Inter‐and Intra‐Laboratory Standardization of TUNEL Assay for Assessment of Sperm DNA Fragmentation

Sajal Gupta1, Rakesh Sharma1, Ashok Agarwal1

1 American Center for Reproductive Medicine, Glickman Urological & Kidney Institute, Cleveland Clinic, Cleveland, Ohio
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 16.11
DOI:  10.1002/cptx.37
Online Posting Date:  November, 2017
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Abstract

The functional aspects of sperm activity such as sperm chromatin integrity and ability to fertilize cannot be characterized by routine semen parameters. Men with unexplained infertility and idiopathic infertility, as well as men with normozoospermic semen profiles, show high DNA fragmentation. Molecular anomalies in the sperm can be detected by a sperm DNA fragmentation (SDF) assay which can be used in adjunct to conventional semen analysis. While the sperm chromatin structure assay (SCSA) remains the “gold standard,” the TUNEL assay using flow cytometry is becoming popular among the different tests that are currently available to measure sperm DNA fragmentation. In this unit, we describe the inter‐laboratory and intra‐laboratory standardization of the TUNEL assay using a benchtop cytometer. The article also provides a step‐by‐step protocol for measuring sperm DNA fragmentation using the TUNEL assay and a bench‐top flow cytometer, and also points out the inherent challenges with this test. © 2017 by John Wiley & Sons, Inc.

Keywords: Accuri C6; flow cytometry; DNA fragmentation; sperm; TUNEL

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Laboratory Measurement of Sperm DNA Fragmentation by TUNEL Assay Using Benchtop Flow Cytometer
  • Basic Protocol 1: Collection of Semen Specimen
  • Support Protocol 1: General Setup of the Benchtop Cytometer
  • Basic Protocol 2: Instrument Quality Control
  • Support Protocol 2: Sample Preparation for TUNEL Assay
  • Support Protocol 3: Preparation of the ‘Positive Control’ Sample
  • Basic Protocol 3: TUNEL Staining with the APO Direct Kit
  • Support Protocol 4: Cleaning and Maintenance of the Benchtop Cytometer
  • Data Analysis
  • Support Protocol 5: Alignment Strategy and Data Analysis in the Collect Tab
  • Support Protocol 6: Data Analysis in “Analyze” Tab
  • Support Protocol 7: Final Result Calculation for Sperm DNA Fragmentation Percent Value and Verification of the Validity of the TUNEL Assay Performed
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Collection of Semen Specimen

  Materials
  • Donor of test specimen
  • Viscosity treatment enzyme, e.g., chymotrypsin (optional)
  • Semen analysis form
  • Wide‐mouth, sterile plastic specimen container

Support Protocol 1: General Setup of the Benchtop Cytometer

  Materials
  • Sheath fluid (blue bottle; BD Biosciences, PN 653156): 0.22‐µm filtered deionized water with or without bacteriostatic concentrate solution; if bacteriostatic concentrate solution is used (optional), add 1 bottle per 1 liter of water
  • Cleaning solution (green bottle; BD Biosciences, PN 653157); dilute 3 ml of cleaning concentrate in 197 ml of filtered deionized water (use the solution within 2 weeks)
  • Decontamination solution (yellow bottle; PN653154); add entire bottle to 180 ml of filtered, deionized water
  • Benchtop flow cytometer (Accuri C6; BD Bioscience; Fig.  )
  • BD Accuri C6 software

Basic Protocol 2: Instrument Quality Control

  Materials
  • Spherotech 8‐peak validation beads (BD Biosciences, cat. no. 653144)
  • 12 × 75–mm tubes
  • Benchtop flow cytometer (Accuri C6; BD Bioscience; Fig.  ), set up as in protocol 2
  • BD Accuri C6 software
  • Computer running Microsoft Excel

Support Protocol 2: Sample Preparation for TUNEL Assay

  Materials
  • Semen sample ( protocol 1)
  • Phosphate‐buffered saline (PBS; APPPENDIX )
  • Sperm counting chamber (Spectrum Technologies, cat. no. SC‐20‐01‐02‐B)
  • Centrifuge

Support Protocol 3: Preparation of the ‘Positive Control’ Sample

  Materials
  • 37% hydrogen peroxide
  • Phosphate‐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°C
  • 70% ethanol, ice cold
  • 50°C heating block
  • Centrifuge

Basic Protocol 3: TUNEL Staining with the APO Direct Kit

  Materials
  • Test samples
  • APO‐DIRECT™ Kit (BD Pharmingen, cat. no. 556381):
    • PI/RNase Staining Buffer
    • Reaction Buffer
    • FITC‐dUTP
    • TdT Enzyme
    • Rinsing Buffer
    • Wash Buffer
    • Negative Control Cells
    • Positive Control Cells
  • 12 × 75–mm polystyrene tubes
  • Benchtop flow cytometer (Accuri C6; BD Bioscience; Fig.  ), set up as in protocol 2
  • BD Accuri C6 software
  • Computer running Microsoft Excel

Support Protocol 4: Cleaning and Maintenance of the Benchtop Cytometer

  Materials
  • BD Accuri C6 software

Support Protocol 5: Alignment Strategy and Data Analysis in the Collect Tab

  Materials
  • BD Accuri C6 software

Support Protocol 6: Data Analysis in “Analyze” Tab

  Materials
  • BD Accuri C6 software
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Figures

Videos

Literature Cited

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Key References
  Agarwal et al. (2016). See above.
  This chapter describes in detail the steps involved in the analysis of semen samples for TUNEL measurement, as well as a short protocol highlighting the main steps.
  Sharma et al. (2016). See above.
  This article describes the measurement of DNA fragmentation in semen samples from healthy controls and infertile men. It also provides the cutoff and sensitivity and specificity of the TUNEL 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 using flow 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 values, and the distribution of DNA fragmentation in infertile men referred to a clinical andrology laboratory.
  Ribeiro et al. (2010). See above.
  This paper describes the standardization of the method and comparison of data across two reference laboratories in different continents using identical semen samples, assay kit, protocol, acquisition settings, and flow cytometers. It validates the TUNEL assay and establishes it as a robust test for measuring DNA fragmentation.
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