DRG International EIA-4937 A ELISA Chromogranin Instruction Manual

DRG International EIA-4937 A ELISA Chromogranin

Instructions for Use

Chromogranin A ELISA

DRG Instruments GmbH, Germany Frauenbergstraße 18, 35039 Marburg
Phone: +49 (0)6421-1700 0, Fax: +49 (0)6421-1700 50 Website: www.drg- diagnostics.de
E-mail: drg@drg-diagnostics.de

Please use only the valid version of the Instructions for Use provided with the kit.

INTRODUCTION

Intended use and principle of the test
Enzyme immunoassay for the quantitative determination of Chromogranin A in serum.
The determination of Chromogranin A helps in the detection of neuroendocrine tumors and is used to assess the course of the cancer treatment.
The quantitative determination of Chromogranin A (CgA) follows the basic principles of the enzyme immunoassay.
First, the Chromogranin A in the samples, controls and standards binds to CgA- specific antibodies fixed to a 96 wells microtiter plate. After incubation and following washing steps, a sandwich is formed by adding CgA antibodies conjugated to horseradish peroxidase. After incubation the wells are washed thoroughly, and the complex bound to the solid phase is detected by using TMB as a substrate resulting in a colour reaction. The reaction is monitored at a wavelength of 450 nm.
By means of a standard curve the CgA concentrations in the samples are determined.
Manual processing of the ELISA is recommended. The use of automatic laboratory equipment is the responsibility of the user.
This in-vitro diagnostic is for professional use only.

Clinical application
Chromogranin A (CgA) is an acid glycoprotein with 439 amino acids that is present in the secretory dense core granules of most neuroendocrine cells [1]. The chromogranin family consists of at least three different water-soluble acidic glycoproteins (CgA, CgB, and secretogranin II, sometimes called Chromogranin C) [1].
Upon stimulation, CgA and other peptide hormones and neuropeptides are released. CgA is also secreted from neuroendocrine-derived tumors [1].
Neuroendocrine tumors (NETs), which originate from neuroendocrine cells, are found widely distributed throughout the body [2]. The most common sites of NET are the lung, stomach, appendix, cecum, duodenum, pancreas, jejunum/ileum, colon and rectum [3]. NET arising from the gastrointestinal tract are collectively known as gastroenteropancreatic neuroendocrine tumors (GEP- NET) and account for approximately 2/3 of incident NET [3]. The annual incidence of NET is estimated as 2 – 5 cases per 100,000 population [2].
CgA is widely expressed throughout the neuroendocrine system and serves as a general biomarker for a wide variety of neuroendocrine tumors [3]. The determination of Chromogranin A helps in the detection of neuroendocrine tumors and is used to assess the course of cancer treatment [3-6].
Therapeutic consequences should never be based on laboratory results alone, even if these results are assessed in accordance with the quality criteria of the method. Any laboratory result is only a part of the total clinical picture of the patient.
Only in cases where the laboratory results are in an acceptable agreement with the overall clinical picture of the patient, it can be used for therapeutic consequences.

