Cardiovascular Disease Risk Tests - Medical Clinical Policy Bulletins (2024)

Number:0381

Table Of Contents

Policy
Applicable CPT / HCPCS / ICD-10 Codes
Background
References

Policy

Scope of Policy

This Clinical Policy Bulletin addresses cardiovascular disease risk tests.

1. Medical Necessity

   1. High-Sensitivity C-Reactive Protein (hs-CRP)

      Aetna considers high-sensitivity C-reactive protein (hs-CRP) testing medically necessary for members who meetallof the following criteria:
      
      1. Member has 2 or more coronary heart disease (CHD) major risk factorsFootnote1*,and
      2. Member has low-density lipoprotein (LDL) cholesterol levels between 100 to 130 mg/dL;and
      3. Member has been judged to be at an intermediate-risk of cardiovascular disease by global risk assessment (i.e., 10 to 20% risk of CHD per 10 years using Framingham point scoringFootnote2**).

      Footnote1*Major risk factors include the following:

      1. Age (men aged 45 years or older; women aged 55 years or older)
      2. Current cigarette smoking
      3. Family history of premature CHD (CHD in male first-degree relative less than 55 years of age; CHD in female first-degree relative less than 65 years of age)
      4. Hypertension (blood pressure [BP] of 140 mm Hg or higher, or on anti-hypertensive medication)
      5. Low high-density lipoprotein (HDL) cholesterol (less than 40 mg/dL).

      Footnote2**Note: Framingham risk scoring for men and women is presented in theAppendixbelow.

      Aetna considers hs-CRP testing experimental and investigational for all other indications, including use as a screening test for the general population and for monitoring response to therapy, because its clinical value for these uses has not been established.

   2. Apolipoprotein B (apo B)

      Aetna considers measurement of apolipoprotein B (apoB) medically necessary for use in high-risk persons with hypercholesterolemia to assess whether additional intense interventions are necessary when LDL cholesterol goals are reached (LDL cholesterol less than70 mg/dL and non-HDL cholesterol less than 100 mg/dL in persons with known cardio-vascular disease (CVD) or diabetes mellitus, or LDL-C less than 100 mg/dL and non-HDL cholesterol less than 130 mg/dL in persons with other risk factors). High-risk persons are those withone or more of the following criteria:

      1. Diabetes mellitus;or
      2. Known CVD;or
      3. Two or more of the following CVD risk factors:
         1. Current cigarette smoking;or
         2. Family history of premature CVD (CHD in male first-degree relative less than 55 years of age; CHD in female first-degree relative less than 65 years of age);or
         3. Hypertension (BP of 140 mm Hg or higher, or on anti-hypertensive medication).

      Aetna considers measurement of apolipoprotein B (apoB) experimental and investigational for all other indications because its clinical value for other indications has not been established.

   3. hom*ocysteine Testing

      Aetna considers hom*ocysteine testing may be medically necessary for the following indications:

      1. Evaluating persons with hom*ocystinuria (cystathionine beta synthase deficiency);
      2. Evaluating persons with coagulation disorders (e.g., unexplained thrombotic disorders such as deep venous thrombosis or pulmonary embolism);and
      3. Evaluating persons with borderline vitamin B12 deficiency.

2. Experimental and Investigational

   1. hom*ocysteine Testing

      Aetna considers hom*ocysteine testing experimental and investigationalfor the following indications:

      1. Assessing CHD or stroke risk and for evaluating women with recurrent pregnancy loss;
      2. hom*ocysteine / lipoprotein(a) testing for evaluation of arterial thrombosis in newborns.

      Aetna considers hom*ocysteine testing experimental and investigational for all other indications because its effectiveness for indications other than the ones listed in Section I above has not been established.

   2. Measurement of Carotid Intima-Media Thickness

      Aetna considers measurement of carotid intima-media thickness experimental and investigational for assessing CHD risk because its effectiveness has not been established.

   3. Noninvasive Measurement of Arterial Elasticity

      Aetna considers noninvasive measurements of arterial elasticity by means of blood pressure waveforms (e.g.,CardioVision MS-2000, CVProfilor, Digital Pulse Analyzer (DPA), DSI Pulse Wave Velocity analysis, Max Pulse and HD/PulseWave CR-2000) and noninvasivecalculation and analysisof central arterial pressure waveforms (SphygmoCor) experimental and investigational for assessing CHD risk becausetheir effectiveness has not been established.

   4. Peripheral Arterial Tonometry

      Aetna considers peripheral arterial tonometry (e.g., the Endo-PAT2000/EndoPAT device) experimental and investigational for assessing CHD because there is insufficient evidence to support the effectiveness of this approach.

   5. Cardiac Stress Testing and Stress Echocardiography

      Aetna considers cardiac stress testing and stress echocardiography experimental and investigational for cardiovascular disease risk assessment in asymptomatic low risk individuals.

   6. Ultrasound of the Upper and Lower Extremity Arteries

      Aetna considers ultrasound of the upper and lowerextremity arteriesexperimental and investigational forscreening of persons without signs or symptoms of peripheral arterial disease.

   7. Venous Ultrasound

      Aetna considers venous ultrasound experimental and investigational for screening of persons without signs or symptoms of peripheral venous disease. and who are not at high risk for venous thromboembolic disorders.

   8. Experimental CHD Risk Tests

      Aetna considers any of the following tests / devicesfor assessing CHD risk experimental and investigational because their clinical value has not been established:

      1. Acarix CADScor System
      2. Activated factor VII
      3. Adiponectin
      4. Algorithmically scored multi-protein biomarker panels (i.e., HART CADhs, HART CVE, HART KD)
      5. Angiotensin gene (CardiaRisk AGT)
      6. Anti-thrombin III
      7. Apelin
      8. Apolipoprotein A-I (apo AI) (Boston Heart HDL Map panel)
      9. Apolipoprotein E (apo E)
      10. Apolipopritein E genotyping
      11. ASCVD risk testing (individual or panel) (eg, c-peptide, islet cell antibodies, nonesterified fatty acids (free fatty acids), proinsulin and total insulin)
      12. B-type natriuretic peptides
      13. CADence System
      14. CARDIO inCode-Score
      15. Carotid ultrasound screening of asymptomatic persons for carotid artery stenosis
      16. Cathepsin S
      17. Chromosome 9 polymorphism 9p21
      18. Circulating microRNAs (e.g., miR-1, miR-16, miR-26a, miR-27a, and miR-29a, miR-133a, and miR-199a-5p; not an all-inclusive list)
      19. Coenzyme Q10 (CoQ10)
      20. Coronary artery reactivity test
      21. Corus CAD Gene Expression Profile
      22. Cystatin-C
      23. Endothelin testing
      24. Factor II (thrombin) (F2 gene)
      25. Factor V Leiden (F5 gene)
      26. Fibrinogen
      27. 4q25 genotype testing (eg, 4q25-AF Risk Genotype Test, Cardio IQ 4q25-AF Risk Genotype Test)
      28. Galectin-3
      29. Genetic testing
      30. GlycA (glycosylated acute phase proteins)
      31. Growth stimulation expressed gene 2 (ST2)
      32. HDL subspecies (LpAI, LpAI/AII and/or HDL3 and HDL2)
      33. Interleukin 6 (IL-6)
      34. Interleukin 6 -174 g/c promoter polymorphism
      35. Interleukin 17 gene polymorphism
      36. Interleukin 18 (IL-18)
      37. Kinesin-like protein6 (KLP6)
      38. LDL gradient gel electrophoresis
      39. LDL subspecies (small and large LDL particles)
      40. Leptin
      41. Lipidomic and metabolomic risk markers
      42. Lipoprotein remnants: intermediate density lipoproteins (IDL) and small density lipoproteins
      43. Lipoprotein(a) (Lp(a)) enzyme immunoassay
      44. Lipoprotein-associated phospholipase A2 (Lp-PLA2) (PLAC)
      45. Liposcale test
      46. Long chain omega-3 fatty acids composition in red blood cell
      47. LPA Intron-25 genotype testing (eg, Cardio IQ Intron-25 Genotype Test, LPA Intron-25 Genotype Test)
      48. MaxPulse testing
      49. Measurement of free fatty acids
      50. Methods to determine vascular age
      51. Mid-regional pro-atrial natriuretic peptide
      52. MIRISK VP test
      53. MTHFR genetic testing
      54. Myeloperoxidase (MPO)
      55. NMR Lipoprofile
      56. OmegaCheck Panel
      57. Osteoprotegerin
      58. Oxidized low-density lipoprotein as a biomarker for cardiovascular disease stratification
      59. Oxidized phospholipids
      60. Peroxisome proliferator-activated receptor
      61. Plasma ceramide
      62. Plasma levels of trimethylamine-N-oxide (TMAO)
      63. Plasminogen activator inhibitor (PAI–1)
      64. Pregnancy-associated plasma protein-A (PAPP-A)
      65. Protein C
      66. Prothrombin gene mutation testing
      67. QuantaFlo System for evaluation of peripheral arterial disease
      68. Receptor for advanced glycosylation end products (RAGE) gene Gly82Ser polymorphism testing
      69. Resistin
      70. Retinol binding protein 4 (RBP4)
      71. Serum sterols (eg, Boston Heart Cholesterol Balance Test)
      72. Singulex SMC testing for risk of cardiac dysfunction and vascular inflammation (eg, SMC Endothelin, SMC IL-6, SMC IL 17A, SMC c TnI and SMC TNF-α)
      73. Skin cholesterol (eg, PREVU)
      74. SLCO1B1 (statin induced myopathy genetic testing)
      75. SNP-based testing (eg, Cardiac Healthy Weight DNA Insight, Healthy Woman DNA Insight Test, Heart Health Genetic Test)
      76. Soluble cell adhesion molecules (e.g., intercellular adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1], E-selectin, and P-selectin)
      77. Thromboxane metabolite(s) testing
      78. Tissue plasminogen activator (tPA)
      79. Toll-like receptor 4 (TLR4) Asp299Gly (rs4986790) polymorphism
      80. Transforming growth factor beta
      81. Troponin I (e.g., PATHFAST cTnI-II)
      82. Tumor necrosis factor-alpha (TNF-a)
      83. Total cholesterol content in red blood cell membranes
      84. Vertical Auto Profile(VAP) with or without vertical lipoprotein particle (VLP) technology
      85. Visfatin
      86. von Willebrand factor antigen level.

      The medical literature does not support the utility of the above tests for screening, diagnosis, or management of CHD.

3. Related Policies

   For coverage criteria for PCSK9 inhibitors (alirocumab (Praluent)), see Pharmacy Clinical Policy Bulletin (PCPB) - PCSK9 Inhibitors.

   See also:

   • CPB 0140 - Genetic Testing
   • CPB 0228 - Cardiac CT, Coronary CT Angiography, Calcium Scoring and CT Fractional Flow Reserve
   • CPB 0348 - Recurrent Pregnancy Loss
   • CPB 0485 - Autonomic Testing / Sudomotor Tests
   • CPB 0525 - Screening for Lipid Disorders
   • CPB 0536 - Vitamin B-12 Therapy
   • CPB 0618 - Brain Natriuretic Peptide Testing
   • CPB 0650 - Polymerase Chain Reaction Testing: Selected Indications
   • CPB 0763 - hom*ocysteine Testing.

Background

Cardiovascular disease (CVD) risk testing is utilized to indicate the chances of having a coronary event. The most common tests to determine cardiac risk are high-density lipoprotein (HDL), low-density lipoprotein (LDL), total cholesterol and triglycerides (often referred to as a basic or standard lipid panel).

Non-traditional risk factors for coronary heart disease (CHD) are used increasingly to determine patient risk, in part because of an assumption that many patients with CHD lack traditional risk factors (e.g., cigarette smoking, diabetes, hyperlipidemia, and hypertension).

Hackman and Anand (2003) summarized existing evidence about the connection between atherosclerotic vascular disease and certain nontraditionalCHDrisk factors (abnormal levels of C-reactive protein [CRP], fibrinogen, lipoprotein(a), and hom*ocysteine [Hcy]). The authors conclude that current evidence does not support the notion that non-traditional risk assessment adds overall value to traditional risk assessment. The authors explained that “for each putative risk factor, there must be prospective controlled trials demonstrating that targeting individuals with elevated levels of these risk factors for proven risk-reducing interventions offers advantages over current methods of targeting therapy (e.g., by cholesterol, diabetes, and blood pressure screening); or selectively and specifically reducing the risk factor reduces hard cardiovascular end points, such as mortality, nonfatal myocardial infarction, and stroke.”

Large prospective studies support screening for traditional risk factors. In one study, Greenland et al (2003) assessed major antecedent risk factors among patients who suffered fatal CHD or non-fatal myocardial infarction (MI) while enrolled in 3 prospective cohort studies involving nearly 400,000 patients (age range of18 to 59). Follow-up ranged from 21 to 30 years. Major risk factors were defined as total cholesterol greater than or equal to 240 mg/dL (greater than or equal to 6.22 mmol/L), systolic blood pressure (BP)greater than or equal to 140 mm Hg, diastolic BP greater than or equal to 90 mm Hg, current cigarette smoking, and diabetes. Of patients age 40 to 59 at baseline who died of CHD during the 3 studies, 90 % to 94 % of women and 87 % to 93 % of men had at least 1 major CHD risk factor. In the 1 study that assessed non-fatal MI, at least 1 major risk factor was present in 87 % of women and 92 % of men age 40 to 59.

In another large study (Khot et al, 2003), researchers analyzed data from more than 120,000 patients enrolled in 14 randomizedcontrolled trials (RCTs) to determine the prevalence of baseline conventional risk factors among CHD patients. Of patients with CHD, 85 % of women and 81 % of men had at least 1 conventional risk factor.

As Cantoand Iskandrian (2003) notes, these data challenge the assumption that "only 50 %" of CHD is attributable to conventional risk factors and emphasize the importance of screening for these risk factors and aggressively treating patients who have them.

An assessment by the BlueCross BlueShield Association Technology Evaluation Center (BCBSA, 2005) provided a framework for the evaluation of the potential clinical utility of putative risk factors for cardiovascular disease. The assessment explained that the strongest evidence of the value of such a test is direct evidence that its measurement to assess cardiovascular disease risk results in improved patient outcomes. In the absence of such evidence, the assessment of the potential clinical utility of a test lies in understanding a chain of logic and the evidence supporting those links in the chain. The potential for clinical utility of a test for assessing cardiovascular disease risk lies in following a chain of logic that relies on evidence regarding the ability of a measurement to predict cardiovascular disease beyond that of current risk prediction methods or models, and evidence regarding the utility of risk prediction to treatment of cardiovascular disease. In order to assess the utility of a test in risk prediction, specific recommendations regarding patient management based on the test results should be stated. he assessment notes that another factor that would be important to consider is the availability and reliability of laboratory measurements.

In a report on the use of non-traditional risk factors in CHD risk assessment, the U.S. Preventive Services Task Force (USPSTF, 2009) stated thatthere is insufficient evidence to recommend the use of non-traditional risk factors to screen asymptomatic individuals with no history of CHD to prevent CHD events. Treatment to prevent CHD events by modifying risk factors is currently based on the Framingham risk model. Risk factors not currently part of the Framingham model (i.e., non-traditional risk factors) include high sensitivity CRP (hs-CRP), ankle-brachial index (ABI), leukocyte count, fasting blood glucose level, periodontal disease, carotid intima-media thickness, electron beam computed tomography,Hcy level, and lipoprotein(a) level.

To determine if non-traditional risk factors could play a role in determining those at high- risk for CHD, the USPSTF reviewed the published literature and found the availability and validity of the evidence varied considerably (USPSTF, 2009). They said there is insufficient evidence to determine the percentage of intermediate-risk individuals who would be re-classified by screening with non-traditional risk factors, other than hs-CRP and ABI. For individuals re-classified as high-risk on the basis of hs-CRP or ABI scores, data are not available to determine whether they benefit from additional treatments. In addition, there is not enough information available about the benefits and harms of using non-traditional risk factors in screening. Potential harms include lifelong use of medications without proven benefit and psychological and other harms from being mis-classified in a higher risk category. The USPSTF stated that clinicians should continue to use the Framingham model to assess CHD risk and guide risk-based preventive therapy (USPSTF, 2009).

High Sensitivity C-Reactive Protein (hs-CRP)

C-reactive protein (CRP) is produced by the liver. An elevated CRP level may be indicative of inflammation (nonspecific location). hs-CRP can detect the slight elevations in serum CRP that are associated with coronary artery disease (CAD), which can be within the normal range.

It has been theorized that certain markers of inflammation –both systemic and local – may play a role in the development of atherosclerosis. High sensitivityCRP (hs-CRP) is one systemic marker of inflammation that has been intensively studied and identified as an independent risk factor for coronary artery disease (CAD). Of current inflammatory markers identified, hs-CRP has the analyte and assay characteristics most conducive for use in practice. A Writing Group convened by the American Heart Association and the Centers for Disease Control and Prevention (Pearson et al, 2003) endorsed the optional use of hs-CRP to identify persons without known cardiovascular disease who are at intermediate risk (10 to 20% risk of coronary heart disease over the next 10 years). For these patients, the results of hs-CRP testing may help guide considerations of further evaluation (e.g., imaging, exercise testing) or therapy (e.g., drug therapies with lipid-lowering, anti-platelet, or cardio-protective agents). The Writing Group noted, however, that the benefits of such therapy based on this strategy remain uncertain. High-sensitivity CRP testing is not necessary in high-risk patients who have a 10-year risk of greater than 20 %, as these patients already qualify for intensive medical interventions. Individuals at low-risk (less than 10% per 10 years) will be unlikely to have a high-risk (greater than 20 %) identified through hs-CRP testing. The Writing group recommended screening average risk (10-year risk less than 10 %) for hs-CRP for purposes of cardiovascular risk assessment. The Writing Group stated that hs-CRP also may be useful in estimating prognosis in patients who need secondary preventive care, such as those with stable coronary disease or acute coronary syndromes and those who have underdone percutaneous coronary interventions. The Writing Group posited that this information may be useful in patient counseling because it offers motivation to comply with proven secondary preventive interventions. However, the Writing Group noted that the utility of hs-CRP in secondary prevention is more limited because current guidelines for secondary prevention generally recommend, without measuring hs-CRP, the aggressive application of secondary preventive interventions. The Writing Group recommends measurement of hs-CRP be performed twice (averaging results), optimally2 weeks apart, fasting or non-fasting in metabolically stable patients. Patients with an average hs-CRP level greater than 3.0 mg/dL are considered to be at high relative risk of CHD. Patients with an average hs-CRP level less than 1 mg/L are at low relative risk, and patients with an hs-CRP level between 1.0 and 3.0 mg/L are at average relative risk. If hs-CRP level is greater than 10 mg/dL, the Writing Group recommends that testing should be repeated and the patient examined for sources of infections or inflammation. The Writing group recommended against the measurement of inflammatory markers other than hs-CRP (cytokines, other acute-phase reactants) for determination of coronary risk in addition to hs-CRP.

In an analysis of Women’s Health Study participants, including hs-CRP in cardiovascular disease (CVD)-risk prediction improved the predictive accuracy in non-diabetic women whose traditional 10-year CVD risk was at least 5 %. Cook et al (2006) compared risk-prediction models that include or do not include hs-CRP. The models were applied to 15,048 Women’s Health Study participants who were age 45 or older and free of cardiovascular disease and cancer at baseline. During a mean follow-up of 10 years, 390 women developed CVD. For accurately predicting CVD events, hs-CRP was out-matched only by older age, current smoking, and high blood pressure among traditional Framingham variables. Non-diabetic women were classified according to their 10-year risk for CVD in a model without CRP. Adding CRP to the model substantially improved predictive accuracy for women with an initial 10-year CVD risk of at least 5 %. The gain in accuracy was greatest among women initially classified in the 5 % to 9.9 % risk range: 21.3 % of those women were re-classified in a more accurate risk category when CRP was included in the risk-prediction model (11.9 % moved down a risk category (toless than5 %) and 9.5 % moved up a risk category (to 10 % to 19.9 %)). Accounting for the predictive value of older age, smoking, and high BP lessened the predictive contribution of CRP but still left CRP ahead of any cholesterol parameter (total, LDL, or HDL).

In a nested, case-control study of 122 cases and 244 controls drawn from a cohort of Women's Health Study participants, Ridker et al (2000) assessed the risk for CVD according to levels of4 inflammatory markers:hs-CRP, serum amyloid A, interleukin-6, and soluble intercellular adhesion molecule type-1 (sICAM-1). hom*ocysteine and several lipid and lipoprotein fractions (including apolipoprotein A-I, apolipoprotein B-100, lipoprotein(a), total cholesterol and HDL cholesterol) were measured. Outcomes included fatal CHD, non-fatal MI, stroke, or coronary re-vascularization procedures. Overall, hs-CRP showed the strongest univariate association with all markers studied. Although several other markers studies were univariate predictors of CVD, hs-CRP was the only novel plasma marker that predicted risk in multi-variate analysis. Total cholesterol-to-HDL ratio also predicted risk in multi-variate analysis.

Yeh (2005) noted that as a clinical tool for assessment of cardiovascular risk, hs-CRP testing enhances information provided by lipid screening or global risk assessment. While statin therapy and other interventions can reduce hs-CRP, whether or not such reductions can actually prevent cardiovascular events is being investigated. This is in agreement with the observation of Nambi and Ballantyne (2005) who stated that studies are now under way to evaluate if targeting patients with high CRP and low LDL cholesterol will have any impact on future cardiovascular events and survival and whether changes in CRP correlate to event reduction.

