GENERAL VETERINARY PATHOLOGY

GENERAL VETERINARY PATHOLOGY

EXPERIMENT NO. 1

POST-MORTEM TECHNIQUES/ EXAMINATION

            Necropsy or Post – mortem examination means systemic examination of animal after death which is conducted by Veterinarian or Veterinary Pathologists to ascertain the cause and nature of disease in fatal cases of disease. The term autopsy is preferred in human medicine for PM examination and necropsy in Veterinary Medicine.

Autopsy means seeing with ones own eyes.

Necropsy means seeing a corpse.

Autopsist  is one who conducts the PM examination.

Objectives of the P.M. Examination

1) To know the cause of death of animals.

2) It is done in the veterolegal/ insurance aspects.

3) To study the epidemiology status of various diseases in the particular area/region.

4) To know change in the disease scenario of a particular disease in an area.

TYPES OF NECROPSY

a. Where no necropsy is conducted.

            If the blood smear from ear vein (cattle, sheep and goat ) or smear from edematous fluid from throat or abdominal region ( pigs, horse) reveals anthrax bacilli no PM should be conducted on the carcass since the organisms are aerobic spore formers. The spores survive as long as 18 years.

S. No.              Particulars                 Anthrax bacilli                            Anthracoids

1.                     Organism                     Bacillus anthracis                          Other than B anthracis

2.                     Capsule                       Predominantly pink stain             Less predominant

3.                     Spores                         Absent                                           Present

4.                     Length of chain           Short-usually 2 to 3 organism       Long chains

5.                     End of bacilli              Truncated                                      Rounded

b. Partial necropsy

            In case of rabies only the brain of the carcass is examined for diagnosis. Here only a part of the body ( head ) is opened for the purpose. Other parts of the body are not opened.

c. Complete necropsy

All parts of the body are thoroughly examined o arrive at a etiological diagnosis.

d. Cosmetic necropsy

            Examination of the carcass is done with very less mutilation. Cuttings and incisions are sewed together and the body is washed to appear as nearly intact as possible. It is done in case of pet and wild animals.

PRECAUTIONS

1.         Obtain permission of the owner in writing before PME.

2.         Request from local police is must in Vetro-legal cases.

3.         Conduct PM as early as possible to avoid putrefaction.

4.         Examine the smear from peripheral blood to rule out anthrax. Besides anthrax bacilli examination of blood smear may reveal blood parasites, other  bacteria and or PM invaders.

5.         PME should be done in day time to appreciate the accurate changes in the colour of the tissues. This is not possible with artificial light.   

6.         Conduct PM far away from animal houses and preferably in a Government land to avoid litigation.

7.         Obtain history, symptoms and treatment done etc.,

8.         Wear gloves, masks, apron and gum boots to avoid contact with Zoonotic agents.

9.         Record the PM findings immediately.

10.       Burry the carcass in deep ditches layered with lime. Carcasses can be burnt to ashes if incinerator is available.

INSTRUMENTS REQUIRED

            Small and large scissors with either pointed or rounded ends, chisel and hammer, curved scissors, small and large knives, scalpel and blades, bone cutters and saw ( small and large), small and large forceps, toothed forceps, hand lens, rubber or latex hand gloves, masks, Bunsen burner or spirit lamp or stoves, spatula, syringe and needles                                                                    (Tuberculin syringe), sterilizer, autoclave, spirit or alcohol, cotton and cotton swabs sterilized, sterilized vials, Petri dishes and test tubes, Pasteur pipettes and rubber bulbs, tissue fixatives 10 % formalin, formal saline, buffered neutral formalin, small or large stainless steel trays, monocular / binocular microscope.

            Clean glass slides and  cover slips, normal saline and glycerine- saline, different staining solutions, Ziehl Neelsen, Giemsa and Wrights and Leishman stain, match boxes, rubber apron, disinfectants including dettol, savlon, phenyl, iodophore etc, fly repellents, bowl for cold water, water container, small screw – top, water0tight jars, blotting paper, staining rack, sticker labels, marking pen, glass marking pencils, slide tray, monopan balance, physical balance / Jeweler’s balance, kitchen balance max 5 kg with weight, measuring tape cm / inches, aluminum foil, adhesive tapes, towel soaps, measuring cylinder, centrifuge, record, ice box / flask, transport media for bacteria, transport media for virus, antibiotics solutions ( penicillin, streptomycin, gentamycin and mycostatin), polypropylene bags, mask0disposable, thread, gum boots, camera with film, postmortem stainless steel top table, rexin /rubber sheets, vials with anticoagulants.

STEPS IN NECROPSY

1.         Record the kind of animal and to whom it belongs.

2.         Write the precise of the case.

3.         Carry the external examination of the carcass. Then proceed to internal examination.

4.         Secure the carcass on its back.

5.         Make incision in the midventral line from chin to anus going round about the external genatalia in male and the incision is also make to medial aspect of all legs and flay the skin.

6.         Examine the subcutaneous tissue.

7.         Open the cavities of the body. Look for exudates or transudes etc.

8.         Examine the position of the organs.

9.         Separate lungs from heart and palpate for any abnormalities. Incise and examine the lungs.

10.       Examine the pericardial sac. Open the pericardium, examine the nature of contents.

11.       Cut through heart and the vessels. Examine the wall and chambers for the nature of contents, valves and the lumen of vessels.

12.       Examine the diaphragm.

13.       Examine the abdominal visceral organs – liver, spleen, kidneys, adrenals, pancreas before and after incising the organs.

14.       Open the bile ducts and gall bladder and examine them.

15.       Divide the kidney symmetrically by longitudinal incision. Remove the capsule, examine the cortex, medulla and pelvis.

16.       Open the mouth to examine the gum, tooth, tongue and buccal cavity. Then open and examine oesophagus.

17.       Open the nasal cavity and examine. Examine the pharynx, larynx, trachea and bronchi.

18.       Open the stomach / fore stomach and abomasums ( ruminants) and examine the nature of contents and the wall.

19.       Open the intestine. Examine the contents and the wall.

20.       Open and examine the urinary bladder for the nature of contents and the wall.

21.       Examine the generative organs.

22.       Open the skull and vertebral column to examine the brain and spinal cord.

23.       Examine the skeleton and musculature.

24.       Record the findings.

25.       Summarize the appearance found.

26.       Collect suitable material for microbiological, histopathological, parasitological and chemical examination as required.

27.       Arrive at an etiological diagnosis based on the PM finding and the results of the material examined.

Post –Mortem examination of vetero-legal cases

The post-mortem examination of veterolegal cases is performed as described above. However, following points must be kept in mind while doing post-mortem examination and preparing the report.

1.         For veterolegal cases, post-mortem should be done by written signed request letter from police officer not below the rank of Inspector or Executive Magistrate.

2.         The P.M. examination of wild animals should be conducted as a special case. One should conduct the P.M. examination only when the DFO or higher officer is making request for post-mortem examination. It should be noted that all the viscera including skin, bones, teeth etc., are returned to the person who requested for the necropsy and no item should be left behind.

3.         Always collect maximum information on history, date & time of death of animal & treatment given. Determine the time of death on the basis of rigor mortis, autolysis, putrefaction, pseudomelanosis etc.,

4.         Animal identification including species, breed, age and number or mark must be clearly noted before conduct of P.M. It is especially necessary in insured animal as well as in religiously disputed cases.

5.         All the lesions present on the skin surface should be clearly defined as laceration, wound,, trauma, incision, erosion, vesicle, ulcer and if there is suspected sharp wound or bullet injury. Also state its depth and width as the case may be.

6.         If the case is suspected for toxic condition/poisoning, try to mention the type of poison in your report. This will help the police authorities to establish / confirm the type of toxin/poison in forensic laboratory.

7.         In case of dispute over calf born dead and calf born alive, a piece of lung should be placed in water. The lung piece will sink in water in case of atelectasis neonatum, while it will float if the calf was born alive.

8.         P.M. examinations should be conducted in day light only.

9.         At the time of P.M. examination, outsiders should not be allowed.

10.       There should be no unnecessary delay in doing P. M. examination.

11.       The P.M. examination should be done thoroughly and complete and it should be done in presence of police authority only.

12.       Fill the P.M. report clearly in neat handwriting and in clear language. Avoid writing general sentences. Be specific in your findings and conclusions. Sign the report with date and always keep a copy of report with you for record and future evidences in the court of law.

13.       All the details observed by the Veterinarian should be carefully and correctly noted in P.M. report.

P.M. ARTIFACTS

              P.M. artifacts are the changes introduced in the dead animals after death and are not related with natural physiological state of the body or tissue  in health or disease to which body might have been subjects.

             

P.M. artifacts may be classified as

1.           Putrefaction

2.           Environmental artifacts

              - P.M. Burning

              - P.M. Corrosion

              - P.M. Maceration

3.           Third party artifacts

              - Animals, birds, insects, maggots etc.,

              - Emergency treatment just prior & after death

              - Deliberate mutilation of dead body.

4.           Miscellaneous artifacts

              Mutilation of dead animals is a criminal offence. Criminal mutilation may be attempted to destroy the mark of identification. The following points must be kept in mind.

a) First of all ascertain the age, species, sex of animal, if not possible to ascertain, then report it accordingly.

b) Try to find out marks of identification, probable time of death and ascertain the cause of death.

c) If the body is in the form of fragments, then ascertain that fragments belongs to same animal.

d) Carry out the following tests if the facilities are available.

              i) Microscopic examination of blood and blood grouping

              ii) Microscopic examination of hair

              iii) Precipitation test for species identification.

EXPERIMENT NO. 2

POST-MORTEM EXMINATION REPORT -A

NOTE OF POST-MORTEM EXAMINATION OF………………....…………………

BELONGING TO ……………………………………………………………………….....

