Intended use

The NG-Test hCG WB is a rapid visual immunoassay for the qualitative presumptive detection of human chorionic gonadotropin in human whole blood to aid in the early detection of pregnancy.The NG-Test hCG WB has a sensitivity of 10mIU/mL

Summary and Explanation 

Human chorionic gonadotropin (hCG) is a glycoprotein secreted by viable placenta tissue during pregnancy. In normal pregnancy,hCG can be detected in whole blood as early as 7 to 10 days after conception. hCG levels continue to rise very rapidly, frequently exceeding 100 mIU/mL make it an excellent marker for confirming pregnancy. The test utilizes monoclonal antibodies to hCG.

Principle of the test

The NG-Test hCG WB is a rapid chromatographic immunoassay for the qualitative detection of Human Chorionic gonadotropin (hCG) in whole blood. The test utilizes monoclonal antibodies to hCG to selectively detect elevated levels of hCG in whole blood. The assay is conducted dispensing an adequate volume of the blood specimen into the sample well of the cassette. A buffer solution is added to the well to facilitate the reagent capillary action across the strip held in the cassette. The sample then migrates across a membrane toward the results window where the labeled hCG complex is captured at a test line region containing immobilized monoclonal anti-hCG. Control region, whatever the result should show a colored band. It indicates that the test has been performed correctly. The appearance of two red lines, one at T (Test) and the other at C (Control) indicates the presence of hCG in the sample. If a detectable level of Hcg (<10 mIU/ml) is not present, only the control band will appear in the result window.

Reagents and material provided

Each kit contains 10 individual pouches containing:  
•    1 cassette, each sealed in a foil pouch with dessiccant. 
•    1 calibrated micropipettes capable delivering 20µl accurately.
•    1 lancet.
•    1 alcohol prep pad.
•    1 Buffer solution in plastic dropper bottle.
•    2 traceability patient labels.
•    1 package insert.

Material required but not provided 

•    Timer.  
•    Disposable gloves.

Precautions

•    For Professional in vitro diagnostic use only.
•    Do not use after the expiration date.
•    The test device should remain in the sealed pouches until use.
•    Handle all specimens as potentially infectious.
•    The test device should be discarded in a suitable biohazardous waste container after testing.
•    The test device should not be reused.

Storage and stability

Store as packaged in the sealed pouch at  4-30°C.Test devices are stable until the expiration date printed on the kit or foil pouch. DO NOT FREEZE.

Specimen collection

Fresh blood from finger prick / puncture should be used as a test specimen. However, fresh anti coagulated whole blood may also be used as a test sample and EDTA or Heparin or Oxalate can be used as suitable anticoagulant. The specimen should be collected in a clean glass or plastic container. If immediate testing is not possible then the specimen may be stored at 2-8°C for up to 72 hours before testing. Clotted or contaminated blood samples should not be used for performing the test.

Procedure

1.    Wear protective gloves.
2.    Bring the kit components to room temperature before testing.
3.    In case the pouch has been stored at 4-8°C, allow at least 10 minutes for the device to come to room temperature.
4.    Open the pouch and remove the device. Once opened, the device        must be used immediately.
5.    For easy blood sample collection the patient should maintain the  arm hang down for at least 3 minutes. Cleanse with alcohol swab the area of the 3rd or 4th finger of the left hand and let dry before pricking.  
6.    Open the lancet and prick the lateral side of the patient’s fingertip to obtain a drop of blood (20μl).  Using gentle pressure, massage the finger towards the fingertip to encourage a drop of blood to form.
7.    Hold the capillary micropipette horizontaly, and touch with the tip of the micropipette the blood sample. 
CAUTION: Filling is automatic by capilary action. Do not squeeze the tube while sampling.
8.    To expel the sample, place the tip of the micropipette on the Sample pad hole marked “S” and squeeze the micropipette bulb.                                                                                                Transfer to the sample pad must be immediate in order to avoid sample clotting.
9.    Add 2 drops of buffer solution (2x40µl) into the Reagent hole marked “R”.
10.    Read the results at 5 minutes and interprete as below.  
NOTE: Do not interprete the test results after 10 minutes. (Results may change.)
     
                                                                                           
Interpretation of results 
                                                                   
Negative result

If there is only one red color band in the control region (C), this indicates that the specimen does not contain a detectable level of hCG and should be interpreted as a negative result

Positive result
If there are two red color bands one in the control region (C) and another in the test region (T), this indicates that the specimen contains hCG at a concentration equal to or greater than 10 mIU/mL and should be interpreted as positive result.

