Basic pathology

1. TISSUE SPECIMEN COLLECTION,FIXATION
AND PROCESSING

2. Objectives
 By the end of this session you will be able to
1- Describe Tissue specimen collection techniques
2- Describe tissues specimen fixation process
3- Describe the methods of tissue specimen processing

3. Tissue specimen collection
Introduction
 Tissue specimen collection is a collection of
tissue, cells or body fluids
for laboratory investigations to aid
diagnosis and/or treatment of medical
conditions.

4. Types of Specimen collection
 There are 2 types of specimen collection
1- Tissue Biopsy specimen collection
2- Cytological specimen collection

5. Tissue biopsy collection
Biopsy
 A biopsy is a procedure that removes a sample of
cells, tissue or organ from the body to be examined
 Biopsy should be performed only after the initial
clinical evaluation is complete
 The biopsy should provide adequate material for
diagnosis

6. Types of biopsy
 There are 3 main types of biopsy.
1.Needle biopsy – in which only a core of tissue is
removed e.g Percutaneous liver or kidney
2.Excisional biopsy – in which the entire lesion and
a small cuff of surrounding normal tissue is removed
3.Incisional biopsy – in which the tumour is
partially excised and the wound carefully closed

7. Types of biopsies cont--
 The choice of biopsy is dictated primarily by the
SIZE and LOCATION of the mass.
 Excisional biopsy is ideal for small lesions
 Incisional biopsy is more suitable for large lesions
where total excision cannot be easily done and
where it would distort anatomic landmarks.

8. Needle biopsy cont--
 Although needle biopsy is “Theoretically”
acceptable for small or large lesions, it should be
restricted for the following reasons:-
 The specimen is less likely to be representative of
the lesion as a whole, and thus may lead to errors
in diagnosis and grading.
 It may not yield sufficient material for additional
stains or electron microscopy studies if necessary.

9. Needle Biopsy cont…
 The interpretation of needle biopsies is particularly
risky in those situations where the pathologist relies
more heavily on the pattern of the lesion than the
features of individual cells

10. Example of biopsy :
Lymph node Biopsy
The biopsy taken from a lymph node
INDICATIONS:
There are 5 main reasons for performing a lymph
node biopsy which are;
1. To make a diagnosis in a case of persistent,
unexplained lymph node enlargement.

11. Indication of lymphnode biopsy cont--
2- To make a diagnosis or assist in the investigation
of a patient who has unexplained symptoms such
as, (fever, loss of weight) accompanied by
lymphadenopathy.
3- To confirm a diagnosis already suspected on other
grounds, such as clinical history and physical
examination highly suggestive of a malignant
lymphoma or where the primary is known (Breast
cancer etc) –in order to plan management.

12. Indications for LN biopsy cont--
4-To assess the extent of spread of known malignant
disease (staging).
5- To monitor the progress of disease in patients with
malignant lymphoma.

13. TECHNIQUES FOR LYMPHNODE
BIOPSY:
 Remove intact node;
The node should be subjected to minimum trauma in
the process of removal
Trauma does the following;
- Tears the capsule
- Disrupts the architecture of the node and
- Distorts the lymphnode cells, producing the so -
called “stringly artefact” of nuclei

14. Techniques for LN biopsy cont--
 The choice of node for biopsy.
 If there is a single node, there is no choice, remove
it
 The choice is needed when there are multiple
lymphadenopathy.
- Inguinal node:- these nodes should be avoided
where possible in adults because they so often
show scarring or other evidence of past-
lymphadenitis which may complicate
interpretation.
- Axillary nodes:- these nodes not infrequently show
fat involution of there centres, for this reason
CERVICAL NODES are generally preferred.

15. Techniques for LN biopsy cont--
 The most accessible node is not always the best one
to remove
 The best node from the point of the pathologist is
the largest one available
 If there are multiple enlarged nodes, the removal of
several nodes may give more information than can
be obtained from a single node.

