While a woman’s body always produces a small amount of human luteinizing hormone (LH), there is a sudden increase of this hormone during the middle of the menstrual cycle. This increase of the LH level, called the LH surge, promotes the release of a mature egg from the ovaries (ovulation). For most women, ovulation will occur within 24-36 hours after the peak of the LH surge. Immediately after ovulation, the egg is capable of being fertilized for a short time (approx. 12 hours).
It is recommended to determine the LH concentration for a time period of approximately 6 days. The starting day of the testing period depends on the length of the menstrual cycle and is individually calculated. During this time period the LH Test will detect the LH surge thus allowing the prediction of the fertile days of the cycle.
The LH test is a semiquantitative immunoassay that is visually interpreted by the comparison of line intensities that appear on the membrane in the presence of LH in the urine sample. LH is detected with the aid of specific antibodies against the hormone. After the addition of the sample, a color-labelled antibody specifically binds to LH if it is present in the sample. When the LH-antibody-complexes migrate upward on the membrane by capillary action, they are captured with the aid of another specific antibody at the test result line region (T) of the test. A red test result line is generated the color intensity of which is dependent on the concentration of LH. In contrast to the test result line (T) the color intensity of the control line (C) is the same in all assays. By comparing the color intensity of the test result line (T) to the control line (C) the user can estimate the concentration of LH present in the sample and can predict if the LH peak has been reached and when ovulation takes place. If no LH is present the color labelled antibody cannot bind at the test result line region. No red test result line is formed. The control line (C) that will always appear in the control line region also serves as an internal control and indicates that proper volume of specimen has been added and membrane wicking has occurred.
Comparison of the color intensities of control line and test result line allows a semiquantitative estimation of the LH concentration of the sample.
• 40 individually pouched test devices
• 40 disposable pipettes included in the pouch
• 1 package insert
• Clean, dry containers for the collection urine samples
• Stop watch or timer
STORAGE AND STABILITY
• The kit should be stored at 2-30°C until the expiry date printed on the sealed pouch.
• The test must remain in the sealed pouch until use.
• Do not freeze.
SPECIMEN COLLECTION AND STORAGE
Calculation of the initial testing date
The initial testing date for a complete 6 day testing period depends on the average length of the last normal menstrual cycles. The first day when bleeding begins in the menstrual period is counted as day 1.
The chart below demonstrates the correlation between the starting day and the length of the menstrual cycle. Beginning with the starting day and on the following 5 days the LH concentration is determined by using 1 test per day.
If the menstrual cycle is shorter or longer than in the chart above, the initial testing date must be calculated by the physician. If the cycles are irregular it might be necessary to prolong the 6 day testing period for several days.
Example for a typical 28 day cycle:
Collection of the specimen
• The LH test is intended for use with human urine specimens only.
• Do not use the first urine in the morning. Any other convenient time of the day can be chosen to collect urine. Approximately 2 hours prior to urine collection the liquid intake should be reduced. An excessive uptake of liquid prior to testing might result in a dilution of the urine causing false negative results. Urine should be collected at about the same time each day for the entire testing period.
• Turbid specimens should be centrifuged, filtered, or allowed to settle and only the clear supernatant should be used for testing. use the first urine in the morning. Any other convenient time of the day can be chosen to collect urine. Approximately 2 hours prior to urine collection the liquid intake should be reduced. An excessive uptake of liquid prior to testing might result in a dilution of the urine causing false negative results. Urine should be collected at about the same time each day for the entire testing period.
• Urine specimens must be collected in clean, dry containers. New specimen collection containers should be used for each sample to avoid cross contaminations.
• Preferably testing should be performed immediately after specimen collection. Do not leave specimens at room temperature for prolonged periods. Specimens may be stored at 2-8°C for up to 24 hours. Do not freeze. For best results, test the urine sample on the same day.
• Refrigerated specimens must be brought to room temperature prior to testing.
• If specimens are to be shipped, pack them in compliance with all applicable regulations for transportation of etiological agents.
Bring tests, specimens, and/or controls to room temperature (15-30°C) before use.
1. Remove the test from its sealed pouch, and place it on a clean, level surface. Label the device with patient or control identification. For best results, the assay should be performed within one hour.
2. Add 2-3 drops of specimen (approximately 80-120 μl) directly into the specimen well (S) using the provided pipette. Start the timer.
Avoid trapping air bubbles in the specimen well (S), and do not add any liquid to the result area in the center of the device. As the test begins to work, color will migrate across the result area.
3. Wait for the colored line(s) to appear. The result should be read at 5 minutes. Do not interpret the result later than 10 minutes after sample addition.
INTERPRETATION OF RESULTS
For reading of the result, the pattern of lines that have appeared at the result area is interpreted. In case there are two lines their color intensities are compared. Do not read the result later than 10 minutes after sample addition as the two lines might become darker at different paces over the time so that a correct semiquantitative interpretation is no longer possible.
During the follicle phase, the values for LH are around 5-15 mIU/ml. During the LH surge shortly before ovulation takes place, values up to 200 mIU/ml are reached. During the luteal phase, the LH concentration decreases again to values of 5-10 mIU/ml. After the onset of menopause LH concentrations are continuously elevated and frequently reach values of >20 mIU/ml (Thomas 2005).
The performance of the assay has been determined with urine samples with known LH concentrations from female patients. The LH Test showed the following performance characteristics:
Diagnostic Sensitivity: > 99 %
Diagnostic Specificity: 97.0 %
Positive Predictive Value: 87.5 %
Negative Predictive Value: > 99 %
Reproducibility: 97.5 %
Range of measurement
The LH test is calibrated to the 2nd International Standard for LH, NIBSC Code 80/552. Faint test result lines are generally seen at LH concentration of 5 mIU LH / ml. The color intensity of the C Line is set in a way that it equals the intensitiy of the the test result line at a concentration of 35 mIU LH / ml. So the cut-off of the assay for positive test results is 35 mIU LH /ml.
No prozone effect (high dose hook effect) is observed up to a concentration of 10,000 mIU LH/ml so under physiological conditions no high dose hook effect is to be expected.
The specificity of the LH test was determined in cross reactivity studies with known amounts human Chorionic Gonadotropin (hCG), Follicle Stimulating Hormone (hFSH) and Thyroid Stimulating Hormone (hTSH). When tested at more fold physiologically expected concentrations, all hormones gave clear negative result. So in healthy, non-pregnant individuals no cross reactivities with the structurally related hormones hCG, hFSH, and hTSH are to be expected.