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Tumor Markers: PSA 
  
Prostate Specific
Antigen

   โพสเตจ สเปซิฟิค แอนติเจน

 Tumor Markers 
  ตัวช่วยบ่งชี้ภาวะการเกิด
  มะเร็งของอวัยวะต่างๆ
PSA
   ภาวะมะเร็งต่อมลูกหมาก
CEA
   ภาวะมะเร็งลำไส้
AFP
   ภาวะมะเร็งตับ
b-HCG
   
CA125
  
Ovarian Cancer 
   Antigen
CA 15-3
  
Breast Cancer 
   Antigen
CA19-9
 
Gastrointestinal 
  Cancer Antigen
AP
  Acid Phosphatase

  มะเร็งต่อมลูกหมาก
     Prostate Cancer

 
ต่อมลูกหมากโต
     Benign Prostatic
     Hyperplasia

   วิธีทดสอบ/วินิจฉัยทางแล็ป
 
     ( ห้องปฏิบัติการชันสูตร ) 
   
Reference test procedure
 
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  Tumor Markers: PSA  Prostate Specific Antigen      
Tumor Antigens : Prostate-Specific Antigen
Include markers defined by both monoclonal antibodies and polyclonal antisera, often the so called oncofetal antigens. The oncofetal substances, present in embryo or fetus, diminish to low levels in the adult but reappear in the tumor.

The PSA screening test is a blood test that looks for a specific tumor marker. In general, tumor markers are produced by the tumor itself or by our body in response to the presence of cancer or non-cancerous conditions. If a tumor marker level is higher than normal, the patient is examined more closely to look for cancer or other conditions. The most commonly tested tumor marker for the prostate gland is prostate specific antigen. It is normally present in low levels in the blood of all adult men. The normal range is 0 to 4 ng/ml.

PSA is prostate-specific, not cancer-specific. A variety of conditions can raise PSA levels: prostatitis (prostate inflammation), benign prostatic hypertrophy (prostate enlargement), and prostate cancer. PSA levels can also be influenced by a number of other things. Some prostate glands normally produce more PSA than others. PSA levels tend to increase with age. And, PSA levels can vary with race: African Americans often have higher PSA levels; Asian men often have lower PSA levels.

PSA seems to have the capability of achieving at least one of the characteristics of an ideal tumor marker- tissue specificity; it is found in normal prostatic epithelium and secretions but not in other tissues. It is a glycoprotein, whose function may be to lyse the seminal clot.

PSA is highly sensitive for the pesence of prostatic cancer. The elevation correlated with stage and tumor volume. It is predictive of recurrence and response to treatment. Finally, the antigen has prognostic value in patients with very high values prior to surgery are likely to relapse.

Unfortunately, PSA is detectable in normal men and often is elevated in benign prostatic hypertrophy, which may limit its value as a screening tool for prostate cancer. A recent study has shown that PSA combined with rectal exam is a better method of detecting prostate cancer than rectal exam alone.

Age-Specific Reference for Serum PSA (Reference Range [ng/ml])
Age Range (Years)
Black
Caucasians
Japanese
40 - 49
0.0 - 2.0
0.0 - 2.5
0.0 - 2.0
50 - 59
0.0 - 4.0
0.0 - 3.5
0.0 - 3.0
60 - 69
0.0 - 4.5
0.0 - 4.5
0.0 - 4.0
70 - 79
0.0 - 5.5
0.0 - 6.5
0.0 - 5.0

 

Enzyme Immunoassay for the Quantitative Determination of Prostate Specific Antigen (PSA) in Human Serum

FOR IN VITRO DIAGNOSTIC USE ONLY
Store at 2 to 8ฐC.

PRODUCT NAMES
PSA Enzyme Immunoassay

INTENDED USE
For the quantitative determination of the Cancer Antigen PSA concentration in human serum.

