Measurement of ROS by Chemiluminescence Assay

Herein we describe the details of measurement of ROS in unprocessed, i.e., liquefied seminal ejaculate without any further processing by chemiluminescence assay.

1. Equipment and material

(a) Disposable polystyrene tubes with caps (15-mL)

(b) Eppendorf pipettes (5-, 10-, 50-, and 1,000-mL)

(d) Desk-top centrifuge

(e) Disposable MicroCell slides

(f) Dimethyl sulfoxide (DMSO; Catalog # D8779, Sigma Chemical Co., St. Louis, MO)

(g) Luminol (5-amino-2,3 dihydro-1,4 phthalazinedione; Catalog # A8511, Sigma Chemical Co., St. Louis, MO)

(h) Polystyrene Round bottom tubes (6-mL)

(i) Luminometer (Model: LKB Autoplus 953)

(j) Dulbecco's Phosphate-buffered saline solution 1x (PBS-1x; Catalog #9235, Irvine Scientific, Santa Ana, CA)

2. Preparation of reagents

(a) Stock luminol (100 mM): Weigh 177.09 mg of luminol and add it to 10 mL of DMSO solution in a polystyrene tube. The tube must be covered in an aluminum foil due to the light sensitivity of the luminol. It can be stored at room temperature in the dark until the expiration date.

(b) Working luminol (5 mM): Mix 20 mL luminol stock solution with 380 mL DMSO in a foil-covered polystyrene tube. This must be done prior to every use. Store the solution at room temperature in the dark until needed.

(c) DMSO solution: Provided ready to use; store at room temperature until the expiration date.

3. Specimen preparation

Upon arrival of the semen specimen, allow it to liquefy in the incubator at 37°C for approximately 20 min. A complete record of patient's name, allocated identity number, period of sexual abstinence, and date and time of collection is noted. Manual semen analysis is performed for assessment of sperm concentration and motility. First, allow the semen sample to undergo liquefaction for 20 min in a 37°C incubator. Then record the initial physical characteristics such as volume, pH, and color and manually verify sperm count and motility. After complete liquefaction is ensured, set up the luminometer for ROS measurement.

4. ROS measurement by luminometer

This procedure is performed in a dark room. Set up the luminometer and the computer attached to it (Fig. 13.2a-c).

Fig. 13.2 Autolumat 953 plus luminometer used in the measurement of ROS by chemilumines-cence assay. (a) External view and (b) internal view. Multiple tubes can be loaded simultaneously for measuring ROS. (c) The luminometer can be connected with the computer and a monitor and all the steps can be observed on the screen

Fig. 13.2 Autolumat 953 plus luminometer used in the measurement of ROS by chemilumines-cence assay. (a) External view and (b) internal view. Multiple tubes can be loaded simultaneously for measuring ROS. (c) The luminometer can be connected with the computer and a monitor and all the steps can be observed on the screen

Table 13.3 Setup for the measurement of ROS

Specimen

Probe luminol

Hydrogen

Labeled tubes (no.)

PBS-lx (mL)

volume (mL)

(5 mM) (mL)

peroxide (mL)

Blank (tubes S1-3)

400

-

-

Negative control (tubes S4-6)

400

-

10

-

Patient (tubes S78)

-

400

10

-

Positive control (tubes S9-11)

400

10

50

(a) Label 11 Falcon tubes (12 x 75 mm) in duplicates and add the following reagents as indicated in Table 13.3 (Fig. 13.3).

Note: To avoid contamination, change pipette tips after each addition.

(b) Gently vortex the tubes to mix the aliquots uniformly.

Fig. 13.3 Preparing the tubes for the ROS measurement. A total of11 tubes are labeled from S1 to 12: Blank, negative control, patient sample, and positive control. Luminol is added only to all tubes except blank. Hydrogen peroxide is added only to the positive control

(c) Place all the labeled tubes in the luminometer in the following order: Blank (tubes labeled 1-3).

(d) Negative control (tubes labeled 4-6), patient sample (tubes labeled 7-8), and positive control (tubes labeled 9-11) (Fig. 13.3).

5. Instrument setup

(a) Turn on the instrument and the computer. 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:

- "Measurement Name" (Initials, Date, Analyte, and Measurement).

- It will show "Measurement Definition-on the 'Tool bar.'"

- Click "Luminometer Measurement" protocol and from the drop menu click on "Rep. assay."

Define each "Parameter" as follows:

Read time

Background read time Total time Cycle time

Delay "Inj M read (s)" Injector M (mL) Temperature (°C) Temperature control (0 = OFF)

- Press "Save"

(c) From the "Setup" menu, select "Assay Definition" and then "New Assay": It will ask for the following:

- "Assay Name" (Initials, Date, Analyte, Assay). Click "OK."

- Select "Measurement Method" and from the drop-down menu select the measurement from Step 2a above.

- Go to "Column Menu." Hide everything except the following: Sample ID

Status RLU mean Read date Read time

- Go to "Sample Type" menu and select "Normal."

- Go to file, "New" click "Workload" Press "OK."

(d) Save your "Work Load" (Date, Initial, Sample or experiment ID) in "Work Load" file.

(e) Click "File name"

(f) After saving the "Work Load," the name of the file will show in the "Title Bar."

(g) The specimens are ready to be analyzed. 6. Analyzing the samples

(a) Load the tubes into the instrument and click "Start." It will start scanning for tubes.

(b) After scanning, it will show how many tubes are detected by the instrument in each batch, press "Next."

(c) Select the "Assay Type" and type file name and then click "Finish."

