Tadrous P.J. Diagnostic criteria handbook in histopathology: a surgical pathology vade mecum

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Osteoarticular 339

Exceptions: ↑ cellularity and cytol. atypia may occur in benign tumours of growing children, small

tubular bones of the hands and feet, well-circ sub-periosteal tumours, soft tissue chondromas and

chondromas in multiple enchondromatoses. Bland cytology can occur in sternal chondrosarcomas

Arch.:  invasion (permeation) is the single most useful criterion = ‘wall-to-wall’ ﬁlling of pre-

existing marrow space with partial erosion of the lamellar trabeculae (not just occ. nodules

separated from the main mass by marrow ± surrounded by lamellar bone as occurs in

benign enchondromas)

 lack of clonal clustering of cells

Grading (determines the likelihood of metastasis):

G1. lobular hyaline matrix, bland cytology, ≈ no mitoses/necrosis

G2. myxoid/necrotic foci, ↑ cellularity at lobule periphery, mild cytol. atypia, mitoses <2/10hpf

G3. extensive myxoid/necrosis, ↑ periph. lobule cellularity, obvious cytol. atypia, mitoses ≥2

Borderline chondrosarcoma = clin. and radiol. suggestive but no ﬁrm histological malignant features

Prog: complete excision at 1

operation with clear margins (and no spillage/seeding) gives best hope

d/dg chondroblastic osteosarcoma (vide supra, see also p. 336 and de-diff chondrosarcoma below)

d/dg degenerative cartilage: radiological signs of degenerative joint disease, joint involvement (rare in

chondrosarcoma), clonal proliferations and other features of OA are all evidence against chondrosar-

coma

d/dg chondromyxoid ﬁbroma (q.v.): this also has chondroid lobules with peripheral hypercellularity

d/dg chordoma: usu. a problem at either end of the spinal column, see p. 184

De-differentiated chondrosarcoma

Lowgrade cartilage tumour sharply demarcated from a high grade soft tissue sarcoma (or osteosarcoma)

d/dg chondroblastic osteosarcoma: but this has pleomorphic cartilage that is admixed (also age and

CPC)

d/dg mesenchymal chondrosarcoma: but this has a monomorphic Ewingoid sarcoma component

Clear cell chondrosarcoma

Good prog., teens to old age, assoc

with epiphysis/diaphysis, radiol: no buttressing/cortical erosion

Cytology: clear cells (glycogen), well-deﬁned cell borders, central nuclei (± binucleate / prominent

nucleolus)

Arch.: vague lobules (± periph. osteoclast-like giant cells or central osteoid/bone)/sheets/trabeculae

Chondroid is not obvious and foci of typical chondrosarcoma are only present in 50% of cases

Vascular ﬁbrous septa, ABC-like areas, chondroblastoma-like polygonal cells ± chicken-wire Ca

Immuno of clear cells: S100 +ve, CK −ve, EMA −ve

d/dg chondroblastoma, osteoblastoma, GCT of bone or ABC

d/dg metastatic CCC has opposite immuno and lacks the other features

Mesenchymal chondrosarcoma

Poor prog, young adult, 1

◦

bone (any site), 1

◦

extraskeletal (esp. head and neck), radiol ‘popcorn’

SBRCT with islands of low grade chondrosarcoma – the interface may be abrupt or gradual.

Immuno: CD99 ±ve

± Pushing margin (with vascular plugs), a HPC-like vasculature and a ﬁbrillary matrix (! not d/dg

osteoid)

d/dg small cell osteosarcoma (p. 337) and de-diff chondrosarcoma (vide supra)

d/dg lymphoma (matrix and immuno)

d/dg Ewing’s sarcoma: matrix +/ spindling (may occur in mesenchymal chondrosarcoma) excludes

Ewing’s

d/dg mesenchymal hamartoma occurs in infants, shows osteoblastic bone formation (not just ossiﬁcation

of chondroid) and does not have malig. SBRCT elements or chondrosarcoma radiol.

Articular Pathology

Differential Diagnosis of Pigments in Joints/Synovium

Haemosiderin: inﬂam

arthritides, trauma, haemophilia, haemochromatosis, haemangioma and PVNS

Tetracyclines result in brown bone (cf. cartilage/synovium)

Osmicated fat in the synovium

Metal deposits (see ‘Joint Prosthesis Pathology’, p. 341)

Ochronosis (polymerised homogentisic acid in articular cartilage in patients with alkaptonuria)

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Osteoarticular 340

Differential Diagnosis of Neutrophils in Joints/Synovium

Sepsis (see p. 341 and pp. 375–376)

Crystals (for details of polarisation analysis, see p. 375):

 monosodium urate (gout, water-soluble, murexide reaction +ve) deposits in superﬁcial artic-

ular cartilage or as soft tissue masses (tophi) with a prominent giant cell reaction; pale pink

on H&E (d/dg amyloid) but may contain Ca

or cholesterol clefts; may occur together with

CPPD

 Ca

pyrophosphate (CPPD, pseudogout, removed by decalciﬁcation, murexide −ve) deposits

in the cartilage (in OA and chondrocalcinosis) or the superﬁcial layers of the synovium,

basophilic on H&E and may lack a giant cell reaction. Occasionally associated with some

metabolic diseases (e.g. hyperparathyroidism), a familial tendency and haemochromatosis

