Reactive Oxygen Species (ROS) Measurement

Abstract

Free oxygen radicals such as superoxide anion (O₂¯), hydrogen peroxide (H₂O₂), hypochlorite (OHCl), and hydroxyl radical (OH) are called reactive oxygen species (ROS). Free radicals have a very short half-life and are continuously produced, mainly by white blood cells (WBC) and especially by granulocytes and abnormal sperm. Excessive amounts of ROS impair sperm quality. ROS levels can be measured with the chemiluminescence method using luminol as a probe.

1 Introduction

Free oxygen radicals such as superoxide anion (O₂¯), hydrogen peroxide (H₂O₂), hypochlorite (OHCl), and hydroxyl radical (OH) are called reactive oxygen species (ROS) [1, 2]. Free radicals have a very short half-life and are continuously produced, mainly by white blood cells (WBC) and especially by granulocytes and abnormal sperm [3]. Excessive amounts of ROS impair sperm quality. ROS levels can be measured with the chemiluminescence method using luminol as a probe.

2 Principle

Luminol is extremely sensitive and reacts with a variety of ROS at neutral pH [4]. It can measure extracellular and intracellular ROS. The free radical combines with luminol to produce a light signal that is converted to an electrical signal (photon) by a luminometer. The number of free radicals produced is measured as relative light units/s/10⁶ sperm [5].

3 Specimen Collection

The patient collects a semen sample by masturbation and then ejaculates into a sterile container. The collection should occur after 48–72 h of sexual abstinence. The patient’s name, medical record number, and date of collection should be recorded on the specimen container.

4 Equipment and Materials

• A. Disposable polystyrene tubes with caps (15 mL)

• B. Pipettes (5 and 10 μL)

• C. Pipettes (1, 2, and 10 mL)

• D. Centrifuge

• E. Computer-assisted semen analyzer (CASA)

• F. Disposable sperm counting chamber

• G. Dimethyl sulfoxide (DMSO)

• H. Luminol (5-amino-2,3dehydro-1,4 phthalazinedione)

• I. Polystyrene round-bottom tubes (6 mL)

• J. Luminometer (Model: AutoLumat plus LB 953, Oakridge, TN)

• K. Dulbecco’s phosphate-buffered saline solution 1× (PBS)

5 Reagent Preparation

• A. Luminol stock solution: 100 mM solution—weigh out 177.09 mg of luminol and add it to 10 mL of DMSO solution in a polystyrene tube. The tube needs to be covered with aluminum foil due to light sensitivity of the luminol. This solution can be stored at room temperature until expiration date.

• B. Working luminol: 5 mM solution—mix 20 μL of the luminol stock solution with 380 μL DMSO in a foil-covered polystyrene tube (Fig. 21.1). Make fresh prior to use. Store at room temperature until needed. Stable for 24 h if not exposed to light.

  Fig. 21.1

  

  Working luminol solution; wrapped in foil due to light sensitivity. [Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography © 2015. All Rights Reserved]

• C. DMSO solution: Provided ready to use. Store in dark container and at room temperature until expiration date (Fig. 21.2).

  Fig. 21.2

  

  Bottle of dimethyl sulfoxide (DMSO) . [Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography © 2015. All Rights Reserved]

6 Specimen Preparation

• A. Allow the semen sample to undergo liquefaction in the 37 °C incubator for 20 min.

• B. Record the patient name, medical record number, period of sexual abstinence, date and time of specimen collection, and age of specimen when the semen analysis is performed.

• C. Record the initial physical characteristics such as volume, pH, color, etc.

• D. Load 5 μL of the well-mixed semen onto a sperm counting chamber.

• E. Perform semen analysis using manual sperm concentration and motility. Also count round cells and perform the Endtz test if needed.

7 ROS Determination

• A. Set up the luminometer and computer attached to it (Fig. 21.3a–c).

  Fig. 21.3

  

  Autolumat 953 plus luminometer used in the measurement of ROS by chemiluminescence assay. (a) External view and (b) internal view. Multiple tubes can be loaded simultaneously for measuring ROS. (c) The luminometer can be connected with a computer and monitor. [Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography © 2015. All Rights Reserved]

• B. Label 11 tubes (12 × 75 mm) in triplicate and add the reagents indicated in Table 21.1 (Fig. 21.4).

