Basic

Histologic & Cytologic

Techniques
Rocco LaSala, MD, D(ABMM)
Director Clinical Microbiology
Associate Professor of Pathology
plasala@hsc.wvu.edu
 Outline
• Overview
o Definitions
o Similarities/differences with clinical microbiology lab

• Techniques
o Specimen collection
o Fixation
o Specimen processing/slide preparation

• Stains
o Bacterial
o Mycobacterial
o Fungal
o Protozoal/parasitic
o Immunologic/Hybridization
No Disclosures
 Definitions
cyto - [suffix, combining form] /ˈsʌɪtəʊ/ - of a cell or cells
German zyt-, zyto-, from Greek kytos ‘hollow vessel’

• Cytopathology – study of abnormal or diseased cells

o Examination of exfoliated or aspirated cells with little
to no architectural frame of reference

histo - [suffix, combining form] /ˈhɪstəʊ/- relating to organic tissue

from Greek histos ‘web, tissue’

• Histopathology – study of abnormal or diseased tissues

o Examination of organ system tissues with near
complete in vitro architectural framework intact

Both generally focuson host (e.g. human) cells and tissues

 Definitions

• Cytopathology – study of abnormal or diseased host cells

• Clinical microbiology – study of non-host (i.e. pathogenic microbial) cells

Many techniques shared between disciplines

Collection
 Specimen Collection
Cytopathology
• Fine needle aspiration
o Palpation guided vs. radiographically guided
o 22-25 gauge needle (0.4 – 0.26 mm)
- Fewer complications (e.g. bleeding) compared to core needle biopsy
- Less “invasive” than surgical excision
o lymph node, visceral nodules, abscesses, etc.

• Fluids, ____-centesis
o Para – peritoneal fluid
o Thora – pleural fluid
o Pericardio – pericardial
o CSF, synovial, cyst

• Exfoliative/abrasive surface sampling

o Brushings/scrapings – cervical, bronchial, pancreatic, biliary
o Lavage/washings – alveoli, bladder
o Naturally shed – mesothelial cells in effusion
 Specimen Collection
Histopathology

• Biopsy (“to view organic material”)

o En bloc removal of some/all damaged/diseased tissue
o 12-18 gauge needle “core” (2.2-0.8 mm)
- More “invasive” than FNA,
- Less difficult than resection
- Often used for initial diagnosis
o Suspected tumors – breast, lung, liver, prostate,
brain, etc.

o Excisional
- Small surgical sampling of lesion
- Potential candidate for intraoperative frozen sectioning

• Resection
o Complete surgical removal
o Most any specimen type
 Specimen Collection
Microbiology

• Aspirates – lymph node, abscess, etc.

• Fluids – peritoneal, pleural, pericardial, CSF, synovial, etc.
• Surfaces – skin, mucosal, “wound” scrapings, brushings . . . swabs
• Tissues – biopsies, resections (usually homogenized)

Most everything . . .
Fixation
 Fixation
General
• Specimen fixation performed for different purposes
o Adherence to glass slide
o Preservation of morphologic detail, cellular or architectural
o Maintain molecular integrity (e.g. antigenicity, lipid content, etc.)
o Prevent autolyis and putrefaction
o Promotes long term storage

• Effects of fixation
o Cellular death
o Stabilization of macromolecules via cross-linking or precipitation
o Facilitate dye uptake

• Types of fixatives
o Heat
o Chemical
 Fixation
Cytopathology

• Two general approaches to cytological smear fixation

• Alcohol-based
o Ethanol, methanol, Cytolyte®, spray fixatives
o Used for specimen preservation OR immediately upon
smear preparation
o Minimal effect on cell size
o Maintains nuclear chromatin detail
o Used with Pap stain

• Air-drying
o Smear prepared and allowed to dry
o Artificial cell enlargement and poor nuclear detail
o Better visualization of cytoplasmic contents and
background material
o Used with Romanowsky-type stains
 Fixation
Histopathology

• Single general approach to specimen fixation

• Aldehydes
o Neutral buffered formalin, formaldehyde, guteraldehyde
o Specimen immersed and/or perfused
o Stiffens tissue and facilitates dissection
o Maintains overall architecture
o Used with most every stain

• Miscellaneous
o Picric acid (e.g. Bouin’s)
o Mercurials (e.g. Zenker’s, B5)
 Fixation
Microbiology

• Heat or methanol
o Improves adherence of specimen to glass slide
o Methanol (chemical fixative) may also improve
staining characteristics of bacteria

• PVA, SAF, etc.

o Analogous to cytological specimens . . .
o Improves cellular morphologic detail

• NOT terribly useful for organism

cultivation
Processing
 Processing
Cytopathology
• Manual slide preparation
o Similar techniques used by Hematology and Microbiology
o Smear, squash, crush, stir, etc.
o Immediately fix or allow to air dray
o On-site assessment sometimes available
Cytopathology Processing
• Cytocentrifugation
o Concentrates specimen (and background)
o More commonly air-dried
o Mostly superseded by “liquid-based” methods

• Liquid based cytology

o ThinPrep (Hologic) and SurePath (BD) both FDA cleared
o Initially developed for cervical cytology (Pap test)
o Specimen placed in preservative vial
o Automated processor agitates, then filters
specimen onto slide
o Removes background debris and
distributes cells evenly
o Use of either system is associated with
significantly higher sensitivity in cervical
cytology screening
Cytopathology Processing
• Liquid based cytology

