Laboratory #
Lipid Studies
Laboratory #: Lipid Studies
Skills= 20
Objectives:
Upon completion of this exercise, the student will be able to:
1. Define hyperlipoproteinemias.
2. Identify and state the purpose of the reagents used in the procedures.
3. State reference ranges for lipids of clinical significance.
4. Perform cholesterol, triglyceride, and LDL procedures as instructed.
5. Calculate and interpret HDL.
6. Determine acceptability of the run by analyzing and evaluating control results.
Materials:
1. Clinical Chemistry analyzer (For on-site ACC Students- the Alera)
2. Pipets
3. Reagents
4. Sample cups
5. Sample seglets
6. QC materials (Level 1 and Level 2)
7. Lecture notes:
References:
Alera Package inserts
Purpose:
Lipids (cholesterol and triglyceride) circulate in the blood associated with proteins in the
form of water-soluble complexes termed lipoproteins. Sufficient elevation in the
concentration of any lipoprotein can result in hyperlipoproteinemia, a metabolic disorder
which may be inborn or due to a number of acquired conditions including endocrine
disorders, specific organ failures, or external causes.
Hyperlipoproteinemias are not disease entities, but are groups of disorders that affect
plasma lipid and lipoprotein concentrations. Disorders which are know n to cause
hyperlipoproteinemia include diabetes mellitus, nephrosis, and biliary obstruction.
Elevated levels of plasma triglycerides and cholesterol are also associated with risk
factors related to atherosclerosis.
Procedures:
Cholesterol
Purpose:
Cholesterol originates from both endogenous and exogenous sources. It is the basic
component from which steroid hormones are produced. About two-thirds of serum
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MLAB 2401
Laboratory #
Lipid Studies
cholesterol is esterified. Serum cholesterol is an important aid in the diagnosis of
hyperlipoproteinemias. Elevated levels of cholesterol are often predictive of an
atherosclerotic process. The process can be monitored by observing changes in the
serum cholesterol levels. Elevated levels may also be observed with hepatic,
hypothyroid, and certain nephritic disorders.
Principle:
The Alera Cholesterol assay measures the sum of both free cholesterol and cholesterol
acyl esters by enzymatic methods.
In this assay, cholesterol esters in serum are completely hydrolyzed by cholesterol
esterase to free cholesterol and free fatty acids. The cholesterol liberated by the
esterase, plus any endogenous free cholesterol, are both oxidized by cholesterol
oxidase to yield hydrogen peroxide. The hydrogen peroxide than acts to oxidatively
couple p-hydroxybenzoic acid and 4- amin-oantipyrine in a reaction catalyzed by
peridase, producing a red colored complex which absorbs strongly at 505 nm. The
amount of chromogen formed, determined by measuring the increase in absorbance
bichromatically at 505nm/647 nm, is directly proportional to the cholesterol
concentration.
This procedure is linear from 4- 600 mg/dL.
Specimen:
Blood should be collected following a 12-hour fast. Non-hemolyzed serum is preferred.
Serum should be separated from the cells within 2 hours of collection.
Cholesterol is stable for 5-7 days at 4 oC.
Reference Range:
Risk Classification
Desirable
Borderline
High
Units (mg/dL)
<200
200-239
>240
Units (mmol/L)
<5.18
5.18-6.19
>6.22
Triglycerides
Purpose:
Triglycerides are the predominant form of glycerol ester found in plasma. In the
circulatory system, they are transported with other lipophilic components in complexes
called lipoproteins and chylomicrons. Triglyceride assays are used to diagnose genetic
and metabolic disorders, as well as in risk assessment for atherosclerosis.
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MLAB 2401
Laboratory #
Lipid Studies
Principle:
The Alera triglyceride assay is a modification of the Fossati and Prencipe enzymatic
method. This method uses glycerol phosphate oxidase and a modified Trinder reaction.
Triglycerides in the serum are hydrolyzed by lipase to form glycerol and free fatty acids.
In the presence of ATP and glycerol kinase, the glycerol is converted to glycerol-1phosphate and the ATP to ADP. The glycerol-1-phosphate is oxidized by glycerol
phosphate oxidase to yield hydrogen peroxide. The hydrogen peroxide then acts to
oxidately couple p-chlorophenol and 4-aminoantipyrine in a reaction catalyzed by
peroxidase, producing a red colored complex which absorbs strongly at 505 nm. The
amount of chromogen formed, determined by measuring the increase in absorbance
bichromatically at 505 nm/692 nm, is directly proportional to the triglyceride
concentration.
