12418485 Diagnostic Clinical Chemistry

DIAGNOSTIC IN CLINICAL CHEMISTRY I

MKEB 2404

DATE:

17 JANUARY 2007

TITLE:

• MEASUREMENT OF LIPID

AIMS & OBJECTIVES:

• To understand the principles involved in cholesterol measurement

• To measure the cholesterol level in the samples given.

MEASUREMENT OF CHOLESTEROL

• CHOD-PAP method

○ Test principle

Cholesterol esterase

Cholesterol ester + HbO ---------------------► cholesterol + RCOOH (fatty acid)

Cholesterol oxidase

Cholesterol + 02 _______________► Cholesteroi-3-one + H202

Peroxidase

2H2O2 + 4-aminophenazone + phenol -----------► 4 - (p-benzoquinone - monoimino) -

phenazone + 4H20

SAMPLE:

• Serum, heparinized plasma or EDTA plasma

PROCEDURES:

• Wavelength: 500nm

• Cuvette: 1 cm light path

1 | P a g e

• Incubation temperature: 20-25°C or 37°C

• Measure against reagent blank

• One reagent blank is sufficient for each assay series.

Pipette into test tubes

Blank Sample/control/standardDistilled water 0.01 ml -Sample material - 0.01 mlCholesterol Reagent 1.00 ml 1.00 ml

• Mix, and incubate blank and sample for 20 min at 20-25°C

• Or 10 min at 37°C.

• Read absorbance of sample against reagent blank within 1 hour.

RESULTS:

TUBES ABSORBANCE READINGNORMAL CONTROL 0.150

STANDARD 0.232SAMPLE 1 0.201SAMPLE 2 0.242

CALCULATIONS:

Cholesterol (mg/dl) = A sample / A Std. X Conc. Std. (mg/dl)

Cholesterol (mmol/l) = Cholesterol (mg/dl) X 0.02586

• Concentration Standard (mg/dL) : 200 mg/dL

• Standard Absorbance : 0.232

TUBES CALCULATION

NORMAL CONTROL Cholesterol (mg/dL)

○ A sample / A Std. X Conc. Std.

2 | P a g e

(mg/dl)

○ 0.150 /0.232 X 200

○ 129.31 mg/dL

Cholesterol (mmol/l)

○ Cholesterol (mg/dl) X 0.02586

○ 129.31 X 0.02586

○ 3.34 mmol/L

SAMPLE 1

Cholesterol (mg/dL)


(mg/dl)

○ 0.201 /0.232 X 200

○ 173.28 mg/dL



○ 173.28 X 0.02586

○ 3.34 mmol/L

SAMPLE 2

Cholesterol (mg/dL)


(mg/dl)

○ 0.242 /0.232 X 200

○ 208.62 mg/dL



○ 208.62 X 0.02586

○ 5.39 mmol/L

CONTROL RANGE:

• Cholesterol: 164.35 +/- 14.76 (2SD) mg/dL.

DISCUSSION:

3 | P a g e

On this experiment, method CHOD-PAP is for the quantitative

determination of total cholesterol in serum and plasma. The CHOL method is

based on the principle first described by Stadtman. Cholesterol esterase (CE)

catalyzes the hydrolysis of cholesterol esters to produce free cholesterol which,

along with preexisting free cholesterol, is oxidized in a reaction catalyzed by

cholesterol oxidase (CO) to form cholest-4-ene-3-one and hydrogen peroxide.

In the presence of horseradish peroxidase (HPO), the hydrogen peroxide

thus formed is used to oxidize N,N-diethylaniline-HCI/4-aminoantipyrine (DEA-

HCI/AAP) to produce a chromophore that absorbs at 500 nm. The absorbance is

due to oxidized DEA-HCI/AAP and directly proportional to the total cholesterol

concentration.

TUBES INTERPRETATION

NORMAL CONTROL• The result shown low than

cholesterol range.

SAMPLE 1& SAMPLE 2• The result shown high than

cholesterol range.

Below are the list interferences or error during the experiment.

INTERFERENCES & ERRORBILIRUBIN • Bilirubin (conjugated) of 8.1

mg/dL [139 umol/L] and

bilirubin (unconjugated) of 9.4

mg/dL [161 nmol/L] decrease

the CHOL result by 15 mg/dL

[0.4 mmol/L] at CHOL

concentration of 150 mg/dL [3.9

mmol/L].

