Final Practical

Principle of Total Cholesterol Test
Enzymatic method using cholesterol esterase to cleave the cholesterol esters in order to free cholesterol and fatty acids. Cholesterol oxidase then oxidizes the 3-OH group of the free cholesterol plus any performed cholesterol to form hydrogen peroxide. Hydrogen peroxide is measured in a reaction catalyzed by peroxidase for form a colored dye, which can be measured at 500nm. The intensity of the red color is directly proportional to the total cholesterol concentration.
Critical steps in the Total Cholesterol Test
The critical steps in the procedure involve measuring 1.0 mL of correct reagent (cholesterol reagent) into each tube and then adding 0.01 mL of appropriate liquid to the tube. The tubes have to mixed and incubated in a heating block for 5 minutes. The intensity of the color is then measured at 500nm within 60 minutes at the end of incubation time.
Sources of Error in the total cholesterol test
The sources of error involve grossly hemolyzed and icteric specimens, and addition of fluoride or oxalate to the tubes as an anticoagulant.
Cholesterol analysis procedure
Label 5 tubes (B, S, C1, C2 and P) and then add 1.0 mL (1000 µL, 1 0 0 0 on the P-1000) of cholesterol reagent into each tube. Then add 0.01 mL (10 µL, 0 0 1 0 on the P-20) of appropriate fluid into each tube. Cover, mix and incubate for 5 minutes at 37°C. Blank the spectrophotometer at 500 nm, then read and record all absorbances within 60 minutes after incubation time. Determine cholesterol concentration via beers law (Cu = Au/As x Cs).
Why should samples with total cholesterol levels greater than 750 mg/dL be diluted and re-assayed?
Samples with total cholesterol greater than 750 mg/dL should be diluted and re-assayed because 750 mg/dL is the limit of linearity for this procedure. The procedure is not longer accurate at a concentration above 750 mg/dL.
Explain the process of diluting and re-assaying
The sample should be diluted with 1 part sample 2 parts isotonic saline. After dilution, the procedure should be repeated with the new diluted sample. After the concentration has been calculated, the results would have to be multiplied by the dilution factor (3) in order to obtain the correct results for release to the doctor.
Which serum lipoprotein fraction carries most of the cholesterol?
The low density lipoprotein fraction carries most of the cholesterol (42%)
What percentage of cholesterol in the blood is esterified?
Cholesteryl esters, which are byproducts of cholesterol esterification by LCAT, make up 70% of the total cholesterol in the plasma.
List 3 clinical conditions associated with elevated cholesterol levels.
Three conditions associated with elevated cholesterol levels are Coronary Heart Disease, Stroke, and Type 2 Diabetes.
The Freidewald formula for determining the LDL cholesterol level, incorporating the total cholesterol value.
LDL = Total Cholesterol – HDL – (Triglycerides/5)
Describe proper specimen collection and handling for total cholesterol measurement.
The proper specimen is plasma or serum a purple top EDTA tube following a 12 hour fast. The specimens should be stored for up to 4 days at 2-8°C, if frozen at -20°C can be held for 3 months
How are lipoprotein fractions classified
relative amounts of lipids and proteins.
Chylomicrons
least dense of tall the lipoproteins. Their core lipids are 86% triglycerides and they transport exogenous triglycerides to the tissues
VLDLs
slightly denser than chylomicrons. They also contain mostly triglycerides, but higher ratios of the other core lipids esters than chylomicrons. They aid in transporting endogenous triglycerides and cholesterol in the liver to the tissues.
IDL
short-lived lipoproteins which are undetectable in blood. They transport endogenous triglycerides and cholesteryl esters.
LDL
known as the bad cholesterol because they are made of 42% cholesterol. They transport cholesteryl esters to the cells.
HDL
known as the good cholesterol because they remove cholesterol from the tissues by transporting it to the liver for excretion. They are half lipids and half lipoprotein
Describe the clinical significance of HDL cholesterol measurements.
The clinical significance of HDL cholesterol measurements is to determine the HDL concentration in the plasma or serum. HDL is measured as a way of monitoring risk for coronary heart disease.
Two emerging methods for the separation of lipoproteins
NMR spectroscopy and density gradient ultracentrifugation
NMR spectroscopy for the separation of lipoproteins
NMR spectroscopy detects lipoprotein-associated fatty acyl methyl and Methylene groups. The signals on the NMR spectrometer from each sub fraction are resolved mathematically. The advantage to NMR is that it only takes a few minutes to analyze the samples, and it only requires 0.5 mL of serum. The disadvantages are that is required fresh samples, specialized equipment, and expertise which correlates to more money spent on this test.
density gradient ultracentrifugation for the separation of lipoproteins
Density gradient ultracentrifugation measures cholesterol continuously in the fractions eluted from the gradient. The advantages are that density gradient ultracentrifugation can measure HDL, LDL, VLDL, IDL, and Lp (a), while NMR can only measure HDL, LDL and VLDL. The disadvantage to this technique is that it requires training, expertise and special equipment.
Triglyceride 271 mg/dL
Total Cholesterol 228 mg/dL
HDL Cholesterol 35 mg/dL
LDL = total cholesterol – HDL – (triglycerides/5)
LDL = 228 mg/dL – 35 mg/dL – ((271 mg/dL)/5)
LDL = 228-35-54.2
LDL= 138.3 mg/dL
The principle of the test for HDL cholesterol
The HDL cholesterol reagent is to quantify the amount of cholesterol in the HDL fraction of the serum or plasma. Non-HDL lipoproteins are precipitated and removed by magnesium chloride/dextran sulfate (precipitating reagent). After centrifugation the HDL remains in the supernatant. The enzymatic method, as described in the cholesterol analysis is then used to determine the concentration of cholesterol in non-HDL fraction remaining after centrifugation and precipitation.
Describe specimen collection and storage procedures for HDL.
Serum or EDTA plasma should be obtained after a 12 hour fast. It should be stored at 2-8°C if not promptly analyzed. It can be stored for 4 days at 2-8°C and 7-14 days at -20°C.
List sources of error for HDL analysis method
The sources of error for this method are the quality of sedimentation, in that incomplete precipitation and sedimentation may occur. Also freezing the samples may increase cholesterol values.
HDL Cholesterol Analysis procedure
Label three tubes (C1, C2 and P). Pipette 0.5 mL (500 µL, 0 5 0 0 on P-1000) of sample into respective tubes. Add 0.5 mL (50 µL, 0 0 5 0 on P-200) of HDL precipitating reagent into each tube. Cover, vortex, and allow to stand at room temperature for 5 minutes. Vortex again and centrifuge for 10 minutes at 1000 x g)

Label five tubes (B, S, C1, C2 and P). Pipette 1 mL (1000 µL, 1 0 0 0 on P-1000) of cholesterol reagent into each tube. Add 0.0025 mL (25 µL, 0 2 5 on P-200) of respective fluid into each tube. Add supernatant from centrifuged tubes to P, C1 and C2. Cover, mix and incubate for 5 minutes at 37°C. Read and record absorbances at 500nm within 60 minutes of incubation time.