INTRODUCTION
Human prostate-specific antigen (PSA) is a serine protease, a single chain glycoprotein with a molecular weight of approximately 34,000 daltons containing 7% carbohydrate by weight. PSA is immunologically specific for prostatic tissue, it is present in normal, benign hyperplastic, and malignant prostatic tissue, in metastatic prostatic carcinoma, and also in prostatic fluid and seminal plasma. PSA is not present in any other normal tissue obtained from men, nor is it produced by cancers of the breast, lung, colon, rectum, stomach, pancreas or thyroid. Besides, it is functionally and immunologically different from prostatic acid phosphatase (PAP). 
Elevated serum PSA concentrations have been reported in patients with prostate cancer, benign prostatic hypertrophy, or inflammatory conditions of other adjacent genitourinary tissues, but not in apparently healthy men, men with non-prostatic carcinoma, apparently healthy women, or women with cancer. Reports have suggested that serum PSA is one of the most useful tumor markers in oncology. It may serves as an accurate marker for assessing response to treatment in patients with prostatic cancer. Therefore, measurement of serum PSA concentrations can be an important tool in monitoring patients with prostatic cancer and in determining the potential and actual effectiveness of surgery or other therapies.
Recent studies also indicate that PSA measurements can enhance early prostate cancer detection when combined with digital rectal examination (DRE).
PRINCIPLE OF THE TEST

The PSA ELISA test is based on the principle of a solid phase enzyme-linked immunosorbent assay. The assay system utilizes a rabbit anti-PSA antibody directed against intact PSA for solid phase immobilization (on the microtiter wells). A monoclonal anti-PSA antibody conjugated to horseradish peroxidase (HRPO) is in the 

antibody-enzyme conjugate solution. The test sample is allowed to react first with the immobilized rabbit antibody at room temperature for 60 minutes. The wells are washed to remove any unbound antigen. The monoclonal anti-PSA-HRPO conjugate is then reacted with the immobilized antigen for 60 minutes at room temperature resulting in the PSA molecules being sandwiched between the solid phase and enzyme-linked antibodies. The wells are washed with water to remove unbound-labeled antibodies. A solution of TMB Reagent is added and incubated at room temperature for 20 minutes, resulting in the development of a blue color. The color development is stopped with the addition of Stop Solution changing the color to yellow. The concentration of PSA is directly proportional to the color intensity of the test sample. Absorbance is measured spectrophotometrically at 450 nm. 

REAGENTS

Materials provided with the kits:

* Rabbit anti-PSA coated microtiter plate with 96 wells.
* Zero Buffer, 7 ml.
* Reference standard containing 0, 2, 4, 15, 60, and 120 ng/ml PSA, lyophilized. 1 set.
* Enzyme Conjugate Reagent, 12 ml.
* TMB Reagent (one step), 11 ml.
* Stop Solution (1N HCl), 11 ml.

Materials required but not provided:

* Precision pipettes: 0.02, 0.05, 0.10, 0.20, and 1.0 ml.
* Disposable pipette tips.
* Distilled water.
* Vortex mixer or equivalent.
* Absorbent paper or paper towel.
* Graph paper.
* A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-2 OD or greater at 450nm

SPECIMEN COLLECTION AND PREPARATION
Serum should be prepared from a whole blood specimen obtained by acceptable medical techniques. This kit is for use with serum samples without additives only.

STORAGE OF TEST KIT 
AND INSTRUMENTATION


Unopened test kits should be stored at 2-8*C upon receipt and the microtiter plate should be kept in a sealed bag with desiccants to minimize exposure to damp air. Opened test kits will remain stable until the expiration date shown, provided it is stored as described above. A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-2 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

REAGENT PREPARATION
1. All reagents should be brought to room temperature (18-25*C) before use. 
2. Reconstitute each lyophilized standard with 1.0 ml distilled water. Allow the reconstituted material to stand for at least 20 minutes and mix gently. Reconstituted standards should be stored sealed at 2-8*C.

