DRG HYE-5361 Analyzer For Immunoassay And Clinical Chemistry Instructions

  Instructions for Use

Insulin

  HYE-5361

    40 tests

DRG Instruments GmbH , Germany
Frauenbergstraße. 18, D-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

Distributed by:

DRG International, Inc. , USA
841 Mountain Ave., Springfield, NJ 07081
Phone: 973-564-7555, Fax: 973-564-7556
Website: www.drg-international.com
E-mail: corp@drg-international.com

DRG Insulin HYE-5361

ASSAY PROTOCOL BARCODE (APB)

(Version 2.61 Software or later)

HYE-5361– v2.61.1

The barcode must be used to install the assay protocol into the DRG:HYBRiD-XL software via the SCAN NEW LOT page.

Please refer to section 3: Routine Procedures: “Installing a new assay product” of the User Manual v2.60 or later.

1 INTRODUCTION

1.1 Intended Use

The DRG:HYBRiD-XL Insulin is an enzyme immunoassay for the quantitative in vitro diagnostic measurement of insulin in serum and plasma (heparin and EDTA plasma).
Only for use with the DRG:HYBRiD-XL Analyzer.

1.2 Summary and Explanation

Insulin is the principal hormone responsible for the control of glucose metabolism. It is synthesized in the β cells of the islets of Langerhans as the precursor, proinsulin, which is processed to form C-peptide and insulin. Both are secreted in equimolar amounts into the portal circulation. The mature insulin molecule comprises two polypeptide chains, the A chain and B chain (21 and 30 amino acids respectively). The two chains are linked by two inter-chain disulphide bridges. There is also an intra-chain disulphide bridge in the A chain.
Secretion of insulin is mainly controlled by plasma glucose concentration, and the hormone has a number of important metabolic actions. Its principal function is to control the uptake and utilisation of glucose in peripheral tissues via the glucose transporters (1,2). This and other hypoglycaemic activities, such as the inhibition of hepatic gluconeogenesis and glycogenolysis are counteracted by the hyperglycaemic hormones including glucagon, epinephrine (adrenaline), growth hormone and cortisol.
Insulin concentrations are severely reduced in insulin-dependent diabetes mellitus (IDDM) and some other conditions such as hypopituitarism (1). Insulin levels are raised in non-insulin-dependent diabetes mellitus (NIDDM), obesity, insulinoma and some endocrine dysfunctions such as Cushing’s syndrome and acromegaly (1,4).

2 PRINCIPLE OF THE TEST

The DRG:HYBRiD-XL Insulin Kit is a solid phase enzyme-linked immunosorbent assay (ELISA) based on the sandwich principle.
The antibody coated wells (ACW) of the reagent cartridges are coated with a monoclonal (mouse) antibody directed towards a unique antigenic site of the insulin molecule.

An aliquot of patient sample containing endogenous insulin is incubated in the coated well with Enzyme Conjugate, which is an anti-insulin antibody conjugated with biotin.

After incubation the unbound conjugate is washed off.
Bound biotinylated antibodies are detected by consequent incubation of a Streptavidin-HRP Enzyme Complex.
After incubation the unbound streptavidin-biotin complex is washed off.
The amount of bound peroxidase conjugate is proportional to the concentration of insulin in the sample.
Having added the substrate solution, the intensity of color developed is proportional to the concentration of insulin in the patient sample.

3 WARNINGS AND PRECAUTIONS

1. This kit is for in vitro diagnostic use only.
   For professional use only.

2. This kit can only be used in combination with the DRG:HYBRiD-XL Analyzer

3. Before starting the assay, read the instructions completely and carefully. Use the valid version of the package insert provided with the kit. Be sure that everything is clear and understood.

4. Do not remove, exchange, discard or damage any of the barcode labels provided with each kit and its components. All barcodes build an integral system for the kit lot.

5. Respect the general safety measures for use of laboratory reagents.

6. All reagents of this test kit which contain human serum or plasma have been tested and confirmed negative for HIV I/II, HBsAg and HCV by FDA approved procedures. All reagents, however, should be treated as potential biohazards in use and for disposal.

7. Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.

8. Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.

9. Wear appropriate disposable gloves when handling specimens and reagents. Microbial contamination of reagents or specimens may cause false results.

10. Handling should be done in accordance with the procedures defined by an appropriate national biohazard safety guideline or regulation.

