RX SERIES LATEX ENHANCED IMMUNOTURBIDIMETRIC ASSAY FOR THE

MEASUREMENT OF LIPOPROTEIN (A) ON THE RX MONACO ANALYSER

INTRODUCTION

What is Lipoprotein(a)?

Lipoprotein(a), also known as Lp(a) or lipoprotein-

little-a, are particles that are made in the liver

and found in the blood plasma that transport

cholesterol, phospholipids, triglycerides and

apolipoproteins. The amount that is present in

your body is mainly determined by genetics and

varies widely hence, the levels of Lp(a) can vary

up to 1000-fold between individuals. Lipoprotein

is assembled from a cholesterol rich LDL particle

and two protein molecules; including apoliporotein

apo(a) and apolipoprotein B100. Although the size

of apolipoprotein B100 is the same for everyone

the size of apo(a) is genetically determined and

differs between individuals and can vary widely.

Having a high level of Lp(a) (greater than 500mg/L

or 120nmol/) increases your risk of cardiovascular

disease (CVD). Levels of Lp(a) do not change

throughout life and are usually unaffected by lifestyle

or the environment. According to the British Heart

Foundation, an average of 420 people die from CVD

each day which equates to one death every three

minutes.

What is Lp(a) used for?

Although having some cholesterol and Lp(a) in

your blood is normal, elevated concentrations of

Lp(a) are a risk factor for coronary heart disease

(CHD), a heart attack, peripheral vascular disease,

aortic stenosis, blood clots and stroke. Therefore

measuring Lp(a) offers the possibility of early

diagnosis and risk of CVD. It has been estimated

that one in five people globally have a high level of

Lp(a) and remain unaware despite high Lp(a) being

a common condition. Additionally around 30% of

those diagnosed with Familial Hypercholesterolemia

have high levels of Lp(a). If a parent has been

diagnosed with high Lp(a) then their children will

have a one in two chance of inheriting it also. An

Lp(a) test is recommended for families where heart

attacks and stroke are common, especially at a

young age and where no additional risk factors are

known to be a contributing factor.

Yet despite the above evidence and the

recommendation of screening for elevated Lp(a)

from the European Atherosclerosis Society,

measurement of Lp(a) is not conducted in clinical

practice. This is largely due to the major challenge

associated with Lp(a) measurement and the size

variation of apo(a) with Lp(a). Dependent upon the

size of apo(a) in the assays calibrator, many assays

under or overestimate apo(a) size in the patient

sample which can lead to patient misclassification.

The Randox assay is one of the only methodologies

on the market that detects the non-variable part of

the Lp(a) molecule and therefore suffers minimal

size related bias; providing accurate and consistent

results. The Randox Lp(a) kit is standardised to

the WHO/IFCC reference material SRM 2B and

is closest in terms of agreement to the ELISA

reference method.

Clinical significance of Lp(a)

Lp(a) determination is intended for use in

conjunction with clinical evaluation, patient risk

assessment and other lipid tests to evaluate

disorders of lipid metabolism and to assess

coronary heart disease risk in specific populations.

Evidence suggests that between 60-70% of African

populations are estimated to have between 250-

300mg/L levels of Lp(a) putting them at greater

risk of developing CVD compared to Chinese

and Japanese populations; where only 30% of the

population is estimated to have between 250-

300mg/L levels of Lp(a).

Scientific Study

This study reports the development of an

immunoturbidimetric assay kit with enhanced

precision and accuracy for the measurement of

Lp(a) in human serum and plasma applied to the

fully automated bench top/floor standing analyser

RX monaco. The assay is applied to the fully

automated RX series analysers and is also suitable

for use on many mainstream clinical chemistry

platforms. This is of value as an accurate, stable

and convenient tool for the determination of

Lipoprotein(a) using these automated systems.

Methodology

Agglutination occurs due to an antigen-antibody

reaction between Lp(a) in a sample and anti-

Lp(a) antibody adsorbed to latex particles. This

is detected as an absorbance change at 700 nm

proportional to the concentration of Lp(a) in the

sample. Concentrations are calculated from a multi-

point calibration. On-board and calibration stabilities

were tested by storing the reagents uncapped

on the analyser for a period of 28 days. Within-

run and total precision were assessed by testing

serum samples at defined medical decision levels,

4 replicates twice a day for 20 days. Correlation

studies were conducted using another commercially

available clinical chemistry system.

Prozone was not observed up to 493 mg/dL

RESULTS

Sensitivity and linearity

12/196/LPA

Reagent on-board stability

12/001/LPA

Assay Sensitivity Assay Linearity

Lp(a) 6.18 mg/dL 103 mg/dL

Specimen Type nMean Within-Run Precision Total Precision

mg/dL SD %CV SD %CV

QC Level 1 80 17.71 0.49 2.8 0.66 3.7

Patient sample 80 24.00 0.98 4.1 1.43 5.9

Serum pool 80 29.25 0.6 2.0 0.71 2.4

Calibrator L5 80 72.29 1.27 1.8 2.65 3.7

Specimen Type: Within-Run Precision and Total Precision

Analyte 16 mg/dL 40 mg/dL

Haemoglobin 1000 mg/dL 1000 mg/dL

Free Bilirubin 60 mg/dL 60 mg/dL

Conjugate Bilirubin 60 mg/dL 60 mg/dL

Triglycerides 2000 mg/dL 2000 mg/dL

Intralipid® 2000 mg/dL 2000 mg/dL

Correlation

This method (Y) was compared with another

commercially available method (X) and the following

linear regression equation obtained:

Y = 0.97X + 0.59

and a correlation coefficient of r = 1.00

40 patient samples were analysed spanning the range

6.51 mg/dL to 101.97 mg/dL.

