Pathology

1. Histopathology & Cytopathology
Gus Lusack

2. Histology, Histopathology, Histochemistry,
Immunocytochemistry & Cytopathology
• What are they? - A mixture of anatomy, biochemistry and physiology.
• Histology - the microscopic study of normal tissue and its structure.
• Histopathology - as above but looking at diseased tissue. This
includes diseases such as infection, inflammation and cancer.
• Histochemistry - this is the study of known substances within the
structural framework, as opposed to using ‘dyes’ for histology, the
chemistry is understood.
• Immunocytochemistry - localisation of specific protein or antigen in
cells using primary antibodies
• Cytopathology – is closely allied to histopathology but distinct as it
involves analysis of cell preparations

3. What does Histopathology involve?
• Patients give consent to have
histopathology specimens
according to Human Tissue Act
2004
• The results are communicated to
the patients by the requesting
clinicians
• Majority of the diagnoses are
based on the assessment of
haematoxylin and eosin (H&E)-
stained sections of formalin-fixed,
paraffin wax-embedded tissue
Steps from specimen to report
 Fixation
 Specimen Collection, transportation
and receipt
 Tissue selection and description
 Tissue processing
 Embedding
 Microtomy
 Staining and mounting
 Quality assurance
 Reporting
 Computer entry & report dispatch

4. The Main Disease Processes 1
Protective response to foreign agents:
Microorganisms: bacteria, fungi, viruses, parasites
Physical agents: trauma, chemicals, radiation (UV, Radiotherapy), temperature
Inflammation
Organ / Tissue Tissues Inflammation
Brain Meninges Meningitis
Mouth Gum (gingiva)
Tonsil
Gingivitis
Tonsilitis
Gastrointestinal tract Oesophagus
Stomach
Appendix
Colon
Oesophagitis
Gastritis
Appendicitis
Colitis
Lung Bronchi
Lung
Bronchitis
Pneumonitis (pneumonia)
Breast Breast Mastitis
Liver Liver Hepatitis
Bladder Bladder mucosa Cystitis
Skin Dermis Dermatitis
Musculoskeletal
system
Tendon
Joints
Tendonitis
Arthritis

5. The Main Disease Processes 2
• Neoplasia means new growth
o Ability to grow and enlarge unless it is treated
o Sometimes referred to as tumour (swelling)
o Classified into benign and malignant types
Neoplasia
Benign Malignant
Size Small Large
Borders Well defined Ill (poorly) defined
Differentiation Resembles tissue of
origin
Variable
Growth rate Slow Rapid
Mitotic figures Rare Common
Necrosis No Yes
Invasion No Yes
Metastasis No Yes

6. The main disease processes 3
Neoplasia
Benign Malignant
Epithelial
Squamous Squamous cell papilloma Squamous cell carcinoma
Transitional Transitional cell papilloma Transitional cell carcinoma
Glandular Adenoma Adenocarcinoma
Mesenchymal
Fibrous tissue Fibroma Fibrosarcoma
Adipose tissue Lipoma Liposarcoma
Blood vessels Haemangioma Angiosarcoma
Cartilage Chondroma Chondrosarcoma
Bone Osteoma Osteosarcoma
Classification of benign and malignant neoplasms

7. Types of biopsy
submitted for
histopathology

8. Dealing with Dead Tissue –
Preparation:
Whole tissue
Fixation
Dehydration
Clearing
Isolated Cells Freezing
Decalcification
Impregnation
Embedding
Microtomy
Staining
Microscopy

9. The Histopathology Laboratory

10. Haematoxylin and Eosin (H & E) Stain
• H & E is the most widely used:
oHaematoxylin is used as a nuclear stain – shows chromatin pattern
in a distinct blue/black
oIt requires oxidation to haematein (formalin pigment)
oEosin is an acid dye – red colour which complements haematoxylin

11. Haematoxylin:
Staining and Differentiation

12. Automated Tissue Processing & Staining
XY Stainer
Coverslipper
Leica Microsystems
Thermo
enclosed
tissue
processor
Thermo
microwave
tissue
processor

13. Non-Melanoma Skin Cancer (NMSC)
Squamous Cell Carcinomas (SCCs)
H&E stained Normal Skin H&E stained SCC

14. Special Stains
Alcian blue -acid mucins
Periodic acid Schiff (PAS) –
Glycogen and neutral mucins
Toluidine blue – Heparin
Diastase PAS – Neutral mucins
Colon
mucus secreting
glands stomach
Cervix
mast cells
Elastic van Gieson – elastin
Lung
Masson Trichrome
Collagen – blue
Muscle, RBC - red

15. Immunocytochemistry in Histopathology
Indirect immunocytochemistry Direct immunocytochemistry
/ Fluorescent Tag / Enzyme Tag
Colon carcinoma – Anti-Cytokeratin 10 antibody
Streptavidin Poly-HRP Conjugate
Anti-Cytokeratin antibody – Fluorescent Tag

