NEUROBEHAVIOR AND SPECIAL SENSES SYSTEM LAB ACTIVITY OF CLINICAL PATHOLOGY II CSF EXAMINATIONS: A. LEUKOCYTE COUNT B.

LEUKOCYTE DIFFERENTIAL COUNT C. TUMOR MARKERS IN BRAIN TUMORS

DEPARTMENT OF CLINICAL PATHOLOGY MEDICAL FACULTY UNIVERSITAS PADJADJARAN BANDUNG 2007

A. CSF LEUKOCYTE COUNT I. OBJECTIVE

At the end of the activity the students will understand and can describe about: 1. How to perform the CSF leukocyte count 2. The interpretation of the results 3. The interfering factors which can affect the result

II. INTRODUCTION
Normally only small amount of leukocyte can be found (<6 cell/mL CSF), and usually consists of mononuclear (MN) cells (lymphocytes or an occasional monocyte).

III. HOMEWORK ASSIGNMENT:

To be collected to your tutor at the day of lab activity 1. Z, 4 yrs old boy, was suspected had meningeal TB, but the results of CSF examinations were normal; why? 2. Why do we have to count all leukocyte cells in the whole area of the counting chamber? 3. What are the differences between Improved Neubauer and Fuchs-Rosenthal counting chamber? 4. In brain tumor case, the protein level in the CSF is increased, why?

IV. MEASUREMENT/ EXAMINATION

PREPARATION: REAGENT: Crystal violet 0.1 g Glacial acetic acid 10.0 cc Distilled water 90.0 cc Filter the solution, because the reagent should be free from any particles SAMPLE: Shake the CSF well, place 0.4cc in a small test tube or a watch glass; so the remaining CSF may not be contaminated by the reagent

PROCEDURE: Draw up the reagent in the leukocyte pipette (Thoma) to the mark 1, and fill with CSF to the mark 11 Mix by (rotatory) shaking the pipette well and then discard first 2-3 drops Place a drop on each side of double counting chamber of Improved Neubauer and wait for 2 minutes for the cells to settle Count the cells in the whole areas of the counting chamber 18 x 1 x 1 x 0.1 mm3 The number of cells counted is then multiplied by 0.6 = the number of cells/mm3 IMPROVED NEUBAUER CELL COUNTING CHAMBER

LEUKOCYTES COUNT : ALL 9 AREAS OF = 1 X 1 X 0.1 mm3; THE VOLUME TO BE EXAMINED= 9 X 1 X 1 X 0.1 mm3 = 0.9 mm3

FUCH`S-ROSENTHAL CELL COUNTING CHAMBER

1 2 3 4 The square of the chamber = 4mm x 4mm The depth= 0.2mm The volume to be examined: 16 x 0.2 mm 3= 3.2 mm 3

10

11

12

16

15

14

13

A Fuchs-Rosenthal counting chamber may be used instead of Improved Neubauer counting chamber The cells must be settled 5 minutes before counting The ruled area covers 16 mm2, and 0.2 mm depth The number of cells counted is multiplied by 0.35 = the number of cells/mm3

INTERPRETATION:
Normal count : 1-8/mm3 Increased count : an irritative or inflammatory lesion of the brain, spinal cord or meningens

RESULT: DISCUSSION:

B. LEUKOCYTE DIFFERENTIAL COUNT MEASUREMENT/ EXAMINATION: METHODOLOGY:

Thin smear of the CSF sediment on glass slide

PREPARATIONS:
Reagent: ethanol absolute; Giemsa stain Differential count sheet paper

PROCEDURES:
Centrifuge CSF for 10-15 minutes at medium-rate speed (1500 -3000 rpm). Pour off the supernatant, and make thin smear of sediment on a glass-slide. Dry the slide in the air without heating it Stain with Giemsa`s reagent (Fixate the slide with absolute methanol for 2-3 minutes, pour off the methanol excess, flooded the slide with Giemsa stain solution for 20-30 minutes and rinse with water. Put the slide up sided, and let it dry. Count and tabulate up to 100 leukocyte (or the entire amount of leukocyte when the amount is <100 cells). Report the result in percentage. NOTE: When CSF is xanthochromic from patient <2 months old, examine for toxoplasma in the PMN or monocytes

REFERENCE VALUES:
Normally: only mononuclear cells (MN) i.e. lymphocyte or an occasional monocyte and an occasional endothelial cells from the lining of the pia-arachnoidal spaces are found

INTERPRETATION: Please, see the table CSF FINDINGS IN DISEASES RESULT: DISCUSSION:
. .