Evidence from the JUPITER trial suggests that, for people choosing to start statin therapy, reduction in both LDL cholesterol and hsCRP are indicators of successful treatment with statins (Ridker et al, 2009). In an analysis of 15,548 initially healthy men and women participating in the JUPITER trial (87 % of full cohort),investigators prospectively assessed the effects of rosuvastatin versus placebo on rates of non-fatal myocardial infarction, non-fatal stroke, admission for unstable angina, arterial re-vascularisation, or cardiovascular deathduring a maximum follow-up of 5 years (median of1.9 years). Compared with placebo, participants allocated to rosuvastatin who achieved LDL cholesterol less than 1.8 mmol/L had a 55 % reduction in vascular events, and those achieving hsCRP less than 2 mg/L a 62 % reduction. Although LDL cholesterol and hs-CRP reductions were only weakly correlated in individual patients (r values < 0.15),the investigators reported a 65 % reduction in vascular events in participants allocated to rosuvastatin who achieved both LDL cholesterol less than 1.8 mmol/L and hs-CRP less than 2 mg/L, versus a 33 % reduction in those who achieved1 or neither target. In participants who achieved LDL cholesterol less than 1.8 mmol/L and hs-CRP less than 1 mg/L,the investigators founda 79 % reduction. The investigators reported that achieved hs-CRP concentrations were predictive of event rates irrespective of the lipid endpoint used, including the apolipoprotein B to apolipoprotein AI ratio (Ridker et al, 2009).

A meta-analysis found that hsCRP concentration has continuous associations with the risk of coronary heart disease, ischemic stroke, and vascular mortality (Emerging Risk Factors Collaboration, 2010). Investigators assessed the associations of hs-CRP concentration with risk of vascular and non-vascular outcomes under different circ*mstances. Investigators meta-analyzed individual records of 160,309 people without a history of vascular disease (i.e., 1.31 million person-years at risk, 27,769 fatal or non-fatal disease outcomes) from 54 long-term prospective studies. Within-study regression analyses were adjusted for within-person variation in risk factor levels. The investigators found thatlog(e) hs-CRP concentration was linearly associated with several conventional risk factors and inflammatory markers, and nearly log-linearly with the risk of ischemic vascular disease and non-vascular mortality. Risk ratios (RRs) for coronary heart disease per 1 standard deviationhigher log(e) hs-CRP concentration (3-fold higher) were 1.63 (95 % confidence interval (CI): 1.51 to 1.76) when initially adjusted for age and sex only, and 1.37 (1.27 to 1.48) when adjusted further for conventional risk factors; 1.44 (1.32 to 1.57) and 1.27 (1.15 to 1.40) for ischemic stroke; 1.71 (1.53 to 1.91) and 1.55 (1.37 to 1.76) for vascular mortality; and 1.55 (1.41 to 1.69) and 1.54 (1.40 to 1.68) for non-vascular mortality. The investigatorsnoted that RRs were largely unchanged after exclusion of smokers or initial follow-up. After further adjustment for fibrinogen, the corresponding RRs were 1.23 (1.07 to 1.42) for coronary heart disease; 1.32 (1.18 to 1.49) for ischemic stroke; 1.34 (1.18 to 1.52) for vascular mortality; and 1.34 (1.20 to 1.50) for non-vascular mortality. The investigators concluded that hs-CRP concentration has continuous associations with the risk of coronary heart disease, ischemic stroke, vascular mortality, and death from several cancers and lung disease that are each of broadly similar size. The investigators noted that therelevance of hs-CRP to such a range of disorders is unclear. The investigators found that associations with ischemic vascular disease depend considerably on conventional risk factors and other markers of inflammation.

According to guidelines from the National Academy of Clinical Biochemistry (2009), if global risk is intermediate and uncertainty remains as to the use of preventive therapies, hs-CRP measurement might be useful for further stratification into a higher or lower risk category.Guidelines from the American College of Cardiology/American Heart Association (2010) also addressthe selection of patients for statin therapy, stating it can be useful in men 50 years or olderand women 60 years of age or olderwith LDL-C less than 130 mg/dL; not on lipid-lowering, hormone replacement, or immunosuppressant therapy; without clinical coronary heart disease, diabetes, chronic kidney disease, severe inflammatory conditions, or contraindications to statins.

Guidelines from the Canadian Cardiovascular Society (2009, 2013) state that the measurement of hs-CRP is being recommended in men older than 50 years and women older than 60 years of age who are at intermediate risk (10% to 19%) according to their Framinghamrisk score and who do not otherwise qualify for lipid-lowering therapy (i.e., if their LDL-C is less than 3.5 mmol/L). The guidelines explain that the rationale for measuring hs-CRP specifically in these individuals is that we now have class I evidence for the benefit of statin therapy in such individuals, if their hs-CRP is greater than 2.0 mg/L. The guidelinesfound that data from theJUPITER studyshow that statin therapy reduces cardiovascular events (hazard ratio 0.56 [95% CI 0.46 to 0.69]; P<0.00001). The guidelines note, because hs-CRP can be elevated during acute illness, clinical judgment should be exercised in the interpretation of any single measurement of hs-CRP. Canadian Cardiovascular Society guidelines (2013) state that those subjects who meet JUPITER criteria (men greater than50 years andwomen greater than60 years of age and CRP greater than or equal to2 mg/L and LDL greater than 3.5 mmol/L) could be considered for treatment based on the results of that study.

An American Heart Association statement on nontraditional risk factors and biomarkers in cardiovascular disease in youth (Balagopal, et al., 2011) stated: "There currently is no clinical role for measuring CRP routinely in children when assessing or considering therapy for CVD risk factors." The AHA statement explains that, although numerous studies suggest that CRP is elevated in children with higher CVD risk, correlates with the progression of atherosclerotic changes, and tracks, albeit weakly, over 21 years from childhood to adulthood independently of other metabolic and conventional cardiovascular risk factors, it is not yet clear whether high CRP levels during childhood and adolescence lead to an increased risk of CVD in later life. The AHA stated that lifestyle interventions have been shown to decrease CRP in children, and statins reduce CRP in adults. "However, minimal information is available on the effect of statins on CRP in children and youth and, importantly whether lowering CRP in children per se would modify preclinical disease or CVD outcomes."

An assessmentprepared for the Agency for Healthcare Research and Quality (Helfand, et al., 2009) found that, "across all of the criteria listed in the table, C-reactive and electron beam computed tomography scan had the strongest evidence for an independent effect in intermediate-risk individuals, and both reclassify some individuals as high-risk."

An National Heart Lung and Blood Institute (2012) guideline on cardiovascular disease risk in children and adolescentsfound insufficient evidence to recommend the measurement of inflammatory markers in youths.

The American Association of Clinical Endocrinologists (2012) have a 2b recommendation for the use ofhs-CRP to stratify CVD risk in patients with a standard risk assessment that is borderline, or in those with an LDL-C concentration less than 130 mg/dL.

A European consensus guideline (2012)included a strong recommendation thaths-CRP should not be measured in asymptomatic low-risk individuals and high-risk patients to assess 10-year risk of CVD. The guideline included a weak recommendation that high-sensitivity CRP may be measured as part of refined risk assessment in patients with an unusual or moderate CVD risk profile.

Lipoprotein (a) Enzyme Immunoassay

Lipoprotein(a) testing (Lp[a])is an LDL cholesterol particle that is attached to a special protein called apo A. Elevated levels in the blood are purportedly linked to a greater likelihood of atherosclerosis and heart attacks.

The lipoprotein(a) (Lp(a)) enzyme immunoassay have been promoted as an important determinantof CHD risk, and as a guide to drug and diet therapy in patients with established CAD.

Although there is evidence for an association ofLp(a) with cardiovascular disease, there are no data to suggest that more aggressive risk factor modification would improve patient-oriented health outcomes (Pejicand Jamieson, 2007). Furthermore,it is very difficult to modifyLp(a). Some studies suggest that it can be lowered using high doses of niacin, neomycin, or estrogen in women (e.g., Gurakar et al, 1985).

Braunwald et al states “because Lp(a) measurement is not a widely available laboratory determination and the clinical significance of alterations in Lp(a) is not known, the NCEP [National Cholesterol Education Program] does not recommend the routine measurement of this lipoprotein at this time.”

Prospective studies that evaluated Lp(a) as a predictor of cardiovascular events have had conflicting results. Some studies suggested that Lp(a) was an independent risk factor for CHD (Bostom et al, 1994; Bostom et al, 1996; Schaefer et al, 1994; Nguyen et al, 1997; Wald et al, 1994; Cremer et al, 1994; Schwartzman et al, 1998; Ariyo et al, 2003; Shai et al, 2003), while others showed no significant association (Coleman et al, 1992; Ridker et al, 1993; Jauhiainen et al, 1991; Cantin et al, 1998; Nishino et al, 2000). A meta-analysis of 5,436 patients followed for at least1 year concluded that elevated Lp(a) is associated with increased cardiovascular risk (relative risk 1.6; 95 % CI: 1.4 to 1.8) (Danesh et al, 2000).

Hackam and Anand (2003) systematically reviewed the evidence for Lp(a) and concluded that “the use of Lp(a) as a screening tool has some limitations.” Although they identified moderate evidence for its role as an independent risk factor, they found minimal information on its incremental risk, and no prospective clinical outcome studies evaluating its role in management.

Although some studies have linked elevated serum levels ofLp(a) to cardiovascular risk, the clinical utility of this marker has not been established. Suk Danik et al (2006) analyzed data available from a cohort of about 28,000 participants followed for 10 years in the Women’s Health Study. Blood samples that had been frozen at study entry were tested for lipoprotein(a), and incident cardiovascular events were documented during the follow-up period. A total of 26 % of the women had lipoprotein(a) levels greater than 30 mg/dL, which is the level currently considered to confer increased cardiovascular risk. However, only the women in the highest quintile with respect to lipoprotein(a) level (greater than or equal to 44 mg/dL) were more likely to experience cardiovascular events than women in the lowest quintile (hazard ratio [HR], 1.47); thus, a threshold effect was seen. Overall, women with the highest rates of cardiovascular disease were those who had lipoprotein(a) levels at or above the 90th percentile and LDL-C levels at or above the median. These findings indicate that routinely measuring lipoprotein(a) is of little benefit for most women. However, lipoprotein(a) testing might be helpful in the clinical management of women who are at particularly high-risk or who have already experienced a cardiovascular event despite having few or no traditional risk factors. Since lipoprotein(a) is not decreased by lipid-lowering therapies, the mainstay of therapy for cardiovascular risk is still aggressive control of LDL-C levels with a statin or niacin, regardless of a woman’s lipoprotein(a) level.

A study by Ariyo et al (2003) of the predictive value of Lp(a) in the elderly (age greater than 65 years) found that lipoprotein(a) levels have prognostic value for stroke and death in men, but not for CHD in men or for any major vascular outcome in women. However, even the links for stroke and death in men were evident only in the highest compared with the lowest quintile, not in intermediate quintiles. Ariyo et al (2003) prospectively studied 3,972 Cardiovascular Health Study participants (minimum age of 65) who had Lp(a) measurements taken at baseline and did not have vascular disease. Overall, mean baseline Lp(a) levels were slightly higher among women (4.4 mg/dL) than among men (3.9 mg/dL). Median follow-up was 7.4 years. Study participants were placed into quintiles of Lp(a) level (lowest, 0.1 to 1.2 mg/dL; highest, 8.2 to 47.5 mg/dL). In analyses adjusted for other vascular-disease risk factors, elderly women in the highest Lp(a) quintile were no more likely to experience stroke,CHD, death from vascular causes, or death from any cause than were elderly women in the lowest quintile. However, compared with elderly men in the lowest Lp(a) quintile, elderly men in the highest quintile were significantly more likely to experience stroke (HR, 2.92), death from vascular causes (HR, 2.09), and death from any cause (HR, 1.60), but not CHD. The authors concluded that, overall, these results do not appear to support routine measurement of Lp(a) levels in elderly persons.

A meta-analysis found independent but modest associations of Lp(a) concentration with risk of CHD and stroke (Emerging Risk Factors Collaboration, 2009). To assess the relationship of Lp(a) concentration with risk of major vascular and non-vascular outcomes, the investigators examined long-term prospective studies that recorded Lp(a) concentration and subsequent major vascular morbidity and/or cause-specific mortality published between January 1970 and March 2009. Individual records were provided for each of 126,634 participants in 36 prospective studies. During 1.3 million person-years of follow-up, 22,076 first-ever fatal or non-fatal vascular disease outcomes or non-vascular deaths were recorded, including 9,336 CHD outcomes, 1,903 ischemic strokes, 338 hemorrhagic strokes, 751 unclassified strokes, 1,091 other vascular deaths, 8,114 nonvascular deaths, and 242 deaths of unknown cause. Within-study regression analyses were adjusted for within-person variation and combined using meta-analysis. Analyses excluded participants with known pre-existing CHD or stroke at baseline. The investigators reported thatLp(a) concentration was weakly correlated with several conventional vascular risk factors and it was highly consistent within individuals over several years. The investigators also found that associations of Lp(a) with CHD risk were broadly continuous in shape. In the 24 cohort studies, the rates of CHD in the top and bottom thirds of baseline Lp(a) distributions, respectively, were 5.6 (95 % CI: 5.4 to 5.9) per 1,000 person-years and 4.4 (95 % CI: 4.2 to 4.6) per 1,000 person-years. The risk ratio for CHD, adjusted for age and sex only, was 1.16 (95 % CI: 1.11 to 1.22) per 3.5-fold higher usual Lp(a) concentration (i.e., per 1 standard deviation), and it was 1.13 (95 % CI: 1.09 to 1.18) following further adjustment for lipids and other conventional risk factors. The corresponding adjusted risk ratios were 1.10 (95 % CI: 1.02 to 1.18) for ischemic stroke, 1.01 (95 % CI: 0.98 to 1.05) for the aggregate of non-vascular mortality, 1.00 (95 % CI: 0.97 to 1.04) for cancer deaths, and 1.00 (95 % CI: 0.95 to 1.06) for non-vascular deaths other than cancer.

A genetic association study identified2 single nucleotide polymorphisms that were strongly associated with both an increased level of Lp(a) lipoprotein and an increased risk for coronary artery disease, providingsupport for a causal role of Lp(a) lipoprotein in CAD (Clarke et al, 2009). Investigators assessed 2,100 candidate genes in 3,145 case patients with CAD and 3,352 controls. Single-nucleotide polymorphisms (SNPs) mapped to3 chromosomal regions (6q26-27, 9p21, and 1p13) associated with Lp(a) lipoprotein weresignificantly associated with CAD risk. An accompanying editorial (Katherisan, 2009) stated: "Although the appropriate role of plasma Lp(a) lipoprotein in risk assessment remains a subject of debate, there is likely to be increased enthusiasm for measuring plasma Lp(a) lipoprotein levels (and possibly LPA genetic variants) to assess the risk of coronary disease. Additional studies are needed to determine whether knowledge regarding Lp(a) lipoprotein will prove to be clinically useful with respect to risk discrimination, calibration, or reclassification." In particular, the editorialist stated: "To close the loop for plasma Lp(a) lipoprotein from a curiosity to a causal risk factor, a therapeutic intervention that selectively lowers the plasma Lp(a) lipoprotein level will need to be tested in a randomized clinical trial" (Katherisan, 2009).

In a nested case-control study, lipoprotein(a) was found to add little to standard lipid measures and CRP in predicting development of peripheral arterial disease. Ridker et al (2001) had access to baseline plasma samples from 14,916 healthy men from the Physicians' Health Study. Samples from 140 cases who developed symptomatic peripheral arterial disease (PAD) during 9-year follow-up were compared with samples from 140 controls (matched by age, smoking status, and length of follow-up) who did not develop PAD. Eleven standard and novel biomarkers were analyzed. Most biomarkers were significant independent predictors of PAD. Ratio of total cholesterol (TC) to HDL cholesterol was the strongest lipid predictor (adjusted relative risk, 3.9; 95% CI: 1.7 to 8.6); CRP was the strongest non-lipid predictor (adjusted RR, 2.8; 95 % CI: 1.3 to 5.9). In a separate analysis of which novel biomarkers would enhance the predictive power of standard lipid measures (TC and TC/HDL ratio), the inflammatory markers (fibrinogen and CRP) were the only ones to add to it significantly (CRP even more than fibrinogen). As expected, lipoprotein(a) andHcy added little, asdid LDL cholesterol, apolipoprotein A-1, and apolipoprotein B-100.

No universally accepted, standardized method for determination for Lp(a) exists, althougha working group of the International Federation of Clinical Chemistry demonstrated the inaccuracy of Lp(a) values determined by methods sensitive to apo(a) size and recommended the widespread implementation of a proposed reference material for those Lp(a) assays that are validated to be unaffected by apo(a) size heterogeneity (Tate et al, 1998; Tate et al, 1999; Marcovina et al, 2000). Lipoprotein(a) concentrations are unaffected by most available lipid-lowering therapies, with the exception of high-dose nicotinic acid, which is often poorly tolerated. This has made it difficult to demonstrate that Lp(a) plays a direct role in vascular disease, since large-scale controlled intervention studies examining the reduction of Lp(a) and hard cardiovascular end points have not been performed. Lastly, the incremental predictive value of Lp(a) measurement additive to that of traditional screening methods for global risk assessment has not been formally studied.

There is no uniform guideline recommendation for the use of Lp(a) in assessment of cardiovascular disease risk. The U.S. Preventive Services Task Force (USPSTF, 2009)does not recommend the use ofLp(a) for cardiovascular screening. The USPSTF (2009)concluded that there is insufficient evidence to recommend the use of lipoprotein(a) level to screen asymptomatic individuals with no history of CHD to prevent CHD events.

An assessment prepared for the Agency for Healthcare Quality and Research (Helfand, et al., 2009) concluded that "lipoprotein(a) probably provides independent information about coronary heart disease risk, but data about their prevalence and impact when added to Framingham risk score in intermediate-risk individuals are limited."

An assessment by the National Academy of Clinical Biochemistry (Cooper et al, 2009) stated that lipoprotein (a) screening is not warranted for primary prevention and assessment of cardiovascular risk. However, if risk is intermediate (10 % to 20 %) and uncertainty remains as to the use of preventive therapies such as statins or aspirin, then lipoprotein (a) measurement "may be done at the physician’s discretion." The assessment also stated that, after global risk assessment, lipoprotein (a) measurements in patients with a strong family history of premature CVD "may be useful" for identifying individuals having a genetic predisposition of CVD. The assessment stated, however, thatbenefits of therapies based on lipoprotein (a) concentrations are uncertain. If both lipoprotein (a) and LDL-C are highly increased, "an attempt can be made at the physician’s discretion to lower lipoprotein (a) level by lowering the elevated LDL-C." The assessment stated that there is insufficient evidence to support therapeutic monitoring of lipoprotein (a) levels for evaluating the effects of treatment. The assessment also stated that population routine testing for small size apolipoprotein (a) is not warranted.

A consensus statement by the American College of Cardiology (ACC) and the American Diabetes Association (ADA) (Brunzell et al, 2008) concluded that the clinical utility of routine measurement of Lp(a) is unclear, although more aggressive control of other lipoprotein paramters may be warranted in those with high concentrations of Lp(a).

A European consensus statement (2012) found thathigh concentrations of Lp(a) are associated with increased risk of CHD and ischemic stroke, although there is no randomized intervention showing that reducing Lp(a) decreases CVD risk. The guidelines concluded that there is no justification for screening the general population for Lp(a) at present, and no evidence that any value should be considered as a target.

Canadian Cardiovascular Society guidelines (2013) state that measurement of Lp(a) might be of value in additional risk assessment particularly in individuals with a family history of premature vascular disease and familial hypercholesterolemia. The guidelines, however, make no recommendation for use of Lp(a) in cardioavascular disease risk assessment.

Guidelines from the American Academy of Clinical Endocrinology (2012) state that testing for lipoprotein (a) isnot generally recommended, although it may provide useful information to ascribe risk in white patients with CAD or in those with an unexplained family history of early CAD.

Guidelines from the National Heart Lung and Blood Institute (2012) on cardiovascular disease in children and adolescents states thatthere is currently no medication therapy specific for elevated Lp(a), and similar to isolated low HDL–C levels, management may focus on addressing other risk factors and on more aggressively managing concomitant elevations of LDL–C, TG, and non-HDL–C. In adults, niacin will lower Lp(a) approximately 15 percent, but this has not been studied in children.

The Emerging Risk Factors Collaboration (Di Angelantonio, et al., 2012) found, in a study of individuals without known CVD, the addition of information on the combination of apolipoprotein B and A-I, lipoprotein(a), or lipoprotein-associated phospholipase A2 mass to risk scores containing total cholesterol and HDL-C led to slight improvement in CVD prediction. Individual records were available for 165,544 participants without baseline CVD in 37 prospective cohorts (calendar years of recruitment: 1968-2007) with up to 15,126 incident fatal or nonfatal CVD outcomes (10,132 CHD and 4994 stroke outcomes) during a median follow-up of 10.4 years (interquartile range, 7.6-14 years). The investigators assessed discrimination of CVD outcomes and reclassification of participants across predicted 10-year risk categories of low (<10%), intermediate (10%-<20%), and high (≥20%) risk. The addition of information on various lipid-related markers to total cholesterol, HDL-C, and other conventional risk factors yielded improvement in the model's discrimination: C-index change, 0.0006 (95% CI, 0.0002-0.0009) for the combination of apolipoprotein B and A-I; 0.0016 (95% CI, 0.0009-0.0023) for lipoprotein(a); and 0.0018 (95% CI, 0.0010-0.0026) for lipoprotein-associated phospholipase A2 mass. Net reclassification improvements were less than 1% with the addition of each of these markers to risk scores containing conventional risk factors.The investigatorsestimated that for 100,000 adults aged 40 years or older, 15,436 would be initially classified at intermediate risk using conventional risk factors alone. Additional testing with a combination of apolipoprotein B and A-I would reclassify 1.1%; lipoprotein(a), 4.1%; and lipoprotein-associated phospholipase A2 mass, 2.7% of people to a 20% or higher predicted CVD risk category and, therefore, in need of statin treatment under Adult Treatment Panel III guidelines.