ASSAY PROCEDURE
1. Secure the desired number of coated wells in the holder.
2. Dispense 50*l of standards, specimens, and controls into appropriate wells. 
3. Dispense 50*l of Zero Buffer into each well.
4. Thoroughly mix for 30 seconds. It is very important to have a complete mixing in this setup.
5. Incubate at room temperature (18-25*C) for 60 minutes. 
6. Remove the incubation mixture by emptying plate contents into a waste container.
7. Rinse and empty the microtiter wells 5 times with distilled or deionized water. (Please do not use tap water.)
8. Strike the wells sharply onto absorbent paper or paper towels to remove all residual water droplets.
9. Dispense 100*l of Enzyme Conjugate Reagent into each well. Gently mix for 5 seconds.
10. Incubate at room temperature for 60 minutes. 
11. Remove the incubation mixture by emptying plate contents into a waste container.
12. Rinse and empty the microtiter wells 5 times with distilled or deionized water. (Please do not use tap water.)
13. Strike the wells sharply onto absorbent paper to remove residual water droplets.
14. Dispense 100*l TMB Reagent into each well. Gently mix for 5 seconds.
15. Incubate at room temperature for 20 minutes.
16. Stop the reaction by adding 100*l of Stop Solution to each well.
17. Gently mix for 30 seconds. It is important to make sure that all the blue color changes to yellow color completely.
18. Using a microtiter plate reader, read the optical density at 450nm within 15 minutes.

CALCULATION OF RESULTS
1. Calculate the average absorbance values (A450) for each set of reference standards, control, and samples.
2. Construct a standard curve by plotting the mean absorbance obtained for each reference standard against its concentration in ng/ml on linear graph paper, with absorbance on the vertical (y) axis and concentration on the horizontal (x) axis.
3. Using the mean absorbance value for each sample, determine the corresponding concentration of PSA in ng/ml from the standard curve.


EXAMPLE OF STANDARD CURVE
Results of a typical standard run with optical density readings at 450nm shown in the Y axis against PSA concentrations shown in the X axis. This standard curve is for the purpose of illustration only, and should not be used to calculate unknowns. Each user should obtain his or her own data and standard curve. 

PSA (ng/ml)

Absorbance (450nm)

Corrected

Absorbance (450nm)

0

0.079

0.000

2

0.163

0.084

4

0.250

0.171

15

0.708

0.629

60

2.034

1.955

120

3.043

2.964

 




EXPECTED VALUES AND SENSITIVITY
Healthy males are expected to have PSA values below 4ng/ml The minimum detectable concentration of PSA in this assay is estimated to be 0.25ng/ml.

LIMITATIONS OF THE PROCEDURE
1. Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of the package insert instructions and with adherence to good laboratory practice.
2. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings. 
3. The results obtained from the use of this kit should be used only as an adjunct to other diagnostic procedures and information available to the physician.