1.         CLASS OF ANIMAL:          

SEX / BREED:

            DESCRIPTIVE MARKS:

2.         DATE & TIME OF DEATH 

3.         DATE & TIME OF CONDUCTING P.M

4.         HISTORY:

5.         CONDITION OF BODY:

            (Rigor mortis, V. m.m., orifices, wound if any , any other abnoemality)

6.         MOUTH & ORAL CAVITY

7.         THORACIC CAVITY

8.         HEART

9.         LARYNX & TRACHEA

10.       LUNGS

11.       ABDOMIANL CAVITY & DIAPHRAGM

12.       LIVER

13.       SPLEEN

14.       INTESTINE

15.       KIDNEYS, URETERS & URINARY BLADDER

16.       REPRODUCTIVE SYSTEM

17.       ENDOCRINE GLANDS

18.       MUSCULATURE

19.       SKELETON

20.       BRAIN, SPINAL CORD & MENINGES

21.       LABORATORY EXAM

            ORGANS FOR HISTOPATHOLOGY

22.       DIAGNOSIS & REMARKS

                                                                                                                                                           

                                                                  

EXPERIMENT NO. 3

POST-MORTEM EXMINATION REPORT -B

NOTE OF POST-MORTEM EXAMINATION OF………………....……………………..

BELONGING TO ……………………………………………………………………….....

1.         CLASS OF ANIMAL:          

SEX / BREED:

            DESCRIPTIVE MARKS:

2.         DATE & TIME OF DEATH 

3.         DATE & TIME OF CONDUCTING P.M

4.         HISTORY:

5.         CONDITION OF BODY:

            (Rigor mortis, V. m.m., orifices, wound if any , any other abnoemality)

6.         MOUTH & ORAL CAVITY

7.         THORACIC CAVITY

8.         HEART

9.         LARYNX & TRACHEA

10.       LUNGS

11.       ABDOMIANL CAVITY & DIAPHRAGM

12.       LIVER

13.       SPLEEN

14.       INTESTINE

15.       KIDNEYS, URETERS & URINARY BLADDER

16.       REPRODUCTIVE SYSTEM

17.       ENDOCRINE GLANDS

18.       MUSCULATURE

19.       SKELETON

20.       BRAIN, SPINAL CORD & MENINGES

21.       LABORATORY EXAM

            ORGANS FOR HISTOPATHOLOGY

22.       DIAGNOSIS & REMARKS

                                                                                   

                                                                                                                                                            

EXPERIMENT NO. 4

POST-MORTEM EXMINATION REPORT -C

NOTE OF POST-MORTEM EXAMINATION OF………………....……………………..

BELONGING TO ……………………………………………………………………….....

1.         CLASS OF ANIMAL:          

SEX / BREED:

            DESCRIPTIVE MARKS:

2.         DATE & TIME OF DEATH 

3.         DATE & TIME OF CONDUCTING P.M

4.         HISTORY:

5.         CONDITION OF BODY:

            (Rigor mortis, V. m.m., orifices, wound if any , any other abnoemality)

6.         MOUTH & ORAL CAVITY

7.         THORACIC CAVITY

8.         HEART

9.         LARYNX & TRACHEA

10.       LUNGS

11.       ABDOMIANL CAVITY & DIAPHRAGM

12.       LIVER

13.       SPLEEN

14.       INTESTINE

15.       KIDNEYS, URETERS & URINARY BLADDER

16.       REPRODUCTIVE SYSTEM

17.       ENDOCRINE GLANDS

18.       MUSCULATURE

19.       SKELETON

20.       BRAIN, SPINAL CORD & MENINGES

21.       LABORATORY EXAM

            ORGANS FOR HISTOPATHOLOGY

22.       DIAGNOSIS & REMARKS

                                                                                             

           

EXPERIMENT NO.  5

STUDY OF GROSS PATHOLOGICAL SPECIMENS & RECOGNITION OF PATHOLOGICAL LESIONS

CARDIOVASCULAR  SYSTEM

                                   

1. Hypertrophy – Heart

2. Traumatic pericarditis                    

           

3. Suppurative pericarditis

4. Fibrinous pericarditis                     

5. Haemopericardium

6. Atrophy of  Heart

7. Haemorrhages  - Endocardium                              

8. Hydropericardium

DIGESTIVE SYSTEM

1. Abscess – Liver

2. Focal necrosis – liver

3. Ulcer- stomach                                           

4. Traumatic reticulitis

5. Tongue ulcer

 6. Abomasal ulcer

7. Johne’s Disease – Intestine (Chronic Enteritis)

8. Verminous intestine

9. Diphtheric enteritis                        

10. Infarction of liver

11. Amyloid infiltration liver

RESPIRATORY SYSTEM

1. Suppurative pneumonia                             

2. Broncho pneumonia

3. Tuberculosis nodule (Chronic inflammation)                     

4. Anthracosis – lung

 5. Gangrenous Pneumonia

URINARY SYSTEM

1. Abscess – kidney                                       

 2. Hydronephrosis

3. Hypoplasia – kidney                                  

4. Gout – Kidney

5. Hypoplasia – kidney

6. Hypertrophy – kidney                                

7. Atrophy – kidney

8. Haemorrhagic cystitis                                

9. Fatty infiltration – kidney

10. Infarction -kidney

GENITAL SYSTEM

1. Pyometra uterus                                         

2. Hydrometra salphinx

3. Hypoplasia testes                                         

4. Orchitis – testes                                         

5. Endometritis – uterus.

EXPERIMENT NO. 6

COLLECTION OF MORBID MATERIALS FOR PATHOLOGICAL DIAGNOSIS

            Materials are collected from dead or live animals for microbiological, histopathological, parasitological and chemical examination to arrive at a correct diagnosis.

A) BLOOD SMEAR

            Blood smear means peripheral blood smear. It is prepared from blood of ear vein; air dried and fixed on methanol for one minute. It can also be prepared from heart blood.      (Peripheral blood smear is preferred). Heart blood if not clotted can be drawn using sterile Pasteur pipette for microbiological examination.

B) PUS SMEAR

Prepare thin smear by spreading pus evenly on a slide with a sterile scalpel.

(or)

Place a small quantity of pus on middle of slide.

Place second slide on the first one.

Press gently the two slides together so as to spread the pus.

Draw the slide apart.

Air dry, label, pack & dispatch.

C) TISSUE / IMPRESSION / EXUDATE SMEAR

Cut out a small piece of organ.

Hold it in a pair of forceps.

Blot the surfaces with blotting paper.

Take impression smear of the cut surfaces on middle third of slide.

Label & dispatch.

D) BLOOD

Anticoagulants are used while collection of blood for haematology.

1. Sodium citrate - 3 mg/ml of blood

2. Ammonium and potassium oxalate mixture  

Mixture of ammonium oxalate 6 parts and potassium oxalate 4 parts are used at a concentration of 2 mg/ml of blood. 

3. Ehtylene diamine tetraacetic acid (EDTA) - 1 to 2 mg/ml of blood

4. Heparin - 0.2 mg / ml of the blood

5. Sodium fluoride - 1 mg/ml of blood

For serum, collect blood as such in test tube & place in cool place and collect the serum in vials for serum biochemistry.

E) COLLECTION OF MATERIALS FOR MICROBIOLOGICAL EXAMINATION

Material (swabs or tissues or contents of lesion and tubular organs) for microbiological examination should be collected in aseptic conditions. The materials obtained can be sent without preservatives i.e. on ice or in transport medium.

1.         Collect materials from areas showing lesions and from sites where the organisms concentrated. Example: Vesicles in the mouth in FMD; Skin lesions in pox; Lymph nodes in Theileriosis, Strangles or Glanders.

2.         Always collects material from fresh carcasses. In case of poultry, birds in advanced stage of ailment is sacrificed  and materials are collected.

3.         Use freshly sterilized equipments and containers.

4.         Collected materials are sent in refrigerated condition ( + 4 ^oC ) on ice.

5.         Materials may also be sent using preservatives.

1) Bacteriology

            Amines transport medium with charcoal or Stuart transport medium without charcoal.

AMINES TRANSPORT MEDIUM (with charcoal)

( Difco/Oxoid/Himedia/Span diagnostics)

            Suspend 20 g powder in 1 liter of distilled water. Bring to the boil to dissolve the agar completely. Distribute into small, screw cap bottles, stirring meanwhile to keep the charcoal evenly suspended. Screw down the caps firmly on the completely filled bottles. Sterilize by autoclaving at 121 ^oC for 15 minutes.  Store in a cool place. Use sterile cotton  swabs on wooden sticks to collect the specimen. Push the swab down one third of the medium depth and cut the stick so that when the cap is screwed down, the swab is forced to the bottom of the medium. Make sure the cap is screwed firmly on the bottle and keep cool during the transport period.

STUART TRANSPORT MEDIUM ( without charcoal )

( Difco/Oxoid/Himedia/Span diagnostics)

Suspend 16 g powder in 1 liter of distilled water. Bring to boil to dissolve completely and dispense into screw –capped ¼ oz ‘Bijou’ bottles. Fill each bottle to the brim, tighten the cap and sterilize by autoclaving at 121 oC for 15 minutes.  

Preparation of charcoal swab:

1.                  Prepare swab by rolling absorbent cotton-wool on wooden sticks.

2.                  Boil the swab in a phosphate buffer solution pH 7.4 of the following composition.

Disodium hydrogen phosphate                       0.81 g

Pottassium dihydrogen phosphate                  0.18 g

Distilled water                                                            100 ml.

3.                  Immediately dip the swabs into a 1 % suspension of charcoal ( Bacteriological ).

4.                  Place in cotton – wool plugged test tubes & dry at 100 ^oC and sterilize in the hot air oven for 1 hour at 160 ^oC.

Transport of swabs:

After collection of the specimen, place the swab in the middle of a ‘Bijou’ bottle of Stuart’s transport medium. Break off the stick, replace the screw cap tightly and store the bottle in a cool place.

2) VIROLOGY

Dispatch the material within 24 hours under refrigerated condition (+4 ^oC) on ice.

