DRG Instruments EIA-4482 96 5-Hiaa Urine Elisa Instruction Manual

DRG Instruments EIA-4482 96 5-Hiaa Urine Elisa

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

Introduced modifications

The following changes have been made in comparison to the previous version:
Detailed editorial revision.
The changes described below were made in both IFUs available in English as well as in Deutsch.
1.1 Intended use and principle of the test| The intended use and principle of the test contain few changes in the wording.
1.2  Clinical application| Contains the change of format for the reference citation.
2| Procedural cautions, guidelines, warnings, and limitations| Point number 16 & 21 contain the addition of the sentence.
3| Storage and stability| Addition of the sentences.
4| Materials| In the materials section, some changes in the Symbols and Hazardous ingredients were added. The Methylation Reagent bottle cap has been replaced by a black color instead of white. Regarding the Hazardous and precautionary statements, some were amended, added, or removed.
4.3 Additional materials required but not provided in the kit| Some materials were removed from the materials required list.
5| Sample collection, handling, and storage| The addition of Handling in the heading.
6| Test procedure| Addition and deletion of sentences and words
6.3  Methylation| The change of symbols.
7| Calculation of results| The addition and removal of sentences and words and change of brackets in the formula for calculating.
7.2  Typical standard curve| The change of the citation format and word.
9| Assay characteristics| The assay characteristics were amended, removed, or added.
“Distributed by” on the front page was replaced by a symbol. “SYMBOLS USED” at the end of the file updated.

INTRODUCTION

Intended use and principle of the test

• Enzyme immunoassay for the quantitative determination of 5-Hydroxyindolacetic acid (5-HIAA) in urine.
• The determination of 5-HIAA helps in the diagnosis of carcinoids.
• The quantitative determination of 5-HIAA follows the basic principles of a competitive enzyme immunoassay.
• First, 5-HIAA is chemically derivatized by a methylation step. The subsequent competitive ELISA uses the microtiter plate format. The antigen is bound to the solid phase of the microtiter plate. The methylated analyte in the standards, controls, and samples compete with the solid phase bound analyte for a fixed number of antibody binding sites. After the system is in equilibrium, free antigen and free antigen-antibody complexes are removed by washing. The antibody bound to the solid phase is detected by an anti-rabbit IgG – peroxidase conjugate using TMB as a substrate resulting in a color reaction. The reaction is monitored at a wavelength of 450 nm.
• Quantification of unknown samples is achieved by comparing their absorbance with a reference curve prepared with known standard concentrations.
• Manual processing of the ELISA is recommended. The use of automatic laboratory equipment is the responsibility of the user.
• This in-vitro diagnostic device is for professional use only.

Clinical application

• 5-Hydroxyindolacetic acid (5-HIAA) is a metabolite of the serotonin pathway [1, 2]. Serotonin and its major urinary metabolite 5-HIAA, is produced in excess by most enterochromaffin cells from carcinoid tumors, especially those associated with carcinoid syndrome. Several studies and publications show that analysis of urinary 5-HIAA is important in the diagnosis of carcinoid patients [1 – 8].
• Therapeutic consequences should never be based on laboratory results alone, even if these results are assessed under 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 instructions 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. 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 that 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 the 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 your eyes with an abundant volume of water and skin with soap and abundant water. Rinse contaminated items before use.
17. For information about hazardous substances included in the kit please refer to the Safety Data Sheet (SDS). The Safety Data Sheet for this product is available 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 under 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.

Limitations
Any inappropriate handling of samples or modification of this test might influence the results.

Interfering substances and proper handling of specimens
Please note the sample collection! It cannot be excluded that high acid concentrations lead to incorrect results.

Drug and food interferences

Foods generally rich in serotonin such as bananas, pineapple, plums, kiwi fruit, tomatoes, avocados, various nuts, and chocolate should be avoided a few days before sample collection. Drugs/substances such as imipramine, isoniazid, isocarboxazid, methyldopa, levodopa, MAO-inhibitors, general OTC medication, alcohol, paracetamol, diazepam, oxprenolol, atenolol, phenothiazines, indomethacin, naproxen, reserpine, glyceryl-guaiacolate influence urinary 5-HIAA levels and should be discontinued a few days before.

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 the 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. Make sure that the Methylation Reagent is recapped immediately after pipetting.

