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

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color-plates December 8, 2007 16:39 Char Count= 0

Layers of the Normal Chorion & Amnion Intertwin Membranes

Cellular

& Reticular

Trophoblastic

Decidual (incl.

blood vessels)

AMNION

(avascular)

Epithelial

Compact

Fibroblastic

Spongy

CHORION

DIAMNIOTIC MONOCHORIONIC

DIAMNIOTIC DICHORIONIC

Inset is x3

cf. main picture

(chorion is present between the amniotic layers)

FIGURE 5.3

FIGURE 8.1

FIGURE 11.3

color-plates December 8, 2007 16:39 Char Count= 0

fovea

lamina cribrosa

sclerae

RETINA

CORNEA

choroid

(vascular & pigmented)

KEY (Retina)

1. Bruch's (basement) membrane

2. Retinal pigment epithelium (RPE)

3. Layer of rods & cones (outer segment)

4. Layer of rods & cones (inner segment)

5. Outer nuclear layer

6. Outer plexiform layer

7. Inner nuclear layer

8. Inner plexiform layer

9. Ganglion cell layer (scant to many nuclei thick)

10. (Optic) Nerve fibre layer (with retinal vessels)

OLM = Outer limiting membrane

endothelium

epithelium (non-keratinised)

basement membrane

(PAS

ve)

Bowman's layer / membrane

(PAS

ve)

Descemet's (basement) membrane

stroma (avascular)

ora serrata

SCLERA

retinal vessels

OLM

FIGURE 14.1

Sertoli cells

Spermatozoa

Spermatids

1° Spermatocytes

Pale & Dark Type A Spermatogonia Type B Spermatogonia

FIGURE 16.1

cde

FIGURE 17.1

intestine

lateral

ala

excretory

column

canaliculus

BAYLISASCARIS

TOXOCARA

nucleus

FIGURE 23.1

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Exam Advice 1

1. Advice for Exam Candidates

Introduction

There is no ‘secret’ or ‘technique’ that will ensure success in the MRCPath exam and by far the best way to

prepare is to do lots and lots and lots of routine diagnostic and autopsy work in as many busy general and

specialist hospitals as you can over the years of your training. You should attend one of the major diagnostic

histopathology courses and one of the major cytopathology courses close to your exam because they help

ﬁll gaps in your knowledge and give you conﬁdence. Going over slide collections is tempting but is often

not very helpful because slide collections are made up of fascinomas whereas the exam is made up of

routine surgicals.

The examiner is looking for evidence that you will be a safe and effective pathologist when

left to your own devices. Safe means that you know your limitations (know when to refer or defer) and

have a mature approach to diagnosis i.e. you make diagnoses based on a combination of multiple factors:

clinical, constellations of morphological criteria, ancillary results, etc. with due consideration of appropriate

differentials and with regard to the consequences of your decisions. Diagnosis by picture-matching or

putting undue emphasis on a single feature is not appropriate for consultant-standard candidates. Effective

means you have sufﬁcient knowledge and experience to be able to make a conﬁdent diagnosis in the

majority of the cases – anyone can muddle through by sitting on the fence or referring every case – but this

can cause harm by means of delayed diagnosis (= delay in getting appropriate treatment) or by causing

the patient to undergo unnecessary repeat diagnostic clinical procedures.

As a trainee, try to avoid the comfort of staying in one institution for longer than 18 months.

Moving around gives you a broader vision, you learn new ‘tricks’ from new colleagues and new ways

of approaching the same conditions. This builds breadth of experience rather than depth – and both are

important.

It is vitally important to ensure that you keep good timing – many have failed because they

haven’t given themselves time to answer all the questions or study all the cases. Take every opportunity

you get to do mock exams and ensure that you are strict with yourself over timing. Take a watch or clock

with you to the exam (but not one with an audible alarm). You are also well advised to check in advance

the quality and build of the microscope available in the centre and consider taking your own instrument to

the exam: making diagnoses in the pressure of the exam is stressful enough without having to ﬁddle with

an unfamiliar objective turret, ﬁeld of view, focus mechanism and maladjusted illumination system.

