Clinical studies show that new ‘cell-free’ test can predict cardiovascular disease risk better than standard HDL cholesterol test
Researchers from the National Institutes of Health (NIH) have developed a diagnostic assay that measures how well high-density lipoprotein (HDL)—the so-called “good” cholesterol—is working in the body. And their findings could lead to new clinical laboratory tests that supplement standard HDL level testing to better determine a person’s risk for heart disease.
Cholesterol tests are among the most commonly performed assays by clinical laboratories. A new test that reveals how well HDL is working in the body would certainly boost a medical laboratory’s test requisition volume.
“Measuring HDL function is limited to research labs and isn’t conducive to large-scale testing by routine clinical laboratories. To try to solve that problem, researchers from NHLBI’s Lipoprotein Metabolism Laboratory created a new diagnostic test,” noted an NHLBI news release.
“This is going to quicken the pace of basic research,” said Edward B. Neufeld, PhD, who along with guest researcher Masaki Sato, PhD, developed the test. “It increases the number of samples that you can study. It increases the number of experiments you can do.”
Such a new cholesterol test would quickly become one of the most commonly performed clinical lab tests because just about every American who has a physical gets cholesterol tests as part of that process.
“Other people may modify this or come up with better versions, which is fine with us,” Edward Neufeld, PhD (above), NHLBI Staff Scientist, said in a news release. “We just really wanted to tackle this problem of evaluating HDL function.” Clinical laboratories may soon have a new cholesterol test to supplement standard HDL level testing. (Photo copyright: ResearchGate.)
Faster Answers Needed about HDL
According to the NIH, the goal should go beyond measuring level of HDL as part of a person’s annual physical. What is also needed is finding out whether HDL cholesterol is effectively doing certain tasks, such as removing extra cholesterol from arteries and transporting it to the liver.
The NHLBI’s new cell-free test may make it possible to step up large-scale clinical testing of HDL function, according to the news release. As it stands now, HDL function study has been limited to research labs where testing involves “harvesting cells in the lab [which] can take days to process,” according to NIH Record.
“Most studies to date that have assessed CAD (coronary artery disease) risk by HDL functionality still use the CEC (cellular cholesterol efflux capacity) in vitro assay and are based on the use of radioisotopes (3H-cholesterol) and cultured cells, which is very labor intensive and impractical to do in a clinical laboratory,” the researchers wrote in The Journal of Clinical Investigation. They also pointed out that CEC batch-to-batch variability does not fit clinical laboratories’ need for standardization.
Advantages of NHLBI’s Test
To overcome these barriers, the NHLBI researchers created an HDL-specific phospholipid efflux (HDL-SPE) assay that has certain advantages over current HDL function assessments done in research labs.
According to the NIH, the HDP-SPE assay:
Is easy to replicate in clinical labs.
Is more suited to automation and large samples.
Offers up results in about an hour.
Is a better predictor of cardiovascular disease risk than HDL cholesterol testing for CAD risk.
“We developed a cell-free, HDL-specific phospholipid efflux assay for the assessment of CAD risk on the basis of HDL functionality in whole plasma or serum. One of the main advantages of the HDL-SPE assay is that it can be readily automated, unlike the various CEC assays currently in use,” the authors noted in their paper.
Here is how the test is performed, according to the NIH:
Plasma with HDL is separated from the patient’s blood.
“Plasma is added to donor particles coated with a lipid mixture resembling plaque and a fluorescent-tagged phospholipid” that only HDL can remove.
The fluorescent signal by HDL is then measured.
A bright signal suggests optimal HDL lipid removal function, while a dim light means reduced function.
The test builds on the scientists’ previous findings and data. In creating the new assay they drew on data from:
A study of 50 severe CAD and 50 non-CAD people.
A Japanese study of 70 CAD and 154 non-CAD participants.
Examined association of HDL-SPE with cardiovascular disease in a study of 340 patients and 340 controls.
