1. How to manage concentration of sperm while processing the sample for evaluation of DNA fragmentation?
If the sperm count in the semen sample is more, than dilute the sample with Phosphate Buffer Saline (PBS) or sperm preparation media to the concentration about 15-20 million per ml. And if the count is less, then centrifuge the semen sample at 800-1000 rpm for 10 minutes and then proceed for slide preparation.
2. What type of semen sample can be analysis with the help of this kit?
The kit is applicable to fresh, raw, diluted, frozen/thawed and processed semen sample.
3. How to maintain prescribed temperature during the slide preparation?
To maintain 90°C for melting of gel one can use boiling water bath or dry bath and to maintain 22°C cooling plates.
4. How to prevent solidification of gel in Agarose gel tube?
After heating the gel tube at 90°C, do not allow the gel temperature to go below 37°C, as gel has the property to get solidify when it gets cool down after digestion. Proceed for slide preparation immediately, when the vial temperature reaches nearly 37°C.
5. How to maintain integrity of gel during procedure?
Uneven gel distribution and bubble formation on the slide while placing the coverslip may disturb the integrity of the gel.
After placing sample over the slide maintain proper temperature and time to get the gel solidify properly.
Jerky overlay of the solution might affect the integrity of the gel so gently overlay the solution.
6. How to manage staining of the slide?
Always prepare fresh stain before proceeding for slide staining. If the staining is too dark then destain the slide completely with 100% ethanol, air dry and again stain by decreasing the time for staining but maintaining rest of the conditions same. If the staining is light then destain the slide with 100% ethanol and then restain by increasing the time for staining.
7. What could be the reason for no halo observed after slide preparation?
Avoid adding of semen sample when the gel in Agarose Gel Tube is too hot as high temperature affects DNA of the sperms. Also avoid vigorous mixing of the semen sample and bubble formation in gel as it generates stress which ultimately affects the integrity of DNA.
8. What is DNA Fragmentation Index (DFI)?
DFI (DNA Fragmentation Index) is the ratio expressed as percentage of sperm having fragmented DNA to the total sperm count.DNA fragmentation in sperm may be a cause of male infertility which cannot be detected by routine semen parameters like sperm concentration, motility analysis and morphology assessment.
9. How to calculate Sperm DNA Fragmentation (SDF) Value?
Following equation can be used to calculate SDF value:
SDF Value (%) = (Number of fragmented Sperms scored / Total number of Sperms scored) X 100
10. How to interpret the SDF value obtained after slide scored?
The values obtain after SDF calculation must correspond with the earlier clinical and laboratory findings of the respective semen sample.
|≤ 15%||Excellent to Good sperm DNA integrity|
|> 15% to ≤ 30%||Good to Fair sperm DNA integrity|
|> 30%||Fair to very Poor sperm DNA integrity|
The above interpretation range is based on SCSA method.
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