Lecture 15: Cell Blocks and Immunohistochemistry (IHC) in Cytopathology

Cell Blocking
• To process body fluids as cell block for microscopic examination.
• Materials form fluids of the body may be examined either by smear techniques or by preparation of tissue blocks to
maximize detection of the neoplastic, inflammatory, and/or infectious lesions in the body fluids and other tissue juices.
• This is a method where in fluids undergo preparation before actual embedding in paraffin.

Specimens used
• Fine Needle Aspiration Cytology (FNAC)/ Fine Needle Aspiration Biopsy (FNAB) - can usually be gotten from the breast or
thyroid
• Sputum
• Urine
• Effusion fluids
• Lavages & Washings

Specimen Receipt
Identity Verification
• In the laboratory, identity verification must be emphasized

Proper ID on both specimen and requisition forms Specimen source

Date and time collected Submitting physician’s signature
Unique identifier/laboratory number
• All of these must match the patient’s requisition form to the specimen

Specimen Handling
Specimen Adequacy Assessment
• Know when to reject the specimen
• Primarily, improper labeling is already one reason for rejecting a specimen

Universal precautions
• Treat all samples as infectious
• Always wear PPE when handling specimens

Gross Description
• Volume (45 mL)
• Transparency (clear, cloudy, hazy)
• Color (amber)
• Extras (brush tip identified, presence of coagulum, no presence of coagulum)

Sample Preparation Techniques

There are four sample preparation techniques in cytopathology:
• Direct smears
• Liquid based preparation
o Thinprep™ or Surepath™
• Cytospins
• Cell blocks

Thin Prep Non-Gynecological Preparation

1. Collection of the sample
2. Concentrate by using centrifugation for 10 minutes
3. Pour off the supernatant and resuspend the cell palette
4. Add an appropriate amount of specimen to preservative solution bile
5. Run the sample on a ThinPrep™ 2000 Processor using sequence 2 or ThinPrep™ 5000 Processor using sequence non-
gynecological
 An example of a microscopic views of a specimen using the thin preparation (Left) in comparison with the cell block (right) and the direct smears
(middle)

Sure Path Non-Gynecological Preparation

1. Combine into one 50mL tube
2. Automated sample transfer, centrifugation, aspiration, and decanting for
cytologic preparation
3. Run on the BD Totalys™ SlidePrep
• The difference in Sure Path is that it is already automated

Cell Blocking
• Introduced in 1896 using colloidin embedding medium
• Accepted in 1940s
• CB techniques have evolved
• Basic protocol similar in all techniques
• Cell blocks are cytologic preparations that are processed as paraffin embedded blocks in a manner comparable to formalin-
fixed paraffin-embedded tissue in surgical pathology
Application
• This protocol can be used on any nongynecologic specimen, most commonly:
o Serous effusions
o Pelvic/abdominal washes
o Fine needle aspirations
o Liquid based specimens (urine, sputum)
• Procedure for cell block should be applied if there is visible sediment after being centrifuged
• Cell block prepared for IHC and molecular studies

Effectiveness of Cell Block Technique

• One of the constraints of the conventional FNA smear is the limited material available for adjuvant diagnostic investigations
including immunocytochemistry.
• The cell block technique employs the retrieval of small tissue fragments from a FNA specimen which are processed to form a
paraffin block.
• It is widely accepted that cell block technique increases the cellular yield and improves diagnostic accuracy.

Cell Block Clot and Cell Block Cell Block Plasma Colloidin Cellient™ Histogel/Agar Thermo
Technique Scrape Pellet Pellet Thrombin Method Automated Method Shandon™
Alcohol Formalin Method System Cytoblock™
Fixation Fixation System
Advantages Inexpensive. Inexpensive, Inexpensive, Simple, low Good Great for Concentration Good
easy, and easy, and cost, clean cellular small/scanty of cells in cellular
No rapid rapid background yield samples, crisp Histogel for yield due to
additional method method for ancillary architecture. an adequate cell
equipment studies Great for sample. suspensions
required. Good for Good for cells with Fully
FNAs of any FNAs of any Suitable for scant automated Good cellular Good for
type of type of FNAs of any cellularity processing. preservation small/
fluids fluids type and scanty
fluids Consistent samples
results.
Eliminates
No cross the need for
contamination tissue
wrapping
and loss of
 scanty
samples
Disadvantages Does not Cellular Cellular Cross Time Time Tedious Time
work well yield viable yield contamination consuming consuming process consuming
with small variable from plasma method of method of (Histogel has method of
samples Limited data and thrombin preparation preparation to be preparation
on IHC due Optimal (45 minutes converted to
Crush to alcohol results for Uneven Toxic ether for each cell liquid state) Cost of kit
artifact is fixation IHC and concentration fumes for block for
common molecular of cells storage preparation) preparation
studies of cell
Variable Expensive blocks
cellularity
Training of
Histology for
cutting thin
blocks

