Cardiovascular Disease (Non-traditional Risk Markers) - Risk Markers

Indications for Testing

Known intermediate to high risk for CVD

Laboratory Testing

hs-CRP assay
- Should be used for atherosclerotic risk assessment
  - Order in patients with intermediate 10-year risk (10-year predicted risk ≥5%) as assessed by Framingham risk score (see Framingham Cardiac Risk calculator); otherwise, this test is not recommended
  - Combine with Lp-PLA₂ to increase accuracy of risk prediction
- hs-CRP results used to assign risk
  - <1.0 mg/L = low risk
  - 1.1-3.0 mg/L = average risk
  - 3.1-9.9 mg/L = high risk
  - ≥10 mg/L = very high risk
- If initial value is >3.0 but <10 mg/L, repeat in 2 weeks
Lp-PLA₂
- Use for atherosclerotic risk assessment
- Order in addition to hs-CRP in patients with intermediate to high 10-year risk (defined as borderline LDL-C 131 mg/dL or HDL-C 39 mg/dL) as assessed by Framingham risk score
Chronic kidney markers
- No recommended testing if 10-year predicted risk as assessed by Framingham risk score is <5%
  - Estimated GFR and microalbumin testing in individuals with hypertension, diabetes mellitus (DM), CVD and/or family history of CVD
  - Perform GFR and serum creatinine for all patients >65 years
APOE mutations
- Consider for primary hypercholesterolemias/hyperlipidemias diagnosis
- Addition of APOE to existing risk models does not enhance predictive power of models; do not use routinely for assessment of cardiac risk
Apo A-1 and B
- Addition of Apo A-1 and B to existing risk models does not enhance predictive power of models; do not use routinely for assessment of cardiac risk
  - May be used in addition to LDL-C monitoring as a non-HDL-C marker (LDL+IDL+VLDL) in patients with serum triglycerides ≥200 mg/dL
    - Apo B/Apo A-1 ratio may also be used as cholesterol/HDL-C ratio when used in scenario above
  - APOB mutation assessment is appropriate in inherited hypercholesterolemias
Homocysteine
- Addition of homocysteine to existing risk models does not enhance predictive power of models; do not use routinely for assessment of cardiac risk
  - Homocysteine levels
    - ≤10 μmol/L – desirable
    - >10 μmol/L to <15 μmol/L – intermediate
    - ≥15 to 30 μmol/L – high
    - ≥30 μmol/L – very high
Lp(a)
- Addition of Lp(a) to existing risk models does not enhance predictive power of models; do not use routinely for assessment of cardiac risk
- Possible clinical uses (in patients with intermediate risk category)
  - Established atherosclerotic disease with normal lipid profiles
  - Recurrent arterial stenosis
  - Hyperlipidemia refractory to therapy
NTproBNP/BNP
- Addition of NTproBNP/BNP to existing risk models does not enhance the predictive power of models; do not use routinely for assessment of cardiac risk

Most biomarkers are not recommended for routine use or for risk stratification of CVD and should not be ordered prior to determining Framingham risk score
May be useful in patients with intermediate 10-year risk using Framingham risk score of 10-20% for further risk stratification

Insufficient data to recommend use of most current non-traditional markers for monitoring therapy
Apo B can be used as an alternative to non-HDL-C levels in monitoring
- Goal is 80 mg/dL for patients with CVD or diabetes mellitus and one risk factor; 90 mg/dL if no CVD but 2 risk factors

Cardiovascular disease (CVD) is a major cause of morbidity and mortality in the U.S. Novel biomarkers can be valuable additions to standard risk factors for CVD.

For further clinical background information regarding cardiovascular disease, refer to cardiovascular disease (traditional risk markers)