O'Donoghue et al (2014) evaluated the prognostic utility of Lp(a) in individuals with CAD. Plasma Lp(a) was measured in 6,708 subjects with CAD from 3 studies; data were then combined with 8 previously published studies for a total of 18,978 subjects. Across the 3 studies, increasing levels of Lp(a) were not associated with the risk of CV events when modeled as a continuous variable (odds ratio [OR]: 1.03 per log-transformed SD, 95 % CI: 0.96 to 1.11) or by quintile (Q5:Q1 OR: 1.05, 95 % CI: 0.83 to 1.34). When data were combined with previously published studies of Lp(a) in secondary prevention, subjects with Lp(a) levels in the highest quantile were at increased risk of CV events (OR: 1.40, 95 % CI: 1.15 to 1.71), but with significant between-study heterogeneity (p = 0.001). When stratified on the basis of LDL cholesterol, the association between Lp(a) and CV events was significant in studies in which average LDL cholesterol was greater than or equal to 130 mg/dl (OR: 1.46, 95 % CI: 1.23 to 1.73, p < 0.001), whereas this relationship did not achieve statistical significance for studies with an average LDL cholesterol less than 130 mg/dl (OR: 1.20, 95 % CI: 0.90 to 1.60, p = 0.21). The authors concluded that Lp(a) is significantly associated with the risk of CV events in patients with established CAD; however, there exists marked heterogeneity across trials. In particular, the prognostic value of Lp(a) in patients with low cholesterol levels remains unclear. The authors stated that “although the current study demonstrates that patients with established CAD who have a high level of Lp(a) are at an increased risk of subsequent major adverse cardiovascular events (MACE), the marked heterogeneity between studies raises questions regarding the value of Lp(a) as a clinically useful biomarker for risk assessment, particularly among patients with well-controlled LDL cholesterol. Moreover, although Lp(a) may directly contribute to CHD, there is currently insufficient evidence to suggest that Lp(a) levels above a discrete cut point should be used to guide therapy or that treatment will translate into improved clinical outcomes”.

Apo [Apolipoprotein] B Testing

An apolipoprotein is any of various proteins that combines with a lipid to form a lipoprotein, such as HDL or LDL. Apolipoproteins are important in the transport of cholesterol in the body and the regulation of the level of cholesterol in cells and blood. Apolipoprotein B (apo B)is the primary apolipoprotein of LDL, which is responsible for carrying cholesterol to tissues.

Each LDL particle has one molecule of apo B per particle. Therefore, the apo B concentration is an indirect measurement of the number of LDL particles, in contrast to LDL cholesterol, which is simply a measure of the cholesterol contained within the LDL. Because apo B is a marker for LDL particle number, the greater or higher the apo B level suggests an increased level of small, dense LDL particles which are thought to be especially atherogenic.

Guidelines from the ACC and the ADA recommend the use of apoB in persons at elevated cardiometabolic risk to assess whether additional intense interventions are necessary when LDL cholesterol goals are reached (Brunzell et al, 2008). According to these guidelines, high-risk persons are those with known CVD, diabetes, or multiple CVD risk factors (i.e., smoking, hypertension, family history of premature CVD). The American Association of Clinical Chemistry has issued similar recommendations regarding the use of apoB (Contois et al, 2009).

The INTERHEART study found the apo B:apo A-1 to be a stronger predictor of MIthan their cholesterol counterparts (McQueen et al, 2008). In this study, 12,461 patients with acuteMI from the world’s major regions and ethnic groups were compared with 14,637 age- and sex-matched controls to assess the contributions of various cardiovascular risk factors. Investigators obtained non-fasting blood samples from 9,345 cases and 12,120 controls and measured cholesterol fractions and apolipoproteins to determine their respective predictive values. Ratios were stronger predictors of MI than were individual components, and apolipoproteins were better predictors than their cholesterol counterparts. Theapo B:apo A-1 ratio was the strongest predictor, with a population-attributable risk of 54 %, compared with risks of 37 % for LDL/HDL and 32 % for total cholesterol/HDL. A 1-standard-deviation increase inapo B:apo A-1was associated with an odds ratio of 1.59 for MI, compared with 1.17 for an equivalent increase in total cholesterol/HDL. The results were similar for both sexes and across all ethnic groups and ages.

Apo B testing has not been validated as a tool for risk assessment in the general population. Astudy found that measuring apo B and apo A-I, the main structural proteins of atherogenic and antiatherogenic lipoproteins and particles, adds little to existing measures ofCADrisk assessment and discrimination in the general population. van der Steeg et al (2007) measured apolipoprotein and lipid levels for 869 cases (individuals who developed fatal or nonfatal CAD) and 1,511 matched controls (individuals who remained CAD-free) over a mean follow-up of 6 years. Upon enrollment, participants were 45 to 79 years old and apparently healthy. Occurrence of CAD during follow-up was determined using a regional health authority database (hospitalizations) and U.K. Office of National Statistics records (deaths). The apo B:apo A-I ratio was associated with future CAD events independent of traditional lipid values, including total cholesterol:HDL cholesterol ratio (adjusted odds ratio, 1.85), and independent of the Framingham risk score (OR, 1.77). However, the apo B:apo A-I ratio did no better than lipid values in discriminating between individuals who would and would not develop CAD, and it added little to the predictive value of the Framingham risk score. In addition, this ratio incorrectly classified 41 % of cases and 50 % of controls.

A large, population-based, cohort study suggests that the apo B:apo A-1 ratio has little clinical utility in predicting incidentCHD in the general population, and that measuring total cholesterol and HDL appears to suffice to determine heart disease risk (Ingelsson et al, 2007). Investigators used a variety of techniques to evaluate the relative utility of apo B, apolipoprotein A-1 (apo A-1), serum total cholesterol, HDL cholesterol, LDL cholesterol, non-HDL cholesterol, and3 lipid ratios in determining risk for CHD, as well as the relative ability of these measures to reclassify CHD risk. More than 3,300 middle-aged, white participants in the Framingham Offspring Study withoutCVD were followed for a median of 15 years. A total of 291 first CHD events occurred, 198 of them in men. In men, elevations in non-HDL cholesterol, apo B, total cholesterol:HDL ratio, LDL:HDL ratio, and apo B:apo A-1 ratio were all significantly associated with increased CHD risk to a similar degree. Elevated apo A-1 and HDL were likewise associated with reduced CHD risk. Women had results similar to those in men except that decreased apo A-1 was not significantly associated with incident CHD. In sex-specific analyses, elevated LDL and total cholesterol were not significantly associated with increased CHD risk in either men or women, perhaps owing to the lack of statistical power of these substudies. In men, total cholesterol:HDL and apo B:apo A-1 ratios both improved reclassification of 10-year risk for CHD; however, the difference between the two was not significant. In women, neither lipid ratio improved CHD risk reclassification.

Canadian Cardiovascular Society guidelines (2009, 2013) recommend apoB as the primary alternate target to LDL-C. The guidelines explain that, based on the available evidence, many experts have concluded that apoB is a better marker than LDL-C for the risk of vascular disease and a better index of the adequacy of LDL-lowering therapy than LDL-C.The guidelines also note that there now appears to be less laboratory error in the determination of apoB than LDL-C, particularly in patients with hypertriglyceridemia, and all clinical laboratories could easily and inexpensively provide standardized measurements of apoB. The guidelines state, however, that not all experts are fully convinced that apoB should be measured routinely and, in any case, apoB is not presently being measured in most clinical laboratories. Consequently, a substantial educational effort for patients and physicians would be required for the most effective introduction of apoB into widespread clinical practice. The guidelines conclude that, despite these reservations, all would agree that physicians who wish to use apoB in their clinical care should be encouraged to do so. Furthermore, the present compromise approach represents a positive transitional phase in the assessment of lipid parameters to improve the prevention of CVD through the clinical measurement of apoB. The guidelines state that apoB target for high-risk subjects is less than 0.80 g/L.

Guidelines from the British Columbia Medical Services Commission (2008)states thatapolipoprotein B (apoB) should be considered for follow-up testing in high-risk patients who are undergoing treatment for hypercholesterolemia (but not for other dyslipidemias). The guidelines state that other lipid tests are not required if using apoB for follow-up.

Guidelines from the American Association of Clinical Endocrinologists (2012) recommend apo B measurements to assess the success of LDL-C–lowering therapy. The guidelines note that LDL particle number as reflected by apo B is a more potent measure of cardiovascular disease (CVD) risk than LDL-C and LDL particle size (e.g., small, dense LDL).

A European consensus statement (2012)reported that, because apoB levels have so frequently been measured in outcome studies in parallel with LDL cholesterol, apoB can be substituted for LDL cholesterol, but it does not add further to the risk assessment.The guidelines found that, based on the available evidence, it appears that apoB is a similar risk marker to LDL cholesterol and a better index of the adequacy of LDL-lowering therapy. Also, there appears to be less laboratory error in the determination of apoB than LDL cholesterol, particularly in patients with hypertriglyceridemia, and laboratories could easily and inexpensively provide standardized measurements of apoB. The guideline stated, however, that apoB is not presently being measured in most laboratories but, if measured, it should be less than 80 and less than100 mg/dL for subjects with very high or high CVD risk, respectively.

Further study is needed to determine the usefulness of apolipoprotein B measurement as an adjunct to risk evaluation by routine lipid measurements in the general population. An assessment prepared for the Agency for Healthcare Research and Quality (Helfand, et al., 2009) concluded that "the contribution of ApoB ...to risk assessment for a first ASCVD event is uncertain at present."

There is emerging evidence of a relationship between apo B and stroke risk. Bhatia et al (2006) assessed the relationships between various lipid subfractions andischemic strokerisk in a cohort of 261 patients after transient ischemic attack (TIA). During 10 years of follow-up, 45 patients experienced ischemic stroke. Apolipoprotein B (Apo B) and Apo B/Apo A1 ratio were the only predictors of stroke.

Standards of Care from the American Diabetes Association (2014) state that some experts recommend a greater focus on non– HDL cholesterol, apolipoprotein B (apoB), or lipoprotein particle measurements to assess residual CVD risk in statin-treated patients who are likely to have small LDL particles, such as people with diabetes, but it is unclear whether clinical management would change with these measurements.

A Working Group of the American Association for Clinical Chemistry (Cole, et al., 2013)found that, in most studies, both apoB and LDL particle number were comparable in association with clinical outcomes, and nearly equivalent in their ability to assess risk for cardiovascular disease. The Working Group stated thatapo B appears to be the preferable biomarker for guideline adoption because of its availability, scalability, standardization, and relatively low cost.

The National Heart, Lung, and Blood Institute’s expert panel on integrated guidelines for cardiovascular health and risk reduction in children and adolescents (2011) stated that “In terms of other lipid measurements:

1. at this time, most but not all studies indicate that measurement of apolipoprotein B (apoB) and apolipoprotein A-1 (apoA–1) for universal screening provides no additional advantage over measuring non-HDL–C, LDL–C, and HDL–C;
2. measurement of lipoprotein(a) (Lp[a]) is useful in the assessment of children with both hemorrhagic and ischemic stroke;
3. in offspring of a parent with premature CVD and no other identifiable risk factors, elevations of apoB, apoA–1, and Lp(a) have been noted; and
4. measurement of lipoprotein subclasses and their sizes by advanced lipoprotein testing has not been shown to have sufficient clinical utility in children at this time (Grade B)”.

Also, UpToDate reviews on “Overview of the possible risk factors for cardiovascular disease” (Wilson, 2014a) and “Estimation of cardiovascular risk in an individual patient without known cardiovascular disease” (Wilson 2014b) do not mention the use of apolipoprotein A-1 (apoA-1) as a management tool.

The Institute for Clinical Systems Improvement’s clinical practice guideline on “Diagnosis and initial treatment of ischemic stroke” (Anderson et al, 2012) did not mention the measurements of markers of cholesterol production (lathosterol and desmosterol) and absorption (beta-sitosterol, campesterol, and cholestanol).

Also, UpToDate reviews on “Overview of the possible risk factors for cardiovascular disease” (Wilson, 2014a) and “Estimation of cardiovascular risk in an individual patient without known cardiovascular disease” (Wilson 2014b) do not mention measurements of markers of cholesterol production (lathosterol and desmosterol) and absorption (beta-sitosterol, campesterol, and cholestanol) as a management tools.

An Endocrine Society practice guideline (Berglund, et al., 2012) states that "The Task Force suggests that measurement of apolipoprotein B (apoB) or lipoprotein(a) [Lp(a)] levels can be of value, whereas measurement of other apolipoprotein levels has little clinical value."

The Emerging Risk Factors Collaboration (Di Angelantonio, et al., 2012) found, in a study of individuals without known CVD, the addition of information on the combination of apolipoprotein B and A-I to risk scores containing total cholesterol and HDL-C led to slight improvement in CVD prediction.The investigators estimated that for 100,000 adults aged 40 years or older, 15,436 would be initially classified at intermediate risk using conventional risk factors alone..The investigators estimated that additional testing with a combination of apolipoprotein B and A-I would reclassify 1.1% of people to a 20% or higher predicted CVD risk category and, therefore, in need of statin treatment under Adult Treatment Panel III guidelines.

Guidelines from the American College of Cardiology and the American Heart Association (Goff, et al., 2014) state that "the contribution of ApoB ...to risk assessment for a first ASCVD event is uncertain at present."

Apolipoprotein E (apo E) Testing

Apolipoprotein E (apo E)is atype of lipoprotein that is a major component of very low density lipoproteins (VLDL). Apo E is essential for the normal catabolism (breaking down) of triglyceride-rich lipoprotein constituents (components). A major function of VLDL is to remove excess cholesterol from the blood and carry it to the liver for processing.

Apo E is essential in the metabolism of cholesterol and triglycerides and helps to clear chyomicrons and very-low-density lipoproteins. Apo E has been studied for many years for its involvement in CVD. Apo E polymorphisms have functional effects on lipoprotein metabolism, and has been studied in disorders associated with elevated cholesterol levels and lipid derangements. The common isoforms of apolipoprotein E (apoE), E2, E3, and E4,have been found tobedeterminants of plasma lipid concentrations, and1 allele of the apoE gene,the epsilon4 (E4)allele is associated withan increasedrisk of coronary heart disease. In addition,the apoE4allele is being investigated as a potential risk factor for Alzheimer's disease and stroke.

Several small studies and an earlier review have demonstrated variation in cholesterol levels and coronary disease risk associated with apo E isoforms. The literature on apo E and CVDwas reviewed by Eichner et al (2002); the investigators concluded that the apo E genotype yields poor predictive values when screening for clinically defined atherosclerosis despite positive, but modest associations with plaque and coronary heart disease outcomes. The value of apo E testing in the diagnosis and management of CHD needs further evaluation.

One study found that smoking increases the risk of coronary heart disease in men of all apo E genotypes, but particularly in men carrying the epsilon4 allele. Humphries et al (2001)investigated whether the effect of smoking on coronary heart disease risk is affected by APOE genotype. The investigatorsenrolled 3,052 middle-aged men who were free of coronary heart disease for prospective cardiovascular surveillance in the second Northwick Park Heart Study (NPHSII). Compared with never-smokers, risk of coronary heart disease in ex-smokers was 1.34 (95 % CI: 0.86 to 2.08) and in smokers it was 1.94 (1.25 to 3.01). This risk was independent of other classic risk factors. In never-smokers, risk was closely similar in men with different genotypes. Risk in men hom*ozygous for the epsilon3 allele was 1.74 (1.10 to 2.77) in ex-smokers and 1.68 (1.01 to 2.83) in smokers, whereas in men carrying the epsilon4 allele risk was 0.84 (0.40 to 1.75) and 3.17 (1.82 to 5.50), respectively, with no significant differences in risk in the epsilon2 carriers. For the epsilon3 group, the genotype effect on risk was no longer significant after adjustment for classic risk factors (including plasma lipids). However, even after adjustment, smokers who were carriers of the epsilon4 allele, showed significantly raised risk of coronary heart disease compared with the non-smoking group (2.79, 1.59 to 4.91, epsilon4-smoking interaction p = 0.007). An accompanying editorial pointed out that it is important to determine how much of the variation in risk for CHD is attributable to the effects of apoE, in order to evaluate the importance of screening forapoE genotype (Wangand Mahaney, 2001).

Bennett et al (2007)conducted a meta-analysis to assess the relation of apo E genotypes to LDL cholesterol (LDL-C) and coronary disease risk. The researchers identified 82 studies of lipid levels (involving data on some 86,000 healthy participants) and 121 studies of coronary outcomes (involving data on some 38,000 cases and 83,000 controls) from both published and unreported sources. Pooling the lipid studies, researchers found a roughly linear relation toward increasing LDL-C levels when apo E genotypes were ordered 2/2, 2/3, 2/4, 3/3, 3/4, 4/4. Participants with the 2/2 genotype had LDL-C levels that were 31 % lower than those with the 4/4 genotype. The associations were weaker between apo E alleles and triglyceride levels or HDL cholesterol levels. Turning to the coronary outcome studies, when the researchers used patients with the most common allele– 3/3– as a reference, they found that carriers of the 2 allele had a 20 % lower risk for coronary disease, while those with the 4 allele had a 6 % increase in risk. Compared with individuals with the most common allele, those with the 2/2 genotype appear to have a 20 % lower risk for coronary heart disease, while those with the 4/4 genotype appear to have a slightly higher risk. A commentator stated that these results are interesting, but the low prevalence of the 2 allele (about 7 % in Western populations) and its association with the development of Parkinson disease make the consequences of these results– and the utility and feasibility of routine screening– uncertain (Foody, 2007).

Available evidence indicates that apo E genotype is a poor predictor of ischemic stoke. Sturgeon and colleagues examined whether apo E genotype alters the risk for ischemic stroke, as previous studies examining whether apo E genotype alters the risk for stroke have yielded conflicting results. In this study, 14,679 individuals in the Atherosclerosis Risk in Communities (ARIC) study were genotyped for apo E. During more than 183,569 person-years of follow-up, 498 participants had an ischemic stroke. After stratifications by sex and race and adjustments for non-lipid risk factors for stroke, no significant relation between apo E genotype and stroke was identified, except for a lower risk associated with APOE-epsilon-2 compared with APOE-epsilon -3 in black women only. The investigators concluded that the apo E genotype is at most a weak factor for ischemic stroke.

The American Association of Clinical Chemistry (AACC, 2009) has stated that the test for apo E is not widely used and it's clinical usefulness is still being researched. Guidelines from the American Association of Clinical Endocrinologists (2012)has a grade 2B recommendationthat assessment of apo AI "may be useful in certain cases."The AACE guidelines state thata normal apo AI level in a patient with low HDL-C suggests the existence of an adequate number of HDL-C particles that contain less cholesterol and may be an indication of less risk.

hom*ocysteine Testing

hom*ocysteine (Hcy) is an amino acid that is found normally in the body. hom*ocysteine is used by the bodyto make protein and to build and maintain tissue.Studies suggest that high blood levels of this substance may increase a person's chance of developing heart disease, stroke, and peripheral artery disease (PAD). It is believed that high levels ofHcy may damage arteries, may make blood more likely to clot, and may make blood vessels less flexible. It is also suggested that treatment consisting of high doses of folic acid, vitamins B6 and B12 decreases a patient'sHcy levels and thus decreases their risk of CVD. However, published study results in the medical literature are conflicting; therefore the usefulness of Hcy testing in reducing CVDrisk and improving patient outcomes has not been demonstrated. ATP III noted the uncertainty about the strength of the relation betweenHcy and CHD, a lack of clinical trials showing that supplemental B vitamins will reduce risk for CHD, and the relatively low prevalence of elevatedHcy in the U.S. population.

In a structured evidence review, Hackam and Anand (2003) found moderate evidence thatHcy is an independent risk predictor of coronary heart, cerebrovascular and peripheral vascular disease. However, the authors found only minimal evidence that Hcy contributes incrementally to risk prediction. The authors also stated that it is unclear whether elevatedHcy is causal or simply a marker of atherosclerotic vascular disease. The authors found few, if any, controlled studies to evaluate risk-reduction strategies for these4 factors. Hackman and Anand (2003) stated “[w]hether hom*ocysteine is causative in the pathogenesis of atherosclerosis, is related to other confounding cardiovascular risk factors, or is a marker of existing vascular disease will have to await the completion of a number of large, randomized controlled trials studying the effect of hom*ocysteine-lowering vitamins on cardiovascular end points.”

An assessment by the Institute for Clinical Systems Improvement (ICSI, 2003) concluded that “[t]he relevance of studies of [plasma hom*ocysteine] as a risk factor for cardiovascular disease is unclear given the decreasing [plasma hom*ocysteine] levels as a result of mandatory folic acid supplementation. It remains unproven whether lowered [plasma hom*ocysteine] levels will result in reduced morbidity and mortality from cardiovascular disease.”

