Discussion

In this relatively large series of consecutive CSF samples, we confirm that the yield of CSF flow cytometry is low in patients with undifferentiated neurologic symptoms when compared against the gold standard of brain biopsy or longitudinal clinical follow-up for 2 years. No patient without a previous history of hematologic malignancy or findings of abnormal enhancement on MRI had positive CSF flow cytometry.

The relatively high number (25) of patients with negative CSF flow cytometry that went on to be diagnosed with CNS lymphoma via subsequent biopsy demonstrates the lack of overall sensitivity (13.8%) of the test, which further argues against it being used as a first-line screening test for CNS lymphoma in patients with no clinical or radiological suspicion. In our study, 8 patients from the 720 cases of negative CSF flow cytometry went on to have CNS lymphoma diagnosed via serial CSF flow cytometry examination. Apart from these cases, all patients underwent clinical chart review which extended 2 years post-CSF analysis and no further clinical or laboratory diagnoses of CNS lymphoma occurred, making it unlikely the remaining negative CSF flow cases were false negatives. This supports previous work suggesting limited utility of CSF flow cytometry for the diagnosis of CNS lymphoma outside of patients with a previous history of hematologic malignancy or suspicious radiologic findings.^{Reference Pittman, Treese and Chen5,Reference Kovach, DeLelys and Kelliher10–Reference Roma, Garcia and Avagnina12}

In Pittman et al., CSF analysis of 102 patients who had no history of lymphoma and no suspicious radiologic findings revealed that 0/102 (0%) had positive CSF flow cytometry.^{Reference Pittman, Treese and Chen5} Only one patient from this group (0.8%) was later diagnosed with primary CNS lymphoma on tissue histology after two negative CSF flow cytometry and one negative cytology.^{Reference Pittman, Treese and Chen5} Other reports corroborate these findings, showing only positive flow cytometry in 1/71 (1.4%),^{Reference Craig, Ohori and Gorrill11} 1/18 (5.6%),^{Reference Roma, Garcia and Avagnina12} and 1/118 (0.8%)^{Reference Kovach, DeLelys and Kelliher10} of samples collected from patients without history of hematolymphoid malignancy. These patients had various undifferentiated neurological symptoms listed as indications for the CSF flow cytometry in 35/71 (49.3%),^{Reference Craig, Ohori and Gorrill11} 118/118 (100.0%),^{Reference Kovach, DeLelys and Kelliher10} without prior history of hematological malignancy. The number of patients with undifferentiated neurological symptoms without prior hematological malignancy was not indicated specifically in the other two reports,^{Reference Pittman, Treese and Chen5,Reference Roma, Garcia and Avagnina12} but 291/373 (78%) of all samples had undifferentiated neurological symptoms in Pittman et al.^{Reference Pittman, Treese and Chen5}

Prior authors have proposed a triage algorithm where CSF flow cytometry and cytology are only utilized when certain clinical criteria are met to employ rational and appropriate use of laboratory services.^{Reference Pittman, Treese and Chen5} Some suggest only utilizing flow cytometry when there is a history of a hematologic malignancy, or when there are atypical cells found on cytology that are suspicious or worrisome for a hematologic malignancy.^{Reference Hsi13}

Our findings support this algorithm in that there was a cytopathological correlation with atypical lymphoid, atypical and malignant cells found on cytopathology corresponding to a higher likelihood of a positive CSF flow cytometry result. A negative (i.e., normal) CSF cytology would make the likelihood of the CSF flow cytometry being positive less likely. Thus, we suggest that adopting a similar testing algorithm that includes taking into account a prior hematological malignancy, abnormal enhancement on MRI, and abnormal cells on cytopathology would increase the diagnostic yield of CSF flow cytometry (Figure 2).

Figure 2: Proposed algorithm of CSF flow cytometry testing. Adapted from Pittman et al.^{Reference Pittman, Treese and Chen5}, used with permission.

One of the common criticisms of such an approach is that sequential testing of CSF is that this would lead to multiple lumbar punctures and thus discomfort for the patient. We understand that this algorithm may be difficult to implement at the clinician level due to this reason. However, there are also challenges with having the receiving laboratory adopt the algorithm. CSF samples for flow cytometry typically must be performed within 24 h of CSF collection in order to yield accurate results. CSF cytology often takes longer than this to be reported and, thus, holding CSF fluid for potential CSF flow cytometry testing is not feasible. However, if the CSF cytology is abnormal, performing another lumbar puncture for CSF flow cytometry is still less invasive than a brain biopsy to confirm the diagnosis of CNS lymphoma, so repeated lumbar punctures in low risk patients is likely justified. The exact best method to adopt this algorithm for CSF flow cytometry testing is yet to be determined and its implementation may be topic for further studies.

