Sperm DNA - BioMEDIA

Kit to asses sperm DNA fragmentation

SD-100

Sperm DNA

Kit to asses sperm DNA fragmentation

A. USAGE:

To assessment sperm DNA fragmentation index (SDFI) using bright field microscopy.

B. CONTENTS:

C. PRECAUTIONS:

All patient samples should be considered potentially infectious and the user must wear protective gloves, eye protection, face mask and laboratory coats when performing the test and take off contaminated parts immediately.
After contact with skin wash immediately with water and soap.
The test should be discarded properly after use (biohazard).
Do not use after expiration date, which appears on the package label.

D. WARNINGS:

Care should be taken to avoid contact with skin or eyes, and to prevent inhalation.
Work under air removal environment.
Do not release the products used into the environment.

E. STORAGE:

F. PEPARATION OF AGAROSE:

Place the agarose vial tube into the float and melt using a water bath or a beaker with water on a hot plate at 95 – 100 °C for 5 minutes or until it is completely melted then quickly divide the agarose into Eppendorf tubes with 100 µl each.

G. PREPARATION OF SLIDE: (Set the kit at room temperature during the whole process)

To avoid sperm crowding in high concentration sample dilute the sperm sample in an appropriate sperm wash medium to reach 20 million/ml.
Take 50 µl from sperm sample, mix with 100 µl melted agarose at 37 °C in Eppendorf tubes.
Add the 10 µl prepared sample to the slide with coverslip then transfer to cold surface (ex. Fridge) at 4°C for 5 minutes to solidify the agarose.
Take the slide out of the fridge and remove the coverslip by sliding it off gently.

H. PROCESSING THE SLIDE: (All the processing must be performed at room temperature)

Apply Denaturation solution on the slide making sure it is fully covered by the reagent during the whole process, incubate for 7 minutes, and then remove the reagent.
Apply Lysis solution on the slide making sure it is fully immersed, incubate for 20 minutes, and then remove the reagent.
Wash the slide with distilled water for 5 minutes.
Dehydrate with 70 % ethanol for 2 minutes.
Drain with 100 % ethanol for 2 minutes.

NOTE:

Processed slides may be kept in slide boxes at room temperature in a dry and dark place for several months.

I. STAINING PROCESS:

Apply Stain (A) for 10 minutes, aspirate the excess of stain (A) by pipette from one end of slide and let thin layer (1:3 of amount) of stain (A) before adding stain (B).
Apply Stain (B) for 10 minutes, slip the slide at one side and drying gradually to removing excess of stain (B).
Let the slide to dry for 5 minutes.

J. SDFI CALCULATIONS:

SDFI (%) = Fragmented sperms / Total number of sperms x 100

Non fragmented sperm:
Big halo: halo width is similar or higher than the diameter of the core.
Medium halo: halo width is between those with large and with very small halo.
Fragmented sperm:
Small halo: halo width is similar or smaller than 1/3 of the diameter of the core.
No halo: those that show no halo.
Degraded: those that show no halo and present a core irregularly or weakly stained.