5. BODY FLUIDS
The intrathoracic and intraperitoneai organs are covered by a single layer
of mesothelial cells, which is continuous with the lining of the thoracic and
peritoneal cavities
The potential space between the two layers of epithelium contains a small
amount of lubricating fluid
An accumulation of fluid due to an imbalance between fluid production and
reabsorbtion is called an effusion
CAUSES ARE :
1. Increase hydrostatic pressure.
2. Decrease colloidal osmotic pressure.
Effusion can be of two type :
1.TRANSUDATIVE
2.EXUDATIVE
6.
7. TRANSPORTATION
The fluid must be transported to the laboratory as soon
as possible.
If for unavoidable reasons processing cannot be done
immediately or within 2 hrs, then the fluid should be
refrigerated at 4 degree celcius and transported on
ice/cold box to arrest degeneration of cells. Specimen
should not be allowed to freeze
Routine cytological evaluation is possible upto 48 hrs,
but cell morphology gets compromised after 24 hrs.
8.
9. COLLECTION OF FLUID SAMPLE
THE FLUID IS COLLECTED IN A STERILE CLEAN DRY
CONTAINER WITH PROPER LABELLING AND
IDENTIFICATION WHICH INCLUDE NAME, AND HOSPITAL
NO OF THE PATIENT
REJECTION CRITERIA-
1. Improperly labelled samples
2. Dirty ,cracked and broken containers without proper lid
3. Inadequate amount of sample
10. RECOMMENDED VOLUME FOR
CYTOLOGICAL EVALUATION
Atleast 20-30 ml is optimal
If cell block preparation is required, 30-50 ml is optimal
Low cellularity specimen would need higher volume of
sample.
11. ANTICOAGULATION
Samples with high protein content and those which are blood
tinged or hemorrhagic anticoagulant prevents clotting of sample
The choice of anticoagulants are –
1. HEPARIN (3-5 IU/ml of fluid). Syringe is rinsed in HEPARIN.
2. CITRATE
3. EDTA
4. AMMONIUM OXALATE(1%).9 parts fluid +1part
anticoagulant
12. 1.SPUTUM
2.BRONCHIAL ASPIRATE
3.MUCOCELE FLUID
HIGH MUCUS
CONTENT
Maximum time
to be kept :
24 hrs if
refrigerated
EFFUSION FLUID
HIGH
PROTIEN
CONTENT
Can be
preserved for
24 -48 hrs
without
refrigeration
CSF/URINE
LOW MUCUS
OR PROTIEN
CONTENT
Maximum time
can be kept :
1-2 hrs
14. GROSS EVALUATION OF THE
SPECIMEN
Following point should be noted:
1.QUANTITY - ml/Ltr
2. COLOR – clear/straw /brown/red/chylous/red/purulent
3.CONSISTENCY – serous/mucoid/gelatinous/thick copious/tar
like
4.TEMPRATURE – room temperature/refrigerated
5.CLOTTING- YES/NO
15. IN CASE OF HEMORRHAGIC FLUID
Hemolysis is achived by any of the following:
1.CARNOY”S FIXATIVE
2.Glacial acetic acid
3. Saline rehydration
4.Saponin
18. CENTRIFUGATION
1. In a solution particle whose density is
higher than that of the solvent
sink(sediment) and particle that are
lighter than it float to the top.
2. The greater the difference in density
the faster they move.
3.If there is no difference in density the
particles stay steady.
20. CYTOCENTRIFUGATION
Cytocentrifugation with the Cytospin allows for the cells to be
placed onto a vertical slide ,while the fluid suspension is
absorbed by surrounding absorbent paper
21. CYTOCENTRIFUGATION
PROCEDURE:
Take two 50 ml conical centrifuge tube
Place it in the centrifuge machine and run it for 2500 rpm for 5 min
Decant the supernatent
A pipette is used to resuspend the sediment
Assemble the holder ,slide, filter card and funnel
Pour 5-8 drops(if clear fluid) or 1-2 drops (cloudy fluid) into the chamber
Specimen are spin at 800 rpm for 5 min on low acceleration
once complete ,the slide clip unit is opened ,and the filter card and slide is removed
Air dried slide is stained with MGG and wet fixed is stained with PAP
22. LIQUID BASED CYTOLOGY
Cytology through a liquid medium
Samples from various sources are collected or rinsed in a
preservative solution ; the cell suspension is then evenly distributed
as a monolayer on a glass slide
TYPES
FDA approved:
Two most popular method are used are :
1. ThinPrep (methanol based) and
2. Surepath (ethanol based)
23. CELL BLOCK
A cell block is a method of preparing cytological material so that it
can be processed, sectioned, stained and viewed as a histology
section
It contain aspirated materials embedded in paraffin that broaden the
diagnostic value of cytology specimen
26. NEED FOR CELL BLOCK
1.cell block can increase both diagnostic sensitivity and specificity (through
both cellular morphology and ancillary testing )
2.It requires minimal effort and is cost efficient.