MATERIALS

Contents of the kit

| REAC- PLATE| Reaction Plate – ready to use
---|---|---
Content:| 1 x 96 well plate, empty, in a resealable pouch
|
| FOILS| Adhesive Foil – ready to use
Content:| Adhesive foils in a resealable pouch
Number:| 1 x 4 foils|
| |
| WASH-CONC 50x| Wash Buffer Concentrate – concentrated 50x
Content:| Buffer with a non-ionic detergent and physiological pH
Volume:| 1 x 20 mL/vial, purple cap|
| |
| CONJUGATE| Enzyme Conjugate – ready to use
Content:| Goat anti-rabbit immunoglobulins conjugated with peroxidase
Volume:| 1 x 12 mL/vial, red cap|
Description:| Species is goat|
| |
| DILUENT| Diluent – ready to use
Content:| Acidic buffer with non-mercury preservative
Volume:| 1 x 22 mL/vial, white cap|
| |
| SUBSTRATE| Substrate – ready to use
Content:| Chromogenic substrate containing 3,3’,5,5’-tetramethylbenzidine, substrate buffer and hydrogen peroxide
Volume:| 1 x 12 mL/vial, black cap|
| |
| STOP- SOLN| Stop Solution – ready to use
Content:| 0.25 M sulfuric acid|
Volume:| 1 x 12 mL/vial, grey cap|
| Ш SER 5-HIAA| Serotonin 5-HIAA Microtiter Strips – ready to use
---|---|---
Content:| 1 x 96 well (12×8) antigen pre-coated microwell plate in a resealable pouch with desiccant
|
| 5-HIAA-AS| 5-HIAA Antiserum – ready to use
Content:| Rabbit anti-5-HIAA antibody, blue colored
Volume:| 1 x 6 mL/vial, blue cap|
Description:| Species is rabbit|
| |
| ASSAY- BUFF| Assay Buffer – ready to use
Content:| TRIS containing buffer with non-mercury preservative
Volume:| 2 x 55 mL/vial, green cap|
| |
| METHYL- BUFF| Methylation Buffer – ready to use
Content:| Methanol and dimethyl sulfoxide
Volume:| 1 x 11 mL/vial, brown cap|
Hazards pictograms:| **
Signal word:| GHS02 GHS06 GHS08
Hazardous| Danger
Ingredients:| Methanol
Hazard|
Statements:| H301+H311+H331 Toxic if swallowed, in contact with skin or if inhaled.
| H370 Causes damage to organs (eye, central nervous system).
Precautionary|
Statements:| P260 Do not breathe fume/gas/mist/vapors/spray.
| P280 Wear protective gloves/protective clothing/eye protection.
| P308+P311 IF exposed or concerned: Call a POISON CENTER or doctor.
| P403+P233 Store in a well-ventilated place. Keep the container tightly closed.
| P501 Dispose of contents/container to an authorized waste collection point.
|
| METHYL- READ| Methylation Reagent – ready to use
Content:| Methylation reagent in hexane
Volume:| 1 x 2.25 mL/vial, black cap|
Hazards pictograms:| **
| GHS02 GHS06 GHS08 GHS09
Signal word:| Danger
Hazardous ingredients:| Hexane, branched and linear, (Trimethylsilyl)diazomethane.
Hazard| H304 May be fatal if swallowed and enters the airways.
Statements:| H330 Fatal if inhaled
| H350 May cause cancer.
| H361f Suspected of damaging fertility.
Precautionary| H370 Causes damage to organs (lungs, inhalation).
Statements:| ****

P201 Obtain special instructions before use.

| P260 Do not breathe mist/vapors/spray.
| P280 Wear protective gloves/protective clothing/eye protection/face protection.
| P304+P340 IF INHALED: Remove person to fresh air and keep comfortable for breathing.
| P310 Immediately call a POISON CENTER or doctor.
| P331 Do NOT induce vomiting.

Calibration and Controls

Additional materials are required but not provided in the kit

• Reaction tubes, at least 3 ml, Polypropylene/Polystyrol
• Absorbent material (paper towel)
• Water (deionized, distilled, or ultra-pure)

Additional equipment is required but not provided in the kit

• Calibrated precision pipettes to dispense volumes between 20 – 300 µL; 1 mL
• 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
• Ventilated hood

SAMPLE COLLECTION, HANDLING AND STORAGE

24-hour urine

• A 24-hour urine sample is used for analysis.
• Over a defined period of 24 hours, all urine is collected in a bottle with acid (10 mL – 15 mL 6 M hydrochloric acid) provided for stabilization and the total volume is noted for evaluation of the results. During the collection period, the collected sample must always be stored in a cool place protected from light (2 °C – 8 °C).

