An unusual fluid. The advantages of cross discipline training.

Introduction

A 70 year old male was admitted to NSH with a two week history of intermittent confusion, occipital headache, nausea, vomiting and periods of expressive aphasia. In 2005 he had been diagnosed with Non-Hodgkin's diffuse large B cell lymphoma involving the pleura and bone marrow. This had been successfully treated with chemotherapy and he had been in complete remission for 2 years. On this current admission he was afebrile. Cardiovascular, respiratory and abdominal examinations were normal. Neurological examination revealed fluctuating confusion and intermittent expressive dysphasia. A computerised tomography (CT) scan of the head was normal.

A CSF sample was obtained and sent to the NSH microbiology laboratory for examination and culture. This showed a white blood cell (WBC) count of 223x106/L (normal range 0-5x106/L) with a differential of 97% lymphocytes and 3% polymorphonuclear neutrophils (PMN), a protein of 0.75g/L (normal range 0.15-0.45g/L), and glucose of 0.7mmol/L (normal range 2.8-4.4mmol/L). Based on these results, and the lack of growth on culture media, samples were sent off for numerous additional tests including polymerase chain reaction (PCR) for TB and HSV. Two days later another CSF sample was obtained. This showed a WBC count of 504x106/L, a protein of 1.09g/L, and glucose of 0.6mmol/L, again with a lymphocytic predominance. In this case the medical laboratory scientist involved in the CSF examination had a background in cytology and recognised the cells in the differential as abnormal and referred the sample to cytology.

Cytological examination produced a diagnosis of malignant meningitis with a diffuse large B-cell non-Hodgkin's lymphoma, consistent with a relapse of his previously treated malignancy. The patient has subsequently been treated with intrathecal chemotherapy with evidence of response.

Basics of cytology

Although it takes years to become fully proficient in cytology a few basic rules of thumb usable even on Gram stains and differential stains which yield nuclear detail, can indicate if a sample should be forwarded to cytology. (Note these pictures are from samples of the patient's CSF, the basic criteria can be applied to all cells, but in this case lymphocytes are predominant).

1) Cellularity- Is the sample more cellular than normal (containing more cells), are there unusual cells, or clusters of cells, and are they chaotically arranged? Some samples, such as CSF, are usually almost acellular, although a few WBC or RBC is not uncommon. This sample shows far more cells than normal (Figure 1).

2) Cellular morphology- Are the cells typical of what should be in the sample? Variations in cellular size and shape are indicative of an abnormality; some cells may be highly atypical (Figure 2).

3) Nuclear/cytoplasmic ratio- Neoplastic cells show an increase in nuclear size relative to the total cell size, compared to normal cells of that type; some cells will show little or no cytoplasm. Note that this is not as helpful with lymphocytes, as they can intrinsically show a variable N/C ratio.

4) Multinucleation- Cells normally only have a single nucleus. Actively replicating cells may show more than one, but multiple cells showing multinucleation is atypical, any multinucleation of lymphocytes in the CSF may be regarded as abnormal (Figure 3, C). NB- lymphomas do not normally exhibit multinucleation, and you should endeavour to be certain the cell is not a histiocyte.

5) Nuclear outline- Typically nuclei are rounded with a smooth regular outline, neoplastic cells often show irregular nuclear outline with a rough nuclear membrane (Figure 4, D and E).

Typical abnormalities include "clefts" (voids between lobes of a nucleus as seen at D), "rat bites" (deep indentations in the nuclei as if a bite has been taken out), and "blebs" (large bulges out from the nuclei).

6) Nucleoli- These represent active cells and appear as darker blue patches in the nucleus in these images, and are not in themselves indicative of neoplasia; however the presence of multiple nucleoli, especially those with irregular outlines, and those showing variation in size and number of nucleoli within nuclei are significant (Figure 4, E).

7) Mitotic figures- These are indicative of rapidly dividing cells. These are unusual in normal cells, and the numbers are indicative of the severity of malignancy. Any mitoses in lymphocytes in the CSF can be regarded as abnormal. (Figure 5, F).

Discussion

This case illustrates the advantage of cross disciplinary training in the laboratory. When the patient's CSF was initially examined in microbiology, a diagnosis of viral meningitis was suspected. When the second sample was then examined by a medical laboratory scientist with experience in cytology, the lymphocytes in the CSF were recognised as being highly abnormal and inconsistent with the diagnosis of viral meningoencephalitis. The specimen was then formally examined by the cytology department and the correct diagnosis of abnormal cells consistent with a malignant meningitis was made. The patient was then able to receive appropriate therapy. Although the patient appears to have responded to treatment for his relapsed NHL, the diagnosis of malignant meningitis was somewhat delayed and he was subjected to a number of expensive and unnecessary tests in the interim.