Conventional ThinPrep
SurePath
Pap smear

• Now commonly used most specimen types

 Processing
Cytopathology

• Cell block
o Specimen centrifuged
o Cell button transferred for
“histological” processing
o Embedded, sectioned and stained
just as with tissue

Pap Stain, FNA Romanawsky Stain, FNA H&E Stain, Cell Block
 Processing
Histopathology

• Gross assessment, dissection, cassette submission

o Macroscopic observations (e.g. appearance, size, pathology, etc.) of biopsied
or surgically resected tissue
o Small specimens (e.g. core biopsies, skin biopsies) completely submitted
o Specific sites of large specimens strategically selected for microscopic
analysis
o Biopsies processed similarly, but en toto
 Processing
Histopathology

• Paraffin infiltration
o Usually performed by automated processor
o Series of dehydration steps (ethanol)
o Clearance of ethanol (xylene)
o Infiltration of tissue with molten paraffin

• Block embedding
o Performed at embedding station
o Paraffinized tissue transferred to mold filled
with molten wax
o Proper orientation CRUCIAL
o Once cool, specimen ready to section
 Processing
Histopathology

• Sectioning
o Performed on microtome
o Sequential sections (4-6 µm thick) form “ribbons”
o Ribbons floated in water bath and transferred to glass
slide
 Processing
Histopathology
Stains
 Stains
Standard

• Hematoxylin and eosin (H&E)

o Routinely used in histology
o Nuclear material – basophilic - blue
o Cytoplasmic material – eosinophilic - pink

• Papanicolaou
o Routinely used in cytology
o Developed for squamous epithelium, but good all
around utility
o Variant of H&E

• Romanowsky
o Often used in cytology (and hematology)
o Includes Geimsa, Wright, May-Grünwald, Diff Quik, etc.
o Nuclear material – purple to gray
o Cytoplasm – red to pink
 Stains
Bacterial

• Gram stain
o Crystal violet, iodine, acid-alcohol decolorization, safranin
counter
o Performed on cytology smears no differently than microbiology
smears

• Tissue Gram stain

o Brown and Brenn – background pink-red (highlights Gram positives)
o Brown and Hopps – background yellow (highlights Gram negatives)

Brown and Hopps

Brown and Brenn

Some bacteria do NOT stain:

1. Spirochetes
2. Mycobacteria
3. Fastidious Gram negatives (e.g.
HACEK, Bartonella)
 Stains
Bacterial

• Silver impregnation stains

o Examples – Steiner, Warthin-Starry, Dieterle
o Used for spirochetes, Helicobacter, Bartonella, etc.
o Yellow-gold background – easily overdeveloped
 Stains
Mycobacterial

• Kinyoun (cold) or Ziehl-Neelsen (hot)

o Carbol fuscin, decolorization, Methylene blue counter
o Performed on cytology smears and histologic sections similarly
to microbiology smears

• Auramine/Rhodamine
o Occasionally used on histologic sections
 Stains
Aerobic Actinomycetes

• Modified Kinyoun
o Carbol fuscin, Methylene blue counter, weaker acid decolonization

• Fite-Faraco
o Developed for M. leprae, but will highlight partially acid fast organisms
 Stains
Protozoa

• Giemsa, Wright, Diff Quik, etc.

o Both pH dependent and can be difficult to perfect
o Used in hematology +/- cytology

• Tissue Giemsa
o Generally not as useful as standard Giemsa

• Iron hematoxylin, Wheatley’s trichrome

o No good histologic equivalent
 Stains
Fungi

• Gomori’s Methenamine Silver (GMS)

o Malachite green background
o Easily overdeveloped
o Fungal cell wall oxidized with acid, resulting (aldehydes) reduce silver

• Periodic Acid Schiff (PAS)

o Stains carbohydrate molecules
o Usually less background staining than GMS
 Stains
And bacteria?

• Gomori’s Methenamine Silver (GMS)

• Periodic Acid Schiff (PAS)

• Generally regarded as “fungal” stains, these may also highlight
bacteria

GMS + Nocardia PAS + Whipple’s Bacillus

 Stains
Fungi

• Mucicarmine
o Mucopolysaccharide stain
o Used for mucin producing cells
o Stains Cryptococcal capsule

• Fontana-Masson
o Melanin stain
o Used for melanocytes
o Stains dematiaceous fungi +/- Cryptococcus
 Stains
Immunologic/Hybridization

• Analogous to DFA/IFA in microbiology

o Primary or secondary antibody conjugated with
enzyme of fluorophore
o Immunohistochemistry (IHC) for tissues,
Immuoncytochemistry for cells
o Staining localized to epitope distribution

• In situ Hybridization
o Similar to IHC but uses nucleic acid complemtary
binding rather than monoclonal antibody
 Conclusions
1. Collection of specimens for diagnostic histo/cyto pathology
is really no different than for clinical microbiology

2. Slide preparation in cytology very similar to that in

microbiology, whereas tissue preparation for histology is
very different

3. The menu of histo- and cytochemical stains used for

microorganisms is much longer than in clinical microbiology

4. Clinical microbiologists are really also cytologists

(but so much more!!)
Questions?