COLLECTION. PRESERVATION AND DISPATCH OF MATERIAL TO FORENSIC /PATHOLOGICAL LABORATORY EXAMINATIONS IN VETROLEGAL CASES
Post no-533 Dt-15/01/2018
Compiled & shared by-DR RAJESH KUMAR SINGH ,JAMSHEDPUR, 9431309542,rajeshsinghvet@gmail.com
All the pathological processes bring about the morphological and other subtle alterations in tissues/organs. These alterations/lesions are suggestive of probable etiological agent(s) and in others indicate the ill-effects of the disease/ailments. In study of pathology, as the difference in structure between diseased and normal tissues is often slight, the accurate and precise diagnosis of many lesions encountered depend upon not only the theoretical knowledge of fundamentals but also the carefulness and precision with which the technique/methodology is followed at every stage. That is, from collection of tissues/lesions, preservation and despatch while forwarding the materials to the laboratory for further processing and their interpretation.
It is obligatory in each and every important case, the morbid materials should be fully representative of the organs and the lesion for laboratory investigation as well as it would be available as a referral material to the professionals in veterinary field. Most valuable specimens, is at times wasted on its receipt at the laboratory in state of either petrifaction or incomplete preservation due to error in the methodology employed.
For meaningful results, the sample must have following qualities:
1. Correct sampling
2. Correct preservation
3. Correct labelling and identification
A. Collection
Tissue should be collected as far as possible immediately after death of animals to avoid autolytic changes.
a. Selection: Following criteria may be considered
(i) Tissue-fully representative of the organ and the lesion.
(ii) Youngest the lesion present or the junction of lesion adjacent appareutly healthy
tissue.
(iii) Two or 3 such areas should be selected to enable the study for ageing process.
Similar thing for biopsy (tumours etc.)
b. Excision: Selected area of organ/lesion should be incised and 3-4 slices should be made, with care that each piece represents the lesion to be diagnosed.
c. Size of the tissue: Very thin sections not exceeding 0.5cm. in thickness, ensure satisfactory fixation and preservation. If large mass of material is desired to be sent then slices are made not exceeding the above thickness.
B. Preservation
(i) Receptacles: Wide mouth glass bottles of varying capacity should be preferred. Penicillin vials could be used provided pieces of tissues of desired thickness and measurement are taken conveniently.
(ii) Fixing and preservative fluid: For routine the best is 10% formalin solution in water or saline Nervous tissue-pieces of tissues from different portion of brain in suspected rabies cases, absolute alcohol or methylated spirit may be used. Special preservative like Zenker’s fluid etc may be used whenever necessary and available.
Quantity: 20-50 times more preservative may be used as such as the bulk of material with exception of the chromium-osmium fixative (where 5-10 times of the bulk of the tissues)
C. Dispatch
(i) Labelling: Done by putting a paper label marked by ordinary lead pencil inside the specimen and similar label pasted on outside the bottle. The label should carry the information- (a) Case number, (b) Name of the organ. A piece of absorbent cotton should invariably be placed to keep the tissue moist, in case the bottle is broken during transit.
(ii) Sealing: Mouth of bottle sealed by paraffin so as to make it water light.
(iii) Packing: Sealed and labelled receptacles containing specimens packed in suitable container along with history sheet. Enough packing material should be provided to ensure safe delivery of the contents in the laboratory.
Precaution in forwarding the materials
The following methods are erroneous and must be avoided:
a. Sending large pieces of tissue such as whole organ. Only surface would be
preserved and interior undergo autolysis sending the tissue unfit for exam.
b. Forcing large pieces of tissues or whole organ in small receptacles-with harding
effect of fixation like formalin-impossible to remove the material without breaking bottle, moreover tissues are mutilated.
c. Sending material from centre of an extensive lesion like suppuration-which will
not give maximum information.
d. Materials sent without proper labelling and history-likely to mislead diagnosis of
the case.
Particulars of materials/specimens
Following clinical and morbid materials are to be collected as per under mentioned particulars:
1. Blood: Blood is examined for different purposes as listed below :
(i) Preparation of blood smears of films for microscopic examination: The ear vein is the most convenient site for taking blood when required for the preparation of smears or films. In poultry, blood is taken from comb or wattles or wing veins.