In diagnostic practice, knowledge (of what diseases exist and what criteria deﬁne them) is more

important than a visual memory for pictures. The histopathology of any one disease entity is deﬁned by

the presence of a set of morphological (± clinical) criteria. Because any one or more of these criteria may

dominate in a given instance of that disease, the overall histological picture may look dramatically different

to a picture of the same disease occurring in another patient – the criteria for making that diagnosis, however,

are the same. This is why pattern matching for diagnosis is potentially dangerous (pattern matching is only

useful to recognise the presence or absence of any individual morphological criterion) and that is why this

book concentrates on deﬁning diagnostic criteria rather than illustrations of ‘typical lesions’. Knowledge

is built up over the years by getting into a habit of reading, teaching and doing. Knowledge is just as

important for the practical exam as it is for the written exam – and even more so for real life diagnostic

practice. A suggested reading list is given in the ‘General Bibliography’ of this book (see page xxiv).

There follows some generaladviceonapproaching the practical aspects ofthe MRCPathexam. The

examis in the process of changing as postgraduate medical education in the UK also undergoes restructuring

towards ‘run through’ training with a deﬁned curriculum and competency-based assessments. The moves

are towards standardisation, centralisation and modularisation. In particular the autopsy component has

been separated off from the main exam as autopsy training becomes more specialised. Gynae cytology may

eventually also become a separate module. For details of the latest exam structures and what’s expected of

candidates see the documents posted on the RCPath website, www.rcpath.org.uk.

At the end of this chapter I suggest a small (and incomplete) list of conditions you should always

think about before arriving at a diagnosis (the ‘Never Forget Group’) and ﬁnally, for the desperate, I provide

a list of mnemonics found throughout this book.

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

JWBK208-01 December 8, 2007 15:57 Char Count= 0

Exam Advice 2

The Diagnostic Slides

Short Surgical Pathology Cases (usu. 20 cases)

You get 9 minutes to write a structured report for each one:

◦

description,

◦

diagnosis,

◦

clinical

comment. You don’t need all three for straightforward cases (obviously,

◦

is essential).

Avoid lists of stains – if you feel the need for further stains or procedures always state why each stain

is needed and what you would expect to see (i.e. how it will help you decide amongst the differentials).

In the set of 20 surgical short cases it would be unusual to put in a case that needs lots of extra stains /

procedures to arrive at a preferred diagnosis (this is the point of the long cases).

Avoid lists of differentials and always attempt to give a preferred diagnosis. If your conﬁdence level

in your preferred diagnosis is low, say so, and discuss the most likely alternatives with reasons for

and against. It is highly frowned upon for a differential to span the benign – malignant divide. This is

clinically a useless position for a pathologist to maintain. If you are really stuck in this decision it may

be a case you will have to ‘refer for second opinion’ but you can’t do this too often (see below).

Use formal language in your reports without anecdotes or use of ‘note style’ writing. For example: ‘On

this section alone my preferred diagnosis is X rather than Y. However, my degree of conﬁdence is not

high and, given the importance of making the distinction between X and Y in this case, I would like

to ...’ [continue, for example, with one of the following]:

 ‘...examine further levels to look for [state the features that will help]’

 ‘...perform a Congo red for amyloid to substantiate the H&E ﬁndings’

 ‘...refer the case for a second opinion’

 ‘...further sub-classify the lesion by making reference to a major dermatopathology textbook

[or, better still, state a precise reference]’.

An exam pass-standard candidate should be able to give a preferred or deﬁnite diagnosis for most of

the 20 cases (at least 17 of the 20 as a general rule of thumb).

Long Surgical Pathology Cases

These will be something like a renal, bone marrow trephine or liver biopsy with special stains or a

tumour with special stains. Special stains may include tinctorial, immuno & EM ± macro photo.

Compose a formal and structured report as you would normally do in routine practice. Give your

preferred diagnosis ± a limited differential with reasons (as described for the short cases above).

Show the examiner that you are able to interpret the signiﬁcance of the special stains: positivity,

negativity, strength / grade and distribution / pattern of staining, artefactual changes, etc.

You may get an unexpected result in the specials – the examiners are trying to see how you cope, e.g.

a probable follicular lymphoma with a negative CD10 stain.