Prospective clinical studies have failed to demonstrate beneficial effects ofHcy- lowering therapy on CVD. An international randomized trial involved 5,522 patients with histories of documented vascular disease (coronary, cerebrovascular, or peripheral) or with diabetes plus another risk factor. Patients received either a combination pill (containing folic acid, vitamin B6, and vitamin B12 or placebo daily (HOPE 2 Investigators, 2006). After 5 years, meanHcy levels were about 25 % lower in the vitamin group than in the placebo group. However, no significant difference was found between groups in the primary endpoint of MI, stroke, or cardiovascular death (18.8% versus 19.8 %; p = 0.41) or in various secondary outcomes. Importantly, vitamin B supplementation did not benefit patients with the highest baselineHcy levels or patients from countries without mandatory folate fortification of food.

In a secondary prevention randomized trial from Norway (Bonaa et al, 2006), 3,749 patients with MI during the preceding 7 days received vitamin B supplements or placebo. During an average follow-up of 3 years, vitamin supplementation conferred no benefit for any clinical outcome.

A randomized controlled clinical trialfound noeffect of treatment with folic acid, vitamin B12 and vitamin B6 for secondary prevention in patients with coronary artery disease or aortic valve stenosis (Ebbing et al, 2008). The researchers reported on a randomized, double-blind controlled trial conducted in the2 university hospitals in western Norway in between 1999 and 2006. A total of 3,096 adult participants undergoing coronary angiographywere randomized. At baseline, 59.3 % had double- or triple-vessel disease, 83.7 % had stable angina pectoris, and 14.9 % had acute coronary syndromes. Study participants were randomly assigned to 1 of 4 groups receiving daily oral treatment with folic acid plus vitamin B12 andvitamin B6; folic acid plus vitamin B12; vitamin B6 alone; or placebo (n = 780). The primary end point of this study was a composite of all-cause death, non-fatal acute MI, acute hospitalization for unstable angina pectoris, and non-fatal thromboembolic stroke. Mean plasma totalHcy concentration was reduced by 30 % after 1 year of treatment in the groups receiving folic acid and vitamin B12. The trial was terminated early because of concern among participants due to preliminary results from a contemporaneous Norwegian trial suggesting adverse effects from the intervention. During a median 38 months of follow-up, the primary end point was experienced by a total of 422 participants (13.7 %): 219 participants (14.2 %) receiving folic acid/vitamin B12versus 203 (13.1 %) not receiving such treatment (HR, 1.09; 95 %CI: 0.90 to 1.32;p = 0.36) and 200 participants (13.0 %) receiving vitamin B6versus 222 (14.3 %) not receiving vitamin B6 (HR, 0.90; 95 %CI: 0.74 to 1.09;p = 0.28). The investigators concluded that this trial did not find an effect of treatment with folic acid, vitamin B12 or vitamin B6 on total mortality or cardiovascular events. The researchers concluded that "[o]ur findings do not support the use of B vitamins as secondary prevention in patients with coronary artery disease."

A randomized trials amongwomenwith and withoutpre-existing CVD failed to support benefits of B-vitamin supplementation on cardiovascular risk (Albert et al, 2008). Within an ongoing RCT of antioxidant vitamins, 5,442 women who were U.S. health professionals aged 42 years or older, with either a history of CVD or 3 or more coronary risk factors, were enrolled in a randomized, double-blind, placebo-controlled trial to receive a combination pill containing folic acid, vitamin B6, and vitamin B12 or a matching placebo, and were treated for 7.3 years from April 1998 through July 2005. The primary endpoint of the study was acomposite outcome of MI, stroke, coronary re-vascularization, or CVD mortality. Compared with placebo, a total of 796 women experienced a confirmed CVD event (406 in the active group and 390 in the placebo group). Patients receiving active vitamin treatment had similar risk for the composite CVD primary end point (226.9/10,000 person-yearsversus 219.2/10,000 person-years for the activeversus placebo group; relative risk (RR), 1.03; 95 % CI: 0.90 to 1.19;p = 0.65), as well as for the secondary outcomes including MI (34.5/10,000 person-years versus 39.5/10,000 person-years; RR, 0.87; 95 % CI: 0.63 to 1.22;p = 0.42), stroke (41.9/10,000 person-yearsversus 36.8/10,000 person-years; RR, 1.14; 95 % CI: 0.82 to 1.57;p = 0.44), and CVD mortality (50.3/10,000 person-yearsversus 49.6/10,000 person-years; RR, 1.01; 95 % CI: 0.76 to 1.35;p = 0.93). In a blood substudy, geometric mean plasmaHcy level was decreased by 18.5 % (95 % CI: 12.5 % to 24.1 %;p < 0.001) in the active group (n = 150) over that observed in the placebo group (n = 150), for a difference of 2.27 micromol/L (95 % CI: 1.54 to 2.96 micromol/L). The researchers concluded that, after 7.3 years of treatment and follow-up, a combination pill of folic acid, vitamin B6, and vitamin B12 did not reduce a combined end point of total cardiovascular events among high-risk women, despite significant Hcy lowering.

Despite the biological plausibility of lower plasma Hcy levels improving endothelial function,a RCT showed no benefit, and actual harm, from B-vitamin supplementation in patients with diabetic nephropathy (House et al, 2010). Hyper-hom*ocysteinemia is frequently observed in patients with diabetic nephropathy. B-vitamin therapy (folic acid, vitamin B(6), and vitamin B(12)) has been shown to lower the plasma concentration of Hcy. In order to determine whether B-vitamin therapy can slow progression of diabetic nephropathy and prevent vascular complications, investigators conducteda multi-center, randomized, double-blind, placebo-controlled trial (Diabetic Intervention with Vitamins to Improve Nephropathy [DIVINe]) at 5 university medical centers in Canada between May 2001 and July 2007 (House et al, 2010). The study involved 238 participants who had type 1 or 2 diabetes and a clinical diagnosis of diabetic nephropathy. Subjects were randomly assigned to receiveB vitamins containing folic acid, vitamin B6, and vitamin B12, or matching placebo. The main outcome measure was a change in radionuclide glomerular filtration rate (GFR) between baseline and 36 months. Secondary outcomes were dialysis and a composite of MI, stroke, re-vascularization, and all-cause mortality. Plasma totalHcy was also measured. The mean (SD) follow-up during the trial was 31.9 (14.4) months; enrollment was ended early by the data and safety monitoring board. At 36 months, the mean decrease in GFR was significantly greater in B-vitamin recipients than in non-recipients, even though plasmaHcy levels declined substantially in treated patients and rose in controls. Treated patients also incurred roughly double the risk for adversecardiovascular events as did controls. At 36 months, radionuclide GFR decreased by a mean (SE) of 16.5 (1.7) mL/min/1.73 m(2) in the B-vitamin group compared with 10.7 (1.7) mL/min/1.73 m(2) in the placebo group (mean difference, -5.8; 95 % CI: -10.6 to -1.1; P = .02). There was no difference in requirement of dialysis (HR, 1.1; 95 % CI: 0.4 to 2.6;p = 0.88). The composite outcome occurred more often in the B-vitamin group (HR, 2.0; 95 % CI: 1.0 to 4.0;p = 0.04). Plasma total Hcy decreased by a mean (SE) of 2.2 (0.4) micromol/L at 36 months in the B-vitamin group compared with a mean (SE) increase of 2.6 (0.4) micromol/L in the placebo group (mean difference, -4.8; 95 % CI: -6.1 to -3.7;p < 0.001, in favor of B vitamins). The authors concluded that, among patients with diabetic nephropathy, high doses of B vitamins compared with placebo resulted in a greater decrease in GFR and an increase in vascular events. Commenting on this study, Schwenk (2010) stated, "[g]iven that most other trials also have shown that B-vitamin supplementation does not prevent stroke and CV disease, such supplements should be avoided unless patient subgroups that derive benefit are identified in future clinical trials."

A long-term RCT involving survivors of MI found that substantial long-term reductions in bloodHcy levels with folic acid and vitamin B12 supplementation did not have beneficial effects on vascular outcomes (Study of the Effectiveness of Additional Reductions in Cholesterol and hom*ocysteine (SEARCH) Collaborative Group, 2010). In this double-blind RCT of 12,064 survivors of MI in secondary care hospitals in the United Kingdom between 1998 and 2008, subjects were randomized to2 mg folic acid plus 1 mg vitamin B12 dailyor tomatching placebo. Study endpoints were first major vascular event, defined as major coronary event (coronary death, MI, or coronary re-vascularization), fatal or non-fatal stroke, or non-coronary re-vascularization. The investigators reported that allocation to the study vitamins reducedHcy by a mean of 3.8 µmol/L (28 %). During 6.7 years of follow-up, major vascular events occurred in 1,537 of 6,033 participants (25.5 %) allocated folic acid plus vitamin B12versus 1,493 of 6,031 participants (24.8 %) allocated placebo (risk ratio [RR], 1.04; 95 % CI: 0.97 to 1.12;p = 0.28). The investigators foundno apparent effects on major coronary events (vitamins, 1,229 [20.4 %],versus placebo, 1,185 [19.6 %]; RR, 1.05; 95 % CI: 0.97 to 1.13), stroke (vitamins, 269 [4.5 %],versus placebo, 265 [4.4 %]; RR, 1.02; 95 % CI: 0.86 to 1.21), or non-coronary revascularizations (vitamins, 178 [3.0 %],versus placebo, 152 [2.5 %]; RR, 1.18; 95 % CI: 0.95 to 1.46). The investigators did not findsignificant differences in the numbers of deaths attributed to vascular causes (vitamins, 578 [9.6 %],versus placebo, 559 [9.3 %]) or non-vascular causes (vitamins, 405 [6.7 %],versus placebo, 392 [6.5 %]). An accompanying commentary by Schwenk (2010) stated: "These results, and those of the seven prior major trials, should end what seems to be an unjustified persistence by many clinicians to recommend folate supplementation to prevent CV disease. Clinical efforts should focus on modification of CV risk factors, for which evidence supports improved outcomes."

These results are consistent with earlier RCTs ofHcy lowering therapy forCVD. In a multi-center double-blind randomized study, Toole et al (2004) enrolled 3,680 patients with non-disabling, non-embolic ischemic strokes and totalHcy levels above the 25th percentile for the North American stroke population. Patients received either high- doses of Hcy-lowering vitamins (2.5 mg folic acid, 25 mg pyridoxine, and 0.4 mg cobalamin) or low doses that would not be expected to lowerHcy significantly (20 µg, 200 µg, and 6 µg, respectively). During 2 years of follow-up, mean total Hcy decreased from 13.4 µmol/L to about 11 µmol/L in the high-dose group and changed only minimally in the control group. However, no reductions were noted in rates of recurrent stroke, coronary events, or death. Even in the subgroup with the highest Hcy levels, high-dose therapy was ineffective.

In an open-label, prospective trial from the Netherlands, Liem et al (2003) randomized 593 consecutive outpatients with CADto folic acid or to standard care. All had been taking statins for at least 3 months. The2 groups had similar baseline characteristics, including mean plasmaHcy levels of 12 µmol/L. By 3 months,Hcy levels had decreased among folic-acid recipients (by 18 %) but not among controls. By a mean follow-up of 24 months, clinical vascular events (i.e., death, MI, stroke, invasive coronary procedures, vascular surgery) had occurred at similar rates in folic-acid (12.3 %) and standard-care (11.2 %) recipients; the similarity also was evident among patients in the highest quartile of baselineHcy level (greater than 13.7 µmol/L). In multi-variate analysis, poor creatinine clearance was a more important cardiovascular risk factor than elevatedHcy level was.

Routine testing forHcy is also not supported in persons with venous thromboembolism. In a secondary analysis of a previously published multi-national RCT designed to assess the effect of Hcy-lowering therapy on the risk for arterial disease (Ray et al, 2007), investigators studied whether daily folate (2.5 mg) and vitamins B6 (50 mg) and B12 (1 mg) affected the risk for symptomatic deep venous thrombosis or pulmonary embolism. Subjects were 5,522 adults (age 55 years and older) with arterial vascular disease, diabetes, and at least1 other CVD risk factor. During a mean follow-up of 5 years,Hcy levels decreased more in the vitamin-therapy group than in the placebo group. However, the incidence of venous thromboembolism did not differ between the vitamin-therapy and placebo groups, both overall and among the quartile with the highestHcy levels (i.e., greater than 13.8 µmol /L) at baseline.

These results were similar to an earlier secondary prevention trial ofHcy for venous thromboembolism (VTE). In the first randomized trial ofHcy therapy to prevent recurrent VTE, den Heijer et al (2007) enrolled 701 patients with recent VTE (either proximal deep-vein thrombosis or pulmonary embolism), but without major predisposing risk factors such as recent surgery or immobilization. At baseline, 50 % the patients had hyper-hom*ocysteinemia (mean, 15.5 µmol/L), and50 %had normal levels (mean, 9.0 µmol/L). Patients were randomized to receive a B-vitamin supplement (5 mg folic acid, 0.4 mg B12, and 50 mg B6) or placebo, in addition to standard anti-coagulation. During 2.5 years of follow-up, the overall incidence of recurrent VTE was not significantly different in the B-vitamin and placebo groups (5.4% versus. 6.4 %). In hyper-hom*ocysteinemic patients, the incidence of recurrent venous thromboembolism was non-significantly higher in B-vitamin recipients than in placebo recipients (6.7% versus 6.0 %); in those with normal Hcy, the incidence of recurrent VTE was non-significantly lower in B-vitamin recipients (4.1 % versus 7.0 %). The authors noted that their study might have been under-powered to detect a small beneficial effect. However, they also speculate that Hcy's observed epidemiologic association with venous thromboembolism might in fact be mediated by some other thrombophilic factor that is correlated with Hcy.

An American Heart Association Science Advisory (Malinow et al, 1999) has concluded: "Although there is considerable epidemiological evidence for a relationship between plasma hom*ocyst(e)ine and cardiovascular disease, not all prospective studies have supported such a relationship …. Until results of controlled clinical trials become available, population-wide screening is not recommended…. Such treatment (supplemental vitamins) is still considered experimental, pending results from intervention trials showing that hom*ocyst(e)ine lowering favorably affects the evolution of arterial occlusive diseases."

A consensus statement from the ACC and the ADA (Brunzell et al, 2008) reported that Hcy testing has been evaluated to determine its prognostic significance in CVD. However, the independent predictive value ofHcy testing and its clinical utility are unclear.

The National Academy of Clinical Biochemistry (Cooperand Pfeiffer, 2009) stated that "we conclude that the clinical application of Hcy measurement for risk assessment of primary prevention of CVD is currently uncertain."

An assessment prepared for the Agency for Healthcare Research and Quality (Helfand, et al., 2009) found that "hom*ocysteine ...probably provide[s] independent information about coronary heart disease risk, but data about their prevalence and impact when added to Framingham risk score in intermediate-risk individuals are limited."

The U.S. Preventive Services Task Force (USPSTF, 2009) stated that there is insufficient evidence to recommend the use ofHcy to screen asymptomatic individuals with no history of CHD to prevent CHD events.

A statement issued by the American Heart Association (AHA, 2010, 2014) states that the AHA does not consider high Hcy levels in the blood to be a major risk factor for cardiovascular disease. The AHA states that a causal link between Hcy levels and atherosclerosis has not been established.

Canadian Cardiovascular Society guidelines (2009, 2013) make no recommendation for hom*ocysteine testing for assessment of cardiovascular disease risk in asymptomatic persons.

Guidelines from the AmericanAssociation of Clinical Endocrinology (2012) does not recommend the routine measurement of hom*ocysteine, noting that several studies have shown no benefit to intervention.

Guidelines from the Royal Australian College of General Practitioners (2012)reported that the value of hom*ocysteine as a risk factor for CHD is uncertain and published RCTs show no evidence of benefit by lowering levels of hom*ocysteine.

Summarizing the evidence for use of hom*ocysteine, a European consensus guideline (2012) stated that hom*ocysteine has shown precision as an independent risk factor for cardiovascular disease. The guidelines state thatmagnitude of hom*ocysteine's effect on risk is modest, and consistency is often lacking, mainly due to nutritional, metabolic (e.g. renal disease), and lifestyle confounders. The guidelines note that, in addition, intervention studies using B vitamins to reduce plasma hom*ocysteine have proven inefficient in reducing risk of cardiovascular disease. The guidelines conclude that, together with the cost of the test, hom*ocysteine remains a "second-line" marker forcardiovascular diseaserisk estimation. The guidelines include a strong recommendation that hom*ocysteine should not be measured to monitor cardiovascular disease risk prevention. The guidelinesinclude a weak recommendationthat hom*ocysteine may be measured as part of a refined risk assessment in patients with an unusual or moderate CVD risk profile.

Veeranna et al (2011) examinedif adding Hcy to a model-based on traditional CVD risk factors improves risk classification. These researchers performed a post-hoc analysis of the MESA (Multi-Ethnic Study of Atherosclerosis) and NHANES III (National Health and Nutrition Examination Survey III) datasets. hom*ocysteine was used to predict composite CVD and hard CHD events in the MESA study and CVD and CHD mortality in the NHANES III survey using adjusted Cox-proportional hazard analysis. Re-classification of CHD events was performed using a net reclassification improvement (NRI) index with a Framingham risk score (FRS) model with and without Hcy. hom*ocysteine level (greater than 15 μmol/L) significantly predicted CVD (adjusted hazard ratio [aHR]: 1.79, 95 % CI: 1.19 to 1.95; p = 0.006) and CHD events (aHR: 2.22, 95 % CI: 1.20 to 4.09; p = 0.01) in the MESA trial and CVD (aHR: 2.72, 95 % CI: 2.01 to 3.68; p < 0.001) and CHD mortality (aHR: 2.61, 95 % CI: 1.83 to 3.73; p < 0.001) in the NHANES III, after adjustments for traditional risk factors and CRP. The level of Hcy, when added to FRS, significantly re-classified 12.9 % and 18.3 % of the overall and 21.2 % and 19.2 % of the intermediate-risk population from the MESA and NHANES cohorts, respectively. The categoryless NRI also showed significant re-classification in both MESA (NRI: 0.35, 95 % CI: 0.17 to 0.53; p < 0.001) and NHANES III (NRI: 0.57, 95 % CI: 0.43 to 0.71; p < 0.001) datasets. The authors concluded that from these 2 disparate population cohorts,they found that addition of Hcy level to FRS significantly improved risk prediction, especially in individuals at intermediate-risk for CHD events.

In an editorial that accompanied the aforementioned study, Mangoni and Woodman (2011) stated that "[i]f Hcy is to be used as a screening tool in primary prevention, it is imperative that further trials are conducted in low- and intermediate-risk patients without previous CVD. Only then can the real value of measuring Hcy as a nontraditional CVD risk factor or risk marker be quantified".

Intermediate Density Lipoproteins

Lipoprotein remnants testingmeasures triglyceride-rich lipoproteins that include intermediate density lipoproteins (IDL) and VLDL. It is proposed that lipoprotein remnants penetrate arterial walls more easily than larger lipoproteins and may be independent risk factors for CVD.

Data from the Framingham Study have suggested that remnant-like particle cholesterol (RLP-C) (intermediate density lipoproteins)is an independent risk factor forCVD in women, and studies have shown that hormone therapy can lower RLP-C levels in healthy post-menopausal women.

The Women's Angiographic Vitaminand Estrogen (WAVE) trial(Bittner et al, 2004) examined whetherhormone therapycan reduce RLP-C and RLP-triglyceride (TG) levels in women with coronary artery disease, and whether these factors predict disease progression. WAVE was a randomized, placebo-controlled, clinical trial ofhormone therapy(conjugated equine estrogen or estrogen plus medroxyprogesterone acetate) and antioxidants in 423 post-menopausal women with angiographic coronary disease; follow-up angiography at 2.8 years showed no benefit withhormone therapyor antioxidants, and no interaction between the two. The WAVE investigators also easured RLP-C and RLP-TG levels in a subset of 397 women. Mean RLP values among the WAVE participants were very high, corresponding to the 90th percentiles in the Framingham cohort. In multi-variate analyses, RLP-C and RLP-TG levels were not related to waist-hip ratio, body mass index (BMI), smoking status, or use of lipid-lowering agents. Compared with placebo,hormone therapydid not significantly reduce RLP levels. Neither baseline RLP levels nor changes in the levels predicted angiographic findings at the end of the study.

The National Cholesterol Education Program Adult Treatment Panel III (ATPIII) Guidelines (2002) state that lipoprotein remnants, including intermediate density lipoproteins (IDLs), as well as very-low-density lipoproteins (VLDL) and small density lipoproteins, have been shown to be atherogenic through several lines of evidence. According to ATPIII, “prospective studies relating various measures to CHD risk are limited, and measurement with specific assays cannot be recommended for routine practice.” The ATPIII panel concluded, however, that the most readily available method of measuring atherogenic triglyceride-rich lipoproteins is measurement of VLDL. A consensus statement by the ACC and the ADA(Brunzell et al, 2008) noted that, although small dense LDL has been shown to be particularly atherogenic, the association of small LDL and cardiovascular disease may simply reflect the increased number of LDL particles in patients with small LDL.

According to guidelines from the American College of Cardiology and the American Heart Association (2010), measurement of lipid parameters, includingparticle size and density, beyond a standard fasting lipid profile is not recommended for cardiovascular risk assessment in asymptomatic adults.

HDL Subspecies

Lipoprotein subfraction testing is testing that separates two of the commonly measured lipoprotein fractions, HDL and LDL, into subclasses based on their size, density and/or electrical charge. HDL subclass testingis suggested to provide information regarding CVD risk when utilized with standard lipoprotein tests, such as total cholesterol, HDL and LDL testing.

