Study question Is Myeloperoxidase Kit, validated for blood but not for semen, a valuable alternative for Leucoscreen to differentiate round cells in semen? Summary answer Myeloperoxidase Kit is a non-toxic alternative for LeucoScreen to differentiate round cells in semen, with the advantage of assessing a 1:2 dilution of semen. What is known already As stated in the WHO laboratory manual, the routine assay of peroxidase activity is useful as screening technique for quantifying the leucocyte population in semen samples. The clinically relevant neutrophil leucocytes stain peroxidase positive and can hereby be differentiated from spermatids and spermatocytes. The commercially available kit, Leucoscreen (Fertipro, Beernem, Belgium) uses carcinogenic ortho-toluidine and was therefore withdrawn from the market and an alternative, LeucoScreen Plus, was suggested. A disadvantage of LeucoScreen Plus is the necessity to dilute the sample 1:6. Testing an alternative inexpensive, quick and not carcinogenic method to differentiate round cells without excessive dilutions, is worth investigating. Study design, size, duration Intra- and inter-variability of Myeloperoxidase Kit (Ral Diagnostics, MLS NV, Menen, Belgium) was assessed by evaluating 2 semen samples 10 times by one technician, and 10 semen samples by 2 technicians, respectively. The stability of the working solution for 5 days was tested on 10 semen samples. The accuracy was tested on 80 samples by comparing Myeloperoxidase Kit with LeucoScreen. The study was performed from July till November 2020. Participants/materials, setting, methods Working solution was made from the Myeloperoxidase Kit (3/5 bottles): 1 ml alphanaphtol solution, 4 ml pyronine solution and 1 droplet hydrogen peroxide. Semen samples brought to the Andrology Lab for diagnostic evaluation and presenting with round cells were included. To differentiate peroxidase positive leucocytes (dark pink with dark granules) from other round cells (unstained or pink), 10 µl solution was mixed with 10 µl of semen and assessed with phase contrast at 400x. Main results and the role of chance The intra-variability of round cell differentiation was acceptable with %CV 6.6% for sample 1 (mean ± SD: 30.8% ± 2.04%) and 4.1% for sample 2 (mean ± SD: 56.7% ± 2.31%), both lower than the 10% criterium. The inter-variability was acceptable according to Passing&Bablok (95%CI Intercept A: -1.25 to 0.36; 95%CI Slope B: 0.92 to 1.11) and Spearman rank correlation coefficient (r = 0.983 with P < 0.001). The stability of the working solution after 5 days, kept at room temperature during the day and at 4 °C at night, compared with freshly made working solution was adequate according to Passing&Bablok (95%CI Intercept A: -2.33 to 0.47; 95%CI Slope B: 0.85 to 1.17) and Spearman rank correlation coefficient (r = 0.991 with P < 0.0001). Also the accuracy could be accepted as the Bland-Altman plot shows a substantial agreement between Leucoscreen and Myeloperoxidase Kit (difference 0.6%) without a proportional bias. Limitations, reasons for caution Granules in neutrophils stain differently with Myeloperoxidase Kit, with Leucoscreen and with Leucoscreen Plus, which requires a short adaptation when starting with a new kit. Wider implications of the findings The use of Myeloperoxidase Kit to differentiate round cells in semen is cheap, non-toxic and easy. It is a valuable asset in the andrology lab because semen samples with low round cell concentrations can be counted in a 1:2 dilution and the working solution can be kept for 5 days. Trial registration number None

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O-136 髓过氧化物酶试剂盒作为 LeucoScreen 区分精液样本中圆形细胞的有价值替代品

研究问题 髓过氧化物酶试剂盒是否适用于血液但未经过精液验证，是 Leucoscreen 区分精液中圆形细胞的有价值的替代品吗？摘要回答髓过氧化物酶试剂盒是 LeucoScreen 用于区分精液中圆形细胞的无毒替代品，其优势在于可以评估 1:2 稀释的精液。已知情况 如 WHO 实验室手册中所述，过氧化物酶活性的常规测定可用作定量精液样本中白细胞群的筛选技术。临床相关的嗜中性白细胞染色过氧化物酶阳性，因此可以与精子细胞和精母细胞区分开来。市售试剂盒 Leucoscreen (Fertipro, Beernem, Belgium) 使用致癌邻甲苯胺，因此从市场上撤出，替代品 LeucoScreen Plus，有人建议。LeucoScreen Plus 的一个缺点是必须按 1:6 稀释样品。测试一种替代的廉价、快速且不致癌的方法来区分圆形细胞而无需过度稀释，值得研究。髓过氧化物酶试剂盒（Ral Diagnostics，MLS NV，Menen，Belgium）的研究设计、大小、持续时间的内部和内部变异性通过由一名技术人员评估 2 个精液样本 10 次和由 2 名技术人员分别评估 10 个精液样本来评估。在 10 个精液样本上测试了工作溶液 5 天的稳定性。通过比较髓过氧化物酶试剂盒与 LeucoScreen 对 80 个样本的准确性进行了测试。该研究于 2020 年 7 月至 2020 年 11 月进行。参与者/材料、设置、方法工作溶液由髓过氧化物酶试剂盒（3/5 瓶）制成：1 ml alphanaphtol 溶液，4 ml pyronine 溶液和 1 滴过氧化氢。包括带到男科实验室进行诊断评估并呈现圆形细胞的精液样本。为了区分过氧化物酶阳性白细胞（深粉色和深色颗粒）与其他圆形细胞（未染色或粉色），将 10 µl 溶液与 10 µl 精液混合，并在 400x 下使用相差评估。主要结果和机会的作用 圆形细胞分化的内部变异性是可以接受的，样本 1 的 %CV 为 6.6%（平均值 ± SD：30.8% ± 2.04%）和样本 2 的 4.1%（平均值 ± SD：56.7% ± 2.31%），均低于 10% 的标准。根据 Passing&Bablok（95%CI 截距 A：-1.25 到 0.36；95%CI 斜率 B：0.92 到 1.11）和 Spearman 等级相关系数（r = 0.983，P < 0.001），相互变异性是可以接受的。根据 Passing&Bablok（95%CI 截距 A：-2.33 至 0.47；95% CI 斜率 B：0.85 至 1.17）和 Spearman 等级相关系数（r = 0.991，P < 0.0001）。准确度也可以接受，因为 Bland-Altman 图显示 Leucoscreen 和髓过氧化物酶试剂盒（差异 0.6%）之间存在显着一致性，没有比例偏差。限制，谨慎的原因 粒过氧化物酶试剂盒、Leucoscreen 和 Leucoscreen Plus 对中性粒细胞颗粒的染色不同，在开始使用新试剂盒时需要短暂适应。研究结果的更广泛意义使用髓过氧化物酶试剂盒来区分精液中的圆形细胞很便宜，无毒且容易。它是男科实验室的宝贵资产，因为可以在 1:2 稀释液中计数低圆形细胞浓度的精液样本，并且工作溶液可以保存 5 天。试用注册号 无