  Table 21.1 Set up for the measurement of ROS

  Fig. 21.4

  

  Setup for reactive oxygen species testing: three blank controls, three negative controls, two patient sample tubes, and three positive controls. [Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography © 2015. All Rights Reserved]

  Note: All readings must be performed in the dark.

• C. It is important that the instrument settings are in place before adding reagents to the tube and loading samples.

• D. Add reagents to the bottom of the tubes and not on the side. Vortex to ensure that the luminol mixes with the rest of the reagent/sample.

• E. The pipette tip should be changed when adding reagent/sample to each tube.

• F. Gently vortex the tubes to mix the aliquots uniformly. Avoid bubbles.

• G. Place all the labeled tubes in the luminometer in the following order: blank (tubes labeled 1–3), negative control (tubes labeled 4–6), test sample (tubes labeled 7–8), and positive control (tubes labeled 9–11) (Figs. 21.5 and 21.6) [4, 5].

  Fig. 21.5

  

  Preparing the tubes for ROS measurement. A total of 11 tubes are labeled from S1–S11: blank, negative control, test sample, and positive control. Luminol is added to all tubes except the blank. Hydrogen peroxide is added only to the positive control

  Fig. 21.6

  

  Proper placement of tubes into luminometer . [Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography © 2015. All Rights Reserved]

8 Instrument Setup

• A. From the desktop, click on “Berthold tube master” icon to start the program.

• B. From the “Setup menu,” select “Measurement Definition” and then “New Measurement.” You will be prompted to the following:

  • a. “Measurement Name” (initials, date, measurement, and patient initials, e.g. LH 3-13-13ROSXX). Copy and click “OK.”

  • b. It will show “Measurement Definition” on the “Toolbar” (initials, date, analyte and measurement, patient initials; e.g. RK 3-13-13 ROS XX).

  • c. Under “Luminometer Measurement protocol,” select “Rep. assay” from the drop-down menu.

  • d. Next, define each of the “Parameters” as follows:

    • i. Read time: 1 s.

    • ii. Background read time: 0 s.

    • iii. Total time: 900 s.

    • iv. Cycle time 30 s.

    • v. Delay “Inj M read (s)”: 0 s.

    • vi. “Injector M (μL)”: 0 s.

    • vii. “Temperature (°C)”: 37 °C.

    • viii. “Temperature control (0 = OFF)”: 1 = ON.

  • e. “Save.”

• C. From the “Setup” menu, select “Assay Definition” and then “New Assay.”

  It will ask for the following:

  • i. “Assay Name” (initials, date, analyte and measurement, patient initials). Click “OK” or paste and click “OK.”

  • ii. Under “Measurement Method” and from the drop-down menu, select the measurement (e.g., RK 3-13-13 ROS XX) from Step 2a above.

  • iii. Go to “Column Menu” and hide everything except the following:

  • iv. “Sample ID.”

  • v. “Status.”

  • vi. “RLU mean.”

  • vii. “Read date.”

  • viii. “Read time.”

• D. Go to “Sample Type” menu and select “Normal.” Press “OK.”

• E. Go to file, click “New,” click “Workload,” and press “OK.”

• F. Go to “Save As” and save in an appropriate folder such as “Clinical ROS” folder.

• G. Save your “Work Load” (initials, date, analyte and measurement, patient initials) in “Clinical ROS” “Work Load” file.

• H. After saving the “Work Load,” the name of the file will show in the “Title Bar.” The samples are ready to be analyzed.

9 Analyzing the Samples

• A. After the tubes have been loaded, click “Start.” The luminometer will start scanning for tubes.

• B. After scanning, the monitor will show how many tubes are detected by the instrument in each batch; press “Next.”

• C. Select the “Assay Type.” Click “Next” and then click “Finish.”

• D. The “Excel spreadsheet” will open.

• E. Measurement of the tubes will start.

• F. Do not touch the computer during this time.

• G. Wait (3–5 min) to make sure everything is working fine.

