Torsdag den 5 mars 2015

Vad är det för skillnad mellan

hematologi och cytologi?

Cytologi med patientfall

Keng-Ling Wallin Med.Dr

Is a set of laboratory tests that examine a

sample of the fluid surrounding the brain and the

spinal cord.

CSF is the ultrafiltrate of plasma.

Its charactersitics are:

a) It is clear and colourless

b) It contains glucose, electrolytes, amino acids

and other substances depending of its

clinical condition

c) It has low protein content and low in

cellularity

Due to its nature and it being linked to several

pathological processes, intrepretation of the

CSF analyses can be a challenging.

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Cerebrospinal Fluid (CSF) analysis:

CSF is withdrawn from the subarachnoid space

through a needle – ”lumbar puncture” or spinal

tap.

Several tubes of CSF are often collected on site,

and at different times/period:

First tube – clinical chemistry and serological

analysis

Last two tubes - hematology and microbiology,

cytology is performed only when indicated.

Several taps made near the site of

suspected LM to obtain representative

samples.

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Lumbar puncture

CSF is invaluable as a diagnostic aid in the evaluation of inflammatory conditions,

infectious or non-infectious subarachnoid haemorrhage and in leptomeningeal

metastasis.

CSF samples must be transported to the laboratory < 1 hr

Cytological - processed within 2 hrs after puncture (due to pre-fixation)

Hematology – processed within 30 min

”Important to use glass tubes only during collection of CSF!” (no longer

valid according to the consensus guidelines in Multiple Sclerosis

International, 2011)

Color

pH

Appearance

Sp Gravity

Clot formation

Total solids

PO2

Cellularity

Colorless

7.28 – 7.32

Clear

1.003 – 1.004

No clot on standing

0.85 – 1.70 g/dL

40- 44 mmHg

Accellular (few lymphocyes

and monocytes at resting

stage, and ependymal cells)

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Characteristics of normal and ”abnormal” CSF

Reddish tinct

Altered

Turbit

Altered

Microfibrils

Elevated

>160 mmHg

Cellular

Walbert & Groves Future Oncology, 2010, 6:287-297

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The disease states that can involve the CSF

High false negative rate

in cytology. 50% whose

lumbar puncture have

”normal values” and no

malignant cells still have

LM

To further increase

specificity, it is

recommended that patients

be examined with MRI

(nagnetic resonance

imaging) prior to lumbar

puncture.

Inflammation and other

inflammatory neuropathies

non-inflammatory disease

infections (bacterial, viral, fungal)

multiple sclerosis (autoimmune

polyneuropathy

neoplasia (primary or secondary)

A task force (EFNS- the European Federation of the

Neurological Societies) has been established to evaluate

the theoretical background and to provide guidelines for

clinical use in routine CSF analysis.

The purpose of these guidelines was to produce

recommendations on

a) how to use the set of CSF parameters in different clinical

settings

b) to show how different constellations of these variables

correlate with different diseases of the nervous system

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Guidelines on routine Cerebrospinal fluid (CSF) analysis

Routine parameters for analysis are determination of protein, albumin,

immunoglobulin, glucose, lactate, cellular changes, specific antigen and

antibody testing.

Deisenhammer et al., European Journal of Neurology 2006, 13:913-922

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Leptomeningeal metastases (LM) is the metastatic dissemination of malignant

cells to the leptomeninges and the subarachnoid spaces of the CNS.

The first report of a LM based on cytology was made in 1904* and since then it

has been used for doagnosis of numerous neoplastic diseases. *(Dufour MH, Meningite sarcomateuse diffuse avec envahissement de la Moelle et des Racines: Cytologie

positive et special du liquid cephalorachidien. Rev Neurol (Paris) 1904(12):104–106)

Deisenhammer et al., European Journal of Neurology 2006, 13:913-922

Total protein analysis:

• Newborns: high protein levels and decrease gradually within the

first year and maintained at low levers throughout childhood

• Adults: the protein concentration increase with age

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National cancer register – Socialstyrelsen The most common cancer incident cases in Sweden 2013

43368 cancer cases

5-(8)% of all cancers present with LM.

