HISTOPATHOLOG

Y LABORATORY
OVERVIEW
Prepared by:
Mrs. Raghad M. Al-Buraey
PURPOSE

• To give an overview on Histopathology laboratory,

its sections and tests examinations being performed
 Services Offered by Histopathology
Laboratory

Pathology has a major role in the diagnosis of cancer, monitoring

subsequent treatment and in supporting the general care of cancer
patients who may be suffering not only from their tumors and its
complications but also other pathologies.

Being connected with teaching hospital, training is one of the goals

and objectives of the lab. Students from College of Applied Medical
Sciences and Health College, residents and trainees from other
hospitals/institutions are being trained in this faculty.

Participating and collaborating for approved medical research through

the approval of the Laboratory Administration.
 Tests Performed in
Histopathology Laboratory

•Tissue Processing.
•Routine Hematoxylin & Eosin staining.
•Routine Histochemistry (17 special stain).
•Immunohistochemistry ( more than 100 markers)
•Direct Immunofluorescence (8 markers)
•Frozen Section. (Intra-Operative Consultation)
 Accessioning

• All specimens must be delivered to the

laboratory immediately.
• Requisition forms
• Specimen container
• Specimen rejection criteria
 Accessioning
cont.

• All specimens must be received fixed in 10%

neutral buffered formalin EXCEPT for the
following:
Frozen section-unfixed, fresh state.
Immunofluorescence – (Renal and Skin
biopsies) either in saline or liquid
nitrogen.
Electron microscopy- 2.5%
glutaraldehyde solution in the cold after
excision
Special Procedure for Specific
Specimens

Renal biopsies
•Advance notice must be sent to the laboratory.
• The specimen will be examined under the dissecting
microscope for the presence of glomeruli ;then divided into 3
parts, one frozen for immunofluorescence, one immersed in
glutaraldehyde for EM study, and one fixed in formalin for
paraffin processing.
 Testicular biopsies Bouin’s fluid used as a fixative.
 Breast Lump: The surgical margins should be oriented by
sutures.
 Special Procedure for Specific
Specimens
cont.

 Mastectomy: The apical nodes should be indicated by

sutures. Superior margin to be indicated by a separate suture
in case of simple mastectomy (without axillary dissection).
 Sentinel lymph node/nodes identified by the surgeon and
removed, will be submitted to the laboratory in a separate
container, with a requisition. Surgeons are requested to
provide all relevant information on the requisition, including
procedure type, date and time of injection, location of lymph
node/nodes, tracers used and radioactivity count.
 Special Procedure for Specific
Specimens
cont.

 Perinodal fat - Whenever possible, include

surrounding fat. This will ensure the integrity of
the lymph node capsules and also assess the
tumor spread beyond the lymph node capsule.
 Infective Specimen - Known infective specimen
should be clearly marked with red Marker (e.g.
patient’s with hepatitis, HIV, T.B., etc.).
 
Special Procedure for Specific
Specimens
cont.

 Bowel Resection - The proximal or distal excised

margins should be oriented by a suture.
 Skin Biopsies intended for immunofluorescent are
received frozen in liquid nitrogen from the
dermatology department then stored in the deep
freezer for immunofluorescence studies.
Type of Specimen Received
1. Referral slides/blocks
2. Surgical specimen to be “Exempt from Pathology
Examination” as approved by the Chairperson Department of
Pathology in conjunction with the hospital administration and
medical staff departments, are :
• Foreign bodies (e.g. bullets, medical devices, IUD, valve
rings).
• Hair, finger nails, toe nails removed for cosmetic reason.
• Teeth, dental appliances.
• Lens (i.e. cataract).
Type of Specimen Received
cont.

• Calculi.
• Nasal septum cartilage and bone (soft tissue is
submitted for microscopic examination).
• Foreskin from circumcision (10 years old and
younger).
• Mammary implant.
• Skin from face-lifts.
• Orthopedic materials.
Type of Specimen Received
cont.

3. Specimens accepted for “Gross Description Only”

• Torn Meniscus.
• Bone fragments from non-pathologic fracture.
• Ear cartilage.
• Fetus.
• Irrespective of any exemptions, Microscopic
examination should be performed whenever there is a
request by the attending physician, or at the discretion
of the pathologist when indicated by the clinical
history or gross findings.
 Grossing

• All surgical specimens are dissected and described by

pathology Resident.
 Tissue Processing

• The aim of tissue processing is to embed the tissue in a

solid medium firm enough to support the tissue and give
it sufficient rigidity to enable thin sections to be cut, and
yet soft enough not to damage the knife or tissue.

• Instrument used:
• Excelsior AS THERMO
• VIP-6 SAKURA
 Embedding

• Processed tissue specimens are embedded in

molten paraffin wax the using the tissue embedding
machine

• Instrument used:
HISTOSTAR THERMO
TISSUE-TEK5 EMBEDDING MACHINE
 SECTIONING

•Embedded paraffin blocks are trimmed to expose the whole

surface of the tissue using a microtome.
•Cool immersed surface
•Thin sections of 2-4 microns (um) are sectioned. (Some tissues for
special stains e.g. Congo red stain may require thicker sections).
•Float out in a water bath
•Picked unto a glass slide labeled with the Pathology number of
each specimen
• Allow to drain and dry.
 SECTIONING cont.

