'Inter-and Intra-Laboratory Standardization of TUNEL Assay ......couples. SDF affects ART outcomes...

UNIT 16.11Inter-and Intra-LaboratoryStandardization of TUNEL Assay forAssessment of Sperm DNA FragmentationSajal Gupta,1 Rakesh Sharma,1 and Ashok Agarwal1

1American Center for Reproductive Medicine, Glickman Urological & Kidney Institute,Cleveland Clinic, Cleveland, Ohio

The functional aspects of sperm activity such as sperm chromatin integrityand ability to fertilize cannot be characterized by routine semen parameters.Men with unexplained infertility and idiopathic infertility, as well as men withnormozoospermic semen profiles, show high DNA fragmentation. Molecularanomalies in the sperm can be detected by a sperm DNA fragmentation (SDF)assay which can be used in adjunct to conventional semen analysis. Whilethe sperm chromatin structure assay (SCSA) remains the “gold standard,” theTUNEL assay using flow cytometry is becoming popular among the differenttests that are currently available to measure sperm DNA fragmentation. Inthis unit, we describe the inter-laboratory and intra-laboratory standardizationof the TUNEL assay using a benchtop cytometer. The article also providesa step-by-step protocol for measuring sperm DNA fragmentation using theTUNEL assay and a bench-top flow cytometer, and also points out the inherentchallenges with this test. C© 2017 by John Wiley & Sons, Inc.

Keywords: Accuri C6 � flow cytometry � DNA fragmentation � sperm �

TUNEL

How to cite this article:Gupta, S., Sharma, R., & Agarwal, A. (2017). Inter-and

intra-laboratory standardization of TUNEL assay for assessment ofsperm DNA fragmentation. Current Protocols in Toxicology, 74,

16.11.1–16.11.22. doi: 10.1002/cptx.37

INTRODUCTION

Male reproductive dysfunction is a key issue and public health concern that leads toreproductive disorders such as male infertility, miscarriages, recurrent pregnancy loss,and anomalies of the newborn (Aitken, Smith, Jobling, Baker, & De Iuliis, 2014). Thepoor predictive value of conventional semen analysis is attributed to the high inter- andintra-observer variability in assessment of routine semen parameters such as sperm count,motility, and morphology (Guzick et al., 2001; Keel, 2006; Tielemans, Heederik, Burdorf,Loomis, & Habbema, 1997), and also to inherent variability in the semen parameterswithin subjects (Keel, 2006). In this unit, we present step-by-step protocols establishedfor the measurement of sperm DNA fragmentation (SDF) in human spermatozoa usinga benchtop flow cytometer. We provide the details of sample collection, processing,and staining using the TUNEL assay. Also included are steps for data acquisition andanalysis, and details of quality control for the cytometer. These steps should enable otherandrology laboratories to follow the protocols and standardize them in their laboratorysettings, and establish reference ranges for the TUNEL assay applicable to their patientpopulation.

Current Protocols in Toxicology 16.11.1–16.11.22, November 2017Published online November 2017 in Wiley Online Library (wileyonlinelibrary.com).doi: 10.1002/cptx.37Copyright C© 2017 John Wiley & Sons, Inc.

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Importance of DNA Fragmentation in Male Infertility

Clinical relevance with ART outcomes

Molecular biology techniques have evolved over the past few decades. There are severalstate-of-the-art techniques that are utilized to assess sperm DNA fragmentation (Irvineet al., 2000). The assessment of SDF or DNA integrity is a reflection of the integrityof the genetic material that a spermatozoon is going to transmit to the offspring. SpermDNA fragmentation is predictive of both natural and assisted fertility. Elevated levels ofsperm DNA fragmentation assessed by SCSA were associated with a high odds ratio offailure to conceive naturally (Zini, 2011), as well as a longer time interval to conceptionor reduced fecundity for couples planning their first pregnancy (Spano et al., 2000). SDFassessment is proposed to be an integral part of the algorithm for management of infertilecouples. SDF affects ART outcomes with impact on all types of assisted conceptiontechniques such as, IUI, IVF, and ICSI, being associated with high pregnancy loss afterIVF or ICSI (Duran, Morshedi, Taylor, & Oehninger, 2002; Jin et al., 2015; Zhao, Zhang,Wang, & Li, 2014).

STRATEGIC PLANNING

Validation of the Inter- and Intra-Laboratory Standardization of the TUNELProtocol

Several literature reports have highlighted the need for standardization of the TUNELassay across laboratories as well as within a laboratory (Muratori et al., 2008; Ribas-Maynou et al., 2013; Sharma et al., 2010; Sharma, Ahmad, Esteves, & Agarwal, 2016;Sharma, Masaki, & Agarwal, 2013). This is due to large variability reported in terms ofdiagnostic accuracy and precision of the sperm DNA fragmentation results assessed bythe TUNEL assay in different studies. The differences in results obtained across differentlaboratories are due to different techniques of sample preparation, fixation, and protocolsfor staining, and differences in the instruments, as well as in the instrument settings fordata acquisition and analysis.

