 Test vials or cuvettes.

PROCEDURES
 Timer. (MANUAL)
 100°C heating bath. 1. Label test tubes: blank, standard, control,
 Control serum, spectrophotometer. patient, etc.
TOTAL LIPID REAGENT SET 2. Transfer 10 ul (0.010 ml) of sample to its
PRECAUTIONS: respective vial, discharging sample into
(COLORIMEYRIC METHOD)  Exercise the normal precautions required bottom of vial.
for handling of all laboratory reagents. 3. Carefully transfer 1.0 ml of concentrated
 Pipetting by mouth is not recommended for sulfuric acid to each vial and mix thoroughly
INTENDED USE so that the entire sample is dissolved.
For the quantitative determination of the total lipid any laboratory reagent. Concentrated
sulfuric acid causes severe burns. 4. Place all vials in 100°C heating bath for 20
index in serum using the sulfo-phospho-vanillin minutes.
colorimetric method.  Exercise extreme care in handling this
chemical. 5. Remove all vials and place all vials in cold
 Concentrated sulfuric acid must be ACS, water for 3-5 minutes
INTRODUCTION 6. Add 2.0 ml of total lipid color reagent down
In blood, at least 95% of the lipids exist in Reagent Grade that is non-yellowed.
the side of each vial, mix by swirling, and
combination with protein. These lipoproteins can be place in cold-water bath for 15 minutes.
quantitated by disrupting this complex. This test is STORAGE AND STABILITY
 Store all reagents at room temperature. (NOTE: Mixing concentrated sulfuric acid
used as a screening method for hyperlipidemia. The with an aqueous solution generates a large
sulfo-phospho-vanillin (SPV) method for the  All reagents are stable until the expiration
amount of heat, therefore exercise
colorimetric determination of the serum total lipids date indicated on the individual bottle.
appropriate care.)
2
was described by Charbrol et al and modified by  Signs of reagent deterioration:
7. Set wavelength of spectrophotometer at
several investigators by omitting phosphoric acid,  Physical appearance
530nm and zero the instrument with the
shortening reaction time, and increasing reagent Slight brown color appearing in the color
blank. Read and record the absorbance of all
stability. Our reagent is based on all of these reagent, appearance of turbidity or
vials. (Wavelength range: 500-550nm).
modifications. crystals that will not readily dissolve are
signs of reagent deterioration.
NOTES
PRINCIPLE  Control assays
1. The Final color is stable for at least 60
Lipids react with sulfuric acid to form carbonium Failure to obtain accurate results in the
minutes.
ions, which subsequently react with the vanillin assay of control materials may indicate
2. Samples with values above 1,300 mg /dl
phosphate ester to yield a purple complex that is reagent deterioration.
should be dilut 1:1 with saline, re-assayed,
measured photometrically. and the results multiplied by two.
SPECIMEN COLLECTION
3. Hemolyzed serum samples must be
MATERIALS 1. Collect whole blood by venipuncture
avoided, as they will
REAGENTS and allow clotting. Centrifuge and
cause falsely elevated results.
 Total lipid color reagent: vanillin, potassium remove serum. Samples should be
4 Thorough mixing is crucial in this method
monobasic phosphate, preservative. collected with patient fasting for 12
because of the viscosity of the concentrated
 Total lipid standard (600 mg/dl): potassium hours.
sulfuric acid employed in the reaction.
oleate equivalent to 600 mg /dl of total lipids 2. Serum may be kept at room
when run according to the instructions with temperature for four hours and at
CALCULATIONS
this procedure. refrigerator temperature (2 - 8°C) for 48
Use the absorbance readings of the STANDARD and
MATERIALS REQUIRED BUT NOT PROVIDED hours. Serum should not be frozen.
UNKNOWN(S) to calculate total lipid values as follows:
 Concentrated sulfuric acid. 3. Hemolysis must be avoided.
 Sample and reagent, pipettes. A = Absorbance
 A (UNKNOWN) x Conc. of STD Sensitivity:
A (STANDARD) (mg/dl) Based on an instrument of A = 0.001, this procedure
has a sensitivity of 3 mg /dl.
= Total Lipid in UNKNOWN (mg/dl)
Comparison Study:
Example: A study performed between this procedure and an
A (patient) = 0.45 identical procedure resulted in a coefficient of
A (standard) = 0.50 correlation of 0.98 with a regression equation of y =
Concentration of standard = 600 mg /dl 1.0x-2.76.
With in Run
0.45 x 600 mg/dl Mean(mg/dl) S.D CV(%)
= 540 mg/dl 0.5 Normal 650 27 401%
Abnormal 1130 60 5.3%
EXPECTED VALUES Run to Run
400-800 mg /dl Mean(mg/dl) S.D CV(%)
Normal 634 38 5.9%
QUALITY CONTROL Abnormal 1138 74 6.5%
 It is recommended that controls be included
in each set of assays. Commercially available REFERENCES
control material with established total lipid 1. Boutwell, J.H.M., U.S. D.H.E.W.
values may be used for quality control. pamphlet (1972).
 The assigned value of the control material 2. Chabrol, E and Charonnet, R, Presse
must be confirmed by the chosen application. Med, 45:1713 (1937).
Failure to obtain the proper range of values 3. Zoellner, N and Kirsch, K, Z.fur die
in the assay of control material may indicate Gesampte Exp Med, 135:545 (1962).
either reagent deterioration, instrument 4. Frings, CS and Dunn, RT, Am J Clin. Path.,
malfunction, or procedural errors. 53:89 (1970).
5. Knight, JA et al, Clin. Chem., 19:199
LIMITATIONS (1972).
Heptane, ethyl benzene, tetrahydrofuran, detergents 6. Jacobs, SL and Henry, RJ, Clin. Chem.
and soaps are known to interfere with the sulfo- Acta, 7:270 (1962).
phospho-vanillin method. Alcohols with a carbon
chain greater than C2 will interfere. The reaction is
specific only for unsaturated compounds. Saturated
fatty acids do not react. For these reasons, Knight et ATLAS MEDICAL
5
al have suggested that it would be more appropriate William James House, Cowley Rd,
to refer to the results as an "index of total lipids" Cambridge, CB4 4WX, UK
when the method is utilized. Tel: ++44 (0) 1223 858 910
Fax: ++44 (0) 1223 858 524
PERFORMANCE CHARACTERISTICS
Linearity: PPI364A01
1300 mg/dl Rev B (21.01.2010)