HDL comprises several components and subfractions that also have been related to CHD risk. While HDL cholesterol is the risk indicator most often used, HDL subfractions (lipoprotein AI (LpAI) and lipoprotein AI/AII (LpAI/AII) and/or HDL3 and HDL2) have also been used for risk prediction. ATPIII concluded, however, that the superiority of HDL subspecies over HDL cholesterol has not been demonstrated in large, prospective studies. Consequently, ATPIII did not recommend the routine measurement of HDL subspecies in CHD risk assessment. A consensus statement by the ACC and the ADA (Brunzell et al, 2008) state that measurements of HDL subfractions appear to provide little clinical value beyond measurements of HDL cholesterol.

LDL Subspecies (LDL Particle Sizes) and LDL Particle Number

LDL subclass testingis suggested as part of an overall risk assessment for CVD, this test measures the cholesterol content of lipoprotein particles in the blood and determines the LDL particle size and/or density pattern.

Density gradient ultracentrifugation (Vertical Autoprofile (VAP) test) measures the relative distribution of cholesterol within various lipoprotein subfractions, quantifying the cholesterol content of VLDL, IDL, LDL, lipoprotein(a), and HDL subclasses (Mora, 2009). The VAPI also determines the predominant LDL size distribution (eg, A, AB, or B phenotype) but does not provide concentrations of the lipoprotein particles themselves. ApoB is also provided, although it is not measured directly.Some labs offer vertical lipoprotein particle (VLP) technology included with the VAP test to further analyze CVD risk. The VLP technology purportedly reports a true particle number (LDL-P), a proposed biomarker for increased risk of heart disease and stroke.

Nuclear Magnetic Resonance (NMR)Spectroscopy (Liposcience)is based on the concept that each lipoprotein particle in plasma of a given size has its own characteristic lipid methyl group nuclear magnetic resonance (NMR) signal (Mora, 2009). Particle concentrations of lipoprotein subfractions of different size are obtained from the measured amplitudes of their lipid methyl group NMR signals. Lipoprotein particle sizes are then derived from the sum of the diameter of each subclass multiplied by its relative mass percentage based on the amplitude of its methyl NMR signal. The NMR LipoProfile simultaneously quantifies lipoprotein concentrations of VLDL, IDL, LDL, and HDL particles and their subfractions, each expressed as a lipoprotein particle concentration (number of particles per liter) or as an average particle size for each of VLDL, LDL, and HDL.

The gradient gel electrophoresismethoddetermines the distribution of LDL size phenotype by proprietary segmented polyacrylamide gradient gels, which separate lipoproteins in a gradient gel on the basis of their size and, to a lesser extent, their charge (Mora, 2009). Pattern A corresponds to large LDL particles; B to small, dense LDL particles; and AB to an intermediate phenotype. This method gives the relative, or predominant, distribution of lipoprotein particles as determined by the predominant peak particle size.

LDL gradient gel electrophoresis (GGE) has been promoted as an important determinantof CHD risk, and as a guide to drug and diet therapy in patients with established CAD. The measurement of LDL subclass patterns may be useful in elucidating possible atherogenic dyslipemia in patients who have no abnormalities in conventional measurement (total cholesterol, HDL, LDL, and triglycerides). However, the therapeutic usefulness of discovering such subclass abnormalities has not been substantiated.

Ion mobility analysis measures both the size and concentrations of lipoprotein particle subclasses on the basis of gas-phase differential electric mobility.

A number of studies have reported that both larger low-density lipoprotein (LDL) particle size and smaller LDL particle sizes are more atherogenic than intermediate-sized particles, and these particles at the extremes of LDL size may be associated with coronary heart disease (CHD) risk. It is thought that LDL subspecies at both extremes of LDL size and density distribution have a reduced LDL receptor affinity.

Musunuru, et al. (2009) tested whether combinations of lipoprotein subfractions independently predict cardiovascular disease in a prospective cohort of 4594 initially healthy men and women (the Malmö Diet and Cancer Study, mean follow-up 12.2 years, 377 incident cardiovascular events). Plasma lipoproteins and lipoprotein subfractions were measured at baseline with a novel high-resolution ion mobility technique. Principal component analysis (PCA) of subfraction concentrations identified 3 major independent (ie, zero correlation) components of CVD risk, one representing LDL-associated risk, a second representing HDL-associated protection, and the third representing a pattern of decreased large HDL, increased small/medium LDL, and increased triglycerides. The last corresponds to the previously described "atherogenic lipoprotein phenotype." Several genes that may underlie this phenotype-CETP, LIPC, GALNT2, MLXIPL, APOA1/A5, LPL-are suggested by SNPs associated with the combination of small/medium LDL and large HDL. The investigators concluded that principal component analysis on lipoprotein subfractions yielded three independent components of CVD risk. Genetic analyses suggest these components represent independent mechanistic pathways for development of CVD.

ATPIII stated that although the presence of small LDL particles has been associated with an increased risk of CHD, the extent to which small LDL particles predict CHD independent of other risk factors is “controversial.” It has been argued by Campos et al (2002), based on epidemiologic evidence, that the relationship between small LDL and CHD found in some studies is probably due to its correlation with other lipoprotein risk factors, and that small LDL is not an independent risk factor for CHD.

Campos et al (2002) demonstrated in a prospective cohort study that large LDL size is a potential statistically significant predictor of coronary events. Large LDL particles are thought to be large because of high cholesterol ester content. However, Campos reported that the relationship between LDL particle size and coronary events was not present among members of the cohort who were treated with pravastatin, perhaps because pravastatin acts by reducing the size of LDL particles. The author concluded that identifying patients on the basis of LDL size may not be useful clinically, since effective treatment for elevated LDL cholesterol concentrations also effectively treats risk associated with large LDL.

Commenting on LDL particle size, a consensus statement from the ACC and the ADAstated: "The size of LDL particles can also be measured. As small dense LDL particles seem to be particularly atherogenic, assessment of particle size has intuitive appeal. Both LDL particle concentration and LDL size are important predictors of CVD. However, the Multi-Ethnic Study of Atherosclerosis suggested that on multi-variate analyses, both small and large LDL were strongly associated with carotid intima-media thickness [Mora et al, 2007], while the Veterans Affairs High-Density Lipoprotein Cholesterol Intervention Trial (VA-HIT) showed that both were significantly related to coronary heart disease (CHD) events [Otvos et al, 2006]. The association of small LDL and CVD may simply reflect the increased number of LDL particles in patients with small LDL. Hence, it is unclear whether LDL particle size measurements add value to measurement of LDL particle concentration" (Brunzell et al, 2008).

The ACC/ADA consensus statement recommended ApoB measurement over measurement of particle number with NMR (Brunzell et al, 2008): "Limitations of the clinical utility of NMR measurement of LDL particle number or size include the facts that the technique is not widely available and that it is currently relatively expensive. In addition, there is a need for more independent data confirming the accuracy of the method and whether its CVD predictive power is consistent across various ethnicities, ages, and conditions that affect lipid metabolism."

An assessment by the California Technology Assessment Forum (CTAF) (Walsh, 2008)of LDL particle number as assessed by NMR spectroscopy concluded that this test did not meet CTAF's assessment criteria. The CTAFassessment stated that there wereno studies addressing whether or not treated LDL particle levels affected clinical outcomes.

A systematic evidence review ofLDL subfractions, including the methods of gradient gel electrophoresis,NMR spectroscopy, and ultra-centrifugation,prepared for the Federal Agency for Healthcare Research and Quality (AHRQ)concluded that "the data do not adequately answer the question of how strongly LDL subfraction information is associated with CVD [cardiovascular disease], in relation to other known and putative risk factors. In summary, none of the LDL subfraction measurements have definitively been demonstrated to add to the ability to discriminate between individuals who are at higher versus lower risks of cardiovascular events compared to commonly used predictors, such as LDL and HDL cholesterol" (Balk et al, 2008). The AHRQ report stated thatit has yet to be determined if cardiac disease risk assessment and treatment decisions would be improved by adding LDL subfraction (subclass) measurements (Balk et al, 2008).

An assessment by the National Academy of Clinical Biochemistry (Wilson et al, 2009) concluded that lipoprotein subclasses have been shown to be related to the development of initialCHD events, but the data analyses of existing studies are generally not adequate to show added benefit over standard risk assessment for primary prevention. The assessmentfound that there are alsoinsufficient data that measurement of lipoprotein subclasses over time is useful to evaluate the effects of treatments. The assessment also noted that several methods are available to assess lipoprotein subclasses, and that standardization is needed for this technology.

The NACB assessment on LDL particle concentrationand subclasses(including measurement by gradient gel electrophoresis) (Wilson et al, 2009) concluded: "Lipoprotein subclasses, especially the number or concentration of small, dense LDL particles, have been shown to be related to the development of initial CHD events, but the data analyses of existing studies are generally not adequate to show added benefit over standard risk assessment for primary prevention."

There is inadequate evidence that LDL subclassification by electrophoresis improves outcomes of patients with cardiovascular disease. According to the guidelines of the National Cholesterol Education Program, electrophoretic methods “cannot be recommended as procedures of choice for measuring LDL-cholesterol.”

Furthermore, guideline from the National Academy of Clinical Biochemistry (Myers, 2009) does not support LDL subclass testing.

According to guidelines from the American College of Cardiology and the American Heart Association (2010), measurement of lipid parameters, including particle size and density, beyond a standard fasting lipid profile is not recommended for cardiovascular risk assessment in asymptomatic adults. Guidelines from the Canadian Cardiovascular Society (2013) recommend measurement of ApoB or non-HDL-C as alternative targets, and make no recommendation for use of other measures of lipid particle number.

Guidelines from the National Heart Lung and Blood Institute (2012) on cardiovascular disease in children and adolescents concluded that measurement of lipoprotein subclasses and their sizes by advanced lipoprotein testing has not been shown to have sufficient clinical utility in children at this time. The guidelines state that the plasma levels of VLDL–C, LDL–C, and HDL–C subclasses and their sizes have been determined in children and adolescents by nuclear magnetic resonance spectroscopy and by vertical-spin density-gradient ultracentrifugation in research studies, but cutpoints derived from these methods for the diagnosis and treatment of dyslipidemia in youths are not currently available.

Guidelines on prevention of cardiovascular disease in women from the American Heart Association (Mosca, et al., 2011) state that the role that novel CVD risk biomarkers, includingadvanced lipid testing, should play in risk assessment and in delineation of appropriate preventive interventions is not yet well defined.

A special report of an AACC Working Group on apoB and NMR Lipoprofile for measuring particle number (Cole, et al., 2013) concluded: "Currently, in the opinion of this Working Group on Best Practices, apo B appears to be the preferred biomarker for guideline adoption because of its widespread availability, scalability, standardization, and relatively low cost."

Standards of Care from the American Diabetes Association (2013) state that some experts recommend a greater focus on non–HDL cholesterol, apolipoprotein B (apoB), or lipoprotein particle measurements to assess residual CVD risk in statin-treated patients who are likely to have small LDL particles, such as people with diabetes, but it is unclear whether clinical management would change with these measurements.

An Endocrine Society Clinical Practice Guideline on hypertriglyceridemia (Brunzell, et al., 2012) states that "The Task Force recommends against the routine measurement of lipoprotein particle heterogeneity in patients with hypertriglyceridemia."

According to guidelines from the American College of Cardiology and the American Heart Association (2010), measurement of lipid parameters, includingparticle size and density, beyond a standard fasting lipid profile is not recommended for cardiovascular risk assessment in asymptomatic adults.

Guidelines on prevention of cardiovascular disease in women from the American Heart Association (Mosca, et al., 2011) state that the role that novel CVD risk biomarkers, includingadvanced lipid testing, should play in risk assessment and in delineation of appropriate preventive interventions is not yet well defined.

Guidelines from the National Heart Lung and Blood Institute (2012) on cardiovascular disease in children and adolescents concluded that measurement of lipoprotein subclasses and their sizes by advanced lipoprotein testing has not been shown to have sufficient clinical utility in children at this time.

Angiotensin Gene

Angiotensin gene polymorphisms have been associated with CVDrisk and certain forms of hypertension. Certain AGT polymorphisms have been associated with responsiveness of BPto sodium restriction and ACE inhibitors, so that analysis of the AGT gene may have the potential to help individualize therapy by predicting patients' responsiveness to certain anti-hypertensive interventions. CardiaRisk AGT from Myriad Genetics Laboratories is a laboratory test that analyzes the angiotensinogen gene. The value of analyzing angiotensin gene polymorphisms in altering the management and improving outcomes of patients has not been demonstrated in prospective clinical studies.

Fibrinogen

Fibrinogen is a circulating glycoprotein in the blood that helps blood clot. Too much fibrinogen may promote excessive clumping of platelets. This can cause clots to form in an artery, which may lead to heart attack or stroke. Fibrinogen has been suggested as a possible indicator of inflammation that accompanies atherosclerosis.

Fibrinogen acts at the final step in the coagulation response to vascular and tissue injury, and epidemiological data support an independent association between elevated levels of fibrinogen and cardiovascular morbidity and mortality.

In a structured evidence review, Hackman and Anand (2003) found moderate evidence that fibrinogen is an independent risk predictor for atherosclerotic disease (CHD, cerebrovascular disease, and peripheral vascular disease). However, they found minimal evidence that fibrinogen is an incremental risk predictor. Hackam and Anand (2003) identified only 1 study that examined the additive yield of screening for fibrinogen. The authors noted that precise and validated tests are not available for fibrinogen. In addition, they concluded that it is unclear whether fibrinogen is causal or are simply markers of atherosclerotic vascular disease. The investigators found, few, if any, controlled studies evaluating risk-reduction strategies for fibrinogen or any of the other novel risk factors that they evaluated. The investigators concluded that “clinical trials are necessary before it can be determined whether fibrinogen has a causal role in atherothrombosis or is merely a marker of the degree of vascular damage taking place.”

A consensus statement from the ACC and the ADA(Brunzell et al, 2008) stated that the independent predictive power and clinical utlity of fibrinogen measurement is unclear. A guideline from the National Academy of Clinical Biochemistry (Cushman et al, 2009) stated that: "There are sufficient data that fibrinogen is an independent marker of CVD risk; however. because of analytical concerns, insufficient assay standardization, and uncertainty in identifying treatment strategies, measurement is not recommended for this application."

The American Heart Association (Balagopal, et al., 2011)statement on nontraditional risk factors and biomarkers for cardiovascular disease in youth concluded: "Although studies in children suggest the presence of a prothrombotic state in obese children at an early age, the role of fibrinogen...as potential markers of CVD risk needs to be confirmed in longitudinal studies; a cause-and-effect relationship cannot be assigned at present in children."

Guidelines from the AmericanAssociation of ClinicalEndocrinologists (2012) state thatfibrinogen screening in the general population is not recommended because fibrinogen levels can vary among ethnic groups. Furthermore, factors unrelated to CAD may affect fibrinogen levels and no standard measurement assay exists.

The Emerging Risk Factors Collaboration (Kaptoge, et al., 2012) analyzed individual records of 52 prospective cohort studies with 246,669 participants without a history of CVD to investigate the value of adding fibrinogen levels to conventional risk factors for the prediction of cardiovascular risk. The analysis showed that adding information of an inflammation biomarker to the standard risk factors used to predict 10-year risk of first cardiovascular event leads to a very small but statistically significant increase in the C-statistics (0.0027 for fibrinogen).

A European consensus guideline (2012) included a strong recommendation that fibrinogen should not be measured in asymptomatic low-risk individuals and high-risk patients to assess 10-year risk of CVD. The guidelines included a weak recommendation that fibrinogen may be measured as part of refined risk assessment in patients with an unusual or moderate CVD risk profile.

European guidelines (2012) identified several issues with measurement of fibrinogenfor cardiovascular disease risk, including:

1. multiplicity of confounders: dependence on other classical major risk factors;
2. lack of precision: narrow diagnostic window forfibrinogenlevel and risk of CVD;
3. lack of specificity: similar level of risk for other non-cardiovascular causes of morbidity and mortality (e.g. other low-grade inflammatory diseases);
4. lack of dose–effect or causality relationship between changes infibrinogen level and risk of CVD; and
5. lack of specific therapeutic strategies or agents targeting circulatingfibrinogen and showing reduction in CVD incidence.

The guideline noted that similar observations could be made for high-sensitivity C-reactive protein. The guidelines also noted theirhigher cost of test compared with classical biological risk factors (e.g. blood glucose and lipids).

Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) (PLAC)

Lipoprotein-associated phospholipase A2 (Lp-PLA2 or PLAC) testingis an enzyme immunoassay for the quantitative determination of Lp-PLA2 in plasma; used in conjunction with clinical evaluation and individual risk assessment as a suggested aid in predicting risk for coronary heart disease (CHD).

Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an enzyme that can hydrolyze oxidized phospholipids to generate lysophosphatidylcholine and oxidized fatty acids, which have pro-inflammatory properties (Ballantyne et al, 2004). Based on a 510(k) premarket notification, the U.S. Food and Drug Administration has cleared for marketing the PLAC Test (diaDexus, Inc., South San Francisco, CA), an enzyme immunoassay for the quantitative determination of Lp-PLA2 in plasma.

Data regarding the association between Lp-PLA2 level and incidence of cardiovascular events are conflicting (Persson et al, 2008). Somelarge prospective clinical studies have found lipoprotein-associated phospholipase A2 (Lp-PLA2) to be an independent risk factor for CAD(e.g., Packard et al, 2000; Blake et al, 2001; Ballantyne et al, 2004), although another large study (Women's Health Study) found that the predictivity of Lp-PLA2 was no longer statistically significant after adjustment for other risk factors (Blake et al, 2001).

Other studies have failed to find an association between Lp-PLA2 and various cardiac disease endpoints(e.g., Kardys et al, 2006; Allison et al, 2006; Kardys et al, 2007; Rana et al, 2011; Oldgren et al, 2007). Rana et al (2011) examined the contribution of physical activity and abdominal obesity to the variation in Lp-PLA2 and other inflammatory biomarkers and incident CHD. In a prospective case-control study nested in the European Prospective Investigation into Cancer and Nutrition-Norfolk cohort,the examined the associations between circulating levels or activity oflipoprotein-associated phospholipase A2 (Lp-PLA2) and other inflammatory markers and CHD risk over a 10-year period among healthy men and women (45 to 79 years of age). A total of 1,002 cases who developed fatal or non-fatal CHD were matched to 1,859 controls on the basis of age, sex, and enrollment period. After adjusting for waist circumference, physical activity, smoking, diabetes, systolic blood pressure, low-density lipoprotein and high-density lipoprotein cholesterol levels, and further adjusted for hormone replacement therapy in women, Lp-PLA2 was not associated with an increased CHD risk.

A meta-analysis foundLp-PLA2 to be significantly associated with CVD (Garza et al, 2007). The researchers reported that therisk estimate appears to be relatively unaffected by adjustment for conventional CVD risk factors. The researchersreported an unadjusted odds ratio of 1.51 (95 % CI: 1.30 to 1.75) for the association between elevated Lp-PLA2 and CVD. When adjusted for traditional CVD risk factors and CRP, the odds ratio was 1.60 (95 % CI: 1.36 to 1.89). An accompanying editorial noted: "Although meta-analytic confirmation of this association is notable, clinicians must not 'jump the gun.' Important questions should be answered before Lp-PLA2 is incorporated into clinical practice, and the authors acknowledge this fully in their discussion (Steinber and Mayer, 2007). The editorialist explained thatone of these questions is whether measurement of Lp-PLA2 yields additional predictive power beyond that already provided by an assessment of traditional cardiovascular risk factors and by current scoring systems such as the Framingham Risk Score. The editorialist stated that, given the weak association between Lp-PLA2 and CVD, this seems unlikely. The editorialist explained that, if a patient's baseline probability of CVD is 50 %, plotting an odds ratio of 1.60 on a Bayesian nomogram results in a posterior probability of about 59 %, a relatively small increase. "Such small changes in probability rarely translate into changes in patient management or reclassification of patients into different risk groups." The editorialist also stated that the operating characteristics of the FDA-cleared test for Lp-PLA2, the PLAC test (diaDexus Inc, San Francisco, CA), have not been adequately established (Steinbergand Mayer, 2007). The editorialist argued that decisions about the utility of a novel biomarker should not be based solely on measurements of association, such as odds ratios or relative risk. Instead, clinical decision making should be guided by the performance characteristics of the diagnostic test that measures the biomarker. The editorialist stated that test characteristics can vary significantly between patient populations. The positive and negative likelihood ratios of the PLAC test for patients at low-, intermediate-, and high-risk of various cardiovascular outcomes need to be clarified if the test is to be used in these populations. Furthermore, prospective studies need to be performed to determine whether the use of the PLAC test, or any other test of Lp-PLA2, leads to meaningful changes in patient management. "As mentioned previously, the weak association between Lp-PLA2 and CVD makes this unlikely." The editorialist also explained that the fact that Lp-PLA2 is associated with CVD does not mean it can be relied on as a surrogate marker of morbidity or mortality in clinical trials (Steinbergand Mayer, 2007). Clinical trials of drug therapy will surely track Lp-PLA2 levels, but they must also measure clinical outcomes. The editorialist also questioned whetherwide-spread statin use, which has changed and grown considerably since many of the patients inprevious studies were enrolled, is already offsetting the small increased risk of CVD that elevated Lp-PLA2 might confer. "This question highlights a critical goal for researchers of Lp-PLA2 drug therapy – randomized controlled trials must be performed against background therapy that reflects current practice." The editorialist explained that, not until this work is done will we know if lowering Lp-PLA2 with targeted drug therapy is good for patients. The editorialist concluded thatLp-PLA2should not be used for screening or risk stratification until further study. Regarding Lp-PLA2 specific drug therapy, "healthy skepticism is advised." "Responsible clinicians will resist the temptation to prescribe on the basis of pharmaceutical claims and inadequate information and wait for solid data instead."

