FLOW CHART

1. Collect the sample

Allow to liquefy at 37°C
Dilute the sample to concentration
37°C 11. Drain excess solution
CANfrag : CA-001/10

maximum 15-20 million per (Kit to assess Sperm DNA Fragmentation)

milliliter
2. Melt Agarose Gel at 90°C in 90°C 12. Wash with 20 ml of distilled Introduction:
boiling water for 2 minute 2 min water The principle of the kit is based on interpreting the difference between fragmented and intact DNA in spermatozoa. The
spermatozoa embedded in agarose matrix are overlaid on the coated slide and exposed to acid solution followed by lysis solution.
After exposure, the spermatozoa with non-fragmented DNA shows the dispersed halos of DNA while the spermatozoa with
fragmented DNA shows minimal or no halos. Fresh, raw, diluted, frozen/thawed and processed semen can be analyzed by this kit.
3. Place the melted agarose at 37°C 13. Drain excess solution Moreover the test is easy to interpret, fast, reliable, reproducible and equivalent to SCSA.
for 5 minutes 5 min Materials and Reagents Provided:
Coated Slides 10 No. Dehydrating Solution 2 14 ml
Agarose Gel tubes 10 No. Dehydrating Solution 3 14 ml
Dilution tubes 20 No. Stain Solution 08 ml
4. Transfer 40µl of prepared sample 14. Dehydrate sequentially by Acid Denaturant 12 ml Stain dilution buffer 04 ml
to Melted Agarose Gel and mix Dehydrating Solution 1, 2 min
Lysis Solution 12 ml Coverslips (25 mm X 50 mm) 2 Box
gently Dehydrating Solution 2,
Dehydrating Solution 3 for 2 Dehydrating Solution 1 14 ml Float 01 No.
minutes each Reagents and Equipment required but not provided:
PBS (Phosphate Buffered Saline) / Sperm preparation media Micropipettes
5. Place 150µl of mixed cell 15. Drain excess solution Distilled water Micropipette Tips
suspension on Coated slide Allow to air-dry Couplin Jar Water bath
Disposable Gloves and mask Dry bath / Tube warmer / Incubator
Pencil Refrigerator
6. Cover the slide with the 8°C 16. Prepare working stain solution Syringe/ Dropper Bright field Microscope
coverslip 4°C 5 min by mixing 300µl of stain and Storage and Stability:
Transfer the slide at 4°C - 8°C 100µl of dilution buffer The kit must be stored at 05-25°C temperature.
for 5 minutes
The Reagent bottles should be tightly capped immediately after use.
The kit reagents beyond the labeled expiration date should not be used for sample processing.
7. Gently remove coverslip by sliding 17. Stain slide with 250µl of working Precautions:
it off stain solution. For research use only.
1. All samples and reagents must be treated as potentially hazardous.
2. The performer must wear disposable gloves, eye protectors and laboratory gowns when performing the process.
3. The solutions and reagents must be discarded in a proper biohazard container at the end of process.
22°C 4. One should not eat, drink, or smoke in area where the sample and kit reagents are handled.
8. Incubate with Acid Denaturant 18. Spread the stain evenly by
Solution for 7 minutes at 22°C 7 min gentle tilting. 3 min Procedure:
Continue staining for 3 minutes. Perform analysis as soon as possible after liquefaction of fresh sample or thawed cryopreserved sample.
A. Sample Preparation:
Collect the sample in the sterile semen collection jar. Allow the sample to get liquefy at 37ºC, avoid manual liquification if possible,
and then perform sperm count. If required, dilute the sample with PBS / sperm preparation media to the concentration about 15-20
9. Drain excess solution 19. Wash the stained slide with tap million/ml.
water in couplin Jar/beaker NOTE: The sample dilutions should be carried out in the DILUTION TUBES provided within the kit.
B. Slide processing:
Note: All the steps should be performed on the even surface. Always use separate micropipette tips for different reagents.
1. Place the provided Agarose Gel tube in float and incubate in boiling water at 90 oC -100 oC for 2 minutes or until gel melts. Then
10. Incubate with Lysis Solution for 20. Drain the excess water and allow cool down this tube at 37°C for 5 minutes.
20 minutes at room temperature 20 min to air-dry and observe under
microscope 2. Add 40 µl of semen sample from sample preparation tube to melted Agarose Gel tube and mix gently to avoid bubble formation.
3. Place 150 µl of suspension on the coated slide, cover with provided coverslip by avoiding air bubbles and transfer the slide at
4oC to 8°C for 5 minute to solidify the gel.
For research use only
4. Take the slide out, and then gently remove coverslip by sliding it off. Note : Precaution should be taken during removal of Positive control slide: Omit step 5 of slide processing and the follow the remaining step of the procedure
coverslip from the slide such that gel integrity is not affected.
Negative control slide: After step 4 of slide processing, overlay100µl of 300µM H2O2* and incubate for 5 minutes 4°C.
5. Place the slide on even surface and overlay 1ml of Acid Denaturant. Incubate at 22oC for 7 minutes and drain the solution Discard the solution and process with step 5 onwards as per instruction
completely after incubation. Note : Overlay the solution in such a way that it should cover the entire gel.
* Not provided with the kit.
6. Overlay 1 ml of Lysis Solution and incubate for 20 minutes at room temperature. Drain the solution completely after incubation.
Note: The positive and the negative control slides will be provided on request.