• Bilirubin (conjugated) of 12.8

mg/dL [219 umol/L] and

bilirubin (unconjugated) of 14.7

mg/dL [251 nmol/L] decrease

the CHOL result by 25 mg/dL

[0.7 mmol/L] at CHOL

4 | P a g e

concentration of 250 mg/dL [6.5

mmol/L].

DRUGS

• The following substances have a

negligible effect (<10 mg/dL [0.3

mmol/L]) on the CHOL method

at the concentrations.

○ Acetaminophen

○ Ampicillin

○ Diazepam

○ Digoxin

○ EDTA

○ Ethanol

○ Gentamicin

○ Hemoglobin

○ Lipemia

○ Nortriptyline

○ Phenobarbital

○ Phenytoin

○ Salicylate

○ Theophylline

STATINS

• Interference from the statins at

the concentrations indicated,

when added to a 151 mg/dL [3.9

mmol/L] CHOL serum pool

respectively, is less than 10%.

○ Atorvastatin

○ Lovastatin

○ PravastatinANTICOAGULANT • Potassium Oxalate/Sodium

Fluoride can decrease cholesterol

results an average of 12%.

• Li Heparin can depress

cholesterol results by an average 5 | P a g e

of 4 mg/dL [0.1 mmol/L] at a

level of 200 mg/dL [5.2

mmol/L].

REAGENT & SAMPLE

• Date due of the reagent.

• Not fresh samples.

• Incubation time not correct,

either prolong or shorten.

HUMAN ERROR• Pipetting error.

• Sample tubes not mixing well.

MEASUREMENT OF HDL-CHOLESTEROL

6 | P a g e

• Test principle

Chylomicrons VLDL and LDL are precipitated by adding

phosphotungstic acid and magnesium ions to the sample. Centrifugation

leaves only the HDL in the supernatant; their cholesterol content is

determined enzymatically.

• Sample material

○ Serum

○ Heparinized plasma

○ EDTA plasma

* Serum must be separated from the blood clot as rapidly as possible.

• Sample preparation (Semi micro assay)

○ Precipitation

○ Mix, and let stand for 10 min at room temperature, then centrifuge:

15 min at 4000 rpm or more

2 min at 12000 rpm.

○ After centrifugation separate the clear supernatant within 2 hours and

determine the cholesterol content by the CHOD-PAP method.

○ The supernatant may be stored for up to five days at +4 to 25°C.

Pipette into test tubesSample/control/standard 0.2 mlPrecipitant 0.2 ml

○ Mix, and let stand for 10 min at room temperature, then centrifuge:

15 min at 4000 rpm or more

2 min at 12000 rpm.

○ After centrifugation separate the clear supernatant within 2 hours and

determine the cholesterol content by the CHOD-PAP method.

○ The supernatant may be stored for up to five days at +4 to 25°C.

• Assay procedure

○ Determination of cholesterol using the CHOP-PAP method.

Wavelength: 500nm.

Cuvette: 1 cm light path.

Incubation temperature: 20-25°C or 37°C.

Measure against reagent blank.

7 | P a g e

One reagent blank is sufficient for each assay series.


Blank Sample/control/standardDistilled water 0.01 ml -Sample material - 0.01 mlCholesterol Reagent 1.00 ml 1.00 ml

• Mix, and incubate blank and sample for 10 min at 20-25°C or 5 min at 37°C.

• Read absorbance of sample against reagent blank within 1 hour.

CALCULATIONS:

• Calculation of HDL cholesterol

HDL-C(mg/dl) = A sample / A Std. X Conc. Std. (mg/dl)

8 | P a g e

• Calculation of LDL cholesterol mg/dL

LDL cholesterol= total cholesterol – triglycerides / 5 - HDL cholesterol

• Calculation of LDL cholesterol mmol/L

LDL cholesterol= total cholesterol – triglycerides / 2.2 - HDL cholesterol



PRECAUTION:

• The values obtained are reliable, provided that;

○ No chylomicrons are present in the sample.

○ The triglyceride concentration does not exceed 400 mg/dl

○ The sample does not show signs of type III hyperlipoproteinemia.