You may get an impossibly difﬁcult case – this is deliberate and you are not expected to give the

diagnosis but the examiner wants to see how you handle something you can’t do. This will be where

your experience and judgement – or lack of it – will really show.

Cytology

Gynae short cases: at the end of your report you should give an appropriate recommendation for

management based on the patient’s age and previous smear history. State if inadequate and why.

Non-gynae short cases should result in clear and unambiguous results. State if inadequate and why.

For the long cases bear in mind problems of interpreting special stains and ensure you have the necessary

controls.

Make sure you are aware of (and familiar with) the common pitfalls: atrophic smear vs. severe

dyskaryosis, decoy cells in urine vs. malignant cells, etc., etc., etc.

The Viva Voce and Macroscopic Pathology exam

Display effective communication skills: address the questioner, look them in the eye, don’t mumble,

use clear expressive and articulate language, don’t wafﬂe and don’t ﬁght.

If you don’t know the answer to a question be honest and up-front. Say you don’t know about that

particular thing but what you do know is... [something closely related to the question] – and let the

examiner stop you if they want to. Don’t use ‘politician speak’ or weasel words to try and ‘fool’ the

examiner: the examiner will not be fooled – and neither will they be impressed.

The viva is part of an OSPE with a strict marking scheme so you cannot use it to admit ‘mistakes’ you

made in the practical (.

. there’s no point discussing the cases with your fellow candidates).

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Exam Advice 3

The Autopsy

General

This will be done on a separate day to the rest of the exam and possibly at a different centre.

You will be allowed up to 3 hours to conduct the autopsy (excluding presentation and write-up).

Review the notes and consent form and write a summary: age, date of death, clinical history.

Conduct a risk assessment (use the local form / questionnaire where available).

Ask the examiner about arrangements for contacting clinicians / students to attend the presentation.

Discuss any special requirements anticipated (e.g. X-rays for neonates, microbiology, FS facilities).

Do not criticise the instruments – although ask for others if required.

On external examination: check identity, check for LNp and – ! – remember to check the back of the

body.

Health and safety is very important, slackness here can easily fail you:

 maintain an orderly instrument layout and demonstrate safe handling of them

 be clean and tidy at all times

 do not leave pools of blood in the body cavities – rinse and sponge out.

Evisceration

Consider taking ascitic ﬂuid for culture if there is intra-abdominal sepsis.

Remember to check for pneumothorax.

The MTO may remove the cranium but you could be expected to remove the brain.

Remove the diaphragm intact by cutting it ﬂush with the thoracic wall.

After removing the organs, clean the inside and outside of the body and check the inside of the rib cage

for fractures (haemorrhage) and check for scoliosis / crush fractures of the spine.

Ask if it is routine to remove the femur. [NB: This is not expected in the current MRCPath exam.]

Ask if you are expected to ﬁx the brain (the preferred option) or dissect it fresh.

Organ Systems

Show the examiner you have a good-quality dissecting technique: open both iliac veins down to the

femorals at the level of the great saphenous vein; keep the pericardium (and display it); assess the skull

thickness for Paget’s disease; don’t spill gastric contents or bile (open these structures into containers);

don’t leave part of the right atrium behind (you will have trouble demonstrating the SA node when

asked). Also, it is generally not good if the examiner opens the 1

part of the duodenum for you to reveal

the ulcers you missed! Remember the carotid and vertebral arteries (at least inspect the intracranial

portions of the vertebrals [the current MRCPath doesn’t require a full dissection]).

Show that you can think of things relevant to the clinical history or PM ﬁndings: remember the lymph

nodes, bone marrow and tonsils in patients with lymphoproliferative disease; be prepared to comment on

the renal arteries in someone with HT; take CSF by syringe from the 3

ventricle for microbiology

if there is reason to suspect meningitis or brain abscess.

Show you have a good knowledge base: know your normal weights and measures, be prepared to

discuss specialist dissection techniques and their indications (inﬂation of the lungs, vertebral arteries,

conducting system, middle ear, etc.), issues of health and safety, consent and the Law (e.g. the Human

Tissue Act, the Coroner’s rules and when to refer a case to the Coroner), macro staining methods (for

MI, amyloid, iron, etc.), toxicology, the future of autopsy (minimally invasive, radiologically assisted,

sub-specialised, etc.), mortuary design and other topical issues.