(ii) For cultural examination, transmission experiments and other studies: In cows, buffaloes, horses, sheep and goats, blood is preferably taken from the jugular vein. From pig, blood is usually obtained by cutting the end of the tail or tip of the ear, or from anterior vena cava. in dogs and cats, from the saphenous or radial vein and from fowls, brachial vein (on the ventral surface of the wing). The site should be prepared by removing hair (feathers) and disinfecting it with spirit. Aseptic precautions are taken while collecting for cultural examination and transmission experiments. Blood is collected by a Pasteur pipette or sterile syringe. From post-mortem cases, blood is collected from right ventricle.
For serological studies, serum is procured by allowing the blood to clot, keeping it in .a sloping position. Serum is removed in clean tube. To preserve serum, mertheolate in 1:10,000 may be added. For haematological studies, blood is not allowed to clot using anticoagulant (sodium citrate, potassium oxlate or EDTA). One part of anti-coagulant is added to 9 parts of blood.
2. Milk: Milk for bacteriological examinations collected in sterile container. For isolation, milk is collected after discarding the first few streams of milk. For tuberculous and brucella organisms, the strippings are preferred.
3. Faeces: Sample for examination of parasitic eggs or coccidial cocysts are collected from the rectum or at the time of defecation.
4. Urine: Urine is collected at the time of micturition or obtained with the help of a clean and sterile catheter.
5. Pus: Smear is made on a clean glass slide. For cultural examination, it is obtained directly from the abscess in a sterile container, or sterile swab is used.
6. Nasal/throat/uterine discharges: To be collected on sterile cotton swabs, sealed in screw cap test tubes.
7. Exudates/transudates and body fluids: Collection has to be made with the help of sterile pipettes or syringes and to be despatched in leak proof screw cap containers.
8. Tissue, blood and exudates smears: Tissue smears are to be prepared from cut surfaces of organs and fixed over a flame. Blood/exudates smears are drawn as thin film and fixed over a flame or in methyl alcohol.
9. Intestinal contents: Intestinal loop with contents tied at both ends are collected and preserved either on ice or 25% glycerine saline. Intestinal contents may also be collected directly in screw cap in test tubes and preserved with few drops of chloroform.
10. Hairs and skin scrapings: Hairs and skin scrapings are to be collected from the margins of cutaneous lesions with a blunt scalpel and packed in non-absorbent paper or dry screw cap vials/test tubes.
Preservation of materials for specific examination 1.
Bacteriological/Mycological
Blood and exudates: are to be collected aseptically in sterile Pasteur pipettes, tubes or vials without any preservative and to be transported over ice.
(ii) Tissues: Pieces of affected organs with lesions should be collected under sterile
precaution and preserved in 25% glycerine saline or on ice.
2. Virological
(i) Blood and exudates: to be collected aseptically without any chemical preservative
and to be stored and transported on ice. In some viral infections (e.g. Blue Tongue, blood may be collected on Heparin or Oxalate citrate buffer/OCG).
(ii) Tissues: Pieces of affected organs are to be collected either on ice or 50% buffer
glycerine saline.
3. Parasitological
(i) For identification of parasite and helminth ova 4 to 10% formalin.
(ii) For coccidial oocysts 2.5% Potassium dichromate solution.
4. Serological tests: Serum samples to be preserved with 4 to 5% Phenol or 0.1%
merthiolate (1 part preservative in 9 parts serum).
5. Histopathological
(i) For routine and general histopathological examination tissue pieces are to be
collected in 10% formal saline (collection to be done in wide mouth bottles with
10 times the volume of tissues).
(ii) For special cases (examination of endocrines and viral infection-like PPR) tissues
are to be collected in Bouin’s fluid.
SPECIMENS REQUIRED FOR LABORATORY EXAMINATIONS
I. INFECTIOUS DISEASES
A. Bacterial
1. Anthrax: In case of horse, pig, dog and cat, smears from sanguinous discharge/
excretion of natural orifices as well. Swab of blood (from the ear vein) or exudates. Pieces of skin or spleen for Ascoli’s test (if putrefaction has set it).
2. Actinomycosis and Actinobacillosis: Pus smears from the deeper part of the
lesions. Affected organs tissue both on ice and in 10% formal saline.
3. Bacillary haemoglobinurea: Portions of liver showing lesions on ice and in formal
saline separately.
4. Black disease: Duplicate samples of necrotic portion of liver on ice and in formal
saline (10%).
5. Black quarter and Malignant oedema: Smears of haemorrhagic muscle
exudates from a freshly dead animal. Portion of affected muscle, air dried (or on
ice) and in formalin (10%) separately.
6. Botulism: Suspected food and intestinal contents on ice.
7. Brucellosis: Blood and serum samples. Quarter milk samples. In case of abortion,
foetus, or foetal stomach contents and heart blood on ice.