 Ca

hydroxyapatite may be seen in Milwaukee shoulder, trauma, hyperparathyroidism, sys-

temic malignancies, PSS and CRF

Acute/active RhA and some seronegative arthritides (esp. Beh¸cet’s disease)

Haemorrhage

Granulomatous Arthritis and the d/dg of Histiocytic Inﬁltrates

Bacteria: TB (may be non-caseating in synovium), atypical mycobacteria and brucellosis

Fungi (thorn synovitis may also show thorn-related foreign body)

Rheumatoid nodules: rarely occur in the synovium (d/dg hemigranulomas = rheumatoid nodule-like

lesions that appear to spill their contents into the joint, seen in OA and some seronegative arthritides)

Sarcoid (rarely affects joints) these may also present as non-speciﬁc



Crohn’s disease chronic inﬂam

without granulomas

Foreign body granulomas e.g. detritus synovitis (see ‘Joint Prosthesis Pathology’, p. 341)

d/dg histiocytic inﬁltrates: HX, multicentric reticulohistiocytosis, Whipple’s disease

Osteoarthritis and Degenerative/Regenerative Cartilage

Clin. and radiol. evidence of degenerative joint disease, usu. polyarticular unless 2

◦

Tangential ﬂaking ± vertical ﬁbrillation ± blurred/irregular/duplicated tide mark

Loss of MPS basophilia esp. in the interterritorial and superﬁcial zones

‘Clonal’ proliferation of chondrocytes → multicellular groups ± dead (‘ghost’) chondrocytes

Growth of cellular ﬁbrocartilage over the surface of the articular cartilage from the joint periphery

Synovium may show a reactive detritus synovitis with villiform hypertrophy and hyperplasia, chronic

inﬂam

± haemosiderin (!d/dg RhA, PVNS or infective/other causes of granulomas) ± pseudogout

Peripheral osteophytes ± concentric osteocartilagenous loose bodies (d/dg synovial chondromatosis)

Subchondral bone sclerosis/necrosis ± degenerative subchondral cyst formation (ganglion-like)

d/dg chondrosarcoma (pp. 338–339)

Rheumatoid Arthritis and Other Chronic Inﬂam

Arthropathies

Polyarticular incl. the TMJ and cervical spine (odontoid peg or vertebral bodies → fusion/slippage)

Villous hypertrophy and lymphoplasmacytic inﬁltrate and germinal centres (M > T-cells > B-cells)

± Giant cells derived from synovial lining M (rheumatoid nodules are rare in the synovium)

Superﬁcial ﬁbrin (→ rice bodies) and PMN (d/dg infection which can also occur) ± haemosiderin

Pyrophosphate crystals are very rare in RhA (but can occur)

Cartilage:  superﬁcial: Swiss cheese vacuolation (= enlarged chondrocyte lacunae) usu. occurs in

cartilage covered by a pannus of granulation tissue and inﬂamed synovium

 deep: local undermining by chronic inﬂam

tissue in the sub-chondral bone

Lymphoid hyperplasia in sub-chondral marrow fat spaces

d/dg: other conditions that may have similar chronic inﬂam

to RhA incl.:

 seronegative arthritis: PSS, SLE, psoriatic arthritis, ankylosing spondylitis (see also ‘Beh¸cet’s

Arthritis’, below)

 Reiter’s arthritis (has more PMN and may show vessels plugged by platelets and granulocytes)

 Lyme disease: stain for spirochaetes (Borrelia burgdorferi) which may be found in the walls

of synovial vessels, some of which show obliterative changes (unlike RhA)

 OA (some of the more inﬂammatory cases overlap with RhA – look for other features of OA)

 syphilis: a non-speciﬁc chronic inﬂam

arthritis (± miliary gummas)

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Osteoarticular 341

Beh¸cet’s Arthritis

Villous hypertrophy of the synovium with surface ﬁbrin ± ulceration

Underlying vascular and ﬁbrous connective tissue with many PMN and mixed inﬂam

cells

d/dg: sepsis/crystals/active phase of RhA

Joint Prosthesis Pathology (Pathology of the Periprosthetic Soft Tissue Membrane)

Gradual wear → particulate matter from prosthesis → M and inﬂam

→ loosening of prosthesis

At surgery for a loose prosthesis, Bx for FS may be sent to decide:

 ? infective → surgeon will remove prosthesis and wait until the infection has cleared before

replacement

 ? wear debris inﬂam

only → surgeon will replace the joint immediately

Type 1 periprosthetic membrane – wear debris inﬂammation

Def

: >20% of the membrane surface is lined by M +/ multinucleate giant cells

Holes that may be large bubbly spaces or small (≈ 80 ) round holes (± residual debris ≤ 2 in

∅=barium sulphate or zirconium) ± foamy M

Polymethylmethacrylate bone cement or unreacted cement monomer beads (soluble in solvents but

stain with oil red O if preserved in fresh FS. If not dissolved, may show as basophilic grape-like clusters

on parafﬁn H&E with mild birefringence

Tiny black opaque particles (round/sharp-edged), may show a birefringent halo: metal (e.g. titanium)

