Tools & Techniques Diagnostics & prognostics

An Inside Look at the CNS

At a Glance

  • Current tests for problems of the brain and spine can be invasive or require significant time investment to obtain results
  • Patients with neurological symptoms often can’t wait for an answer – so they’re treated based on early evidence
  • CSF cytokine profiling is rapid and performed on spinal fluid samples routinely obtained from patients with serious CNS disorders
  • Performing such a procedure early in a patient’s presentation can optimize treatment and spare the patient unnecessary additional tests

A patient presents with neurological symptoms – perhaps headache, numbness, or functional difficulties. It’s clear that this is an urgent situation, but the patient obviously can’t be treated appropriately without knowing the underlying cause of their symptoms. But even the most basic questions – for example, “Is the problem a brain infection or cancer?” – can be time-consuming and require a brain biopsy to answer.

By examining the cytokine patterns in the cerebrospinal fluid (CSF) of various central nervous system (CNS) diseases ... my colleagues and I are essentially decoding what the body has already figured out.

The immune system has a fascinatingly precise and fairly reproducible response to various tissue threats: a cytokine cascade. By examining the cytokine patterns in the cerebrospinal fluid (CSF) of various central nervous system (CNS) diseases during my residency training in pathology at Thomas Jefferson University Hospital, my colleagues and I are essentially decoding what the body has already figured out. Based on the presence or absence – and relative levels – of these immune mediators, the cytokine profile reveals what the immune system has probably encountered. The faster we can make such a diagnosis, the better the outcome, and our new test allows rapid triage of these diseases to assess the most appropriate next steps in care. It’s a big-picture approach to diagnosis; instead of asking the direct question (“What is it?”), we are instead asking, “What is happening?” by observing how the environment reacts. We are learning a tremendous amount by looking at CNS diseases from this perspective and, after validating this work with large sample sizes, we will be closer to helping patients in real time.

Making the differential diagnosis

The current standard of care for diagnosis of a tumor involving the central nervous system (CNS) is a brain biopsy, followed by histopathologic analysis of the tissue sample. But even cases deemed likely to be a brain tumor prior to biopsy are sometimes found to be not a neoplasm but a neuroinflammatory process (either a CNS infection or an autoimmune disease). A brain biopsy is an extremely invasive process. If the pathologic process can be determined in another way (for example, if the patient’s cytokine profile suggests an infectious agent rather than a tumor or an autoimmune process), then we can spare the patient a brain biopsy and instead perform further testing for the specific pathogen (see Clinical Case 1).

Clinical Case 1

A young teenager was admitted to the hospital with signs and symptoms of severe encephalitis. The clinical suspicion was viral encephalitis. Analysis of serum, stool, sputum, and CSF for a variety of pathogens including enterovirus was negative on multiple samples from each site. Brain biopsy was performed and revealed encephalitis, and PCR of the brain tissue was positive for enterovirus.

We characterized the CSF cytokine profile of enterovirus in an earlier paper (1). If more in-depth CSF analysis, including cytokine profiling, had been performed in this case, the pattern might have suggested a viral infection. The patient might have been spared an invasive brain biopsy and treatment with approved antiviral agents might have been initiated earlier.

Another common disease, CNS lymphoma, often goes undiagnosed following CSF cytology, flow cytometry, and B cell clonal analysis. The clinical and radiologic differential for CNS disease that turns out to be lymphoma often includes both infection and autoimmune disorders. CSF cytokine profiles may prove helpful in such cases prior to biopsy.

To identify a CNS disease state as infectious, you may find CSF parameters including cell count, protein, and glucose concentrations valuable. But some pathogens, such as the human parechovirus (HpeV), do not elicit a marked cellular response, so the virus as a cause of illness in a patient may be missed. HPeV is the second most common cause of meningitis in neonates worldwide, so the ability to rapidly and accurately identify it is important – a task with which an inflammatory CSF cytokine profile can help.

Other options

There are a number of ways to investigate suspected infectious disease in the CNS – but none of them is without its downfall. There can be overlap in terms of CSF cell count, glucose, and protein concentrations between CNS infections and other brain and spinal cord diseases. Rapid special stains and smears to discern bacterial and fungal organisms in the fluid can be helpful, but may be negative. Identification of an infectious organism after growth in culture can take days or longer for positive results. PCR for viral infections is rapid, but only works if testing for the causative virus. Molecular techniques to identify viral pathogens are extremely powerful, but require clinicians to know the precise virus that they are looking for – and these assays can still return a negative result (see Clinical Case 2). Additionally, only the more common CNS viral pathogens are usually included in tests, limiting our ability to diagnose patients with rare or emerging CNS infections.

Clinical Case 2

A young adult in his early 20s presented with signs of cerebellar dysfunction. MRI revealed a contrast-enhancing mass in the cerebellum, thought by radiology and the neurosurgeons to be a malignant neoplasm. Brain biopsy showed an inflammatory process – but no tumor. The patient improved with just supportive therapy.