Presentation and Writing a Report

Periodically clean, dry and arrange the organs and instruments during the presentation.

Start with the history then, in order: cause of death in ONS format →predisposing pathology → other

major ﬁndings → trivia. Avoid lists of negatives. Demonstrate good interpretative skills.

Be slick (e.g. you should be able to demonstrate the coronary arteries swiftly) and point speciﬁcally to

pathology with a probe. Your manual dexterity (throughout the autopsy) is part of the assessment.

Demonstrate good communication skills and show a good rapport with the clinicians.

After presentation write the report and block index (ask the examiner for details – some may allow you

to dictate). Remember to put the cause of death in the ONS format (for those over 28 days old) and do

not use modes of dying as a substitute for a cause of death (see Chapter 25: Autopsy).

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Exam Advice 4

The Frozen Sections Exam

Give a clear and unambiguous answer that will help the management – not a detailed report / diagnosis.

If you are really stuck, you may ‘defer to parafﬁn’ but do this once too often and you will fail. Although

in real life there is the possibility of requesting further levels on a FS or more tissue from the surgeon,

the cases chosen for the exam are unlikely to require this.

Remember FS artefacts (e.g. the lack of lacunar cells in NSHL or Orphan Annie nuclei in PTC) and

be prepared to mention the possibility and utility of imprint cytology.

The ‘Never forget’ Group

Don’t make a diagnosis until you’ve considered the following – the mnemonic, ‘CAMMeLS’, will help

you to remember:

Chemotherapy / radiotherapy / inﬂammatory atypia

Amyloid

Melanoma (1

◦

)

Metastatic / 2

◦

carcinomas (e.g. RCC metastatic to mucosae, skin or bone)

Leukaemia: CLL, chloroma / ‘granulocytic sarcoma’ (AML / CGL)

Sarcoid / Crohn’s (incl. extra-intestinal Crohn’s) / reaction to malignancy: when faced with true