CONCLUSION:

Table: CSF
PRESSURE DISEASE

FINDINGS IN DISEASES
CELLS QUALITATIVE PROTEIN (globulin) QUANTITATIVE PROTEIN (mg/dl) GLUCOSE (mg/dl)

Normal Pneumococcus meningitis TBC Meningitis

(mm of water) 100-150 Greatly increased

APPEARANCE

Clear, colorless, no clot Turbid to yellow, Clot (+) Clear, opalescent, or white. Fibrin web Clear-milky occ. fibrin clot Normal, occ. Fibrin clot Normal or sl. turbid

0-8 MN Acute: slight increase Less acute: 100-5000 95% PMN Children Early 10-100 Late100-1000 70-90% MN Adult fewer cells Pre-paralytic 15-2000 PMN paralytic 10-100 MN 10-200 all monocytes; <10 in 3050% cases Ruptured 10-100; 7075% PMN; unruptured 5-30; 90-95% MN Normal or 10-80 Normal or increase in MN 70-90% cases: normal; others: 5-50 MN

0 ++ to +++

25-45 (A/G ratio1:5) 100-400

40-70 60% of blood glucose 0-10

500-1000

to +++

30-400

15-20

Acute Poliomyelitis Epidemic encephalitis Brain abscess

Usually increased Usually 200 or more Usually increased

to ++

Pre-paralytic 25-60, paralytic 60-300 25-60

Normal

Above normal 65-120 Normal or increased

to +

30-100

Brain tumors Spinal cord compression Multiple sclerosis

Usually increased Normal or decreased Normal or decreased

Normal or xanthochromic Clear or xanthochromic Normal

to ++ + to ++++

50-200 Complete block 3002000; Partial block 45-300 30-80 (10-49% cases : above normal)

40-100 Normal

0 to +

Normal

PRESSURE DISEASE

Subarachnoid hemorrhage or cerebral injury

(mm of water) Slightly increased

APPEARANCE

CELLS

QUALITATIVE PROTEIN (globulin)

QUANTITATIVE PROTEIN (mg/dl)

GLUCOSE (mg/dl)

Bloody or xanthochromic

Cells increased due to blood

+ to ++

45-200 or higher

Normal or increased

REFERENCE:
1. Henry JB; Clinical Diagnosis and Management by Laboratory Method; 19th ed 1996; W.B. Saunders Co 2. Breuninger CM, Wittig P; Diagnostics- An A to Z Guide to Laboratory Tests and Diagnostic Procedures; 2001, Springhouse Corp, Pennsylvania 3. Gaedeke MK, Laboratory and Diagnostic Tests Handbook, 1996, AddisonWesley, The Benjamin/Cummings Publishing Co 4. Hepler, OE; .Manual of clinical Laboratory method, 1968

C. TUMOR MARKERS IN BRAIN TUMORS I. OBJECTIVE

At the end of the activity the students will understand and can describe about: 1. The kinds of tumor markers in the serum of the patients with brain tumor 2. The interpretation of the results 3. The interfering factors which can affect the result

II. INTRODUCTION
Molecular mechanism of CNS tumors remains poorly understood. The brain is physiologically different than other organs in the body; it has blood brain barrier (BBB), blood flow auto regulation, lack of lymphatic drainage, lack of regenerative capacity. Because of the BBB, the use of serum tumor markers is still in debates. THE USES OF TUMOR MARKERS 1. Diagnosis : primary or metastasis tumors 2. Follow-up/ response to therapy 3. Prognosis 4. Will permit new therapies in the future which are more directly against the specific etiology

SAMPLES: brain parenchyma; CSF PRIMARY TUMORS OF CNS Most common WHO : Pylotic & other low-grade astrocytomas (Gr I) Fibrillary astrocytomas (Gr II) Anaplastic astrocytomas (Gr III) Glioblastoma multiform (Gr IV) 50% DIAGNOSIS & MONITORING PROCEDURES Best : MRI CT-scan Surgical/ needle biopsy Immunohistochemical (IHC) (tissue) Serum and CSF tumor markers, especially in pediatric cases CSF LEVELS OF AFP, HCG & PLACENTAL ALP Intracranial non-germinomatous germ cell tumors 1. AFP: Immature teratoma () Embryonal carcinoma (+) Endodermic sinus tumor (+++) 2. HCG: Germinoma () Embryonal carcinoma (++) Choriocarcinoma (+++) 3. Placental ALP: germinoma (++) SPECIFIC MARKERS: 1. Proliferating cell nuclear antigen (PCNA) 2. Ki-67/MIB-1,preferred method in assessing proliferation potential of brain tumors 3. Chromosome 1p and 19q 4. O6-methyl-guanine-DNA methyltransferase 5. Epidermal growth factor receptor (EGFR) 6. p53 and Mouse Double Minute-2 (MDM-2) 7. Matrix metalloproteinase (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) 8. Telomerase 9. Neurotrophin receptor kinase C METASTATIC TUMORS TO THE BRAIN Most patients present with multiple metastases Numerous oncoprotein have been evaluated in brain metastases :i.e. p53, bcl-2, MMPs, ecadherin, CD44, nerve growth factor The use of tumor markers : monitoring of the brain metastases

p53 : not specific, because it is so commonly mutated Cell adhesion molecule CD44, (techniques : RT-PCR, IHC); has a role as homing device to the brain MMPs and TIMPs GT1B (another cell adhesion molecule); appears to target the brain as a site of metastasis, lineage-specific?

NOVEL TUMOR MARKERS OF THE BRAIN Vascular endothelial growth factor (VEGF) - Detectable in CSF - Differs astrocytic and nonastrocytic tumors Recoverin (protein A) - Detectable in serum - May be useful as glioma tumor marker

REFERENCE: - http://www.medscape.com/viewarticle/47002 - Tumor markers in primary and metastatic brain tumor, Arnold SM, Patchell RA