Although it was not used in clinical decision-making, CSF cytokine profiling in this patient revealed a profile suggesting a viral infection. A validated CSF cytokine analysis test might be helpful in such patients in the future and allow them to avoid the stress of potential misdiagnosis with a CNS malignancy.

Cytokine profiling can identify patients who need urgent therapy, assist with treatment selection, and direct additional testing. Therefore, analysis of the CSF sample obtained when the patient comes to the hospital – or at any time during their admission – could allow us to treat each patient’s affliction in the fastest and most appropriate way.

How it works

Analyzing the body’s cytokine response to infections and other pathologic CNS processes is tapping into the central nervous system’s innate immune system response to cellular injury – be that an infectious pathogen, a neoplasm, or an autoimmune disorder. The measured cytokine profile reflects how the immune system is responding to a particular insult at the time the CSF is obtained.

The fluid is obtained via lumbar puncture as part of the clinical workup of a patient for a possible CNS disorder. In our new study (2), cytokine levels (pg/mL) were measured in undiluted patient CSF using human cytokine/chemokine magnetic bead panel plates on a multiplex assay instrument. The test requires a total 100 μL of CSF for running samples in duplicate, and samples are run with appropriate controls and standard curves. Each time we completed the run of a 96-well plate, the data was transferred automatically to an Excel file for further analysis.

Each run analyzes 41 cytokines, with results available for review within two hours. In experimental animal models, pro-inflammatory cytokine levels are elevated as soon as two hours after exposure to pathogenic bacteria. We previously detected markedly elevated CSF cytokine levels with virus-specific changes in samples obtain within several hours following onset of symptoms of meningitis in neonate and infant patients with CNS viral infections. Why such young patients? Children are especially susceptible to meningitis and encephalitis – and even when they display symptoms of severe illness, they are not able to tell the clinical team how they feel (for instance, if their neck feels stiff or the light hurts their eyes). The threshold for performing a lumbar puncture in these very young patients is therefore much lower than in older patients, so it’s a common procedure when a baby arrives in the emergency department with symptoms that may be sepsis or meningitis. The ability to quickly determine whether symptoms represent a CNS infection or a systemic process could potentially save children’s lives.

Moving into the mainstream

In many clinical situations, it is important to know as soon as possible whether or not a patient who presents with acute or recent onset of neurologic disease has an infectious CNS process. For example, patients coming to the emergency department with new-onset seizures and a CT or MRI negative for mass lesions or cerebrovascular event may be worked up extensively for infectious disorders. The workup may include tests for infections (viral, fungal, and bacterial) and autoimmune processes – the latter of which are investigated using CSF analysis for autoimmune antibodies. This test in particular is expensive, but those for infectious disease also consume time and resources. A CSF cytokine test performed early in the patient’s care may help determine which specific tests should be ordered for the patient, improving overall utilization management.

A cytokine profile that points toward (or away from) an infectious process can help the neuropathologist decide which immunohistochemical stains and molecular tests to perform on the biopsy tissue.

In some cases, early analysis of CSF cytokines might spare a patient an unnecessary biopsy if they indicate a treatable infectious process when the clinical and radiologic differential also include a metastatic tumor or a glioma. Even in patients who have already had a biopsy, CSF cytokine analysis can be helpful. The histologic findings in biopsies from patients with autoimmune encephalitis can be indistinguishable from those of viral encephalitis. A cytokine profile that points toward (or away from) an infectious process can help the neuropathologist decide which immunohistochemical stains and molecular tests to perform on the biopsy tissue.

Our next step is to formally validate the use of CSF cytokine profiles to distinguish infectious from noninfectious CNS disorders by analyzing a much larger number of patient samples. We also plan to expand our analysis to include additional infectious pathogens and a wider range of noninfectious processes. Further studies will include additional cytokines that might help us identify profiles that can distinguish not just bacterial from fungal infections – not just viral from non-viral infections – but even distinguish between virus types. We would eventually like to obtain FDA approval for our test and partner with a manufacturer of cytokine analysis systems to develop an instrument ideal for the efficient analysis of patient samples in an acute setting.

Hopefully, our future analysis of cytokine profiles in a wider range of infections – including rare and emerging CNS infections and noninfectious processes – will reveal important information about the pathogenic mechanisms of these disorders. For example, CSF analysis in patients with new-onset seizures in the absence of either infection or mass lesion may reveal important information about possible immune factors in epilepsy.

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About the Authors
Danielle Fortuna

Assistant Professor of Clinical Pathology and Laboratory Medicine at the Perelman School of Medicine, University of Pennsylvania.

Mark Curtis

Associate Professor and Director of Neuropathology and the Residency Program at Thomas Jefferson University, Philadelphia, USA.

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