16. APPLICATION OF LYMPHNODE
BIOPSY:
A: FRESH TISSUE:
Bacteriological or virological studies.
Imprint preparation
Cell suspension
Cryostat sections (frozen sections):
Rapid diagnosis

17. FRESH TISSUE LN biopsy cont--
Evaluation of measurement of immunoglobulin
fractions
B: FIXED TISSUE:
Resin embedding
Electron microscopy
Paraffin embeding
Routine stains
Chloroacetate esterase
Immunoperoxidase staining methods

18. IMPRINT(TOUCH) PREPARATIONS
OF LN BIOPSY:
 Fresh lymphnode
 Cut lymphnode into halves
 Remove excess blood or fluid by soft absorbent paper
 Cut surface is slightly touched on to a clean, grease free-
glass slide
 Staining of imprint;
 Routine stain
 PAS( Periodic Acid Schiff Stain)- for glycogen granules
(positive stain abolished by prior diastase digestion)
- immunoglobulin in cells of plasma lineage
- mucin in identifying signet ring carcinoma

19. Imprint staining of LN cont--
Oil red – for cytoplasmic lipid (in Burkitt’s
lymphoma and other high grade lymphomas)
 Enzyme methods:-
Acid phosphatase – for histiocytes, T cells and
hiary cells
Acid esterase – for T lymphocytes
Chloroacetate esterase – for neutrophil
granulocytes and their precursors, mast cells

20. Principle of tissues Biopsy specimen
Collection
 Biopsy should be performed only after the initial
clinical evaluation is complete
 The biopsy should provide adequate material for
diagnosis
 The biopsy should be planned in such a way that
the entire biopsy tract is excised by the subsequent
excision if required.

21. Principle Cont……
 The biopsy incision should be kept small (1-2cm)
so that it can easily be incorporated in the
definitive excision
 On extremities it should be placed in a vertical
rather than horizontal direction
 Bleeding should be kept to an absolute minimum
because it represents a potential source of tumour
spread between tissue compartments
 Care should be extended to obtain viable tumour
rather than necrotic or reactive tissue.

22. Containers for tissue collection
 When tissues are collected they should be put in an
appropriate container.
 In cases of tissue biopsies the containers have the
following qualities:-
The mouth of the containers (bottle or plastics)
should be wide enough such that the tissue passes
without being squeezed or compressed.
The size of the bottle should be wide enough to
accommodate the specimen

23. Containers for tissue collection cont--

The container should contain an appropriate
fixative and the volume of a fixative should be 10-
20 times that of a specimen.

The container should be labeled properly to
include name, type of tissue, date of taking
specimen.

The container should be accompanied by a request
form which bears the relevant clinical data, brief
relevant clinical history and examination findings
and provisional diagnosis or previously confirmed
diagnosis.

24. CYTOLOGICAL Tissue Specimen
Collection
Exfoliative cytology
Refers to the examination of cells that are shed
spontaneously into fluid or secretions
Examples:-
- sputum, CSF, urine, vaginal secretions
- Effusions in the body cavities (pleura, pericardium,
peritoneum
- Nipple discharge, vitreous humors

25. Exfoliative cytology cont--
 The number of cells in such specimens is usually
not sufficient to enable an adequate assessment by
direct smearing of the fluid on glass slide
In cytology laboratories, the use of various cell
concentration techniques, such as centrifugation or
membrane filtration, to prepare smears is used.
Subsequently the smears are stained by one or
more of the several methods, the most popular of
which is the Papanicolaou (Pap) stain

26. Cytological methods cont--

Abrasive cytology:

This encompasses methods by which cells are
disloged by various tools from body surfaces (skin,
mucous membranes, serous membranes)

Examples:-
-
Cervical smear (Pap), using a spatula or smal brush
and immediately fixed in 50:50 Ethylalcohol

27. Examples of abrasive cytology cont--
-
Endoscopic brushings of the mucosal surfaces of the
Gastrointestinal, Respiratory and urinary tracts.
-
Balloon technique for obtaining cells from the oesophagus
-
The scraping of cutaneous, oral (buccal smear- for sex
determination-Barr bodies), vaginal, or conjunctival lesions
to detect herpesvirus inclusions
-
Washings (lavage) of mucosal or serosal surfaces during
endoscopy or open surgery may be considered a combined
exfoliative and abrasive method, since it samples both cells
that are shed spontaneously and those disloged
mechanically

28. Abrasive cytology cont--

Cells obtained by scraping or brushing are directly
smeared on glass slides and fixed immediately in
95% ethanol or with a spray fixative before staining.

Lavage specimens are sent to the laboratory for cell
concentration, as described in exfoliative cytology
above.