In a prospective U.S. cohort study (Cook et al, 2006), researchers assessed whether adding measurements of Lp-PLA2 or any of 18 other novel risk factors to traditional risk factors (age, race, sex, HDL and total cholesterol levels, systolic BP, use of anti-hypertensive agents, and smoking and diabetes status) improved prediction of incident coronary heart disease among nearly 16,000 adults (age 45 years or older). The authors found that, although Lp-PLA2 showed a statistically significant increase in predictive value compared with traditional risk factors only, this increase was not clinically important. The accompanying editorialist commented that, given that only1 in 3 people with elevated blood pressure or cholesterol levels achieves adequate control, clinician should focus on treatment and control of traditional risk factors. The authors concluded that, for now, routine screening of Lp-PLA2 levels seems unwarranted.

An analysis of the Atherosclerosis Risk in Communities Study, whichassessed the association of 19 novel risk factors with coronary heart disease in a cohort of 15,792 adults, found that measurement of Lp-PLA2in that population addedvery little to the 5-year predicted risk of a coronary heart disease event based on assessment of traditional risk factors (Folsom et al, 2006). Although Lp-PLA2was among the novel risk factors that added the most to the area under the receiver operating curve (AUC), Lp-PLA2 resulted in a verysmall increase in theAUC of only 0.006. The authors concludedthat routine measurement of Lp-PLA2 and othernovel markers is not warranted for risk assessment. The authors stated that, on the other hand,their findings reinforce the utility of major, modifiable risk factor assessment to identify individuals at risk for CHD for preventive action.

There is insufficient evidence that Lp-PLA2 is useful inreducing risk of stroke. Ballantyne et al (2005) evaluated the ability of Lp-PLA2 and C-reactive protein to predict stroke cases in a manner that is statistically independent from traditional risk factors. The authors use data from the Atherosclerosis Risk in Communities (ARIC) Study, a high-quality prospective follow-up of healthy U.S. adults with standardized risk factor measurements as well as stored blood samples that facilitated analysis of the potential new risk predictors. As expected from prior research on stroke risk, race, hypertension, diabetes, systolic and diastolic blood pressure, and triglyceride and HDL-C levels were each individually associated with higher stroke risk. The investigators reported an association of higher Lp-PLA2 and CRP levels with increased stroke risk in statistical models adjusted for the major traditional risk factors. In the highest tertile, CRP level was associated with higher stroke risk by about 2-fold, although confidence intervals were wide. For Lp-PLA2 levels in the top tertile, with adjustment for traditional risk factors and CRP, stroke risk was higher by about 2-fold as well. Thus, the investigators found that the Lp-PLA2 level was a moderately strong stroke risk predictor, and its association with stroke in this study was statistically independent of traditional risk factors as well as the inflammatory marker CRP. In unadjusted analyses, apparently healthy middle-aged people with high levels of both CRP and Lp-PLA2 (highest tertiles of both) had a stroke risk 11-fold higher than people with low levels of both. The authors speculated that Lp-PLA2 and CRP levels may be complementary to traditional risk factors for identifying middle-aged individuals at increased risk for stroke.

The accompanying editorialists explained, however, that from the Ballantyne et al study, it is unclear how useful CRP or Lp-PLA2 level will be for improving risk prediction versus traditional risk factors alone (Greenlandand O'Malley, 2005). The editorialists explained that, simply showing statistical independence is not adequate for demonstrating clinical utility for risk prediction. "Hazard ratios and p values are useful for demonstrating statistical associations, but they fail to show whether the new marker is truly capable of making a major impact on risk prediction or risk discrimination." The editorialists explained that one helpful way to determine additive utility of a new test is through the use of receiver operating characteristic (ROC) curves and AUC information. The editorialist noted that, unfortunately, Ballantyne et al did not report AUC or ROC information. However, based on statistical analytic findings reported elsewhere, individual tests with relative risks of only 2.0 to 3.0 "are simply not capable of increasing the AUC to a clinically significant degree." The editorial concluded that "[t]o date, this search for new cardiovascular risk markers has not led to any test that can be recommended as a routine measurement beyond that of traditional risk factors."

A cohort study found no significant gain of Lp-PLA2 and minimal gains of other novel biomarkersover conventional biomarkers in predicting future cardiovascular events in a low-to-moderate risk community based population. Melander et al (2009) reported on a cohort study of 5,067persons without cardiovascular disease from Malmö, Sweden, who attended a baseline examination between 1991 and 1994. Participants underwent measurement of Lp-PLA2,CRP, cystatin C, midregional proadrenomedullin (MR-proADM), mid-regional proatrial natriuretic peptide, and N-terminal pro-B-type natriuretic peptide (N-BNP) and underwent follow-up until 2006 using the Swedish national hospital discharge and cause-of-death registers and the Stroke in Malmö register for first cardiovascular events (MI, stroke, coronary death). During median follow-up of 12.8 years, there were 418 cardiovascular and 230 coronary events. Lp-PLA2 did not have astatistically significant relationship to cardiovascular events or coronary events, andwas not retained in backwared elimination models for cardiovascular events and coronary events. Models with conventional risk factors had C statistics of 0.758 (95 % CI: 0.734 to 0.781) and 0.760 (0.730 to 0.789) for cardiovascular and coronary events, respectively. Biomarkers retained in backward-elimination models were CRP and N-BNP for cardiovascular events and MR-proADM and N-BNP for coronary events, which increased the C statistic by 0.007 (p = 0.04) and 0.009 (p = 0.08), respectively. The investigators reported that theproportion of participants reclassified was modest (8 % for cardiovascular risk, 5 % for coronary risk). Net re-classification improvement was non-significant for cardiovascular events (0.0 %; 95 % CI: -4.3 % to 4.3 %) and coronary events (4.7 %; 95 % CI: -0.76 % to 10.1 %). Greater improvements were observed in analyses restricted to intermediate-risk individuals (cardiovascular events: 7.4 %; 95 % CI: 0.7 % to 14.1 %;p = 0.03; coronary events: 14.6 %; 95 % CI: 5.0 % to 24.2 %;p = 0.003). However, correct re-classification was almost entirely confined to down-classification of individuals without events rather than up-classification of those with events. Inthis cohort of some 5,000 participants initially free of CVD and followed almost 13 years, the novel biomarkers improved prediction scores "only minimally," resulting in the re-assignment of only 1 % of participants to a higher risk group (Melander et al, 2009).

A meta-analysis found associations of circulating Lp-PLA2 mass and activity with risk of coronary heart disease, stroke, and mortality under different circ*mstances (Lp-PLA(2) Studies Collaboration, 2010).The investigatorsconducted ameta-analysis of39 studiesto calculate risk ratios (RRs) per 1standard deviation (SD)higher value of Lp-PLA2. The investigators found relative risks for coronary heart disease, adjusted for conventional risk factors,of 1.10 (95 % CI : 1.05 to 1.16) with Lp-PLA2 activity and 1.11 (1.07 to 1.16) with Lp-PLA2 mass. Relative risks for ischemic stroke were 1.08 (0.97 to 1.20) for LpPLA2 activity and 1.14 (1.02 to 1.27) forLpPLA2 mass. Relative risks were 1.16 (1.09 to 1.24) and 1.13 (1.05 to 1.22) for vascular mortality; and 1.10 (1.04 to 1.17) and 1.10 (1.03 to 1.18) for non-vascular mortality, respectively. Although the researchers acknowledge that further research is required in to this area, they suggest, “Randomised trials of potent reversible pharmacological inhibitors of Lp-PLA2 activity should help to establish whether modification of Lp-PLA2 can reverse vascular risk.” An accompanying editorialstated that these analyses suggest that increased Lp-PLA2 activity is associated with higher risk of coronary heart disease (Rosenson, 2010). The editorialist noted, however, that the predictive valueof Lp-PLA2 activity wasweaker with higher apolipoprotein B concentrations; lower concentrations of apolipoprotein B (0·85 mg/L for the mean in the lowest tertile) were associated with higher risk (1·23 [95 % CI: 1·14 to 1·33] per 1-SD change in Lp-PLA2 activity) than were apolipoprotein B concentrations in the higher two tertiles (1·09 [1·01 to 1·19] and 1·11 [1·03 to 1·19], respectively). The editorialist stated thatfuture studies that evaluate the cardiovascular risks associated with Lp-PLA2 activity and/or mass should at least adjust for apolipoprotein B concentrations, and small LDL-particle concentration. The editorialist stated that these analyses are important to fully understand the contribution of increased Lp-PLA2 activity and/or mass to future risk of cardiovascular events beyond the risk obtained from quantification of LDL particles. "Clinically, the independent contribution of Lp-PLA2 concentrations or activity for risk stratification beyond the association with small LDL-particle concentration awaits the results of randomised trials that are designed to investigate whether selective and reversible inhibition of this pathway reduces cardiovascular events."

Lp-PLA2 is also being investigated for predicting outcome in acute ischemic stroke. Elkind et al (2006) reported on apopulation-based study of stroke risk factors in 467 patients with first ischemic stroke. The studywas undertaken to determine whether levels of hs-CRP andLp-PLA2 predict risk of stroke recurrence, other vascular events, and death. The investigators found that levels of Lp-PLA2 and hs-CRP were weakly correlated (r = 0.09;p = 0.045). High-sensitivity CRP, but not Lp-PLA2, was associated with stroke severity. After adjusting for age, sex, race and ethnicity, history of coronary artery disease, diabetes mellitus, hypertension, hyperlipidemia, atrial fibrillation, smoking, and hs-CRP level, compared with the lowest quartile of Lp-PLA2, those in the highest quartile had an increased risk of recurrent stroke (adjusted HR, 2.08; 95 %CI: 1.04 to 4.18) and of the combined outcome of recurrent stroke, MI, or vascular death (adjusted HR, 1.86; 95 %CI: 1.01 to 3.42). The researchers reported that, after adjusting for confounders, hs-CRP was not associated with risk of recurrent stroke or recurrent stroke, MI, or vascular death but was associated with risk of death (adjusted HR, 2.11; 95 %CI: 1.18 to 3.75).

Whiteley et al (2009) reported on a systematic review of the evidence relating Lp-PLA2 and other blood markers and prognosis in ischemic stroke. The investigatorssearched Medline and EMBASE from 1966 to January 2007 for studies of blood markers in patients with ischemic stroke and an assessment of outcome (death, disability, or handicap). The investigators found 82 studies of 41 blood markers that met inclusion criteria, including1 study of Lp-PLA2 (citing Elkind et al, 2006). The researchers found that, although blood biomarkers might provide useful information to improve the prediction of outcome after acute ischemic stroke,the review showed that many studies were subject to bias. The researchersfound that although some markers had some predictive ability, none of the studies was able to demonstrate that the biomarker added predictive power to a validated clinical model. The reseachers concluded that the clinical usefulness of blood biomarkers for predicting prognosis in the setting of ischemic stroke has yet to be established.

Few studies have investigated the role of elevatedLp-PLA2 with stroke risk (Wassertheil-Smoller et al, 2008). Wassertheil-Smaller and colleagues (2008) assessed the relationship between Lp-PLA2 and the risk of incident ischemic stroke in 929 stroke patients and 935 control subjects in the Hormones and Biomarkers Predicting Stroke Study, a nested case-control study from the Women's Health Initiative Observational Study. Mean (SD) levels of Lp-PLA2 were significantly higher among case subjects (309.0 [97.1]) than control subjects (296.3 [87.3]; p < 0.01). Odds ratio for ischemic stroke for the highest quartile of Lp-PLA2, compared with lowest, controlling for multiple covariates, was 1.08 (95 % CI: 0.75 to 1.55). However, among 1,137 nonusers of hormone therapy at baseline, the corresponding odds ratio was 1.55 (95 % CI: 1.05 to 2.28), whereas there was no significant association among 737 hormone users (odds ratio: 0.70; 95 % CI: 0.42 to 1.17;p for interaction = 0.055). Moreover, among non-hormone users, women with high CRP and high Lp-PLA2 had more than twice the risk of stroke (odds ratio: 2.26; 95 % CI: 1.55 to 3.35) compared with women low levels in both biomarkers. Furthermore, different stroke cases were identified as high-risk by Lp-PLA2 rather than by CRP. The investigators concluded that Lp-PLA(2) was associated with incident ischemic stroke independently of CRP and traditional cardiovascular risk factors among non-users of hormone therapy with highest risk in those who had both high CRP and high Lp-PLA2.

Persson et al (2008)reported on aprospectivepopulation-based studyexploring the relationship between baseline Lp-PLA2 activity and mass, respectively, on levels and incidence of first CHD and ischemic stroke. Lp-PLA2 activity and mass were assessed in 5,393 (60 % women) subjects who participated in the Malmo Diet and Cancer Study cardiovascular program during 1991 to 1994. In all, 347 subjects had an event (195 CHD and 152 ischemic strokes) during the follow-up period (mean 10.6 +/- 1.7 years). In an age-, sex- and CV risk factors-adjusted Cox regression analysis, comparing top to bottom tertile of Lp-PLA2 activity, the relative risk [RR; 95 % CI): for incident CHD and ischemic stroke events were 1.48; 0.92 to 2.37 and RR: 1.94; 1.15 to 3.26, respectively. The corresponding figures for Lp-PLA2 mass were 0.95; 0.65 to 1.40 and RR: 1.92; 1.20 to 3.10. The investigatorsconcluded that elevated levels of Lp-PLA2 activity and mass, respectively, were in this study, independently of established risk factors related to the incidence of ischemic stroke but after adjustment for lipids not significant related to incident CHD.

Nambi et al (2009) reported on aprospective case-cohort (n = 949) study in 12,762 persons in the Atherosclerosis Risk in Communities (ARIC) study, to determinewhether Lp-PLA2 and hs-CRP levels improved the AUC for 5-year ischemic stroke risk. The investigators alsoexamined how Lp-PLA2 and hs-CRP levels altered classification of individuals into low-, intermediate-, or high-risk categories compared with traditional risk factors. In a model using traditional risk factors alone, the AUC was 0.732. The addition of the biomarkers increased the AUC modestly,by 0.011 for hs-CRP alone, 0.020 for Lp-PLA2 alone, and 0.042 when hs-CRP, Lp-PLA2, and its interaction term were added. The investigators reported that, with the use of traditional risk factors to assess 5-year risk for ischemic stroke, 86 % of participants were categorized as low- risk (less than2 %); 11 %, intermediate-risk (2 % to 5 %); and 3 %, high-risk (greater than5 %). The addition of hs-CRP, Lp-PLA2, and their interaction to the model re-classified 4 %, 39 %, and 34 % of the low-, intermediate- and high-risk categories, respectively. The investigators stated that, based ontheir analysis, the addition of both hs-CRP and Lp-PLA2 seems to satisfy the statistical requirements for a test to improve risk prediction. The investigators stated, however, that the more important question is whether the improvement conferred by the addition of the marker is clinically important and cost-effective. The investigators noted that the addition of hs-CRP and Lp-PLA2 did change risk categories in approximately 13 % ofthe study population. "It would be ideal to validate our findings in other cohorts, conduct studies to examine if changes in therapy secondary to such a risk stratification scheme will improve ischemic stroke prevention, and examine cost-effectiveness of such a strategy."

Randomized clinical studies of statin therapy for hyperlipidemic personshave shown lower incidence of stroke in the placebo group (Armarenco and Labreuche, 2009); prospective randomized studies of statins for prevention ofrecurrence in stroke and TIA have shown marginal effects(Manktelowand Potter, 2009). However, it is not known whether treatment with statins would reduce stroke risk in a subset of normo-lipidemic patients for whom statin therapy would otherwise not be indicated. In addition,a number of studies have also shown that certain drugs can have an impact on Lp-PLA2 levels; these studies, however, do not demonstrate whether changes in Lp-PLA2 can improve outcomes when used as a target of treatment.

There is a lack of evidence from prospective clinical studies that incorporation of Lp-PLA2 testing in cardiovascular risk assessment improves clinical outcomes. ATPIII guidelines do not include a recommendation for Lp-PLAC testing in assessment ofCADrisk. Guidelines from the American Heart Association and the American Stroke Association (Goldstein et al, 2006) on primary prevention of ischemic stroke state: "No recommendations about Lp-PLA2 modification can be made because of an absence of outcome studies showing clinical benefit with reduction in its blood levels." Aconsensus statement from the American College of Cardiology and the American Diabetes Association on management of patients with cardiometabolic risk makes no mention of Lp-PLA2 (Brunzell et al, 2008). The American Association of Clinical Chemistry (AACC, 2009) has stated that Lp-PLA2 is not widely available, and, "while the findings from recent studies support the potential usefulness of Lp-PLA2 in CHD and ischemic stroke risk assessment, its ultimate clinical utility has yet to be established."Canadian Cardiovascular Society guidelines (Genest, et al., 2009) do not recommend Lp-PLA2 for screening for heart disease risk. The American College of Cardiology and the American Heart Association(2010)examined Lp-PLA2 and concluded that it might be reasonable for assessment in intermediate-risk asymptomatic adults. This was a class IIb recommendation, indicating that the recommendation's usefulness/efficacy is less well established.

European consensus guidelines (2012) state that the magnitude of Lp-PLA2's effect on risk remains modest at the level of the general population; study limitations or bias are present. The guidelines state that LpPLA2 remains a "second-line" marker for CVD risk estimation. The guidelines suggest that LpPLA2may be measured as part of a refined risk assessment in patients at high risk of a recurrent acute atherothrombotic event. This is a class IIb recommendation, indicating that the recommendation's usefulness/efficacy is less well established.

The American Stroke Association and the American Heart Association (Goldstein, et al., 2011) also rendered a class IIb recommendation for the use of Lp-PLA2. "Measurement of inflammatory markers such as hs-CRP or Lp-PLA2 in patients without CVD may be considered to identify patients who may be at increased risk of stroke, although their effectiveness (ie, usefulness in routine clinical practice) is not well established."

Guidelines from the American College of Clinical Endocrinology (2012)has a grade 2B recommendation to usehighly sensitive CRP to stratify CVD risk in patients with a standard risk assessment that is borderline, or in those with an LDL-C concentration less than 130 mg/dL, and tomeasure Lp-PLA2 when it is necessary to further stratify a patient’s CVD risk.

Other guidelines make no recommendation for measurement of Lp-PLA2 (New Zealand Guidelines Group, 2009; National Vascular Diseae Prevention Alliance, 2009; Lindsay, et al., 2010; National Vascular Disease Prevention Alliance, 2012). An National Heart Lung and Blood Institute (2012) guideline on cardiovascular disease risk in children and adolescentsfound insufficient evidence to recommend the measurement of inflammatory markers in youths.

An ad-hocpanel of Lp-PLA2 investigators recommended consensus guidelines for Lp-PLA2 use in clinical practice (Davidson et al, 2008). The panel recommended Lp-PLA2 testing as an adjunct to traditional risk factors in determining the target goal for lipid treatment in correlation with absolute risk. The panel did not recommend Lp-PLA2 testing as a screening tool for low-risk patients. Commenting on these guidelines, Ali and Madjid (2009) stated that it is to be noted that these recommendations are based on consensus, and that more evidence is needed to determine the exact clinical approach for use of Lp-PLA2 as a screening test and as part of a treatment regimen.

Bertoia et al (2013) examined the prospective association between oxidation-specific biomarkers, primarily oxidized phospholipids (OxPL) on apolipoprotein B-100-containing lipoproteins (OxPL/apoB) and lipoprotein (a) [Lp(a)], and risk of PAD. These researchers examined, as secondary analyses, indirect measures of oxidized lipoproteins, including autoantibodies to malondialdehyde-modified low-density lipoprotein (MDA-LDL) and apolipoprotein B-100 immune complexes (ApoB-IC). The study population included 2 parallel nested case-control studies of 143 men within the Health Professionals Follow-up Study (1994 to 2008) and 144 women within the Nurses' Health Study (1990 to 2010) with incident confirmed cases of clinically significant PAD, matched 1:3 to control subjects. Levels of OxPL/apoB were positively associated with risk of PAD in men and women: pooled relative risk: 1.37, 95 % CI: 1.19 to 1.58 for each 1-SD increase after adjusting age, smoking, fasting status, month of blood draw, lipids, BMI, and other cardiovascular disease risk factors. Lipoprotein (a) was similarly associated with risk of PAD (pooled adjusted relative risk: 1.36; 95 % CI: 1.18 to 1.57 for each 1-SD increase). Autoantibodies to MDA-LDL and ApoB-IC were not consistently associated with risk of PAD. The authors concluded that OxPL/apoB were positively associated with risk of PAD in men and women. The major lipoprotein carrier of OxPL, Lp(a), was also associated with risk of PAD, reinforcing the key role of OxPL in the pathophysiology of atherosclerosis mediated by Lp(a).

The main drawbacks of this study included:

1. because the NHS and HPFS studies contain predominantly white subjects, it is unclear if these findings can be generalized to minority populations, some of whom are at increased risk for PAD,
2. it is possible that some control subjects have undiagnosed PAD, and
3. these finding alone cannot definitely separate OxPL and Lp(a) as individual determinants of PAD, given their inherent biological inter-relationship.

The authors stated that “Future research should continue to explore the mechanisms that link oxidation to risk of PAD and test whether modifiable risk factors, potentially including novel therapies that reduce levels of OxPL, might prevent the development of artherosclerotic diseases such as PAD”.

The Emerging Risk Factors Collaboration (Di Angelantonio, et al., 2012) found, in a study of individuals without known CVD, the addition of information onLp-PLA2 to risk scores containing total cholesterol and HDL-C led to slight improvement in CVD prediction. Individual The investigators estimated that for 100,000 adults aged 40 years or older, 15,436 would be initially classified at intermediate risk using conventional risk factors alone. Additional testing with Lp-PLA2 would reclassify 2.7% of people to a 20% or higher predicted CVD risk category and, therefore, in need of statin treatment under Adult Treatment Panel III guidelines.