Conducting the standardization experiment requires that two to three measurementsfor each subject be planned and recorded. Two independent observers are requiredto conduct the measurements both from the same subjects and from different subjects.Evaluation of inter- and intra-observer variability is done using all TUNEL measurements(Sharma et al., 2010). The difference in the results between the two observers (inter-observer variability) is analyzed by assessing the difference in the values between thetwo observers. The differences in the different readings by the same observer for thesame samples need to be calculated to record intra-observer variability. Our group hasestablished the reference ranges for the TUNEL assay to discriminate between infertileand fertile males (Sharma et al., 2010). We published a study on validation of the benchtopflow cytometer for TUNEL assay (Sharma et al., 2016; Sharma et al., 2010). We havealso reported the standardization of the TUNEL assay across laboratories and validatedTUNEL as a reproducible and robust assay within and across laboratories (Ribeiro et al.,2017). This study was performed to standardize the TUNEL assay in two establishedlaboratories across two continents. The same samples were assessed by two independent,experienced observers. Furthermore, identical TUNEL protocols and lot numbers of kits,as well as similar acquisition settings with an identical template, were utilized for thestandardization, with comparable results for both labs. We have established, for the firsttime, a standardized protocol for use in different andrology laboratories across the globe.

LABORATORY MEASUREMENT OF SPERM DNA FRAGMENTATION BYTUNEL ASSAY USING BENCHTOP FLOW CYTOMETER

This TUNEL assay protocol defines the procedure for sperm DNA fragmentation assess-ment (Figure 16.11.1) with the benchtop BD Accuri C6 flow cytometer (Figure 16.11.2;

Sperm DNAFragmentation by

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Supplement 74 Current Protocols in Toxicology

Figure 16.11.1 Schematic of the DNA staining by the TUNEL assay.

Figure 16.11.2 Setup of a benchtop flow cytometer.

Agarwal, Gupta, & Sharma, 2016). Apoptosis in the spermatozoa results in activation ofendonucleases, and these enzymes induce sperm DNA fragmentation (Muratori, Marchi-ani, Maggi, Forti, & Baldi, 2006). High-order sperm chromatin is broken down intosmaller DNA fragments of �50 kb by the endonucleases. The DNA strand breaks arelabeled by the fluorescein isothiocyanate deoxyuridine triphosphate (FITC-dUTP) stain.This is accomplished in the presence of the template-independent enzyme terminal de-oxyribonucleotidyl transferase (TdT), which helps transfer the deoxyribonucleotides tothe 3′-hydroxyl (3′-OH) end of the single- and double-strand breaks. The intensity oflabeling is proportional to the number of DNA strand break sites.

BASICPROTOCOL 1

Collection of Semen Specimen

Materials

Donor of test specimenViscosity treatment enzyme, e.g., chymotrypsin (optional)


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Current Protocols in Toxicology Supplement 74

Semen analysis formWide-mouth, sterile plastic specimen container

1. Collect the semen sample after a minimum of 48 hr and not more than 72 hr of sexualabstinence. A notation is made of the patient name, medical record number, and daysof abstinence on the semen analysis form.

2. The sample should be collected by masturbation only. It should be collected into aclean, wide-mouth sterile plastic specimen container. All lubricants should be avoided.

3. Make a note on the semen analysis worksheet of any unusual collection conditionsfor the sample or any unusual findings such as sperm agglutination, high viscosity, orlarge amount of cellular debris. The sample should be incubated at 37°C for 20 min forcomplete liquefaction before being used in the TUNEL assay (see Support Protocol2).

For viscous samples, a viscosity treatment enzyme such as chymotrypsin should be addedand the sample placed in the incubator for additional 10 min for complete liquefaction.

SUPPORTPROTOCOL 1

General Setup of the Benchtop Cytometer

The major components of the flow cytometer are (1) optical assembly, (2) blue laser, (3)red laser, (4) sheath pump, and (5) waste pump and in-line sheath filter.

Materials

Sheath fluid (blue bottle; BD Biosciences, PN 653156): 0.22-µm filtered deionizedwater with or without bacteriostatic concentrate solution; if bacteriostaticconcentrate solution is used (optional), add 1 bottle per 1 liter of water

Cleaning solution (green bottle; BD Biosciences, PN 653157); dilute 3 ml ofcleaning concentrate in 197 ml of filtered deionized water (use the solutionwithin 2 weeks)

Decontamination solution (yellow bottle; PN653154); add entire bottle to 180 mlof filtered, deionized water

Benchtop flow cytometer (Accuri C6; BD Bioscience; Fig. 16.11.2)BD Accuri C6 software

1. Open the software by double-clicking the “BD Accuri C6 software” icon on thedesktop.

2. Inspect all the reagent bottles to ensure that the fluid levels are adequate.

3. The waste bottle should be empty.

4. The sheath fluid, cleaning solution, and decontamination bottles must be full.

5. Turn on the cytometer by pressing the power button firmly.

6. While starting the software the “traffic light” will turn yellow. This is an indicationthat the peristaltic pump has started to run.

7. Allow 5 min for the fluidics line to get flushed with the sheath fluid.

8. Wait for the cytometer software light to turn green, indicating that the C6 Accuri isconnected and ready.

9. Flush the tubing to remove any bubbles from the cytometer system.

10. Place a 0.22 µm-filtered deionized (DI) water tube on the sheath injection port (SIP).

11. Run a cycle with criteria selected as “Run with limits.”


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12. Select “Fluidics” speed as “Fast.”

13. After selection of the above criteria, click the “Run” button.

14. Leave the SIP tube on the tube holder. Save the file as “Flush.”

BASICPROTOCOL 2

Instrument Quality Control

Quality control for the FL1, FL2, and FL3 channels is performed and validated with8-peak beads. The 8-peak beads are 3.2-µm particles excited by the blue laser. The beadsemit light at eight different wavelengths. The validation of the benchtop flow cytometeris done by running the 8-peak beads and determining the coefficient of variation (CV)and mean fluorescence intensity (MFI) each time the instrument is used. These can beplotted as CV and MFI in the Levy-Jennings chart.

