Comparative Study for the measurement of HDL Cholesterol between a direct Assay and a Precipitation Method

 

Senan Ibrahim¹*, Mohammad Imad Khayat², Mohamad Ayman Awama³

¹Postgraduate Student, Department of Biochemistry and Microbiology, Faculty of Pharmacy, Tishreen University, Lattakia, Syria

²Assistant Professor, Department of Laboratory Medicine, Faculty of medicine, Tishreen University, Lattakia, Syria

³Teacher, Department of Biochemistry and Microbiology, Faculty of Pharmacy, Tishreen University, Lattakia, Syria

 

ABSTRACT:

Background: The semiautomatic devices used in conventional laboratories rely on the precipitation method in measuring HDL-C concentrations, while the automated devices often used in hospitals and modern chemical laboratories use the direct method. It is of a particular importance for the clinical laboratory to provide accurate, reliable, and easy –to- perform measurement of HDL-C, especially at the decision points of 35 and 60 mg/dl. A significant problem with HDL precipitation methods is the interference from elevated triglyceride levels. Objectives: To know if the results of precipitation method in measuring HDL-C concentrations, match the results of the direct method, we compared the precipitation method with the direct assay according to the difference in triglyceride levels. Design and methods: 211 serum samples were collected from patients who has fasted overnight. HDL-C levels measured by the routine dextran sulfate-Mg²⁺ precipitation compared with the direct assay as a reference method. Results: HDL-C precipitation method interferences with triglyceride levels more than 300 mg/dl. The strongest correlation between direct HDL-C and sedimentary HDL-C was found when the level of TG concentration was less than 100mg/dl (P value = 0.0001, r = o.81), and the weakest relationship was found when the concentration of TG was greater than 300mg/dl (P value = 0.0417, r = 0.47). The coefficient of variation in the values of sedimentary HDL-C was high when the values of TG greater than 300mg/dl (CV =0.47). Conclusions: The results of the HDL-C values obtained in the precipitation method adopted by the semiautomatic devices comparable to the results of HDL-C values obtained directly by the automated devices when the concentration of triglycerides less than 300 mg/dl.

 

 

 

INTRODUCTION:

Epidemiological and clinical studies have clearly shown the inverse correlation between serum high density lipoprotein cholesterol (HDL-C) concentration and the risk of atherosclerotic diseases. Therefore, HDL-C concentration <35 mg/dl are thought to be a cardiovascular risk factor.

 

According to the guidelines of National Cholesterol Education Program Adult Treatment Panel II (NCEP ATP II) for the diagnosis and treatment of hypercholesterolemia in adults, HDL-C should be measured along with total cholesterol (TC) at the initial screening stage [1-3]. Moreover, high HDL-C (>60mg/dl) is a negative risk factor, thereby compensating for the presence of a positive risk factor. Therefore, it is of a particular importance for the clinical laboratory to provide accurate, reliable, and easy –to- perform measurement of HDL-C, especially at the decision points of 35 and 60 mg/dl.

 

Common methods for separating HDL from other lipoproteins involve chemical precipitation of apo B –containing lipoproteins from fresh plasma or serum, centrifugation, and quantification of the cholesterol in the supernatant , a procedure which is labor-intensive, prevents full automation and requires a long volume of sample. Large samples are not always available, especially in children, and subjects who are often submitted to an extension laboratory analysis, as with patients with end-stage renal disease on chronic hemodialysis. Additionally, in the presence of high plasma triglycerides (TG), commonly found in patients with renal diseases, apo B-containing particles do not completely precipitate, accounting for the falsely increased results for HDL-C.

 

The recently developed direct or homogeneous assays for HDL-C, also called third generation assays, are fully automated and require only a small sample volume. Although the reagent costs are higher than the conventional methods, their advantages in every-day practice and their reliability have been extensively discussed [4-13].

 

We wanted to know if the results of the semiautomatic devices used in conventional laboratories, which rely on the precipitation method in measuring HDL-C concentrations, match the results of the automated devices often used in hospitals and modern chemical laboratories that use the direct method?

 

MATERIALS AND METHODS:

Samples:

For the method comparison study we obtained 211 blood samples from the daily pool of new specimens received at clinical chemistry laboratories, including Tishreen University Hospital and AL-Assad Hospital in Lattakia , Syria.

 

Blood samples were collected from patients who has fasted overnight.The samples were centrifuged at 2000g for 15 min. The serum samples thus obtained were processed immediately, and total cholesterol, and HDL-C were measured within 1 day.

 

Triglyceride values were obtained from patient's files at laboratories.

 

Total Cholesterol measurement:

Total Cholesterol was determined enzymatically with the cholesterol oxidase / peroxidase method.

 

Direct (Homogeneous) Assay:

Reagent 1 contains synthetic polymers and polyanions which stabilize VLDL, LDL and chylomicrons and shield their cholesterol. Reagent 2 a detergent destroys the HDL structure releasing cholesterol, which is then quantified by cholesterol esterase, cholesterol oxidase, peroxidase, and 4-aminoantipyrene present, as detailed in the manufacture's instructions.

