preparation of working solution and semen sample for leukocyte analysis

Peroksidaz Pozitif Lökosit Hücreleri için Semen Analizi

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.

Yazar Spotlight: Semende Lökosit Analizi için Basit ve Uygun Maliyetli Bir Yöntem 

Semende Peroksidaz Pozitif Lökositlerin İncelenmesi

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Preparation of Working Solution and Semen Sample for Leukocyte Analysis  

Lökosit analizi için çalışma solüsyonu ve semen örneği hazırlanması

Başlamak için, çalışma çözeltisini hazırlamak için reaktifleri toplayın ve oda sıcaklığına yerleştirin. Ayrıca, çalışma solüsyonunu saklamak ve ışıktan korumak için opak bir şişe hazırlayın. Daha sonra 100 mikrolitre amonyum klorür çözeltisini, 200 mikrolitrelik bir transfer pipeti kullanarak opak reaktif şişesine aktarın.Ayrıca 100 mikrolitre sodyum EDTA çözeltisini reaktif şişesine aktarın ve pipetleyerek karıştırın. Daha sonra, opak reaktif şişesine 900 mikrolitre ortotoluidin substrat çözeltisi ekleyin ve pipetleme ile hafifçe karıştırın. Daha sonra beş mikrolitre hidrojen peroksit çözeltisini reaktif şişesine aktarın.1000 mikrolitrelik bir transfer pipeti kullanarak, çözeltiyi pipetleyerek hafifçe karıştırın. Semen örneklerini hazırlamak için sabit sıcaklık standını açın ve 37 santigrat dereceye kadar ısıtın. Ayrıca, polimer bazlı kabın ağırlığına göre tartının darasını alın.Semen örneğini aldıktan sonra, numuneyi tartın ve polimer bazlı kabın dış duvarına tanımlama kodu, toplama süresi, yoksunluk süresi gibi ayrıntılı bilgileri kaydedin. Ardından, semen sıvılaşması için semen örneğini sabit sıcaklık standına yerleştirin. Semen örneğini polimer bazlı bir pipete çekin ve semen örneği tamamen sıvılaşana kadar 30 dakika boyunca her beş dakikada bir sıvılaşma olup olmadığını kontrol edin.

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

Research

JoVE Journal

Medicine

Preparation of Working Solution and Semen Sample for Leukocyte Analysis

83 Görüntüleme. Başlamak için, çalışma çözeltisini hazırlamak için reaktifleri toplayın ve oda sıcaklığına yerleştirin. Ayrıca, çalışma solüsyonunu saklamak ve ışıktan korumak için opak bir şişe hazırlayın. Daha sonra 100 mikrolitre amonyum klorür çözeltisini, 200 mikrolitrelik bir transfer pipeti kullanarak opak reaktif şişesine aktarın.Ayrıca 100 mikrolitre sodyum EDTA çözeltisini reaktif şişesine aktarın ve pipetleyerek karıştırın. Daha sonra, opak reakti...

Video: 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 ...