Bluish or transparent, birefringent, may be thread-like: HMW polyethylene/other plastics

Non-birefringent but oil red O +ve (in parafﬁn sections): submicronic polyethylene

Refractile bosselated yellowish material, not birefringent: Silastic



Silicone granulomas with birefringent particles: silicone elastomers

Tiny (≈ 1) birefringent grey particles in M but few/no giant cells: ceramics

Thin black rods of variable length: carbon ﬁbre

Type 2 periprosthetic membrane – infective

Def

: >10 PMN/10 hpf (because you don’t see PMN with wear debris alone) – useful criterion on FS

Lower grade infection may not have so many PMN but PMN are present with oedema, ﬁbroblasts and

proliferated capillaries

Small lymphocyte aggregates with assoc

plasma cells are also suggestive of low grade infection

Multinucleated giant cells are sparse

Type 3 periprosthetic membrane – mixed wear debris/infective

≈ Equal proportions of the membrane are affected by Type 1 and Type 2 morphology

Type 4 periprosthetic membrane – indeterminate reactive changes

Collagen and ﬁbrosis-rich, cell-poor

± Mature granulation tissue (but no sig. PMN inﬁltrate cf. d/dg infective Type 2)

May have a partial lining of ﬁbrin or synovium (! do not mis-diagnose as Type 1 M)

Synovial Chondromatosis

A metaplastic process, usu. in a single large joint; radiol. may show erosion of the articular surface

Nodules of chondroid matrix with ‘clonal clusters’ of proliferating chondrocytes occurs within the

synovium, these eventually pedunculate and break off to form many loose bodies in the joint

The nodules may display a synovial lining and contain focal Ca

May be hypercellular with binucleate cells and mitoses

Long-standing loose bodies may acquire outer concentric lamellae of ﬁbrocartilage

d/dg chondrosarcoma: the clonal clusters are unusual for chondrosarcoma which rarely affects the joint

space. Synovial chondrosarcoma is very rare, is solid/myxoid and extends beyond the joint capsule or

permeates the bone

d/dg osteochondral loose bodies in severe/rapid degenerative joint disease: the lack of clin./radiol.

evidence of severe degenerative joint disease and the presence of ‘clonal clusters’

Pigmented Villonodular (Teno)Synovitis (PVNS) – incl. GCT of Tendon Sheath

Types: diffuse (large joints, 25% recur)/nodular (‘GCT of tendon sheath’, small joints, rarely recur)

Nodular arch. (lobulated contour with ﬁbrous septa) – early nodules may have mitoses ++ centrally

with peripheral xanthomatoid ‘maturation’ (nuclei shrink and cytoplasm gets lipidised)

± Stromal hyaline or torsion necrosis

Pseudoglandular cleft-like spaces lined by synoviocytes (d/dg synovial sarcoma)

Histiocytoid mononuclear cells (modiﬁed synoviocytes) ± occ. bizarre cells

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Osteoarticular 342

Multinucleated giant cells (fusion of mononuclear cells) – nuclei are variably open to pyknotic

Xanthoma cell groups (pale islands)

Haemosiderin is found throughout the lesion (this is the ‘pigment’)

Malignancy in GCT of tendon sheath:

 benign GCT with an obviously malignant component (i.e. pleomorphism, diffuse inﬁltration,

mitoses abnormal or >10/10hpf, few giant cells, tumour necrosis, lack of maturation)

 GCT with benign histology but malignant behaviour (e.g. metastases)

 both are very rare so consider the d/dg before making the diagnosis

Malignancy in PVNS: lobular invasive growth, necrosis and prominent nucleoli.

Immuno: mononuclear cells are +ve for CD68 and (focally) desmin

d/dg ! distinguish PVNS from chronic haemorrhage (which is not Rx by surgery): in haemorrhage the

haemosiderin is near the surface of the fronds, in PVNS it is throughout the lesion (which is cellular)

d/dg: inﬂammatory myxohyaline tumour (=acral myxohyaline ﬁbroblastic sarcoma) has scattered large

bizarre virocytes/RS-like cells; it usu. occurs on the hands – see ‘Myxoﬁbrosarcoma’ on p. 312.

d/dg: tendinous xanthoma (multiple, giant cells sparse, cholesterol clefts ++, hyperlipidaemia)

d/dg ﬁbroma of tendon sheath may have occ. giant cells and foam cells (and is possibly related to GCT)

Adventitious Bursa

A bursa not connected to a joint cavity but forming over bony prominences

Fibrous wall ± a synovial lining (formed by metaplasia of connective tissue) with mucoid contents

Juxta-articular Myxoma

See p. 327.

Synovial Fluid Analysis

See pp. 375–376.

Bibliography

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Pathology, 59 (6), 635–638.

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13 (eds P.P. Anthony and R.N.W. MacSween), Churchill Livingstone, Edinburgh, pp. 255–269.

Dahlin, D.H. and Unni, K.K. (1986) Bone Tumors. General Aspects and Data on 8,542 Cases,4

edn, Charles C. Thomas, Springfield, Illinois.

Fletcher, C.D.M., Unni, K.K. and Mertens, F. (eds) (2002) WHO Classification of Tumours: Pathology & Genetics Tumours of Soft Tissue and Bone,

edn, IARC Press, Lyon.