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Lymphoma

Lymphomas are neoplasms (cancers) of lymphatic cell origin. They can arise from B or T cell lineages. Mature B-cell neoplasms are clonal proliferations of cells at various stages of B-cell differentiation and the progenitor cells can range from mature plasma cells to naive B-cells. Each stage of differentiation leads to a specific form of lymphoma. B cell lymphomas are most commonly follicular or diffuse large cell lymphomas, and comprise over 90% of lymphoid neoplasms world wide (1). Diffuse large B-cell lymphomas constitute 30-40% of adult non-Hodgkin's lymphomas, with the median age at diagnosis being 70-80 years, although the disease can be seen in children. Morphologically they can be divided into centroblastic, immunoblastic, histocytic rich or anaplastic varieties, depending on the cell line of origin (1), and while aggressive may be successfully treated with multiagent chemotherapy.

Lymphomas may be characterised through immunophenotyping, a process by which the cells are distinguished through the attachment of labelled antibodies raised to cell surface antigens (typically Cluster Designation or CD antigens), to these antigens. This is useful to distinguish between malignant and benign lymphoid proliferations, between B- and T-cell processes, and between sub-categories of B- and T-cell lymphomas. However in this case as it is a recurring lymphoma it would probably not be necessary to repeat it.

Malignant meningitis

This is also known as meningeal carcinomatosis, and can be caused by primary neuro ectodermal tumours such as neuroblastoma, tumours of the haemopoietic system such as lymphoma, or solid tumours metastasizing to the leptomeninges (e.g. lung, breast, gastrointestinal tumours, and melanoma (2, 3)). In up to 50% of patients the site of the primary tumour is unknown and the disease may manifest up to 20 years after a primary tumour.

Clinical findings of malignant meningitis may include pain (radicular discomfort, headache, neck or back pain), mental state abnormalities, weakness and occasionally seizures (3, 4). Radiographic studies such and contrast-enhanced MRI may give a clue to the diagnosis. The diagnosis is most often made fortuitously when CSF is obtained to investigate such neurological symptoms (2)

The classic CSF findings include a high opening pressure, a high protein level and low glucose (3) and raised white blood cell count. The microscopy may show tumour cells; these are often bizarre and may occur in clumps. .The finding of such malignant cells in the CSF is pathognomonic, however the sensitivity of CSF is 80-95% and it may be necessary to obtain further samples to confirm the diagnosis. It is important that large volumes of CSF are withdrawn (10mls minimum), and specimens should be processed promptly (within 1hr to ensure well preserved cells are available for evaluation to improve the yield. If the patient has a known primary tumour then proteins specific to this primary tumour may be sought.

The prognosis of metastatic meningitis is poor, primarily because the malignant disease is well advanced. The treatment that is principally used is craniospinal irradiation and intrathecal chemotherapy, as the blood brain barrier prevents accumulation in the CSF of therapeutic concentrations of most drugs administered by other routes (2).

Conclusions

Although malignant meningitis is an unusual diagnosis, medical laboratory scientists, especially those in microbiology, routinely deal with fluids that may contain neoplastic cells. They should be trained to recognise abnormal cytological features as described previously, such as unusually high cellularity that would then prompt them to seek a formal cytological examination for the specimen. The ability to determine that a fluid may contain neoplastic cells, and that it should be forwarded for cytological exam, is important in making a rapid and accurate diagnosis.

Acknowledgments

Dr Stephen Allpress, Dr Jan Craik, and Dr Sanjeev Chunilal, North Shore Hospital, Auckland.

References

(1.) Jaffe ES, Harris NL, Stein H, Vardiman JW. World Health Organization. Classification of Tumours, Pathology and Genetics, Tumours of Haematopoietic and Lymphoid Tissues. IARC Press, Lyon, France, 2001.

(2.) Peckham M, Pinedo HM, Verones U. Oxford Textbook of Oncology (Vol. 2). Oxford Medical Publications 1995.

(3.) Demopoulos A, Posner JB. Pathophysiology and clinical features of leptomeningeal metastases (carcinomatous meningitis). Up To Date (http://Uptodate.com).

(4.) Norden A, Hochberg E, Hochberg F. Secondary involvement of the central nervous system by non-Hodgkin's lymphoma. Up To Date (http://Uptodate.com).

G Gibbs, BSc, DipSci, BMLS

K M Read, BSc, MBChB, FRACP

Microbiology, North Shore Hospital, Auckland