If delay is expected store at – 70 ^oC ( -20 to 25 ^oC  is also satisfactory but infectivity is reduced in certain cases.)

Dry ice ( solid CO₂ ) can also used. Keep the specimens in tightly capped container to avoid deleterious effect of  CO₂ ).

Preservatives like phosphate buffer saline (PBS) 50 % glycerol saline  or virus transport medium may also be used.

Antibiotics are added to the different media. The quantity varies with specimens. Material with higher bacterial load e.g. intestine, intestinal contents, lung require highest concentration of antibiotics.

1.         Benzyl penicillin 1000 to 10000 IU/ml.

2.         Streptomycin 1 mg to 10 mg / ml.

3.         Gentamycin ( Autoclaving does not destroy the antibiotics ) up to 250mg/ml

4.         Mycostatin

Phosphate buffered saline ( pH 7.3 )

            NaCl                                        8 g

            K₂HPO₄                                  1.4 g

            KH₂PO₄                                  0.3 g

            Distilled water                        1000 ml

            (free from chlorine)    

            PBS is a very useful general diluents and suspending fluid

Glycerol saline (50%)

            Equal amounts of glycerine and saline ( 0.85 % NaCl )

            Autoclave at 121 ^oC for 15 min.

Tissue culture medium

Hank’s Balanced salt solution (BSS)

            Stock Solution A

            1          NaCl                                        160 g

                        KCl                                         8 g

                        MgSO₄.7H₂O                          2 g

                        MgCl₂.6H₂                              2 g

                        Distilled water                                    800 ml

            2          CaCl2                                      2.8 g

                        Distilled water                                    100 ml

Mix these two solutions slowly and make up volume to 1000 ml with ditilled water. Add 2 ml chloroform and store at 4 ^oC.

Stock solution B

Na₂HPO₄.13H₂O                    3.04 g

KH₂PO₄                                  1.2 g

Glucose                                   20 g

Distilled water                        800 ml

                                                                       

            When chemicals have dissolved add 100 ml of 0.4 % phenol red in NaOH. Make up volume to 1000 ml with distilled water. Add 2 ml chloroform and store at 4 ^oC Phenol red in 325 ml NaOH 0.1 mol/liter. Make up to 1000 ml with distilled water. Distribution in 20 ml amounts. Autoclave for 15 min at 121 ^oC. Omit phenol red for fluorochrome detection of mycoplasmas.

For use

            Stock Solution A                                100 ml

            Stock Solution B                                 100 ml

            Distilled water                                                800 ml

Dispense in 100 ml amounts. Sterilize by autoclaving at 121 ^oC for 20 min. Store at 4 ^oC.

F) Histopathological Examination

1.         For routine histopathological examination, 10 % formalin is used as preservative for collection of the various organs.

2.         The material should be properly fixed when it is in a fresh condition before being dispatched.

3.         The aim of fixation is to set or fix the tissues in normal condition & to prevent post-mortem changes.

4.         The quantity of the formalin used should  not be less than 10 times the volume of the material.

5.         Cut the tissue into thin sections, each around 0.5 to 1 cm in thickness so that the fixative may penetrate and kill the  cells quickly.

6.         Abnormal portion should be collected with adjoining normal portion of each organ (80:20).

7.         In case it becomes necessary to send a large mass of material for examination, it should be accompanied by some small slices of the organ or lesion cut out and fixed  separately.

G) COLLECTION OF MATERIALS IN POISONING CASES FOR CHEMICAL EXAMINATION IN GENERAL

1.         A successful toxicological examination requires appropriate specimens & thorough history, including clinical signs, treatment, PM findings & circumstances involved.

2.         Use clean wide mouthed, colourless glass bottles fitted with glass stoppers- capacity one liter.

3.         Preservatives used are rectified spirit, methylated spirit, saturated solutions of common salts. Amongst these, saturated solutions of common salts is commonly used as preservative for collection of the organs in poisoning cases.

4.         Rectified spirit is contra-indicated in cases of suspected poisoning by alcohol, phosphorus, paraldehyde, acetic acid or carbolic acid other drugs of phenol groups.  Methylated spirit  or saturated solutions of common salts may be used if rectified spirit is not available.

5.         Slash the viscera or cut them into small pieces to ensure penetration of the preservative.

6.         The bottles are filled only upto two-thirds of the height and not to the full height to avoid bursting due to gas formation on decomposition.

7.         Apply motor grease, Vaseline or any other suitable grease to stopper ( to prevent sticking) and secure by tape or string and seal the bottle.

8.         Paste the label bearing the identification mark of the animal and viscera.

9.         A sample of preservative used should be kept separately ( rectified or methylated spirit or saturated common salt solution – 100 ml).

10.       Keep the bottles in a card board box and seal it.

11.       Keep the card board box in wooden box. Lock the wooden box and seal it. All bottles and pockets should be carefully sealed and closed in such a manner that they cannot be opened without destroying the seal.

12.       Paste the address label (covering the key hole). Use separate box for different cases and send them by railway parcel to the chemical examiner or Forensic Sciences Laboratory. Better to send it by a messenger.

13.       All these should be done in the presence of Veterinary Officer.

14.       The covering letter, details of material collected, railway receipt, postmortem report along with complete history, details of seal and the request from the District Magistrate with the report from police should be sent separately by registered post to the Chemical Examiner.

MATERIAL TO BE COLLECTED

1.         Stomach and its contents ( one kg.).

2.         Any suspicious substances in the stomach and intestine.

3.         Upper part of the small intestine with its contents ( About one kg)

4.         Portion of the liver, not less than 500 g or the whole liver.

5.         Spleen or its portion, if large.

6.         One kidney

7.         Urine and faeces in preservative kept separately. Thymol is used for preserving urine if rectified spirit is contra-indicated.

8.         Heart and portion of brain if poisoning by nux vomica or strychnine.

9.         Lung tissues and blood from the heart cavity without adding preservatives in cases of suspected poisoning of carbon monoxide, coal gas, hydrocyanic acid, alcohol or chloroform. CSF in alcohol.

10.       Portion of  skin and subcutaneous tissue in suspected poisoning by s/c injection.

11.       Portion of the long bones in suspected cases of sub acute or chronic poisoning by arsenic and antimony ( especially in case of extreme putrefaction or body is exhumed after long burial)

12.       Hair is suspected cases of subacute or chronic poisoning by minerals ( most minerals are eliminated through hair)

13.       Genitalia-uterus and its appendages and upper part of vagina in suspected case of criminal abortion.

            Item 1 and Item (s) in 7 to 13 are preserved separately;

            Item 2 to 6 are preserved together.

14.       Dry dung without addition of spirit.

            If not forwarded to Chemical Examiner, the viscera and other articles are preserved for a period of six months and are destroyed with the consent of District magistrate.

Organs to be collected in different poisoning cases

Sr. No	Poison/Agent	Organ / sample to be collected
1	Arsenic a) Acute b) Chronic	Liver, Kidney, stomach part & intestinal content. Horn, hooves, urine.
2	Alkaloid	Liver, urine, brain, & stomach content
3	Copper	Liver, kidney, blood, stomach content & 500gm of suspected feed sample.
4	Cyanide	Oxalated blood, liver, stomach content & 500 gm of  suspected feed sample.
5	Chlorinated hydrocarbons	Liver, fat & tissue
6	Hydrocyanic acid	Ruminal contents in airtight container, blood & muscle
7	Lead	Liver, kidney, urine, bone & reticular contents.
8	Mercury	Kidney, liver & 500 gm of suspected feed sample.
9	Zinc	Kidney, liver & 500 gm of suspected feed sample.
10	Nitrate/nitrite	Stomach content with chloroform or 10% formalin in airtight container, 2 ml of blood in 4 ml. of PBS, urine.
11	Phosphorus	Liver, blood, stomach content & muscle
12	Oxalate	Kidney & suspected feed sample
13	Rodenticide	Liver & urine
14	Sodium chloride	Brain, liver & stomach contents.
15	Strychnine	Blood, kidney & urine
16	Organophosphate compounds	Liver, blood & muscle
17	Fluorides	Bone of mandible, teeth & urine.

Quantity of sample to be collected

Sr. No.	Organ/sample	Quantity
1	Blood heparinised in live animal Blood clots in dead animals	30-50 ml
2	Brian	Entire
3	Fat	200 gm
4	Hair	10-20 gm
5	Liver	250- 500 gm
6	Stomach contents	500 gm in large animal All available in small animals
7	Urine	All available
8	Kidney	100-200 gm
9	Feed sample	250 – 500 gm
10	Water sample	250 – 500 ml
11	Dry/green grass	500 gm

EXPERIMENT NO: 7

Techniques for preservation and dispatch of materials

FIXATION AND PRESERVATION

Qualities of fixatives

1.         It must be cheaper and easily available.

2.         It should have quick penetrating power.       

3.         It should not interfere with processing and staining.

4.         It should not be harmful to the persons who use it.

Purpose of fixation

1.         To harden the tissues by coagulating the cell protein and thus facilitating section cutting.

2.         To stop autolysis by destroying the intracellular enzymes.

3.         To preserve the structure of tissue.

4.         To arrest the shrinkage of tissue.

            For routine paraffin technique the formal saline (10 % solution ) is used as a fixative.

Formaline ( 40 % formaldehyde solution )     -----      100 ml.

Sodium chloride                                              -----      8.5 gm.

Distilled water                                                            -----      900 ml.

Duration of fixation                                        -----      24 - 48 hrs.

Different fixatives specially used for staining of tissue or cell constituents are as follows

1.         Lipids                                                              :           Buffered formaline

2.         Glycogen and other carbohydrates                 :           a. Formaline alcohol

                                                                                                b. Carnoy’s fluid

                                                                                                c. Alcoholic Bouin’s fluid.

3.         Enzymes and hormones                                  :           Chilled alcohol at ice cold        

                                                                                                temp for 2-4 hrs.

