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

पेरोक्सीडेज-पॉजिटिव ल्यूकोसाइट कोशिकाओं के लिए वीर्य का विश्लेषण

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.

लेखक स्पॉटलाइट: वीर्य में ल्यूकोसाइट विश्लेषण के लिए एक सरल और लागत प्रभावी विधि 

वीर्य में पेरोक्सीडेज-पॉजिटिव ल्यूकोसाइट्स की परीक्षा

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  

ल्यूकोसाइट विश्लेषण के लिए कार्य समाधान और वीर्य नमूना तैयार करना

शुरू करने के लिए, काम कर रहे समाधान तैयार करने के लिए अभिकर्मकों को इकट्ठा करने और कमरे के तापमान पर उन्हें जगह है. इसके अलावा, काम करने वाले समाधान को स्टोर करने और इसे प्रकाश से बचाने के लिए एक अपारदर्शी बोतल तैयार करें। फिर अमोनियम क्लोराइड समाधान के 100 माइक्रोलीटर को 200 माइक्रोलीटर ट्रांसफर पिपेट का उपयोग करके अपारदर्शी अभिकर्मक बोतल में स्थानांतरित करें।सोडियम EDTA समाधान के 100 माइक्रोलीटर को अभिकर्मक बोतल में भी स्थानांतरित करें और पाइपिंग द्वारा मिलाएं। इसके बाद, अपारदर्शी अभिकर्मक बोतल में ऑर्थोटोलुइडीन सब्सट्रेट समाधान के 900 माइक्रोलीटर जोड़ें, और धीरे से पाइपिंग द्वारा मिलाएं। फिर हाइड्रोजन पेरोक्साइड समाधान के पांच माइक्रोलीटर को अभिकर्मक बोतल में स्थानांतरित करें।1000 माइक्रोलीटर ट्रांसफर पिपेट का उपयोग करके, धीरे से पाइपिंग द्वारा समाधान मिलाएं। वीर्य के नमूने तैयार करने के लिए, निरंतर तापमान स्टैंड चालू करें और इसे 37 डिग्री सेल्सियस तक गर्म करें। इसके अलावा, बहुलक आधारित पात्र के वजन के लिए वजन पैमाने को टारें।वीर्य का नमूना प्राप्त होने पर, नमूना का वजन करें और बहुलक आधारित पात्र की बाहरी दीवार पर पहचान कोड, संग्रह समय, संयम अवधि जैसी विस्तृत जानकारी रिकॉर्ड करें। अगला, वीर्य द्रवीकरण के लिए निरंतर तापमान स्टैंड पर वीर्य का नमूना रखें। वीर्य का नमूना एक बहुलक आधारित विंदुक में ड्रा करें, और वीर्य नमूना पूरी तरह से तरलीकृत होने तक 30 मिनट के लिए हर पांच मिनट में द्रवीकरण की जांच करें।

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 ...