Materials

Spherotech 8-peak validation beads (BD Biosciences, cat. no. 653144)12 × 75–mm tubesBenchtop flow cytometer (Accuri C6; BD Bioscience; Fig. 16.11.2), set up as in

Support Protocol 1BD Accuri C6 softwareComputer running Microsoft Excel

Preparation of 8-peak beads

1. Use a 12 × 75–mm tubes and label it as “8-Peak QC Beads.” Also mark the date ofpreparation.

2. Add 1 ml of deionized water to each of the tubes.

3. Vortex each of the bead vials provided by manufacturer for 5 sec. Place four dropsof 8-peak beads in the tube and vortex. Cover the tube with an aluminum foil.

Preparation for the run of the 8-peak QC beads

4. Double click and open the 8-peak bead template (Figure 16.11.3).

5. Turn on the cytometer by pressing the power button located in front of the cytometer.

6. A green light will be displayed under the ‘Collect’ tab, indicating that the machineis ready for sample acquisition.

7. Start the acquisition by clicking on the well “A1.”

8. Place a tube with 2 ml of 0.22-µm deionized water on the SIP.

9. Check “Run with limits” and set the time limit to “15 min.”

10. Set “Fluidics” speed to “Fast.”

11. “Click” the “Run” button.

12. The software will prompt to “Save” the file.

13. After completion of the “Run,” place the tube with deionized water on the SIP.

Acquisition of the 8-peak bead data

14. Select an empty field from left heading towards the right with selecting one well ata time from A1 to H12.

15. Enter in the empty space above the wells the acquisition date for the 8-peak beadsas “8 peak-beads—‘date’—‘technician initials’.”


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Figure 16.11.3 8-peak quality-control beads as seen after analysis in software; the CV of thebrightest peak (M3, M6, M9) is measured.

16. The acquisition is performed under the “Collect tab.”

17. Unselect the “Time” check-box next to “Min.” and “Sec.”

18. Select the “Events” check-box and check the “50,000” option in the “Events” field.

19. From the drop-down menu, click on “Ungated sample.”

20. Set “Fluidics” speed to “Slow.”

21. Mix the 8-peak QC bead suspension by vortexing the tube.

22. Remove tube of deionized water from the SIP.

23. Place the “8-Peak QC Bead” tube under the SIP.

24. Click the “RUN” button to start the acquisition.

25. Save the file as “8 Peak QC—‘date’—‘technician initials’.”

26. After the cytometer has recorded 50,000 events, acquisition will stop.

27. When the run is finished, remove “8-Peak QC Bead” tube from SIP and clean theSIP using a lint-free wipe.

28. Place the tube containing 2 ml of deionized water on the SIP.

Ending the run

29. With the 2-ml tube of deionized water on the SIP, select an empty well in the BDAccuri software.

30. Check “Time” and set the time to “2 min.”

31. Set “Fluidics” speed to “Fast.”


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32. Click the “Run” button.

33. When the run is finished, place the tube with 2 ml of deionized water on the SIP.

34. Before running any other samples, click “delete events” to erase the data collectionfrom the water run.

35. If shutting down instrument, proceed to the “Machine Shutdown” section run witha 10% solution of bleach for 2 min, followed by the deionized water run.

Analyzing the 8-peak bead acquisition data

36. The analysis is done in the “Collect” tab only.

37. Select the well (example: well A1) where the data was acquired for the 8-peak beadsrun.

38. Adjust the R1 gate to include 75% to 85% of all events.

39. In the first plot—‘FSC-H’ on the x axis and ‘SSC-H’ on the y axis—click on theborder of the ‘R1’ gate. The border will become bold and handles will appear toadjust the gate settings.

40. Include all the “Singlets” or the main bead population, making sure to exclude allthe doublets which appear as light-gray dots.

41. FL1-H, FL2-H, and FL3-H must be gated on R1.

42. Measure the CV of the brightest peak (right most peak) of the FL1-H, FL2-H, andFL3-H histograms (Figure 16.11.3).

Criteria for successful 8-peak bead QC

The CV for all three peaks must be less than 5% for validation of the three channels ofthe instrument.

43. To select the brightest peak, use the zoom tool over the histogram and zoom in onthe brightest peak in the FL1-H histogram.

44. The ‘M1’ marker is adjusted tightly around the brightest peak.

45. The above two steps need to be repeated around the FL2-H and FL3-H histogramsas well.

46. Save this template for future runs of the 8-peak quality control.

Performance tracking of 8–peak bead quality control

47. Open the file for the acquisition data obtained from the 8-peak bead run. Highlightall the statistics that need to be copied and transferred to the Excel spread sheet.In the “statistics column selector,” check the boxes for the mean and CV of thebrightest peak (M3, M6, and M9) for the following parameters: FL1-H, FL2-H, andFL3-H. The Levy-Jennings chart gets populated by the data and the data is saved.

SUPPORTPROTOCOL 2

Sample Preparation for TUNEL Assay

Materials

Semen sample (Basic Protocol 1)Phosphate-buffered saline (PBS; APPPENDIX 2A)

Sperm counting chamber (Spectrum Technologies, cat. no. SC-20-01-02-B)Centrifuge


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1. Semen sample is kept in the incubator for 20 min at 37°C to undergo liquefaction.