8. Chronic Respiratory Disease (CRD): Affected live birds or dead birds packed
on ice. Serum samples.
9. Enterotoxaemia: Smears from bowel mucous membrane and intestinal loop or
samples of ingesta from duodenum, jejunum and ileum. Ingesta to be preserved with 3-4 drops of chloroform (formalin should not be used).
10. Fowl cholera: Affected birds (chronic cases). Heart blood, liver and spleen on
ice.
11. Fowl Spirochaetosis: Sick birds or blood smears from such birds.
12. Fowl typhoid: Heart blood, liver and spleen on ice or recently dead bird on ice.
13. Glanders: Swabs from nasal and skin lesions. Lungs and other organs showing
lesions on ice and in formal saline (10%).
14. Johne’s disease: Smears of rectal mucosa scrapings. From dead animals,
portions of intestine showing lesions and associated lymph nodes on ice and in
formal saline separately.
15. Leptospirosis: Portions of kidney and liver on ice and in formal saline (10%).
Blood (collected during febrile stage of the disease) on ice. Freshly voided urine on ice. Serum samples.
16. Listeriosis: In the case of abortion, foetus, or foetal stomach and heart, on ice.
In the cases of encephalitis (circling disease) or septicaemia, portions of brain,
liver, spleen and kidney on ice and in formal saline separately.
17. Pasteurellosis, Haemorrhagic Septicaemia and Swine plague: Blood smears
or exudates obtained with the help of sterile syringe and needle from oedematous
swelling. Heart blood, portions of liver, spleen, kidney and lungs showing
pneumonic lesions on ice. Long bones packed in charcoal. Pieces of lung and
other affected organs in 10% formal saline.
18. Contagious Bovine or Contagious Caprine Pleuropneumonia (CBPP/CCPP):
Blood, serum portions of affected lungs and associated lymph nodes on ice and
also in formal saline (10%) pleural fluid.
19. Pneumonia due to other infectious agents: Portion of diseased lungs and
associated lymph nodes on ice, portion of lung in glycerine saline and portion of
diseased lung in formal 10% saline. Blood and serum.
20. Pollurum disease: Blood sera from adult birds. Freshly dead chick or heart
blood and liver, on ice. Pieces of affected organs in 10% formal saline.
21. Pyosepticaemia neonatorum (septicaemia in new born animals) and
Paratyphoid in animals: Mesenteric lymph nodes and portions of kidney, spleen and liver on ice. Portion of bowel in formal saline (10%).
22. Swine erysipelas: In acute cases, heart blood, portions of liver, kidney and
spleen on ice. In chronic cases, affected joints (without opening) on ice. Organs
showing lesions in formal saline (10%) serum samples.
23. Tuberculosis: Portions of lesions unpreserved on ice or in 25% glycerine saline
and in formal saline (10%).
24. Ulcerative lymphangitis and Caseous lymphadenitis: Swabs and smears from
the lesions. In case of caseous lymphdenitis, pieces of affected organs and lymph
nodes also on ice or 25% glycerine saline and in formal saline (10%).
25. Vibriosis: Foetus or foetal stomach over ice. Portion of placenta and uterine
discharge on ice.
B. Viral and Rickettsial
26. African horse sickness: Blood, spleen and lungs in 50% glycerine containing
0.5% sodium citrate and 0.5% carbolic acid. Blood in EDTA. Blood for serum samples.
27. Avian encephalomyelitis and Infectious porcine encephalomyelitis (Technen
disease): Sick birds or animals. Brain and spinal cord in formal saline (10%)
and buffered glycerine separately. Serum from convalescent cases.
28. Avian leucosis complex. Affected birds or portions of liver, spleen and nerve if
involved and other organs showing lesions in formal saline.
29. Canine distemper. Pieces of liver and spleen on ice. Portions of urinary bladder,
kidney, liver, lung and trachea in formal saline.
30. Encephalitides arthropod borne: St. Louis, Western equine, Eastern equine,
Japanese-B, Venezulean, Russian spring summer and Louping ill: Serum
from acute and convalescent cases. Defibrinated or oxalated blood, portions of
brain including medulla and spinal cord from freshly dead carcasses in buffered
glycerine as well as in formal saline.
31. Equine abortion: Portions of placenta and foetal organs (liver and spleen) in
glycerine saline. 50% foetal stomach and heart blood on ice. Pieces of liver,
spleen, lungs and trachea of foetus in formal saline. 10% serum of the mare.