16. Molecular Diagnostics in Histopathology
• In situ Hybridisation
oPolymerase chain reaction (PCR)
oSouthern blotting
In situ Hybridisation (ISH)
Fluorescence In situ Hybridisation (FISH)
Breast cancer cells- multiple copies of
HER2 gene (Red)
In situ Hybridisation (ISH)
Squamous cell carcinoma (SCC) – probes
for Human Papillomavirus (HPV) DNA

17. Review and Reporting by Histopathologist
• Check request form and slides match
• H&E stained sections are examined and assessed for adequacy
of sectioning and staining
• Identify tissue type
• Biopsy assessed for adequacy of sampling, size and
preservation
• A systematic approach to analyse tissue type and disease
process – e.g. skin biopsy is assessed by observing the overall
architecture of tissue (epidermis, the epidermal-dermal interface,
the dermis and subcutaneous tissue)
• Assessment of cellular composition and identify pathological
changes
• Identify the disease process

18. Cytopathology
•Cervical screening
•Cytology of urine
•Lower Respiratory tract cytology
•Serous effusions
•Basic semen analysis (Andrology)

19. Cervical Screening
• Prevention of carcinoma of the uterine cervix
• Cervical carcinomas:
oSquamous cell carcinoma derived from ectocervix
oAdenocarcinoma derived from glandular epithelial cells of
endocervix
oThe precursors are termed as intraepithelial- abnormal cells
remain within the epithelium
oCervical carcinomas are caused by specific types of Human
papillomaviruses (HPVs)
oMost infections resolve within 6-18 months
oSome persist and increase the risk
oIt takes about 20 years from HPV infection to invasion of the
lesion

20. Cervical Screening
• The NHS screening programme;
• For women aged 25 to 49 - 3 years interval
• For women aged 50 to 64 – 5 years interval
• Samples (Transformation Zone) usually taken in
GP practice
• Most popular staining method for wet-fixed preparation is Papanicolaou
technique –Pap smear (George Papanicolaou 1940s)
• Haematoxylin to stain the nuclei of cells and two cytoplasmic
counterstains – orange G and eosin azure

21. Cervical Screening
Liquid Based Cytology (LBC)
• A number of points about the PAP smear:
o LBC involves immediately dispersing the cells in a liquid transport medium
o Monolayer (thin layer) preparation – facilitating the automated evaluation
of individual cells
o Membrane filtration/density gradient centrifugation - Reduce the number of
lucocytes (WBCs) and erythrocytes (RBCs)
o 6% reduction in tests considered inadequate for reporting in 2009 –
reduced the need for many repeat tests

22. Normal Cervical Cytology
• Chromatin pattern and distribution are the most
useful features that distinguish normal cells from
neoplastic cells
• Nuclear shape e.g. round, oval, irregular
• Regularity of the nuclear membrane, e.g. smooth,
wrinkled, angular
• Intensity of nuclear staining, e.g. normochromasia,
hyperchromasia (darkly stained) and
hypochromasia (pale staining)

23. Normal Cervical Cytology
Metaplastic cells
Endometrial cells

24. Abnormal Cervical Cytology
Mild dyskaryosis
A normal cell undergoing mitosis A abnormal cell undergoing mitosis
Normal CIN1 CIN2 CIN3
Tripolar mitosis
Moderate dyskaryosis Severe dyskaryosis

25. Abnormal Cervical Cytology
(a) A ‘tadpole’ cell (the pink cell left of
centre) and a fibre cell (right of
centre).
(b) Cytoplasmic keratinisation in
malignant squamous cells
(c) A microbiopsy of severe
dyskaryotic cells
(d) Tumour diathesis
o Koilocytosis is caused by HPV
o Superficial squamous cells with large
o perinuclear clear space
o Thickened uneven rim of dense
o cytoplasm – wire loop appearance
o Binucleation / multinucleation
o Dyskeratosis is usually present

26. Cytological features suggestive of
invasive carcinoma
• Numerous dyskeratotic cells
• Cellular pleomorphism (bizarre-shaped, fibre-shaped, tadpole
shaped)
• Very coarse aggregates of nuclear chromatin
• Large, irregular, sometimes multiple nucleoli
• Cytoplasmic keratinisation – presence of anucleate
fragments of keratinsed cytoplasm
• Tissue fragments composed of dyskeratotic cells
• Tumour diathesis – mixture of necrotic cell debris,
inflammatory exudate and blood

27. Cytological pitfalls
Potential false positives
Immature squamous metaplastic
cells mimic moderate/severe
dyskeratosis
Endometrial cells- appear as
hyperchromatic cells with high
N:C ratio
Histiocytes – mimic of dyskaryosis
Potential false negatives
Small cell dyskaryosis Pale dyskaryosis – less chromatin
Intensity compared to adjacent
polymorphs
A microbiopsy / hyperchromatic
crowded cell group