4.         Nucleic acids ( DNA/ RNA )                         :           a. Zenker’s fluid

                                                                                                b. Helly’s fluid

                                                                                                c. Heidenhan’s fixative

5.         Connective tissue                                            :           a. Helly’s fluid

                                                                                                b. Boiun’s fluid

6.         Chromosomes                                                 :           Carnoy’s fluid

7.         Proteins                                                           :           Formal saline 10 %

8.         Glandular tissue                                              :           Bouin’s fluid.

For bony and calcified tissue

            The removal of lime salts from bones is called as decalcification. It is done with acids. The process of decalcification can be accelerated by higher temp.  But it should not be done at above 50 ^oC. It is better to destroy the bony tissue at room temp for 4 days.

1.         Saw the bone into small discs of 3-5 mm thickness.

2.         Fix such pieces in 10 % buffered formaline.

3.         Wash the tissue to remove excess of formaline.

            There are four methods of decalcification:

a)         Acid method                           b)         Iron exchange resins

c)         Electrical ionization                d)         Chelating method

            The most commonly used method is acid method. For decalcification the fixed tissue is hanged in the acid solution till the calcarious matter is dissolved. The acid is formed as follows.

Formaline (commercial 40 %)  -          5 ml

Nitric acid                                 -         5 -10 ml

Water                                        -         80 - 85 ml.

            Change the solution once or twice daily. Treat till the bone becomes soft and decalcified. It requires 4 or more days (some time 15 days) for decalcification. When decalcified it can be easily punctured with needle. Wash in water and transfer to 70 % alcohol directly.

Dispatch of materials

Label the bottle with following description.

---------------------------------------------------------------------------------------

Name of the Institute / Dispensary                  Name of owner

P.M. No._______ ,                                          Dt.________

Description of the animal:

Species: ______,          Breed: _______,     Age : ______,     Sex :_____

Tissue collected:                                                       Suspected for:

------------------------------------------------------------------------------------

Special note should be dropped in the bottle so that by any reason the lable is spoiled, the no. on the cheat can be read. This should be written by lead pencil or a ball pen if fixative contains water.

EXPERIMENT NO. 8

HISTOPATHOLOGICAL TECHNIQUES – TISSUE PROCESSING

& SECTION CUTTING

            It is the science or an art of preparing organs, tissues or tissue components for microscopic observations and study, this techniques includes the study of the tissues collected from a living animal at the time of operation (biopsy) or from recently dead animal at post mortem.

            Histopathological technique includes several processes viz.,

1.         Collection and preservation of tissues (Fixation).

2.         Processing of tissues for microscopic examination.

                        a) Washing                              b) Dehydration

                        c) Clearing                               d) Embedding and block making.

3.         Staining of mounted sections.

COLLECTION OF TISSUES FOR HISTOPATHOLOGICAL EXAMINATION

1.         Tissues should be cut by a sharp instrument without laceration.

2.         Tissues should not be pressed or handled in such a way that there is mutilation of the tissues.

3.         Tissues should be collected immediately after death of animal within 4-6 hrs. Otherwise putrefactive changes set in.

4.         Tissues collected for histopathological examination should be cut in such a way that 80% tissue is abnormal and 20 % tissue is apparently normal for comparison purpose and more than 2-3 cm in thickness.

5.         Prior to fixation wash the collected tissue.

6.         The tissues should be collected in a wide mouth bottle containing adequate amount of fixative (ratio of tissue) to preservative should be 1:20 (w/v).

7.         Absorbent cotton or a filter paper should be kept at the bottom of bottle so that preservatives penetrate well even from bottom.

8.         Lung or floating tissues should be covered with absorbent cotton.

9.         Label the bottle with all details.

PROCESSING OF TISSUE

            Cut thin pieces of tissue not exceeding 3 - 5 mm thickness and keep it in tissue capsule along with marking with lead pencil only.

Washing

            Tissue capsule containing tissue is them kept in running tap water to remove the fixatives. Mouth of the beaker should be covered by tied muscline cloth. Flow of water should be slow to prevent agitation of water and help to remove old water. Tissues are kept in washing in running tap water overnight for at least 12 hours to remove the formalin from tissue.

Dehydration

           

            The dehydration is necessary because the paraffin will not penetrate the tissue in presence of water. So from the tap water tissue is kept on a filter paper to remove excess of water and then dehydration is done in ascending grades of alcohol as follows:

                        50% alcohol                -----      1 hr.

                       

                        70% alcohol                -----      1 hr.

                        80% alcohol                -----      1 hr.

                        90% alcohol                -----      1hr.

                        95% alcohol                -----      1 hr.

                        (Rectified spirit)

                        -----do---------- 2          -----      1 hr.

                        Absolute alcohol-1      -----      1 hr.

                        -------do-------- 2          -----      1 hr.

            This removes water from the tissue. Grades of alcohol are used so that tissues are not spoiled.

Clearing

            It is removal of alcohol. This dealcoholisation facilitates penetration of paraffin used for embedding.

                        alcohol  :  xylol (50 : 50)                     - 1 /2 hr.

                        xylol    1                                              - 20 min.

                        xylol    2                                              - 20 min.

            Clearing through xylol is quick process, the tissue becomes clear transparent. If tissue appears cloudy it means there is water so dehydration will have to be done again. If the tissues left in xylene for more time than scheduled time they become hard. Cedar wood oil is also a good clearing agent and tissues can be kept for more time in it without damage.

Embedding

            After dehydration and clearing the tissues are ready for embedding. It consists of impregnating the tissue completely with an embedding in paraffin or celloidins.

            The paraffin embedding method is most commonly used method. It makes the tissue hard and easy to cut. Generally paraffin of melting point 58-60 ^oC & 60-62 ^oC are used in winter and summer respectively. The paraffin is kept in cups or kartory marked 1, 2 and 3. The number of changes may vary with size and consistency of specimen of tissue. The nervous tissue, skin connective tissue as tendons and bones require more time than other tissue. 3 or 4 changes of 15 – 30 min. each are given routinely. If they are kept for more time they become hard.

Block making

            For making the blocks two ‘L’ shaped brass moulds are used. These are arranged over a porcelain tile. The tile and inner side of ‘L’ moulds is smeared with glycerine so that blocks will be removed easily. Two ‘L’ moulds are adjusted in such a way as to make rectangle. 

            The molten paraffine is poured into ‘L’ moulds and then the tissues from last cup of paraffine is transferred into moulds. The tissue is so oriented that the cutting surface of the tissue is touching to the porcelain tile. The block thus formed is removed from ‘L’ moulds. It is then trimmed and labeled. The paraffine block should be stored in a cool place, preferably lower chamber refrigerator.

Section cutting

            The section cutting is done with the help of a microtome machine. The surface opposite to the cutting surface of paraffine block is pressed firmly on heated surface of metal block – holder or checkmate and then transferred to cold water. In the microtome machine sharp knife is fixed. The angle is set so that a straight ribbon of section will cut.

            Fix the block holder to microtome machine and tighten all the adjusting screws so that cutting surface will be parallel to knife edge. Set the thickness gauge (routinely 3-5 micron). Operate the microtome machine till a ribbon of complete section is obtained. The clean glass sides are numbered with diamond pencil and smeared with Mayer’s albumin.

            The ribbon is carefully detached by needle and transferred to thermostatically controlled tissue flotation bath, adjusted to 45-50 ^oC. The sections are then spread on water in flotation bath; see that there are no folds, wrinkles or scores of cutting knife. The sections are collected on slide by dipping the smeared slide straightly perpendicular to the section in the flotation bath. These slides are kept in oven for overnight at 37 ^oC to dry. Now the slides are ready for staining.

Mayer’s albumin

            Egg albumin (White of fresh egg)                  -           100 ml.

            Glycerol ( BDH )                                            -           100 ml.

            Thymol crestal                                     -           0.5 gm.

            Allow to stand for 2 days with occasional shaking. Filter through a thick gauge and label. Store in cool place.

FROZEN SECTION

            The tissue is hardened by rapid freezing with CO₂ gas or an ethyl chloride spray and sections are cut immediately with a freezing microtome. Except fixation no preliminary manipulations are required. Thus a section can be obtained within few minutes. A frozen section is therefore particularly useful in quick histological diagnosis, of a suspected cancer of any disease while the patient is still on operation table and it enables the surgeon to plan the operation accordingly. A frozen section is also useful for positive demonstration of amyloid, glycogen, enzymes in the tissues.

CRYOSTAT

            It is a microtome enclosed in a refrigerator chamber ( -30^oC ) but can be operated from outside. The tissue can be rapidly frozen and cut with a cold knife. The special advantage is that the tissue is kept cold even during actual act of cutting and artifacts caused by fixation, autolysis and heat are avoided. Moreover, antigens, antibodies and enzymes in the tissues are well preserved and can be demonstrated insight by special techniques.

Sections for electron microscopy

            The tissue is embedded in polyester or an epoxy resin like Araldite and cut on an ultra microtome to obtain sections not more than 1 / 10 in thickness. Ultra microtome is motor driven its knife is either of a diamond or of a glass.

EXPERIMENT NO. 9

HISTOPATHOLOGICAL TECHNIQUES – STAINING & IDENTIFICATION

OF MICROSCOPIC LESIONS 

            After the sections are taken on slide and they are allowed to remained for 24 hrs. so that they adhere to the slide firmly. Slide can be dried in oven for 1 / 2 hr. Solutions are kept in coupline jars.

Steps involved in H & E staining are as follows.

1.         Keep the slides for 5 min. in xylol - 1

2.         Remove and keep for 5 min. in xylol -2 during this process the paraffin is removed.

3.         Keep the slides for 5 min. in absolute alcohol -1.

4.         Remove and keep the slides for 5 min. in rectified spirit - 1.

5.         Then keep in rectified spirit - 2 for 5 min.

6.         The slides are kept then in 80 % alcohol for 5 min.

7.         Remove and keep the slides for 5 min. in 70 % alcohol.

8.         Then in 50 % alcohol for 5 min.  during this process xylol is removed.

9.         Keep the slide in distilled water for 3-5 min.

10.       Keep the slide in heamatoxylene solution for 10 – 20 min.

            If heamatoxylene is old less time will require. It will stain nuclei and cytoplasm blue.

