【Product Name】

General Health Profile

【Packing Specification】

1 Disc / Sample

【Instrument】 See the InSight V-CHEM chemistry analyser Operator’s Manual for complete information on use of the analyser. 【Intended Use】 The General Health Profile used with the InSight V-CHEM chemistry analyser is intended to be used for the in vitro quantitative determination of total Protein (TP), albumin (ALB), total bilirubin (TBIL), alanine aminotransferase (ALT), blood urea (BUN), creatinine (CRE), amylase (AMY), creatinekinase (CK), calcium (Ca2+), phosphorus (P), alkaline Phosphatase (ALP), glucose (GLU), and total cholesterol (CHOL)in heparinised whole blood, heparinised plasma, or serum in a clinical laboratory setting or point of care location. The General Health Profile and the InSight V-CHEM chemistry analyser comprise an in vitro diagnostic system that aids the physician in the following disorders: liver and gall bladder diseases, urinary system diseases, carbohydrate metabolism disorders, lipid metabolism disorders, cardiovascular disease and pancreatic diseases. 【Test Principles】 This product, which is based on spectrophotometry, is used to quantitatively determine the concentration or activity of the 13 biochemical indicators in the sample. The test principles are as follows: (1) Total Protein (TP) The total protein method is a Biuret reaction, the protein solution is treated with cupric [Cu(II)] ions in a strong alkaline medium. The Cu(II) ions react with peptide bonds between the carbonyl oxygen and amide nitrogen atoms to form a coloured Cu-protein complex. The amount of total protein present in the sample is directly proportional to the absorbance of the Cu-protein complex. The total protein test is an endpoint reaction and the absorbance is measured as the difference in absorbance between 550 nm and 800 nm.

- Total Protein + Cu(II) ⎯ OH⎯⎯ →Cu-Protein Complex

(2) Albumin (ALB) Bromcresol green (BCG), when bound with albumin, changes from a yellow to green colour. The absorbance maximum changes with the colour shift.

BCG + Albumin ⎯ pH4⎯⎯.2→Albumin Complex

Bound albumin is proportional to the concentration of albumin in the sample. This is an endpoint reaction that is measured as the difference in absorbance between 630 nm and 700 nm. (3) Total Bilirubin (TBIL) In the enzyme procedure, bilirubin is oxidised by bilirubin oxidase (BOD) into biliverdin. Bilirubin is quantitated as the difference in absorbance between 450 nm and 546 nm. The initial absorbance of this endpoint reaction is determined from the bilirubin blank cuvette and the final absorbance is obtained from the bilirubin test cuvette. The amount of bilirubin in the sample is proportional to the difference between the initial and final absorbance measurements.

BOD Bilirubin + O2 ⎯ ⎯⎯ → Biliverdin + H2O

(4) Alanine Aminotransferase (ALT) ALT catalyses the transfer of an amino group from L-alanine to ɑ-ketoglutarate to form L-glutamate and pyruvate. Lactate dehydrogenase catalyses the conversion of pyruvate to lactate. Concomitantly, NADH is oxidised to NAD+, as illustrated in the following reaction scheme. L-Alanine + ɑ-Ketoglutarate ⎯ ALT⎯⎯ →L-Glutamate + Pyruvate

Pyruvate + NADH + H+ ⎯ LDH⎯⎯ → Lactate + NAD+ The rate of change of the absorbance difference between 340 nm and 405 nm is due to the conversion of NADH to NAD+ and is directly proportional to the amount of ALT present in the sample. (5) Urea(BUN) In the coupled-enzyme reaction, urease hydrolyses urea into ammonia and carbon dioxide. Upon combining ammonia with 2-oxoglutarate and reduced nicotinamide adenine dinucleotide (NADH), the enzyme glutamate dehydrogenase (GLDH) oxidizes NADH to NAD+.

Urease BUN + H2O ⎯ ⎯⎯ → NH3 + CO2

GLDH + NH3 + 2-Oxoglutarate + NADH ⎯ ⎯⎯ → L-Glutamate + H2O + NAD The rate of change of the absorbance difference between 340 nm and 405 nm is caused by the conversion of NADH to NAD+ and is directly proportional to the amount of urea present in the sample. (6) Creatinine (CRE) In the coupled enzyme reactions, creatinineamidohydrolase (CAH) hydrolyses creatinine to creatine. A second enzyme, creatineamidinohydrolase (CRH), catalyses the formation of sarcosine from creatine. Sarcosine oxidase (SAO) causes the oxidation of sarcosine to glycine, formaldehyde and hydrogen peroxide (H2O2). Peroxidase (POD) catalyses the reaction among the hydrogen peroxide, N-Ethyl-N-(2- hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt (FDAOS) and 4-aminoantipyrine (4-AAP) into a red quinoneimine dye. Potassium ferrocyanide and ascorbate oxidase are added to the reaction mixture to minimise the potential interference of bilirubin and ascorbic acid, respectively.

