Clinical Chemistry Lab Practical Procedure Info

Pipetting, Accuracy, & Precision: Principle

To calibrate a pipette while checking its accuracy and precision

Pipetting, Accuracy, & Precision: Enzymes/Chemicals

Distilled water & EDTA whole blood

Pipetting, Accuracy, & Precision: Chemical reaction

No chemical reaction, simply the displacement of air to aspirate and dispense a liquid

Pipetting, Accuracy, & Precision: Signal Measured

The mass/weight measured in grams via a balance.

Pipetting, Accuracy, & Precision: Clinical Utality

Quality control

Pipetting, Accuracy, & Precision: Reference Range

Accuracy should be 99-101% and CV less than 1%

Pipetting, Accuracy, & Precision: Equation Accureacy

%Error=[ (measured value - True Values) / (True Value) ] X 100

Pipetting, Accuracy, & Precision: Equation CV

precision = [ (standard Deviation) / (Mean) ] X 100

Glucose oxidase method: Principle

Glucose oxidase to gluconic acid + H2O2 + chromogen forms color (via peroxidase)

Glucose oxidase method: Enzymes/Chemical

Glucose oxidase, peroxidase, chromogen

Glucose oxidase method: Signal Measured

Color change (abs 500nm)

Glucose oxidase method: Clinical utility

Diagnosis/monitoring of diabetes, hypoglycemia, hyperglycemia

Glucose oxidase method: Reference range

70-99 mg/dL

Glucose oxidase method: Equation

(abs sample / abs standard) x Con. Stand

Glucose hexokinase Method: Principle

Glucose phosphorylated by hexokinase; G6P oxidized by G6PD + NADP+ -> NADPH

Glucose hexokinase Method: Enzymes/Chemicals

Hexokinase, G6PD, NADP+

Glucose hexokinase Method: Signal Measured

NADPH production (abs 340nm)

Glucose hexokinase Method: Clinical utility

Accurate measurement of blood glucose

Glucose hexokinase Method: Reference range

70-99 mg/dL

Glucose hexokinase Method: Equations

[(Sample abs - Blank abs) / (standard abs - Blank abs)] x Conc. Stnd

Total Protein (Biuret method): Principle

Proteins react with Cu^2+ ions in alkaline solution -> violet complex

Total Protein (Biuret method): Enzymes/Chemicals

Copper sulfate, NaOH

Total Protein (Biuret method): Signal Measured

Violet color (abs 540nm)

Total Protein (Biuret method): Clinical utility

assess nutritional status, liver/ kidney disease

Total Protein (Biuret method): Reference range

6.4-8.3 g/dL

Total Protein (Biuret method): Equations

(abs sample / abs standard) x Con. Stand

Total Protein & Albumin Calculated Globulin Equation

= TP - Alb

Total Protein & Albumin A/G Ratio Equation

= (Alb / globulin) = (Alb / TP - Alb)

Albumin (dye-binding method): Principle

Albumin binds dye (e.g., BCG or BCP) -> color complex

Albumin (dye-binding method): Enzymes/Chemicals

Bromocresol green (BCG) or purple (BCP)

Albumin (dye-binding method): Signal Measured

Color change (absorbance 628 nm)

Albumin (dye-binding method): Clinical utility

Assess liver function, nutritional status

Albumin (dye-binding method): Reference range

3.5-5.0 g/dL

Albumin (dye-binding method): Equations

(abs sample / abs standard) x Con. Stand

Total Cholesterol: Principle

Cholesterol esters hydrolyzed -> free cholesterol -> oxidized -> H2O2 + chromogen = color

Total Cholesterol: Enzymes/Chemicals

Cholesterol esterase, cholesterol oxidase, peroxidase

Total Cholesterol: Signal Measured

Color change (absorbance 500 nm)

Total Cholesterol: Clinical utility

Assess risk of cardiovascular disease

Total Cholesterol: Reference range

< 200 mg/dL

Total Cholesterol: Equation Total Cholesterol

= (abs sample/ abs standard) x Conc. Stnd

Total Cholesterol: Equation HDL

= (abs sample/ abs standard) x Conc. Stnd x 2

Total Cholesterol: Equation LDL

= (Ttl. Chol) - (HDL) - (Trigs /5)