This procedure is linear from 8- 1000 mg/dL.
Specimen:
Blood should be collected following a 12-hour fast. Non-hemolyzed serum is preferred.
Serum should be separated from the cells within 2 hours of collection. Blood collection
tubes, including stoppers must be free of glycerol contamination.
Triglyceride is stable for 5-7 days at 4 oC.
Reference Range:
Risk Classification
Normal
Borderline
High
Units (mg/dL)
<150
150-199
200-499
Units (mmol/L)
<1.70
1.7-2.2
2.3-5.6
LDL-C
Purpose:
60-70 %Of the total plasma cholesterol is carried by the LDL cholesterol lipoprotein
fraction. There is a strong association between increased LDL cholesterol in serum and
the incidence of coronary artery disease. LDL is elevated in primary and secondary
hyperlipoproteinemia.
Principle:
In the Alera LDL-C procedure, detergents in the LDL buffer solubilize only the non-LDL
lipoprotein particles in the serum. The cholesterol released is consumed by cholesterol
esterase and cholesterol oxidase in a non-color fprming reaction. A second detergent
present in the LDL color reagent solubilizes the remaining LDL particles and a
chromogenic coupler allows for color formation. The amount of color formed,
determined by measuring the increase in absorbance bichromatically at 544/692 nm is
directly proportional to the LDL cholesterol concentration.
The procedure is linear from 3- 800 mg/dL.
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MLAB 2401
Laboratory #
Lipid Studies
Specimen:
Non-hemolyzed serum is preferred. Serum should be separated from the cells within 2
hours of collection.
LDL cholesterol is stable for 5-7 days at 4 oC.
Reference Range:
Risk Classification
Desirable
Borderline
High
Units (mg/dL)
<130
130-159
>160
Units (mmol/L)
<3.37
3.37-4.12
>4.14
Quality Control:
Two levels of controls should be run every day of patient testing, prior to reporting any
patient results. In addition, controls are also run when new bottles of reagent are
loaded, when recalibration is performed, or when major instrument maintenance is
performed.
Procedure:
1. The instructor will deliver a specimen and requisition to each student.
2. An appointed group will perform daily maintenance and quality control on the
Alera.
3. Each student will log the daily QC onto his/her report sheet.
4. Each student will program his/her sample demographics into the Alera, then load
the sample on the instrument.
5. Following analysis, each student will review the printout, looking for critical
values, and flags.
6. If no follow-up is needed, the student will attach the printout with his/her name
and date to the report sheet and turn into instructor.
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MLAB 2401
Laboratory #
Lipid Studies
Student Name:______________
Date:____________________
Laboratory: Lipid Studies
Report Form
Skills= 20
1. Log the lot number, expiration date, control range, and your results from running
the QC in the table. Indicate whether you are “in control”. (10 points)
Lot/Exp
Date
Level 1:
Test
Quality Control
Alera Results
Controls’ Range of
Expected Results
In
control?
Yes/No
Chol
Trig
LDL
Level 2:
Chol
Trig
LDL
**Note: include units
2. Based on the above information, can you run your patient? If not, why? ( 1 point)
3. Attach your patient printout to your report form. Make sure your name, and
today’s date are written on the printout. ( 5 points)
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MLAB 2401
Laboratory #
Lipid Studies
Laboratory #: Lipid Studies
Study Questions
Points= 11
Student Name:_______________
Date:_______________________
Unless otherwise stated, each question is worth one (1) point.
1. What is the specimen of choice for lipid studies?
2. Briefly define hyperlipoproteinemias.
3. Triglycerides are hydrolyzed by the enzyme ______________.
4. During your run of QC this morning, the LDL was deemed out of control and
service has been called to evaluate the issue. In the meantime, one of the
physicians wants you to calculate the LDL’s on his patients by the Friedewald
equation. Using your lecture notes, state the Friedewald equation.
5. Using the Friedewald equation, calculate the LDL from the data below.
 Cholesterol= 200 mg/dL
 Triglycerides= 150 mg/dL
 HDL= 15 mg/dL
Using the course lecture notes, answer the following.
6. What 4 analytes are considered part of a lipid panel/profile? (2 points)
7. What substance appears on the serum as a creamy top layer?
8.
Under what circumstances can HDL NOT be performed?
9.
Name four (4) factors that affect the measurement of cholesterol. (2 points)
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MLAB 2401