epithelioid granulomas

Mnemonics

Mnemonic Index

Condition/entity Mnemonic Chapter

‘Never forget’ group of diseases CAMMeLS Chapter 1: Advice for Exam

Candidates

Atheroma (complications of) CUT Chapter 6: Vascular

Abbreviated list of T-cell / NK lymphomas: THE PSALM Chapter 10: Lymphoreticular

Morphological types of myoepithelial cells: SPEC Chapter 11: Alimentary Tract

Neuroendocrine cell tumours: EDGE Chapter 11: Alimentary Tract

Causes of malabsorption: FIDLES BV Chapter 11: Alimentary Tract

Drug effects in the small bowel VACU Chapter 11: Alimentary Tract

Drug effects in the large bowel PUMICE Chapter 11: Alimentary Tract

Hepatocellular carcinoma variants: PC STAGS Chapter 12: Liver, Biliary Tract

and Pancreas

Nephrotic and nephritic syndromes: POH / OHO Chapter 15: Renal Medicine

Causes of 2

◦

membranous GN: SIND Chapter 15: Renal Medicine

Causes of 2

◦

FSGN: S.H.I.P. Chapter 15: Renal Medicine

Causes of nephrotic syndrome DASHIN Chapter 15: Renal Medicine

Inﬁltrating lobular carcinoma of the breast: CAST Chapter 18: Breast

MEN 1 (Wermer’s syndrome): All the ‘P’s Chapter 19: Endocrine

Painful lumps in skin / subcutis: TEABAGSPEND Chapter 20: Skin

Leukocytoclastic vasculitis (associations): MAID Chapter 20: Skin

Blistering diseases of the skin: PErHEPS Chapter 20: Skin

Distribution of bullous pemphigoid: WOLF Chapter 20: Skin

Epidermolysis bullosa acquisita (associations): ABC Chapter 20: Skin

Carcinomas metastatic to bone: The 5 ‘B’s Chapter 22: Osteoarticular

Albright’s syndrome: All the ‘P’s Chapter 22: Osteoarticular

Sarcomas positive for cytokeratins (CK): PEARLS Chapter 21: Soft Tissues

Malignant ﬁbrous histiocytoma (MFH): MAGIC Skin Chapter 21: Soft Tissues

Kaposi sarcoma (clinical types): CELTA Chapter 21: Soft Tissues

Kaposi sarcoma (classical type): WIELDA Chapter 21: Soft Tissues

Kaposi sarcoma (endemic type): SMACS Chapter 21: Soft Tissues

Kaposi sarcoma (lymphadenopathic type): RAW Chapter 21: Soft Tissues

Kaposi sarcoma (transplant associated): LAFS Chapter 21: Soft Tissues

Kaposi sarcoma (AIDS associated): MDM Chapter 21: Soft Tissues

Causes of shock CHAOS Chapter 25: Autopsy

JWBK208-01 December 8, 2007 15:57 Char Count= 0

Exam Advice 5

Bibliography

Stamp, G.W.H. and Wright, N.A. (1990) Advanced Histopathology,1

edn, Springer-Verlag, Berlin & Heidelberg.

Weir, J., Benbow, E.W. and McMahon, R.F.T. (2004) How to pass and how to fail the MRCPath in histopathology part 2, ACP News (Winter 2004),

39–43.

Web sites

www.rcpath.org.uk (accessed April 2006) MRCPath Part 2 examination – Autopsy module: Guidelines for examiners and candidates (2004), Royal

College of Pathologists.

JWBK208-02 December 8, 2007 21:25 Char Count= 0

Histotechniques 6

2. Histological Techniques

K¨ohler Illumination

K¨ohler Illumination for Photography and Image Analysis

Setting up the microscope for K¨ohler illumination

For even illumination and optimum contrast do the following.

Condenser

Diaphragm (CD)

Field

Diaphragm (FD)

Condenser

focus control

FIGURE 2.1 Controls relevant to

K¨ohler illumination

1. Put a specimen on the stage, fully open both the ﬁeld diaphragm

(FD) and the condenser diaphragm (CD) and focus the image at low

power (e.g. ×10 or the lowest power at which full ﬁeld illumination

is achieved without the need to ‘ﬂip down’ part of the condenser lens

system. This is because the full set of condenser optics is needed

for the following steps).

2. Close the FD and adjust the condenser focus control (! not the

main microscope focus control) until you see a sharp image of the

FD.

3. Centre the image of the FD by adjusting the condenser centration

screwsusu. found near the base of the condenser. Some microscopes

have a locking screw which must be loosened before adjusting

the centration screws. It should be re-tightened when centration is

complete.

4. Adjust the FD aperture such that its image just disappears beyond the ﬁeld of view. As the aperture of

the FD is increased you may ﬁnd it easier to make ﬁne adjustments to the condenser centration.

5. Now adjust the CD aperture to match the numerical aperture of the objective. This may be done by

either a direct or indirect method:

 Direct method: remove one of the eyepieces and look down at the aperture of the objective with your

eye ≈ 5–10cm away from the eyepiece holder tube. Adjust the CD until its boundary is just within the

aperture of the objective.

 Indirect method: leave the eyepieces in place and look at the specimen. Slowly close the CD until a

slight drop in image brightness is ﬁrst perceived.

6. Adjust brightness and colour-balance using the illumination power control or ﬁlters only – do not adjust

the diaphragms or condenser focus because this will alter image quality / sharpness. For imaging work,

note that a change of objective or any of the controls above will alter the amount of light getting through

to the imaging device – so re-calibration / re-metering will be necessary.

7. Whenever you change objective you should repeat steps 4 and 5 above to match the new ﬁeld of view

and objective numerical aperture. If you change the slide you may also need to repeat steps 1 and 2 because

the preparation may be of slightly different optical thickness. [NB: low power objectives may require some

modiﬁcation of the condenser such as swinging a lens out or, with very low power (≤×2) the condenser

may have to be removed altogether or the CD fully opened or a condenser diffuser inserted.]