11. Do not use reagents beyond expiry date as shown on the kit labels.

12. Unused reagent cartridges must be stored at 2 °C to 8 °C in the sealed foil pouch with desiccant provided.

13. Optimal test results are only obtained when using calibrated pipettes.

14. Do not mix or use components from kits with different lot numbers. It is advised not to interchange reagent cartridges of different kits even of the same lot. The kits may have been shipped or stored under different conditions and the binding characteristics of the wells in the reagent cartridges may differ slightly.

15. Some reagents contain Proclin 300, BND and/or MIT as preservatives. In case of contact with eyes or skin, flush immediately with water.

16. TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with an abundant amount of water and skin with soap and plenty of water. Wash contaminated objects before reusing them. If inhaled, take the person to open air.

17. Chemicals and prepared or used reagents have to be treated as hazardous waste according to the national biohazard safety guidelines or regulations.

18. For information on hazardous substances included in the kit please refer to Safety Data Sheets.
    For professional users the Safety Data Sheet for this product is available upon request directly from DRG.

4 REAGENTS

4.1 Reagents provided

4.1.1 Reagent Cartridges

40 pieces containing the following:
− Antibody Coated Well (ACW)
coated with anti-insulin antibody (monoclonal).
− Enzyme Conjugate, 120 µL,
Anti-insulin antibody conjugated with biotin;
Contains non-mercury preservative.
− Enzyme Complex, 170 µL,
streptavidin coupled to horseradish peroxidase;
Contains non-mercury preservative.
− Substrate Solution, 270 µL
Tetramethylbenzidine (TMB).

4.1.2 Re-Calibrator 1 & 2

2 vials, 1 mL each, ready to use;
For re-calibration of the quantitative DRG:HYBRiD-XL Insulin test.
Concentrations are lot-specific.
Conversion: µIU/mL x 0.0433 = ng/mL, ng/mL x 23.09 = µIU/mL

Re-Calibrators are standardised against the following reference material:
WHO International Standard Insulin, human (NIBSC code 83/500) Contain non- mercury preservative.

4.1.3 Control 1 & 2

2 vials, 1 mL each, ready to use;
For control values and ranges please refer to the bar code on vial label or to the QC-Datasheet.
Contain non-mercury preservative.

4.2 Materials required but not provided

− General needed laboratory equipment
− Ultra-pure water
DRG recommends to use Clinical Laboratory Reagent Water (CLRW) according to CLSI guideline 3C-A4 with the following specifications:
Resistivity at 25 °C [MΩ·cm]: > 10
Conductivity at 25 °C [μS·cm−1]: < 0.1
Total Organic Carbon/p.p.b.[μg/L] : < 50
Colloids [μg/mL]: <0.05
− HYB-5252 DRG:HYBRiD-XL Analyzer
− HYI-5392: System Solution 5L, 5000 mL;
(Instrument Feed Water according to CLSI guideline 3C-A4 with the following specification can also be used:
Resistivity at 25 °C [MΩ·cm]: > 1
Conductivity at 25 °C [μS·cm−1]: < 1
Total Organic Carbon/p.p.b.[μg/L] : < 200
Colloids [μg/mL]: <0.1)
− HYI-6234: Wash Buffer, 10x concentrate, 100 mL
− HYI-5395: Needle Cleaning Solution, 30 mL. Cleaning solution for the pipetting needle (daily and weekly maintenance, see also user manual)
− HYI-5387: Cuvettes, (2 x 360 pieces)

For use of the Secondary Sample Holder for secondary tubes the following tubes are required:
− HYI-5391: Sample Tubes (Secondary), 2500 pcs.

4.3 Storage Conditions

All kit components should be stored at 2 °C to 8 °C to ensure product performance until the defined expiry date.

When stored at 2 °C to 8 °C, unopened kits will retain reactivity until expiration date. Do not use reagents beyond this date.
− Cartridges (stored at 2 °C to 8 °C) in the supplied and unopened zip/foil bags will retain reactivity until expiration date.
− Unopened Re-Calibrators and Controls (stored at 2 °C to 8 °C) will retain reactivity until expiration date.

Opened reagents and the reagent cartridges must be stored at 2 °C to 8 °C.

Once the plastic bag has been opened, care should be taken to tightly close it again along with the supplied desiccant bag.
Immediately after end of each run the Re-Calibrator and Control vials have to be removed from the instrument, tightly capped and stored at 2 °C to 8 °C.
− Unused cartridges in opened zip/foil bags (stored at 2 °C to 8 °C) will retain reactivity until expiration date, if stored as described above.
− Pierced or open cartridges must be disposed of immediately.
− Opened Re-Calibrators and Controls (stored at 2 °C to 8 °C) will retain reactivity for 8 weeks.