“We have established the HDL-SPE assay for assessment of the functional ability of HDL to efflux phospholipids. Our combined data consistently show that our relatively simple HDL-SPE assay captures a pathophysiologically relevant parameter of HDL function that is at least equivalent to the CEC assay in its association with prevalent and incident CAD,” the researchers concluded in The Journal of Clinical Investigation.
Test May Be Subject to New FDA Rule
While HDL cardiovascular-related research is moving forward, studies aimed at the therapeutic side need to pick up, NIH noted.
“Someday we may have a drug that modulates HDL and turns out to be beneficial, but right now we don’t have that,” said Alan Remaley MD, PhD, NHLBI Senior Investigator and Head of the Lipoprotein Metabolism Laboratory, in the news release.
It may be years before the HDL-SPE test is used in medical settings, the researchers acknowledged, adding that more studies are needed with inclusion of different ethnicities.
Additionally, in light of the recently released US Food and Drug Administration (FDA) final rule on regulation of laboratory developed tests (LDT), the company licensed to bring the test to market may need to submit the HDL-SPE assay to the FDA for premarket review and clearance. That could lengthen the time required for the developers to comply with the FDA before the test is used by doctors and clinical laboratories in patient care.
Newly-defined Cardiovascular-Kidney-Metabolic Syndrome (CKM) means physicians will be in close collaboration with clinical laboratories to make accurate diagnoses
In a presidential advisory, the AHA defines a newly described systemic health disorder called Cardiovascular-Kidney-Metabolic Syndrome (CKM). The syndrome “is a systemic disorder characterized by pathophysiological interactions among metabolic risk factors, CKD (chronic kidney disease), and the cardiovascular system leading to multi-organ failure and a high rate of adverse cardiovascular outcomes.”
A CKM diagnosis, which is meant to identify patients who are at high risk of dying from heart disease, is based on a combination of risk factors, including:
weight problems,
issues with blood pressure, cholesterol, and/or blood sugar,
reduced kidney function.
CKM is a new term and doctors will be ordering medical laboratory tests associated with diagnosing patients with multiple symptoms to see if they match this diagnosis. Thus, clinical laboratory managers and pathologists will want to follow the adoption/implementation of this new recommendation.
“The advisory addresses the connections among these conditions with a particular focus on identifying people at early stages of CKM syndrome,” said Chiadi Ndumele, MD, PhD (above), Associate Professor of Medicine at Johns Hopkins University and one of the authors of the AHA paper, in a news release. “Screening for kidney and metabolic disease will help us start protective therapies earlier to most effectively prevent heart disease and best manage existing heart disease.” Clinical laboratories will play a key role in those screenings and in diagnosis of the new syndrome. (Photo copyright: Johns Hopkins University.)
Stages of CKM Syndrome
In its presidential advisory, the AHA wrote, “Cardiovascular-Kidney-Metabolic (CKM) syndrome is defined as a health disorder attributable to connections among obesity, diabetes, chronic kidney disease (CKD), and cardiovascular disease (CVD), including heart failure, atrial fibrillation, coronary heart disease, stroke, and peripheral artery disease. CKM syndrome includes those at risk for CVD and those with existing CVD.”
The five stages of CKM syndrome, which the AHA provided to give a framework for patients to work towards regression of the syndrome, are:
Stage 0: No CKM risk factors. Individuals should be screened every three to five years for blood pressure, cholesterol, and blood sugar levels, and for maintaining a healthy body weight.
Stage 1: Excess body fat and/or an unhealthy distribution of body fat, such as abdominal obesity, and/or impaired glucose tolerance or prediabetes. Patients have risk factors such as weight problems or prediabetes and are encouraged to make healthy lifestyle changes and try to lose at least 5% of their body weight.
Stage 2: Metabolic risk factors and kidney disease. Includes people who already have Type 2 diabetes, high blood pressure, high triglyceride levels, and/or kidney disease. Medications that target kidney function, lower blood sugar, and which help with weight loss should be considered at this stage to prevent diseases of the heart and blood vessels or kidney failure.