PROCEDURAL CAUTIONS, GUIDELINES, WARNINGS AND LIMITATIONS

Procedural cautions, guidelines and warnings

1. This kit is intended for professional use only. Users should have a thorough understanding of this protocol for the successful use of this kit. Only the test instruction provided with the kit is valid and must be used to run the assay. Reliable performance will only be attained by strict and careful adherence to the instructions provided.
2. This assay was validated for a certain type of sample as indicated in Intended Use (please refer to Chapter 1). Any off-label use of this kit is in the responsibility of the user and the manufacturer cannot be held liable.
3. The principles of Good Laboratory Practice (GLP) must be followed.
4. In order to reduce exposure to potentially harmful substances, wear lab coats, disposable protective gloves and protective glasses where necessary.
5. If serious incidents should occur in connection with this product, they should be reported to the manufacturer and the competent national authorities.
6. All kit reagents and specimens should be brought to room temperature and mixed gently but thoroughly before use. For dilution or reconstitution purposes, use deionized, distilled, or ultra-pure water. Avoid repeated freezing and thawing of reagents and specimens.
7. The microplate contains snap-off strips. Unused wells must be stored at 2 °C – 8 C in the sealed foil pouch with desiccant and used in the frame provided. Microtiter strips which are removed from the frame for usage should be marked accordingly to avoid any mix-up.
8. Duplicate determination of sample is highly recommended.
9. Once the test has been started, all steps should be completed without interruption. Make sure that the required reagents, materials, and devices are prepared for use at the appropriate time.
10. Incubation times do influence the results. All wells should be handled in the same order and time intervals.
11. To avoid cross-contamination of reagents, use new disposable pipette tips for dispensing each reagent, sample, standard and control.
12. A standard curve must be established for each run.
13. The controls should be included in each run and fall within established confidence limits. The confidence limits are listed in the QC-Report provided with the kit.
14. Do not mix kit components with different lot numbers within a test and do not use reagents beyond expiry date as shown on the kit labels.
15. Avoid contact with Stop Solution containing 0.25 M H2SO4. It may cause skin irritation and burns. In case of contact with eyes or skin, rinse off immediately with water.
16. TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with an abundant volume of water and skin with soap and abundant water. Rinse contaminated items before reuse.
17. For information about hazardous substances included in the kit please refer to Safety Data Sheet (SDS). The Safety Data Sheet for this product is made available directly on the website of the manufacturer or upon request.
18. Kit reagents must be regarded as hazardous waste and disposed of according to national regulations.
19. The expected reference values reported in this test instruction are only indicative. It is recommended that each laboratory establishes its own reference intervals.
20. In case of any severe damage to the test kit or components, the manufacturer has to be informed in writing, at the latest, one week after receiving the kit. Severely damaged single components must not be used for a test run. They must be stored properly until the manufacturer decides what to do with them. If it is decided that they are no longer suitable for measurements, they must be disposed of in accordance with national regulations.
21. The results obtained with this test kit should not be taken as the sole reason for any therapeutic consequence but must be correlated to other diagnostic tests and clinical observations.
22. Reagents of this kit which contain human serum or plasma have been tested and confirmed negative for HIV I/II, HBsAg and HCV by approved procedures. All reagents however, should be treated as potential biohazards in use and for disposal.

Limitations
Any inappropriate handling of samples or modification of this test might influence the results.
In about 10 % of the samples used in the method comparison, a discrepancy was detected between the Kryptor CgA II and the ELISA measurements. These were exclusively samples whose CgA concentrations were in the range of 350 to 900 µg/L.
The sequence of the specific antibodies used was checked for possible cross- reactions. Even if no significant cross-reactivities could be detected, it cannot be excluded that in rare individual cases and depending on medication or disease status, influences on the values may occur.
If the Chromogranin A determination is used as part of a patient’s follow-up, we therefore recommend the following procedure:

• A patient’s sample should always be examined using the same method during the course of his treatment.
• In case of abnormalities during the follow-up, it should be investigated whether changes in medication or lifestyle have taken place.

If you have any further questions, please contact the manufacturer.

Interfering substances
Serum samples containing precipitates or fibrin strands might cause inaccurate results.
Biotin (up to 1200 ng/mL), hemolytic samples (up to 1 mg/mL hemoglobin), icteric samples (up to 50 mg/dL bilirubin) and lipemic samples (up to 1700 mg/dL triglycerides) have no influence on the assay results.
When in doubt, it is recommended that hemolytic, icteric, and lipemic samples not be used in the assay.

Drug interferences
Medications like proton pump inhibitors, selective serotonin reuptake inhibitors, histamine type-2 receptor antagonists and somatostatin analogues can influence CgA level in serum.

High-Dose-Hook effect
No hook effect was observed in this test.

STORAGE AND STABILITY

Store kit and reagents at 2 °C – 8 °C until expiration date.
Do not use kit and components beyond the expiry date indicated on the kit labels.
Once opened the reagents are stable for 2 months when stored at 2 °C – 8 °C.
Once the resealable pouch of the ELISA plate has been opened, care should be taken to close it tightly again including the desiccant.

MATERIALS

Content of the kit

Calibration and Controls

Standards and Controls – ready to use

| Component| Colour/Cap| Concentration [µg/L] CgA| Volume/Vial
| STANDARD A| white| 0| 1 mL
| STANDARD B| yellow| 30| 1 mL
| STANDARD C| orange| 110| 1 mL
| STANDARD D| blue| 450| 1 mL
| STANDARD E| grey| 900| 1 mL
| CONTROL 1| green| Refer to QC-Report for expected value and acceptable range.| 1 mL
| CONTROL 2| red| 1 mL
Content:| Assay Buffer with a defined quantity of human Chromogranin A and stabilizing protein
Description:| Chromogranin A is derived from human, the stabilizing protein is from bovine origin

Additional materials required but not provided in the kit

• Water (deionized, distilled, or ultra-pure)
• Absorbent material (paper towel)