Biomarkers of cardiovascular disease

Cardiovascular Biomarkers for the Detection of Cardiovascular Disease
Atheroscleroses	Traditional markers: Total cholesterol LDL-C/HDL-C Total cholesterol/HDL-C ratio Triglycerides Novel markers: Lp(a) Homocysteine Apolipoproteins (Apo A, B, E)
Inflammation	Novel markers: Acute phase reactants hsCRP SAA Cytokines IL-6, IL-8, IL-18 TNF-α receptors 1&2 Leukocytes (neutrophils)/platelets, endothelium ICAM-1 VCAM-1 P-selectin, E-selectin Lp-PLA₂ MPO CD40L MCP-2 Coagulation/fibrinolysis Factor VIII vWF PIGF PAPP-A
Ischemia/Necrosis	Traditional markers: Troponins I & T CK-MB Lactate Myoglobin Novel markers: FABP GP-BB IMA FFAu WB-Chol Sphingomyosin MDA-LDL-C
Myocardial Dysfunction	Traditional markers: NTproBNP/BNP Novel markers: ST2
BNP = b-type natriuretic peptide; CD40L = CD40 ligand; CK-MB = creatine kinase-muscle and brain; CRP = C-reactive protein; hs-CRP = high-sensitivity CRP; FABP = fatty acid binding protein; FFAu = unbound free fatty acid; GP-BB = glycogen phosphorylase - brain; IL = interleukin; HDL-C = high-density lipoprotein-cholesterol; ICAM = inter-cellular adhesion molecule; IMA = ischemia-modified albumin; LD = lactate dehydrogenase; LDL-C = low-density lipoprotein-cholesterol; Lp(a) = lipoprotein a; Lp-PLA₂ = lipoprotein-associated phospholipase A2; MCP-2 = monocyte chemoattractant protein-2; MDA-LDL-C = malondialdehyde-modified LDL-C; MPO = myeloperoxidase; NTproBNP = N-terminal proBNP; PAPP-A = pregnancy-associated plasma protein A; PIGF = placental-derived growth factor; SAA = serum amyloid A; ST2 = soluble member of the interleukin-1 receptor family; TNF-α = tumor necrosis factor alpha; VCAM = vascular cell adhesion molecule; vWF = von Willebrand Factor; WB-Chol = whole blood choline

Recent Focus in Research for Novel Markers of CVD

hs-CRP

hs-CRP
CRP is the most sensitive of the acute phase reactant proteins and is elevated in the following Acute insults Elevates <2 hours in Myocardial infarction Trauma Surgery Infection Peaks and begins to decrease within 48 hours of acute insult if no other inflammatory event occurs Arterial disease hs-CRP is a marker of existing arterial disease and future risk Healthy individuals with hs-CRP results in highest quartile (upper 25%) have 2-fold greater risk of developing atherosclerotic disease as compared with results in the lowest quartile Basal CRP concentration remains stable over long periods of time

hs-CRP

CRP is the most sensitive of the acute phase reactant proteins and is elevated in the following

Acute insults
- Elevates <2 hours in
  - Myocardial infarction
  - Trauma
  - Surgery
  - Infection
- Peaks and begins to decrease within 48 hours of acute insult if no other inflammatory event occurs
Arterial disease
- hs-CRP is a marker of existing arterial disease and future risk
- Healthy individuals with hs-CRP results in highest quartile (upper 25%) have 2-fold greater risk of developing atherosclerotic disease as compared with results in the lowest quartile
- Basal CRP concentration remains stable over long periods of time

Apolipoproteins

Apolipoproteins
Apo A1 Primarily found in HDL-C where it activates lecithin acyltransferase, resulting in the removal of free cholesterol from extrahepatic tissues Elevated Apo A1 levels may protect against CVD and peripheral vascular disease Reduced levels of Apo A1 are one of the most reliable predictors of CVD
Apo B Main apolipoprotein of LDL-C and chylomicrons Apo B makes cholesterol soluble for transport, leading to arterial deposition Increased levels of LDL-C are associated with an increased risk of CVD Autosomal dominant familial hypercholesterolemia type B is caused by mutations in the APOB gene 40% of male and 20% of female APOB mutation heterozygotes develop CVD or hypercholesterolemia Homozygotes or compound heterozygotes for two APOB mutations are at highest risk for hypercholesterolemia and CVD Apo B is a surrogate marker for measuring non-LDL-C
Apo E Present on plasma lipoproteins, including chylomicrons, very low density lipoprotein (VLDL), and HDL-C Serves as the ligand for lipoprotein receptors and plays an important role in lipoprotein metabolism Given their differing receptor affinities, the Apo E isoforms alter plasma lipoprotein concentrations to varying degrees APOEE2* allele is associated with lower LDL-C and type III hyperlipoproteinemia APOEE4* allele is associated with increased total and LDL-C Both E2 and E4 alleles are associated with increased plasma triglyceride concentrations

Apolipoproteins

Apo A1

Primarily found in HDL-C where it activates lecithin acyltransferase, resulting in the removal of free cholesterol from extrahepatic tissues
Elevated Apo A1 levels may protect against CVD and peripheral vascular disease
Reduced levels of Apo A1 are one of the most reliable predictors of CVD

