INTRODUCTION:

A study on the kits used for cholesterol estimation using CHOD PAP method has been carried out. The present investigation has been undertaken to establish the optimum incubation time to reach the end point of the reaction. Also an attempt is made to confirm the status of the reaction at the end point.

MATERIAL AND METHODS:

The present study has been carried out at 200 mg/dl concentration of the cholesterol. The commercial manufacturers of the kits have recommended different time intervals as “incubation time”. For attaining end point of the assay, the results are tabulated in table below, by adhering to protocol ¹ . The absorption was recorded at wavelength 505nm on Screen Master- 3000 biological analyzer for a period of 0 to 1220 seconds (0 to 20 minutes) at an interval of 50 seconds.

All the tubes are maintained at constant temperature of 37^o C in a temperature regulated flow cell. Each assay was repeated thrice such that the average of the three was considered for further evaluation.

Table I

Manufacturer’s recommended optimum time interval for end point assay
Kit	At 37^o C	At room temperature(15-30^o C )
A	5 minutes	15 minutes
B	5 minutes	15 minutes
C	5 minutes	15 minutes
D	5 minutes	15 minutes

Protocol for end point study:

All the reagents are brought to 37^o C before assay is carried out. Thus incubation at 37^o C and absorbance was recorded from 0 to 1200 seconds. The results are summarised as under:

Additions	Blank	Tube 1(L)
Cholesterol working reagent (µl)	1000	1000
Normal Saline (µl)	10	-
Low Standard (200 mg/dl)	-	10

TABLE II: REACTION-INCUBATION TIME INTERVAL
TIME(MIN)	A	B	C	D
0	0.043	0.165	0.041	0.058
1	0.415	0.445	0.382	0.123
2	0.437	0.461	0.389	0.182
3	0.438	0.457	0.392	0.265
4	0.436	0.453	0.392	0.324
5	0.436	0.47	0.392	0.349
6	0.435	0.471	0.393	0.366
7	0.435	0.469	0.392	0.37
8	0.434	0.466	0.393	0.376
9	0.434	0.465	0.395	0.379
10	0.434	0.465	0.408	0.38
11	0.434	0.47	0.395	0.38
12	0.432	0.471	0.395	0.381
13	0.435	0.468	0.393	0.381
14	0.435	0.468	0.394	0.381
15	0.434	0.472	0.395	0.38
16	0.433	0.47	0.394	0.381
17	0.433	0.471	0.394	0.379
18	0.433	0.471	0.394	0.379
19	0.432	0.471	0.394	0.378
20	0.432	0.472	0.394	0.378
25	0.43	0.474	0.391	0.375
30	0.428	0.478	0.393	0.372
35	0.424	0.478	0.388	0.367
40	0.421	0.479	0.384	0.36
45	0.413	0.47	0.377	0.355
50	0.412	0.476	0.382	0.357
55	0.412	0.476	0.38	0.352
60	0.409	0.476	0.376	0.351
65	0.408	0.478	0.376	0.35
70	0.407	0.478	0.375	0.346
75	0.403	0.478	0.373	0.342
80	0.4	0.477	0.372	0.341
85	0.4	0.48	0.372	0.338
90	0.4	0.48	0.37	0.333
95	0.398	0.479	0.368	0.33
100	0.398	0.479	0.368	0.321
105	0.396	0.479	0.367	0.315
110	0.395	0.481	0.367	0.312
115	0.394	0.486	0.367	0.309
120	0.391	0.484	0.365	0.309

TABLE III: REACTION (INCUBATION) TIME INTERVAL
	min	OBSERVATIONS
		KIT-A	KIT-B	KIT-C	KIT-D
Blank nm (B)	0	0.043	0.165	0.041	0.058
KITS nm (T)	5	0.436	0.47	0.392	0.349
Reaction Aborted (f) nm	20	0.432	0.472	0.394	0.378
EP=(T-B) nm		0.393	0.305	0.351	0.32
f-B=F (nm)		0.389	0.307	0.353	0.291
F-EP (nm)		-0.004	0.002	0.002	-0.029
%ERROR		-1.02	0.66	0.57	-9.06
S.D.		0.00	0.00	0.00	0.02
EXPECTED (mg/dl)		200	200	200	200
DERIVED (mg/dl)		197.96	201.31	201.14	181.88

RESULT:

The kinetics of the reaction mixture was studied at concentration of 200mg/dl. The observations are recorded in table II and their statistics in table III. This evaluation was done by following protocol of Trender P(1969) for 200mg/dl concentration on all the four in study kits. It is observed that evaluated concentration was well within the recommended linearity range as claimed by the manufacturers.

DISCUSSION:

As per the claims of the manufacturers the endpoint for all the four kits A,B,C and D is five minutes. The table I gives the manufacturer’s recommended optimum time interval for end point assay. The kinetic reaction for Kit D suggests an incomplete reaction since optical density is still climbing beyond recommended end point. This observation was also validated by the statistics in table III. This table calculates difference between the readings of two time points difference (DIFF),% variance (%VAR), SD(Standard Deviation) and % error ². Out of all these statistics the % error is most revealing since the % error recorded for kits A,B, C and D is -1.02, 0.66,0.57 and -9.06 respectively at extended time of 20 minutes from the initiation of the reaction is pertinent to note that the manufacturer mentions the reading can be taken at any time after the manufacturer’s recommended end point and before one hour ³. In this experiment we have recorded readings upto 20 minutes and do not know how it will behave beyond such time. The % error is plotted in chart above for 200 mg/dl concentration of cholesterol. Kits B and C show upward trend whereas kits A and D show the reverse, that is, downward trend. The downward trend is attributed to poor colour stability and there could be variety of reasons which need to be explored.

REFERENCES:

1. Trender P, Annual Clin. Biochem. 6:24 (1969).

2. Westgard J O, Hunt M R. Use and interpretation common statistical test in medical comparison studies. Clinical Chemistry, 19:49-57(1973)

3. Westgard J O, Precision and accuracy, concepts and assessment by method evaluation testing. CRC critical reviews in clinical lab sciences 13: 283-330(1981a)

Received on 24.07.2013 Modified on 18.08.2013

Asian J. Research Chem. 6(10): October 2013; Page 950-951