28. Cytology of Urine
• Sample types:
ovoided urine
oIleal conduit urine – urinary diversion for bladder cancer
patients
oCatheter urine
oBladder washings – not a common source
• Benign changes - bladder could be infected by bacteria,
fungi, viruses or parasites
• Useful test for identification of high-grade urothelial cancer

29. High-Grade Urothelial Carcinoma
(a) Increased cellularity
(b) Isolated malignant cells
(c) Clusters of malignant
cell with hyperchromatic
nuclei
(d) Malignant cells showing
pleomorphism
(e) Very high nuclear to
cytoplasmic ratio
(f & g) Marked nuclear
hyperchromasia
(h) Coarse chromatin pattern
(i) These two cells have
coarse and regular
chromatin pattern
(j) Angular nuclear outline
(k) Prominent nucleoli seen

30. Cytology of the Lower Respiratory Tract
• Sample types:
oSputum – mixture of mucus and cells
oBronchial brushings
oBronchial washings
oTransbronchial fine needle aspiration
oTransthorasic fine needle aspiration
• Infections: bacterial (pneumonia), TB (Mycobacterium
tuberculosis), Herpes simplex virus
• Lung cancer: small cell lung cancer & bronchial carcinoid

31. Cytology of the Lower Respiratory Tract
• Bacterial infection
Pneumonia sputum sample –mixture
of inflammatory cells in necrotic debris
Actinomyces – filamentous branching
bacteria, often found – no significance
Bronchial washing sample from a
patient confirmed with TB –giant cells
(the nuclei are at one pole)
Necrotic debris from a TB patient

32. Serous effusions

33. Serous Effusions - Adenocarcinoma
• The most common malignancy in effusions
• May present as papillary (nipple –like protrusion) or acina (gland-
like)
• Multilayered clusters of cells
• Nuclei - pleomorphism, enlargement, hyperchromasia
(a) Cluster of malignant cells
(b) Breast carcinoma showing
spherical clusters
(c) Ovarian carcinoma
showing papillary and
acinar clusters; (d)
psammoma body; (e)
vacuolated malignant cells
(f) Lung carcinoma showing
signet ring formation

34. Basic semen analysis (Andrology)
• A number of risk factors associated with poor semen quality
• History of reproductive tract infections / sexually transmitted
diseases
• Exposure to environmental pollutants
• Athletes exposed to conditions which may lower sperm
counts
• Alcohol abuse/ use of tobacco/ recreational drugs
• Malignancy
• Medicinal drugs, especially steroids
• Previous testicular surgery / recent trauma

35. Basic Semen Analysis
• Two sample types;
oPost-vasectomy
oInfertility
• Equipment : An improved Neubauer Haemocytometer &
Microscope
Infertility
1. Liquefaction 2. Appearance
3. Volume 4. Viscosity
5. pH 6. Motility
7. Morphology (% of normal sperm)
8. Sperm count and leucocytes count (millions per ml)
9. Anti sperm antibodies – potential cause of infertility
10. Vitality (% of live sperm)

36. Basic Semen Analysis
Normal Sperm
Test Parameter Lower Reference Values WHO
Semen volume 1.5 ml or more
Total number of sperm
per ejaculate
39 million
Sperm concentration 15 million sperm per ml
Total motility 40%
Progressive motility 32%
Vitality (live) 58%
Morphology (% normal) 4%
pH 7.2 or higher
White blood cells Fewer than I million per ml
Sperm antibodies <50% of sperm reactive
Abnormal sperm morphology

37. Quality Assurance (QA) 1
Internal Quality Control (IQC):
• Ensure measures and procedures employed are
performed to expected standards
• Use known positive controls in histochemistry or with
special stains
• e.g. A control slide known to contain tubercle bacilli with every batch of
slides stained with a Ziehl Neelson (ZN) stain
• Slides from patients are submitted to a Histopathologist
for reporting only if the control slide is positive

38. Quality Assurance 2
• External Quality Assessment (EQA)
• Membership of an external QA scheme is a requirement for UKAS ISO
15189 accreditation
• Over 140 schemes operate from 24 centres based at major hospitals, research
institutions and universities
• Cover qualitative and interpretive investigations in andrology, clinical chemistry,
genetics, haematology, histopathology, immunology and microbiology
• Participants receive independent, objective and impartial reports – identify
weaknesses and take appropriate action
• In histopathology;
• Covers diagnostic aspects of the service – UK NEQAS Breast Screening
Pathology
• Covers technical aspects – UK NEQAS Cellular Pathology Technique and
UK NEQAS Immunocytochemistry and In situ hybridisation
National External Quality Assessment Scheme (NEQAS)
www.ukneqas.org.uk

39. Exercise 1
• Write short notes on the following:
• Carcinoma
• Adenocarcinoma
• Sarcoma
• Leukaemia
• Lymphoma
• Myeloma
• UKAS ISO15189 Accreditation
• Human Tissue Act 2004
(Word count:1500)