Additional equipment required but not provided in the kit

• Calibrated precision pipettes to dispense volumes between 20 – 400 µL
• Microtiter plate washing device (manual, semi-automated or automated)
• ELISA reader capable of reading absorbance at 450 nm and if possible 620 – 650 nm
• Microtiter plate shaker (shaking amplitude 3 mm; approx. 600 rpm)
• Vortex mixer

SAMPLE COLLECTION, HANDLING AND STORAGE

Serum
Collect blood by venipuncture, allow to clot, and separate serum by centrifugation according to manufacturer’s instructions. Do not centrifuge before complete clotting has occurred. Patients receiving anticoagulant therapy may require increased clotting time.
When in doubt, it is recommended that hemolytic, icteric, and lipemic samples not be used in the assay (see 2.2.1).
Storage: Up to 2 days at 2 °C – 8 °C; storage for a longer period (up to 6 months) at -20 °C.
Repeated freezing and thawing should be avoided.

TEST PROCEDURE

Allow all reagents and samples to reach room temperature and mix thoroughly by gentle inversion before use. Number the microwell plates (Microtiter Strips which are removed from the frame for usage should be marked accordingly to avoid any mix-up). Duplicate determinations are recommended.
The binding of the antisera and of the enzyme conjugate and the activity of the enzyme are temperature dependent. The higher the temperature, the higher the absorption values will be. Varying incubation times will have similar influences on the absorbance. The optimal temperature during the enzyme immunoassay is between 20 °C – 25 °C.
The use of a microtiter plate shaker with the following specifications is mandatory: shaking amplitude 3 mm;
approx. 600 rpm. Shaking with differing settings might influence the results.

Preparation of reagents and further notes

Wash Buffer
Dilute the 20 mL Wash Buffer Concentrate WASH-CONC 50X with water to a final volume of 1000 mL.
Storage: 2 months at 2 °C – 8 °C

Chromogranin A Microtiter Strips
In rare cases residues of the blocking and stabilizing reagent can be seen in the wells as small, white dots or lines. These residues do not influence the quality of the product.

Preparation of samples – Dilution

1. Prior to use, the serum samples have to be diluted 1+20 with ASSAY-BUFF,  e.g. 20 µL of serum sample + 400 µL of ASSAY-BUFF.
   Serum samples which have been found off-curve should also be diluted accordingly with ASSAY-BUFF  and reassayed.

Chromogranin A ELISA

1. Pipette 50 µL of the standards, controls and diluted samples into the appropriate wells of the Chromogranin A Microtiter Strips and incubate 1 h at RT (20 °C – 25 °C) on a shaker (approx. 600 rpm).
2. Discard or aspirate the content of the wells. Wash the plate 4 times by adding 300 µL of Wash Buffer, discarding the content and blotting dry each time by tapping the inverted plate on absorbent material.
3. Pipette 50 µL of the CONJUGATE  into all wells and incubate 1 h at RT (20 °C – 25 °C) on a shaker (approx. 600  rpm).
4. Discard or aspirate the content of the wells. Wash the plate 4 times by adding 300 µL of Wash Buffer, discarding the content and blotting dry each time by tapping the inverted plate on absorbent material.
5. Pipette 100 µL of the SUBSTRATE  into all wells.
6. Incubate for 25 ± 5 min at RT (20 °C – 25 °C) on a shaker (approx. 600 rpm). Avoid exposure to direct sunlight!
7. Add 100 µL of the STOP-SOLN  to all wells and shake the microtiter plate shortly.
8. Read the absorbance of the solution in the wells within 10 minutes, using a microtiter plate reader set to 450 nm (if available a reference wavelength between 620 nm and 650 nm is recommended).

CALCULATION OF RESULTS

Measuring range

| Chromogranin A in serum
---|---
2.3 – 900 µg/L

The standard curve, which can be used to determine the concentration of the unknown samples, is obtained by plotting the absorbance readings (calculate the mean absorbance) of the standards (linear, y-axis) against the corresponding standard concentrations (logarithmic, x-axis) using a concentration of 0.001 µg/L for Standard A (this alignment is mandatory because of the logarithmic presentation of the data).
Use a non-linear regression for curve fitting (e.g. 4-parameter, marquardt).
The concentrations of the samples and controls can be read directly from the standard curve.
Samples found with concentrations higher than the highest standard (Standard E) should be diluted accordingly with ASSAY-BUFF and must be re-assayed.