 

Both reagents are ready to use. The calibration value for cholesterol standard is 60 mg/dl. The required serum sample volume is 12µl.

 

PTA assay (precipitation method):

The samples were mixed with a precipitating solution containing phosphotungstic acid and Mgcl2. After incubation and precipitation at room temperature, samples were centrifuged, and the supernatant was collected for assays.

 

HDL-C was determined with an enzymatic endpoint assay using cholesterol oxidase, peroxidase, and a chromogenic reaction with 4-aminophenazone. The assay was carried out according to the application protocol provided by the manufacturer.

 

Statistical analysis:

Analyze ANOVA was applied to study the relationship between HDL-C and TG concentration. Pearson's Coefficient Correlation (r) was used to study the association between direct and sedimentary HDL-C at each level of TG concentration. The differences at the threshold of significance P ≤ 0.05 were considered statistically significant. Statistic analysis was carried out with help of statistical program Stata (version 6.0).

 

RESULTS:

Table 1 shows the mean, standard deviation SD, and variance coefficient CV for total cholesterol, HDL-C and TG in the samples involved in the research.

 

Table 1: Descriptive statistics

 

 

We divided the samples into four groups according to the concentration of triglycerides (Table 2 ).

 

 

Table 2 : Distribution of samples by concentration of TG

 

 

 

Study of the relationship between concentration of HDL-C and triglycerides concentration:

Relationship between sedimentary HDL-C and concentration of TG.

Table 3: Relationship between sedimentary HDL-C and concentration of TG

There was no statistically significant relationship between the sedimentary HDL-C and TG concentration (P value > 0.05 ).

 

But it can be observed that the coefficient of variation in the values of sedimentary HDL-C is high when the values of TG greater than 300.

 

Relationship between Direct HDL-C and concentration of TG

 

Table 4: Relationship between Direct HDL-C and concentration of TG

 

As for the direct concentration of HDL-C and its relation to triglycerides concentration, we observed a statistically significant relationship between direct HDL-C concentration and triglycerides concentration (statistical significance 0.0001 ).

 

We found a gradual decrease in the mean direct HDL-C concentration with increased triglycerides concentration. it can be observed that the contrast coefficient in direct HDL-C values was less different between different levels of triglycerides.

 

Figure 1 show a comparison between the mean concentration of both direct HDL-C and sedimentary HDL-C in each triglycerides level.

 

 

Figure 1: a comparison between the mean concentration of both direct HDL-C and sedimentary HDL-C in each triglycerides level

We observed that the mean concentration of sedimentary HDL-C is less than the mean direct HDL-C concentration when the TG concentration level is less than 200mg/dl. The mean sedimentary HDL-C concentration is equal to the mean direct HDL-C concentration when the TG concentration level is between 200 and 300mg/dl. While the average sedimentary HDL-C concentration is higher than the direct HDL-C concentration when the level of TG concentration is more than 300mg/dl.

 

The association between the direct HDL-C and sedimentary HDL-C concentration within each triglyceride level:

We then studied the association between direct HDL-C and sedimentary HDL-C concentration within each triglyceride level. The strongest correlation between direct HDL-C and sedimentary HDL-C was found when the level of TG concentration was less than 100mg/dl ( the correlation coefficient was 0.81) and the weakest relationship was found when the concentration of TG was greater than 300mg/dl ( correlation coefficient 0.47 ). (Table 5), (figure 2 A, B, C, D)

 

Table 5: the association between the direct HDL-C and sedimentary HDL-C concentration within each triglyceride level

 

Figure 2 A

TG<100

 

Figure 2 B

100<TG<200

 

Figure 2 C

200<TG<300

 

Figure 2 D

TG>300

Figure 2: the association between the direct HDL-C and sedimentary HDL-C concentration within each triglyceride level

 

DISCUSSION:

We reported that the HDL-C values assayed by the precipitation method were lower than those by the direct assay when the concentration of TG is less than 200 mg/dl. The precipitating reagent containing dextran sulfate, sodium phosphotungstate and Mgcl2 readily precipitates apo E-rich HDL, often found in large –size HDL, which is readily precipitated [7, 15]. From the present examinations of the supernatants obtained by the precipitation method , we speculate that the commercial reagents precipitate part of HDL with normal size, even in routine samples , because of high concentration of Mgcl2, which was necessary to precipitate non-HDL lipoproteins completely in samples with high TG values [15].

 

The reason for the high HDL-C values when TG concentration is higher than 300 mg/dl is that when triglyceride-rich lipoproteins (chylomicrons and VLDL) are present, the low density of lipoproteins can prevent them from sedimenting. this incomplete sedimentation results of over estimation of HDL cholesterol [16].

 

CONCLUSIONS:

The results of the HDL-C values obtained in the precipitation method adopted by the semiautomatic advices used in conventional laboratories are comparable to the results of HDL-C values obtained directly by the automated devices used in hospitals and modern laboratories, when the concentration of triglycerides less than 300 mg/dl.

 

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Received on 01.11.2017         Modified on 07.12.2017

Accepted on 19.12.2017      © RJPT All right reserved