Freemont, A.J. (2003) Joint disease and the pathologist, in Progress in Pathology, Vol. 6 (eds N. Kirkham and N.A. Shepherd), Greenwich Medical

Media, Ltd, London, pp. 79–100.

Ghadially, F.N. (1998) Diagnostic ultrastructural pathology: A self-evaluation and self-teaching manual.2

edn, Butterworth-Heinemann, Boston.

Malcolm, A.J. (2004) Ewing’s tumour – examplar of defining a tumour. ACP News (Winter 2004), pp. 11–12.

Morawietz, L., Classen, R-A., Schr¨oder, J.H. et al. (2006) Proposal for a histopathological consensus classification of the periprosthetic interface

membrane. Journal of Clinical Pathology, 59 (6), 591–597.

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Reid, R.P. and Catto, M.E. (1987) Some unusual tumours of cartilage and pseudosarcoma of the hand, in Recent Advances in Histopathology,

Vol. 13 (eds P.P. Anthony and R.N.W. MacSween), Churchill Livingstone, Edinburgh, pp. 68–70.

Revell, P.A. (1987) The synovial biopsy, in Recent Advances in Histopathology, Vol. 13 (eds P.P. Anthony and R.N.W. MacSween), Churchill

Livingstone, Edinburgh, pp. 79–94.

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235–246.

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cytologic findings. Cytopathology, 7 (3), 211–217.

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Infection and Immunity 343

23. Infection and Immunity

Introduction

This is a morphological guide for practising histopathologists and not a strict taxonomical classiﬁcation.

Some bacteria e.g. Gardnerella and Haemophilus are classiﬁed as coccobacilli here when a microbiologist

would call them short bacilli (and they may indeed grow as proper bacilli under some conditions). Some

bacteria here are placed in categories that aid morphological recognition in tissues rather than according to

strict taxonomy (e.g. Yersinia is placed under ‘coccobacilli’ when it actually belongs to the coliform group

of enterobacteria). Further information on some site-selective infections (e.g. rhinosporidiosis or infective

colitis/hepatitis) are provided in other chapters.

The numerous amoebae, helminthes, ﬂies, etc. that parasitise man are too numerous to do any

kind of justice to in a book of this sort. Here I give some morphological features of some of the least rare

parasites encountered in UK practice but specialist text-books are an essential tool for those working where

such protozoan and metazoan infections occur – see the Bibliography for examples.

Bacteria

Morphological Characterization

Cocci

Gram +ve  clusters, (e.g. Staphylococcus)

 pairs (diplococci e.g. pneumococcus)

 chains (e.g. Streptococcus)

Gram −ve e.g. Neisseria gonorrhoeae and meningitidis are ‘kidney bean diplococci’ usu. within PMN

Bacilli

Gram +ve  spore-forming

 central spores: (e.g. Bacillus anthracis [anthrax] or cereus [food poisoning,

ophthalmitis])

 eccentric spores: (e.g. Clostridium tetani [terminal spore], perfringens [thick

bacilli, no spore

], botulinum or difﬁcile [sub-terminal and free-ﬂoating

spores])

 non-sporulating (e.g. Corynebacterium diphtheriae [club-shaped short rods], Listeria

spp., Propionobacterium spp. and Lactobacillus spp.)

Gram −ve  straight (e.g. coliforms [E. coli, Shigella, Salmonella, Klebsiella and Proteus spp.]),

Legionella spp., Pseudomonas spp. [may be Gram and Brown-Brenn invisible, so use

Brown-Hopps])

 curved (Vibrio spp., Campylobacter spp., Helicobacter spp., Gastrospirillum spp.)

Coccobacilli

Gram −ve e.g. Bartonella spp. (some may form chains), Bordetella spp., Brucella spp., Calymatobac-

terium granulomatis (usu. in pairs and surrounded by a clear ‘capsule’ on Giemsa within a M to form

the Donovan bodies

of granuloma venerium

), Haemophilus inﬂuenzae, Yersinia spp., mycoplasmas,

Rickettsia spp., Coxiella spp., Francisella spp.: use Brown-Hopps, silver or Giemsa

Gram +ve e.g. some Vibrio spp.

Gram variable: e.g. Gardnerella vaginalis

NB: many coccobacillary species are seen within cells (epithelial, macrophage or PMN)

Filamentous bacilli

Actinomyces (silver and Gram +ve, branching 1 thick ﬁlaments, endogenous organism, forms radi-

ating aggregates (macro = yellow ‘sulphur granules’), no ↑ incidence in the immunosuppressed)

Nocardia (Gram +ve exogenous actinomycete, usu. scattered in the tissues and seen in the immuno-

suppressed)

NB: spore forming bacilli may not always show spores in tissue e.g. it is rare to see spores in tissue-situated Clostridium perfringens

but they are seen in soil or cultured samples

not to be confused with the amastigote forms of Leishmania spp. (= Leishman-Donovan bodies)

also called granuloma inguinale : these are not to be confused with LGV (a chlamydial disease)

Diagnostic Criteria Handbook in Histopathology: A Surgical Pathology Vade Mecum. Paul J. Tadrous