3.Moreover tissue preserved in cell block can be stored easily.
27. PLASMA THROMBIN METHOD
PRINCIPLE : To enmesh the cellular material
1.Aspirate is taken in a test tube
2.Centrifugation at 300G for 10 min
3.Cell pellet + supernatant
4.Supernatent is decanted off
5.Cell pellet is resuspended in 3 drops of plasma
6.3 drops of thromboplastin is added and mixed
7.3 drops of calcium chloride is added and mixed gently
8.Allow to stand undisturbed for 15 -20 minutes
9.The pellet is then embedded in paraffin
10.cutting
11.Staining
28.
29. PLASMA THROMBIN METHOD
ADVANTAGES DISADVANTAGES
SIMPLE, LOW COST CROSS CONTAMINATION FROM
PLASMA AND THROMBIN
EASY AVAILABILITY OF
REAGENTS
UNEVEN CONCENTRATION OF
CELLS
OPTIMAL CYTOMORPHOLOGY
CLEAN BACKGROUND FOR
ANCILLARY STUDIES
30. HISTOGEL METHOD
PRINCIPLE: The concentrated sediment is supported by a cell
adjuvant.
HistoGel is a modified agar .It is useful in situations where no visible
sediment is seen after initial centrifugation ,since the gel permeates
the cells and traps them in a solid matrix.
LIMITATIONS Include:
1.LOW CELLULARITY
2.POOR VISUALISATION OF CELLULAR MATERIAL
3.TEDIOUS PROCESS AS HISTOGEL AGAR MUST BE
MAINTAINED AT A TEMPRATURE ABOVE ITS GELLING
TEMPRATURE
31.
32. BACTERIAL AGAR METHOD
Agar solidifies below 50 degree celcius ,this property of agar
is used to form a solid cell pellet
Similar to histogel method
33. COLLODION BAG METHOD
Collodion is a commercially available liquid polymer.
It is a nitrocellulose solution that is used to coat conical specimen tubes
before centrifugation in order to capture the cell pellet
A conical centrifuge tube is filled with the collodion solution and then poured
out,leaving a thin layer of solution coating the test tube wall
This forms a membrane bag inside the tube once it is dried.
Serous fluid is poured into the collodion lined tube and centrifuge to form a
cell pellet
The membrane bag containing cell pellet is gently removed from the tube
The pellet end is tied off from liquid end and excess bag is cut off.
Cell pellet in the bag is then processed as tissue .
34.
35. CELLIENT AUTOMATED CELL BLOCK
SYSTEM
The cellient automated cell block system concentrates, processes,
and embeds loose cells into a paraffin block ready for sectioning.
User variability is minimized and time is reduced.
Cells concentrated into a well in the cassette by vacuum, and
subsequent washes with alcohol and xylene fix and clear the
specimen
The cell button is then infiltrated with paraffin in the same cassette.
The entire process takes approx. 45 minutes
The instrument can accommodate only one specimen block at a time
,so high volume labs may require multiple instruments
The standard process uses alcohol fixation. However formalin fixed
protocol is available, giving the user options in case immunochemistry
may be required.
36. CELL BLOCK
ADVANTAGES DISADVANTAGE
1. Cell blocks are
simple,reproducible,and readily available
in routine labs
1.Compared to routine smears it
takes longer time.
2.No necessity of biopsy 2.Distortion artifacts.
3.Increased cellularity 3.Sparse cellularity
4.Better morphological and architectural
patterns,increased diagnostic yields
5.Unlimited storage of sample
6.Application of ancillary studies(ICC
and molecular testing)