29. FINE NEEDLE ASPIRATION
CYTOLOGY (FNAC).

Aspiration cytology is a technique that uses cells
obtained by aspiration under negative pressure
through a thin gauge needle

Virtually any organ or tissue can be sampled by
fine needle aspiration:-

Superficial organs e.g. thyroid, breast, lymph node,
prostate, skin, soft tissues

Deep organs, such as the lung, mediastinum, liver,
pancreas, kidney, adrenal gland, and
retroperitoneum, are aspirated with guidance by
fluoroscopy, computed tomography,or ultrasound

30. ADVANTAGES OF CYTOPATHOLOGY

Less trauma:-

There are fewer complications, such as
haemorrhage or perforation

A large sampling surface:-

A large sampling surface is available for cytologic
methods
e.g. this may be particularly important in endoscopic
procedure and in the assessment of intraperitoneal
spread of cancer cells during laparotomy

31. Advantages of cytopathology cont--

Tumour that are difficult to access by biopsy may be
sampled by cytologic methods.
e.g – CSF to diagnose meningeal carcinomatosis
- brushing or washing of gasrtointestinal tract
stricture that doe not permit passage of the biopsy
instrument

32. Advantages of cytopathology cont--

A rapid diagnosis:-

This is one of the major advantages of cytologic
methods

Direct smears and fine needle aspirates can be read
within a few minutes of collection

Greatest convenience:-

In most instances, no prior preparation of the
patient is necessary, and the sampling is done as
an office procedure.

33. Advantages of cytopatholgy

An increased detection rate of malignancy:
e.g. in endoscopic procedures is achieved by
combining cytologic sampling (washingor brushing)
with biopsy

A greater cost effectiveness:-
- there is a low cost when using cytology as a method
of cancer detection compared to tissue biopsy
method

34. LIMITATIONS OF CYTOPATHOLOGY:

Classification of the type of tumour

Classification is generally more difficult with
cytologic samples than with the biopsy specimens
because of;
- the small size of cytologic samples
- the loss of tissue pattern

Cytologic interpretation relies heavily on
morphologic alterations of individual cells

35. Limitations of cytopatholgy
 The small size of the specimen:-
May preclude accurate classification of some
neoplasms with mixed elements such as;
- adenosquamous carcinoma
- carcinosarcoma
- synovial sarcoma, if only one component of the
tumour is sampled

36. Limitations of cytopathology cont--
 The extent and depth of invasion:-
Cannot be assessed by cytologic examination ( e.g.
in-situ vs invasive carcinoma)

37. TISSUE FIXATION AND PROCESSING

38. Tissue Specimen Fixation
Tissue fixation
is a process to preserve tissues permanently as
life-like state as possible.

39. When to do
Fixation should be carried out as soon as possible after
removal of the tissues (in the case of surgical
pathology) or soon after death (with autopsy) to
prevent autolysis.

40. Why fixation
 Cells and tissue begin to undergo two major changes
shortly after death or removal from the body,
1. Putrefaction is due to invasion of cells or tissue by
bacteria/fungal which generally disseminate from
alimentary tract and spread quickly into surrounding
organs.
2.Autolysis is due to the action of enzymes from the dead
cell. This phenomenon occurs chiefly in the central nervous
system and the endocrine system.

41.  These result into breakdown and ultimate
destruction.
 These changes may be retarded by low temperatures
or prevented by the use of chemical fixatives.
 THE ULTIMATE AIM IS PREPERATION OF A
GOOD THIN STAINED SPECIMEN

42. Fixatives
 Definitions
A fixative is substance that will preserve the shape,
structure, relationship and chemical constituents of
tissues and cells after death

43.  A variety of fixatives are available for use, depending
on the type of tissue present and features to be
demonstrated
 There are two types of classification;
 1- According to mechanism of action
 2- According to number of substances composing it

44. Classification according to mechanism of
action
1. Aldehydes – Formaldehyde (formalin),
Glutaraldehyde
2. Mercurials – Zenker, B5
3. Alcohols – Carnoy’s, Ethanol, Methanol
4. Oxidizing agents – Osmium tetra oxide,
Potassium permanganate,
Potassium dichromate
5. Picrates – Bowin’s

45. Formaldehydes Glutaraldehyde
 Formalin is used for all routine
surgical pathology and autopsy
tissue.
 Tissue is fixed by cross-linkages
formed in the proteins, particularly
between lysine residues.
 Formaldehyde is good for
immunoperoxidase techniques.
 Formalin penetrates tissue well, but
is relatively slow.
 The standard solution is 10% neutral
buffered formalin.
 A buffer prevents acidity that would
promote autolysis and cause
precipitation of formol-heme
pigment in the tissues
 Causes deformation of
alpha-helix structure in
proteins so is not good for
immunoperoxidase staining.
 It fixes very quick so is good
for electron microscopy.
 It penetrates very poorly,
but gives best overall
cytoplasmic and nuclear
detail.
 The standard solution is a
2% buffered glutaraldehyde
Properties of fixatives and mechanisms of action