Apo B

Main apolipoprotein of LDL-C and chylomicrons
Apo B makes cholesterol soluble for transport, leading to arterial deposition
Increased levels of LDL-C are associated with an increased risk of CVD
Autosomal dominant familial hypercholesterolemia type B is caused by mutations in the APOB gene
40% of male and 20% of female APOB mutation heterozygotes develop CVD or hypercholesterolemia
Homozygotes or compound heterozygotes for two APOB mutations are at highest risk for hypercholesterolemia and CVD
Apo B is a surrogate marker for measuring non-LDL-C

Apo E

Present on plasma lipoproteins, including chylomicrons, very low density lipoprotein (VLDL), and HDL-C
Serves as the ligand for lipoprotein receptors and plays an important role in lipoprotein metabolism
Given their differing receptor affinities, the Apo E isoforms alter plasma lipoprotein concentrations to varying degrees
APOE*E2 allele is associated with lower LDL-C and type III hyperlipoproteinemia
APOE*E4 allele is associated with increased total and LDL-C
Both E2 and E4 alleles are associated with increased plasma triglyceride concentrations

Lp(a)

Lp(a)
Heterogenous family of lipoprotein molecules consisting of an Apo A molecule attached to an Apo B-100 and a lipid-rich, LDL-like core Concentrations that are generally very low are largely under genetic control Elevated concentrations associated with increased risk of myocardial infarction, stroke, and CVD

Homocysteine

Homocysteine
Homocysteine is an amino acid that exists in the blood Moderately elevated homocysteine is an independent predictor for atherosclerosis, CVD and thromboembolism Risk of atherosclerosis increases progressively with increasing concentration of homocysteine 10% of total risk may be attributable to elevated plasma homocysteine Risk ratio of 1.18 for each 5 µmol/L homocysteine elevation (Humphrey 2008) Methylenetetrahydrofolate reductase (MTHFR) is the enzyme involved in homocysteine metabolism Enzyme inactivation causes an increase in plasma homocysteine Most common genetic risk factors for hyperhomocysteinemia are MTHFR mutations C677T and A1298C C677T homozygosity associated with 3-fold risk of early CVD Very high concentrations are associated with homocystinuria, a rare inborn error of metabolism Concentrations usually >50 μmol/L Associated with ocular, skeletal, central nervous system and vascular abnormalities High risk for pulmonary embolism, stroke and myocardial infarction

Homocysteine

Homocysteine is an amino acid that exists in the blood
Moderately elevated homocysteine is an independent predictor for atherosclerosis, CVD and thromboembolism
Risk of atherosclerosis increases progressively with increasing concentration of homocysteine
- 10% of total risk may be attributable to elevated plasma homocysteine
- Risk ratio of 1.18 for each 5 µmol/L homocysteine elevation (Humphrey 2008)
Methylenetetrahydrofolate reductase (MTHFR) is the enzyme involved in homocysteine metabolism
- Enzyme inactivation causes an increase in plasma homocysteine
- Most common genetic risk factors for hyperhomocysteinemia are MTHFR mutations C677T and A1298C
  - C677T homozygosity associated with 3-fold risk of early CVD
Very high concentrations are associated with homocystinuria, a rare inborn error of metabolism
- Concentrations usually >50 μmol/L
- Associated with ocular, skeletal, central nervous system and vascular abnormalities
- High risk for pulmonary embolism, stroke and myocardial infarction

Lp-PLA2

Lp-PLA₂
Lipase enzyme located primarily on the surface of circulating LDL-C Also known as platelet-activating factor acetylhydrolase High levels associated with ischemic stroke; may be useful in risk assessment Proatherogenic Lp-PLA₂ hydrolyzes LDL-C with resulting formation of lysophosphatidylcholine Lysophosphatidylcholine is an attractant for macrophages, T-cells and vascular smooth muscle cells Antiatherogenic Functions to hydrolyze platelet activating factor Arterial disease Independent marker of moderate- and high-risk populations for CVD Use in combination with hs-CRP to increase risk prediction May be most useful in moderate- and high-risk groups with metabolic syndrome Has been found to be associated with ischemic stroke; may be useful in risk assessment