CAH Creatinine + H2O ⎯ ⎯⎯ →Creatine

CRH Creatine + H2O ⎯ ⎯⎯ →Sarcosine + Urea

SAO Sarcosine + H2O + O2 ⎯ ⎯⎯ → Glycine + Formaldehyde + H2O2

POD H2O2 + FDAOS + 4-AAP ⎯ ⎯⎯ →Red Quinoneimine Dye + H2O Two cuvettes are used to determine the concentration of creatinine in the sample. Endogenous creatine is measured in the blank cuvette, which is subtracted from the combined endogenous creatine and the creatine formed from the enzyme reactions in the test cuvette. Once the endogenous creatine is eliminated from the calculations, the concentration of creatinine is proportional to the intensity of the red colour produced. The endpoint reaction is measured as the difference in absorbance between 550 nm and 700 nm. (7) Amylase (AMY) In the coupled-enzyme reaction, amylase hydrolyses Ethylidene-nitrophenol-maltgglucoside into smaller molecules malt oligomeric glycoside. ɑ-glucosidase (ɑ-GLU) catalyses oligomeric glycoside to produce p-nitrophenolc and glucose. The absorbance of this rate reaction is measured at 405 nm. The production is directly proportional to the amylase activity in the sample.

AMY E-4-NP-G7+ H2O ⎯ ⎯⎯ →E-G5 + G2-4-NP+E-G4 + G3-4-NP+E-G3+G4-4-NP

-GLU G2-4-NP+G3-4-NP+G4-4-NP+H2O ⎯ ⎯ ⎯ →4-NP+GLU

(8) Glucose (GLU) The reaction of glucose with adenosine triphosphate (ATP)catalysed by hexokinase (HK), produces glucose-6-phosphate (G-6-P) and adenosine diphosphate (ADP). Glucose-6-phosphate dehydrogenase (G-6-PD) catalyses the reaction of G-6-P into 6-phosphogluconate and the reduction of nicotinamide adenine dinucleotide phosphate (NADP) to NADPH.

HK Glucose + ATP ⎯ ⎯ →Glucose-6-Phosphate + ADP

G-6-P + NADP ⎯ G⎯-6-⎯PD→6-Phosphogluconate + NADPH + H+ The absorbance is measured dichromatically at 340 nm and 405 nm. The production of NADPH is directly proportional to the amount of glucose present in the sample. (9) Total Cholesterol (CHOL) The reaction of CHOL is an enzymatic end-point method that uses cholesterol esterase (CE) and Cholesterol oxidase (COD). Peroxidase (POD) catalyses the reaction among the hydrogen peroxide, N- Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylanilineodium salt (TOOS) and 4-aminoantipyrine (4-AAP) into a red quinoneimine dye.

CE Cholesterol Esters + H2O ⎯ ⎯ → Cholesterol + Fatty Acids

COD Cholesterol +O2 ⎯ ⎯⎯ →Cholestenone+ H2O2

POD H2O2+4-AAP+TOOS ⎯ ⎯⎯ →Quinoneimine dye +H2O

(10) Calcium (Ca2+) Calcium in the patient sample binds with Arsenazo III to form a calcium-dye complex. Ca2+ + Arsenazo III ⎯⎯ → Ca2+-Arsenazo III Complex It is an endpoint reaction. The amount of total calcium in the sample is proportional to the absorbance.

(11) Phosphorus (P) The reaction of inorganic phosphorus with inosine, catalysed by purine nucleoside phosphorylase (PNP), produces hypoxanthine and ribose phosphate. The hypoxanthine is then oxidised to uric acid (UA) catalysed by Xanthine oxidase (XOD). Peroxidase (POD) catalyses the reaction among the hydrogen peroxide, N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline odium salt (TOOS) and 4- aminoantipyrine (4-AAP) into a red quinoneimine dye. P + Inosine ⎯ PNP⎯⎯ → Hypoxanthine + Ribose Phosphate

XOD Hypoxanthine + O2 +H2O ⎯ ⎯⎯ → UA + H2O2

POD H2O2 + 4-AAP + TOOS ⎯ ⎯⎯ →Quinoneimine dye + H2O

(12) Creatine Kinase (CK) Creatine kinase catalyses the formation of creatine and adenosine triphosphate (ATP) from creatine phosphate and adenosine diphosphate (ADP). With hexokinase (HK) as a catalyst, ATP reacts with D- glucose to form ADP and D-glucose-6-phosphate (G-6-P), which is reacted with nicotinamide adenine dinucleotide phosphate (NADP) in the presence of glucose-6-phosphate dehydrogenase (G-6-PD) to produce 6-Phosphogluconate (6-PG) and NADPH. The formation of NADPH is measured as a change in absorbance at 340 nm relative to 405 nm. This absorbance change is directly proportional to creatine kinase activity in the sample.

CK Creatine phosphate + ADP ⎯ ⎯ →Creatine + ATP

HK ATP + D-glucose ⎯ ⎯ →ADP + G-6-P

G-6-PD G-6-P + NADP ⎯ ⎯ ⎯ → 6-Phosphogluconate + NADPH

(13) Alkaline Phosphatase (ALP) Under the catalysis of ALP, the Phosphoric acid on nitrobenzene (4-NNP) was turned into Para nitro phenol (4-NP).4-NP shows a yellow colour in alkaline solution. At the wavelength of 405nm, the ALP activity can be calculated by monitoring the absorbance change rate.