11.       Wash in water to remove excess of the heamatoxylene and differentiate in 0.5 % acid water to remove heamatoxylene  from cytoplasm.

12.       Wash in running water for 10 -15 min. to remove acid from sections.

13.       Counter stain with alcoholic eosin 0.5 % for 2 mins.

14.       Blot the slide gently, then keep in 70 % alcohol for 5 mins.

15.       80 % alcohol for 5 mins.

16.       90 % alcohol for 5 mins.

17.       95 % alcohol -1  for 5 mins.

18.       95 % alcohol -2  for 5 mins.

19.       Absolute alcohol -1  for 5 mins.

20.       Absolute alcohol -2  for 5 mins.

21.       Xylol  -1  for 5 mins.

22.       Xylol  -2  for 5 mins.

23.       Clear the slides with muslin cloth and mount the section in PDX mountant as follows :

            Place a clean cover slip on a blotting paper; put a drop of DPX mountant on the center. Invert the slide on coverslip in such a way that the section is dipped in DPX. Press the slide so that DPX spread and covers the section. Remove air bubbles by manipulating with mounting needle.

24.       Result

Nuclei  - Blue in colour          

Cytoplasm – Pink in colour

Mayer’s Haematoxylene

                        Haematoxylene                       :           1 gm

                        Distilled water                        :           100 ml

                       

                        Amm. / Pot. Alum                   :           50 gm

                        Sodium iodate                         :           0.2 gm

                        Citric acid                               :           1 gm

                        Chloral hydrate                       :           50 gm

Staining time                           :           as a progressive stain 15 mins. &

as a regressive stain  40 – 60 mins.

Eosin:

1.         1 % stock solution of alcohol eosin

            Eosin Y (Water soluble)                      :           1 gm.

            Distilled water                                                :           20 ml. 

            Dissolved and add alcohol 95 %        :           80 ml.

2.         Working solution

            Eosin stock solution                            :           1 part

            Alcohol 80 %                                      :           3 parts

            Add a drop of glacial acetic acid in the coupling jar while staining. This makes the eosin stain more stable in further dehydration and clearing.

EXPERIMENT NO. 10

REVISION OF HISTOLOGY OF IMPORTANT ORGANS

A)        LIVER

i)          Each hepatic lobules having polyhedral or prismation structure.

ii)         Central or intralobular vein present in each hepatic lobule.

iii)        Hepatic cords radiates from the central vein towards the periphery.

iv)        The hepatic cells are polygonal in shape.

v)         Each hepatic cord is made up of two rows of the hepatic cords.

vi)        Bile canaliculi is seen in between the two rows of the hepatic cords.

vii)       The space in between two adjacent cord is called as sinusoidal space.

viii)      Space of disc is the space between the sinusoidal epithelium and hepatic cord cells.

ix)        The sinusoids are incompletely lined by reticuloendothelial cells – Kupffer’s cells.

x)         Note the hepatic triad at an interlobular septum where three lobules come together.

xi)        Hepatic triad consists of interlobular branch of the portal vein, hepatic artery and bile duct.

B)        KIDNEY

1.         Tubular gland made up of a number of microscopic units of uriniferous tubules or nephrons.

2.         Glomerulus is surrounded by a Bowman’s capsule, proximal convoluted tubules and distal convoluted tubules.

3.         Proximal convoluted tubules are numerous and having small lumen and brush borders.

4.         No intertubular space present.

5.         Glomeruli are more in cortical portion than medullary portion of kidney.

C)        HEART

1.         Cardiac muscle fibers branch without much change in diameter.

2.         Cross section is less prominent and nucleus is central.

3.         More irregular thicker cross striations seen at intervals at branching of muscle called inter calated discs.

D)        LUNG

1.         Lung section consists of bronchi, bronchioles and alveoli.

2.         Bronchi or bronchioles lined by pseudostratified columnar epithelium but cartilage and glands are lacking in bronchioles.

3.         A branch of pulmonary artery accompanies the respiratory bronchioles.

4.         Capillary plexus with connective tissue and fibroblasts are seen in intra alveolar septa.

E)        LYMPH NODE

1.         Parenchyma divided into cortex and medulla.

2.         Cortex is separated from the capsule by the marginal sinus.

3.         Cortex consists of lymphatic nodules.

4.         Lymphatic nodules incompletely separated by trabaculae and trabacular sinuses.

5.         Lymphocytes form the cell cord called medullary cords in medullary portion.

6.         Trabacular sinuses surround these cords.

F)        SPLEEN

1.         Spleen does not have cortex and medulla.

2.         Splenic nodules are seen throughout the parenchyma of spleen.

3.         Each splenic nodule is accompanied by a branch of trabacular artery called as central artery.

4.         Capsule and trabaculae in spleen are thicker than in lymphnodes.

5.         Central artery accompanies each splenic nodules where as lymph node devoid of it.

G)        SKIN

1.         Skin is composed of two principle layers – outer epidermis and inner epidermis or corium.

2.         The epidermis is stratified and composed of five layers or zones.

a)         Stratum cornium                                 b)         Stratum lucidum

c)         Stratum granulosum                            d)         Stratum germinativum

Stratum germinativum consists of :

i)          Stratum spinosum                   ii)         Stratum basalis or basal cell layer.

3.         Epidermis is devoid of blood capillaries and nerves.

H)        CARTILAGE

1.         Cartialge consists of chondrocytes which are located in lacunae and matrix of the intercellular substances.

2.         Chondrocytes in fibrocartilage are round and without processes.

3.         In hyaline cartilage collagenic fibers are more while in the elastic cartilage elastic fibers are more.

I)         BONE

1.         Bone consists of matrix, in which there are cavities which contain the osteocytes.

2.         Osteocytes are either flat or oval in shape.

3.         Osteoblasts are larger than osteocytes and osteoclasts are located within cavities.

4.         Lamillae surrounds the vascular channels in a cylendrical arrangements referred as Haversian system or osteon.

5.         Each system consists of  4 – 20 tubes arranged around central vessels canal known as Haversian canal.

6.         In between Haversian system interstitial lamellae present.

J)         TESTES

1.         Testes have the following layers:

a)         Tunica vaganalis – consist of simple squamous epithelium.

b)         Tunica albugenia – consists of collagenic fibers few elastic fibers and blood vessels.

c)         Septula testes and mediastinal testes – consists of collagenicfibers and loose connective tissue with many elastic fibers.

d)         Seminiferous tubules – lined by sertoli cells with irregular out lines, narrow apex and broad base.

K)        OVARIES

1.         Consists of outer cortex and central medulla.

2.         Cortex is lined by       

a) Germinal epithelium (Simple cuboidal or squamous epithelim).

b) Tunica albugenia with dense network of irregular collagen fibers.           

c) Follicles primary primary follicles are located beneath the second layer. Growing follicles are located in the deep layer of cortex.

L)        PANCREAS

1.         Exocrine portion consists of acini which have pyramidal cells with round basal nucleus and condensed chromatin.

2.         Endocrine part consists of islets surrounded by reticular fibers.

3.         Islets contain various cells types.

4.         Pancreas is surrounded by common connective tissue capsule consisting of collagen fibers.

GASTROINTESTINAL TRACT (GIT)

1.         There are four major layers of GIT.

a)         Mucosa            b) Submucosa              c) Muscularis mucosae            d) Adventetia.

M)       OESOPHAGUS

1.         Epithelium is simple squamous type and villi are absent.

2.         Submucosa contains loose connective tissue blood vessels & lymphatic, nerves & seromucosal gland.

3.         Muscularis mucosae contains inner circular & outer longitudinal muscles.

STOMACH

1.         Mucosae of glandular stomach has three regions.

a) Cardiac        b) Fundic         &         c) Pyloric.

2.         Epithelium is stratified squamous and villi are absent.

3.         Lamina propria contains gastric glands.

4.         Submucosa of rumen is papillary, omasum has longitudinal laminae.

N)        SMALL INTESTINE

1.         Epithelium is tall columnar with numerous goblet cells.

2.         At the base of villi are crypts of Liberkhun with paneth cells.

3.         Submucosa of duodenum contains Brunner’s gland and Payer’s patches are present in ilium.

4.         Muscularis mucosae contains inner circular & outer longitudinal muscles.

O)        LARGE INTESTINE

1.         The villi are absent.

2.         Intestinal glands are longer straight and compact.

3.         Goblet cells are numerous but paneth cells are absent.

4.         Lymphatic nodules are increased in number.

EXPERIMENT NO. 11

DISTURBUNSENCE OF CIRCULATION

a)         ACUTE CONGESTION – LUNG:

1.         Note the alveolar capillaries containing more erythrocytes.

b)         CHRONIC VENOUS CONGESTION – LIVER (Nutmeg liver)

1.         Central vein is distended with the blood.

2.         Sinusoids around central vein are distended.

3.         Hepatic cords are atrophied which appear fine pink.

c)         CHRONIC VENOUS CONGESTION - KIDNEY

1.         Note the congestion of glomeruli , inter tubular vessels & straight veins of medulla

2.         Convoluted tubules show cloudy swelling of the cell epithelial granular and hyaline casts in the lumen.

THROMBOSIS – LIVER

1.         Note a net work of fibrin in the hepatic artery attached to their walls.

2.         The network contains RBCs, WBCs and platelets which under go   homogenization.

PULMONARY EDEMA

1.         Note homogenous pink staining area in the alveoli of lung without cells.

INTESTINAL HAEMORRHAGES

1.         Note the extravasation of the blood in the mucosa of bowel.

2.         Infiltration of mucosa & submucosa by the inflammatory cells.

INFARCTS – KIDNEY

1.         Tubular epithelium is swollen & contains coarse granules.

2.         Infarcted area appears as wedge shaped.

EXPERIMENT NO. 12

DISTURBANCES OF CELL METABOLISM

Cellular swelling, hydropic & hyaline degeneration, mucinous & mucoid degeneration, amyloid infiltration, fatty changes, necrosis & calcification