NOTES:

• The values obtained are reliable, provided that;

○ The supernatant obtained on centrifugation must be clear.

○ If the sample has a high triglyceride content (above 1000 mg/dl),

lipoprotein precipitation may be incomplete (cloudy supernatant), or part of

the precipitate may float on the surface.

○ In these cases, dilute the sample 1 + 1 with 0.9% NaCI solution and repeat

the precipitation step.

○ The result of the cholesterol assay must then be multiplied by 2.

RESULTS:



TUBES CALCULATION

9 | P a g e

NORMAL CONTROL

HDL-CHOLESTEROL (mg/dL)


(mg/dl)

○ 0.294 /0.787 X 200

○ 74.71 mg/dL

HDL-CHOLESTEROL (mmol/l)

○ HDL-Cholesterol (mg/dl) X 0.02586

○ 74.71 X 0.02586

○ 1.93 mmol/L

SAMPLE 1



(mg/dl)

○ 0.125 /0.787 X 200

○ 31.77 mg/dL



○ 31.778 X 0.02586

○ 0.82 mmol/L

SAMPLE 2



(mg/dl)

○ 0.119 /0.787 X 200

○ 30.24 mg/dL



○ 30.24 X 0.02586

○ 0.78 mmol/L

CONTROL RANGE:

• HDL-Cholesterol: 43.12 +/- 7.99 (2SD) mg/dL.

DISCUSSION:

10 | P a g e

On this experiment, method HDL is for the quantitative determination of high

density lipoprotein cholesterol (HDL-C) in serum and plasma. Plasma lipoproteins are

spherical particles containing varying amounts of cholesterol, triglycerides, phospholipids

and proteins.

The phospholipid, free cholesterol and protein constitute the outer surface of the

lipoprotein particle, while the inner core contains mostly esterified cholesterol and

triglyceride. These particles serve to solubilize and transport cholesterol and triglyceride in

the bloodstream.

The relative proportions of protein and lipid determine the density of these

lipoproteins and provide a basis on which to begin their classification. These classes are:

chylomicron, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and

high-density lipoprotein (HDL).

The principle role of HDL in lipid metabolism is the uptake and transport of

cholesterol from peripheral tissues to the liver through a process known as reverse

cholesterol transport (a proposed cardioprotective mechanism).

Low HDL-C levels are associated with an increased risk of coronary heart disease and

coronary artery disease. Hence, the determination of serum HDL-C is a useful tool in

identifying high-risk patients. The reference method for the quantitation of HDL-C

combines ultracentrifugation and chemical precipitation to separate HDL from other

lipoproteins, followed by cholesterol measurement using the Abell-Kendall assay.

This method is too time consuming and labor intensive for use in routine analysis.

Therefore, most laboratories utilize one of several methods for selective precipitation and

removal of LDL and VLDL, followed by the enzymatic measurement of HDL-C in the

supernatant fraction. Since these methods require off-line pretreatment and separation

steps the assay procedures cannot be fully automated. As a result, routine determination of

HDL-C has suffered from long handling times and poor reproducibility.


NORMAL CONTROL• The result shown high than

HDL-C cholesterol range.

SAMPLE 1& SAMPLE 2• The result shown low than

HDL-C cholesterol range.

Below are the list interferences or error during the experiment.

INTERFERENCES & ERROR

11 | P a g e

DRUGS • Interference from the following

substances at concentrations

indicated, when added to a 40

mg/dL [1.04 mmol/L] HDL-C

serum pool, is less than 10%.

○ Albumin

○ Amikacin

○ Ampicillin

○ Ascorbic Acid

○ Atorvastatin

○ Caffeine

○ Carbamazepine

○ Chloramphenicol

○ Chlordiazepoxide

○ Chlorpromazine

○ Cimetidine

○ Clofibrate

○ Creatinine

○ Furosemide

○ Gemfibrozil

○ Heparin (sodium)

○ Ibuprofen

○ Immunoglobulin G (IgG)

○ LDL-Cholesterol

○ Lidocaine

○ Lithium Chloride

○ Lovastatin

○ Niacin

○ Nicotine

○ Penicillin G

○ Pentobarbital

12 | P a g e

○ Phenytoin

○ Protein, Total

○ Rheumatoid Factor

○ Urea

○ Uric Acid

SAMPLING

• Interference from the following

substances at concentrations

indicated, when added to a 40

mg/dL [1.04 mmol/L] HDL-C

serum pool, is less than

10%.Serum or plasma should be

removed from cells within 2

hours of venipuncture

• Serum or plasma should be

removed from cells within 2

hours of venipuncture.