Limited data
on IHC due to
alcohol
fixation

Plasma Thrombin Technique

• Cell block prepared by adding cell button to plasma and thrombin
• Cell material collects in clot

1. Effusion sample is centrifuged

2. Decant the supernatant and remove excess supernatant
3. Add plasma to sediment and stir
4. Add thrombin to the sediment
5. Clot formation is placed in 10% formalin forming a paraffin block

Agar or other gelatin techniques

• Concentrated sediment supported in agar or Histogel
• Cell pellet formed then processed in paraffin

1. The effusion sample is centrifuged separating the supernatant and the sediment
2. Decant the supernatant
3. Dry the excess supernatant over filter paper so that the remaining sample in the tube will be the sediment
4. Add a few drops of Molten Histogel™ and gently stir to permit mixing of the Histogel and the sediment
5. Transfer the cold and solidified Histogel to the tissue processing cassette
6. Process for paraffin embedding

Collodion bag technique

• Concentrated sediment supported in agar or histogel
• Cell pellet formed then processed in paraffin

1. Fill the tube with collodion

2. Let stand in a warm place for 10 minutes
3. Puncture the top joint layer of the collodion and decant the collodion in another container to reuse.
4. Keep the Pyrex tubes inverted
5. Let the collodion coat on the wall of the tubes, dry completely, and store in a cool place
6. Add 10mL Cell Pellel suspension to the collodion bag attached to the wall of the Pyrex tube
7. Top up with saline
8. Centrifuge at 1500 RPM for 8 minutes
9. Discard the supernatant
10. Loosen the collodion bag carefully from the wall of the Pyrex tube with forceps
11. Roll the bag along the length, starting from the bottom without disturbing the pellet
12. Wrap the rolled collodion bag with pellet in tissue paper
13. Keep in B-5 fixative for 2 minutes. Soak in 70% ethanol for 2 minutes to wash the excess B5 fixative.
 14. Put the tissue in a processing cassette to transfer to 10% formalin. Process for paraffin embedding.

Tissue clot technique

• Allow clot to form in lumen of needle tip
• Clot transferred to formalin
• Prevents loss of diagnostic material

1. Allow the clot to form in lumen of needle tip

2. Clot is transferred to formalin
3. This prevents the loss of diagnostic material

Cellient® automated cell block preparation

• This method is already machine generated. All you have to do is insert the
specimen or the sample in the machine.

Shandon™cytoblock™ method
• Uses cytocentrifugation to concentrate cells in Cytospin
• Already available kits of cell block cassettes and reagents

Sedimentation cell block technique

Example of cells of this technique:
• Squamous cell carcinoma - Lung
• Adenacarcinoma - Lung
• Hodgkin’s lymphoma
• Adenocarcinoma – Endometrium

Protocol
• Transfer material to centrifuge tube
• Spin down to achieve a concentrated cell button
• Centrifuge set at 2400 RPM for five minutes
• Pour off supernatant
• Add buffered formalin
• Let material set in tube; 30-60 minutes
• Remove hardened cell button with a metal spatula
• The button placed on histology tissue paper that has been moistened with buffered formalin

Special Instructions; Quality Assurance

• Cell blocks cut at 8μm
• If IHC needs to be performed it is noted on the QC sheet so appropriate number of sections can be cut and ready for IHC
stains
• Cassette is checked by two cytotechnology lab technologists for proper
labeling to ensure Quality Assurance
• Cell block placed in formalin for at least six hours, documented on
requisition with the time stamp

PAP Test
• Thin Prep: Papanicolaou stain
• Cell block: Hematoxylin and Eosin stain

Urine; Bladder Washing

• Thin Prep: Papanicolaou stain
• Cell Block: Hematoxylin and Eosin stain