preparation of working solution and semen sample for leukocyte analysis

Análise de sêmen para células leucocitárias positivas para peroxidase

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.

Autor em destaque: um método simples e econômico para análise de leucócitos em sêmen 

O Exame de Leucócitos Peroxidase Positivos no Sêmen

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  

Preparação de solução de trabalho e amostra de sêmen para análise leucocitária

Para começar, recolha os reagentes para preparar a solução de trabalho e coloque-os à temperatura ambiente. Além disso, prepare um frasco opaco para armazenar a solução de trabalho e protegê-la da luz. Em seguida, transfira 100 microlitros da solução de cloreto de amônio para o frasco de reagente opaco usando uma pipeta de transferência de 200 microlitros.Transfira também 100 microlitros da solução de EDTA de sódio para o frasco de reagente e misture por pipetagem. Em seguida, adicione 900 microlitros da solução de substrato ortotoluidina no frasco de reagente opaco e misture suavemente por pipetagem. Em seguida, transfira cinco microlitros da solução de peróxido de hidrogênio para o frasco de reagente.Usando uma pipeta de transferência de 1000 microlitros, misture suavemente a solução por pipetagem. Para preparar as amostras de sêmen, ligue o suporte de temperatura constante e aqueça-o até 37 graus Celsius. Além disso, tare a balança para o peso do recipiente à base de polímero.Após o recebimento da amostra de sêmen, pesar a amostra e registrar informações detalhadas, como código de identificação, tempo de coleta, duração da abstinência na parede externa do recipiente à base de polímero. Em seguida, coloque a amostra de sêmen no suporte de temperatura constante para liquefação do sêmen. Retirar a amostra de sémen para uma pipeta à base de polímero e verificar a liquefação de cinco em cinco minutos durante 30 minutos até que a amostra de sémen esteja totalmente liqueficada.

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Research

JoVE Journal

Medicine

Preparation of Working Solution and Semen Sample for Leukocyte Analysis

75 visualizações.  The Third Affiliated Hospital of Sun Yat-sen University. Para começar, recolha os reagentes para preparar a solução de trabalho e coloque-os à temperatura ambiente. Além disso, prepare um frasco opaco para armazenar a solução de trabalho e protegê-la da luz. Em seguida, transfira 100 microlitros da solução de cloreto de amônio para o frasco de reagente opaco usando uma pipeta de transferência de 200 microlitros.Transfira também 100 microlitros da solução de EDTA de sódio para o frasco de reagente e misture por pipetagem. Em s...

Vídeo: 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 ...