REFERENCES
1 Hara, M. and Kimura, H. Two prostate-specific antigens, gamma-seminoprotein and beta-microseminoprotein. J. Lab. Clin. Med. 113:541-548;1989.
2 Yuan, J.J.; Coplen, D.E.; Petros, J.A.; Figenshau, R.S.; Ratliff, T.L.; Smith, D.S. and Catalona, W.J. Effects of rectal examination, prostatic massage, ultrasonography and needle biopsy on serum prostate specific antigen levels. J. Urol. 147:810-814; 1992.
3 Wang, M.C.; Papsidero, L.D.; Kuriyama, M.; Valenzuela, L.A.; Murphy, G.P. and Chu, T.M. Prostatic antigen: a new potential marker for prostatic cancer. Prostate 2:89-93; 1981.
4 Stowell, L.I.; Sharman, I.E. and Hamel, K. An Enzyme-Linked Immunosorbent Assay ( ELISA ) for Prostate-specific antigen. Forensic Science Intern. 50:125-138; 1991.
5 Frankel, A.E.; Rouse, R.V.;Wang, M.C.; Chu, T.M. and Herzenberg, L.A. Monoclonal antibodies to a human prostate antigen. Canc. Res. 42:3714; 1982.
6 Benson, M.C.; Whang, I.S.; Pantuck, A.; Ring, K.; Kaplan, S.A.; Olsson, C.A. and Cooner, W.H. Prostate specific antigen density: a means of distinguishing benign prostatic hypertrophy and prostate cancer. J. Urol. 147:815-816; 1992.
7 Gorman, C. The private pain of prostate cancer. Time 10(5):77- 80; 1992.
8 Walsh, P.C. Why make an early diagnosis of prostate cancer. J. Urol. 147:853-854; 1992.
9 Labrie, F.; Dupont, A.; Suburu, R.; Cusan, L.; Tremblay, M.; Gomez, J-L and Emond, J. Serum prostate specific antigen as pre-screening test for prostate cancer. J. Urol. 147:846-852; 1992.
10 McCarthy, R.C.; Jakubowski, H.V. and Markowitz, H. Human prostate acid phosphatase : purification, characterization, and optimization of conditions for radioimmunoassay. Clin. Chim. Acta. 132:287-293; 1983.
11 Heller, J.E. Prostatic acid phosphase: its current clinical status. J. Urol. 137:1091-1099; 1987.
12 Filella, X.; Molina, R.; Umbert, J.J.B.; Bedini, J.L. and Ballesta, A.M. Clinical usefulness of prostate-specific antigen. Tumor Biol. 11:289-294; 1990.
13 Shin, W.J.; Gross, K.; Mitchell, B.; Collins, J.; Wierzbinski, B.; Magoun, S. and Ryo, U.Y. Prostate adenocarcinoma using Gleason scores correlates with prostate-specific antigen and prostate acid phosphatase measurements. J. Nat. Med. Assoc. 84:1049-1050; 1992.
14 Wirth, M.P. and Frohmuller, H.G. Prostate-specific antigen and prostate acid phosphatase in the detection of early prostate cancer and in the prediction of regional lymph node metastases. Eur. Urol. 21:263-268; 1992.
15 Campbell, M.L. More cancer found with sensitive PSA assay. Urol. Times. 20:10; 1992.
16 Vessella, R.L.; Noteboom, J. and Lange, P.H. Evaluation of the Abbott IMx Automated immunoassay of Prostate-Specific Antigen. Clin. Chem. 38:2044-2054; 1992.
17 Brawer, M.K.; Chetner, M.P.; Beatie, J.; Buchner, D.M.; Vessella, R.L. and Lange, P. H. Screening for prostatic carcinoma with prostate specific antigen. J. Urol. 147:841-845; 1992.
18 Benson, M.C. Whang, I.S.; Olsson, C.A.; McMahon, D.J. and Cooner, W.H. The use of prostate specific antigen density to enhance the predictive value of intermediate levels of serum prostate specific antigen. J. Urol. 147:817-821; 1992.
19 Oesterling, J.E. and Hanno, P.M. PSA still finding niches in cancer diagnosis. Urol. Times 20:13-18; 1992.
20 Babaian, R.J.; Fritsche, H.A. and Evans, R.B. Prostate-specific antigen and the prostate gland volume: correlation and clinical application. J. Clin. Lab. Anal. 4:135-137; 1990.

Rapid PSA TEST 

FOR THE QUANTITATIVE ASSESSMENT OF HUMAN PSA IN
HUMAN SERUM

For in vitro Diagnostic Use

INTENDED USE
The RapidPSA test is an immunochromatography based one step in vitro test. It is designed for the rapid semi-quantitative determination of human prostate specific antigen (PSA) in serum specimens. 

SUMMARY AND EXPLANATION 
Prostate cancer is the one of the most common types of cancer found in man. The incidence of prostate cancer increases with age and accounts for a growing number of newly diagnosed patients. Prostate specific antigen (PSA) is produced primarily in the prostate gland and is secreted into the prostate ducts and at ejaculation serves to liquefy the seminal coagulum. Virtually all healthy males under 50 years of age have PSA concentration under 4.0 ng/ml. If PSA level is above 20 ng/ml, the patient most likely to have prostate cancer. Some studies indicated that elevated total PSA levels are found in serum from patients who have prostate cancer cells metastasized throughout their bodies. Other studies indicated that Free PSA, which can not foam a complex with serine protease tends to be more abundant in patients with benign prostatic hyperplasia. RapidPSA test use antibodies which can equally recognize both free PSA and PSA-ACT complex. 