Foot and mouth disease, Vescicular stomatitis and Vesicular exanthema:
Epithelium from the vesicles along with fluid in phosphate buffered glycerine and in saline tissue culture medium containing antibiotics. Paired sera samples at early and late stages. Pieces of oral tissue including tongue and meat in 10% formal saline.
33. Infectious bronchitis: Lungs including trachea, spleen and heart blood in
buffered glycerine and on ice. Serum samples particularly from old cases. Lungs
and trachea in formal saline (10%).
34. Infectious laryngotracheitis: Live birds in early stages of the disease or trachea
including tracheal exudates (from early sacrificed case) in 50% glycerine saline.
Portion of trachea in formal saline.
35. Pox in sheep, goats, pigs and cattle and Contagious pustular dermatitis
(Contagious ecthyma) in sheep and goats: Scabs in 50% glycerine saline or unpreserved. Pieces of skin and other organs with lesion in 10% formal saline. Blood (at febrile stage).
36. Fowl pox. Scabs in 50% glycerine saline or unpreserved. Cutaneous lesion in
10% formal saline.
37. Pseudorabies: Brain and spinal cord in glycerine saline as well as in formal
saline (10%).
38. Psittacosis and Ornithosis: Serum from acute and convalescent cases. Whole
blood on ice. Dead or sacrificed bird wrapped with Lysol soaked cloth and packed
over ice. Pieces of affected organs in 10% formal saline.
39. Rabies: Head packed over ice. Brain half of it, divided longitudinally in formal
saline 10% and half in 50% glycerine saline.
40. Ranikhet disease (New Castle disease): Freshly dead carcase packed over
ice, or brain, spleen and liver in glycerine saline.
41. Rinderpest and Mucosal disease complex: Defibrinated or citrated heparinised
blood when the animal is running high temperature. Pieces of lymph nodes in
mesenteric tonsils and spleen on ice or in buffered glycerine. Pieces of spleen
lymph node, intestine, oral mucosa, liver, lungs and kidney in 15% buffered saline
or Bouin’s fluid.
42. Swine fever Defibrinated blood from living animals. Heart, blood, liver, tonsils
and pancreas and spleen on ice, Organs (including brain) showing lesions in
formal saline. 10%
43. Swine influenza and Viral pneumonia of pigs: Portion of lung showing
pneumonia patch on ice, in buffered glycerine (50%) and in formal saline (10%)
separately. Material should be collected after destroying the animal in early stage
of the disease.
44. Blue tongue: Blood in heparin or OCG, Heart blood, bone marrow, pieces of
spleen, liver on ice. Serum sample paired skeletal muscles and heart muscle in
10% formalin.
45. Peste des petits ruminants (PPR): Eye, mouth and rectal swabs on ice, about
10 ml. or more blood at the height of body temperature in anti coagulant. Pieces of spleen (10- 30 gm.), small intestine, mesenteric lymph nodes and lung on ice or buffered glycerine. Pieces of affected lung, spleen, small intestine (Payer’s patches), mesenteric lymph nodes in 10% formalin saline. Materials from 5 or 6 or more animals be collected and dispatched for better picture of diseases/ outbreaks.
C. Parasitic
46. Anaplasmosis: Thin blood films, citrated or oxalated blood from sick animals.
47. Coccidiosis: Faeces in formalin and for in pot. Dichromate solution. Portion of
intestine showing lesions in formal saline.
48. Piroplasmosis: Thin blood smears, heart blood, spleen and kidney smears from
dead animals. Citrated or oxalated blood.
49. Theileriosis: Thin blood films. Smears or biopsy material from prescapular lymph
node. Portions of lymph node, liver and kidney on ice and in formal saline 10%.
50. Trichmoniasis: Uterine discharge (collected within 24 hours of abortion or during
heat period) on ice.
51. Trypanosomiasis: Citrated blood. Blood films. In camel, cattle and buffaloes,
serum as well.
52. Parasitic infections: Faecal samples in formalin. From dead animals,
unpreserved stomach or abomasum and small and large intestine, if possible, otherwise their contents in formalin. In case of lambs, kids, piglets and fowls, whole carcass or sick animal itself.
53. Mange: Scabs and deep skin scrapings.
D. Fungal
54. Fungal diseases (in general): Portions of organs/tissues showing lesions on
ice and in formal saline 10% separately.
55. Aspergillosis of fowls (Brooder’s pneumonia): Affected birds. Lungs along
with caseous masses from the air sacs over ice. Lungs in formal saline 10%.