46. Mercurials Alcohols

 Fix tissue by an unknown
mechanism.
 They contain mercuric chloride
and include such well-known
fixatives as B-5 and Zenker's.
 These fixatives penetrate
relatively poorly and cause some
tissue hardness, but are fast and
give excellent nuclear detail.
 Their best application is for
fixation of hematopoietic and
reticuloendothelial tissues.
 Since they contain mercury, they
must be disposed of carefully.
 Including methyl alcohol
(methanol) and ethyl
alcohol (ethanol), are
protein denaturants and are
not used routinely for
tissues because they cause
too much brittleness and
hardness.
 However, they are very good
for cytologic smears because
they act quickly and give
good nuclear detail.
……cont..

47. …..cont..
Oxidizing agents Picrates
 Include permanganate fixatives
(potassium permanganate),
dichromate fixatives
(potassium dichromate), and
osmium tetroxide.
 They cross-link proteins, but
cause extensive denaturation.
 Some of them have specialized
applications, but are used very
infrequently.
 Include fixatives with picric
acid.
 Foremost among these is
Bouin's solution.
 It has an unknown mechanism
of action.
 It does almost as well as
mercurials with nuclear detail
but does not cause as much
hardness.
 It stains everything it touches
yellow, including skin.

48. Mechanisms of Fixation
 1- Denaturation : Alcohols (Methanol)
 2- Addition and cross link formation. Formaldehydes
& Oxidixing agents
 3- Unknown Mechanism: Mercurial and Picric acids

49. Classification according to number of
substances making it
1. Simple fixative: It is an individual fixative
composed only by one substance.
2. Compound fixative: It is the solution resulting
from the mixing of two or more fixatives, in
order to obtain the combine effect of their
individual action upon the cell and tissue
constituents.
e.g. Susa fluid, Zenker’s solution, Bowin’s

50. Methods of Fixation
 Immersion fixation: Tissue added to fixative
(Specimen)
 Infusion fixation: Intraluminal viceral injection of
fixative
 Perfusion fixation: Intravascular injection of
fixative (perfusion of the vascular system) (whole
organ )
 Vapour Fixation (paraformaldehyde and osmium
tetroxide can be used to vapour-fix freeze-dried
tissues)

51. Factors affecting fixation
1- Buffering
Fixation is best carried out close to neutral pH,
in the range of 6-8. Hypoxia of tissues lowers
the pH, so there must be buffering capacity in
the fixative to prevent excessive acidity. Acidity
favors formation of formalin-heme pigment
that appears as black, polarizable deposits in
tissue. Common buffers include phosphate,
bicarbonate, cacodylate, and veronal.
Commercial formalin is buffered with
phosphate at a pH of 7.

52. 2-Penetration
Penetration of tissues depends upon the
diffusability of each individual fixative, which is
a constant. Formalin and alcohol penetrate the
best, and glutaraldehyde the worst. Mercurials
and others are somewhere in between. One way
to get around this problem is sectioning the
tissues thinly (2 to 3 mm). Penetration into a
thin section will occur more rapidly than for a
thick section.

53. 3-Volume
The volume of fixative is important. There
should be a 10:1 ratio of fixative to tissue.
Obviously, we often get away with less than
this, but may not get ideal fixation. One way to
partially solve the problem is to change the
fixative at intervals to avoid exhaustion of the
fixative. Agitation of the specimen in the
fixative will also enhance fixation.

54. 4-Temperature
Increasing the temperature, as with all
chemical reactions, will increase the speed of
fixation, as long as you don't cook the tissue.
Hot formalin will fix tissues faster, and this is
often the first step on an automated tissue
processor.

55. 4- Concentration
Concentration of fixative should be adjusted down to the lowest
level possible, because you will expend less money for the
fixative. Formalin is best at 10%; glutaraldehyde is generally
made up at 0.25% to 4%. Too high a concentration may
adversely affect the tissues and produce artefact similar to
excessive heat.

56. 6-Time interval
The faster you can get the tissue and fix it, the
better. Artefact will be introduced by drying, so
if tissue is left out, please keep it moist with
saline. The longer you wait, the more cellular
organelles will be lost and the more nuclear
shrinkage and artefactual clumping will occur.