2. After liquefaction, sample is evaluated for volume, sperm concentration, total spermcount, sperm motility, and round cell concentration.

Total sperm count is calculated as: concentration × sample volume.

Sperm motility is calculated as: [(Average number of motile sperm)/(Average number oftotal sperm)] × 100

Round cell concentration (using a 20× objective) is calculated as: (Average number ofround cells) × (20)/(100 round cells) × 106 cells

3. Aliquot 5 µl of the sample with the appropriate pipet. The 5 µl of sample is loadedinto the fixed cell chamber well.

4. Assess the sperm concentration by counting sperm in five different fields.

5. The sample volume for TUNEL needs to be adjusted to 2.5 × 106/ml. This can beachieved by the following formula:

(2.5 × 1000 µl)/[sperm concentration (106/ml)] = x µl.

6. Save two tubes each for the test sample, negative control, and positive controlsamples. If the sample is inadequate, a single tube may be saved for the negativeand the positive control sample.

7. Label tubes with the following information:

TUNELPatient nameMedical record numberDate.

8. Aliquot the required volume for an adjusted sperm concentration of 2.5 × 106/mlcells to each of the four tubes.

9. Spin the aliquotted sample 7 min at 300 × g, 25°C.

10. Remove the supernatant after the spin.

11. Replace the supernatant with 1 ml PBS

12. Centrifuge 7 min at 300 × g, 25°C.

13. Remove the supernatant and replace with 1 ml PBS.

SUPPORTPROTOCOL 3

Preparation of the ‘Positive Control’ Sample

Materials

37% hydrogen peroxidePhosphate-buffered saline (PBS; APPENDIX 2A)Positive control (semen sample from healthy donor)3.7% paraformaldehyde: add 90.0 ml of phosphate buffered saline (PBS) pH 7.4

(APPENDIX 2A) to 10.0 ml of formaldehyde (37%); stored at 4°C70% ethanol, ice cold

50°C heating blockCentrifuge


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Prepare cells for fixation/permeabilization

1. Add 100 µl of 37% solution hydrogen peroxide to 1400 µl of PBS prepare a 1:15dilution of H2O2.

2. Add and suspend the sperm cells in 1 ml of the diluted H2O2 solution.

3. Place the sperm cell resuspended in H2O2 on a heating block at 50°C for 60 min.

4. After incubation, centrifuge the tube for 7 min at 300 × g, 25°C.

5. Aspirate the supernatant with a transfer pipet and resuspend in 1 ml PBS andcentrifuge 7 min at 300 × g, 25°C.

6. Remove the supernatant and replace with 1 ml PBS. Along with the ‘Test’ and‘Negative’ control sample tubes, repeat centrifugation step for 7 min at 300 × g,25°C.

7. Remove the supernatant and replace with 1 ml PBS.

Fixation and permeabilization

Fixation of the sperm cells is done with paraformaldehyde.

8. The supernatant from the ‘Test’ sample, ‘Negative’, and Positive’ control samples isremoved after centrifugation 7 min at 300 × g, 25°C, followed by addition of 1 mlof 3.7% paraformaldehyde solution.

9. Incubate the samples at room temperature for 15 min.

10. Centrifuge the samples 4 min at 300 × g, 25°C.

11. Carefully aspirate the paraformaldehyde and replace it with 1 ml of PBS.


13. Aspirate the supernatant and replace with 1 ml of ice-cold 70% ethanol. Place thesample at 4°C for 15 to 30 min.

14. Perform a second wash with PBS at 25°C.

BASICPROTOCOL 3

TUNEL Staining with the APO Direct Kit

Materials

Test samplesAPO-DIRECTTM Kit (BD Pharmingen, cat. no. 556381):

PI/RNase Staining BufferReaction BufferFITC-dUTPTdT EnzymeRinsing BufferWash BufferNegative Control CellsPositive Control Cells

12 × 75–mm polystyrene tubesBenchtop flow cytometer (Accuri C6; BD Bioscience; Fig. 16.11.2), set up as in

Support Protocol 1BD Accuri C6 softwareComputer running Microsoft Excel Male

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Table 16.11.1 Components of the Staining Solution

Staining solution 1 Assay 6 Assays 12 Assays

Reaction buffer (green cap) 10.00 µl 60.00 µl 120.00 µl

TdT enzyme (yellow cap) 0.75 µl 4.50 µl 9.00 µl

FITC-dUTP (orange cap) 8.00 µl 48.00 µl 96.00 µl

Distilled H2O 32.25 µl 193.5 µl 387.00 µl

Total volume 51.00 µl 306.00 µl 612.00 µl

Prepare samples and tubes for TUNEL assay

The negative and positive controls are provided as kit components.

1. The negative controls, positive control, and test samples should be mixed well byvortexing them.

2. Aliquot 2 ml of the well-mixed kit control suspensions into 12 × 75–mm polystyrenetubes.

The 2 ml of suspension contains approximately 1 × 106 cells/ ml.

3. Include internal controls—both positive and negative semen samples—with eachrun.

These are semen samples with known DNA fragmentation.

4. The kit control samples, test samples, and internal control samples should be cen-trifuged for 7 min at 300 × g, 25°C.