56. Ring worm: Skin scrapings from the lesions, including some hair roots,
unpreserved in a tightly stopper container.
II. TUMOURS
Entire tumour, if small in size (less than 0.5 inch in diameter) or sliced portion
of it may be fixed in 10% formalin solution.
III. NON-INFECTIOUS DISEASES
1. Rickets:
a. 5 ml serum with a drop of saline.
b. 50-100 g bone as such.
c. Costo-chondral junction., vetrebra, long bone and tooth in 10% formal saline.
2. Osteodystrophic fibrosa:
a. 5 ml serum with a drop of saline.
b. 50-100 g bone as such
c. Mandible and long bone in 10% formal saline.
3. Osteomalacia:
a. 5 ml serum with a drop of saline
b. 50-100 g bone as such
c. Long bone and Vertebra in 10% formal saline.
4. Sway back/enzootic ataxia:
a. 10% oxalated blood
b. 10 g of liver tissue in rectified spirit.
c. Brain, spinal cord, heart, liver, spleen and kidney in 10% formal saline.
5. Goitre:
a. 20 ml blood plasma
b. Thyroid gland, both in rectified spirit and 10% formal saline.
6. Fluorosis:
a. 20 ml blood plasma.
b. 100 ml of fresh urine and 500 ml of suspected drinking water.
c. Average size piece of pelvic bone and molar teeth as such.
d. Pieces of bone preferably having exostosis, vertebra and costo-chondral junction
in 10% formal saline.
7. Ketosis:
a. 10 ml of serum.
b. Pieces of liver, kidney and heart in formalin.
8. Alkali disease (Selenosis):
a. As for goitre
b. 50-100 g of liver and kidney in rectified spirit.
c. 5-10 g hair from live animal.
9. Parakeratosis:
a. As for goitre
b. 50-100 g each of kidney, pancreas, liver in rectified saline, oesophagus and
testes in 10% formalin.
10. Diabetes mellitus:
a. 5 ml of blood with sodium fluoride as anticoagulant.
b. Pancreas, liver and kidney in 10% formai saline.
11. Muscular dystrophy:
a. 20 ml of blood plasma
b. Heart and skeletal muscle
separately.
12. Avitaminosis-A:
a. 10 ml citrated blood.
b. Liver 10 g in 40 ml of 10% KOH solution.
c. Parotid gland (in bovines), trachea, oesophagus, lung, kidney, optic nerve and
testes in 10% formal saline.
13. Athyminosis: Blood serum for the estimation of thiamine and pyruvic acid.
14. Molybdenum Toxicity:
a. 10 to 20m1 oxalated whole blood
b. 5-10 g each of liver, kidney and bone preserved in rectified spirit.
15. Iron toxicity/deficiency:
a. Whole blood.
b. 5-10 g of liver, preserved in rectified spirit and bone marrow in 10% formal saline.
16. Poisoning (Chemical/Plant): Duplicate samples of (i) pieces of liver, spleen,
kidney and other organs showing lesions. (ii) Pieces of stomach wall and, stomach
contents and (iii) Portion of the bowel showing lesions, alongwith the contents, in
COLLECTION, PRESERVATION AND DISPATCH OF MATERIAL TO FORENSIC LABORATORY
The collection, preservation and dispatch of different tissues/organs, fluids and viscera should be done as described in section 4 of appendix. However, in veterolegal cases, these materials should be sent to forensic laboratory under sealed packings. In the suspected cases of toxic condition or• poisoning, the stomach and intestinal contents should be sent after proper ligation at both the ends and sent it in ice to avoid putrefaction. Besides, samples of blood, liver, spleen and kidneys should be sent in separate container. All the materials should be collected in leak• proof glass or plastic bottles. Tissues for histopathology must be collected in• 10% formalin or formol saline, this can be sent to laboratory under normal temperature. The materials suspected for toxicity should be• sent in ice without adding any preservative. The bottles or containers should be sealed and• labelled properly indicating the name of owner, identification of animal (number, name, mark etc.), type of tissue collected and
preservative used. The examination requested and disease or poisoning suspected should also be written. A copy with details of post-mortem report and• containing above information should be sent separately under separate cover. The address of the forensic laboratory should• be clearly written. All the containers should be packed with cloth• and sealed with sealing wax and should preferably be sent through person in order to avoid any breakage in transit. One copy of the forwarding letter should be• kept in file for future reference and one copy should accompany the material and one copy should be sent by post. The forwarding letter bearing number and date should have the information about materials sent, type of preservative used, type of examination requested and identification of animals including other details of owner.