ALP 4-NNP ⎯ ⎯⎯ → Acyl phosphate + 4-NP

【Principle of Operation】 Refer to the InSight V-CHEM chemistry analyser for the Principles and Limitations of the Procedure. 【Description of Reagents】 Each General Health Profile contains dry test-specific reagent beads. Each sealing pouch contains a reagent disc and a pack of desiccant. A one-time use disc is for only one sample. Each sealing pouch has a unique code printed on the surface. The ingredient of each General Health Profile are as follows (after redissolution):

Component Quantity

Total protein assay reagent 13.5μL

Albumin assay reagent 13.5μL

Total Bilirubin assay reagent 13.5μL Alanine Aminotransferase assay reagent 13.5μL

Urea assay reagent 13.5μL

Creatinine assay reagent 13.5μL

Creatine Kinase reagent 13.5μL

Glucose assay reagent 13.5μL

Total Cholesterol assay reagent 13.5μL

Phosphorus reagent 13.5μL

Calcium reagent 13.5μL

Amylase reagent 13.5μL

Alkaline Phosphatase reagent 13.5μL

Stabilizer Appropriate amount

【Storage】 Store reagent discs in their sealed pouches at 2-8°C (36-46°F). Do not expose opened or unopened discs to direct sunlight or temperatures above 32°C (90°F). Reagent discs may be used until the expiration date included on the package. The expiration date is also encoded in the unique code printed on the sealing pouch. An error message will appear on the InSight V-CHEM chemistry analyser display if the reagents have expired. A torn or otherwise damaged pouch may allow moisture to reach the unused disc and adversely affect reagent performance. Do not use a disc from a damaged pouch. 【Sample Requirements】 Sample collection techniques are described in the “Sample Collection” section of the InSight V- CHEM chemistry analyser Operator’s Manual. The required sample size is ~100 μL of heparinised whole blood, heparinised plasma, serum or control material. Whole blood samples obtained by venipuncture must be homogeneous before transferring a sample to the reagent disc. Gently invert the collection tube several times just prior to sample transfer. Whole blood venipuncture samples should be run within 60 minutes of collection. Glucose concentrations are affected by the length of time since the patient has eaten and by the type of sample collected from the patient. To accurately determine glucose results, samples should be obtained from a patient who has been fasting for at least 12 hours. The glucose concentration decreases approximately 5- 12 mg/dL in 1 hour in uncentrifuged samples stored at room temperature. Total bilirubin results may be adversely affected by photodegradation. Whole blood samples not run immediately should be stored in the dark for no longer than 60 minutes. If the sample cannot be analysed within that period, it should be separated into plasma or serum and stored in a capped sample tube in the dark at low temperatures. Use only lithium heparin evacuated specimen collection tubes for whole blood or plasma samples. Start the test within 10 minutes of transferring the sample into the reagent disc. 【Procedure】

Materials Required General Health Profile Rotor; InSight V-CHEM chemistry analyser; Transfer pipette (100 μL fixed volume) and tip. Test Procedure The complete sample collection and step-by-step operating procedures are detailed in the InSight V-CHEM chemistry analyser Operator’s Manual. Calibration The InSight V-CHEM chemistry analyser is calibrated by the manufacturer before shipment. The unique code printed on the sealing pouch provides the analyser with disc-specific calibration data. See the InSight V-CHEM chemistry analyser Operator’s Manual. Quality Control See Operator’s Manual of the InSight V-CHEM chemistry analyser. Performance of the InSight V- CHEM chemistry analyser can be verified by running controls. For a list of approved quality control materials with acceptance ranges, please contact Woodley Equipment or your local distributor for technical support. If control results are out of range, repeat. If still out of range, contact Woodley Equipment. Do not report patient results if controls are outside their labelled limits. Results The InSight V-CHEM chemistry analyser automatically calculates and prints the analyte concentrations in the sample. Details of the endpoint and rate reaction calculations are found in the InSight V-CHEM chemistry analyser Operator’s Manual. 【Interpretation of Results】 Physiological interferents (haemolysis, icterus and lipaemia) cause changes in the reported concentrations of some analytes. The sample indices are printed on the bottom of each printout to inform the operator about the abnormal sample. The operator should avoid the haemolysis phenomenon caused by irregular blood collection. The InSight V-CHEM chemistry analyser suppresses any results that are affected by >10% interference from haemolysis, lipaemia or icterus. “HEM”, “LIP”, or “ICT” respectively, is printed on the printout in place of the result. Any result for a particular test that exceeds the assay range should be analysed by another approved test method or sent to a referral laboratory. Do not dilute the sample and run it again on the InSight V- CHEM chemistry analyser. 【Manufacturer】 Company name: Woodley Equipment Company Ltd. Address: Old Station Park Buildings, St. John Street, Horwich, Bolton, Lancashire, BL6 7NY, UK Tel: +44 (0) 1204 669033 Fax: +44 (0) 1204 669034 Website: www.woodleyequipment.com