5. Remove 70% ethanol with a transfer pipet by aspirating it, without disturbing thecellular pellet.

6. Add 1.0 ml of the ‘Wash Buffer’ from the kit (blue cap) and mix well.


8. Aspirate and remove the supernatant from the tubes.

9. Repeat the washing step with the ‘Wash Buffer’ and discard the supernatant.

10. Number all the tubes starting from the ‘Negative controls’, ‘Positive controls’, ‘Testsamples’, and ‘Internal controls’.

Staining for TUNEL assay

11. Count the total number of tubes or test samples including the kit controls and theinternal controls.

12. Prepare stain for an additional five to seven tubes as described in the following steps.

13. Remove “Reaction Buffer” vial (green cap) from 4°C and the TdT (yellow cap) andFITC-dUTP (orange cap) vials from –20°C storage and place them at room temper-ature for 20 min. Give a quick vortex to bring the reagent to the bottom of the vial.

14. Prepare the stain as shown in Table 16.11.1.

15. Add the reagents in the same sequence as indicated in the table.

16. All the steps for the stain preparation must be carried out in the dark.

17. Omit the TdT from the negative controls.

18. Resuspend the pellet in each tube in 50 μl of the ‘Staining Solution’.


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Figure 16.11.4 Representative plot of ‘Negative kit control’.

19. Incubate the sperm suspension for 60 min at 37°C.

20. The tubes should be covered with aluminum foil.

21. After the 60-min incubation, add 1.0 ml of ‘Rinse Buffer’ (red cap) to each tube.Centrifuge the tubes for 7 min at 300 × g, 25°C. Aspirate and remove the supernatant.

22. Repeat the wash with addition of 1 ml of ‘Rinse Buffer’.

23. Repeat the centrifugation step for 7 min at 300 × g, 25°C.

24. Aspirate and discard the supernatant.

25. Resuspend the pellet in 0.5 ml of PI/RNase buffer.

26. Incubate the suspension mixture for 30 min at room temperature.

Run kit controls and acquire data for kit controls

27. Run the kit controls using the “kit control template” (Figures 16.11.4 and 16.11.5).The settings include “Run with limits” for a total of 10,000 events with a slowfluidics speed and threshold set at 80,000 on FSC-H. Data is recorded on four plots:FSC-A/SSC-A, FSC-A/FL2-A, FL2-A/FL2-H, and FL1A/FL2-A. Observe the rightupper quadrant and record the FITC positive as the percent positive value for eachkit control.

Running patient samples

Patient samples are run under the “Collect” tab. Use the standardized data acquisitiontemplate (Figure 16.11.6). The complete acquisition data should be saved in a designatedfolder for patient results.

28. Double-click on the “TUNEL patient template” folder.

29. Wait until the software loads.


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Figure 16.11.5 Representative plot of ‘Positive kit control’.

Figure 16.11.6 Example of template setup for the analysis of the patient sample.

30. Ensure that there is no data in any of the wells in the template file. If there is data inany of the wells, select the “delete all events icon” at the bottom left of the screenand remove the data.

31. Select well “F1” and import the standard sample file (.fcs; Figure 16.11.7).

32. Select the first well “A1.” In the space provided above the well, insert “TUNELpatient result,” technician initials, date, and well number. Hit the Save button.


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Figure 16.11.7 Representation of a “Standard sample alignment.”

33. Begin with tube #1 (first test sample).

34. Remove deionized water tube from the SIP.

35. Vortex the sample and place on the SIP.

36. The run parameters are set as follows for patient samples:

a. “Run with limits”: check ‘10,000 events’.b. “Fluidics” speed: select ‘Slow’.c. Select gate ‘P3 in P1’.d. Threshold: set at 80,000 on ‘FSC-H’.

37. The acquisition of data is begun by clicking on the “Run” button.

38. After 10,000 events, the run will be completed.

39. Remove the tube from the SIP.

40. Use a lint-free wipe to clean the SIP.

41. Vortex and place the second sample on the SIP.

42. Select the next well (A2, and so on) for the next samples.

43. The above steps are repeated for each sample to allow processing of all samples.

44. The data acquisition workspace is saved in a subfolder—e.g., TUNEL Pa-tient Results' ''date'', ''technician initials''.C6. Savethe workspace and close the file.

45. Remove the tube from the SIP and replace with “bleach tube” on the SIP.

46. A “bleach cycle” is run at the end with the following parameters:

a. “Run with limits”: 2 min.b. “Fluidics” speed: Fast.c. Threshold: 80,000 on FSC-H.


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47. Wipe the SIP at the end of the run.

48. Remove the tube and replace it with deionized water tube.

49. Repeat step 46 with deionized water.

50. Follow the shutdown steps at the end of the run.

SUPPORTPROTOCOL 4

Cleaning and Maintenance of the Benchtop Cytometer

The machine requires some elements of maintenance such as SIP cleaning and fluid cycleto be performed daily. The SIP needs to be cleaned daily by performing a back flush.A decontamination of the fluidics line can be done daily by running a decontaminationfluid cycle using deionized water. The fluidic bottle filters and the in-line sheath filtersshould be changed every 2 months. The two peristaltic tubings for the sheath pump andthe waste pump are also changed every 2 months.

DATA ANALYSIS

The dual strategy outlined below is used for data analysis

1. Alignment strategy is performed under the “Collect” tab. A standard sample file isused for alignment of all the samples.

2. Data analysis is performed under the “Analyze” tab. Each sample must be aligned to“Standard sample” under the “Analyze tab.”