Current guidelines recommend that the diagnosis of CNS lymphoma not be made on the basis of CSF flow cytometry alone. The test is meant to be used in conjunction with clinical, imaging, and ideally pathological data to make the diagnosis.^{Reference Fox, Phillips and Smith14} Definitive tissue histopathological diagnosis of CNS lymphoma is preferred prior to initiating chemotherapy given the uncertain specificity and sensitivity of CSF flow cytometry and the need to assess cytomorphology and pattern of tissue involvement by histologic exam. There are instances, however, of when CSF flow cytometry can be the preferred test for diagnosis of CNS lymphoma and we continue to acknowledge its usefulness in the correct clinical context. CSF flow cytometry may be used alone in the absence of a mass that can be biopsied when suspicion of lymphoma is high or in the instance where the mass is in a location where biopsy has a high-surgical risk. In these circumstances, the positive CSF flow cytometry result along with the clinical signs and symptoms may guide the clinician to making the diagnosis of CNS lymphoma and beginning treatment. That being said, the standard diagnostic approach for primary CNS lymphoma remains stereotactic needle brain biopsy when possible.^{Reference Carnevale and Rubenstein15}

On review of our cohort, there were six patients who had negative CSF flow cytometry, absence of enhancing lesions on MRI, and no prior history of a hematopoietic malignancy that went on to have brain biopsy revealing the diagnosis of DLBCL. All six patients had a non-enhancing abnormality on brain MRI or CT. After negative workup, a brain biopsy was pursued to make the diagnosis which was DLBCL in all cases. Two of these cases actually had initial treatment with dexamethasone, leading to initial improvement of their symptoms and imaging abnormalities. However, upon taper of the steroid, they returned with worsened symptoms and imaging abnormalities. Brain biopsy was pursued and led to the diagnosis of DLBCL. Although by following our triage algorithm these patients would not have had CSF flow cytometry sent, it is possible that even with a positive CSF flow cytometry result these patients may have still undergone brain biopsy prior to committing them to a therapeutic course of chemotherapy. As mentioned, confirmation with tissue biopsy is the gold standard for diagnosis of and treatment planning for primary CNS lymphoma.^{Reference Fox, Phillips and Smith14,Reference Carnevale and Rubenstein15}

Others have reviewed serial CSF examinations in diagnosing hematological disease, but have found the addition of repeated CSF flow cytometry in succession is of low yield.^{Reference Scharf, Hanson and Howard16} In this study, 477/613 patients had 2 CSF examinations and 136/613 patients had 3 or greater CSF examinations. Of the 477 patients with two CSF examinations, 2 (0.4%) had a large B cell lymphoma diagnosed on the second CSF flow cytometry exclusively, but none with three or greater CSF examinations (0/136) had a diagnosis of a CNS hematological malignancy found on CSF flow cytometry with subsequent CSF examination. Of these patients who had suspicion of hematological malignancy (but non-diagnostic) on serial CSF examinations, five underwent additional confirmatory tissue biopsy (including brain, bone marrow, and median nerve).^{Reference Scharf, Hanson and Howard16} In our study, out of 54 patients who had two CSF examinations, 3/54 (5.6%) had positive CSF flow cytometry sample on the second CSF examination. Out of 39 patients who had three or greater CSF examinations, 5/39 (12.8%) patients have their positive CSF flow cytometry sample on the third or greater CSF examination. In total, this made up 8/28 (28.6%) of patients who had positive CSF flow cytometry in our study. Of note, however, all eight of these patients had a prior history of hematological malignancy prior to serial CSF examination. Although our yield of serial CSF examinations is higher compared to what is previously reported, all of the patients who had a diagnosis of CNS lymphoma from serial CSF examination came from patients with a prior hematological malignancy. Thus, we agree that serial CSF examination in patients without a prior hematological malignancy is low yield, as none of these patients had positive CSF flow cytometry on serial CSF examination. Alternatively, this potentially makes an argument for serial CSF examinations in only those patients who have a prior history of hematological malignancy. In addition to this, the impact of serial CSF sampling on time to diagnosis and therefore potential treatment delay versus patients who went on to CNS biopsy immediately are questions that future research may help clarify.