(d) The "Excel spreadsheet" will open, measurement of the tubes will start.

(e) Do not touch or change the screen, wait (3-5 min) to make sure everything is working fine.

(f) After finishing measurements, it will ask for "Save Excel Spread Sheet," save it in the "My Document" under "ExcelSheet" folder.

Time

SI

S2

0

6

4

42.3

6

7

72.3

9

7

102.3

6

9

132.3

9

9

162.3

6

6

192.3

6

7

222.3

6

9

252.3

7

9

282.3

6

6

312.3

7

9

342.3

7

7

372 3

4

7

402 3

7

6

432.3

7

10

462.3

7

7

492.3

6

9

522.3

6

7

552.3

6

12

582.3

7

6

612.3

10

7

642.3

9

6

672.3

6

9

702.3

7

7

732.3

9

7

762.3

6

4

792.3

12

7

322.3

6

4

352.3

3

9

882.3

7

6

812.3

7

9 10 13

6 10 12

10 9 10

12 10 7

4 10 10

S6 S7 SS

10 19 12

10 19 12

12 13 9

10 19 12

10 16 12

10 19 15

15 21 13

7 16 13

12 16 9

9 13 12

12 16 12

10 13 12

9 13 10

9 12 10

15 13 10

12 12 9

12 12 9

9 10 18

13 9 27

10 16 9

10 12 33

9 12 12

15 13 10

9 15 10

12 12 12

18 13 10

S9

S10

Sit

1943

1719

2386

1857

1657

2248

1736

1676

2179

1742

1666

2060

1701

1493

2003

1614

1486

1943

1611

1462

1845

1532

1442

1812

1506

1406

1770

1462

1377

1729

1462

1358

1691

1391

1344

1657

1393

1330

1623

1372

1307

1583

1361

1319

1567

1319

1276

1534

1300

1275

1462

1278

1269

1437

1266

1233

1402

1244

1225

1424

1223

1216

1384

1182

1231

1344

1191

1207

1338

1169

1207

1310

1161

1157

1294

1163

1169

1287

1100

1156

1282

1114

1132

1212

1097

1142

1219

1085

1125

1234

1135

1117

1195

Fig. 13.4 A representative display of the readings showing the number of signals generated in each of the above 12 tubes (S1-11). The measurement is for a total of 900 s. As seen here, blanks have the least number of ROS produced and the positive control to which hydrogen peroxide is added has the highest number of ROS generated

(g) Select "Excel Sheet," name the file, and save type as "Measurement Files" (*.txr). Save the "Excel Sheet" (Fig. 13.4).

7. Printing ROS results

(a) Print Excel as well as the "chart 1" (Figs. 13.4 and 13.5).

(b) Close the "Excel sheet"

(c) Print the "Work Load" sheet (Fig. 13.6), save, and close it.

8. Calculating results

(a) Calculate the "average RLU" for Negative control, Samples, and Positive control.

2b) Calculate sample ROS by subtracting its average from negative control 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 (RLU/s/ x 106 sperm)

A typical example of calculating ROS values is illustrated in Fig. 13.6.

Fig. 13.5 A typical graph showing the ROS levels in the 11 tubes (S1-11). As seen here, only positive controls have significantly higher levels of ROS. Those producing low levels (tubes S1-8) of ROS are seen very close to the X axis

Sample

Sample ID

Status

RLU Mean

Read Date

Read Time

1

Done

6269

11/17/2010

1:35:54 PM

2

Blank

Done

6713

11/17/2010

1:35:56 PM

3

Done

6189

11/17/2010

1:35:57 PM

4

Done

8454

11/17/2010

1:35:59 PM

5

Negative Control

Done

8104

11/17/2010

1:36:00 PM

6

Done

9993

11/17/2010

1:36:02 PM

7

Test Sample

Done

12954

11/17/2010

1:36:03 PM

S

Done

11368

11/17/2010

1:36:05 PM

9

Done

1261225

11/17/2010

1:36:06 PM

10

Positive Control

Done

1207794

11/17/2010

1:36:08 PM

11

Done

1458674

11/17/2010

1:36:10 PM

Patient average (PJ = 12161 RLU/sec Sperm count = 12.6 x 106/mL Negative Control average [NCJ - 8850.3 RLU / sec Corrected value = Piv - NC^

= 3310.7 RLU/sec

Corrected ROS = 3310.7 - 262.7 RLU / sec/ x 106 sperms 12.6

Result = ROS positive

Fig. 13.6 A representative print out generated after the ROS measurement is complete. The chart shows the 11 tubes (S1-11), each representative of the type of sampler and the mean RLU value for each tube. At the bottom is also an example illustrating how to calculate the final amount of ROS generated in a given test (patient) sample

Reference values

Normal range: <20 RLU/s/x 106 sperm Critical Values: >20 RLU/s/x 106 sperm

9. Quality control

(a) Criteriafor acceptance: control reads 20 RLU/s/x 106 sperm.

(b) Criteriafor rejection: control reads >20 RLU/s/x 106 sperm. The assay must be repeated and results shown to the Director.

( c) The reagent lot numbers and expiration dates are recorded in the assay Quality Control book located in the laboratory.

Pregnancy Guide

Pregnancy Guide

A Beginner's Guide to Healthy Pregnancy. If you suspect, or know, that you are pregnant, we ho pe you have already visited your doctor. Presuming that you have confirmed your suspicions and that this is your first child, or that you wish to take better care of yourself d uring pregnancy than you did during your other pregnancies; you have come to the right place.

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