Storage

• for a short period up to 7 days is at 2 °C – 8 °C.
• for a longer period up to 6 months is at -20 °C.

Repeated freezing and thawing should be avoided. Avoid direct sunlight!

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. If the product is prepared in parts, unused wells in Reaction Plates should be covered to avoid contamination. After preparation, the used wells must be labeled to prevent double use.

• 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.

• The Methylation Reagent is volatile. If possible, please pipette the Methylation Reagent with a repetitive pipette and make sure that the vial is recapped immediately after pipetting.

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

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

Predilution of standards, controls, and samples

1. Pipette 50 μL of standards, controls, and urine samples into the respective wells of the REAC-PLATE.
2. Pipette 200 μL of the DILUENT into all wells.
3. Shake for 1 min at RT (20 °C – 25 °C) on a shaker (approx. 600 rpm).
   20 μL are needed for the methylation.

Methylation

| Pipette 20 µL of the prediluted standards , controls, and urine samples into the respective reaction tubes.

The following steps 2 – 5 must be performed in a ventilated hood!

---|---
2| Pipette 100 µL of METHYL-BUFF into all reaction tubes.
|
3.| Add 20 µL of METHYL-REAG to each reaction tube and mix each reaction tube immediately after the  addition of the Methylation Reagent.
4.| Cover all reaction tubes and methylate for 20 min at RT (20 °C – 25 °C).
5.| Pipette 1000 µL of ASSAY-BUFF into all reaction tubes.

After this step, the use of a ventilated hood is not necessary anymore!

**| Proceed with the ELISA (Chapter 6.4) immediately** as the methylated standards, controls, and samples are only stable for 1 hour!

5-HIAA ELISA

1.| Pipette 25 µL of the methylated standards, controls, and samples into the appropriate wells of the Ш SER 5-HIAA

microtiter strips.

---|---
2.| Pipette 50 µL of the 5-HIAA-AS into all wells.
|
3.| Cover the plate with FOIL and incubate for 1 h at RT (20 °C – 25 °C) on a shaker (approx. 600 rpm).
4.| Remove the foil. 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 CONJUGATE into all wells.
|
6.| Cover the plate with FOIL and incubate for 1 h at RT (20 °C – 25 °C) on a shaker (approx. 600 rpm).
7.| Remove the foil. 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.
8.

| Pipette 100 µL of the SUBSTRATE into all wells and incubate for 20 – 30 min at RT (20 °C – 25 °C) on a shaker

(approx. 600 rpm).

Avoid exposure to direct sunlight!

9.| Add 100 µL of the STOP-SOLN to all wells and shake the microtiter plate shortly.
10.| Read the absorbance of the solution in the wells within 10 minutes, using a microplate reader set to 450 nm (if available a reference wavelength between 620 nm and 650 nm is recommended).

CALCULATION OF RESULTS

Measuring range

| 5-HIAA
---|---
0.4 – 50 mg/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 mg/L for Standard A (this alignment is mandatory because of the logarithmic presentation of the data). Use non-linear regression for curve fitting (e. g. 4-parameter, marquardt).This assay is a competitive assay. This means: the OD-values are decreasing with increasing concentrations of the analyte. OD-values found below the standard curve correspond to high concentrations of the analyte in the sample and have to be reported as being positive.

Urine samples and controls
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 F) should be diluted accordingly with water (deionized, distilled, or ultra-pure) and must be re-assayed. The total amount of 5-HIAA excreted in urine during 24 h is calculated as follows: mg ⁄24 h = mg/L x L ⁄24 h.
Conversion : 5-HIAA [mg/L] x 5.25 = 5-HIAA [µmol/L]

Expected reference value

• It is strongly recommended that each laboratory should determine its own reference values.
• The expected reference value was determined in a study by Meijer et al 2000 [2]. Normalization is performed on creatinine, which is an indication of urinary dilution.

| 5-HIAA in urine
---|---
Reference value (ULN)| 2.8 mmol/mol creatinine
Typical pathological range| up to 380 mg ∕24 h

Typical standard curve
Example: Do not use for calculation

QUALITY CONTROL

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 Specificity (Cross Reactivity)

Substance

| Cross Reactivity [%]
5-HIAA
Serotonine| 7.6
5-Hydroxy-DL-Tryptophane| 2.3
Tryptamine| < 0.1
Melatonine| < 0.1
5-Methoxytryptamine| < 0.1
DL-Vanillic mandelic acid| < 0.1
Homovanillic acid| < 0.1
Precision

