CELL BLOCK TECHNIQUE

AND ITS UTILITY IN

DIAGNOSIS
Presented by:
Dr. Deep Banerjee
(2nd year resident, Dept. of Pathology, NBMCH)
Moderated by:
Dr. Vaswati Das
(Associate Professor, Dept. of Pathology, NBMCH)
 Any lesion

Cytology Histology

Cell Block • Various

• Exfoliative
Liquid based • CELL
Abrasive BLOCK IS A METHOD OF PREPARING
biopsies
• Tissue
cytology • CYTOLOGIC MATERIAL
Aspiration SO THAT ITsampling
CAN BE
• Architectural features
PROCESSED, SECTIONED,
• Less invasive,STAINED
less cost AND VIEWED
• Archive for reference
AS HISTOLOGIC
• Ancillary tests MATERIAL

• Not suitable for

HOLD TOGETHER OR
• Salvage BLOCKING
of material not PARAFFIN
• Tumour heterogeneity
architectural study – lessBLOCK
stromal
• Material wasted in the contamination
hub of needle or • Invasive
• Formalin fixed
container- Residual, clot • Costly
paraffin embedded
• ROSE available (FFPE) blocks
• Smear needed for each • Specific diagnosis-
ancillary test targeted treatment
HISTORY
• Ascitic fluid sediments
• Embedded in Celloidin
1896
• Sectioned in
microtome

Advent of Centrifuge
1901

1959 Shackelford & Jones- use

of agar

1973 Kaltenbach et al. – Plasma

thrombin method

Hologic Inc
2007 Cellient automated cell
block system
 SPECIMEN COLLECTION

Cytology specimen FLUID SPECIMEN FNAC SPECIMEN

50 ml Fresh in
Buffered
conical sterile
saline
tube container

Serous fluids
Various washings
Urine
Cervical Liquid based specimen
 SPECIMEN FOR CELL BLOCK

Fresh Unfixed specimen / single Formalin fixed or fresh specimen

scattered cells with fatty fragments

Hypocellular Cellular Blood mixed With Clot

Sediment • Nylon mesh- filter fat

Sediment Let clot fix in
concentrated fragments
concentrated Lysis agent 10% formalin
as button, gel • Fix > 2hrs
by added Cut fix clot 2mm
or agar added • No centrifugation – fat float
Centrifugation slices
or centrifuged

Hypocellular
Cellular?
?
 SPECIMEN ADEQUACY:

Cytocrit/Tissuecrit:
Defined as percentage sediment in comparison to supernatant

Hypocellular: <1ml
sediment with
<50% tissuecrit
Supernatant

Centrifuge
2500 rpmi Sediment
3 min
Cellular: >1ml
sediment with >
50ml tissuecrit
Ref : CellBlockistry: Chemistry and art of cell-block making – A
detailed review of various historical options with recent
advances - PMC (nih.gov)
 VARIOUS METHODS OF CELL BLOCKING

COAGULATION based
GEL based methods
methods:
OTHERS:

Chemical based Enzymatic based

• PREFORMED SUPPORTING
MEDIA:
• Gelatin 1. Gell discs with wells
• Albumin- 2. Sponge discs with wells
• Agar 95% alcohol 3. Collodion method
• Clotted • Already processed cytology
• Histogel • Picric acid
specimen preparation:
• Alginate-
• Plasma 1. Scrapped material from
Calcium cytology smears
fibrinogen
• Glucomanan 2. Milipore filters
thrombin
-formalin
with alcohol
 NORMAL SALINE RINSE METHOD:
• Most commonly used
• Cheap
• Easy

METHOD:
Rinse aspiration needle with 20-30ml
normal saline

Processed as regular Preserve in RPMI

paraffin specimen medium

Ancillary
Stained with techniques • Archieve
routine H/E stains • Future
centrifugation
 RESIDUAL CLOT FIXED SEDIMENTATION METHOD:

METHOD

Residual clot in needle hub/tissue

container

Rinsed in 50% Ethanol-buffer

/formalin Fixative

Centrifuge – 4000rpm-6mins- form

pellets

Supernatant discarded
Sediment removed
Put into filter paper

Placed in cassette
Processed as regular paraffin
specimen
 PLASMA THROMBIN METHOD:
DISADVANTAGES:

Principle: • Requires centrifuge

• To enmesh cellular • Material lost on decanting
material in clot • Decreased antigenicity on exposure to saline
• Flimsy button- difficult to align on cutting
METHOD:
surface
• After discarding • Specimen fixatives which inhibit thrombin
supernatant
• For 1 ml sediment-4ml
plasma
• 4 drops thrombin
• Mix – gentle tap, vortexer
• Sit for few minutes , soft
ball forms
https://pubmed.ncbi.nlm.nih.gov/29893770/
• Moved to cassette
• Submit to processing MODIFIED PLASMA THROMBIN METHOD:
Pooled fresh plasma -aliquoted into 2-mL Eppendorf
tubes and the FNA sample directly rinsed into the
plasma. Two drops of reconstituted thrombin are
added and gently mixed. A cell clot is transferred to a
tissue bag, fixed in formalin, and processed.
 The AGAR method