• H. After finishing the measurements, the computer will ask you to “Save” the Excel spreadsheet. Save it in “My Computer,” in a folder under “Clinical ROS.”

• I. Save Berthold measurement “Measurement Files” (*.txr) in the same directory as the Excel spreadsheet using the same name (e.g., RK 3-13-13 ROS XX).

10 Printing ROS Results

• A. Print spreadsheet as well as the “chart 1” and the Berthold sheet (Fig. 21.7).

  Fig. 21.7

  

  A representative display of the readings showing the number of signals generated in each of the above 11 tubes (S1–S11). The measurement is for a total of 900 s. As seen here, blanks have the lowest amount of ROS (RLU), and the positive control, to which hydrogen peroxide was added, has the highest amount of ROS (RLU)

• B. Close the Excel spreadsheet.

• C. Print the “Work Load” sheet (Fig. 21.8). Make sure all three sheets are printed before saving and closing the file.

  Fig. 21.8

  

  A typical graph showing the ROS levels in the 11 tubes (S1–S11). As seen here, only the positive controls have significantly higher levels of ROS. Those producing low levels (Tubes S1–S8) of ROS are seen very close to the X-axis

11 Calculating Results

• A. Calculate the “average RLU” for the negative control, samples, and positive control.

• B. Calculate sample ROS by subtracting the negative control average from its average.

• C. Sample ROS = average “RLU mean” for sample—average “RLU mean” for negative control.

• D. Correct the sample ROS by dividing it with “sperm concentration/mL.”

Corrected sample ROS:

Calculated sample ROS/sperm concentration = XX.X (RLU/s/10⁶ sperm/mL).

A typical example of calculating ROS values is illustrated in Fig. 21.9.

Fig. 21.9

Berthold sheet showing a typical ROS calculation

12 Reference Values

Normal range: <93 RLU/s/10⁶ sperm/mL.

Critical value: ≥93 RLU/s/10⁶ sperm/mL.

13 Quality Control

• A. The reagent lot numbers and expiration dates are recorded on the worksheet and placed in the Quality Control book.

• B. Criteria for rejection: No sperm are present.

14 Factors Affecting ROS Measurement

• A. The luminometer instrument, its calibration, determination of sensitivity, dynamic range, and units used.

• B. The concentration and type of probe used.

• C. The concentration and volume of the semen sample, use of reagent, and temperature of the luminometer.

• D. Semen age (i.e., time to analysis) after sample is collected to the time of ROS measurement.

• E. Viscous samples and poor liquefaction, which may interfere with chemiluminescent signals.

• F. Repeated centrifugation: Artificial increase in chemiluminescent signal because of the shearing forces generated by centrifugation.

• G. Serum albumin: Use of media that contain bovine serum albumin can generate spurious signals in the presence of human seminal plasma.

• H. Medium pH: Luminol is sensitive to pH changes.

• I. Nonspecific interference: Many compounds can artificially increase (cysteine, thiol-containing compounds) or decrease (ascorbate, uric acid) the chemiluminescent signal generated by the spermatozoa. Hence, it is necessary to run sperm-free controls as an integral part of the chemiluminescent assay.

15 Troubleshooting

• A. Clean the interior of the instrument with antistatic spray, especially the chain belt. Keep a container filled with distilled water inside the machine at all times to maintain humidity and reduce static.

• B. Check the instrument background reading from the “rate meter.” It should not be ≥20 RLU. If it is ≥20 RLU, contact the company. If <20 RLU, proceed to the next step.

• C. Check the reagents for contamination. First, check the PBS buffer. Does this resolve the issue? If not, go to the next step.

• D. Next, check the luminol solution. Prepare a fresh luminol solution. Does this resolve the issue? If not, go to the next step.

• E. Prepare luminol in fresh DMSO.

• F. Test multiple runs in triplicate for blank, negative control, and positive control. Use chart below.

  	RLU/s
  No	Blank	Negative control	Positive control
  1
  2
  3
  4

• G. If the issue is resolved, the instrument and reagents are good for new measurements.

• H. If the issue is not resolved, contact the luminometer manufacturer (865-483-1488).

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