Test Glucose Determination Enzymatic-Colorimetric Glucose Oxidase-PAP Method (Trinder Reaction)

Reagent Composition - Glucose oxidase 15IU/mL - Peroxidase (horseradish)1.2IU/mL - Mutarotase 2IU/mL - 4-aminophenazone 0.2mM - Phenol 4mM - Phosphate buffer (pH7.5) - 0.1M non-reactive ingredients and preservatives - Glucose standard 100 mg/dL in aqueous solution Cholesterol reagent: - Cholesterol esterase 150 U/L - Cholesterol oxidase 150 U/L - Peroxidase 2500 U/L - 4-aminoantipyrene 0.3mM - Phenol 15mM - Phosphate buffer pH 6.8 - non reactive stabilizers and preservatives - Cholesterol standard: 200 mg/dL

Specimen - serum

Specimen Requirements - must be free from hemolysis - must be separated from clot promptly - stable for 24 hrs. when stored at 2-8C

Assay Conditions Wavelength: 500 520 nm Optical path: 1 cm Temp: 37C

Linearity 500 mg/dL glucose

Sensitivity Absorbance change of 0.001 at 500 nm = 0.5 mg/dL

Expected Values 75 115 mg/dL or 4.2 6.4 mmol/L

Total Cholesterol Determination Enzymatic Colorimetric Cholesterol Oxidase PAP Method (Trinder Reaction)

nonhemolyze d serum

- Cholesterol in serum is reported stable for seven days at room temperature (18-25 C) and six months when frozen and protected against evaporation - Stable for 3 days at 2-30C - Heparinized plasma should not be used - Gentisic acid affects the enzymatic cholesterol - should be collected following a 24hr fast and separated from the clot ASAP - avoid anticoagulants containing fluoride or oxalate - may be stored for 1 week at 28 C or for 3 months at -20C

W: 500 520 nm O: 1 cm T: 37C

700 mg/dL total cholesterol

Absorbance change of 0.001 at 500 nm = 0.5 mg/dL

Desirable: <200 mg/dL Borderline High: 200 239 mg/dL High: >240 mg/dL

HDL Cholesterol Determination

- 200 g/L polyethylene glycol buffered at pH 10 - Cholesterol (enzyme) reagent - Cholesterol standard 50 mg/dL ( store at 15-30 C should be clear, colorless)

Unhemolyzed serum

W: 520 nm Reaction type: endpoint Reaction: 10 min at 37C Stability of final reaction n prod: w/in 30 min

120 mg/dL

0.8 mg/dL per 0.001 absorbance units

Males: >55 mg/dL Females: > 65 mg/dL

Triglyceride Determination

- good buffer pH 7.0, 50 nM - ATP 0.5 mM - Magnessium salt 12 mM - 4-aminophenazone 0.33 mM - Glycerol kinase (microbial) >250 U/L - Glycerol phosphate oxidase (microbial) >4500 U/L - Peroxidase (horseradish) >2000 U/L - Lipase (microbial) >200,000 U/L - surfactants - stabilizers - preservatives - Sodium azide 0.1% (Store reagent at 2-8, protect from direct light, avoid microbial contamination) - Triglycerides standard 200 mg/dL -

- fresh, clear, unhemolyzed serum

W: 500 nm AT: endpoint SR: 1:101 T: 37 C IncTime: 5 min

1000 mg/dL

1mg/dL=0.001 abs

36-165 mg/dL

BUN Determination (Kinetic: Urease-Glutamate Dehydrogenase Reaction)

- NADH 0.28 mM/L - Urease 3,000 U/L - Glutamate dehydrogenase 15,000 U/L - 2-oxoglutarate 4.0 mM/L - buffer at pH 2.0 - Urea nitrogen standard 20 mg/dL (must be stored at 2-8C prior to reconstitution; after reconstitution, the reagent is stable for 2 days at RT (18-25C) and for 21 days at 2-8C; should be clear and colorless)

serum

Creatinine Determination (Colorimetric Method Jaffe Reaction)

Uric Acid Determination (Colorimetric: Uricase-Trinder Reaction)

- Picric acid 3.3 mM (should be clear and yellow; orange red color -> deterioration) - Base reagent: 0.17 M Sodium Hydroxide, 26mM Sodium Tetraborate, surfactant (must be clear, colorless) - Direct Creatinine Calibrator: Creatinine hydrochloride equiv. to 6.0 mg/dL serum creatinine (clear and colorless; turbidity-> deterioration) ( store at 15-30 C) Uric acid reagent: - 4-aminophenazone 4 mM - 2, 5-dichloro-2-hydroxybenzene sulfonic acid 2mM - stabilizer and surfactant - uric acid (microbial) >150 U/L - Peroxidase (horseradish) 10,000 U/L - Buffer pH 7.5 Uric acid standard (5mg/dL) (stored refrigerated at 2-8C -> stable for 30 days) - Sodium Hydroxide: 600 mmol/L - Potassium Sodium Tartrate: 32 mmol/L - Copper Sulfate: 12 mmol/L - Potassium Iodide: 30 mmol/L - Protein Standard: 5 g/dL - Citrate buffer(pH 4.2): 7.5 mmol/L - Bromcresol green: 0.25 mmol/L - Sodium azide: 0.05% - Albumin Standard: 5 g/dL (stored at 2-8 C; clear yellow/green)

Serum (unhemolyzed )

Serum

- Must be free from hemolysis anticoagulated plasma must not be used - all materials must be free from ammonia and heavy metals - refrigerated serum (2-8 C): stable for 72 hrs unrefrigerated: w/in 8 hrs - 24-hr urine must be preserved with 15g Boric acid - stable for one week at 2-8C or 3 months at frozen temp - protect from direct sunlight - must be unhemolyzed - stable for 3 days at 2-8C and up to 6 months if frozen

W: 340 nm A: enzymatic, kinetic SR: 1:101 T: 37 C Prewarm time: 3 min

80 mg/dL

1.65 8.13 mg/dL

W: 510 nm Filter selection: 490-530 nm R: endpoint IT: 37C ITime: 15 min Sample vol: 0.1 mL Picric acid: 1.5m L Base rgt: 1.5 mL

20 mg/dL

0.025mg/dL per 0.001 abs

Serum creatinine: 0.5-1.7 mg/dL Urine creatinine: 0.61.6 g/24 h Creatinine clearance - Males: 105+/- 20 mL/min - Females: 95 +/- 20 mL/min Males: 3.4-7.0 mg/dL Females: 2.4-5.7 mg/dL

Up to 25 mg/dL

0.001 abs = 0.03 mg/dL

Total Protein Determination (Colorimetric Method-Biuret Reaction)

Serum or heparinized or EDTA plasma

- stable for 1 month at 2-8C or 1 week at 37C Stable for 1 month at 2-8 C or 1 week at 15-25 C

Serum Albumin Determination (Colorimetric Method-Bromcresol Green Dye Binding Reaction)

Serum , heparinized or EDTA plasmas

W:540 nm O: 1 cm T:20 -25 C Measurement against rgt blank W: 630 nm O: 1 cm T: 20-25 C Measurement against rgt blank

12g/dL

Newborn: 4.6-7.0 g/dL Children/adults: 6.68.7 g/dL 3.8-5.1 g/dL

7 g/dL