2007 by John Wiley & Sons, Ltd. ISBN: 978-0-470-51903-5

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Infection and Immunity 344

Tropheryma whippelii is a very short actinomycete but we don’t use Gram stains to diagnose it

d/dg Actinomycosis vs. Botryomycosis:

 actino colonies may be surrounded by radiating eosinophilic ﬂares of Splendore-Hoeppli

 other bacteria (e.g. staphylococci) can cause a mimicking reaction in tissues (= botryomycosis)

so careful high power microscopy of Gram stains should be used

d/dg Actinomycosis vs. Pseudoactinomycotic radiate granules (PAMRAG):

 PAMRAG have refractile crystalline strips and irregular granules

 PAMRAG stain diffusely Gram +ve (ﬁlaments are seen in actino) and are silver −ve

Mycobacteria

Although Gram +ve (and Giemsa +ve in tissue but −ve image in cytology preps) they may not stain

hence the use of ZN or silver (or DPAS for large numbers of atypical mycobactieria). M. leprae is

weakly acid fast so may not stain with ZN hence the need for a Wade-Fite or Fite-Faraco stain

3–5 long, slightly wiggly ± beaded: e.g. M. Tuberculosis, M. bovis, M. leprae

Slightly longer and thicker: atypical mycobacteria (e.g. M. marinum or MAI)

MAI are more beaded cf. TB and tend to be intra-M, aligned in stacks and assoc

with non-necrotising

diffuse M inﬁltration with few discrete granulomas – the latter usu. only in LN or bone marrow

≈20 long, thicker, more crooked and more beaded/banded: some of the atypical mycobacteria (e.g.

M. Kansasii which tends to form cords/serpentine coils and is assoc

with PMN and well-formed

necrotising granulomas unlike d/dg MAI)

Screening a ZN-stained slide for mycobacteria:

 use a thick section (e.g. 10)

 screen for 20 minutes (after which concentration is reduced) at high power (x40 or above)

 focus up and down as mycobacteria may stick up ≈ perpendicular to the plane of the section

Spirochaetes

Stains: silver methods, basophilic (if numerous) and speciﬁc immuno for Borrelia, Leptospira, etc.

Leptospira spp. characteristically have one or both extremities bent back (as a shepherd’s crook)

Borrelia spp. are characteristically variable in length (upto 40) and may show ‘elbow bends’

Treponema pallidum (like Leptospira) are at the shorter end of the spectrum (upto 15)

Chlamydiae

Gram −ve coccoid intracellular bacterioids, may form variably-sized (≈nucleus-size) basophilic glyco-

genated intraepithelial cytoplasmic inclusions stainable by Lugol’s iodine, PAS or Giemsa (granular

texture) but speciﬁc immuno or EM is more useful for diagnosis

Plasma cells +/ PMN are the typical tissue reaction in the female genital tract

Stellate necrotising palisaded granulomas with PMN are seen in the LN in LGV

Follicular conjunctivitis with central necrosis of follicles and peripheral collagenisation in trachoma

Mycoplasma-associated Diseases

Pneumonia (‘primary atypical pneumonia’)

Meningoencephalitis

Haemolytic anaemia

Group A Streptococcus-associated Diseases

Acute follicular tonsillitis/quinsy

Rheumatic fever

ADP GN/RPC GN

Scarlet fever

Infected skin sores of malnourished children

Mycobacterial Pseudotumour

LN, bone marrow, spleen, lung, skin

Storiform spindle cell proliferation

± Mitoses ± vascular proliferation – d/dg KS (! but KS may co-exist with it)

Immuno: +ve for desmin (d/dg a muscle tumour), S100, lysozyme, 

AT; −ve for CD31 and CD34

Syphilis

Fibrosis esp. of capsules of organs and also perivascularly

Chronic inﬂammation esp. plasma cells

Acute inﬂammation esp. PMN inﬁltrating epithelia

Vascular damage esp. endarteritis obliterans

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Infection and Immunity 345

Fungi

Morphological Characterization

Yeast forms

Shape and size: round or oval, with capsule or without, uniform diameter or pleomorphic

Division:  ﬁssion (e.g. Penicillium marneffei – useful in d/dg histoplasmosis)

 budding:  equal/unequal

 thin-necked/broad-based

 single buds/multiple buds

Nucleus: single or multinucleated (may not be visible in routine stains)

Hyphal forms

Thin/thick (uniform/irregular thickness)

Septate (frequent, rare, regularly distributed or not, constrictions present/absent)/non-septate

Branching (dichotomous

or irregular; consider the branch angles)/non-branching

Pigmented/non-pigmented/hyaline/poorly-staining

Presence (often where exposed to air) and type of conidia ± fruiting bodies (conidiophores)

Dimorphic forms

Most pathogenic fungi are dimorphic but few show both their hyphal and yeast stages in tissues

Candida spp. (e.g. C. albicans)

Dimorphic: budding ovoid yeast (budding is single, thin-necked, unequal and yeasts are ≈ 4 )

Thin (≈ 4) septate, branching hyphae (also pseudohyphae i.e. yeast buds that elongate and stay

attached at constriction points but with no true branching)

d/dg Histoplasma (vs. spore-only Candida): granulomas, intracellular yeasts and lack of pseudohyphae