Stage 3: Early cardiovascular disease without symptoms in people with metabolic risk factors or kidney disease or those at high predicted risk for cardiovascular disease. People show signs of disease in their arteries, or have heart function issues, or may have already had a stroke or heart attack or have kidney or heart failure. Medication may also be needed at this stage.
Stage 4: Symptomatic cardiovascular disease in people with excess body fat, metabolic risk factors or kidney disease. In this stage, people are categorized as with or without having kidney failure. May also have already had a heart attack, stroke or heart failure, or cardiovascular conditions such as peripheral artery disease or atrial fibrillation.
“We now have several therapies that prevent both worsening kidney disease and heart disease,” said Chiadi Ndumele, MD, PhD, Associate Professor of Medicine at Johns Hopkins University and one of the authors of the Circulation paper, in a news release. “The advisory provides guidance for healthcare professionals about how and when to use those therapies, and for the medical community and general public about the best ways to prevent and manage CKM syndrome.”
According to an AHA 2023 Statistical Update, one in three adults in the US have three or more risk factors that contribute to cardiovascular disease, metabolic disorders, or kidney disease. While CKM affects nearly every major organ in the body, it has the biggest impact on the cardiovascular system where it can affect the blood vessels, heart muscle function, the rate of fatty buildup in the arteries, electrical impulses in the heart and more.
“There is a need for fundamental changes in how we educate healthcare professionals and the public, how we organize care and how we reimburse care related to CKM syndrome,” Ndumele noted. “Key partnerships among stakeholders are needed to improve access to therapies, to support new care models, and to make it easier for people from diverse communities and circumstances to live healthier lifestyles and to achieve ideal cardiovascular health.”
New AHA Risk Calculator
In November, the AHA announced PREVENT (Predicting risk of cardiovascular disease EVENTs), a tool that doctors can use to assess a person’s risk for heart attack, stroke, and heart failure. The new risk calculator, which incorporates CKM, allows physicians to evaluate younger people as well, and examine their long-term risks for cardiovascular issues.
Doctors can use PREVENT to assess people ages 30 to 79 and predict risk for heart attack, stroke, or heart failure over 10 to 30 years.
“Longer-term estimates are important because short-term or 10-year risk in most young adults is still going to be low. We wanted to think more broadly and apply a life-course perspective,” Khan said. “Providing information on 30-year risk may reveal earlier opportunities for intervention and prevention efforts in younger people.”
According to CDC data, about 695,000 people died of heart disease in the US in 2021. That equates to one in every five deaths. Clinical pathologists will need to understand the AHA recommendations and how doctors will be ordering clinical laboratory tests to determine if a patient has CKM. Then, labs will play a role in helping doctors monitor patients to optimize health and prevent acute episodes that put patients in the hospital.
New dichromatic color scale developed by scientists at the Pacific Northwest National Laboratory could play a role in how slides are stained and how software color-codes digital pathology images in ways that make it easier for human eyes to recognize structures and features of interest
Clinical laboratories, anatomic pathologists, and other specialized diagnostics providers play an essential role in precision medicine. Imagine, however, performing surgical pathology analysis on slides using displays that cannot recreate—or worse, inaccurately display—a range of colors used in the image being analyzed.
As many as 8% of men and 0.5% of women of Northern European ancestry already experience issues discerning colors in the interfaces, information, and world around them due to red-green color blindness according to the National Eye Institute. This can lead to potential for misreadings and medical errors.
Now, research from Pacific Northwest National Laboratory (PNNL) holds the potential to establish a standard colormap that eliminates the impact of red-green colorblindness on visuals. Surgical pathologists, for example, spend much of their days viewing slides and/or digital pathology images. Thus, any new method of illustrating/coloring/highlighting features of interest could eventually prove to be a useful innovation in the specialty of anatomic pathology.