Expected reference values
It is strongly recommended that each laboratory should determine its own reference values.
The expected reference values indicated below are based on method comparison studies to B.R.A.H.M.S Kryptor CgA II. The expected reference value was determined in a study by van Treijen, M.J.C., et al [7].

| Chromogranin A in serum
---|---
Reference value (ULN)| <100 µg/L
Typical pathological range| Up to 143 500 µg/L

Typical standard curve

Example: Do not use for calculation!

CONTROL SAMPLES
It is recommended to use control samples according to national regulations. Use controls at both normal and pathological levels. Commercially obtained control samples should be treated like unknown samples. Control samples should fall within established confidence limits. The confidence limits of the kit controls are indicated on the QC-Report.

ASSAY CHARACTERISTICS

Performance data

Analytical Sensitivity|
---|---
Limit of Blank (LOB)| 0.9 µg/L
Limit of Detection (LOD)| 1.4 µg/L
Limit of Quantification (LOQ)| 2.3 µg/L
Precision

Intra-Assay| Inter-Assay
n = 12| n = 10
Sample| Mean ± SD [µg/L]| CV [%]| Sample| Mean ± SD [µg/L]| CV [%]
1| 43.6 ± 1.2| 2.8| 1| 73.0 ± 3.8| 5.2
2| 73.5 ± 3.0| 4.2| 2| 102 ± 3.5| 3.5
3| 103 ± 3.4| 3.3| 3| 161 ± 5.7| 3.6
4| 161 ± 10.1| 6.3| 4| 300 ± 16.0| 5.3
5| 283 ± 14.6| 5.1|
6| 502 ± 15.9| 3.2
Lot-to-Lot

| Sample| mean ± SD [µg/L]| CV [%]
Chromogranin A in serum (n = 3)| 1| 46.3 ± 2.3| 5.1
2| 111 ± 7.2| 6.5
3| 479 ± 61.8| 12.9
Recovery

| Range [µg/L]| Mean [%]| Range [%]
Chromogranin A| 43.6 – 502| 102| 100 – 104
Linearity

| Serial Dilution up to| Mean [%]| Range [%]
Chromogranin A| 1:64| 92| 91 – 96
Method Comparison: B.R.A.H.M.S Kryptor CgA II| CgA ELISA = 1.05 x (Kryptor CgA II) – 15; R2 = 0.97; n = 57
---|---
Diagnostic Performance GEP-NET [7]

Diagnostic Specificity [%]| Diagnostic Sensitivity [%]| Positive Predictive Value (PPV) [%]| Negative Predictive Value (NPV) [%]
83| 56| 87| 49
Positive Likelihood Ratio (LR+)| Negative Likelihood Ratio (LR-)
3.3| 0.53

Metrological Traceability
The values assigned to the standards and controls of the Chromogranin A ELISA are traceable to the reference method B.R.A.H.M.S Kryptor CgA II.

7.5

• This defines an interval about the measured result that will include the true value with a probability of 95%

10 REFERENCES / LITERATURE /

1. O’Toole, D., et al., ENETS Consensus Guidelines for the Standards of Care in Neuroendocrine Tumors: biochemical
   markers. Neuroendocrinology, 2009. 90(2): p. 194-202.

2. Verbeek, W.H., C.M. Korse, and M.E. Tesselaar, GEP-NETs UPDATE: Secreting gastro-enteropancreatic neuroendocrine tumors and biomarkers. Eur J Endocrinol, 2016. 174(1): p. R1–7.

3. Singh, S. and C. Law, Chromogranin A: a sensitive biomarker for the detection and post-treatment monitoring of gastroenteropancreatic neuroendocrine tumors. Expert Rev Gastroenterol Hepatol, 2012. 6(3): p. 313–34.

4. Louthan, O., Chromogranin a in physiology and oncology. Folia Biol (Praha), 2011. 57(5): p. 173–81.

5. Yang, X., et al., Diagnostic value of circulating chromogranin a for neuroendocrine tumors: a systematic review and meta-analysis. PLoS One, 2015. 10(4): p. e0124884.

6. Corti, A., F. Marcucci, and T. Bachetti, Circulating chromogranin A and its fragments as diagnostic and prognostic disease markers. Pflugers Arch, 2018. 470(1): p. 199–210.

7. van Treijen, M.J.C., et al., Blood Transcript Profiling for the Detection of Neuroendocrine Tumors: Results of a Large Independent Validation Study. Front Endocrinol (Lausanne), 2018. 9: p. 740.

For updated literature or any other information please contact your local supplier.
Once EUDAMED is fully in place, the Summary of Safety and Performance can be found here:
https://ec.europa.eu/tools/eudamed .

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