Intra- Assay| Inter- Assay
n = 24| n = 9
Sample| Mean ± SD [mg/l]| CV [%]| Sample| Mean ± SD [mg/l]| CV [%]
1| 1.1 ± 0.15| 13.3| 1| 11.3 ± 1.3| 11.9
2| 1.9 ± 0.18| 9.3| 2| 4.8 ± 0.6| 12.8
3| 5.3 ± 0.48| 9.0| 3| 3.1 ± 0.3| 8.6
4| 14.3 ± 1.2| 8.7| 4| 7.3 ± 0.8| 10.8
| | | 5| 19.0 ± 2.2| 11.4
Lot-to-Lot

| Sample| Reference Range [mg/L]| Mean ± SD [mg/L]| Mean ± SD Recovery [%]| CV [%]
5-HIAA in artificial matrix (n = 3)| 1| 3.0 – 7.0| 4.9 ± 0.36| 98.0 ± 7.2| 7.4
2| 9.0 – 21.0| 14.6 ± 1.3| 97.3 ± 9.0| 9.2
Recovery

| Range [mg/l]| Range [%]| Mean [%]
Urine| 0.8 – 40.5| 86 – 93| 90
Linearity

| Serial dilution up to| Range [%]| Mean [%]
Urine| 1:10| 98 – 112| 105
Method Comparison: ELISA vs. XLC-MS/MS [1]

Urine| ELISA = 0.9749 * (XLC-MS/MS) – 0,0868; r2 = 0,98; n = 95
Diagnostic Performance [ 2 ]

Diagnostic Specificity [%]| Diagnostic Sensitivity [%]| Positive Predictive Value (PPV) [%]| Negative Predictive Value (NPV) [%]
89| 68| 58| 93
Positive Likelihood Ratio (LR+)| Negative Likelihood Ratio (LR-)
6.2| 0.36

Metrological Traceability
The values assigned to the standards and controls of the 5-HIAA ELISA are traceable to SI Units by weighing with the quality-controlled analyte.

Standards and Controls

| Uncertainty [%]
---|---
1.2
5-HIAA ELISA

Sample| Expanded Uncertainty [%] k = 2*
1| 23.9
2| 25.7
3| 17.4
4| 21.7
5| 22.9

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

REFERENCES/LITERATURE

1. de Jong, W.H., et al., Urinary 5-HIAA measurement using automated on-line solid-phase extraction-high-performance liquid chromatography-tandem mass spectrometry.
   J Chromatogr B Analyt Technol Biomed Life Sci, 2008. 868(1 – 2): p. 28 – 33.

2. Meijer, W.G., et al., Discriminating capacity of indole markers in the diagnosis of carcinoid tumors.
   Clin Chem, 2000. 46(10): p. 1588 – 96.

3. Formica, V., et al., The prognostic role of WHO classification, urinary 5-hydroxyindoleacetic acid and liver function tests in metastatic neuroendocrine carcinomas of the gastroenteropancreatic tract.
   Br J Cancer, 2007. 96(8): p. 1178 – 82.

4. Grouzmann, E., C. Centeno, and P.J. Eugster, Quantification of vanillylmandelic acid, homovanillic acid, and 5-hydroxy indole acetic acid in urine using a dilute-and-shoot and ultra-high pressure liquid chromatography-tandem mass spectrometry method.
   Clin Chem Lab Med, 2018. 56(9): p. 1533 – 1541.

5. Gut, P. and M. Ruchała, Evaluation of 5-hydroxyindoloacetic acid excretion in urine in patients with small intestine neuroendocrine neoplasm and carcinoid syndrome treated with somatostatin analogs.
   Neuro Endocrinol Lett, 2019. 40(7 – 8): p. 315 – 318.

6. Padelli, M., et al., Determination of thresholds values for platelet serotonin and urinary 5-HIAA concentrations for the biological diagnosis of digestive neuroendocrine tumors.
   Ann Biol Clin (Paris), 2019. 77(2): p. 161 – 168.

7. Tirosh, A., et al., Prognostic Utility of 24-Hour Urinary 5-HIAA Doubling Time in Patients With Neuroendocrine Tumors. Endocr Pract, 2018. 24(8): p. 710 – 717.

8. van der Horst-Schrivers, A.N., et al., Persistent low urinary excretion of 5-HIAA is a marker for favorable survival during follow-up in patients with disseminated midgut carcinoid tumors.
   Eur J Cancer, 2007. 43(18): p. 2651 – 7.

For updated literature or any other information please contact your local supplier.

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