 2020 Nov 28;9(12):2553. doi: 10.3390/cells9122553.
(1) Lysosomal storage diseases are rare inherited disorders with no standardized or commercially available tests for biochemical diagnosis. We present factors influencing the quality of enzyme assays for metachromatic leukodystrophy (MLD) and gangliosidoses (GM1; GM2 variants B and 0) and validate the reliability and stability of testing in a retrospective analysis of 725 samples. (2) Patient leukocytes were isolated from ethylene-diamine-tetra-acetic acid (EDTA) blood and separated for subpopulation experiments using density gradient centrifugation or magnetic cell separation. Enzyme activities in whole leukocyte lysate and leukocyte subpopulations were determined. (3) The enzyme activities in leukocyte subpopulations differed significantly. Compared to lymphocytes, the respective enzyme activities were 2.31-4.57-fold higher in monocytes and 1.64-2.81-fold higher in granulocytes. During sample preparation, a considerable amount of the lysosomal enzymes was released from granulocytes. Nevertheless, with the sample preparation method used here, total leukocyte count proved to be more accurate than total protein amount as a reference unit for enzyme activities. Subsequent analysis of 725 individuals showed clear discrimination of enzyme activities in patient samples (48 MLD; 21 gangliosidoses), with a sensitivity of 100% and specificity of 98-99%.
(1) 溶酶体贮积症是罕见的遗传性疾病，目前尚无用于生化诊断的标准化或商业可用检测方法。我们介绍了影响异染性脑白质营养不良（MLD）和神经节苷脂病（GM1；GM2变异型B和0）酶分析质量的因素，并通过对725份样本的回顾性分析验证了检测的可靠性和稳定性。(2) 从乙二胺四乙酸（EDTA）抗凝血中分离患者白细胞，并使用密度梯度离心或磁性细胞分离法分离用于亚群实验。测定全白细胞裂解物和白细胞亚群中的酶活性。(3) 白细胞亚群中的酶活性差异显著。与淋巴细胞相比，单核细胞中相应的酶活性高2.31 - 4.57倍，粒细胞中高1.64 - 2.81倍。在样本制备过程中，相当数量的溶酶体酶从粒细胞中释放出来。然而，采用此处使用的样本制备方法，总白细胞计数被证明比总蛋白量作为酶活性的参考单位更准确。随后对725名个体的分析显示，患者样本（48例MLD；21例神经节苷脂病）中的酶活性有明显区分，灵敏度为100%，特异性为98 - 99%。
