The Core of Diagnosis

Two high-tech laboratories are hubs for diagnosing, treating rare diseases 

For children with rare diseases, figuring out what’s wrong often requires far more than one physician working with a family over a few office visits.

Pinning down diagnoses in complex cases frequently takes an orchestra of experts employing the latest high-tech instruments.

Some of them work in glassy high-rise research towers. Others work in windowless basements with miles of wires and pipes running overhead. Patients rarely see these places or meet these scientists. Yet children from all over the world owe their health to the work these experts do.

At Cincinnati Children’s, two core laboratories – mass spectrometry and flow cytometry – have grown rapidly in recent years. Investigators predict these technologies will become increasingly routine tools for medical practice in years to come.

Mass spectrometry

“We are seeing exponential increases in demand for this technology,” says Kenneth Setchell, PhD, director of the Mass Spectrometry Laboratory, part of the Division of Pathology at Cincinnati Children’s. “We get samples coming in every day from all around the world.”

Mass spectrometry (MS) involves ionizing the molecules in the sample by bombarding them with high-energy electrons, or a beam of atoms, or by generating a charged spray to break them down and determine their mass and chemical structure. The technology has existed for decades, but in more recent years improved instruments and techniques have made MS a more practical tool for clinical diagnosis.

Cincinnati Children’s operates six mass spectrometry instruments, making it one of the largest facilities in pediatric medicine.

The laboratory leads the study of cholesterol, steroid and bile acid metabolism. Under the leadership of Setchell and James Heubi, MD, director, General Clinical Research Center, the research team identified six genetic defects in bile acid production that help explain why some children develop certain forms of liver failure.

Every month, the laboratory receives 60 to 90 urine samples from children across the US and other countries who have unexplained liver disease.

These children are facing death from toxins building up in their systems, and previous testing has failed to explain their liver failure. Before registering these patients on transplant lists, many specialists request one more test – a test that can provide a few children with a dramatically more desirable treatment option.

“Mass spectrometry results in a very precise fingerprint of the chemical composition of a sample,” Setchell says. “It often can provide definitive answers when other testing methods cannot.”

Setchell’s team is renowned internationally for detecting genetic defects in bile acid production. Of nearly 11,000 samples tested so far, 2 percent of the patients have one of the six known bile acid defects. For those children, life can be transformed.

Setchell’s early research demonstrated that children with these gene defects can safely take oral doses of cholic acid, the bile acid their liver cannot make. The treatments trick the liver into registering normal bile acid levels, helping to break a deadly feedback loop of the malfunctioning organ pumping too much of its own toxic version of bile acid.

Setchell founded the mass spectrometry lab at Cincinnati Children’s in 1984 to pursue liver disease research he had begun in England. Since then, other applications have emerged.

One instrument is dedicated exclusively to measuring free T4 (thyroid hormone), which allows precise monitoring of thyroid function. Cincinnati Children’s previously paid a commercial laboratory to run such tests. Now the medical center runs more than 100 tests a day.

Mass spectrometry also is used to diagnose other genetic defects involving organic and fatty acids in infants, in therapeutic drug monitoring, and to gather data for clinical drug trials. Recent projects have included measuring the immunosuppressant sirolimus (rapamycin) and the antiseizure drug, Topamax. The group also has a strong research program profiling various lipids related to Gaucher’s disease.

Flow cytometry

At the Diagnostic Immunology Laboratory (DIL), experts go beyond incorporating the latest science into the art of diagnosis. They use increasingly sophisticated instruments to compose entirely new tests.

This laboratory provides clinical testing for pediatric cancers, blood diseases and other immunologic disorders. It performs more than 15,000 specialty assays a year, including more than 6,000 tests ordered by other institutions.

Of the 38 tests offered, 15 were developed at Cincinnati Children’s. Five of those were introduced in the past two years, and four new tests are in development, says Alexandra Filipovich, MD, director of the Immune Deficiency and Histiocytosis Program.

“It’s constantly expanding. When I came here 15 years ago, we were doing two or three tests,”Filipovich says.

The immune deficiency program treats patients with more than 80 disorders. Many are rare, such as autoimmune lymphoproliferative syndrome (ALPS), X-linked lymphoproliferative syndrome (XLP) and hemophagocytic lymphohistiocytosis (HLH).

“We are the only center in North America that tests for all forms of HLH,” Filipovich says. “One advantage we offer, in contrast to a commercial lab, is that we have the clinical expertise here. Doctors can call and chat with us about what these test results mean.”

The workhorse technology in the immunology lab is flow cytometry, a technique that uses lasers to detect, count and sort specific types of cells as well as to analyze structures within the cells.

These boxy tabletop devices conceal a flurry of microscopic activity. They process fluid samples so that cells travel one at a time past a laser. Refracted light reveals the size and composition of the cell. Proteins tagged with fluorescent compounds also can be counted, providing vital measures of how genetic mutations, drug therapies and other factors affect cells.

Experts at Cincinnati Children’s have recently used these instruments to develop unique tests to track apoptosis, or programmed cell death.

“There are a number of defects in which the apoptosis process fails to function,” Filipovich says. “In extreme circumstances, this can lead to malignancy. We’ve developed assays that look at the process of cell death in different patient populations. That’s not done anywhere else in North America on a clinical basis.”

Flow cytometry also is used heavily in research.

“We have 24 divisions that use the instrumentation,” says Sherry Thornton, PhD, director of the flow cytometry research core.

An emerging research area involves using flow cytometry to study cell signaling.

“We can assess molecular pathways that are activated within a cell,” Thornton says. “You can get at what types of cells are activated by a disease state, how that pathway might be targeted by drugs and whether a drug successfully blocks that pathway.”

A new instrument at Cincinnati Children’s enhances this work. In addition to scatter plots, an imaging cytometer captures a magnified image of targeted cells as they speed through.

“This allows us to visualize cell surface and internal markers. The data can determine whether two signals are co-localized on or in a cell, indicating that proteins are interacting in a specific way,” Thornton says.

More than ‘plug-and-go’

The latest flow cytometry and mass spectrometry instruments are marvels of automation and computer programming. However, these devices still require human expertise.

“Developing a new test requires a lot of validation,” Filipovich says. “Assays need to be reproducible. In many cases it also requires developing age-related normal ranges.”

Establishing a new test can take two years. The process even includes studying the best ways to handle and ship samples.

“It’s not plug-and-go,” Setchell says. “It takes considerable skill to develop these tests, to prepare samples, and to analyze the results. That’s where the art comes in.”