HDL Detail Aid

HDLHDL

HDLHDL

HDL Detail Aid

Accurate and precise measurements of HDL Cholesterol are necessary for the effective diagnosis and treatment of lipid disorders.

CDC Reference method – Ultracentrifugation

The ultracentrifugation method involves the separation

of lipoproteins based on density followed by a cholesterol analysis

step. It is not only tedious and time-consuming but extremely

difficult to perform as separations are highly dependent on the skills

of the technician. Although considered the most accurate method of

HDL determination, its use is limited in routine laboratories.

Randox Direct clearance method

The Randox direct clearance method is superior to other direct

methods on the market and works by completely removing all

non-HDL components (vLDL, Chylomicrons and LDL) in the

first step of the reaction. HDL-Cholesterol is then specifically

measured in the second step of the reaction resulting in a high

degree of accuracy even with abnormal samples

Direct masking methods There are several direct HDL methods available offering several

advantages over the ultracentrifugation reference method as

they do not require sample pre-treatment and enable the rapid

analysis of HDL. Although many of these methods perform

well with normal patient samples their specificity is affected by

samples containing high levels of bilirubin or triglycerides. These

direct methods include:

Wako (Immunoseparation),

Daiichi (Selective Detergent)

Kyowa (PEGME)

Precipitation method

LDL, VLDL and Chylomicrons are precipitated in the first step

of the reaction. HDL is then measured using a total cholesterol

kit. The precipitation method, like the ultracentrifugation method, is

time-consuming and depends greatly on the skills of the technician.

HDL Cholesterol measurementThere are several methods available for the measurement of HDL Cholesterol:

Clearly anyone can get it right with normal samples

LDL LDLCM CM

VLDL VLDL

Buffer release

Surfactant release

Cholesterol Esteraseand

Cholesterol Oxidase

Cholestenone+ H202

Cholestenone+ H202

H20 + 02

Coloured compound

Catalase

Peroxidase

Clearance step

Assay step

Cholesterol Esteraseand

Cholesterol Oxidase

* Sodium Azide prevents catalase action in the assay step

Reaction Principle The assay consists of two distinct reaction steps

Elimination of chylomicrons, VLDL-Cholesterol and LDL-Cholesterol by cholesterol esterase, cholesterol oxidase and subsequently catalase.

Specific measurement of LDL-Cholesterol after release of LDL-Cholesterol by detergents in Reagent 2.

Specificity of Randox Direct Clearance Assay for HDL CholesterolSpecificity of the Randox direct clearance HDL assay was verified against gel filtration. The diagram below shows just how specific the

Randox direct clearance method is for HDL Cholesterol. Our kit was found to only react with the HDL fractions separated by gel filtration.

Performance in Discrepant SamplesA correlation study using 207 samples found six samples with discrepant results. The discrepant samples were analysed using different

methods and compared to the ultracentrifugation reference method. The graphs below show the Randox direct clearance method

compares well to the ultracentrifugation method compared to other direct methods.

Sample 1

0

5

10

15

20

25

Randox Clearance Method

Method 1 Method 2

Ultracentrifugation

Direct method

mg/

dL

Sample 2

Method 1 Method 20

5

10

15

20

25

30

35

40

mg/

dL

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29-10

0

10

20

30

40

50

60

Total Cholesterol Reagent

Randox HDL Cholesterol Reagent

Frac No.

mg/

dL

LDL

HDL

VLDL

Randox Clearance Method

Information correct at time of print. All Randox products are made in the UK. Randox Laboratories Limited is a company registered within Northern Ireland with company number N.I. 15738. VAT Registered Number: GB 353 030 400. Product availability may vary from country to country. Please contact your local Randox representative for information.

Randox Laboratories Limited, 55 Diamond Road, Crumlin, County Antrim, BT29 4QY, United KingdomT +44 (0) 28 9442 2413 F +44 (0) 28 9445 2912 E marketing@randox.com I www.randox.com LT

154

DEC

11

Precipitation method (mg/dL)

(Phosphotungstate-Mg)

Ran

dox

(mg/

dL)

Benefits of Randox Direct Clearance Method for HDL Measurement• Liquidready-to-usereagents

• Accurateresultswithdiscrepantsamples

• HighlyspecificforHDLCholesterol

• Notriglycerideinterferencereported

• Nobilirubininterferencenoticedupto25mg/dL(428μmol/L)

• Excellentprecisioncomparedtoprecipitationmethods

• Excellentagreementwiththeultracentrifugationreferencemethod

• Nosamplepre-treatmentrequired

• 30dayon-boardstability

• Fullyautomated,suitableforuseonawiderangeofclinicalanalysers.

100

80

60

40

20

0

0 20 40 60 80

n=50r=0.9903Y=1.024x.0.9

Ran

dox

(mg/

dL)

Ultracentrifugation method (mg/dL)

100

80

60

40

20

00 20 40 60 80

n=50r=0.9901Y=0.9413x+0.2

HDL Cholesterol - Randox (mg/dl)

HD

L C

hole

ster

ol -

CD

C (

mg/

dL)

0 20 40 60 80 1000

102030405060708090

100

y = 0.979 + 1.13R2 - 0.9787

Correlation with precipitation and ultracentrifugation methodsThe accuracy of our direct clearance method has been verified by direct comparison to the CDC reference method. The graphs

below show the excellent correlation obtained between the Randox direct clearance assay and the precipitation and ultracentrifugation methods.