Name: Staining of Peroxidase-Positive Leukocytes in Semen Samples
Uploaded: 2024-01-19T14:15:00
Description: Watch this Scientific Journal Video about Staining of Peroxidase-Positive Leukocytes in Semen Samples at JoVE.com

staining of peroxidase-positive leukocytes in semen samples

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

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 Staining of Peroxidase-Positive Leukocytes in Semen Samples at JoVE.com

Staining of Peroxidase-Positive Leukocytes in Semen Samples  

Semen Örneklerinde Peroksidaz Pozitif Lökositlerin Boyanması

Başlamak için, toplanan insan spermi örneklerini sıvılaştırın ve bir pastör pipeti kullanarak iyice homojenize edin. Daha sonra 100 mikrolitre semen örneğini bir santrifüj tüpüne aktarın. Bu tüpe 900 mikrolitre boyama solüsyonu ekleyin.Pipetleme ile tekrar tekrar karıştırın ve reaktanı iyice homojenize edin. Oda sıcaklığında 30 dakika boyunca 200 RPM'de sürekli çalkalama için tüpü iki boyutlu bir çalkalayıcı üzerine yerleştirin. Boyama reaksiyonunu beklerken, CASA sistemini kullanarak spermatozoa konsantrasyonunun analizine devam edin.Sistemi açtıktan sonra SCAscope ikonuna çift tıklayarak uygulama yazılımını açınız. Ardından, semen örneğini pipetleyerek iyice karıştırın ve 10 mikrolitreye kadar olan örneği SCA sayım odasına aktarın. Semen örneğinin sürüklenmesinin durması için 60 saniye bekleyin.Ardından, SCA sayma odasını CASA sisteminin numune tutucusuna yerleştirin. Uygulama yazılımında, diyalog kutusuna hastanın bilgilerini girin. Menideki spermatozoa konsantrasyonunu kaydetmek için başlat düğmesine tıklayın.Ardından, 30 dakikalık boyama reaksiyonu tamamlandığında çalkalayıcıyı durdurun. Analizden önce, santrifüj tüpünün içeriğini pipetleme ile karıştırın. 10 mikrolitreye kadar reaktanı sperm sayma odasına aktarın ve özel bir cam kapak kılıfı ile örtün.Sperm sayma odasını mikroskobik sahneye yerleştirin ve optik mikroskobu açın. Ardından, reaktan numunesinin sürüklenmeyi durdurması için 60 saniye bekleyin. 10x objektif lens altında, bir görüş alanı seçmek için rotayı ve ince ayar düğmelerini ayarlayın.Ardından 40x objektif merceğe geçin ve spermatozoa ve peroksidaz pozitif lökositleri gözlemleyin. En az 200 spermatozoa ve kahverengi peroksidaz pozitif lökosit sayısını saymak için elektronik sayaç sistemini kullanın. Bu teknik kullanılarak, semen örneğinde peroksidaz pozitif lökositler boyandı.Peroksidaz pozitif lökositler, boyanmamış, sıradan, yuvarlak hücrelerle kontrast oluşturan kahverengi bir renk tonu sergiledi.

Research

JoVE Journal

Medicine

Staining of Peroxidase-Positive Leukocytes in Semen Samples

The Examination of Peroxidase-Positive Leukocytes in Semen

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