1.         CELLULAR SWELLING: LIVER

a)         LIVER:

1.         Hepatocytes are swollen & appear rounded (normally polygonal).

2.         Sinusoids are narrowed or obliterated.

3.         Hepatocytes have hazy appearance & cytoplasm shows coarse granules.

4.         Nuclei of liver cells do not show any alterations.

5.         Stagnation of bile in bile canaliculi.

b)         CELLULAR SWELLING: KIDNEY

1.         The principle changes are seen in convoluted tubules.

2.         Tubular epithelium is enlarged resulting in narrowing of lumen.

3.         Brush borders are lost (in proximal convoluted tubules)

4.         Cytoplasm show coarse granules & appears hazy.

5.         Swollen cells are stained darker with eosin.

2.         HYDROPIC DEGENERATION: SKIN (FOWL POX)

a)         SKIN FOWL POX:

1.         The cells of stratum spinosum layer of epithelium are ballooned & show      vacuoles.

b)         KIDNEY:

1.         The cells of tubules are swollen & contains one or two vacuoles displacing the nucleus to one side.

2.         The changes is differentiated by using special stains.

3.         HYALINE DEGENERATION

a)         Epithelial hyaline – ( Skin ) :

1.         Stratum corneum is excessively formed.

2.         The affected tissue is homogeneous  & stains pink with eosin.

3.         Seen in vitamin A deficiency.

b)         EPITHELIAL HYALINE – ( SQ. CELL CARCINOMA )

1.         Concentrically laminated structures of epithelial cells known as pearl’s are observed.

2.         The affected tissue stains homogeneous pink.

c)         EPITHELIAL HYALINE – (MAMMARY GLAND)

1.         Note acinar structure of mammary glaned lined by cuboidal cells.

2.         The cuboidal cells lining acini gets desquamated forming rounded masses in center. (corpora amylaceae )

3.         These rounded structures stains pinkish with eosin.

d)         MUSCLE HYALINE – (HEART)

1.         The affected heart muscle shows loss of striation but nuclei present.

2.         The affected tissue stains pink with eosin.

4.         MUCINOUS DEGENERATION – (NASAL SEPTUM)

1.         Nasal mucus membrane shows large number of goblet cells & mucin in the cytoplasm of cells.

2.         Mucin is stained faintly blue with hematoxylin.

5.         MUCOID / MYXOMATOUS DEGENERATION – (C.T.)        

1.         Connective tissue cells are stellate shaped & have many branching processes.

2.         There is more of intercellular bluish staining material.

6.         a) AMYLOID INFILTRATION – (LIVER ) :

1.         Amyloid  ( homogenous pink staining material ) is seen deposited in between the   cords of hepatic cells & lining of sinusoidal space i.e. in the space of disse.

2.         Due to deposition of amyloid hepatocytes under go atrophy.

            b) BACON SPLEEN & SAGO SPLEEN

1.         General amyloidosis of spleen.

2.         Observe entire faint pink staining area in spleen tissue.

7.         GOUT   (URATIC INFILTRATION) : KIDNEY

1.         Kidney tissue showing fan shaped area in H & E stain.

2.         Crystals taken greenish colour by special staining i.e. carmine stain.

8.         FATTY CHANGES - LIVER

1.         Hepatocytes are loaded with variable sizes of fat globules which appears at vacuolated areas in H & E stained sections.

2          Nucleus is pushed to one side if fat droplets are larger in size.         

3.         In special staining by osmic acid, fat globules appears black.

9.         NECROSIS:

a)         COAGULATIVE NECROSIS – LIVER :

1.         Hepatic cords are swollen & cells show coarse granules.

2.         Architecture of the cell is maintained without nucleus in the cord cells.

3.         Entire area under gone this change being eosinophillic stain pink.

b)         COAGULATIVE NECROSIS- KIDNEY :

1.         Tubular epithelium is swollen & contains coarse granules.

2.         Infarcted area appears as wedge shaped.

c)         CASEATION NECROSIS – LIVER ( T. B. )

1.         Necrosed area is surrounded by a capsule.

2.         Cellular & architectural details are lost.

3.         The area appears bluish pink in H & E. stain.

4.         In surrounding area lymphocytes are seen.

10.       ATROPHY- LIVER : ( Amyloid infiltration )

1.         The sinusoids & liver cells are compressed & under gone pressure atrophy due to the amyloid deposition in the space of disse.

EXPERIMENT NO.   13

DISTURBANCES OF PIGMENTS & MINERAL METABOLISM

1. PIGMENT METABOLISM

Bilirubin pigmentation - Liver

1.         Section showing accumulation of yellowish pigment in between the cells or tissue spaces.

HAEMOSIDEROSIS - SPLEEN

1.         Section of spleen showing brownish pigment deposited in the pulp.

2.         In purssian blue reaction or perl’s staining, haemosiderin appears as blue masses due to formation of ferric ferrocyanide complex.

ANTHRACOSIS – LUNG

1.         Note accumulation of black granules (coal particles) in the walls of alveoli (inter alveolar septa)

2. MINERAL METABOLISM

CALCIFICATION HEART

1.         Lime salts stain deep blue with H & E. stain, so note such areas in the section of heart.

CALCIFICATION – KIDNEY

1.         Calcified areas stained deep blue with H & E stain

2.         In special stain by Van kossa the same calcified areas appears blackish or brownish.

EXPERIMENT NO. 14

DISTURBANCES OF CELL GROWTH

Hypoplasia, hyperplasia, atrophy, hypertrophy & metaplasia

1.         HYPOPLASIA (TESTES)

1.         Seen in the paired organs.

2.         Seminiferous tubules appear irregular in size.

3.         The sertoli cells are not conspicuous.

4.         Spermatogenic cells multiplication is greatly reduced.

5.         More of fibrous tissue in between the seminiferous tubules.

2.         HYPERPLASIA (THYROID)

1.         Cuboidal cells lining the acini of gland multiply occupying the lumen.

2.         Very small amount of colloid or no colloid seen in the center of lumen.

3.         HYPERPLASIA & HYPERTROPHY

            (Pulmonary adenomatosis – Lung )

1.         Note the hyperplasia and hypertrophy of the alveolar and bronchial epithelium.

4.         METAPLASIA (TRACHEA)

a)         Trachea:

1.         Ciliated columnar type of epithelium lining of trachea is metastasized to squamous stratified type.

b)         Oesophagus:

EXPERIMENT NO. 15

INFLAMMATION & REPAIR

ACUTE INFLAMMATION

Pericarditis – Heart

1.         Note infiltration of inflammatory cells alongwith exudates in the pericardial sacs.

Myocarditis - Heart muscle

1.         Section shows a large no. of inflammatory cells, i.e. neutrophills  in between the muscle fibers.

2.         Muscle fibers shows degenerative changes.

FIBRINOUS PNEUMONIA – LUNG

1.         Note the deposition of fibrinous material in the alveoli.

2.         Inflammatory exudates contains cells of inflammation.

SUPPURATIVE PNEUMONIA

1.         Bronchi contains exudates with large no. of neutrophills.

SUPPURATIVE INFLAMMATION: KIDNEY / LIVER – ABSCESS

1.         Note circumscribed lesions in liver / kidney tissue.

2.         Center of abscess is structureless.

3.         Large no. of neutrophills in the surrounding area are seen.

HAEMORRHAGIC PNEUMONIA :LUNG

1.         Exudate in alveoli of lung contain predominantly RBCs along with other cells of inflammation.

HAEMORRHAGIC INFLAMMATION : HEART

1.         Muscle fibers in addition to inflammatory cells contains RBCs.

HAEMORRHAGIC INFLAMMATION : INTESTINE

1.         Note the extravaseted blood in the mucosa of the bowel.

2.         Infiltration of the mucsa and submucosa by inflammatory cells.

CHRONIC INFLAMMATION

JHONE’S DISEASE

1.         Mucosa and submucosa are thickened due to infiltration of lymphocytes, macrophages and plasma cells.

2.         Few giant cells may be seen in area of cellular infiltration.

3.         The organisms appears reddish with acid fast staining i.e. Zeil-Neelson’s stain.

4.         The villi becomes blunt.

CUFFING – BRAIN

1.         Observe the infiltration of lymphocytes in the perivascular space of the capillaries ( space of Robin Virchow )

TUBERCULOSIS NODULE : ( Langarhan’s giant cells )

1.         Central bluish calcified mass.

2.         Zone of necrosis surrounded by epitheloid cells.

3.         Layer of lymphocytes, macrophages and giant cells ( Langhan’s type-half moon shape )

4.         Fibrous capsule.

CELLS OF INFLAMMATORY EXUDATE

Note the various cells of inflammation in the blood smear.

A)        NEUTROPHILLS

i)          8 – 10 u in size.

ii)         On an average they are 28 % in blood.

iii)        The nucleus is lobulated i.e. 3- 5 lobes and connected to each other by filaments.

iv)        Cytoplasm contains minute pinkish granules which can be stained with neutral dyes.

B)        BASOPHILLS

i)          8 – 10 u in size.

ii)         Lobulations of the nucleus is not well defined as.

iii)        The cytoplasm contains irregularly scattered large blue granules which mask the nucleus.

iv)        On an average they are 0 – 2 % in the blood.

C)        EOSINOPHILLS

i)          8 – 10 u in size.

ii)         On an average they are  9 % in the blood.

iii)        The nucleus is usually bilobed resembling spectacles.

iv)        Cytoplasm contains characteristics coarse bright pinkish granules.

(like pumgrante seeds.)