4.3.1 On-board Stability

For Re-Calibrators and Controls the on-board stability has been evaluated under controlled laboratory conditions at room temperature (20 °C to 25 °C).
Due to the differences in laboratory environmental conditions and reagent volumes, the on-board stability may deviate from the declared value.

4.4 Reagent Preparation

Bring all reagents, such as controls and re-calibrators, to room temperature (20 °C to 25 °C) prior to use. Reagent Cartridges can be used directly after storage in the refrigerator.

Wash Buffer (10x) (HYI-6234; not included in the kit)
For Wash Buffer (1x) dilute 100 mL of Wash Buffer (10x) with 900 mL ultra-pure water to a final volume of 1000 mL.

4.5 Disposal of the Kit

The disposal of the kit and all used materials/reagents must be performed according to the national regulations. Special information for this product is given in the Safety Data Sheet.

4.6 Damaged Test Kits

In case of any damage to the test kit or components, DRG must be informed in writing, at the latest one week after receiving the kit. Damaged single components should not be used for a test run. They have to be stored until a final solution has been found. After this, they should be disposed of according to the official regulations.

5 SPECIMEN COLLECTION AND PREPARATION

Serum or plasma (EDTA- or heparin plasma) can be used in this assay.
A minimum of 110 µL of sample is needed for one determination. This includes 50 µL sample and 60 µL dead volume.
Attention:

− This test was not verified with blood collection tubes of all available manufacturers.
− Sample Collection Systems of some manufacturers may contain different materials which in isolated cases could affect the test results.
− If primary tubes for sample collection are used, please follow the instructions of the manufacturer.
− Do not use haemolytic, icteric or lipaemic specimens.
− Samples containing precipitates have to be centrifuged prior to the test run.
− Do not use heat inactivated samples.
− Do not use standards or external controls stabilized with azide.

5.1 Specimen Collection

Serum:
Collect blood by venipuncture (e.g. Sarstedt Monovette for serum), allow to clot, and separate serum by centrifugation at room temperature. Do not centrifuge before complete clotting has occurred. Patients receiving anticoagulant therapy may require increased clotting time.

Plasma:
Whole blood should be collected into centrifuge tubes containing anti- coagulant (e.g. Sarstedt Monovette with the appropriate plasma preparation) and centrifuged immediately after collection.

5.2 Specimen Storage and Preparation

Specimens should be capped and may be stored for up to 4 days at 2 °C to 8 °C prior to performing the assay.
Specimens stored for a longer time (up to one year) should be frozen only once at -20 °C prior to the assay. Thawed samples should be inverted several times prior to testing.

5.3 Specimen Dilution

5.3.1 Manual Sample Dilution

If in an initial assay, a specimen is found to contain more than the highest standard, the specimens can be diluted with Sample Diluent* and reassayed as described in Assay Procedure.
For the calculation of the concentrations this dilution factor has to be taken into account.

Example:
a) dilution 1:10: 10 µL sample + 90 µL Sample Diluent (mix thoroughly)
b) dilution 1:100: 10 µL dilution a) 1:10 + 90 µL Sample Diluent (mix thoroughly).

• Sample Diluent for manual dilution is not included in this kit, but can be ordered on request ( HYE-5361-DIL, 20 mL).

6 ASSAY PROCEDURE

6.1 General Remarks

− All reagents, such as controls and re-calibrators and specimens, must be allowed to come to room temperature (20 °C to 25 °C) before use.
All reagents and samples must be mixed without foaming. Reagent Cartridges can be used directly after storage in the refrigerator.
− Samples, controls and re-calibrators should be measured within 2 hours in order to avoid possible evaporation effects.
− The Secondary Sample Holder (HYI-5437) for secondary tubes has the capacity for a maximum of 20 samples including controls and re-calibrators. They all have to be pipetted into the secondary tubes, and the respective barcodes of control/re-calibrator vials and, if available, the sample barcodes have to be read with the external barcode scanner.

6.2 Test Procedure

− The total assay time for DRG:HYBRiD-XL Insulin is 120 minutes.
− To ensure proper operation of the test, the instructions in the user manual for the DRG:HYBRiD-XL should strictly be followed.
− All test specific information required for the correct operation is included in the respective barcodes of the reagents.
Take care not to damage these bar codes!
− It is recommended to tap the bottom of the Cartridge Segments containing the reagent cartridges once on the bench before placing them on the rotor. This is to avoid foam and adhering of the liquid on the sealing of the reagent cartridge.
− Place reagent cartridges on the rotor of the unit. The heating to 37 °C incubation temperature is performed automatically in the unit.

6.3 Calibration

Traceability:
This method was standardized against the following reference material:
WHO International Standard Insulin, human (NIBSC code 83/500).