Holst-Albrechtsen et al (2013) noted that studies indicate that elevated plasma concentrations of Lp-PLA2 is associated with increased risk of cardiovascular disease. Lp-PLA2 seems to play a crucial role in the formation of plaques and acute inflammation, and plasma Lp-PLA2 could therefore potentially be used as a predictor of long-term outcome in ACS patients. To evaluate this, data concerning Lp-PLA2 as a predictor in ACS patients was gathered through a systematic literature review, and studies on this issue were extracted from relevant databases, including PubMed and Cochrane. A total of 14 articles were retrieved, but after thorough evaluation and elimination of irrelevant articles only 7 studies were eligible for the literature review. All studies except 2 showed significant correlation between Lp-PLA2 and CV events in ACS patients. Only 1 study found an independent value to predict CV events 30 days after ACS. Altogether, there was inconsistency in the findings regarding the potential use of Lp-PLA2 and a lack of knowledge on several issues. These investigators stated that Lp-PLA2 seems to give valuable information on which ACS patients are prone to new events and also provides important information on plaque size. However, they stated that more focused studies concerning genetic variations, time-window impact, patients with and without CV risk factors (e.g., diabetes), and treatment effects are needed. The authors concluded that Lp-PLA2 offers new insight in the pathophysiological development of ACS, but until the aforementioned issues are addressed, the biomarker will mainly be of interest in a research setting, not as a predictive parameter in a clinical setting.

Mahmut and colleagues (2014) documented the presence and role of Lp-PLA2 in calcific aortic valve disease (CAVD). These researchers documented the expression of the phospholipase A2 family of genes in aortic valves by using a transcriptomic assay. Messenger ribonucleic acid and protein expression were confirmed in aortic valves explanted from 60 patients by quantitative polymerase chain reaction (qPCR) and immunohistochemistry, respectively. The effect of lysophosphatidylcholine, the product of Lp-PLA2 activity, was documented on the mineralization of valve interstitial cell cultures. Transcriptomic analyses of CAVD and control non-mineralized aortic valves revealed that Lp-PLA2 was increased by 4.2-fold in mineralized aortic valves. Higher expression of Lp-PLA2 in stenotic aortic valves was confirmed by qPCR, immunohistochemistry, and enzymatic Lp-PLA2 activity. The number of Lp-PLA2 transcripts correlated with several indexes of tissue remodeling. In-vitro, lysophosphatidylcholine increased the expression of alkaline phosphatase, the ectonucleotide pyrophosphatase/phosphodiesterase 1 enzyme, sodium-dependent phosphate cotransporter 1 (encoded by the SLC20A1 gene), and osteopontin. These investigators then showed that lysophosphatidylcholine-induced mineralization involved ectonucleotidase enzyme as well as apoptosis through a protein-kinase-A-dependent pathway. The authors concluded that these results demonstrated that Lp-PLA2 is highly expressed in CAVD, and it plays a role in the mineralization of valve interstitial cells. Moreover, they stated that further work is needed to document whether Lp-PLA2 could be considered as a novel target in CAVD. Krintus et al (2014) stated that despite great progress in prevention strategies, pharmacotherapy and interventional treatment of coronary artery disease (CAD), cardiovascular events still constitute the leading cause of mortality and morbidity in the modern world. Traditional risk factors, including hypertension, diabetes mellitus, smoking, obesity, dyslipidemia, and positive family history account for the occurrence of the majority of these events, but not all of them. Adequate risk assessment remains the most challenging in individuals classified into low or intermediate risk categories. Inflammation plays a key role in the initiation and promotion of atherosclerosis and may lead to acute coronary syndrome (ACS) by the induction of plaque instability. For this reason, numerous inflammatory markers have been extensively investigated as potential candidates for the enhancement of cardiovascular risk assessment. These investigators assessed the clinical utility of well-established (C-reactive protein [CRP] and fibrinogen), newer (Lp-PLA2 and myeloperoxidase [MPO]) and novel (growth differentiation factor-15 [GDF-15]) inflammatory markers which, reflect different pathophysiological pathways underlying CAD. Although according to the traditional approach all discussed inflammatory markers were shown to be associated with the risk of future cardiovascular events in individuals with and without CAD, their clear clinical utility remains not fully elucidated. Current recommendations of numerous scientific societies predominantly advocate routine assessment of CRP in healthy people with intermediate cardiovascular risk. However, these recommendations substantially vary in their strength among particular societies. These discrepancies have a multi-factorial background, including:

1. the strong prognostic value of CRP supported by solid scientific evidence and proven to be comparable in magnitude with that of total and high-density lipoprotein cholesterol, or hypertension,
2. favorable analytical characteristics of commercially available CRP assays,
3. lack of CRP specificity and causal relationship between CRP concentration and cardiovascular risk, and
4. CRP dependence on other classical risk factors.

Of major importance, CRP measurement in healthy men greater than or equal to 50 years of age or healthy women greater than or equal to 60 years of age with low-density lipoprotein cholesterol less than 130 mg/dL may be helpful in the selection of patients for statin therapy. Additionally, evaluation of CRP and fibrinogen or Lp-PLA2 may be considered to facilitate risk stratification in ACS patients and in healthy individuals with intermediate cardiovascular risk, respectively. Nevertheless, the clinical utility of CRP requires further investigation in a broad spectrum of CAD patients, while other promising inflammatory markers, particularly GDF-15 and Lp-PLA2, should be tested in individuals both with and without established CAD. These researchers noted that further studies should also focus on novel performance metrics such as measures of discrimination, calibration and reclassification, in order to better address the clinical utility of investigated biomarkers and to avoid misleadingly optimistic results. It also has to be emphasized that, due to the multi-factorial pathogenesis of CAD, detailed risk stratification remains a complex process also involving, beyond assessment of inflammatory biomarkers, the patient's clinical characteristics, results of imaging examinations, electrocardiographic findings and other laboratory parameters (e.g. lipid profile, indices of renal function, markers of left ventricular over-load and fibrosis, and biomarkers of myocardial necrosis, preferably cardiac troponins).

Carotid Intima-Media Thickness

Carotid intima media thickness (IMT) testingmeasures the thickness of the inner two layers of the wall of the carotid artery. The intima is the innermost layer and the media is the middle layer of the arterial wall. An ultrasound image is used to detect carotid IMT which can purportedly diagnose early stages of atherosclerosis, before symptoms occur and assess for drug efficacy. It is thought that a thickening of the carotid intima media confirms the likelihood of atherosclerosis of other arteries, including the coronary and carotid arteries. This led to the theory that carotid IMT could be used to identify persons at high risk for cardiovascular and cerebrovascular disease. Examples of US Food and Drug Administration (FDA) approved IMT devices include ArterioVision andCardioHealth Station.

Carotid ultrasonography measurement of the intimal medial thickness of the carotid arteries has been used to assess the atherosclerotic plaque burden. Increased carotidintimal medial thickneshas beencorrelated with a gradual, graded increase in the risk of future cardiovascular events, but the magnitude of the relationship lessened when traditional risk factors were taken into account (Chambless et al, 1997; Hodis et al, 1998; O'Leary et al, 1999; Simons et al, 1999; Touboul et al, 2000; Bots et al, 2007; Lorenz et al, 2007).

ATPIII reports that the extent of carotid atherosclerosis correlates positively with the severity of coronary atherosclerosis, and that some studies have shown that severity of intimal medial thickness independently correlates with risk for major coronary events. ATPIII states, however, that the predictive power of carotid medial intima thickness for persons without multiple risk factors has not been determined in prospective studies. ATPIII concluded that “its expense, lack of availability, and difficulties with standardization preclude a current recommendation for its use in routine risk assessment for the purpose of modifying intensity of LDL-lowering therapy.”

A consensus statement from the ADAand the ACCobserved that measurements of carotid intima media thickness, as well as measurement of coronary calcification and ankle-brachial index, can detect the presence of so-called subclinical vascular disease, and that patients with documented subclinical atherosclerosis are at increased CVD riskand may be considered candidates for more aggressive therapy. The consensus statement concluded, however, that it is unclear whether such tests improve prediction or clinical decision making in patients with cardiometabolic risk (Brunzell et al, 2008).

The U.S. Preventive Services Task Force (USPSTF, 2009) stated that there is insufficient evidence to recommend the use of carotid intima-media thickness to screen asymptomatic individuals with no history of CHD to prevent CHD events.

American Association of Clinical Endocrinology (2012) guidelinesstate that carotid intima media thickness measurementsshould not be performed routinely, but may be used in certain clinical situations as adjuncts to standard CVD risk factors in an attempt to refine risk stratification and the need for more aggressive preventive strategies. This is a grade 4 recommendation, based upon opinion (D level evidence).

An American Heart Association guideline on cardiovascular disease in women (Mosca, et al., 2011) stated: "Although recent evidence suggests that using imaging modalities such as coronary calcium scoring and carotid ultrasound to demonstrate the presence of advanced atherosclerosis has the greatest utility for reclassifying risk in those (including women) predicted to be at intermediate risk on the basis of short-term risk equations such as the Framingham risk score, their value in improving clinical outcomes has not been established."

An assessment prepared for the Agency for Healthcare Research and Quality (Helfand, et al., 2009) concluded that "carotid intima media thickness ...probably provide[s] independent information about coronary heart disease risk, but data about their prevalence and impact when added to Framingham risk score in intermediate-risk individuals are limited."

Guidelines from the Canadian Cardiovascular Society (Anderson, et al., 2013)noted thata recent metaanalysisfound that carotid intima media measurements added only little to risk reclassification after adjustment for conventional risk factors.

van den Oord et al. (2013) conducted asystematic review and meta-analysis of the evidence on the association of carotid intima media thickness with future cardiovascular events and the additional value ofcarotid intima media thicknessto traditional cardiovascular risk prediction models. The association ofcarotid intima media thickneswith future cardiovascular events and the additional value ofcarotid intima media thicknesswere calculated using random effects analysis. The literature search yielded 1196 articles of which 15 articles provided sufficient data for the meta-analysis. A 1standard deviationincrease incarotid intima media thicknesswas predictive for myocardial infarction (HR 1.26, 95% CI 1.20-1.31) and for stroke (HR 1.31, 95% CI 1.26-1.36). A 0.1 mm increase incarotid intima media thicknesswas predictive for myocardial infarction (HR 1.15, 95% CI 1.12-1.18) and for stroke (HR 1.17, 95% CI 1.15-1.21). The overall performance of risk prediction models did not significantly increase after addition ofcarotid intima media thicknessdata. The areas under the curve increased from 0.726 to 0.729 (p = 0.8). The authors concluded thatcarotid intima media thicknessas measured by B-mode ultrasound is associated with future cardiovascular events. However, the addition ofcarotid intima media thicknessto traditional cardiovascular risk prediction models does not lead to a statistical significantly increase in performance of those models.

Den Ruijter (2012) conducted a metaanalysis to determine whether common carotid intima media thickness has added value in 10-year risk prediction of first-time myocardial infarctions or strokes, above that of the Framingham Risk Score. The authors identified relevant studies through literature searches of databases (PubMed from 1950 to June 2012 and EMBASE from 1980 to June 2012) and expert opinion. The included studies if participants were drawn from the general population, common carotid intima media thickness was measured at baseline, and individuals were followed up for first-time myocardial infarction or stroke. The authors combined individual data into one data set and they performed an individual participant data meta-analysis on individuals without existing cardiovascular disease. The authors included 14 population-based cohorts contributing data for 45,828 individuals. During a median follow-up of 11 years, 4007 first-time myocardial infarctions or strokes occurred. The authors first refitted the risk factors of the Framingham Risk Score and then extended the model with common carotid intima media thickness measurements to estimate the absolute 10-year risks to develop a first-time myocardial infarction or stroke in both models. The C statistic of both models was similar (0.757; 95% CI, 0.749-0.764; and 0.759; 95% CI, 0.752-0.766). The authors found that the net reclassification improvement with the addition of common carotid intima media thickness was small (0.8%; 95% CI, 0.1%-1.6%). In those at intermediate risk, the net reclassification improvement was 3.6% in all individuals (95% CI, 2.7%-4.6%) with no differences between men and women. The authors concluded that the addition of common carotid intima media thickness measurements to the Framingham Risk Score was associated with small improvement in 10-year risk prediction of first-time myocardial infarction or stroke, but this improvement is unlikely to be of clinical importance.

Guidelines from the American College of Cardiology and the American Heart Association (Goff, et al., 2014) state that"routine measurement of CIMT is not recommended in clinical practice for risk assessment for a first ASCVD event."

Carotid Ultrasound Screening

The United States Preventive Services Task Force (USPSTF, 2014) recommends against screening for asymptomatic carotid artery stenosis in the general population of adults without a history of transient ischemic attack, stroke, or other neurologic signs and symptoms. This is a D recommendation, meaning that the USPSTF recommends against this service because there is moderate or high certainty that the service has no net benefit or that the harms outweigh the benefits. The USPSTF observed that the most feasible screening test for carotid artery stenosis (defined as 60% to 99% stenosis) is ultrasonography. The USPSTF stated that, although adequate evidence indicates that this test has high sensitivity and specificity, in practice, ultrasonography yields many false-positive results in the general population, which has a low prevalence of carotid artery stenosis (approximately 0.5% to 1%). The USPSTF also found that there are are no externally validated, reliable tools that can determine who is at increased risk for carotid artery stenosis or for stroke when carotid artery stenosis is present. The USPSTF found that all screening strategies, including ultrasonography with or without confirmatory tests (digital subtraction or magnetic resonance angiography), have imperfect sensitivity and could lead to unnecessary surgery and result in serious harms, including death, stroke, and myocardial infarction. The USPSTF concluded with moderate certainty that the harms of screening for asymptomatic carotid artery stenosis outweigh the benefits.

Measurement of Arterial Elasticity

Arterial elasticity has been shown to decrease with aging and with vascular disease. A number of studies have demonstrated loss of arterial elasticity in persons with CAD, heart failure, hypertension and diabetes.

Arterial stiffness, measured as aortic pulse wave velocity between the carotid and femoral arteries, appears to be a predictor of cardiovascular events (Mattace-Raso et al, 2006; Willum-Hansen et al, 2006). Inthe Rotterdam Study, the adjusted relative risk for coronary disease or stroke in the 2nd and 3rd tertiles was 1.72 and 2.45 compared to the lowest tertile (Mattace-Raso et al, 2006). The predictive value was independent of cardiovascular risk factors, carotid intima-media thickness, and pulse pressure. By contrast, carotid artery distensibility was not independently associated with CVD.

Hypertension Diagnostics, Inc. (HDI, Eagan, MN) has developed a method of analyzing blood pressure waveforms to noninvasively measure the elasticity (compliance) of arteries and arterioles. The HDI CVProfilor and the HD/PulseWave CR-2000 graphs the blood pressure waveform (“pulse contour analysis”) and calculates the elasticity (flexibility) of large and small arteries and arterioles. The CVProfilor obtains blood pressure and waveform data by use of a blood pressure cuff placed on the left upper-arm and a piezoelectric-based, direct contact, acoustical transducer placed over the right radial artery near the wrist. A computer performs a pulse contour analysis of blood pressure waveform data, and generates a report which includes a large artery elasticity index (a measure of capacitative compliance) and a small artery elasticity index (a measurement of oscillatory or reflective compliance). The CVProfilor also provides measurements of standard blood pressure values (systolic, diastolic and mean arterial pressure), heart rate, body surface area (BSA) and BMI. Arterial elasticity has been investigated as an early marker of vascular disease in patients without standard risk factors for CVD. Several studies have examined the impact of various factors on arterial elasticity, and have examined the question of whether arterial elasticity is an independent risk factor for cardiovascular disease. However, there is inadequate evidence from prospective clinical studies demonstrating that non-invasive measurements of arterial elasticity using the CVProfilor alters patient management and improves clinical outcomes. Current guidelines from leading medical professional organizations do not include a recommendation for use of pulse waveform analysis in cardiovascular disease risk assessment.

In a clinical trial, Woodman et al (2005) reported that large and small artery compliance, and stroke volume/pulse pressure (measured by HDI/PulseWave CR-2000), and systemic arterial compliance show poor agreement with central pulse wave velocity, an established measure of central arterial stiffness.

Interleukin 6 -174 g/c Promoter Polymorphism

Inflammation plays an important role in the pathogenesis of atherosclerosis. Interleukin 6 (IL-6) has many inflammatory functions, and the IL-6 -174 g/c promoter polymorphism appears to influence IL-6 levels. Previous findings on the relation between this polymorphism and risk of CVD are inconsistent. Sie and colleagues (2006) examined this polymorphism in relation to risk of CHD in a population-based study and meta-analysis. Subjects (n = 6,434) of the Rotterdam Study were genotyped. Analyses on the relation between genotype and CHD were performed using Cox proportional hazards tests, and the association between genotype and plasma levels of IL-6 and CRP was investigated. All of the analyses were adjusted for age, sex, and common cardiovascular risk factors. A meta-analysis was performed, using a random effects model. No association between genotype and risk of CHD was observed. The polymorphism was not associated with IL-6 levels, but the C-allele was associated with higher CRP levels (p < 0.01). This meta-analysis did not show a significant association between the genotype and risk of CHD. The authors concluded that the polymorphism is not a suitable genetic marker for increased risk of CHD in persons aged 55 years or older.

In men, plasma interleukin-6 (IL-6) concentrations have been shown to be predictive of a futuremyocardial infarcation(Ridker et al, 2000; Woods, et al, 2000), but itscontribution to risk of MIis attenuated significantly when other risk factors are taken into account (Pai et al, 2004).

Myeloperoxidase (MPO)

Myeloperoxidase (MPO) is an enzyme found in white blood cells that is purportedly linked to inflammation and cardiovascular disease. Higher levels of the leukocyte enzyme myeloperoxidase (MPO), which is secreted during acute inflammation and promotes oxidation of lipoproteins, are associated with the presence of coronary disease (Zheng et al, 2001; Zheng et al, 2004) and may be predictive of acute coronary syndrome in patients with chest pain (Brennan et al, 2003). Stefanescu et al (2008) found that patients with stable CAD had increasedCVD risk if plasma MPO levels were elevated and a small study demonstrated that MPO deficiency may protect against CVD (Kutter and Devaquet, 2000). Furthermore, among patients with chronic systolic heart failure (HF), elevated plasma MPO levels have been associated with an increased likelihood of more advanced HF and may be predictive of a higher rate of adverse clinical outcomes (Tang et al, 2007).

Although elevated plasma MPO concentration may be associated with a more advancedCVD risk profile, plasma MPO does not predict mortality independent of other CVD risk factors in patients with stable CAD. There is a lack of scientific evidence regarding how measurements of MPO would affect management of individuals at risk for or patients with CHD. Large randomized controlled studies are needed to ascertain the clinical value of MPO in the management of CHD.

Apolipoprotein A-1

Apolipoprotein A1 (Apo A1)is the major protein constituent ofHDL cholesterol and a relatively abundant plasma protein. Apo-A1 is instrumental in promoting the transfer of cholesterol into the liver where it is metabolized and then excreted from the body via the intestine. Although most guidelines recommend cardiovascular risk assessment based on LDL, measurement ofApo A1has not been established as a clinically useful test at this time. It has not been proven usefulin determining therapy for patients with CAD or dyslipemia.

Apolipoproteins are measured in routine clinical laboratories with the use of immunonephelometric or immunoturbidimetric assays. Importantly, international standards have been developed for apolipoprotein B100 (apoB) and A-1 (Mora, 2009). ApoB reflects the number of potentially atherogenic lipoprotein particles because each particle of very-low-density lipoprotein (VLDL), β-VLDL, intermediate-density lipoprotein (IDL), LDL, and lipoprotein(a) particle carries on its surface 1 apoB100 protein. Most of plasma apoB is found in LDL particles. HDL particles do not carry apoB but instead carry apolipoprotein A-1 (apoA-1). However, apoA-1 does not correspond directly to the concentration of HDL particles in the 1-to-1 fashion seen for apoB100 and LDL particles because an HDL particle may carry >1 apoA-1 protein (Mora, 2009).

While Ridker et al (2005) found that Apo A1predicts cardiovascular disease,it hasno more predictive value than more readily available markers, such as the non-HDL cholesterol level and the ratio of total to HDL cholesterol. In a secondary analysis of a large prospective cohort study involving 15,632 healthy womenin the Women's Health Study,investigators assessed the value of several markers. Subjects were followed for at least 10 years, during which time 464 had first cardiovascular events (MI, ischemic stroke, coronary re-vascularization, or death). After adjustment for age, smoking status, blood pressure, diabetes, and BMI, the hazard ratios for a first cardiovascular event in the most extreme quintiles for each marker (compared with the most favorable quintiles) were as follows: LDL cholesterol level, 1.62; apolipoprotein A-I level, 1.75; total cholesterol level, 2.08; HDL cholesterol level, 2.32; apolipoprotein B level, 2.50; non-HDL cholesterol level, 2.51; CRP level, 2.98. For lipid ratios, thehazard ratioswere: apo B:apo A-I, 3.01; LDL:HDL cholesterol, 3.18; apo B:HDL cholesterol, 3.56; total:HDL cholesterol, 3.81.