D)        LYMPHOCYTES

i)          Generally two types i.e. large lymphocytes and small lymphocytes.

ii)         They are slightly smaller  or larger than neutrophills or eosinophills.

iii)        Contains well defined round darkly stained nucleus occupying nearly whole the cells.

iv)        Cytoplasm is pale blue, scanty and is nothing but more than a ring around the nucleus.

v)         On an average they are 58 % in the blood.

E)        MONOCYTES

i)          16 -32 u in size.

ii)         Nucleus is kidney shaped or horse shoe shaped in adult cells and placed eccentrically.

iii)        Cytoplasm is plentiful.

iv)        On an average they are 1 – 4 % in the blood.

F)        PLASMA CELLS

i)          Rare in peripheral blood.

ii)         Larger than lymphocytes.

iii)        Eccentrically nucleus which show cart wheel arrangement of its chromatin.

iv)        Cytoplasm plentiful and deeply basophilic and may contain a few azurophillic granules.

GRANULATION TISSUE

1.         Numerous small capillary beds are seen.

2.         In between the beds lymphocytes, a few neutrophills and fibroblasts are seen.

EXPERIMENT No. 16

NEOPLASM – GROSS & MICROSCOPIC STUDY

Oncology – Oncos = tumor, logos = study.

Neoplasm (Neo= new, plasm= growth) means new formation or new growth. A mass of tissue formed as a result of abnormal, excessive, uncoordinated, autonomous and purposeless proliferation of cells.

Types:

1) Benign tumours -Adult type

2) Malignant tumours - Embryonic stages

Classification: Benign & Malignant Neoplasm

	Tissue of origin	Benign	Malignant
I	Neoplasms of one parencymal cell type
(i)	Epithelial neoplasms
1.	Squamous epithelium	Papilloma	Squamous cell carcinoma
2.	Transitional epithelium	Papilloma	Transitional cell carcinoma
3.	Glandular epithelium	Adenoma	Adenocarcinoma
4.	Basal cell layer	-	Basal cell carcinoma
5.	Melanoblasts	Melanoma	Melenocarcinoma
6.	Hepatocytes	Liver cell adenoma	Hypatocellularcarcinoma
7.	Placenta	-	Choriocarcinoma
(ii)	Non-epithelial neoplasms (mesenchymal)
1.	Adipose tissue	Lipoma	Liposarcoma
2.	Fibrous tissue (adult)	Fibroma	Fibrosarcoma
3.	Fibrous tissue (embryonic)	Myxoma	Myxosarcoma
4.	Bone	Osteoma	Osteosarcoma
5.	Cartilage	Chondroma	Chondrosarcoma
6.	Smooth muscle	Leiomyoma	Leiomyosarcoma
7.	Skeletal muscle	Rhabdomyoma	Rhabdomyosarcoma
8.	Blood vessels	Hemangioma	Hemangiosarcoma
9.	Lymph vessels	Lymphangioma	Lymphangiosarcoma
10.	Meninges	Meningioma	Invasive meningioma
11.	Lymphoid tissue	Lymphoma	Malignant lymphoma
12.	Brain nerve sheath	Neurofibroma	Neurogenic sarcoma
13.	Brain nerve cell	Ganglioneuroma	Neuroblastoma
14.	Blood cells (lymphocytes)	-	Leukemia
15.	Mesothelium	-	Mesothelioma
II.	Mixed neoplasms
1.	Salivary gland	Mixed salivary neoplasm	Malignant mixed salivary neoplasm
III.	Neoplasms of more than one germ layer
2.	Gonads	Mature teratoma	Immature teratoma

I) Epithelial Neoplasm

1) Papilloma

It is benign epithelial tumors projecting from an epithelial surface and involves squamous, transitional or coloumnar epithelium depending up on the tissue from which it originates.

It is commonly found on the skin and on the buccal mucosa. Some may arise from the mucosa of the intestine and bladder. Skin papilloma  also called as warts & are very common in animals found on the shoulder, head, neck and dewlap mostly as clusers.

Macroscopically: They may be pedunculated as in the papilloma of bladder or have broad base. The surface may be smooth or rough and horny (horn like). Size  small- few mm to 10 cm diameter.

Microscopically: Thick layers of epithelium. Epithelium irregularly arranged & shows finger like projections.

2) Squamous cell carcinoma  

This is malignant tumor of Squamous epithelial cells usually squamous stratified epithelium. It is a common tumor among the cattle in India affecting horn (called as Horn Cancer) and eyes.

Dogs: It is found on the skin of animal in various locations abdomen, prepuce, anus, tail, tongue, legs, ear, eye and lips.

Horse: eye, vulva, penis, hip region, base of tail, prepuce and limbs.

Cattle: eyes, horn, ear, vulva

Macroscopically: tumor is fast growing and has a papillary or cauliflower like appearance having broad base. It is soft and has grayish or pink appearance.

Macroscopically:  In squamous cell carcinoma of skin, all the layers of epidermis may be found. Cells of stratum germinativum proliferates and later on these cells become prickle cells of stratum spinosum. The basal layer is very much thickened. Keratinised layer of the cells is seen in the centre. As layer after layer of kerato-hyaline is deposited concentrically by progressive maturation of proliferating epithelial cells forming “pearls” or “cell-nests” which are characteristics. The neoplastic cells are polyhedral with prickle border and large nuclei. Mitotic figures are seen in large numbers.

3) Adenoma

Benign tumor of glandular epithelium. Adenoma can arise from any glands (with and without ducts) in the body. But they are more frequently seen in the following locations.

Dogs : Mammary, sebaceous, prostrate and bronchial glands.

Horse: Thyroid and sebaceous glands

Cattle: multiple adenoma of pancreas, tumors of adrenal cortex, papillary adenoma of bladder and gall bladder.

Macroscopically – adenomas are nodular and encapsulated. They may be firm to soft, pink in colour, solitary or multiple and clearly demarcated from tissue from which they arise. In hollow organs, like intestine, stomach and bladder they may assume a polypoid shape.

Microscopically – Consists of single layer of columnar or cuboidal epithelium lining an acinus. The secretions do not drain as there is no duct. They accumulate causing atrophy of lining epithelium and thus resulting in cystic dilation of gland resulting in cysadenoma. Those tumors that show intra-dermal pappilary projections are called papillary adenomas. When cystadenoma shows papillary projections, it is called as papillary cysadenoma.

a) Sebaceous gland adenoma

Adenoma arising from the sebaseous glands are common in among aged dogs.

Site :- Skin of head, neck, eye-lids, prepuce, tail and back

Macroscopically: - The tumors are small and lobulated. Greyish-yellow in colour and greasy to touch.

Microscopically- Tumors consists of large polyhedral cells with a cytoplasm containing numerous fat droplets. Nucleus is small, round and central with a fine chromatin and two nucleoli. The cells are grouped into masses or separated by connective tissue stroma forming lobules.  

b) Perianal gland adenoma

Perianal glands ademona is benign  tumor of  modified sebaceous glands located as a ring around the anus.

Site: The tumors are subcutaneous and may be lateral to or above the anus.

Macroscopically: These are single or multi-nodular. They are firm and encapsulated.

Microscopically: The tumor consists of sheets of epithelial cells arranged in lobules which are divided from one another by delicate septa containing blood vessels. The cells are large and polyhedral with faint granular acidophilic cytoplasm. The cells are large and rounded. One or two nucleoli may be seen. Acinar formation may be seen in some tumors.

c) Sweat gland adenoma

Sweat gland adenoma is benign tumour of sweat glands of skin of face and is common in dogs.

Macroscopically : Solid nodules small in size & grey in colour. It is Hard to touch.

Microscopically : Columnar or cuboidal cells arranged in glandular fashion. Acini’s may have lumen or solid. Cells have acidophilic and faintly granular cytoplasm.Nucleus are round or oval. When secretion accumulated, then it is called as cyst adenocarcinoma / cystadenoma.

4) Adenocarcinoma

This is a malignant tumor of glandular epithelium. In these tumors, the cells are anaplastic, lose their polarity, are large and irregular with hyperchromatic nuclei. The acini may contains many layers of cells, which may often shows papillary projections in the lumen. The cells infiltrate under basement membrane and into the surrounding stroma. Mitotic figures are frequent and metastasis is common.

5) Basal cell carcinoma

It is malignant tumor of basal cells of the Malpighian layer of the epidermis or the basal layer of the hair matrix or of sebaceous glands. It is also known as “rodent” or “Jacob’s” ulcer, hair matrix carcinoma or hair cell carcinoma. Incidence: Dog, horses and cats

Site: Head is most common site

Macroscopically: Occurs singly and has broad bas. It is subcutaneous, rounded and frequently encapsulated, firm and covering the skin which may show alopecia or ulcerations.

Microscopically : The cells which are small and round with round or cigar shaped hyperchromatic nuclei may be arranged in sheets or islands with plentiful connective tissue stroma. Mitotic figures  may be seen. Prickle cells are not observed.

6) Melanoma

Melanoma is benign tumor of the specialized cells (melanoblasts) that are capable of producing melanin and mostly arises in skin.

Macroscopically : Vary in size from tiny  pieces to large tumors weighing upto 20 Kgs and are black or brown in colour. They may be rounded, nodular, flat or pedunculated, firm and smooth. On section, shiny black surface is seen.

Microscopically : consist of collection of pigment laden melanoblast. The cells are polyhedral or oval

7) Hepatocellular carcinoma

Tumor arising from hepatic cells.

Macroscopically: Tumors may be small or large. They usually project out on the surface as brownish or greenish nodules. The nodules may be round or ovoid and are usually clearly demarcated from healthy areas.

Microscopically: Hepatic cells arranged usually in columns. These cells are large and polyhedral with usually acidophilic granular cytoplasm but has tendency towards basophillia. The nucleus is large, central and pale staining. Mitotic figures may be numerous. Tumor giant cells may be seen with two or more nuclei. The new growth is devoid of bile ducts and so stasis of bile formed gives the tumor a green colour. Tumor is divided into lobules by connective tissue septa.

