Glucose UV Reagent - Cliniqa

1 33024_01 11/23/09 2008 Cliniqa Corporation. All rights reserved. Glucose UV Reagent Order No. Description R80017 10 x 20 mL R80036 10 x 50 mL R85411 4 x 1000 mL INTENDED USE For the quantitative in vitro enzymatic determination of Glucose in biological fluids. TEST SUMMARY The method for determination of Glucose employing the Hexokinase (HK) Glucose -6-phosphate dehydrogenase (G-6-P-DH) reactions was developed by G. Bucolo for Calbiochem in 1961 and was marketed by the latter Company in 1962 (1). The method is highly specific for Glucose ; the Reagent can be used for the determination of Glucose in serum, plasma, urine and cerebrospinal fluid without prior treatment of these specimens. Common anticoagulants, including fluoride, at the recommended levels, do not interfere. The assay is based on the following reactions: HK Glucose + ATP G-6-P + ADP G-6-P-DH G-6-P + NAD 6-PG + NADH Glucose is phosphorylated by adenosine-5'-triphosphate (ATP), in a reaction catalyzed by HK.

2 The Glucose -6-phosphate (G-6-P) formed is oxidized to 6-phosphogluconate (6-PG) by G-6-P-DH. In this same reaction an equimolar amount of Nicotinamide Adenine Dinucleotide (NAD) is reduced to NADH, with a resulting increase in absorbance at 340 nm. The concentration of Glucose can be calculated from this increase in absorbance, using the millimolar extinction coefficient of reduced NAD (NADH). Reagent COMPOSITION When the Reagent is dissolved in water as per directions, the solution will have the following approximate concentrations: Reactive ingredients: Magnesium aspartate mmol/L Nicotinamide adenine dinucleotide 2 mmol/L Adenosine-5 -triphosphate mmol/L Hexokinase 1500 U/L Glucose -6-phosphate dehydrogenase 1500 U/L Non-reactive ingredients.

3 Buffers, stabilizers and fillers Reagent PREPARATION Dissolve the dry Reagent in vial with volume of deionized or distilled water specified on the vial label. After addition of water, cap vials, without delay, mix the vials by inversion several times. Do not shake. Occasional swirling of the vials will aid solution. Reagent STORAGE AND STABILITY The unreconstituted Reagent is stable for at least 2 years when stored in the refrigerator (2 8 C). The reconstituted Reagent is stable at room temperature (20 25 C) for 24 hours and in the refrigerator (2 8 C) for 30 days. The dry powder in the vials may occasionally appear moist; this is not an indication that water has entered the vials. However, if the Reagent in these vials, after reconstitution, has an absorbance at 340 nm against a blank of water greater than , discard the Reagent If the reconstituted Reagent develops turbidity, this may indicate contamination.

4 Do not use. PRECAUTIONS Good laboratory safety practices should be followed when handling any laboratory Reagent . Refer to a recognized laboratory safety program for additional information. (See GP17-T, Clinical Laboratory Safety; Tentative Guideline (1994), National Committee on Clinical Laboratory Standards, Wayne, PA.) Intended for in vitro diagnostic use only. SPECIMEN COLLECTION, PREPARATION AND STORAGE Glucose is metabolized by erythrocytes at a rate varying, according to temperature and other conditions. This consumption can be as high as 10 mg/dL per hour. It is, therefore, very important that serum and plasma be separated from the formed elements of blood as soon as possible after collection. Hemolyzed samples should not be used. The hexokinase Reagent can be used to determine Glucose in urine without prior treatment (2). INTERFERING SUBSTANCES The hexokinase method has been found to be free of interference from a large number of contaminants (3).

5 Young et al. (4) have published a comprehensive list of drugs and substances which may interfere with in vitro diagnostic assays, including Glucose . MATERIALS REQUIRED BUT NOT PROVIDED 1. Spectrophotometer capable of accurate absorbance measurements at 340 nm. 2. Square cuvettes preferably with a 1 cm light path. 3. Accurate pipettes to measure distilled or deionized water, Reagent and samples. 4. Distilled or deionized water. MATERIALS PROVIDED R80017 Glucose UV 10 20 mL R80036 Glucose UV 10 50 mL R85411 Glucose UV 4 1000 mL TEST PROCEDURE (1:151 sample to Reagent ratio) 1. Set spectrophotometer to 340 nm. 2. Dispense mL of Reagent into a clean, dry cuvette. 3. Adjust spectrophotometer to 0 absorbance with a water blank. Read absorbance of Reagent (A0). 4. Add 10 L of sample to the Reagent . Mix by inversion. 5. After 5 minutes, but not longer than 20 minutes, read the absorbance (A5) of the assay against the same water blank.