A case control study found that the ratio of apolipoprotein B to apolipoprotein A-I was associated with coronary artery disease but added little to existing measures of risk assessment (van der Steeg et al, 2007). United Kingdomresearchers evaluated whether the ratio of apolipoprotein B to apolipoprotein A-I was associated with CAD among 869 adults with CAD and 1,511 controls matched for age, sex, and time of enrollment. The highest quartile of the apolipoprotein ratio was significantly associated with fatal and non-fatal CAD (odds ratio, 1.85) in analyses adjusted for cardiovascular risk factors (sex, diabetes, BMI, smoking, systolic blood pressure, CRP levels, and LDL and HDL cholesterol levels). The ratio also was associated with CAD (odds ratio [OR], 1.77) in analyses adjusted for the Framingham risk score (a well-established algorithm for combining risk factors to predict CAD). However, the total/HDL cholesterol ratio and the apolipoprotein ratio categorized cases and controls similarly. In addition, the proportion of people with CAD who were predicted to have higher risk for CAD was similar when both ratios were used and when the apolipoprotein ratio was added to the Framingham risk score. An editorialist commented that "risk factor proliferation puts patients and clinicians at risk for confusion" (Berkwitsand Guallar, 2007).

A report from the Framingham Offspring Study, a large, population-based, cohort study,found thatapo A-1 ratio has little clinical utility in predicting incident coronary heart disease, and that measuring total cholesterol and HDL appears to suffice to determine heart disease risk (Ingelsson et al, 2007). More than 3,300 middle-aged, white participants in the Framingham Offspring Study without CVD were followed for a median of 15 years. A total of 291 first CHD events occurred, 198 of them in men. In men, elevations in non-HDL cholesterol, apo B, total cholesterol:HDL ratio, LDL:HDL ratio, and apo B:apo A-1 ratio were all significantly associated with increased CHD risk to a similar degree. Elevated apo A-1 and HDL were likewise associated with reduced CHD risk. Women had results similar to those in men except that decreased apo A-1 was not significantly associated with incident CHD. In sex-specific analyses, elevated LDL and total cholesterol were not significantly associated with increased CHD risk in either men or women, perhaps owing to the lack of statistical power of these substudies. In men, total cholesterol:HDL and apo B:apo A-1 ratios both improved re-classification of 10-year risk for CHD; however, the difference between the two was not significant. In women, neither lipid ratio improved CHD risk re-classification.

A large observational study reported that apolipoproteins were better than HDL and LDL in cardiac disease risk assessment (McQueen et al, 2008). In the INTERHEART study, 12,461 patients with acute MI from the world’s major regions and ethnic groups were compared with 14,637 age- and sex-matched controls to assess the contributions of various cardiovascular risk factors. Investigators obtained nonfasting blood samples from 9,345 cases and 12,120 controls and measured cholesterol fractions and apolipoproteins to determine their respective predictive values. The investigators found that ratios were stronger predictors of MI than were individual components, and apolipoproteins were better predictors than their cholesterol counterparts. The Apo B/Apo A1 ratio was the strongest predictor, with a population-attributable risk of 54 %, compared with risks of 37 % for LDL/HDL and 32 % for total cholesterol/HDL. A 1-standard-deviation increase in Apo B/Apo A1 was associated with an odds ratio of 1.59 for MI, compared with 1.17 for an equivalent increase in total cholesterol/HDL. The results were similar for both sexes and across all ethnic groups and ages. The authors argued that Apo B and Apo A1 should be used in clinical practice worldwide for cardiovascular risk assessment. A commentator noted, however, that no prospective evidence indicates that such a change would improve clinical outcomes (Soloway, 2008).

A meta-analysis found no relationship between apo A1 and apo B and stroke risk (Emerging Risk Factors Collaboration, 2009). Anindividual-patient meta-analysis, aimed at providing clear estimates of the vascular risks associated with lipid levels, included 68 prospective studies with data on 302,430 people without vascular disease at baseline; of these, 32 studies provided data on ischemic stroke outcomes in more than 173,000 people. Non-HDL cholesterol levelwas modestly associated with ischemic stroke risk, but triglyceride and HDL cholesterol levels were not associated with either ischemic or hemorrhagic stroke risk. Both non-HDL and HDL cholesterol levels were associated with cardiac risk. Measurement ofapo B andapo A-Idid not add predictive value.

The NCEP report concludes that Apo A1 is not appropriate for routine cardiovascular riskscreening. An ACC/ADA consensus statement (Brunzell et al, 2008) concluded that measurements ofApo A1appears to provide little clinical value beyond measurements of HDL cholesterol.

A European consensus statement (2012)explainsApo A1is the major apoprotein of HDL. The consensus stated that "it is beyond doubt that the apoB:apoA1 ratio is one of the strongest risk markers." The guidelines note, however, thatit is still not established whether this variable should be used as a treatment goal. "As the measurement of apolipoproteins is not available to all physicians in Europe, is more costly than currently used lipid variables, and does not add more information, its use is not as yet generally recommended."

Peripheral Arterial Tonometry

Endothelium plays an important role in the maintenance of vascular homeostasis. Nitric oxide (NO) is the key mediator of endothelial function; it is a potent vasodilator, it inhibits platelet aggregation, vascular smooth muscle cell migration and proliferation, and monocytes adhesion. Cardiovascular risk factors promote development of endothelial dysfunction, characterized by impairment of endothelium-dependent vasodilation (EDV) and by pro-coagulant/pro-inflammatory endothelial activities. The assessment of EDV is a common parameter for testing endothelial function. Endothelium-dependent vasodilation in the coronary arteries is angiographically evaluated by measurement of the vessel response to endothelial agonists, such as acetylcholine (gold standard). A non-invasive technique for the detection of EDV employs the ultrasound evaluation of flow-mediated dilation (FMD) of the brachial artery following reactive hyperemia. A close relation between FMD and coronary vasomotor response to acetylcholine has been reported. Endothelial dysfunction in the coronary circulation may precede development of angiographically evident coronary atherosclerosis; endothelial dysfunction has been also associated with a higher prevalence of CAD and resulted predictive of future cardiovascular events; recently, it has been associated with a higher risk of re-stenosis after coronary stent implantation. Endothelial dysfunction is actually considered a reversible phenomenon; drug therapies with angiotensin converting enzyme (ACE) inhibitors, angiotensin receptor blockers, statins, anti-oxidants agents have shown a beneficial effect on endothelial function (Patti et al, 2005).

Peripheral arterial tonometry (PAT) has been proposed as a non-invasive method to measure endothelial dysfunction and potentially identify patients with early-stage CAD. Endothelial dysfunction is measured by the PAT signal that is obtained using the Endo-PAT2000device (Itamar Medical) and proprietary software. The test involves the measurement of blood flow in the fingertips following compression of the upper arm with an inflatable cuff. The Endo-PA2000 was cleared by the FDA through the 510(k) process in November 2003. It is indicated for use as a diagnostic aid in the detection of coronary artery endothelial dysfunction (positive or negative) using a reactive hyperemia procedure. The device is not intended for use as a screening test in the general patient population. However, there is currently insufficient evidence to support theuse ofPAT in assessing CAD risk.

Kuvin et al (2007) assessed endothelial function in 2 peripheral vascular beds before and during reactive hyperemia in an out-patient clinic setting. The brachial artery was imaged with a portable ultrasound device and changes in vessel diameter were expressed as "% FMD". Pulse wave amplitude of the finger was detected by PAT and PAT hyperemia was defined as the maximal plethysmographic recording compared to baseline. A total of 60 individuals (43 men) were enrolled with an average age 53 +/- 2 years (mean +/- SE). The 31 individuals with more than 2 cardiac risk factors (CRF) had lower FMD (7.0 +/- 1.1 %) and PAT hyperemia (2.1 +/- 0.9) compared to the 29 persons with 0 to 2 CRF (FMD 11.3 +/- 0.8 %, PAT hyperemia 2.4 +/- 0.1; p < 0.05 for both). The 32 individuals with CAD had lower FMD (6.8 +/- 1.1 %) and PAT hyperemia (2.0 +/- 0.1) compared to the 28 individuals without CAD (FMD 11.5 +/- 0.8 %, PAT hyperemia 2.4 +/- 0.1; p < 0.05 for both). Thus, peripheral vascular endothelial function testing in the ambulatory setting correlates with the extent of CAD risk and the presence or absence of CAD. The authors concluded that these data suggested that peripheral vascular endothelial function testing is feasible in ambulatory patients, and this is an important next step in bringing this technology to clinical applicability.

Ghiadoni et al (2008) stated that the endothelium plays a key role in the maintenance of vascular homeostasis. A dysfunctional endothelium is an early marker of the development of atherosclerotic changes and can also contribute to cardiovascular events. Vascular reactivity tests represent the most widely used methods in the clinical assessment of endothelial function and in the last 2 decades, several methodologies were developed to study it non-invasively in the peripheral macro-circulation (conduit arteries) and micro-circulation (resistance arteries and arterioles). These investigators reviewed the most relevant available non-invasive techniques in the research on endothelial function, their advantages and limitations. Flow mediated dilation of the brachial artery by ultrasounds is the most widely used vascular test toascertain endothelium-dependent vasodilation. Other approaches include measurement of micro-circulatory reactive hyperemia by fore-arm venous plethysmography or digital pulse amplitude tonometry, response to beta-2 agonist by applanation tonometry or digital photo-plethysmography and several test by skin laser Doppler. It appears that FMD is the most reproducible test when an appropriate and accurate methodology is applied. Systemic markers proposed as measures of NO biology, inflammatory cytokines, adhesion molecules, or markers of endothelial damage and repair have only a very limited role as a result of biological and assay availability and variability, these factors currently have a limited role in the assessment of individual patients. The optimal methodology for investigating the multi-faceted aspects of endothelial dysfunction is still under debate. Thus, no available test to assess endothelial function has sufficient sensitivity and specificity to be used yet in clinical practice. Only the growing concordant results from different reproducible and reliable non-invasive methods examining endothelial function with different stimuli will support and strengthen experimental findings, thus providing conclusive answers in this area of research.

Chemla and associates (2008) reviewed recent advances in the non-invasive assessment of arterial pressure (indirect methods) in the field of critical care. Automated oscillo-metric measurements under-estimate intra-arterial systolic blood pressure. Digital photo-plethysmography has led to conflicting results, although the obtained respiratory pulse pressure variation correlates with the fluid-challenge-induced changes in stroke volume. Thepulse oximetry photo-plethysmographic signal recorded at the digital or ear level may be useful in monitoring respiratory arterial pressure variations, although technical improvements and clarifications are needed. Arterial tonometry is increasingly used in the cardiovascular field to reconstruct central aortic pressure. A recent study has shown that radial artery tonometry is feasible in hemodynamically stable patients and that peripheral pulse pressure reflects the combined influences of arterial stiffness and stroke volume, especially in elderly patients. The limitations of this technique include the potential bias related to the use of a generalized transfer function and the difficulty in obtaining reliable recordings in hemodynamically unstable patients. The authors concluded that intra-arterial blood pressure must be preferred over non-invasive blood pressure recordings when critical decisions are required. In hemodynamically stable patients, valuable information may be obtained by using non-invasive techniques, amongst which arterial tonometry seems promising.

Burg et al (2009) stated that myocardial ischemia provoked by emotional stress (MSI) in patients with stable CAD predicts major adverse cardiac events. These researchers tested an easily administered, non-invasive technology to identify vulnerability to mental stress ischemia. Patients with documented CAD (n = 68) underwent single photon emission CT myocardial perfusion imaging concurrent with pulse wave amplitude assessment by PAT during a mental stress protocol of sequential rest and anger stress periods. Heart rate and blood pressure were assessed, and blood was drawn for catecholamine assay, during rest and stress. Myocardial ischemia provoked by emotional stress was defined by the presence of a new perfusion defect during anger stress (n = 26) and the ratio of stress to rest PAT response was calculated. Patients with MSI had a significantly lower PAT ratio than those without MSI (0.76 +/- 0.04 versus 0.91 +/- 0.05, p = 0.03). An ROC curve for optimum sensitivity/specificity of PAT ratio as an index of MSI produced a sensitivity of 0.62 and a specificity of 0.63. Among patients taking ACE inhibitors, the sensitivity and specificity of the test increased to 0.86 and 0.73, respectively; 90 % of patients without MSI were correctly identified. The authors concluded that PAT in concert with ACE inhibition may provide a useful approach to assess risk for MSI. They stated that future studies should help determine how best to utilize this approach for risk assessment in the clinical setting.

B-Type Natriuretic Peptides

Brain natriuretic peptide (BNP)is a hormone produced in the body that, when elevated, may be an indication of congestive heart failure (CHF). BNP testing may be used to detect this hormone and aid in the diagnosis of CHF. N-terminal pro-BNP (NT-proBNP) is the precursor molecule for BNP. BNP or NT-proBNP testing has been proposed for the determination of CVD risk and may be included in CVD risk testing panels.

In a systematic review and meta-analysis on B-type natriuretic peptides (BNP)and cardiovascular risk, Di Angelantonioand colleagues(2009) stated that measurement of BNP concentration or its precursor (N-terminal fragment [NT-proBNP]) is recommended in patients with symptoms of left ventricular dysfunction and in other settings, but the relevance of these peptides to CVD in general populations or in patients with stable vascular disease is uncertain. These investigators collated datafrom 40 long-term prospective studies involving a total of 87,474 participants and 10,625 incident CVD outcomes. In a comparison of individuals in the top-third with those in the bottom-third of baseline values of natriuretic peptides, the combined RR, adjusted for several conventional risk factors, was 2.82 (95 % CI: 2.40 to 3.33) for CVD. Analysis of the 6 studies with at least 250 CVD outcomes (which should be less prone to selective reporting than are smaller studies) yielded an adjusted RR of 1.94 (95 % CI: 1.57 to 2.39). Risk ratioswere broadly similar with BNP or NT-proBNP (RR, 2.89 [95 % CI: 1.91 to 4.38] and 2.82 [95 % CI: 2.35 to 3.38], respectively) and by different baseline vascular risk (RR, 2.68 [95 % CI: 2.07 to 3.47] in approximately general populations; RR, 3.35 [95 % CI: 2.38 to 4.72] in people with elevated vascular risk factors; RR, 2.60 [95 % CI, 1.99 to 3.38] in patients with stable CVD). Assay of BNP or NT-proBNP in addition to measurement of conventional CVD risk factors yielded generally modest improvements in risk discrimination. The authors concluded that available prospective studies indicate strong associations between circulating concentration of natriuretic peptides and CVD risk under a range of different circ*mstances. They stated that further investigation is warranted, particularly in large general population studies, to clarify any predictive utility of these markers and to better control for publication bias.

Melander and co-workers (2009)assessed the utility of contemporary biomarkers for predicting cardiovascular risk when added to conventional risk factors. A total of 5,067 participants (mean age of 58 years; 60 % women) without CVD were included in this study. Participants underwent measurement of CRP, cystatin C, Lp-PLA2, mid-regional pro-adrenomedullin (MR-proADM), mid-regional pro-atrial natriuretic peptide, and N-terminal pro-B-type natriuretic peptide (N-BNP) and underwent follow-up using the Swedish national hospital discharge and cause-of-death registers and the Stroke in Malmo register for first cardiovascular events (e.g., MI, stroke, coronary death). Main outcome measures were incident cardiovascular and coronary events. During median follow-up of 12.8 years, there were 418 cardiovascular and 230 coronary events. Models with conventional risk factors had C statistics of 0.758 (95 % CI: 0.734 to 0.781) and 0.760 (0.730 to 0.789) for cardiovascular and coronary events, respectively. Biomarkers retained in backward-elimination models were CRP and N-BNP for cardiovascular events and MR-proADM and N-BNP for coronary events, which increased the C statistic by 0.007 (p = 0.04) and 0.009 (p = 0.08), respectively. The proportion of participants re-classified was modest (8 % for cardiovascular risk, 5 % forcoronary risk). Net re-classification improvement was non-significant for cardiovascular events (0.0 %; 95 % CI: -4.3 % to 4.3 %) and coronary events (4.7 %; 95 % CI: -0.76 % to 10.1 %). Greater improvements were observed in analyses restricted to intermediate-risk individuals (cardiovascular events: 7.4 %; 95 % CI: 0.7 % to 14.1 %;p = 0.03; coronary events: 14.6 %; 95 % CI: 5.0 % to 24.2 %; p = 0.003). However, correct re-classification was almost entirely confined to down-classification of individuals without events rather than up-classification of those with events. The authors concluded that selected biomarkers may be used to predict future cardiovascular events, but the gains over conventional risk factors are minimal. Risk classification improved in intermediate-risk individuals, mainly through the identification of those unlikely to develop events. They stated that "[t]hese data do not exclude a future role for circulating biomarkers as adjuncts to conventional risk factors, nor do they minimize the potential for biomarkers to provide insight into underlying mechanisms of diseases. Several biomarkers studied did lead to shifts in predictive accuracy that were at least statistically significant. The challenge will be to find new cardiovascular biomarkers that alone or in combination with existing biomarkers can bring about improvements in risk assessment that are not just statistically but clinically significant as well". Commenting on this study, Schwenk (2009) concluded that this study "shows that several markers that are associated with CAD and other cardiovascular diseases in high-risk populations do not provide much incremental predictive value over known demographic and clinical risk factors in low-to-moderate risk community-based populations. For now, more-precise personalized approaches to risk stratification and subsequent prevention of cardiovascular disease are not available."

An assessment by the National Academy of Clinical Biochemistry (Christenson et al, 2009) stated thatmeasurement ofB-type natriuretic peptide (BNP) or N-terminal proBNP (NT-proBNP) concentrations forCVD risk assessment in the primary prevention setting is unwarranted. Similarly, guidelines from the American College of Cardiology and the American Heart Association (2010)do notrecommend measurement of natiuretic peptides for CVD risk assessment in asymptomatic adults

Using specific immunoassay and tandem mass spectrometry, Siriwarden et al (2010) showed that a fragment derived from the signal peptide of B-type natriuretic peptide (BNPsp) not only is detectable in cytosolic extracts of explant human heart tissue but also is secreted from the heart into the circulation of healthy individuals. Furthermore, plasma levels of BNPsp in patients with documented acute ST-elevation myocardial infarction (n = 25) rise to peak values (about 3 times higher than the 99th percentile of the normal range) significantly earlier than the currently used biomarkers myoglobin, creatine kinase-MB, and troponin. Preliminary receiver-operating characteristic curve analysis comparing BNPsp concentrations in ST-elevation MI patients and other patient groups was positive (AUC = 0.97; p < 0.001), suggesting that further, more rigorous studies in heterogeneous chest pain patient cohorts are warranted. The authros concluded that these findings demonstrated for the first time that BNPsp exists as a distinct entity in the human circulation and could serve as a new class of circulating biomarker with the potential to accelerate the clinical diagnosis of cardiac ischemia and myocardial infarction.

In an editorial that accompanied the aforementioned article, Ichiki and Burnett (2010) stated that the study was small (n = 25). If the current findings are confirmed, then BNPsp17-26 may markedly increase the armamentarium of cardiac biomarkers for myocardial ischemia and injury. They noted that further studies are needed.

Guidelines from the Royal Australian College of General Practitioners (2012) stated that the evidence for screening for heart failure using BNP is mixed despite its sensitivity and prognostic significance. The guidelines stated that BNP may be useful in excluding the condition in suspected heart failure.

Mid-Regional Pro-Atrial Natriuretic Peptide

The rapid and reliable estimation of prognosis in acute ischemic stroke is pivotal to optimize clinical care. Mid-regional pro-atrial natriuretic peptide (MR-proANP), a recently described, stable fragment of the ANP precursor hormone, may be useful in this setting. In a prospective observational study, Katan and colleagues (2010) examined the prognostic value of MR-proANP in patients with acute ischemic stroke. These rsearcehrs measured MR-proANP on admission in plasma of 362 consecutive patients presenting with acute ischemic stroke. The prognostic value of MR-proANP to predict mortality within 90 days and functional outcome (defined as a modified Rankin Scale of less than or equal to 2 or greater than or equal to 3) was evaluated and compared with the National Institutes of Health Stroke Scale (NIHSS) score. The discriminatory accuracy, calculated with the AUC of the receiver operating characteristics curve, of MR-proANP to predict death was comparable to the NIHSS (AUC: 0.86 [95 % CI: 0.82 to 0.90] and 0.85 [95 % CI: 0.81 to 0.89; p = 0.7]). Combined, the accuracy significantly improved (0.92 [95 % CI: 0.88 to 0.96; p < 0.01]). The AUC of MR-proANP to predict functional outcome was 0.70 (95 % CI: 0.65 to 0.75), similar to the NIHSS (0.75 [95 % CI: 0.70 to 0.80]; p = 0.16). The prognostic value of MR-proANP for both outcomes was independent of the NIHSS. Higher MR-proANP concentrations were found in stroke of cardioembolic etiology. The authors concluded that MR-proANP is a prognostic marker in the acute phase of stroke, improving the discriminatory value of the NIHSS, independently predicting post-stroke mortality and functional outcome.

In an editorial that accompanied the paper by Katan et al, Grangerand Laskowitz (2010) stated that the current study was performed at a single center with only 44 deaths, and the results need to be validated in an independent study. A number of important questions remain. does this biomarker change predicted risk enough to alter recommended therapy? Does use of the biomarker result in improved care and clinical outcomes? And is it cost-effective?

Guidelines from the American College of Cardiology/American Heart Association (2010)and the National Academy of Clinical Biochemistry (2009) do not recommend measurement of natriuretic peptidesfor CVD risk assessment in asymptomatic adults.

Measurement of Long-Chain Omega-3 Fatty Acids in Red Blood Cell Membranes

Long-chain omega-3 fatty acidsare afamily of unsaturated fatty acids that have in common a carbon-carbon double bond in the thir