8) Cholangiocellular carcinoma

Tumor arising from bile ducts.

Macroscopically: The bile ducts carcinomas are small and multiple. They may be round and not encapsulated. Yellowish white in colour.

Microscopically: The tumor consists of acini lined by columnar cells and contains mucin. In some places,  cyst like spaces which may be filled with solid masses of neoplastic cells.  Nucleus located at base of cells. Cells are surrounded by collagen stroma.

 II) Connective tissue tumors

     (Non- epithelial neoplasm)  

1) Lipoma

Lipoma is benign tumor of fat cells.

It is seen in the tissues of subcutis, subserosa, mesentery, and sub-mucosa.

Macroscopically – It is vary in size from small nodules to big masses. They are spherical or may be lobulated or pedunculated or encapsulated and may be firm or soft. On section, surface is oily and translucent and may be white or yellow in colour. 

Microscopically- Tumors consists of closely packed cells. The cells may be polyhedral and contains single large fat globule or several small ones. The nuclei may pushed to periphery.

Variable amount of connective tissues divide the tumor into lobules.

2) Liposarcoma

Liposarcoma is malignant tumor of fat cells.

Macroscopically: these are much more variable in appearance. They may be soft or firm and have a definite nodular appearance.

Microscioically: tumor cells are much more difficult to identify as anaplastic cells have little fat. They may be round, stellate, spindle shaped or polyhedral. Small globules of fat can be identified only with fat stains. The nuclei are very large. Mitotic figures and giant cells are very common.

3) Fibroma

Fibroma is a benign tumor of mature fibrous connective tissue.

Depending upon the amount of collagenous fibres, fibroma is classified as hard ( Fibroma duram) in which fibres predominate or Soft ( Fibroma motle ) in which cells predominate and fibril are few.

Incidence : common in horses and dogs

Site of occurrence : Arise from any place where connective tissue is present. But more often it is found in the subcutis of head, neck, shoulder and legs.

Macroscopically- hard fibroma is round and firm. On section, the surface is dry and white. Soft fibroma are softer and oedematous which reveals a pink surface on section.

Microscopically – Fibroma consists of interlacing bundles of fibrous connective tissue, which runs in all directions. The nuclei of fibroblasts are spindle shaped. Blood vessels and variable numbers of lymphocytes, monocytes  and eiosinophills are seen.

4) Fibrosarcoma

Fibrosarcoma is a malignant tumor arising from fibrous connective tissue. The tumors may arise from the places where fibroma occurs.

It occurs in horses, cattle, dogs and fowls.

Macroscopically – It is irregular in shape and have nodular appearance. These tumors have abundant blood supply. Those found on the body surface, may ulcerate and emit a foul smell.

Microscopically- arrangement of cells are similar to that of a fibroma, but the cells show features of malignancy like darkly staining nuclei. Plentiful blood supply is found.

5) Myxoma

It is benign tumor of the specialized fibrous tissue that are capable of producing mucin.

Macroscopically : Tumors are rounded and appear as a bunch of grapes. They may be encapsulated and are slimy to touch because of mucin content.

Microscopically : The tumors consists of cells which are spindle shaped or stellate with long processes and lying in a basophilic mucinous matrix. Pleomorphism is evident.

6) Osteoma & osteosarcoma

Osteoma is benign and osteosarcoma is malignant and hard tumour of bone.

Flat bones- Compact osteoma

Long bones- Spongy osteoma

Macroscopically

It is found on skull, scapula and pelvic bones which is nodular and encapsulated.

Round or ovoid in shape and white, yellow or pink in colour.

Microscopically

Osteoid tissue with lamellar arrangement is seen.

Osteosarcoma have pleomorphic cells & shows Tumour giant cells, several mitotic figures and blood vessels are seen.

7) Chondroma  

Benign tumor derived from cartilage cells.  Cartilage is formed by the proliferation of fibroblasts of the peri-chondrium and their subsequent differentiation into cartilage cells. Dogs are most commonly affected among animals. Epiphysis of long bones, chondro-costal and chondro-sternal articulations as well as the bronchi, trachea and larynx are mostly affected.

Macroscopically- It is large in size, multinodular, encapsulated, rounded and bluish white. On section, shows translucent appearance. Foci of calcification may be seen.

Microscopically: Consists of round or ovoid cell set in a bluish matrix. These cells arranged singly but not in groups of four or eight as in the normal cartilage. Strands of fibrous tissue separate the tumor into lobules.

8) Chondrosarcoma  is more cellular and its cell often show pleomorphism. At the periphery may be seen the immature spindle shaped cell while at the centre may be found fully differentiated cartilage cells. Mitosis is frequent.

9) Leiomyoma & Leiomyosarcoma

Leiomyoma is a benign and leiomyosarcoma is malignant neoplasm of smooth muscles especially of the hollow organs like – uterus, vagina, intestine, stomach, urinary bladder and oesophagus which is common in cow, dog and fowl.

Macroscopically: Tumors are smaller, encapsulated, firm and lobulated and pink in colour on section.

Microscopically: consists of plain muscle bundles arranged in all directions and planes. The bundles are separated by strands of fibrous tissue in which plentiful blood supply is present. The muscle fibers are spindle shape and are arranged in parallel to each other which have ribbon shaped nucleus with rounded ends (cigar- shaped).

Leiomyosarcoma : have anaplastic cells and  shows mitotic figures.

10) Rhabdomyoma & Rhabdomyosarcoma

Rhabdomyoma is a benign and rhabdomyosarcoma is malignant tumor of skeletal and cardiac muscles and are rare in animals.

Macroscopically : Found in tongue, chest, sternal muscles, neck region, cardiac muscles and have broad based.

Microscopically: Striated muscles grow on different directions and fibrous tissue divides the muscle bundles into lobules.

In Rhabdomyosarcoma, the anaplastic cells are pleomorphic, polyhedral and spindle shaped and tumour giant cells are seen.

11) Haemangioma & haemangiosarcoma

Haemangioma is benign tumour of blood vessels while haemangiosarcoma is malignant tumour of blood vessels.

Incidence: occurs in cattle, horse, sheep, dogs, swine & fowl. Liver is most oftenly affected. Spleen, limbs, perineal region, thorax & vagina are the other parts affected. Depending upon the relative presence of blood spaces and cellular tissue, it is divided in to three varieties.

1) Hemanigioma simplex or Capillary type – in which capillary are more or less in uniform in size.

2) Hemangioma cavernosum or Cavernous Hemangioma- composed of capillaries that are greatly dilated.

3) Hemangioma Hypertrophicum or solid hemangioma – consists of masses of endothelial cells without any vascular lumen.

Macroscopically – These are dark red to purple in colour and soft in consistency. These bleeds when injured.

Microscopically- More vascular type of hemangioma consists of newly formed many capillaries lined by single layer of endothelial cells. Capillaries or blood filled spaces may be seen. Sometimes, lumen may be filled completely with proliferating endothelial cells.

12) Mesothelioma

It is a tumor of mesothelial lining cells of serous cavities specially of peritoneum and pleura. Such tumours are found in thorax and abdomen.

Macroscopically: it is pink in colour, hard, multiple, nodular and scattered in cavity. Found in thorax and/or abdominal cavity

Microscopically: Collection of cells resembling epithelial cells. Cells have acidophilic granular cytoplasm and large vesicular nucleus. Numerous blood vessels in tumor. A core of fibrous tissue present. Mitotic figures may be seen.

13) Mastocytoma

It  is a tumour of mast cells commonly seen in dogs in subcutaneous tissue of hind legs.

Macroscopically: Usually single and have a size of about 8-12 cm diameter, nodular, pedunculated, hard, pink or grey in colour. Ulceration over tumour is common

Microscopically: Pleomorphic mast cells. Inflammatory cells like neutrophils and lymphocytes are also seen. Cells are darkly stained. Cytoplasm is more than in lymphocytes. No or little cytoplasmic granules & few mitotic figures are seen.

14) Lymphoma  : is a benign neoplasm of lymphoid cells primarily found in lymph nodes.

15) Lymphosarcoma / Leukemia / Hodgkin’s Disease:

Malignant tumor of the lymphoid tissue, is common tumor of the dog, cat, cattle and pigs.

Site: Tumor primarily affects the lymph nodes – peripheral and visceral lymph nodes.

Macroscopically- the affected lymph glands are enlarged. These do not exhibit the contrast between the cortex and medulla being diffusely pale and soft. Bone marrow os replaced by the tumor cells and so appears pale. The metastasis that occurs in other organs are nodular and white.

Microscopically:

In Lymphocytic form: The neoplastic cells are the lymphocytes. These may be either normal lymphocytes or immature forms. The immature cell ( lymphoblast) is large and polyhedral with the large nucleus placed eccentrically. The cytoplasm stains blue. Mitotic figures are present.

In Histiocytic type : reticulum cell sarcoma, the cells are large and pleomorphic with a vesicular nucleus which may show indentation or may be triangular. Mitotic figures are frequent. Reed –Sternberg giant cells may be present.

In Hodgkin’s disease type: There is mixture of lymphoid cells, reticulum cells, fibroblasts and eosinophils. The characteristics cell to be seen is the Reed –Sternberg giant cells with two “mirror –image” nuclei.

III) Others

1) Veneral Granuloma

Commonly seen in dog and bitch. It is transmitted by transplantation of tumor cells during coitus and found on the glans penis and prepuce in male and in vagina in female.

Macroscopically: Tumors may be solitary or multiple, small or large, pedunculated or may spread like cauliflower, pink in colour and very soft.

Microscopically: Section reveals a very cellular tumor, comprising of cells, uniform in size and shape. The cells are round to polyhedral, having a finely granular acidophilic cytoplasm. The nucleus is large, round, central and hyperchromatic. Mitotic figures are present.

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