Cryptococcus spp. (e.g. C. neoformans)

Budding round yeast (usu. single, thin-necked, unequal), can form hyphae and pseudohyphae in the

immunosuppressed

Great variability in sizes (5–15 )

Clear halo mucinous capsule (mucicarmine, AB, Hale’s colloidal iron → crenated/‘hairy’ capsule)

Surrounding ‘holes’ give the tissue a spongy, ‘soap bubble’ or ‘pseudoadipose’ look

Dry-variants exist (non-encapsulated) and capsulated cells may not be common in ordinary forms

Small-yeast variants exist (d/dg Histoplasma, etc. but only Cryptococcus has a mucinous capsule and

≈ 50% of histoplasmosis and blastomycosis show some ZN +ve organisms but cryptococci are −ve)

Histoplasma spp. (e.g. H. capsulatum)

Clin.: spores occur in soil and in faeces of bats/chickens; causes a cavitating TB-like disease in the im-

munocompetent and disseminated disease in the immunosuppressed (bones, adrenals, liver, spleen, LN)

Budding ovoid yeast (budding is single and thin-necked)

May be intracellular (e.g. aggregates in M)

Smaller and less variable in size cf. Cryptococcus (2–5)

±Artefactual clear halo (esp. if intracellular) but no true capsule (unlike Cryptococcus)

Large yeast variant (duboisii) exists esp. in Africa (d/dg Cryptococcus, blastomyces, etc.)

d/dg Penicillium, Candida and small-yeast forms of Cryptococcus, Blastomyces and Malassezia; also

Leishmania, Toxoplasma, etc. Histoplasma stains weakly with PAS (other yeasts stain strongly)

Aspergillus spp.

Regularly septate, dichotomously branching hyphae of regular thickness (≈ 4)

Typically each limb of a branch makes an equal angle to the stem to give a ‘Y’ conﬁguration where the

two upper limbs of the ‘Y’ are at an acute angle to each other

Viable hyphae are basophilic, necrotic ones eosinophilic

Conidia (spores) and conidiophores (fruiting bodies) can form in cavities

d/dg Pseudallescheria spp. (their hyphae are identical to aspergillus but their conidia differ)

d/dg fusariosis but these usu. make branches at 90

◦

to the stem (as well as ‘Y’ forms)

Mucormycotic Zygomycosis due to Rhizopus spp.

Irregularly branching hyphae of irregular thickness (upto 20) and irregular orientation

‘dichotomous’ (from the Greek for ‘cut in two’) means that both branches are of equal thickness

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Branches often come off at 90

◦

and are thinner than the stem (non-dichotomous)

Non or pauci-septate (but ﬂimsy and often folded/kinked/twisted and may form ‘tangles of sticky tape’)

Weakly basophilic thin walls but empty inside; weak Grocott staining

Conidia (thick-walled with a deeply basophilic core) may form at the end of hyphae, break off and

cluster (! d/dg non-budding yeasts)

Viruses

Morphological Characterization of Viral Inclusions

Site

Cytoplasmic (e.g. HBV surface Ag, HPV, rabies Negri and lyssa bodies, RSV [usu. paranuclear])

Nuclear (e.g. HBV core, HDV, polyoma, adenovirus, HSV/VZV, parvovirus) – d/dg nuclear clearing

due to biotin (! false +ve immuno) esp. in endodermal epithelia (endometrium, lung, thyroid, etc.)

Both (e.g. HBV core, CMV, measles)

Shape

Round/oval (e.g. HBV, CMV nuclear inclusion, rabies [Negri and lyssa bodies in neurons], RSV)

Irreg. (e.g. measles cytoplasmic inclusions or the ‘exaggerated’ keratohyaline granules in wart HPV)

Clear rim or larger halo (e.g. early adenovirus, late HSV/VZV, HBV surface Ag, RSV, HPV, parvovirus

and CMV nuclear inclusion)

Blurred margin (late adenovirus nuclear inclusion = ‘smudge cell’ [NB: the term ‘smudge cell’ or ‘smear

cell’ is also applied to appearance of some CLL lymphocytes in PB ﬁlms without viral inference])

Multinucleation with (e.g. HSV/VZV, measles in epithelial cell nuclei, RSV in alveolar cell cytoplasm)

or without (e.g. HIV, parainﬂuenza, measles Warthin-Finkeldey cells) inclusion bodies

Staining properties

Basophilic (e.g. cytoplasmic inclusionof CMV, nuclearinclusion of HBV core Ag, adenovirus, polyoma,

early HSV/VZV [diffuse stage])

Amphophilic (examples are as for basophilic; esp. seen in the earlier stages of some inclusions)

Eosinophilic (e.g. nuclear inclusion of CMV [but this may be basophilic], HBV surface Ag, measles,

parvovirus, molluscum pox, RSV, rabies [Negri bodies are well-deﬁned, are upto the size of an RBC and

have an inner more basophilic core; lyssa bodies are less well deﬁned and have less internal structure],

late HSV/VZV [angulated inclusion with halo])

Uniform texture/‘ground glass’: (e.g. adenovirus, HBV, delta, HSV/VZV, measles, early polyoma)

Coarse granular texture (e.g. cytoplasmic CMV inclusion)

Reticular texture (e.g. late polyoma inclusion)

Non-inclusion morphology

Many viruses may not show inclusions but show typical inﬂammatory (e.g. HCV, VHF), reactive (e.g.

