preparation of working solution and semen sample for leukocyte analysis

Peroxidase-Positive Leukocyte Cells에 대한 정액 분석

Infertility has emerged as a global public health issue affecting approximately 15% of couples in their reproductive age. Male factors are contributed by around 50% of total subfertility cases, and nearly 20% to 30% can be attributed solely to male factors1,2,3. Male genital tract infections (MGTIs) constitute one of the significant causes of male infertility, accounting for about 15% of cases4,5.
Most individuals have leukocytes in their semen, constituting 13% of non-spermatozoan cells, with differential proportions being neutrophils 12%, macrophages 0.9%, and lymphocytes 0.1%6,7. According to the World Health Organization (WHO) laboratory manual for the examination and processing of human semen (5th ed.), leukocytospermia is usually defined as the presence of &#62;1 &#215; 106 cells/mL leucocytes in semen8. This condition can be caused by numerous factors such as environmental toxins, substance abuse, varicoceles, MGTIs, etc., all potentially leading to an abnormal increase in leukocyte concentration in semen9. Leukocytes can elevate reactive oxygen species (ROS) levels in semen, causing lipid peroxidation and oxidative damage to protein and DNA, which results in decreased spermatozoa motility, increased spermatozoa morphological abnormalities, elevated spermatozoa DNA fragmentation index, impairment of the spermatozoa acrosome function, and even negative impacts on embryo development10,11.
Currently, andrologists commonly choose to examine round cells in semen or elastase in seminal plasma when identifying genital tract inflammation. However, it&#39;s often challenging to distinguish sloughed spermatogenic cells and reproductive tract epithelial cells, which do not contribute to reducing the indiscriminate and unnecessary use of antibiotics6. The latter examination method is relatively complicated and time-consuming, with slow result reporting, which is not beneficial for early diagnosis and treatment of diseases such as MGTIs. The American Urological Association (AUA) suggests further differentiation between leukocytes and sloughed cells from the genital tract should be made when the round cell concentration in semen analysis is greater than 1 &#215; 106 cells/mL12. Some andrology labs utilize flow cytometry13 or leukocyte antigen (e.g., CD45) immunocytochemistry14 to examine leukocytes. While these methods are precise, they are expensive and time-consuming, making large-scale clinical implementation difficult, especially in developing countries.
Peroxidase is widely distributed in various types of cells and plays a crucial role in the resistance to oxidative stress damage. Myeloperoxidase (MPO) is a member of the peroxidase subfamily that is generally expressed in immune cells. It is most highly expressed in neutrophils&#39; azurophilic granules15,16 and is also expressed in lymphocytes17,18, monocytes, and macrophages19. The concentration of leukocytes, especially neutrophils, in semen can be obtained by examining round cells that are peroxidase-positive7,20. To make the examination process of leukocytes in semen economical, convenient, and efficient, we reengineered the examination method. Assisted by the computer-assisted semen analysis (CASA) system, the concentration of leukocytes can be obtained within 60 min. This new method reduces the patients&#39; examination costs and waiting time for obtaining results, alleviates the workload of lab technicians, and shortens the doctor&#39;s diagnosis and treatment waiting period.

저자 스포트라이트: 정액의 백혈구 분석을 위한 간단하고 비용 효율적인 방법 

정액에서 과산화효소 양성 백혈구 검사

Male reproductive medicine is my primary research focus, and our protocol primarily addresses the issues of speed and cost in analyzing peroxidase-positive leukocytes in semen. Semen is inherently heterogeneous, which greatly affects the stability of semen analysis results. Therefore, it is essential to ensure that semen is liquified and thoroughly mixed before examination.Our protocol combines manual and automatic methods to calculate the concentration of leukocytes by utilizing sperm concentration, which saves time and is more cost-effective compared to the ready counting leukocytes.

