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 van sperma voor peroxidase-positieve leukocytencellen

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.

Auteur in de schijnwerpers: een eenvoudige en kosteneffectieve methode voor leukocytenanalyse in sperma 

Het onderzoek van peroxidase-positieve leukocyten in sperma

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  

Bereiding van werkoplossing en spermamonster voor leukocytenanalyse

Verzamel om te beginnen de reagentia voor het bereiden van de werkoplossing en plaats ze op kamertemperatuur. Maak ook een ondoorzichtige fles klaar om de werkoplossing op te slaan en te beschermen tegen licht. Breng vervolgens 100 microliter van de ammoniumchloride-oplossing over in de ondoorzichtige reagensfles met behulp van een transferpipet van 200 microliter.Breng ook 100 microliter van de natrium-EDTA-oplossing over in de reagensfles en meng door te pipetteren. Voeg vervolgens 900 microliter van de orthotoluïdine-substraatoplossing toe aan de ondoorzichtige reagensfles en meng voorzichtig door te pipetteren. Breng vervolgens vijf microliter van de waterstofperoxide-oplossing over in de reagensfles.Gebruik een transferpipet van 1000 microliter om de oplossing voorzichtig te mengen door te pipetteren. Om de spermamonsters voor te bereiden, zet u de standaard met constante temperatuur aan en verwarmt u deze tot 37 graden Celsius. Tarreer ook de weegschaal voor het gewicht van de op polymeer gebaseerde houder.Weeg het monster na ontvangst van het spermamonster en noteer gedetailleerde informatie zoals identificatiecode, verzameltijd, onthoudingsduur op de buitenwand van de op polymeer gebaseerde houder. Plaats vervolgens het spermamonster op de standaard met constante temperatuur voor het vloeibaar maken van het sperma. Zuig het spermamonster in een pipet op polymeerbasis en controleer gedurende 30 minuten elke vijf minuten op liquefactie totdat het spermamonster volledig vloeibaar is.

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 ...