Lp-PLA₂

Lipase enzyme located primarily on the surface of circulating LDL-C
- Also known as platelet-activating factor acetylhydrolase
High levels associated with ischemic stroke; may be useful in risk assessment
Proatherogenic
- Lp-PLA₂ hydrolyzes LDL-C with resulting formation of lysophosphatidylcholine
- Lysophosphatidylcholine is an attractant for macrophages, T-cells and vascular smooth muscle cells
Antiatherogenic
- Functions to hydrolyze platelet activating factor
Arterial disease
- Independent marker of moderate- and high-risk populations for CVD
- Use in combination with hs-CRP to increase risk prediction
  - May be most useful in moderate- and high-risk groups with metabolic syndrome
- Has been found to be associated with ischemic stroke; may be useful in risk assessment

ST2

ST2
Member of the interleukin-1 receptor family Secreted by myocytes under mechanical strain Elevated concentrations of soluble ST2 occur in patients with heart failure and are strongly predictive of mortality

Kidney disease markers – serum creatinine, cystatin, urine microalbumin

Chronic Kidney Disease (CKD)
Independent kidney disease markers for CVD risk Serum creatinine – estimated glomerular filtration rate (GFR) Refer to GFR calculator <60 mL/min/1.73 m² defined as CKD Urine microalbumin Used in WHO definition of metabolic syndrome; may be strong predictor of CVD risk Cystatin May be a more powerful predictor than GFR, but needs more evaluation

Chronic Kidney Disease (CKD)

Independent kidney disease markers for CVD risk

Serum creatinine – estimated glomerular filtration rate (GFR)
- Refer to GFR calculator
- <60 mL/min/1.73 m² defined as CKD
Urine microalbumin
- Used in WHO definition of metabolic syndrome; may be strong predictor of CVD risk
Cystatin
- May be a more powerful predictor than GFR, but needs more evaluation

Other, less-well-defined markers are also being researched, including IL-6, TNF-α, MPO and CD40L

Tests generally appear in the order most useful for common clinical situations

Test name: C-Reactive Protein, High Sensitivity

ARUP #: 0050182

Methodology: Quantitative Immunoturbidimetric

Use:

Use as a marker of low-level inflammation in CVD risk prediction

Recommend use only if patient has intermediate 10-year risk of >5% using Framingham risk score

Order in conjunction with Lp-PLA₂ to enhance cardiovascular risk prediction

Limitations:

Should not be performed during acute illness

Follow-up:

If first result is >3.0 mg/L but >10 mg/L, recommend repeating test at least 2 weeks later when patient is in metabolically stable state free of infection or acute illness

The lower of the two results should be used to determine patient’s risk

Test name: Apolipoprotein A-1

ARUP #: 0050030

Methodology: Quantitative Nephelometry

Use:

Deficient level of Apo A-1 is highly reliable predictor of CVD

Diagnose analphalipoproteinemia (Tangier disease)

May be used with LDL-C for monitoring

Limitations:

Addition of Apo A-1 to existing risk models does not enhance predictive power of models for most patients

Do not use routinely for assessment of cardiac risk

Test name: Apolipoprotein B/A Ratio

ARUP #: 0050028

Methodology: Quantitative Nephelometry/Calculation

Use:

May be used in addition to LDL-C monitoring as a non-HDL-C marker in patients with serum triglycerides greater than or equal to 200 mg/dL

May also be used as cholesterol/HDL-C ratio

Limitations:

Addition of Apo A-1 and B to existing risk models does not enhance predictive power of models for most patients

Do not use routinely for assessment of cardiac risk

Test name: Apolipoprotein B

ARUP #: 0050029

Methodology: Quantitative Nephelometry

Use: Diagnose abetalipoproteinemia

Limitations:

Addition of Apo B to existing risk models does not enhance predictive power of models

Do not use routinely for assessment of cardiac risk

Test name: Apolipoprotein B (APOB) Mutation Detection

ARUP #: 0055654

Methodology: 3Polymerase Chain Reaction/Fluorescence Monitoring

Use:

Identify an inherited cause for hypercholesterolemia

Confirm diagnosis of familial defective APOB-100 (FDB)

Screen individuals who are at risk for APOB mutation (positive family history of FDB)

Limitations:

Not recommended for nonsymptomatic patients <18 years

Mutations in other genes or other mutations in the APOB gene that may cause familial hypercholesterolemia or increased risk for CVD are not ruled out

Test name: Apolipoprotein E (APOE) 2 Mutations, Cardiovascular Risk

ARUP #: 0055566

Methodology: Polymerase Chain Reaction/Fluorescence Monitoring

Use:

Consider for primary hypercholesterolemias/hyperlipidemias diagnosis

Mutations tested: E2, E3 (normal) and E4 alleles of the APOE gene

Clinical sensitivity: ~5% of individuals with early CVD are homozygous for E2

Limitations:

Addition of APOE to existing risk models does not enhance predictive power of models for most patients

Do not use routinely for assessment of cardiac risk

Low sensitivity and specificity in predicting CVD

APOE genotypes are associated with hyperlipoproteinemia; however, genotype alone is not sufficient for diagnosis of these disorders

Not recommended for nonsymptomatic patients <18 years old

Rare isoforms of APOE will not be detected; if rare alleles are suspected, phenotyping by isoelectric focusing may be indicated

Rare diagnostic errors can occur due to primer site mutations

Test name: Glomerular Filtration Rate, Estimated

ARUP #: 0020725

Methodology: Quantitative Enzymatic/Calculation

Use:

Recommend for individuals with hypertension, DM, CVD or family history of CVD

Test name: Microalbumin, Urine

ARUP #: 0050203

Methodology: Quantitative Immunoturbidimetric

Use:

Recommend for individuals with hypertension, DM, CVD or family history of CVD

Test name: Creatinine, Serum or Plasma

ARUP #: 0020025

Methodology: Quantitative Enzymatic

Use:

Recommend for individuals with hypertension, DM, CVD or family history of CVD

Test name: Lipoprotein-Associated Phospholipase A₂ (PLAC®)

ARUP #: 0081055

Methodology: Quantitative Enzyme-Linked Immunosorbent Assay

Use:

Order in conjunction with hsCRP in patients with intermediate to high 10-year risk to enhance CVD risk prediction

Test name: Lipoprotein (a)

ARUP #: 0099174

Methodology: Quantitative Immunoturbidimetric

Use: Most useful in patients with early CVD or family history of early CVD

Limitations:

Addition to existing risk models does not enhance predictive power of models for most patients

Do not use routinely for assessment of cardiac risk

Test name: Homocysteine, Total

ARUP #: 0099869

Methodology: Quantitative Enzymatic

Use:

Measure homocysteine levels for estimated risk level

Limitations:

Addition to existing risk models does not enhance predictive power of models for most patients

Do not use routinely for assessment of cardiac risk

Fasting sample recommended

Additional Tests Available

Test name: VAP Cholesterol

ARUP #: 0095263

Methodology: Ultra Centrifugation

Comments:

Test name: LDL Subclasses

ARUP #: 0050021

Methodology: Quantitative Electrophoresis

Comments:

Test name: Methylenetetrahydrofolate Reductase (MTHFR) 2 Mutations

ARUP #: 0055655

Methodology: Polymerase Chain Reaction/Fluorescence Monitoring

Comments:

Test name: C-Reactive Protein

ARUP #: 0050180

Methodology: Quantitative Immunoturbidimetric

Comments:

Should not be used for CVD risk assessment

Test name: LDL Cholesterol, Direct

ARUP #: 0020257

Methodology: Quantitative Detergent Solubilization/ Enzymatic

Comments:

Test name: HDL Cholesterol

ARUP #: 0020053

Methodology: Detergent Solubilization, Enzymatic

Comments:

Test name: Lipoprotein Electrophoresis

ARUP #: 0080503

Methodology: Qualitative Electrophoresis/Quantitative Enzymatic /Detergent Solubilization

Comments:

Test name: ST2, Soluble

ARUP #: 2002270

Methodology: Quantitative Enzyme Immunoassay

Comments:

Test name: NMR LipoProfile® Test

ARUP #: 2002043

Methodology: Quantitative Nuclear Magnetic Resonance Spectroscopy

Comments:

Test name: proBrain Natriuretic Peptide, NT

ARUP #: 0050083

Methodology: Quantitative Electrochemiluminescent Immunoassay

Comments:

Test name: Cystatin C, Serum

ARUP #: 0095229

Methodology: Quantitative Nephelometry

Comments:

Guidelines

General

ARUP Institute for Clinical and Experimental Pathology®

Comprehensive Review: April 2011
Last Update: April 2011

Acute Phase Proteins

Cardiovascular Disease (Traditional Risk Markers)

Diabetes Mellitus

Heart Failure

Ischemic Heart Disease

Metabolic Syndrome

Renal Function Markers