 Invalid result
If the control band will not appear the test result is invalid. Insufficient specimen volume or incorrect procedural techniques are the most likely reasons for control line failure. Deterioration of the test kit may have occurred. Repeat the test procedure using a new test. If the problem persists, discontinue using the kit immediately and contact your local distributor.

NOTE: The intensity of the red color in the test line region (T) will vary depending on the concentration of hCG present in the specimen. However, neither the quantitative value nor the rate of increase in hCG can be determined by this qualitative test2.

Quality Control

Internal procedural controls are included in the test. The appearance of the control band confirms sufficient specimen volume and correct procedural technique.
At the read time, a clear background is an internal negative background control. If the test working properly, the background in the result area should be white to light pink and not to interfere with the ability to read the test result.
It is recommended that external hCG controls be run with each new lot. 

Limitations

1.    False negative results may occur when the levels of hCG are below the sensitivity level of the test (10mIU/mL). When pregnancy is still suspected repeat the test 48 hours later.
2.    Very low levels of hCG are present in whole blood specimen shortly after implantation. However, because a significant number of first trimester pregnancies terminate for natural reasons 5, a test result weakly positive should be confirmed by retesting 48 hours later.
3.    A number of conditions other than pregnancy, including trophoblastic disease, testicular tumors, prostate cancer, breast cancer and lung cancer cause elevated levels of hCG 6-7. Therefore, the presence of hCG in whole blood specimen should not be used to diagnose pregnancy unless these conditions have been ruled out.
4.    As with any assay employing mouse antibodies, the possibility exists for interferences by human anti-mouse antibodies (HAMA) in the specimen. Specimens from patients who have received preparations of monoclonal antibodies for diagnosis therapy may contain HAMA. Such specimens may cause false positive or false negative results.
5.    This test provides a presumptive diagnosis for pregnancy. 
A confirmed pregnancy diagnosis should be made by a physician after all clinical and laboratory findings have been evaluated.                   

Expected values

Negative results are expected in healthy non-pregnant women and healthy men. The amount of hCG will vary greatly with gestational age and between individuals. 
The NG-Test hCG WB has a sensitivity of 10mIU/mL and is capable of detecting pregnancy as early as 1 day before expected period date.  

Performance characteristics

Specificity

The addition of LH (300 mUI/mL), FSH (1000 mUI/mL) and TSH        (1000 mUI/L) to negative (0 mIU/mL hCG) and positive (10 mIU/mL hCG) specimens showed no cross-reactivity.
The following potentially interfering substances were added to hCG negative and positive (10 mIU/mL) specimens.

None of the substances at the concentration tested interfered in the assay.
Interfering substances    Concentration level
 Rheumatoid Factor    174 IU/mL
Human anti-mouse antibodies    715 ng/mL
Antinuclear antibodies (centromere type speckled nucleolar and homogeneous)    Titer > 1280
Caffeine    200 µg/mL

Sensitivity

The tests are performed on true samples measured by a reference laboratory.
Fresh blood (sampling <48H) Patient 1; [HCG] <2 mIU/mL)
Given the difficulty of obtaining positive fresh blood samples with hCG concentrations given below, the tests are conducted using sera.
Patient 2: [HCG] = 8 mIU/mL  
Patient 3: [HCG] = 10 mIU/mL 
Patient 4: [HCG] = 11 mIU/mL 

Twenty repetitions are performed on each lot (3 lots) with a blood sample at a concentration of [HCG] <2 mIU/mL or a total of 60 repetitions.
Twelve repetitions are performed on each lot (3 lots) with sera at a concentrations of [HCG] = 8 mIU/mL; [HCG] = 10 mIU/mL and [HCG] = 11 mIU/mL.
Serum samples are performed in blind manner. The person who interprets the results does not know the level of HCG concentration in serum samples.
The sensitivity of NG-hCG test whole blood is 10 mIU/mL with greater than 99.9% probability.

Standardisation

The test has been standardized to the W.H.O Fifth International Standard (07/364).

Hook Effect

No Prozone effect observed up to 500,000 mIU/mL.
The test bands may appear with a less intensive color than expected for samples containing high levels of hCG> 500,000 mIU/mL.

Accuracy

A study included 168 blood samples, 72 positive hCG samples at a concentration of 10 mIU/mL or greater and 96 negative hCG samples provided the following results:

The results demonstrated a 100% overall agreement (for an accuracy of >99%) of the NG-Test hCG WB when compared to the reference method.