RECEIVING OF

SPECIMENS AND
FIXATIVES

- Dr ARUNDHATHI J PANICKER
RECEIVING OF SURGICAL
SPECIMENS
INTRODUCTION
-In a laboratory setting, numerous histological specimens are received
throughout the day for testing. It is important to maintain a systematic
approach to ensure that all samples are accounted for and are being
received and tested appropriately. Without it, there is potential to
misplace or lose samples.
Checklist

• Name of the patient

• Sex and age of patient
• Registration no,opd or indoor no
• Type of sample like appendix or lymph node
-after matching the above points between requisition and form and
label on the sample container, accession no of the histopath should be
given on the requisition form and on the sample container.
Register for reference
• Date
• Accession number
• Patient’s name age sex
• Patients registration no /opd/indoor no
• Type of sample
• Number of samples received from one patient
• Remarks/final diagnosis
Preparation of gross room

• Size of grossing room

• Illumination and ventilation
• Gross station
• Racks to keep the specimens
• Cutting board
• Ready to access to sink with hot and cold water
• Formalin-stock and 10% buffered formalin
• Box of instruments-scissors,forceps,malleable probe,scalpel with blades,long
knife,scale,pins to attach specimens
• Containers with different fixatives
• Bone cutter
• Large disposable bin
• Box with casettes and labels
• Photographic facility
• Refrigerator
• Balance to weigh the gross specimen
• Xray view box
• Other equipment for tissue bank facility
 FIXATIVES
DEFINITION
Substance that preserve the morphological and chemical
characteristics of cells and tissue in life like state
Prevent autolysis and putrefaction changes.
Fixation is the preservation of biological tissues from decay due to
autolysis or putrefaction.
The basic aims of fixation are the
following:
• To preserve the tissue nearest to its living state.
• To prevent change in shape and size of tissue.
• To prevent autolysis
• To make the tissue firm to hard
• To prevent any bacterial growth in the tissue
• To make it possible to have clear stain
• To have better optical quality of the cells
Principle of fixation
• Fixation result in denaturation and coagulation of protein in the
tissues. The fixative have a property of forming cross link between
proteins, thereby forming a gel, keeping everything in their in vivo
relation to each other.
Ideal fixative
• Prevention of autolysis of the cells and tissue
• Prevention of decomposition of the tissue by bacteria
• Maintaining the volume and shape of the cell as far as possible
• Consistently high quality staining particularly routine stain such as
haematoxylin and eosin stain and Papanicolaou’s stain
• Rapid action
• Cheap
• Non toxic
Tissue changes in fixation
• Volume change
• Hardening of tissue
• Interference of staining
• Changes the optical density by fixation
Types of fixation
• CLASSIFICATION
• Nature of fixation
• Chemical properties
• Component present
• Action of tissue protein
Simple fixatives

• Formaldehyde:Commercially available solution contains 35-45% gas

by weight, called as formalin. Formaldehyde is commonly used as 4%
solution, giving 10% formalin for tissue fixation.
• Formalin is most commonly used fixative.It is cheap, penetrates
rapidly and does not over –harden the tissues.
• It converts free amine group to methylene derivatives.
• Artefacts as brown pigment in the tissues.To avoid this buffered
formalin is used.
• Absolute alcohol-it may be used as a fixative as it coagulates protein.
-Due to its dehydrating property it removes water too fast from the
tissues and produces shrinkage of cells and distortion of morphology.it
penetrates slowly and over harden the tissues
• Acetone-Used in the study of enzymes especially phosphatases and
lipases.
• Mercuric chloride-Protein precipitant –shrinkage of tissues.It gives
brown color to the tissues, which is removed by treatment with iodine
• Potassium dichromate-It has a binding effect on protein similar to formalin
• Osmic acid-It is used for fixation of fatty tissues and nerves.
• Chromic acid-It precipitates all proteins and preserve the carbohydrates
• Osmium tetroxide-It gives excellent preservation of cellular details hence
used for electron microscopy
• Picric acid-It precipitates proteins and combine with them to form
picrates. Owing to its explosive nature when dry. Kept under a layer of
water. Thorough washing with water to remove color.
• Potassium dichromate, chromic acid, picric acid- tissues fixed with these
fixatives require through washing before dehydration.
Compound fixatives

• Formal saline
• Formal calcium
• Zenker’s fluid
• Helly’s fluid
• Bouins fluid
Cytological fixatives

NUCLEAR FIXATIVES CYTOPLASMIC FIXATIVES

• Carnoy’s fluid • Champy’s fluid
• Clarke’s fluid • Zenker’s fluid
• Flemings’s fluid • Reagaud’s fluid
Precautions for fixation

• Tissue should be free from excessive blood

• Tissue should be thinly cut in 3-5mm thickness
• Amount of fixative should be 20 times more than the volume of the
tissue
• Tissue with fixative should be in a tightly screw capped bottle
Mechanism of fixation

• Formaldehyde
Formaldehyde in aqueous solution combines with water to form
methylene hydrate, a methylene glycol
Formaldehyde reacts with side chain of protein-hydroxymethyl side
group
Cross linking and methylene bridges formed
• Glutaraldehyde
Aldehyde group react with amino acid and may also cross link
Cross linking is rapid and irreversible
• Methanol and ethanol
Alcohol remove water –strong dehydrating agents-destabilize the
hydrogen bonds and will disrupt the tertiary structure of protein
• Osmium tetroxide
React with 2 unsaturated carbon atoms of lipid and cross link with them
Factors affecting fixation

• Ph of fixative
• Temperature
• Osmolarity of the fixative solution
• Duration of fixation
• Concentration
• Agitation
Secondary fixation

• Aka POSTMORDANTING
• Tissue is fixed by sequential application of 2 fixatives
Used-when primary fixative is not effective
Tissue can withstand action of second harsher fixative
Adv-Tissue can be easily cut and gives better quality of staining
Artefacts

• Formalin pigment-insoluble brownish black pigment-hb

• Use buffered formalin to avoid it
• Methods to remove formalin pigments
-Picric acid method
-Kardasewitsch’s method
-Lille’s method
-Verocay’s method
-Schridde’s method
• MERCURY PIGMENT
Dark brown irregular deposit
To remove-application of iodine converts it into mercuric iodide which is
removed by sodium thiosulphate

• FUZZY STAINING
Due to improper fixation;insuffiecient fixative or too little time in fixative
Nuclear and cytoplasmic details are obscured and the section looks hazy
• PROLONGED FIXATION : shrinkage and empty spaces
• DICHROMATE DEPOSIT
After dichromate fixation ,if not properly washed –chromium salt
Salt + alcohol=yellow brown precipitate
STREAMING ARTIFACT
Precipitation and displacement of glycogen
ZONAL FIXATION
Different zones different fixation-sp too large, poor penetration rate of
reagent, insufficient time in fixative