That will give us 5 x43368/100 = 2168 to (3469) cases of LM/per year

in Sweden. This incidence is often higher, since not all LM patients are diagnossed with LM

before death.

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Cerebrospinal Fluid Involvement in

Mantle Cell Lymphoma (an aggressive

cancer incurable by standard therapy)

A–B, a pleomorphic population of lymphoid cells with

irregular nuclear contours and variable amounts of

cytoplasm was seen in the majority of CSF positive

cases (Wright-stained cytocentrifuge preparation, high

magnification).

Large lymphoid cells with dispersed chromatin,

prominent nucleoli, and scant cytoplasm resembling

other blastic-type proliferations involving the CSF were

observed in the two cases of blastoid mantle cell

lymphoma (MCL) as well as in one case of typical MCL

Valdez et al., Modern Pathology, 2002, 15:1073-9

A review of 108 patients with MCL, 25/108

(23%) had CSF involvement. 9/25 (36%)

had specific radiographic abnormalities.

10/25 (40%) were identified by cytology.

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Metastatic Gastric Adenocarcinoma

The CSF samples were processed by the

Cytospin method and the smears stained

with Papinocolaou stains. Tumor cells

were large with ample cytoplasm,

pleomorphic vesicular nuclei, and

prominent nucleoli. Cytoplasmic mucin

was seen on mucicarmine staining.

33 yr old woman presented with pain in

abdoment. Had ascitis which had

mailgnant cells. Mesentric LN showed

metastatic adenocarcinoma. Endoscopy

revealed an irregular gastric growth. CT

showed foci of vasogenic edema. MRI

showed signs of LM with white matter

edema and nodules.

Bisht S et al., Journal of Cytology, 2008, 25:25-27

Prominent nucleolus

Mucinous cytoplasm

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A. Postcontrast sagittal T1-weighted MRI of the cervical

spine shows diffuse leptomeningeal enhancement along

the surface of the spinal cord. B. Axial contrast-enhanced

CT image through the abdomen demonstrates extensive

ascites and an enhancing midline mass (arrows) adjacent to

the shunt catheter (arrowhead).

A. Nests of anaplastic tumor cells are seen with round

to oval eccentric nuclei, coarse chromatin, prominent

nucleoli and moderate homogeneous cytoplasm (Diff-

Quik). B. Single giant malignant cells with similar

morphology are also seen

Bilic et al. CytoJournal 2005 2:16

FNA

CSF (Diff-Quik)

A. Single bizzare multinucleated neoplastic cells display

"partial wreath-like" nuclear configuration, coarse chromatin

clumping and multiple nucleoli. B. Clusters of smaller tumor

cells showing variation in size, high nuclear to cytoplasm ratio,

scant homogeneous cytoplasm, with even chromatin distribution

and occasional clefting of the nuclear membrane.

CSF (Pap)

Disseminated primary diffuse Leptomeningeal Gliomatosis

Highly pleomorphic

cells from peritoneal

mass

Post-contrast fat-suppressed sagittal

and axial T 1 –weighted image

demonstrates pathologic intrathecal

enhancement at the L5 and S1

levels. Arrows indicate the

pathologic intrathecal enhancement

at the L5 and S1 levels.

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Metastatic squamous cell carcinoma in CSF (1)

56 yr old caucasian male

presented with rapidly

increasing right

submandibular swelling.

Patient manisfest with

several bilateral enlarged LNs.

FNA showed keratinizing squamous cell

carcinoma, probably metastic. Primary

tumor unknown.

3 weeks later loss of vision in right eye,

pain and numbness in arm and both legs.

MRI showed bilateral abnormal

enhancement of optic nerve and lumbar

spine showed nodular thickening.

Ren s et al., Acta Cytologica, 2012, 56:209-213

A) Atypical mononuclear, hyperchromatic

cells with distinct cellular outline (Pap,

cytospin).

B) Positive membranous staining of the

atypical cells with broad-spectrum

cytokeratin (AE1/AE3, cytospin).