HIV) or neoplastic (e.g. EBV, HHV8, HPV) manifestations

HIV and AIDS

AIDS-deﬁning illnesses (ADI)

Molluscum, CMV, HSV, PML

Recurrent bacterial pneumonia, TB, MAI

Severe Candida, PCP, Cryptococcus, Histoplasma, Coccidioides

Toxoplasma, Cryptosporidium, Isospora

SCC cervix, high grade B-cell lymphoma, KS

HIV dementia, HIV wasting syndrome

AIDS and the skin / bone marrow and AIDS lymphadenopathy

See pp. 306–307, p. 100 and p. 119.

AIDS and the GIT

HIV enteropathy:  villous atrophy of the crypt atrophic or crypt hyperplastic type

 ↑crypt apoptoses and IELs (d/dg GVHD)

Opportunistic / other infections (‘gay bowel syndrome’ is an outmoded loosely-deﬁned term relat-

ing to any number of a variety of alimentary tract infections associated with HIV/AIDS e.g. gonor-

rhoea, syphilis, Shigella, Salmonella, amoebiasis, giardiasis, Enterobius, spirochaetosis, Campylobac-

ter, Chlamydia, HPV warts and even viral hepatitis have all been included in this ‘syndrome’)

Oral hairy leukoplakia, cystic LESA, KS, lymphomas

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AIDS-associated malignancies

Sarcomas: KS and angiosarcoma

Lymphomas: HL, NHL and multiple myeloma

◦

brain tumours: gliomas and PNET

Seminoma

Others (e.g. anal and cervical SCC) may be common in pre-AIDS HIV but don’t have a marked

post-AIDS increase in incidence as the above examples do

Parvovirus B19 Related Disease

Infects erythroid precursors and is present in most people (serum IgG is +ve) being asymptomatic

Normal hosts may get immune complex mediated ‘ﬁfth disease’ with a rash in children (‘slapped

cheek’) or arthralgias in adults

The more proliferative the erythroid line (e.g. in the fetus or in sickle cell disease), the more pathogenic

the effect on red cell production culminating in pure red cell aplasia (→ hydrops fetalis in the fetus)

Micro: giant pronormoblasts ± red cell precursors with clear or eosinophilic [nucleolus-like] nuclear

haloed inclusion(s) (but these are not always seen, even in proven infections)

EBV (HHV4) Related Disease

Non-neoplastc: infectious mononucleosis, cytophagic histiocytic panniculitis

Lymphoma: HL, Burkitt’s, angioimmunoblastic, angiocentric T/NK-cell, DLBCL (−ve for HHV8

except in PEL), extranodal lymphomas in HIV,

Post Tx lymphoproliferative disorders (PTLD)

Nasopharyngeal carcinoma

KSHV (HHV8) Related Disease

KS, multicentric Castleman’s, PEL, some plasmablastic lymphomas/microlymphoma and PTLD

Protozoa

Pneumocystis carinii

Cysts are ≈ size of an RBC and may be spherical or partly collapsed (incl. crescentic)

Grocott shows thick wall with a dot-like thickening or comma-like inclusions (1 or 2 thereof)

Giemsa doesn’t stain wall but may show tiny dots in (sporozoites) or outside (trophozoites) the cysts

Giardia lamblia

Trophozoite: a binucleate, amphophilic, DPAS −ve, 10 × 20 ‘tear-drop’ or sickle-shaped ﬂagellate

Cyst: a 10 × 8 ovoid with internal longitudinal linear structures (= axonemes) and rounded structures

(= nuclei, upto 4 thereof and crowded into one end)

The trophozoite is seen in the small bowel and both trophozoite and cyst may be seen in faecal smears

Toxoplasma gondii

Tachyzoites: fast-growing forms in cells and interstitium are 4 ×2, crescentic, haematoxyphil and

Giemsa +ve (but PAS and silver [e.g. Grocott] −ve). They suggest lack of immunity and are always

pathogenic. Immunity slows them into encysted bradyzoites

Bradyzoites are haematoxyphil, Giemsa and PAS +ve (DPAS −ve) dots in a well-deﬁned cyst within

cell cytoplasm (cyst wall is silver +ve). Pathogenicity cannot be assumed without a tissue reaction

Bradyzoite cysts may rupture (→ tissue reaction and bradyzoites become less basophilic) or immuno-

suppression (e.g. steroids) may cause transformation into free tachyzoites

Tissue response (general): lymphocytes, M ± PMN inﬂam

with focal necrosis ± vasculitis or en-

darteritis

LN (characteristic features useful in d/dg): follicular hyperplasia may be prominent, para/intra-germinal

centre microgranulomas (here deﬁned as <25 nuclei), no multinucleated giant cells, sparse (<3 per

section) larger granulomas (typically <50 nuclei and non-necrotising), ± sinus B-cell reaction

Brain: glial nodules (neonates have subependymal lesions with ulceration ± Ca

)

Placenta: bradyzoites in chorion and decidual cells – usu. without a reaction

Others: lung (interstitial pneumonitis), GIT (ulceration), bone marrow (cysts), etc.

d/dg (use immuno to Toxoplasma and serology for IgM):

 fungi (esp. Cryptococcus and Histoplasma)–Toxoplasma are silver −ve

 viruses (CMV cytoplasmic inclusions lack a capsule and are granular rather than distinct dots)

 protozoa (Leishmania, Trypanosoma cruzi, microsporidia)

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Entamoeba histolytica

See p. 143.