In completing their research, the PNNL scientists created an open-source tool called Cmaputil that other researchers can use. Could it enable clinicians and laboratory workers to improve the visibility of critical elements in samples, slides, and other visual data formats used daily at medical laboratories and anatomic pathology groups?
PLOS One published details about the development of the colormap and its potential scientific applications in August.
PNNL’s Cividis Color Scale: A Better Alternative to Rainbow Color Scales?
While the typical rainbow color map draws attention to a chart or image, it is not particularly great at conveying information—especially if the reader is color vision deficient (CVD) or color blind. Yet, despite this, rainbow scales are common in everything from local weather reports and news stories to medical images and medical studies.
Jamie Nuñez, lead author of the PLOS One study and a chemical and biological data analyst at PNNL, told Scientific American, “People like to use rainbow because it catches the eye. But once the eye actually gets there, and people are trying to figure out what’s actually going on inside of the image, that’s kind of where it falls apart.” (Photo copyright: University of Washington.)
PNNL scientists started with the viridis colormap due to “its wide range of colors” and “overall sharpness when overlaid with complex images.” They created an open-source software tool capable of taking existing color scales and simulating the visual effect of red-green color blindness using a mathematical model of human sight. Their software adjusts the scale so that color and brightness vary at a steady rate.
Their adaptions resulted in what they call the “cividis colormap.” It is a blue and yellow scale that provides an accurate change in hue and luminance when compared to changes in the data set. Researchers noted that, to their knowledge, this is the first study to mathematically optimize a colormap specifically for viewing by both those with CVD and those with normal vision.
“Here, we present an example CVD-optimized colormap created with this module that is optimized for viewing by those without a CVD as well as those with red-green colorblindness. This colormap, cividis, enables nearly-identical visual-data interpretation to both groups, is perceptually uniform in hue and brightness, and increases in brightness linearly,” the researchers noted in the PLOS One study.
Example above is of a misleading colormap, taken from the PNNL/PLOS One study. An image of yeast cells is shown in gray scale (left), with a rainbow color scale (middle) and as a person with red-green color blindness sees the rainbow image (right). (Photo/caption copyright: Nuñez JR, Anderton CR, Renslow RS (2018) PLoS ONE 13(7): e0199239/Scientific American.)
The PNNL researchers report that the colormap will soon be ready in a number of tools, including:
According to Scientific American, cividis will be added to the color-scale libraries of roughly a dozen software packages.
“While it may take some time for the full scientific community to both be aware of the need to choose appropriate colormaps and agree on preferred colormaps,” PNNL researchers note, “we hope the code we provide here can help with this transition by allowing others to experiment with the different aspects of colormap design and see how the various characteristics of a colormap affect its interpretation.”
They are concerned that the changing color spaces on future displays may make current colormaps and standards obsolete, as they display colors outside the standard sRGB color space. However, the researchers also note that any change to color spaces could result in an increase in color availability and allow cmaputil to create better-optimized color schemes.
How Cividis and Similar Approaches Might Impact Pathology
While the technology was developed with mass spectrometry and fluid flow analysis in mind, it could prove useful for medical laboratories and specialized diagnostics providers as well—in particular, anatomic pathology and surgical pathology labs.
Coverage of a presentation at the 2011 IEEE Information Visualization Conference by Phys.org highlights a similar concept for diagnosing heart disease. By taking 3D representations of arteries using a rainbow colormap and converting them to 2D projections using a dichromatic black to red colormap, Harvard researchers found their HemoVis tool increased diagnostic accuracy from 39% to 91% in their study.
Technologies and techniques designed for scientific applications often find use in healthcare environments. For anatomic and surgical pathologist and other diagnostics providers, the research from PNNL shows promise for adapting the latest data visualization trends to further improve accuracy, efficiency, and accessibility of medical images, samples, and other complex images used daily in the process of diagnosing disease.