• Must test within 8 hour.

HUMAN ERROR

• Sampling, mixing and

processing samples.

• Cuvettes contain human body

contact.

MEASUREMENT OF TRIGLYCERIDES (GPO-PAP method)

13 | P a g e

Enzymatic hydrolysis of triglyceride with subsequent determination of liberated glycerol

by colorimetry

• Test principle

14 | P a g e

Lipoprotein lipase

Triglycerides + 3H20---------------► glycerol + 3 RCOOH

Glycerokinase

Glycerol + ATP----------------► glycerol-3-phosphate + ADP

Glycerol-3-phosphate-oxidase

Glycerol-3-phosphate + O2 dihydroxyacetone phosphate + H2O2

peroxidase

2 H2O2 + 4 aminophenazone + 4-chlorophenol 4-(p-benzoquinone-mono-

imino)-phenazone + 2H2O + 2 HCL

• Sample material

○ Serum, heparinized

○ EDTA plasma.

• Procedure

○ Wavelength: 500nm

○ Cuvette: 1 cm light path

○ Incubation temperature: 20-25°C

○ Measure against reagent blank

○ One reagent blank is sufficient for each assay series.


Blank Sample/control/standardDistilled water 0.01 ml -Sample material - 0.01 mlTriglycerides Reagent 1.00 ml 1.00 ml

15 | P a g e

• Mix and incubate for 20 min at 20-25°C or 10 min at 37°C.

• Read absorbance of sample against reagent solution within 1 hour.

• Calculation via factor

○ Calculate the concentration (c) of triglycerides as follows:

Triglycerides (mg/dl) = A sample / A Std. X Cone. Std (mg/dl)

Triglycerides (mmol/L) = Triglycerides (mg/dL) X 0.01126

• Calculation via factor

○ Calculate the concentration (c) of triglycerides as follows:

* Please note: To correct for free glycerol subs tract 10 mg/dl (0.11 mmol/T) from

the calculated triglyceride value.

• Clinical interpretation according to the recommendations of the European

Atherosclerosis.

Analytes Reference Range Lipid disorder

Cholesterol

Triglycerides

<200mg/dl

<200 mg/dl No

Cholesterol 200-300 mg/dlYes, if HDL cholesterol

< 35 mg/dl

Cholesterol

Triglycerides

>300 mg/dl

>200 mg/dl Yes

• Values in mmol/l:

TEST UNITSCholesterol: 35 mg/dl = 0.9 mmol/L

200mg/dl=5.2 mmol/L

300mg/dl= 7.8 mmol/LTriglycerides: 200mg/dl= 2.3 mmol/L

16 | P a g e

RESULTS:





• Control range Triglyceride: 130.99 +/ - 14.97 (2SD) mg/dL

TUBES CALCULATION

NORMAL CONTROL

TRIGLYCERIDES (mg/dL)

○ A sample / A Std. X Conc. Std. (mg/dl)

○ 0.377 /0.426 X 200

○ 176.99 mg/dL

TRIGLYCERIDES (mmol/L)

○ Triglycerides (mg/dl) X 0.01126

○ 176.99 X 0.01126

○ 1.99 mmol/L SAMPLE 1 TRIGLYCERIDES (mg/dL)


○ 0.227 /0.426 X 200

○ 106.57 mg/dL



○ 106.57 X 0.01126

○ 1.2 mmol/L

17 | P a g e

SAMPLE 2

TRIGLYCERIDES (mg/dL)


○ 0.403 /0.426 X 200

○ 189.20 mg/dL



○ 189.20 X 0.01126

○ 2.13 mmol/L

TUBES CALCULATION VIA FACTOR

NORMAL CONTROL

LDL CHOLESTEROL (mg/dL)

○ Total Cholesterol - Triglycerides / 5 X

HDL cholesterol (mg/dl)