Watch this Scientific Journal Video about Preparation of Working Solution and Semen Sample for Leukocyte Analysis at JoVE.com

Preparation of Working Solution and Semen Sample for Leukocyte Analysis  

백혈구 분석을 위한 작업 용액 및 정액 샘플 준비

시작하려면 작업 용액을 준비하기 위한 시약을 수집하여 실온에 두십시오. 또한 작업 용액을 보관하고 빛으로부터 보호하기 위해 불투명 병을 준비하십시오. 그런 다음 200 마이크로리터 이송 피펫을 사용하여 100 마이크로리터의 염화암모늄 용액을 불투명 시약 병에 옮깁니다.또한 100 마이크로리터의 나트륨 EDTA 용액을 시약 병에 옮기고 피펫팅으로 혼합합니다. 다음으로, 900 마이크로리터의 오르토톨루이딘 기질 용액을 불투명 시약 병에 넣고 피펫팅하여 부드럽게 혼합합니다. 그런 다음 5 마이크로 리터의 과산화수소 용액을 시약 병에 옮깁니다.1000 마이크로리터 이송 피펫을 사용하여 피펫팅으로 용액을 부드럽게 혼합합니다. 정액 샘플을 준비하려면 항온 스탠드를 켜고 섭씨 37도까지 가열합니다. 또한, 폴리머 기반 용기의 중량에 대한 계량 저울을 용기에 담습니다.정액 샘플을 받으면 샘플의 무게를 측정하고 식별 코드, 수집 시간, 금욕 기간과 같은 자세한 정보를 폴리머 기반 용기의 외벽에 기록합니다. 다음으로, 정액 액화를 위해 정액 샘플을 항온 스탠드에 놓습니다. 정액 샘플을 폴리머 기반 피펫에 넣고 정액 샘플이 완전히 액화될 때까지 30분 동안 5분마다 액화 여부를 확인합니다.

preparation-working-solution-semen-sample-for-leukocyte

Research

JoVE Journal

Medicine

Preparation of Working Solution and Semen Sample for Leukocyte Analysis

75 조회수.  The Third Affiliated Hospital of Sun Yat-sen University. 시작하려면 작업 용액을 준비하기 위한 시약을 수집하여 실온에 두십시오. 또한 작업 용액을 보관하고 빛으로부터 보호하기 위해 불투명 병을 준비하십시오. 그런 다음 200 마이크로리터 이송 피펫을 사용하여 100 마이크로리터의 염화암모늄 용액을 불투명 시약 병에 옮깁니다.또한 100 마이크로리터의 나트륨 EDTA 용액을 시약 병에 옮기고 피펫팅으로 혼합합니다. 다음으...

비디오: Preparation of Working Solution and Semen Sample for Leukocyte Analysis  

The Examination of Peroxidase-Positive Leukocytes in Semen

Leukocytospermia can lead to decreased spermatozoa motility, increased spermatozoa morphological abnormalities, elevated spermatozoa DNA fragmentation index, impairment of the spermatozoa acrosome function, and even affected embryonic development. It is a common andrological disease in clinical practice and one of the important causes of male infertility. When determining whether male reproductive tract inflammation exists, andrologists often choose to examine round cells or seminal plasma elastase in the semen as a clinical diagnostic basis. However, the examination of round cells is easily influenced by sloughed spermatogenic cells and reproductive tract epithelial cells, which do not contribute to reducing the indiscriminate and unnecessary use of antibiotics. At the same time, the detection process of elastase is relatively complicated, time-consuming, and slow in reporting results, which is not beneficial for early diagnosis and treatment of diseases such as male genital tract infections (MGTIs). We have innovatively applied the examination of peroxidase-positive leukocytes in semen assisted by a computer-assisted semen analysis (CASA) system as a diagnostic criterion for leukocytospermia, successfully solving these problems. This examination only requires the addition of the operating fluid consisting of four reagents into the specimen, and the total reaction time at room temperature can be controlled within 20-30 min. With the subsequent smear and microscopic examination, the concentration of peroxidase-positive leukocytes in semen can be obtained within a total of 60 min, which can be used to diagnose whether the inflammation of the male reproductive tract existed.

This paper presents an economical and efficient protocol for examining peroxidase-positive leukocytes in semen. With the assistance of a computer-assisted semen analysis (CASA) system, the concentration of peroxidase-positive leukocytes in semen can be obtained within a total of 60 min, effectively improving the efficiency of andrology laboratory and andrologists.

Leukocytospermia can lead to decreased spermatozoa motility, increased spermatozoa morphological abnormalities, elevated spermatozoa DNA fragmentation ...