SUPPORTPROTOCOL 5

Alignment Strategy and Data Analysis in the Collect Tab

Materials

BD Accuri C6 software

1. Go to File, open workspace, or template. Select the acquisition data saved in theTUNEL template (TUNEL Patient Template).

2. Select an empty well where the standard sample data acquisition file has to beimported.

3. Standard sample should be selected as a sample which has a known percentage ofDNA fragmentation. Go to the “Standard template” and select it.

4. Click on “File” > “import.”

5. Select an open workspace.

6. Save the workspace as TUNEL patient acquisition—data analyzed—technicianinitials—date.

7. A single sample is selected as “Standard sample.”

8. Select the negative peak of the “Standard sample” and use it as a reference to beapplied to all samples for alignment (Figure 16.11.8).

9. Click on the histogram for the “Standard sample.”

10. Change the x-axis parameter from FSC-A to FL1-A.

11. Right click below the x axis (FL1-A) and select the “Virtual gain” module foralignment.

12. Change the gate to P3 in P1 for plot 5. This gate is the same as plot 4, which is aquadrant gate.


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Figure 16.11.8 Aligning a test sample to the standard sample.

13. Select the vertical line icon at the bottom left of the histogram plot.

14. Align the selected blue line to the center of the histogram to obtain 50% cellpopulation on either side.

15. Select the sample to be aligned from the grid of wells.

16. Next, align the blue line to the center of the peak of the selected sample

17. Click the tabs of “Preview” and “Apply.”

18. An asterisk will appear below the histogram. This confirms the alignment of thesample (Figures 16.11.9 and 16.11.10).

19. Hit the Close button. Go to File and hit “Save” after each sample is aligned.


16.11.15


Figure 16.11.9 Applying the alignment to the test sample. This is indicated by an asterisk at thebottom of the histogram confirming the alignment of the sample to the standard file.

SUPPORTPROTOCOL 6

Data Analysis in “Analyze” Tab

Materials


1. The data acquired in the “Collect” tab is utilized for analysis under the “Analyze” tabwithin the Accuri C6 software. Open the “Analyze” tab. Create a set of three plots foreach sample: FSC-A/SSC-A, FSC-A/FL2-A, and FL1-A/FL2-A.

2. Apply the same gating strategy as used in the “Collect” tab (Support Protocol 4).

3. The first plot, “FSC-A/SSC-A” has no gating. The cell population is PX.

4. The second plot “FSC-A/FL2-A” will have the gate PX in all events. The cell popu-lation is PY.

5. The third plot “FL1-A/FL2-A” will have gate of PY in PX in all events.


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Figure 16.11.10 Steps showing the alignment of the sample saved with an asterisk under the histogram plot.

6. Record the percent damage reflected in the upper right quadrant from the “FL1-A/FL2-A “plot” (Figure 16.11.10).

7. Record the preliminary results in the TUNEL Laboratory record form.

SUPPORTPROTOCOL 7

Final Result Calculation for Sperm DNA Fragmentation Percent Value andVerification of the Validity of the TUNEL Assay Performed

Materials


1. Calculate the average value of the “Negative samples” where no TdT was added.

2. Subtract the average negative value form the percent damage for each sample recordedfrom the “FL1-A/FL2-A” in the right upper quadrant of the plot (Figure 16.11.11).

3. The following two conditions need to be verified for the assay to be considered valid:

Figure 16.11.11 Plot in the analyzed mode showing percentage of DNA damage.

16.11.17


a. The spermatozoa “positive control” samples must have higher percentage of spermpositive for TUNEL than percentage of positive spermatozoa in the actual sper-matozoa samples.

b. The positive kit control sample should have greater than 30% of spermatozoapositive for TUNEL.

COMMENTARY

Background InformationUnlike other somatic cells, sperm nuclear

DNA is highly compacted because of its pro-tamine content. DNA fragmentation can be at-tributed to a number of factors such as ox-idative stress (Agarwal et al., 2014; Lewis &Aitken 2005; Sakkas, Seli, Bizzaro, Tarozzi, &Manicardi, 2003), abortive apoptosis (Sakkaset al., 2003), failure to repair DNA strandbreaks (Lewis & Aitken 2005), environmen-tal exposure of sperm DNA to toxins, defec-tive chromatin packaging, and protamine de-ficiency (Aitken et al., 2014). This has led tothe introduction of DNA integrity as an im-portant parameter for clinical assessment ofmale infertility. Sperm DNA fragmentation isa biomarker for evaluation of male infertility.

Accurate assessment of sperm DNA in-tegrity expressed as SDF is a good predic-tor of semen quality (Feijo & Esteves 2014;Ribas-Maynou et al., 2013; Sergerie, Laforest,Bujan, Bissonnette, & Bleau, 2005; Sharmaet al., 2010). There are four main techniquesfor assaying SDF: (1) sperm chromatin struc-ture assay (SCSA), which measures proportionof sperm that are susceptible to DNA dam-age (red fluorescence) compared with thosethat are not (green fluorescence), utilizing flowcytometry acid denaturation (Evenson et al.,1999); (2) TdT-mediated dUTP nick-end la-beling (TUNEL), which measures the inten-sity of fluorescence proportional to the num-ber of DNA stand breaks (Sharma et al., 2016),quantifying the incorporation of fluorescentlylabeled dUTP at the 3′OH strand breaks withTdT (Ribas-Maynou et al., 2013); (3) single-cell gel electrophoresis assay (SCGE, alsoknown as Comet), which measures the dis-placement of the nuclear and tail material uti-lizing fluorescently labeled cells embedded inagarose by electrophoresis after they are lysed(Ribas-Maynou et al., 2014; Ribas-Maynouet al., 2013; Ribas-Maynou et al., 2012; Singh,McCoy, Tice, & Schneider, 1988); and (4)sperm chromatin dispersion (often known asHALO), utilizing microscopy to detect cellswith intact DNA (large halos) versus thosethat have damaged DNA (small or no halo)(Fernandez et al., 2003; Fernandez, Cajigal,

Lopez-Fernandez, & Gosalvez, 2011; Ribas-Maynou et al., 2013).