Each DRG:HYBRiD-XL reagent contains a barcode with the specific information for recalibration of the reagent lot. The Master Curve is printed as a 2-D barcode on the outer label of the kit package and on the QC-Datasheet and has to be scanned with the external barcode scanner prior to the first use of the respective kit lot.

Recalibration is recommended:
− if a new kit lot is used. Each new lot should be verified by running the kit internal re-calibrators and controls before routine use.
− if one or both assay controls are found outside the specified range.
− after 4 weeks of use of the same reagent kit on the unit.

6.4 Calculation of Results

The analyte concentrations are calculated automatically by the DRG:HYBRiD-XL’s system software.

7 QUALITY CONTROL

It is recommended to use control samples according to state and federal regulations. The use of control samples is advised to assure the day to day validity of results.
It is also recommended to participate in national or international Quality Assessment programs in order to ensure the accuracy of the results.

Apply appropriate statistical methods for analysing control values and trends. If the results of the assay do not agree with the established acceptable ranges of control materials, patient results should be considered invalid. In this case, please check the following: expiration dates and storage conditions of reagents, operational reliability of the analyser. In addition, it is indicated to perform a Recalibration.
In case of further questions please contact your local distributor or DRG directly.

7.1 Internal Controls

For Quality Control it is necessary to use the two internal controls provided with each kit.
Acceptance ranges for both internal controls (Control 1 & 2) were established by the manufacturer and are summarized in the QC-Datasheet added to the kit. Note that the expected values and acceptance ranges stated in the QC-Datasheet always refer to the current kit lot.
Internal controls should be run in single determination:
− on a routine basis (e.g. once per 24 h)
− if re-calibration is required (if one or both internal controls are out of range)
− if a new kit lot is used (in order to avoid any negative impact on the kit performance by improper transport or to detect improper storage during transport).

7.2 External Controls

Use controls at both normal and pathological levels.
The control intervals and control ranges for external controls should be adapted to the individual requirements of each laboratory. All results must be within the defined limits.
Each laboratory should establish corrective measures to be taken if values of external controls are not found in the acceptance range.

8 EXPECTED NORMAL VALUES

It is strongly recommended that each laboratory should determine its own normal and pathological values.
In a study conducted with apparently healthy subjects, using the DRG:HYBRiD-XL Insulin the following data were observed:

These values are in concordance with data published elsewhere (3).
The results alone should not be the only reason for any therapeutic consequences. The results should be correlated with other clinical observations and diagnostic tests.

9 LIMITATIONS OF USE

Reliable and reproducible results will be obtained, when the assay procedure is performed with a complete understanding of the instructions for use and with adherence to good laboratory practice.
Any improper handling of samples or modification of this test might influence the results.

10 PERFORMANCE CHARACTERISTICS

10.1 Assay Dynamic Range

The dynamic range of the assay is defined by the limit of detection and the maximum value of the Master Curve.
Values found below the measuring range are indicated as “< 2.21 µIU/mL“.
Values found above the measuring range are indicated as “> 75 µIU/mL”.
The measuring range of the assay is between 2.21 µIU/mL – 75 µIU/mL.

10.2 Specificity of Antibodies (Cross-Reactivity)

The following substances were tested for cross-reactivity of the assay:

Human Insulin                     96 %
Bovine Insulin                 > 100 %
Dog Insulin                           82 %
Rabbit Insulin                       63 %
Rat Insulin                   < 0.0001 %
C-Peptide                     < 0.0001 %
Proinsulin                     < 0.0001 %
Leptin                           < 0.0001 %
IGF-1                           < 0.0001 %
Hepcidin-25                  < 0.0001 %

10.3 Sensitivity

The sensitivity study was designed according to CLSI guideline EP17-A2.

The Limit of Blank (LoB) is 1.61 µIU/mL.
The Limit of Detection (LoD) is 2.21 µIU/mL.
The Limit of Quantification (LoQ) is 2.96 µIU/mL.

10.4 Precision Performance

The precision study was designed according to CLSI guideline EP5-A2.

10.4.1 Within-Run (Intra Assay)

Samples were analysed 2 times daily in duplicates for 20 days on 3 different instruments. CV (%) is given as mean of 40 duplicate CVs. The within-run variability is shown below:

10.4.2 Total Precision (Inter Assay)

Samples were analysed 2 times daily in duplicates for 20 days (n=80) on 3 different instruments. The total precision is shown below:

10.4.3 Inter-Lot

The between-lots variation was determined by 6 measurements of 4 samples with 3 different kit lots.

