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

• Cytopathology – study of abnormal or diseased cells o Examination of exfoliated or aspirated cells with little

to no architectural frame of reference

• Histopathology – study of abnormal or diseased tissues o Examination of organ system tissues with near

complete in vitro architectural framework intact

cyto - [suffix, combining form] /ˈsʌɪtəʊ/ - of a cell or cells German zyt-, zyto-, from Greek kytos ‘hollow vessel’

histo - [suffix, combining form] /ˈhɪstəʊ/- relating to organic tissue from Greek histos ‘web, tissue’

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

Definitions

Many techniques shared between disciplines

Collection

Specimen Collection

• 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

Cytopathology

• 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 Histopathology

• 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 . . .

Specimen Collection Microbiology

Fixation

Fixation

• 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

General

Fixation

• 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

Cytopathology

Fixation

• 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)

Histopathology

Fixation

• 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

Microbiology

Processing

Processing

• 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)

Cytopathology

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

Processing • Liquid based cytology

• Now commonly used most specimen types

Cytopathology

Conventional Pap smear SurePath ThinPrep

Processing

• Cell block o Specimen centrifuged o Cell button transferred for

“histological” processing o Embedded, sectioned and stained

just as with tissue

Cytopathology

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

Processing

• 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

Histopathology

Processing

• 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

Histopathology

Processing

• 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

Histopathology

Processing Histopathology

Stains

Stains

• 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

Standard

Stains

• 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)

Bacterial B

row

n an

d B

renn

Brow

n and Hopps

Some bacteria do NOT stain: 1. Spirochetes 2. Mycobacteria 3. Fastidious Gram negatives (e.g.

HACEK, Bartonella)

Stains

• Silver impregnation stains o Examples – Steiner, Warthin-Starry, Dieterle o Used for spirochetes, Helicobacter, Bartonella, etc. o Yellow-gold background – easily overdeveloped

Bacterial

Stains

• 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

Mycobacterial

Stains

• 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

Aerobic Actinomycetes

Stains

• 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

Protozoa

Stains

• 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

Fungi

Stains

• Gomori’s Methenamine Silver (GMS)

• Periodic Acid Schiff (PAS)

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

And bacteria?

GMS + Nocardia PAS + Whipple’s Bacillus

Stains

• 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

Fungi

Stains

• 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

Immunologic/Hybridization

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?