RapidPSA test is a sandwich immunoassay. When serum sample is added to sample pad, it moves through the conjugate pad and mobilizes gold anti-PSA conjugate that is coated on the conjugate pad. The mixture moves along the membrane by capillary action and reacts with anti-PSA antibody that is coated on the test region. If PSA is present, the result is the formation of a colored band in the test region. The color intensity is dependent on the concentration of PSA in the sample. On the other hand, a light color band will always appear at the control zone. This control band serves as a reference of 4.0 ng/ml of PSA.

MATERIAL PROVIDED
1. RapidPSA Test device


MATERIALS REQUIRED BUT NOT SUPPLIED
1. Serum collection containers.
2. Timer or clock

STORAGE 
Store the test device at 2 to 30oC. Do Not Freeze.

PRECAUTIONS
1. For in vitro diagnostic use only.
2. Do not use product beyond the expiration date.
3. Handle all specimens as potentially infectious.

SPECIMEN COLLECTION AND PREPARATION
1. The serum specimen should be collected under standard laboratory conditions
2. Patient samples performed best when tested immediately after collection. If the sample cannot be tested within 24 hours, freeze until the test can be performed. Allow sample to reach room temperature before proceeding.
3. Sodium azide can be added as a preservative up to 0.1% without effecting the test results. 


QUALITY CONTROL
1. The control band is an internal reagent and procedural control. It will appear if the test has been performed correctly and the reagents are reactive.

PROCEDURE
For Test Strip:
1. Remove the test device from the foil pouch.
2. Immerse the strip into the sample (sample volume should be no less than 150 ml) with the arrow pointing toward the sample. The sample level should not be higher than the arrow pointed line.
3. You may leave the strip in the sample or take the strip out after a minimum of 30 seconds in the sample and lay the strip on a flat non-absorptive surface.
4. Read the result at 5 minutes after adding the sample.

For Test Card:
1. Bring all materials and specimens to room temperature.
2. Remove the test card from the sealed foil pouch.
3. Place the transfer pipette in the specimen and depress the bulb to withdraw a sample.
4. Hold the pipette in a vertical position over the sample well of the test card and deliver 2-3 drops(100-150 ml) of sample into the sample well.
5. Read the result at 5 minutes.

Note: Results after 5 minutes may change and cause false interpretation.

INTERPRETATION OF RESULTS
Positive:
If the color of test band is equal or stronger than that of control band, it indicates the PSA level is equal or higher than the cut-off, 4.0 ng/ml.

Negative:
If only a colored control band appears or the color intensity of the test band is less than athat of control band, it indicates the PSA level is less than cut-off, 4.0 ng/ml.

Invalid result:
The test result is invalid if a colored band does not form in the control region. The sample must be re-tested, using a new test device.

LIMITATIONS OF THE PROCEDURE
1. The test is for in vitro diagnostic use only.
2. The test is limited to the semi-quantitative detection of PSA levels in serum specimen. 
3. Although the test is very accurate in detecting elevated PSA, a low incidence of false positive results can occur.
4. As with all diagnostic tests, a definitive clinical diagnosis should not be based on the result of a single test, but should only be made by the physician after all clinical and laboratory findings have been evaluated.


PERFORMANCE CHARACTERISTICS
Sensitivity:
RapidPSA test can detect PSA in serum with concentration of 4.0 ng/ml or greater. 

Interference testing:
The following substances were added to PSA negative and 4.0 ng/ml PSA spiked serum samples. No interference was found with any of the substances at the following concentrations:

Acetaminophen 20 mg/dl
Bilirubin 10 mg/dl
Triglycerides 250 mg/dl
PAP 1000 ng/ml
HCG 100 IU/ml
Prolactin 1000 ng/ml
Transferrin 5.0 mg/ml




REFERENCES

1. Cancer Facts and Figures-1994. American Cancer Society, Inc. (1994)
2. Lundwall, A. and Lilja, H., FEBS, Lett.,, Vol. 214, 317-222 (1987)
3. Glenski, W. J., et al., Prostate, Vol. 21, 99-110 (1992)
4. Oesterling, J.E., et al., J. Urol., Vol. 139, 766-772 (1988)
5. Barry R., et al., Clin. Chem. Vol. 42, 536-544 (1996)


 

 

  
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