○ 129.31 – 176.99 /5 X 74.71

○ 19.20 mg/dL

LDL CHOLESTEROL (mmol/L)

○ Total Cholesterol - Triglycerides / 2.2 X

HDL cholesterol (mmol/L)

○ 3.34 – 1.93/2.2 – 1.99

○ 0.47 mmol/L

SAMPLE 1

LDL CHOLESTEROL (mg/dL)



○ 173.28 – 31.77 /5 X 106.57

○ 60.36 mg/dL




○ 4.48 – 0.82/2.2 – 1.2

2.91 mmol/L

SAMPLE 2 LDL CHOLESTEROL (mg/dL)

18 | P a g e



○ 208.62 – 30.24 /5 X 189.20

○ 13.37 mg/dL




○ 5.39 – 0.78/2.2 – 2.13

○ 2.91 mmol/L

• The amount of free glycerol, 10 mg/dL (0.11 mmol/L) is subtracted from

the calculated triglyceride value.

SAMPLE TRIGLYCERIDE

(mg/dL)

TRIGLYCERIDE

(mmol/L)

1106.57 – 10

= 96.57 mg/dL

1.2 - 0.11

=1.09 mmol/L

2189.20 – 10

=179.20 mg/dL

2.13 – 0.11

= 2.02 mmol/L

DISCUSSION:

This experiment is a quantitative determination of triglycerides in serum

and plasma. It is useful in diagnosis and treatment of patients with diabetes

mellitus, nephrosis, liver obstruction, other diseases involving lipid metabolism,

or various endocrine disorders.

Triglycerides are water-insoluble lipids consisting of three fatty acids linked

to one glycerol molecule. Triglycerides are transported in the blood as core

constituents of all lipoproteins, but the greatest concentration of these molecules is

carried in the triglycerides-rich chylomicrons and very low density lipoproteins

(VLDL).

Through the action of lipases and bile acids, triglycerides are hydrolyzed

into glycerol and fatty acids which are absorbed by adipose tissue for storage or

19 | P a g e

by other tissues requiring a source of energy. A peak concentration of

chylomicron-associated triglycerides occurs within 3-6 hours after ingestion of a

fat-rich meal; however, the rate of absorption of fats is highly variable, depending

on the individual and dietary composition of the fat. After absorption,

triglycerides are resynthesized in the epithelial cells and combined with

cholesterol and a number of apolipoproteins to form chylomicrons.


NORMAL CONTROL• The result shown high than

cholesterol range.

SAMPLE 1& SAMPLE 2• The result shown low than

cholesterol range.

INTERFERENCES & ERRORDRUGS • The following substances do

not interfere with the TGL

method when present in serum

and plasma at the

concentrations indicated.

Inaccuracies (biases) due to

these substances are less than

10% at triglycerides

concentrations of 200 mg/dL

[2.26 mmol/L].

○ Acetaminophen

○ Albumin

○ Amikacin

○ Ampicillin

○ Ascorbic Acid

○ Bilirubin

○ Caffeine

○ Carbamazepine

○ Chloramphenicol

20 | P a g e

○ Chlordiazepoxide

○ Chlorpromazine

○ Cholesterol

○ Cimetidine

○ Creatinine

○ Dextran 75

○ Diazepam

○ Digoxin

○ Erythromycin

○ Ethanol

○ Ethosuximide

○ Furosemide

○ Gentamicin

○ Sodium Heparin

○ Hemoglobine (monomer)

○ Ibuprofen

○ Lidocaine

○ Lithium (lithium chloride)

○ Nicotine

○ Penicillin G

○ Pentobarbital

○ Phenobarbital

○ Phenytoin

○ Primidone

○ Propoxyphene

○ Protein: Total

○ Rheumatoid factors

○ Salicylic Acid

○ Theophylline

○ Urea

○ Uric Acid

21 | P a g e

Free glycerol & human error

• Cause a positive interference.

• Sampling, mixing and

processing samples.

• Cuvettes contain human body

contact.

CONCLUSION:

1. These routines test that one mostly done as diagnostic tests and it is a

quantitative determination of all lipids either in serum or plasma.

2. These experiments are based on enzymatic reaction.

22 | P a g e

23 | P a g e

24 | P a g e

Documents

12418485 Diagnostic Clinical Chemistry