Each of these tests is related to prop-erties of the DNA damage and providessemi-quantitative estimates only. Howeverthey do not provide information regarding thespecific DNA sequences that may be affected(Ni, Spiess, Schuppe, & Steger, 2016). A sig-nificant association has been reported, utiliz-ing SDF tests, between sperm DNA damageand pregnancy outcomes. High DNA integrityis generally seen in fertile men with normalsemen parameters, whereas infertile men withabnormal semen parameters have low DNA in-tegrity. In addition, many men with normal se-men parameters have abnormal DNA integrity(Spano et al., 2000; Zini et al., 2002). Thereis, however, some evidence to suggest that in-creased DNA fragmentation is associated withreduced fertility. Yet, this evidence is not con-clusive for these tests to be truly predictive offertility status.

The literature reports that 5% of womenexperience two consecutive miscarriages andapproximately 1% suffer three or more con-secutive miscarriages (ASRM Practice Guide-lines, 2012; Rai & Regan 2006), and that theseobservations are linked with sperm DNA dam-age. A number of systematic reviews have ex-amined the effects of SDF on ART outcome(IUI/IVF/ICSI), and the results have been in-conclusive (Bareh et al., 2016; Carlini et al.,2017; Coughlan et al., 2015; Osman et al.,2015; Robinson et al., 2012; Simon, Zini, Dy-achenko, Ciampi, & Carrell, 2017; Zini, 2011).At present, current data does not support aconsistent association between abnormal DNAintegrity and reproductive outcomes achievedthrough natural conception, IUI, IVF, or ICSI.More well-designed studies using standardtechniques are needed for the predictive abilityof SDF in recurrent pregnancy loss.

Alternate kitsEvaluation by Apo-BrdU apoptosis detectionkit

There are other Apo-direct kits avail-able from companies such as Biovision andThermo Fisher. BioVision has a kit on the


TUNEL Assay

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market named “Apo-BrdUTP in situ DNAfragmentation kiD.” The instructions from themanufacturer have to be adapted for sper-matozoa. The kit also includes positive andnegative controls. The DNA labeling is donewith BrdUTP, which binds to the 3′-OH ter-minals of the DNA strand breaks and termi-nal deoxynucleotidyl transferase (TDT). Theantibodies to the BrdUTP molecule are linkedto fluorescein. These will be detected in theFL1 channel (Anzar, He, Buhr, Kroetsch, &Pauls, 2002; Ribeiro et al., 2013). Sampleanalysis is done with a flow cytometer. TheFL3 channel records the propidium iodide flu-orescence. The percentage of TUNEL-positivecells can be obtained from the right up-per quadrant in the FL1-A/FL3-A, 4-quadrantplot.

Historical background of comparabletechniques and description of the alternatetechniques

Both SCSA and TUNEL tests using flowcytometry have been shown to correlate witheach other. TUNEL-based assays show higheraccuracy than the SCD and Comet assay (Cuiet al., 2015).

Critical ParametersFactors that can influence the protocol and

need special attention are:a. Viscous semen samples: These make it

difficult to assess sperm concentration andsubsequent sample preparation for TUNEL as-say. Viscosity can be reduced by treating withchymotrypsin (5 mg) and incubating the sam-ple for an additional 10 min. before examiningfor concentration.

b. Oligozoospermic samples: Samples thathave extremely low sperm concentration (<10× 106 sperm/ml) will require larger samplevolumes. In such cases, it is important to re-move the seminal plasma to avoid clump-ing/fixation of the seminal plasma proteinswith paraformaldehyde. This will make all thesubsequent washing and staining steps diffi-cult. Also it will likely clog the SIP during theanalysis step.

c. Aspiration of the supernatant must bevery carefully done, as sperm will be lost ineach washing and re-suspension step.

d. The reagent volumes that need to bealiquotted for preparing the stain must be care-fully calculated and verified as described toavoid over staining or under staining of thesamples.

e. It is helpful to give a quick spin to theTdT vial to bring the volume to the bottom ofthe vial.

f. The preparation of the stain and all sub-sequent steps must be conducted in indirectlight or in the dark.

g. The incubation step with the stain shouldnot exceed 60 min. at 37°C. The incubationtime can be noted on the aluminum foil.

h. The appropriate volume of ‘staining so-lution’ to prepare for a variable number ofassays is based upon multiples of the com-ponent volumes needed for one assay. Mixonly enough ‘staining solution’ to completethe number of assays prepared per analysis.

i. The ‘staining solution’ is active for ap-proximately 24 hr at 4°C.

j. The cells must be analyzed within 3 hrafter staining. They will start to deteriorate ifleft overnight before analysis.

k. Always run the ‘Kit controls’ and ‘Pa-tient samples’ in the ‘Collect Tab’.

l. The gate should be changed to ‘P3 in P1for sample data acquisition.

m. Do not change the settings in the fourplots.

n. The test must always be validated to con-firm that the test was correctly performed asper the validation criteria provided above.

o. It is important to clean the SIP each timeby clicking on the ‘Back flush’ button and fol-lowing the steps in the manual.

p. The flow cytometer quality control mustbe performed as recommended for optimalperformance of the instrument.