6 6. Subtract A0 from A5 to obtain A. Refer to calculations below. ALTERNATE TEST PROCEDURE (1:101 sample to Reagent ratio) 1. Prepare a Reagent blank by pipetting 1 mL of Reagent into a cuvette. 2. Pipette 1 mL of Reagent into another cuvette. Add 10 L of sample and mix. 3. After 5 minutes, zero the spectrophotometer with the Reagent blank and read the absorbance of the Reagent to which the sample was added. 4. Use this absorbance in the calculations. QUALITY CONTROL Serum controls are recommended to monitor the performance of manual and automated assay procedures, providing a continued screening of the instrument, reagents and technique. Commercially available control material with established values for Glucose concentration may be usd. Assayed Control Serum, Level 1 (Cat. No. R83082) and Level 2 (Cat. No. R83083) are recommended for this purpose. CALCULATIONS TV 100 A = mg/dL Glucose SV 1000 A = Increase in absorbance of the assay.

7 TV = Volume of the assay in mL. = Glucose . 100 = for conversion from mL to dL. = Millimolar extinction coefficient of NADH at 340 nm SV = Sample volume in mL 1000 = to convert g to mg. 33024_01 11/23/09 2008 Cliniqa Corporation. All rights reserved. From this a factor can be derived which, when multiplied by the absorbance of the assay, gives the mg/dL Glucose in the sample. For the test procedure employing 10 L of sample and mL of Reagent this factor is: 437. Example: If the increase in absorbance of the assay is , the Glucose concentration is x 437 = 546 mg/dL. For the procedure employing 10 L of sample and 1 mL of Reagent the factor is: 292. Example: If the increase in absorbance of the assay is , the Glucose concentration is x 292 = 365 mg/dL LIMITATIONS OF THE PROCEDURE The assay is linear to 800 mg/dL (1:151 sample to Reagent ratio) and to 530 mg/dL (1:101 sample to Reagent ratio).

8 Samples with Glucose concentration exceeding these linearities should be diluted with an equal volume of distilled or deionized water and the assay repeated; multiply the results by 2. The alternate test procedure will lower the upper limit of linearity of the method. The absorbance of the sample is disregarded in these procedures as negligible. In the alternative test procedure, where a larger sample to Reagent ratio is employed, the contribution of turbid or icteric samples to the absorbance will be slightly greater. A serum blank can be run, for very accurate determinations, by adding the same volume of sample used in the assay to a volume of sodium chloride solution equal to that of the Reagent employed in the assay. Read the absorbance of this diluted sample against a blank of the same sodium chloride solution and subtract this absorbance from the final absorbance of the assay.

9 Use this corrected absorbance for the calculations. Reagent PERFORMANCE 1. Linearity: The assay is linear to 800 mg/dL in the procedure employing 1:151 sample to Reagent ratio and to 530 mg/dL in the procedure employing the 1:101 sample to Reagent ratio. 2. Correlation: Results of the assays on 79 serum samples ranging in Glucose concentration from 71 to 522 mg/dL using our Reagent were compared with those obtained using Calbiochem Reagent . The correlation coefficient was and the regression equation was y = - 3. Precision: Within Run Mean 83 280 553 SD CV (%) N 8 8 8 Run-to-Run Mean 82 281 541 SD CV (%) N 24 23 23 REFERENCE RANGE The reference range is reported to be 65-110 mg/dL.

10 Each laboratory should establish its own reference range, as more representative of the population in its area of operation. REFERENCES 1. Calbiochem Clinalytical Reagents. Summer Catalog, 1962. 2. Peterson, and Young, , Anal. Biochem. 23:301, 1968 3. Bondar, and Mead, , Clin. Chem. 20:586, 1974. 4. Young, , Pestaner, LD. and Gibberman, V., Clin. Chem. 21:N. 5, 1975. RE-ORDER INFORMATION Glucose UV Reagent For in vitro diagnostic use See package insert for proper use SALES AND TECHNICAL SUPPORT FOR ORDERS AND CUSTOMER SERVICE Cliniqa CORPORATION CEpartner4U 288 Distribution St. San Marcos, CA 92078 USA P: 800 728 5205 +1 760 744 1900 F: +1 760 571 5198 P: 800 728 5205 +1 760 744 1900 F: +1 760 571 5197 Esdoornlaan 13 3951 DB Maarn, The Netherlands P: +31 (0)6 516 536 26 Catalog No. Catalog No.