A) Neck aspirate smears showing diagnostic

keratinizing malignant squamous epithelial

cells in a background with mononuclear

hyperchromatic cells and necrosis. (Diff-

Quick, FNA)

B) Positive membranous staining of the

malignant cells with broad-spectrum

cytokeratin (AE1/AE3, FNA).

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Metastatic squamous cell carcinoma in CSF (2)

A B

CSF

A B

Ren s et al., Acta Cytologica, 2012, 56:209-213

(A) CT image showing an isodense or slightly hyperdense

lesion in the left temporal lobe (CT value: 45 to 61)

(B) MRI image showing a 6-mm abnormal signal in the left

temporal lobe, which was hyperintense on T1-

weighted images,

(C) hypointense on T2-weighted images,

(D) slightly hyperintense on fluid-attenuated inversion

recovery images, and

(E) mild enhancement with gadolinium contrast

(F) Punctate and linear enhancement in the sulci and gyri

of the cerebellum can be observed.

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Leptomeningeal metastases from a primary CNS Melanoma

A 37 yr old woman refered due to horizontal

diplopia and progressive headaches for 2 wks.

She had a medical history of non-frequent

epilepsy as a child. Had scattered melanocytic

nevi with hair when she was born. These nevi

were not darkened in color, enlargement, pruritus

or ulceration. Neurological examination on

admission revealed right eye adduction.

Pan et al., World Journal of Surgical Oncology, 2014, 12:265-269

(A, B) Round or oval tumor cells with different sizes were scattered,

and presented as pseudopodia-like protrusions on cell membranes,

with basophilic characteristics. Cells had round nuclei, large and

distinct nucleoli, coarse and unevenly distributed chromatin, and

particles in the nucleus or cytoplasm. (Liquid-based cytology,

Papanicolaou staining, ×400).

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Primary Melanoma in Cerebrospinal Fluid

Pan et al., World Journal of Surgical Oncology, 2014, 12:265-269

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Temozolomide belongs to a

group of chemotherapy drugs

called alkylating agents. Unlike

many other chemotherapy

drugs, it can reach the brain

from the bloodstream.

Liposomal cytarabine is used

to treat cancer of the lymph

system that has spread to the

brain.

A 42- year old patient was diagnosed with Her2- positive breast cancer in 1994 and under

went therapy. In 2002, an axillary relapse was operated and treated.

In December 2005, a relapse at the left brachial plexus discovered and treated.

In December 2006, the patient developed bladder dysfunction and a slowly progressing

paraparesis. The diagnostic workup revealed diffuse solid spinal metastases and CSF

involvement. In March 2007, focal irradiation was applied, followed by near-continuous

temozolomide 100 mg/m2 day 1-5/7 and liposomal Ara-C every other week for 8 weeks

and then every 4 weeks. The bladder dysfunction improved to normal function and the

paraparesis stabilized with only a slight paresis of the left leg remaining. MRI showed stable

spinal metastases in June 2007. The CSF cell count dropped to normal values and the

CSF was free of tumour cells one single time in August 2007.

In November 2007, MRI showed progression of a preexisting metastasis. Despite rapid

irradiation, the patient developed subtotal paraparesis and underwent further therapy. The

CSF was free of tumor cells in February 2008. Temozolomide was started again and the

combination with liposomal Ara-C continued, because of persistence of malignant CSF

cells, until June 2008. In spite of intensive care of the skin ulcerations, the patient died in

July 2008 from septicaemia without signs of progression of the CNS manifestation, 16.8

months after diagnosis of NM.

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The prognosis of patients with CSF dissemination and solid CNS metastases from

breast cancer is poor. Without specific treatment, the median survival of NM from

solid tumours is 4-6 weeks (Intensive treatment : median overall survival is 4-8

months)

Hoffmann et al., Anticancer Research 2009, 29:5191-5196

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Treatment of Breast Cancer in neoplastic meningitis

Overview of the radiological and cytological course (case 2) during treatment with

temozolomide 100 mg/m2 day 1-5/7 and lumbar liposomal Ara-C every 2-4 weeks. Lower

panels: CSF cytology (Pappenheim, ×1000).

Hoffmann et al., Anticancer Research 2009, 29:5191-5196

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One can go on forever, case efter case…...