It is important that proper quality controlfor reagents and instrument be in place for thetest be precise and accurate. The instrumentquality control using the ‘8 peak’ beads pro-vided by the manufacturer must be performedregularly. In addition, negative and the pos-itive kit controls that are provided with theTUNEL kit must be included with each run.Appropriate negative (no TdT) and positivecontrols (treated with hydrogen peroxide toinduce DNA fragmentation) should also be in-cluded with each run. Internal controls withknown sperm DNA fragmentation are also in-cluded in the run for validation of the assay.

Troubleshooting

Common problems with the protocols, theircauses, and potential solutions

Common problems with contemporary pro-tocols measuring sperm DNA fragmentationinclude the lack of standardized reference val-ues for DNA fragmentation. This is largely


16.11.19


attributed to the variations in the methodologyfor measuring DNA fragmentation.

The four common methods described arethe SCSA, TUNEL, Comet, and SCD as-say. Each of them measures a different end-point/aspects of DNA damage. TUNEL mea-sures a definitive end point: DNA strandbreaks at the 3′ terminal end. Some testsmeasure single-strand breaks; others measuredouble-strand breaks. While SCSA andTUNEL using flow cytometry, which is a ro-bust technique, are objective, there are otherreports using microscopy and manual assess-ment, which are subjective, with counting of200 or 500 cells to derive the percentage ofDNA fragmentation.

The manual methods account for the largevariability in the reference value, which even-tually affects the sensitivity and the specificityof each of these tests. These ultimately influ-ence the overall diagnostic accuracy of thesetests.

In order to overcome some of the potentialdifficulties, it is important that these tests bestandardized in each laboratory and that instru-ment/assay variability as well as observer vari-ability be minimized. These goals can be ac-complished with adequate training, followingcompletely vetted protocols with establishedreference ranges.

Statistical AnalysesAppropriate statistical methods must be

used. The differences in the distribution ofTUNEL-positive sperm DNA fragmentationlevels between infertile patients and donorsshould be assessed using the Wilcoxon rank-sum test for paired samples. For a comparisonbetween patients and donors, an independentsample test must be used. If samples are nor-mally distributed, a parametric test should beused. Once adequate number of subjects havebeen tested for SDF, the receiver operatingcharacteristic (ROC) curve can be generatedto establish the cutoff, sensitivity, specificity,area under the curve, and positive and nega-tive predictive values, as well as the accuracyof the test (Sharma et al., 2016; Sharma et al.,2010).

Alternative statistical approachesTo examine the inter-laboratory standard-

ization of the TUNEL test, after a test fornormal distribution is done, Spearman’s rankcorrelation (non-parametric) can be used todetermine the strength of the relationship be-tween the TUNEL values obtained in eachlaboratory and can also be used to catego-rize the degree of correlation—i.e., moderate,

strong, or very strong (Evans, 1996). The av-erage DNA fragmentation rates can comparedusing the Wilcoxon Signed Rank Test. Thestatistical significance level is set at 5%.

Anticipated ResultsSample concentration should be between

1-5 × 106 sperm for appropriate run andto avoid clumping of the cells. SDF for thenegative kit control should be 3% to 5%, andfor the positive kit control, >30% to 40%, us-ing the apoptosis detection kit. If the controlresults are outside this range, there are issueswith the staining. The percent DNA fragmen-tation values of positive control spermatozoamust be greater than the percent DNA frag-mentation of the spermatozoa samples. Thekit positive control percent DNA fragmenta-tion must be greater than 30%.

Time ConsiderationsAdequate time must be set aside to conduct

this test. Conducting initial semen analysis toexamine sperm concentration and preparationof the test samples along with appropriate neg-ative and the positive controls takes about 2 to3 hr. Processing and staining of samples takesabout 2.5 hr. Finally, the running of the sam-ples by flow cytometer takes about 1 hr. Theanalysis of the data and calculation of spermDNA fragmentation takes another 60 min.

AcknowledgementsThe study was supported by funds from the

American Center for Reproductive Medicine,Cleveland Clinic.

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Key ReferencesAgarwal et al. (2016). See above.This chapter describes in detail the steps involved in

the analysis of semen samples for TUNEL mea-surement, as well as a short protocol highlightingthe main steps.

Sharma et al. (2016). See above.This article describes the measurement of DNA

fragmentation in semen samples from healthycontrols and infertile men. It also providesthe cutoff and sensitivity and specificity of theTUNEL test.

Sharma et al. (2013). See above.This article is a step-by-step guide explaining how

to set up the TUNEL assay as a clinical test usingflow cytometry or fluorescence microscopy.

Sharma et al. (2010). See above.This paper establishes the inter-and intra-observer

and inter-and intra-assay variability, cutoff val-ues, and the distribution of DNA fragmentationin infertile men referred to a clinical andrologylaboratory.

Ribeiro et al. (2010). See above.This paper describes the standardization of the

method and comparison of data across two ref-erence laboratories in different continents usingidentical semen samples, assay kit, protocol, ac-quisition settings, and flow cytometers. It vali-dates the TUNEL assay and establishes it as arobust test for measuring DNA fragmentation.


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