The Randox Lp (a) test kit shows minimum apo

(a) size related bias. Size heterogeneity of apo(a)

can affect to varying degrees the outcome of other

commercially available kits.

Interference

The analytes below were tested up to the following

levels and were found not to interfere at Lp(a)

concentrations of 16 mg/dL and 40 mg/dL.

Analyte Interference Levels at 16mg/dL and 40mg/dL

Correlation Lp(a) assay on RX monaco vs

available clinical chemistry system

FINDINGS The Randox Lp(a) immunoturbidimetric

assay applied to the bench top/floor standing

RX monaco analyser, exhibits high sensitivity and

reproducibility with the added advantage of using

reagents with good stability. This is of value in the

accurate determination of this analyte in human

serum/plasma for clinical and research applications.

The Randox Lp(a) immunoturbidimetric assay is

also available on all fully automated RX analysers

including the RX imola, RX daytona+ and

RX modena.

Further information on the assay and RX analyser

used within this study

Lp(a) features:

• Sample type - Suitable for use with serum

and plasma

• Immunoturbidimetric method - Making it simple

and quick to perform

• Liquid ready-to-use reagents - For ease of use

and convenience

• Excellent stability - All reagents are stable to

expiry date when stored at +2-8ºC or 28 days

on board the analyser at approximately 10°C.

• Extensive measuring range 3-90 mg/dl -

Ensuring concerning levels of Lipoprotein(a) are

comfortably detected

• Superior 5-point calibrator available - Providing

a complete diagnostic testing package

Advantages of the RX analysers for testing:

• Low water consumption

• User friendly software

• High throughput of tests including ISE

(analyser dependent)

• Low sample volume required

• STAT sample functionality

• Dual 5 speed stirrers optimized for each

chemistry reaction

• Reagent micropipette with liquid level sensor

and crash detection

• Liquid level sensor, crash, bubble and clot

detection

Randox Laboratories Ltd, 55 Diamond Road, Crumlin, County Antrim, BT29 4QY, United Kingdom +44 (0) 28 9442 2413 • marketing@randox.com • randox.com/clinical-chemistry-analysers/

Information correct at time of print. Randox Laboratories Ltd is a subsidiary of Randox Holdings Limited a company registered within Northern Ireland with company number N.I. 614690. VAT Registered Number: GB 151 6827 08. Product availability may vary from country to country. Please contact your local Randox representative for information.

Products may be for Research Use Only and not for use in diagnostic procedures in the USA.

REFERENCES

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4. Marcovina, S.M., Albers, J.J., Wijsman, E., Zhang, Z., Chapman, N.H. and Kennedy, H. (1996). Differences in

Lp(a) concentrations and apo(a) polymorphs between black and white Americans. Journal of Lipid Research

37: 2569 – 2585.

5. Jenner, J.L., Ordovas, J.m., Lamon-Fava, S. et al. (1993). Effects of Age, Sex and Menopausal Status on

Plasma Lipoprotein (a) Levels. The Framingham Offspring Study. Circulation 87: 1135 – 1141.

6. Marcovina, S.M., Albers, J.J., Scanu, A.M. et al. (2000) Use of a Reference Material Proposed by the

International Federation of Clinical Chemistry and Laboratory Medicine to Evaluate Analytical Methods for

the Determination of Plasma Lipoprotein (a). Clinical Chemistry 46: (12) 1956 – 1967.

7. Lipoprotein a Foundation. 2018. Understand Inherited Lipoprotein(a). [ONLINE] Available at: http://www.

lipoproteinafoundation.org/?page=UnderstandLpa. [Accessed 10 May 2018].

8. Doc’s Opinion. 2012. Lipoprotein(a). [ONLINE] Available at: https://www.docsopinion.com/health-and-

nutrition/lipids/lipoprotein-a/. [Accessed 10 May 2018].

9. Lipoprotein (a). (2014). 1st ed. [ebook] England: Heart UK - The Cholesterol Charity. Available at: https://

heartuk.org.uk/files/uploads/huk_fs_mfss_lipoprotein_02.pdf [Accessed 10 May 2018].

NOTES:

Information correct at time of print. Randox Laboratories Ltd is a subsidiary of Randox Holdings Limited a company registered within Northern Ireland with company number N.I. 614690. VAT Registered Number: GB 151 6827 08. Product availability may vary from country to country. Please contact your local Randox representative for information. Products may be for Research Use Only and not for use in diagnostic procedures in the USA.