10.5 Recovery

Recovery was determined by adding four increasing concentrations of the analyte to four different patient samples containing different amounts of endogenous analyte. Each sample (non-spiked and spiked) was assayed and analyte concentrations of the samples were calculated from the Master Curve. The percentage recoveries were determined by comparing expected and measured values of the samples.

10.6 Linearity

Four samples containing different amounts of analyte were serially diluted with Sample Diluent. The percentage recovery was calculated by comparing the expected and measured values for the analyte.

10.7 Interfering Substances

Haemoglobin (up to 4 mg/mL), Bilirubin (up to 0.5 mg/mL) and Triglyceride (up to 7.5 mg/mL) have no influence on the assay results.

Until today no substances (drugs) are known to us, which have an influence to the measurement of insulin in a sample.

10.8 High-Dose-Hook Effect

Hook effect was not observed in this test up to a concentration of 1500 µIU/mL of insulin.

11 METHOD COMPARISON

A comparison of DRG:HYBRiD-XL Insulin Test HYE-5361 (y) and the ROCHE Cobas Insulin ECLIA (x) using clinical samples gave the following correlation:

n = 19
r = 0.969
y = 0.968 x – 1.002

12 LEGAL ASPECTS

Only for countries where the declaration of European Conformity (CE mark) is applicable.

12.1 Reliability of Results

The test must be performed exactly as per the manufacturer’s instructions for use. Moreover, the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable national standards and/or laws. This is especially relevant for the use of control reagents. It is important to always include, within the test procedure, a sufficient number of controls for validating the accuracy and precision of the test.
The test results are valid only if all controls are within the specified ranges and if all other test parameters are also within the given assay specifications. In case of any doubt or concern please contact DRG.

12.2 Therapeutic Consequences

Therapeutic consequences should never be based on laboratory results alone even if all test results are in agreement with the items as stated under point 12.1. Any laboratory result is only a part of the total clinical picture of a patient.
Only in cases where the laboratory results are in acceptable agreement with the overall clinical picture of the patient should therapeutic consequences be derived.
The test result itself should never be the sole determinant for deriving any therapeutic consequences.

12.3 Liability

Any modification of the test kit and/or exchange or mixture of any components of different kit lots could negatively affect the intended results and validity of the overall test. Such modification and/or exchanges invalidate any claim for replacement.
Claims submitted due to customer misinterpretation of laboratory results subject to point 12.2 are also invalid. Regardless, in the event of any claim, the manufacturer’s liability is not to exceed the value of the test kit. Any damage caused to the test kit during transportation is not subject to the liability of the manufacturer.

For further information please refer to the User Manual of the DRG:HYBRiD- XL, analyser-specific application sheets, product information and package inserts of all necessary components.

13 REFERENCES

1. McPherson RA and Pincus MR, editors. Henry´s clinical diagnosis and management by laboratory methods. 22nd edition. 2011; 211-225.

2. Thomas L. editor. Labor und Diagnose. 8th edition. 2012; 242-253.

3. Francois A, Maumus S et al. Age and sex-related reference values for serum insulin concentration and its biological determinants in a French healthy population. The Stanislas cohort.
   Clin Chem Lab Med; 2003; 42-1140-9.

4. Marks V, Teale JD. Hypoglycemic disorders.
   Clin Lab. 2005; 21: 79-97.

14 SYMBOLS USED

Symbol|
---|---
| European Conformity
| Consult instructions for use
| In vitro diagnostic medical device
| For research use only
| Catalogue number
| Batch code
| Contains sufficient for tests
| Temperature limit
| Use-by date
| Manufacturer
| Caution*
Distributed by| Distributor
Content| Content
Volume/No.| Volume/No.
Reagent Cartridge| Reagent Cartridge
Re-Calibrator| Re-Calibrator
Control| Control
Enzyme Conjugate| Enzyme Conjugate
Enzyme Complex| Enzyme Complex
Substrate Solution| Substrate Solution
Sample Diluent| Sample Diluent
Reagent 1| Reagent 1
Reagent 2| Reagent 2
Wash Buffer| Wash Buffer
System Solution| System Solution
Needle Cleaning Solution| Needle Cleaning Solution
Denaturation Buffer| Denaturation Buffer
Neutralization Buffer| Neutralization Buffer
Assay Buffer| Assay Buffer
|
|
Secondary Sample Holder| Secondary Sample Holder
Sample Tubes (Secondary)| Sample Tubes (Secondary)
Dilution Cartridge| Dilution Cartridge
Vial Adapter| Vial Adapter

Vers. 4.0                            2024-04-19 – cj

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