HDL Cholesterol: Principle

PEG w/ serum -> LDL/VLDL + HDL supernatant -> colored quinoneimine complex

HDL Cholesterol: Enzymes/Chemicals

Polyethylene Glycol, Cholesterol Esterase, Cholesterol oxidase, Peroxidase, 4-aminoantipyrine

HDL Cholesterol: Signal Measured

500nm absorbance wavelength via spectrophotometry

HDL Cholesterol: Clinical utility

Cardiovascular risk assessment. Calculating LDL

HDL Cholesterol: Reference range

=/> 60

HDL Cholesterol: Equation Unknown conc.

= (Abs unknown / abs std) x Conc. std x dilution factor

Triglycerides: Principle

Triglycerides hydrolyzed -> glycerol -> phosphorylated and oxidized -> H2O2 + chromogen

Triglycerides: Enzymes/Chemicals

Lipase, glycerol kinase, glycerol phosphate oxidase, peroxidase

Triglycerides: Signal Measured

Color change (absorbance 500 nm)

Triglycerides: Clinical utility

Assess lipid metabolism and CVD risk

Triglycerides: Reference range

Triglycerides: Equation

= (abs sample/ abs standard) x Conc. Stnd

BUN: Principle

Urea hydrolyzed by urease -> ammonia -> reacts with indicator -> color

BUN: Enzymes/Chemicals

Urease, glutamate dehydrogenase or Berthelot reagents

BUN: Signal Measured

Color change or NADH disappearance (absorbance ~340 nm)

BUN: Clinical utility

Evaluate kidney function

BUN: Reference range

7-20 mg/dL

BUN: Equation

[(A1 unknown - A2 unknown)/ (A1 Stnd - A2 Stnd)] x Con. BUN Stnd

BUN: Equation BUN:Creatinine

= (creatinine):(BUN) -> fraction to divide for whole number

BUN: Equation BUN:Creatinine Range

10:1 to 20:1

Creatinine (Kinetic Jaffe Method): Principle

Creatinine + Picric acid —alkali—> Creatinine-picrate complex (yellow-orange)

Creatinine (Kinetic Jaffe Method): Enzymes/Chemicals

Picric Acid, Sodium Hydroxide (alkali), surfactant

Creatinine (Kinetic Jaffe Method): Signal Measured

rate of absorbance change at 510nm.

Creatinine (Kinetic Jaffe Method): Clinical utility

Renal function. Indicator of glomerular filtration

Creatinine (Kinetic Jaffe Method): Reference range

0.40 - 1.40 mg/dL

Creatinine (Kinetic Jaffe Method): Equations

=[(A2 test - A1 test)/ (A2 Stnd - A1 Stnd)] x Con. Stnd

Serum Iron: Principle Serum Iron

Fe3+ —acid pH—-> Fe2+ + Ferrozine —> Violet complex

UIBC: Principle

Serum + excess Fe2+ —alkali pH—> unbound Fe2+ + Ferrozine —> Violet complex

TIBC: Principle

Transferrin Saturation: Principle

Serum Iron, UIBC, TIBC, Transferrin Saturation: Enzymes/Chemicals

Hydroxylamine hydrochloride, ferrozine, acetate buffer, tris buffer

Serum Iron, UIBC, TIBC, Transferrin Saturation: Signal Measured

Absorbance of violet ferrozine complex at 560nm

Serum Iron, UIBC, TIBC, Transferrin Saturation: Clinical utility

Diagnosis of Iron-deficiency anemia, and hemochromatosis.

Serum Iron: Reference range

60-150 ug/dL

TIBC: Reference range

250-400 ug/dL

Transferrin Saturation: Reference range

20-55%

Serum Iron: Equation

= [(A2 test - A1 test)/ (A2 Stnd - A1 Stnd)] x Con. Iron Stnd

TIBC: Equation

Serum Iron (ug/dL) + UBIC (ug/dL)

TIBC: Conversion

ug/dL x 0.179 = umol/L

UIBC: Equation

= [(A2 test - A1 test)/ (A2 Stnd - A1 Stnd)] x Con. Iron Stnd

Transferrin Saturation: Equation

= (Serum Fe / TIBC) x 100