Use of AGAR:
Principle: Agar is good choice for tissue embedding because
• Concentrated sediment of its property of hysteresis as it remains solid at
supported by cell 36°C±1.5°C, which remains firm even at 60-65°C thus
holding the tissue firm and oriented in molten paraffin
adjuvant- agar wax. Agar melts only at 87°C±1.5°C, a temperature
• Agar solidifies below 50 range which tissue processing never reaches.
deg centigrade Ref.
https://medicopublication.com/index.php/ijfmt/article
Method:
• Tube of 4% agar- in a
small beaker of water-
heat in microwave to melt
• 4ml of agar – add to
sediment- mix PROS:
• Creates good quality
• Refrigerate- cell button – block
CONS:
• Availibilty of agar
cassette • Various cell in
• Time consuming method
• Routine processing different layers for
optimal visualization
OTHER CELLULAR ADJUVANTS:
• Collodion bag-scant cellularity-nitrocellulose material-block of friable
material eg, brain
• HistoGel- Hydroxyethyl agarose, same as agar method
• Gelatin foam
• Albumin
• Pre-gelatinized starch
 COLLOIDION BAG TECHNIQUE:

Method:
• Colloidion is poured into
tube
• Tube coated completely-
excess colloidion
removed- 8-10 min air
drying inverted position
• Formalin fixed specimen
transferred to coated
tube and centrifuged
• Bag removed from tube
• Placed in cassette
• Processed
 DIFFERENCE BETWEEN VARIOUS CELL BLOCK METHODS:

Cell Block method Advantage Limitation Interference with ancillary

tests

Residual Clot sedimentation • Simple method • Low yield • Not seen as such
method • No adjuvant/equipment • Adequate passes – patient
needed compliance

Agar Method • Low cost • Flimsy button • Over heating may interfere
• Better tissue orientation • Long cooling time with IHC
• Applied to fresh/fixed both

Plasma Thrombin method • Low cost • Flimsy button • No such interference seen
• Simple method • Fixatives interfere with
• High tissuecrit specimen thrombin

Colloidion method No significant advantage • High volatile, inflammable • B5 fixative interferes with
• Safe handling IHC
• Expensive, less available
 UTILITY OF CELL BLOCK OVER CONVENTIONAL SMEAR:

IN FLUID CYTOLOGY IN SOLID ORGAN

1. Spontaneous clots-enmesh
cells
2. Identification of primary site
of malignancy
3. Histologic pattern of cancer Gynecological Lung cytology Breast cytology
4. Psammoma bodies, cytology
collagenous stroma, • Stromal invasion &
Tumor necrosis • Her2neu IHC study
granulation tissue better • Subtyping of lung
differentiate HSIL • PR study
identified carcinoma by IHC • Ethanol may alter
5. Long storage in refrigerator from SCC • EBUS TBNA
• ICC & surrogate samples Core
possible before cell block
marker study for HPV biopsies increase
6. Molecular testing- FISH, in
Sensitivity &
situ PCR
Specificity
7. Polymorphous and • Molecular
monomorphous population alteration study-
of lymphoid cells targeted therapy
 PLEURAL FLUID

CONVENTIONAL SMEAR CELL BLOCK

ASCITIC FLUID
 PERITONEAL FLUID

CONVENTIONAL SMEAR CELL BLOCK

PLEURAL FLUID

METASTATIC DEPOSIT OF LUNG ADENO CA TTF-1 & NAPSIN

The sensitivity and specificity was 100% and 76% respectively. The positive predictive value was 41% and negative
predictive value was 100%. Accuracy rate was 80%
PAP SMEAR CELL BLOCK
A 60 year old
HSIL
lady

P16 STAIN BIOPSY

 FNAC CELL BLOCK
Poorly
45 year old man
differentiated
with lung mass
SCC

P40 +ve tumour cells CK5 & CK6 +ve

 FNAC CELL BLOCK
56 year oldCell
Small male, lung
mass,Cancer
H/O smoking

TTF-1 + ve CD56+ve
 B
R
FNAC E CELL BLOCK
A
S
T

FNAC, DUCTAL CARCINOMA CELL BLOCK, DUCTAL CARCINOMA

ACCURACY OF CELL BLOCK 88.2%
ACCURACY OF FNAC 69.2%
 RECENT ADVANCEMENTS

• In 2007
• Vaccum assisted filtration to
capture cells
• Methanol based solution wash
• Xylene fixed
• Embedded in paraffin
• Ready to process

1. Nano gel with AV marker

2. Subtractive Coordinate Immunoreactivity pattern(SCIP)- IHC
3. Cellient Automated Cell Block

CONS:
45 mins, one block
Methanol interferes with IHC
 TAKE HOME MESSAGE

• Major role in ancillary techniques- IHC, Molecular genetics, Electron microscopy

etc
• Suboptimal cellularity
• Skill based- incomplete pellet, sample loss
• No standard protocol
• Procedure varies in different laboratories
• Newer advancement may prove beneficial
• Cell block as an adjunct to Cytology and Histology
can prove to be the ‘Missing block’ in diagnostic
puzzle