K¨ohler Illumination in Diagnostic Practice

The above method is unnecessary for day-to-day work and may have disadvantages (e.g. any spec of dust

on ﬁlters over the FD may be constantly in the ﬁeld of view causing an irritating distraction). Thus most

pathologists set up their microscope as described above once only and then leave the CD open to that extent

which is appropriate for the highest power objective commonly used (usu. ×40 or ×63), only opening it

further if they go to a higher power (e.g. ×100 oil immersion). Leaving the CD open like this has negligible

detrimental effects for direct viewing at lower power objectives. It is also common to keep the condenser in

a slightly de-focused position in order to get dust particle images blurred enough so as to be unnoticeable.

The FD is usu. left completely open at all times. Precise K¨ohler re-adjustment may, however, be necessary

when using very high power lenses e.g. looking for bacteria in Gram, ZN or CFV preparations.

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

JWBK208-02 December 8, 2007 21:25 Char Count= 0

Histotechniques 7

Counting Mitotic Figures and Ki-67 Proliferation Labelling Index

Only include deﬁnite mitoses in metaphase/anaphase (furry chromosomes and no nuclear membrane).

Scan the section for the most mitotically active area and start the count at that ﬁeld. In some tumours

– e.g. breast – you must count at the growing edge of the tumour (not the centre).

For counts per 10hpf there are two methods:

1. count 3 or 4 sets of 10 ﬁelds and give the highest count;

2. count 100 hpf and divide the count by 10.

For counts per 50hpf count the n˜o. of mitoses in 50 hpf.

An hpf usu. means a ×40 objective with a standard wide ﬁeld ×10 eyepiece. You should state the area

(e.g. in mm

) or diameter (in mm) of your hpf whenever giving a mitotic count in a report.

Mitotic count decreases with time from excision to ﬁxation (up to 50% fewer if >12 hours) so are usu.

higher in frozen sections cf. parafﬁn sections.

For Ki-67, use the most active area and either

◦

count the n˜oof+ve nuclei in 2000 tumour cells and

divide by 20 to givea%or

◦

give the n˜oof+ve tumour cell nuclei per 10 hpf.

Fixation

Formaldehyde

Covalently cross-links peptides to inhibit degradation and ‘ﬁx’ structure

A standard ﬁxative as 4 % aqueous saline solution (=10 % formalin) – cheap and widely available

Good membrane structural preservation for LM (and, to a lesser extent, EM)

Good Ag preservation when combined with Ag retrieval methods (heat, protease, washing, sonic)

Slow tissue penetration rate (≈500 /hour) with even slower optimal ﬁxation rate (≈80 /hour) because

it takes time for the cross-links to form even when formalin is present

Induces ﬂuorescence in biogenic amines (e.g. noradrenaline and melanin precursors) / alters the ﬂuo-

rescence properties of other native tissue structures

Some aqueous molecules / antigens (Ags) can diffuse out of the tissue

If not buffered, varying pH can cause varying artefacts, such as nuclear shrinkage and hyperchromasia

and variable cytoplasmic staining intensity

Can form birefringent formalin pigment in bloodied areas

Increases the weight of specimens (sometimes by almost 10%)

The volume of ﬁxative should be ≥ 10× the specimen volume for adequate ﬁxation

Alcoholic Fixatives

Disrupts hydrophobic bonds → denatures tertiary structure leaving 1

◦

and 2

◦

structure intact

Examples include: 70% ethanol, Carnoy’s (=ethanol, chloroform, acetic acid – excellent for ﬁxing

tissue inks onto specimens at cut-up), formol alcohol, etc.

Faster tissue penetration cf. formalin (.

. good for cytology / rapid process / FS post-ﬁxation)

Better preservation of large peptide Ags and less induced autoﬂuorescence (.

. good for some IF)

Better preservation of nucleic acids (with Carnoy’s)

Worse preservation of membrane structure .

. sometimes combined with acetone (which has better

membrane-preserving properties but worse Ag preservation) e.g. for cytology

Can show worse differential tissue shrinkage artefacts cf. Formalin

Bouin’s

An example of a combination ﬁxative, ingredients: picric acid, formaldehyde, acetic acid

Advantages:  stains tissue yellow .

