Name: Preparation of Working Solution and Semen Sample for Leukocyte Analysis
Uploaded: 2024-01-19T14:15:00
Description: 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

Analyse af sæd for peroxidase-positive leukocytceller

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.

Forfatter Spotlight: En enkel og omkostningseffektiv metode til leukocytanalyse i sæd 

Undersøgelsen af peroxidase-positive leukocytter i sæd

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  

Forberedelse af arbejdsløsning og sædprøve til leukocytanalyse

Til at begynde med skal du samle reagenserne til fremstilling af arbejdsløsningen og placere dem ved stuetemperatur. Forbered også en uigennemsigtig flaske til opbevaring af arbejdsløsningen og beskyt den mod lys. Derefter overføres 100 mikroliter ammoniumchloridopløsning til den uigennemsigtige reagensflaske ved hjælp af en 200 mikroliter overførselspipette.Der overføres også 100 mikroliter natrium EDTA-opløsning til reagensflasken og blandes ved pipettering. Derefter tilsættes 900 mikroliter orthotoluidinsubstratopløsningen i den uigennemsigtige reagensflaske og blandes forsigtigt ved pipettering. Overfør derefter fem mikroliter hydrogenperoxidopløsning til reagensflasken.Brug en 1000 mikroliter overførselspipette til forsigtigt at blande opløsningen ved pipettering. For at forberede sædprøverne skal du tænde det konstante temperaturstativ og opvarme det op til 37 grader Celsius. Tara også vejevægten for vægten af den polymerbaserede beholder.Efter modtagelse af sædprøven vejes prøven, og detaljerede oplysninger såsom identifikationskode, opsamlingstid, varighed af afholdenhed vejes på ydervæggen af den polymerbaserede beholder. Placer derefter sædprøven på stativet med konstant temperatur for sædfortætning. Træk sædprøven i en polymerbaseret pipette, og kontroller for fortætning hvert femte minut i 30 minutter, indtil sædprøven er fuldt flydende.

Research

JoVE Journal

Medicine

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 ...