• Place on a hot plate to sufficiently melt off the paraffin

and at the same time enable the section to adhere to the
slide.
• Instrument used:
-Microtome
-Float bath
-Hot plate
-Cold plate
 STAINING

• The Hematoxylin and Eosin stain is the most commonly used stain in
histology. It is the basis of most diagnosis and the starting point for all other
investigation. Hematoxylin - stain the nucleus blue-purple. Eosin - stain the
cytoplasm and connective tissue element. Together these two stains make
the best general morphological stain in use.
• Instrument used:
-LEICA MULTAISAINER AND COVERSLIPPER
-TISSUE-TEK PRISMA AND FILM SAKURA
 SPECIAL STAINS

• Special staining is performed to visualize selected tissue

elements, entities and microorganisms. Based on classical dye
staining methods, special stains technique provide valuable
information in the evaluation of numerous abnormal or disease
conditions.
• (PAS, AFB, GRAM, Trichrome…)
• Instruments used:
- DAKO ARTISANLINKPRO AUTOSTAINER
- VENTANA BENCHMARK SPECIAL STAINER
N0. SPECIAL STAIN INTENDED USE

1.    
Acid Fast Demonstration of acid-fast Mycobacteria such as M.Tuberculosis, M. Leprose,M.
Kansasii, M Ayium.
2.    
Alcian Blue Evaluation of Acid mucins and mucopolysaccharides

3. Differentiation of neutral and acidic mucopolysaccharides

Alcian Blue / PAS

4.    
Amyloid (Congo Red) Demonstration of Amyloid in tissue sections

5. Demonstration of Parasite and H.Pylori bacteria

Giemsa

6. Demonstrate of fungi
GMS (Grocott Methenamine Silver)

7. Demonstration of Gram-positive and Gram-Negative bacteria

Gram

8. Detection of ferric iron in tissue sections and blood or bone marrow films
Iron (Perl’s)

9. Evaluation of reticulin fibers

Reticulin

10. Evaluation of collagen and muscle fibers

Trichrome (Masson’s)

11. Evaluation of mucin, mucopolysaccharides and capsule of cryptococcus

Mucicarmine

12. Undigested; evaluation of mucopolysaccharides and glycoprotien

PAS

13. Diastase digested; evaluation of glycogen and glycogen storage disease

PAS/ Diastase

14. Evaluation of elastic fiber

Elastic Fiber

15. Demonstration of H.Pylori

Warthin Starry
 Immunohistochemistry (IHC)

•Immunohistochemistry (IHC)
•Immunohistochemistry is the localization of antigens or
proteins in tissue sections using a labeled antibody as specific
reagent through antigen-antibody interactions that are
visualized by a marker such as chromogenic dye.
•Valuable tool for the identification and visualization of tissue 
antigens in biological research and clinical diagnostics. 
•Characterize various biological processes or pathologies, such
as wound-healing, immune response, tissue rejection, and
tissue–biomaterial interactions.
 Direct Immunofluorescence (DIF)

• Direct Immunofluorescence (DIF)

• Direct immunofluorescence staining is a specific type of
immunohistochemistry, usually to detect the presence of
immunoglobulin and complement in the skin and kidney.
A batch of fluorescein isothiocyanate-labelled antibodies
against IgG, IgM, IgA, fibrin and C3 is applied to frozen
sections of fresh tissue and examined by fluorescence
microscopy.
 Direct Immunofluorescence (DIF)

• DIF involves the application of antibody–

fluorophore conjugate molecules to samples of patient tissue
obtained from biopsies.
• These antibody fluorophore conjugates target
abnormal depositions of proteins in the patient’s tissue.
• When exposed to light, the fluorophore emits its own
frequency of light, seen with a microscope. The particular
staining pattern and type of abnormal protein deposition seen
in the tissue sample help diagnose the disease.
 Immunohistochemistry (IHC)
&
Direct Immunofluorescence (DIF)
• DIF is useful in the diagnosis of suspected 
autoimmune disease, connective tissue diseases
 and vasculitis. The staining patterns seen in tissue samples
may be specific to a disease entity or they may need to be
interpreted with the clinical and histological findings.
• The clinical interpretation of any staining, or its absence,
must be complemented by morphological studies and
evaluation of proper controls.
• Instrument used:
- VENTANA BENCHMARK ULTRA IHC
 FROZEN SECTION
(Intra-Operative Consultation)
Rapid Diagnosis

• Intra-operative frozen section examination was established as a

highly reliable and accurate diagnostic procedure for the rapid
histological evaluation of tissue specimen during surgery.
• Frozen section is a rapid test that usually does not exceed 20
minutes. In these cases, fresh specimens are sent to the
Histopathology laboratory.
• By freezing the tissue, it is possible to cut thin sections which
can be stained and viewed by a pathologist within a matter of
minutes, as compared to routine paraffin process which takes
13 hours to complete.
 FROZEN SECTION
(Intra-Operative Consultation)
Rapid Diagnosis
• Pathologist examines the section, writes their diagnosis on
the FS report then relay the results to the surgeon in the O.R.
by telephone or verbally if available.
• This is considered a “STAT” procedure and takes priority
over all other activities of the laboratory.
 FROZEN SECTION
(Intra-Operative Consultation)
Rapid Diagnosis
Table 5.6.5 Optimal Cryostat Cutting Temperature for Unfixed tissues
Tissue Temperature Range
Instruments used : Brain

- NX70 THERMO CRYOSTAT Lymph node

Liver -12 to 160C
- CM1860UV LEICA CRYOSTAT Kidney
- PRESTOCHILL Spleen

(CRYOEMBEDDING MACHINE) Testis

-----------------------------------------------------------------------------------------
Breast (little fat)
Skin (Little fat)
Thyroid - 18 to 300C
Adrenal
Muscle
Prostate
• Why do tissues require fixation?
• What is the most used fixative in
Histology lab?