. easy to ﬁnd LNs or embed small fragments (e.g. brain Bx)

 good nuclear detail (e.g. spermatocytic seminoma)

 small antigens are less soluble → enhanced sensitivity on immuno (e.g. AFP)

 good for ﬁxing India ink onto specimens prior to cutting

Disadvantages:  degrades RNA and DNA .

. not good for ISH / PCR / Feulgen cytometry

 causes undue differential shrinkage (e.g. glomeruli)

 picric acid is explosive when dry .

. can’t use as a ﬁxative in ordinary processors

 more expensive than formalin

 some Ags don’t survive the acid ﬁxation or they show altered staining patterns

(e.g. prostate lumenal cell +vity for the protein product of c-erbB-2 [=the proto-

oncogene of type 2 EGFR, also known as HER2, the rat equivalent being neu]).

JWBK208-02 December 8, 2007 21:25 Char Count= 0

Histotechniques 8

Additives to Standard Fixatives

E.g. mercurials (obsolete due to health and safety), aprotinin and zinc

These enhance peptide antigenicity by inhibiting natural tissue proteases thereby hindering autolysis

Effects of Fixation on Staining

Fat retention: osmium / dichromates > formalin / glutaraldehyde  acetone / OH

Protein retention: formalin / glutaraldehyde > osmium / dichromates > acetone / OH

Enzyme activity: acetone / OH > formalin / glutaraldehyde

Different methods alter the acidophilia:basophilia balance between tissue structures

Fe is leached out by acid ﬁxatives

Effects of resin embedding: tissue and Ag occlusion, dye retention (e.g. due to hydrophobicity)

Formalin reduces colour contrast of many trichrome methods – use Bouin’s ﬁxative (alternatively

pre-treat formalin-ﬁxed tissues with picric acid and trichloroethylene)

Hard Tissues

Decorniﬁcation

Softens tough keratinous tissues e.g. toe-nail

Phenol (outmoded due to health and safety)

Commercial alternatives available

Decalciﬁcation

Acids or Ca

chelating agents – often mixed with formalin for combined decalciﬁcation/ﬁxation

Strong acids (e.g. nitric) are rapid but give worse cytological and antigenic preservation

Weak acids (e.g. formic) are a good compromise between speed and Ag preservation

Chelators (e.g. EDTA) are the slowest but give best Ag / enzymic preservation

surface / superﬁcial decalciﬁcation: bathe the cut-surface of a wax tissue block in HCl

Assessing when Decalciﬁcation is Complete

Standard time – uniform specimens (e.g. trephine Bx) may be known to decalcify within a set time for

a given decalciﬁcation agent and can be standardised for any given lab

X-raying specimens: expensive and has additional health and safety requirements for operators

Chemical tests of residual Ca

in the decalciﬁcation ﬂuid: when changing the ﬂuid, add ammonia and

ammonium oxalate to the spent ﬂuid. If CaOH precipitates out (ﬂuid turns cloudy) the specimen needs

more decalciﬁcation → add fresh decalciﬁcation ﬂuid.

Undecalciﬁed Sections

Sections of bone can be cut with a diamond knife and ground down to histological thinness

This allows routine staining (usu. with von Kossa or Goldner’s method – p. 11) and assessment of

calciﬁcation front and osteoid seam thickness for assessing metabolic bone diseases e.g. osteomalacia

Staining (Principles)

Dye Nomenclature

Mordanted dyes are dyes complexed to metal ions. The metal ion forms a covalent bond with the tissue

thus cementing (mordanting) the dye in place e.g. iron haematoxylin (celestin blue) is used in HVG

because VG is an acidic counterstain and will complex with and remove ordinary haematoxylin

Acid dyes are those whose coloured species are anionic e.g. eosin

Basic dyes are those whose coloured species are cationic e.g. methylene blue or alum haematoxylin

(by means of its Al

mordant)

Neutral dyes: both the anion and cation are coloured e.g. the Romanowsky mixture (p. 10)

Immunohistochemical Methods

Labelling Technologies

1. Fluorophore: very sensitive and can be used for quantitation if used with a direct method but ﬂuorescence

fades with time so no good for permanent preparations