Cryptosporidia spp.

Clin.: like isosporiasis (q.v.). Biliary/respiratory infection is rare and usu. only if immunosuppressed

Histology ranges from near-normal to active inﬂam

(or coeliac-like in the SI) ± regenerative changes

Oocysts (spherical, 5 ) infect epithelial cell cytoplasm and bud onto the lumenal surface being

invested in a cytoplasmic coat (hence they appear larger)

Although deep gland epithelium may be infected, cryptosporidia don’t invade sub-epithelial tissues

Stains: the cysts are basophilic, dark blue with Giemsa and variably acid fast, silver and Gram +ve

Microsporidiosis (Enterocytozoon and Encephalitozoon spp.)

Oral, ocular or inhalation of spores → infection of epithelial, endothelial, stromal and M cells

Clin.: diarrhoea, nausea, weight↓, biliary obstruction, cough, maxillary sinus polyps, haematuria, etc.

Granulomatous interstitial nephritis/prostatitis, diffuse punctate keratoconjunctivitis, etc.

SI may show variable sprue-like changes with disorganisation of epithelium ± vacuolisation

Spores

Are ovoid (1 × 1.5 for intestinal spp., upto 4 × 2 for corneal spp.) with posterior vacuole and dark

nucleus (best seen in toluidine blue semithin sections)

Form clusters in the cytoplasm (apical in enterocytes and may ‘cup’ indent the nucleus)

Staining: usu. basophilic, refractile and variablybirefringent (bluewith Giemsa, pink with Brown-Brenn

and white with Warthin-Starry), Gram +ve (‘hourglass’ shape) and have a PAS +ve polar body

d/dg : EM can help; PCR and immunoassays are also available (serology, urine, etc.)

Isosporiasis (e.g. Isospora belli)

Clin.: watery diarrhoea ± (if immunosuppressed) weight loss, malabsorption, fever, headaches, etc.

Variable villous atrophy of the crypt hyperplastic type (duodenum/jejunum)

Enterocytes become disorganised and ﬂatter with loss of nuclear polarity

Parasite stages are present in the epithelium (usu. closer to the BM): Giemsa, AB, PAS and silver +ve

One ovoid (schizont) or multiple elongated structures (merozoites) in a cluster (total size is ≈ size of

one enterocyte nucleus). This is surrounded by a clear halo (= parasitophorous vacuole)

Macrogametocyte: an oval cell (just larger than an enterocyte nucleus) with central round nucleus,

prominent central nucleolus and surrounded by lipid rich granules in the cytoplasm

Microgametocyte: (≈

size of a goblet cell’s mucin blob) contains many dots (microgametes)

d/dg: if in doubt, EM can help

Plasmodium spp. (Malaria)

Female Anopheles mosquito injects sporozoites that get taken up by hepatocytes where they multiply

to form a schizont (bag of merozoite 1 dots) that ruptures → merozoites infect RBC and undergo

further schizony divisions to form an RBC schizont (via an early signet ‘ring form’) that matures into a

gametocyte (15 long, banana-shaped and expands the RBC in P. falciparum) →taken up by mosquito

(→sexual stages →sporozoites). RBC cytoplasm may have stippled or cleft-like densities

Giemsa stain is best for seeing the nuclear DNA of the parasitic stages

Infected RBC stick to endothelium (via knobs seen on EM) and may cause clogging/ring haemorrhages

Malarial pigment in phagocytes: a birefringent porphyrin-globin conjugate from digested Hb

General: 2

◦

changes of haemolysis, infected RBC, blocked capillaries (et seq.) and malaria pigment

Liver: Kupffer cell expansion with malarial pigment (zone 3 early on but more in zone 1 later)

Brain:  ring haemorrhages

 D¨urck’s granulomas (demyelinated patches and gliosis and infected RBC)

Kidney: effects of haemoglobinuria and ADP GN/membranous GN/membranoproliferative GN

d/dg: morphology of RBC stages on thick (haemolysed) and thin blood ﬁlms: multiple ring forms in one

RBC suggest falciparum malaria (over vivax, ovale or malariae) as does the banana-shaped gametocyte

(others have round ones). Strap-shaped ‘band-form’ early schizonts suggest P. malariae

Leishmania spp. (Visceral Kala-azar and Mucocutaneous Espundia)

A sandﬂy injects the ﬂagellate form into man → parasitises M and looses ﬂagellum (= amastigotes)

They are 4 ovoids containing a double-dot structure on Giemsa (= nucleus + elongated kinetoplast)

Inﬂam

in the skin/mucous membranes may be ulcerative, acute, granulomatous or non-reactive (an-

ergic) with sheets of M; viscera are expanded by infected M (e.g. in the sinusoids)

Granulomas (even necrotising/caseating) may occur in the LN draining mucocutaneous lesions