Final Practical

Magnesium and Calcium Assays Body system
BUN/Creatinine Assays Body system
Bilirubin Assay Body system
Amylase Assay Body system
When working with Beer’s law, what is the nanometer used to measure?
The nanometer is used to measure the wavelength of light of absorbance for the assay.
Bilirubin Principle
Classic diazo reaction of Ehrilich to determine bilirubin level in serum
Bilirubin reagents
diazotized sulfanilic acid (p-benzenediazonium) which couples with direct and indirect bilirubin to produce a color reaction
Bilirubin reactions
Sulfanilic acid in dilute HCl + Sodium Nitrite –> nitrous acid

Nitrous acid is basis for diazotized sulfanilic acid (p-benzenediazonium sulfonate)

Direct Bilirubin + diazotized sulfanilic acid at alkaline pH -> Blue
Indirect Bilirubin + diazotized sulfanilic acid + accelerating agent
–> Blue

Creatinine principle
Jaffe reaction, red-orange color forms when metabolite is treated with alkaline picrate.
Creatinine reagents
Picric acid
Creatinine reaction
Creatinine + Picric acid –> Creatinine-Picrate complex (red-orange)
Total protein principle
: Biuret method depends on the presence of peptide bonds, found in all proteins
Total protein reagents
Cupric acid
Total protein reaction
Cupric ions (CU2+) in alkaline solution + peptide bonds –> colored complex
Amylase principle
Wallenfels modification, using PNPG7 with terminal glucose blocked to reduce spontaneous degradation by reagents
Amylase reagents
Glucosidase, Glycoamylase, and p-Nitrophenyl D-maltoheptaosde (PNPG7)
Amylase reaction
PNPG7 –Amylase> PNPG3
PNPG3—glucoamylase> PNPG1
PNPG1 –glucosidase> glucose + p-nitrophenol (yellow)
Calcium principle
O-cresolphthalein complexone to complex with calcium to form an intense chromophore
Calcium reaction
Calcium reagents
CPC + calcium in alkaline solution –> chromophore
HDL principle
Quantitative determination of cholesterol concentration in HDL fraction of serum or plasma
HDL reagents
Magnesium chloride/dextran sulfate, enzymes
HDL reactions
HDL cholesterol + enzymes –>color
Total cholesterol principle
enzymatic methods
Total cholesterol reagents
cholesterol esterase, cholesterol oxidase, peroxidase
Total cholesterol reaction
Cholesterol Esters + H2O –cholesterol esterase> Cholesterol + Fatty Acids

Cholesterol + O2 –Cholesterol oxidase> Cholest-4-en-3-one + H2O2

H2O2 + 4-Aminophenazone + phenol –peroxidase> Quinoneimine Dye + 2H2O

Forms of calcium
There are three forms of calcium (1) free (ionized) 50% (2) bound to plasma proteins 40% and (3) complexed with small anions 10%.
The origin and breakdown of bilirubin including the enzyme involved
When macrophages remove and phagocytized abnormal red cells, the heme ring is opened and heme is metabolized into biliviridin by heme oxygenase. Biliviridin is reduced by bilirubin reductase to bilirubin. Bilirubin leaves the macrophage and enters the blood stream where it is bound to albumin (indirect bilirubin). Bilirubin in conjugated to glucaronic acid in the hepatocytes by UDP glucaronic transferase and then enters the plasma in small amounts (direct bilirubin).
The regulation of calcium metabolism
Calcium is regulated by parathyroid hormone (PTH) which decreases excretion, calcitonin (CT) which increases excretion and Vitamin D (1, 25 (OH) 2D) which decreases excretion. High calcium levels are absorbed into the bone, and low calcium levels activate PTH which activates conversion of vitamin D into active form. Calcitonin is produced in response to high calcium levels.
The regulation of phosphorus metabolism
Phosphorus is regulated by PTH which decreases resorption in the proximal tubule and increases 1,25 D which stimulated intestinal absorption of phosphate.
Creatinine clearance us used to measure GFR
CrCl= (Urine Volume (ml/min)x UCr)/PCr
Alpha2- globulin
Relationship between the net charge of the proteins in a sample and their electrophorectic mobility of protein fractions.
Rate of electrophorectic mobility is directly proportional to net charge of the proteins, if the protein is very negative it will move faster toward the positive side. Serum proteins carry a negative charge and move toward the anode (+).
The measurement of the intensity of light at selected wavelengths
A physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary whereas the other moves in a definite direction.
An instrumental method for measuring the absorbance, reflectance, or fluorescence of each separated fraction on an electrophorectic strip as it is moved past a measuring optical system.
A technique that uses a nephelometer to measure the number and size of particles in a suspension; a detector is placed at an angle to the incident light beam to measure the intensity of the light that is scattered by the particles.
Amperometric measurement
an electrochemical process where current is measures at a fixed potential difference between the working and reference electrodes in an electrochemical cell
Acute Cirrhosis
decreased albumin, decreased alpha 2, beta Gamma Bridge
Chronic Cirrhosis
decreased albumin, decreased alpha 2, decreased beta
Acute Inflammation
decreased or normal albumin, increased alpha 1, increased alpha 2, increased beta, and decreased or normal Gamma
Nephrotic syndrome
decreased albumin, increased alpha 2, increased beta
Monoclonal Gammopathy
decreased albumin, really increased gamma
Polyclonal Gammopathy
decreased albumin, increased gamma
What effect would an air bubble have on a blood gas measurement
An air bubble would be air exposure which would cause an increase in pH and pO2 and a decrease in pCO2.
Henderson-Hasselbalch equation
pH=pKa+log?(( [A-])/([HA])?)
Henderson-Hasselbalch equation for blood gases
pH=pKa+log( (HCO3-)/(0.031 x pCO2))
Specimen collection for blood gas determination
Whole blood mixed well in syringe containing heparin which is usually collected from hand or arterial line. Venous blood is okay if O2 assessment is not essential. In order to maintain anaerobic conditions air bubbles must be expelled, specimen must be tested with 30 minutes because room air and WBCs will utilize oxygen content.
Calculate a specimen that has been diluted
Solution is too concentrated- dilute one part sample to one part saline (1:2) – multiply results by 2 to compensate for dilution
Specimen interferences Total protein
Gross hemolysis and lipemia
Specimen interferences HDL cholesterol
freezing increases cholesterol values
Specimen interferences Total cholesterol
– bilirubin > 10 mg/dL, gross hemolysis and icteric specimens, fluoride and oxalate anticoagulants
Specimen interferences BUN
anticoagulant with ammonium salts, ammonia contamination of glassware/water, fluorides > 5mg/dL, drugs and metabolites
Specimen interferences Crea
– bilirubin, drugs and substances
Specimen interferences Calcium
calcium contaminated glassware, high bilirubin or hemoglobin, ethanol, organic solvents
Specimen interferences Magnesium
EDTA, glass magnesium contamination, hemolysis
Specimen interferences bilirubin
gross hemolysis, drugs, turbid/lipemic serum
Specimen interferences amylase
drugs and substances

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