57. Secondary fixation
It is the process in which we use two fixatives in
successions.
Commonly after fixation of tissue with
formaldehyde they undergoing to secondary
fixation with mercuric chloride/formaldehyde
solution for a period of hours
Advantages: Sections are more easy to cut and
tissues stain more brilliantly
Disadvantages: It is extracostive and toxic

58. TISSUE PROCESSING
 Processing is usually considered to include:-
Dehydration
Clearing
Infiltration (Embedding)
NB:
 The choice of both the fixative and processing
method should be made either before or immediately
after removal of tissue at surgery or autopsy.

59. ….Cont..
 The choice of processing methodology may influence
the choice of fixative (e.g. frozen section or routine
studies)
 The aim of tissue processing is to embed the tissue in
a solid medium to support the tissue and give
sufficient rigidity to enable thin sections to be cut,
and yet soft enough to enable the knife to cut the
section with little damage to the knife or tissue.

60. …..cont..
: DEHYDRATION:-
The 1st stage in the processing of fixed tissue involves
the REMOVAL OF WATER by a variety of
compounds, many of which are alcohols of
varying strengths and types
-several compounds (alcohols) are hydrophilic and
attract water from the tissue, whereas others affect
dehydration repeated dilution of the tissue fluid.
The dehydration process is frequently began in series
of increasing strength, beginning by immersing the
tissue in for example; 60%, 70%, 95%, absolute
alcohol (almost 100%)

61. …..cont..
II: CLEARING:-

The term relates to the appearance of the tissue after they
have been treated by the fluid chosen to remove
dehydrating agents.

Many of these fluids have similar refractive index to that
of protein, consequently rendering the tissue transparent
(dealcoholization)

The use of clearing agents is necessary when the
dehydrating agent (e.g. alcohol) is NOT MISCIBLE with
IMPREGNATING MEDIUM (e.g paraffin wax)

Thus the essential requirement of a clearing agent is that
it is miscible with dehydrating and embedding agents.

62. …..cont..
Examples of clearing agents include;
- Xylene
- Toluene
- Chloroform
- Benzene
- Cedar wood oil etc.

63. ….cont..
III: INFILTRATION (EMBEDDING):
 After dehydration and clearing, tissue must be
infiltrated with supporting medium.
 The medium is generally referred as embedding
medium which will hold the cells and intercellular
structures relationship while tissue sections are cut

64. ….cont..
 Example of embedding media are; paraffin wax
(most popular), Carbowax, celloidin etc.
 Embedding is also referred to as CASTING or
BLOCKING, which involves enclosing the tissue in
the infiltration medium used for processing and
allowing the medium to solidify.

65. Clinical Application
 Tissue fixation is used to preserve cells and tissue
components to allow for the preparation of thin,
stained sections.
 Rule out benign and malignant lesions
 Diagnosis and treatment of tumours

66. References
 Robbins and Contrans-Pathological Basis Diseases
 Drury RAB, Wallington EA. Carleton's histological technique. 5th ed. New
York: Churchill Livingstone, 1980.
 Carson, Freida L; Christa Hladik (2009). Histotechnology: A Self-
Instructional Text (3 ed.). Hong Kong: American Society for Clinical
Pathology Press. p. 2. ISBN 978-0-89189-581-7.
 Eltoum I, Fredenburgh J, Myers RB, Grizzle WE. Introduction to the theory
and practice of fixation of tissues. J Histotechnol 2001;24;173 -190.
 Winsor L. Tissue processing. In Woods A and Ellis R eds. Laboratory
histopathology. New York: Churchill Livingstone, 1994;4.2-1 - 4.2-39.
 Williams JH, Mepham BL, Wright DH. Tissue preparation for
immunocytochemistry. J Clin Pathol 1997;50;422-428.

67.  Leong AS-Y. Fixation and fixatives. In Woods AE and Ellis RC eds. Laboratory
histopathology. New York: Churchill Livingstone, 1994;4.1-1 - 4.1-26.
 Hopwood D. Fixation and fixatives. In Bancroft J and Stevens A eds. Theory
and practice of histological techniques. New York: Churchill Livingstone, 1996.
 Carson FL. Histotechnology. 2nd ed. Chicago: ASCP Press, 1997.
 Pearse AGE. Histochemistry, theoretical and applied. London: Churchill
Livingstone, 1980

 Clayden EC. Practical section cutting and staining. Edinburgh: Churchill
Livingstone, 1971.
 Hopwood D. Fixation and fixatives. In Bancroft J and Stevens A eds. Theory
and practice of histological techniques. New York: Churchill Livingstone, 1996.
 Carson FL. Histotechnology. 2nd ed. Chicago: ASCP Press, 2007.