7. Gently wash the slide with 20 ml of distilled water with help of syringe or dropper by placing the slide in vertically slanted
position. Precautions:
8. Place the slide on even surface and sequentially dehydrate the slide by overlaying 1 ml of Dehydrating Solution 1, Dehydrating Use disposable gloves while processing the slide.
Solution 2 & Dehydrating Solution 3 and incubate for 2 minutes each. Then completely drain the excess solution. Allow to set the temperature of melted Agarose Gel properly before mixing sample as it causes damage to the sperm cells.
9. Allow to air dry the slide completely. Processed slide can be stored in dark at room temperature for months. Avoid formation of bubbles while pipetting the gel, sample and while loading the mixture on Coated Slide.
10. Stain the slide as followed before scoring. Immediately place the coverslip after loading of gel and sample mixture. Otherwise the drop will solidify on Coated Slide.
C. Staining: Troubleshooting:
Preparation of the working stain:
Take 300µl of Stain solution and add 100µl of dilution buffer in the dilution tube provided in the kit and mix properly. This diluted No. Problem Solution
stain must be used within 1 hour. 1. Gel clumps during Allow the gel to properly melt at 90oC-100oC for 2-5 minutes or do not allow the gel temp to go
Procedure: mixing of sample down below 37°C.
1. Place the processed slide horizontally. 2. Loss of gel integrity Uneven distribution of the gel over the coated slide. Make sure that while placing coverslip over
2. Overlay 250µl of freshly prepared working stain solution on slide and evenly spread the stain by carefully rotating slide. during procedure the drop of gel and semen mix, it gets even distribution over the slide
3. Rock the slide by tilting in to and fro direction for 3 minutes to maintain even distribution of stain over the slide. Solidify the gel properly at 4°C (maintain temperature). Less or more time given for solidifying
hampers integrity.
4. After 3 minutes wash the slide by dipping and moving in a couplin jar or a beaker filled with tap water.
Avoid air bubble while loading mix over slide and coverslip.
5. Place slide in slanting position and allow to air-dry.
Jerky overlay of the solution also affect the integrity
6. Observe the slide under 20 X and 40 X of bright-field microscope.
3. Staining: If the staining is dark, then destain the slide with 100% ethanol and again stain by decreasing
Results and Interpretation: Dark Staining the time of the stain but maintain rest of the conditions as same.
The cells which correspond to spermatozoa along with presence of tail are only taken into consideration for scoring. Minimum 200 Light Stain If the slide is stained very light then destain slide with 100% ethanol and again restain the slide
sperms per sample are to be scored. by increasing the staining time and maintaining rest of the conditions same.
Scoring:
Always prepare the stain fresh before staining the slide.
A B Note: The staining should differentiate between the core and halo of the sperm.
Spermatozoa without DNA fragmentation: Spermatozoa with DNA fragmentation:
rd rd
Halo dispersion greater than or equal to 1/3 width of head. Halo dispersion less than 1/3 width of head or no Halo. 4. No Halo observed Avoid adding of semen sample in melted gel while the temperature of gel is higher than 37°C
after stain
Avoid vigorous mixing of semen sample to avoid stress to sperm cells and bubble formation
Process exactly as per the instruction given. Maintain exact temperature during the procedure
as mentioned in the literature.
5. Too few cells for Concentrate the semen sample by centrifugation at 800-1000 rpm for 10 minutes and then
analysis proceed for slide preparation.
References:
Threshold for frequency of sperm DNA Fragmentation (SDF) have been suggested by Evenson et al. (Evenson and Wixon , Reprod Biomed
Online 12:466-472. 2006)
C. Others: Cells without tail and whose morphology does not correspond to the spermatozoa is considered as other type of cells. R. K. Sharma, T. Said, A. Agarwal (2004), Sperm DNA damage and its clinical relevance in assessing reproductive outcome, Asian J Androl ,
Calculation of Sperm DNA Fragmentation (SDF) value is done as followed: Jun; 6:139-148
SDF Value (%) = Number of Fragmented sperms × 100 Marziyeh Tavalaee, Mohammad Hossein Nasr-Esfahani, Mohammad Reza Deemeh, (2008) International Journal of Fertility and Sterility, Vol 2,
Total number of sperms scored No 1, Pages: 1-8
The values obtained after SDF calculation must correspond with all the earlier clinical and laboratory findings of the respective
semen sample. https://www.scsadiagnostics.com/uploads/content/clinicalreport.pdf
SDF% Interpretation Technical Assistance:
<_ 15% Excellent to Good sperm DNA integrity
> 15% to_< 30% Good to Fair sperm DNA integrity Bioscience

> 30% Fair to Very Poor sperm DNA integrity 1st floor, Shashwat Complex, Naranpura, Ahmedabad, Gujarat, India 380013.
Web: www.candore.in | E-mail: info@candore.in | Call: +91 - 846 008 3121 | +91 - 79 - 2768 3121
Note: Literatures signify that SCSA, SCD and TUNEL Test predict the same level of DNA fragmentation. The above interpretation
range is based on SCSA method. Irritant Toxic Flammable Use by LOT Lot number REF Product reference Manufacturer Consult instruction of use