FIELD GUIDE TO SUBMISSION OF SPECIMEN

[ General Notes ]  [ Laboratory tests ] [ Pathology ] [ Avian Submissions ] [ Parasitology & Hematology ] [ Bacteriology] [ Selection of Tissues For Microbiology ] [ Submission of Samples For Serological Tests ] [ Virology ] [ Serology/Virology ] [ Toxicology ] [ Rabies Submission ]
[ Submission of Specimens According to Main Clinical Signs ]

INTRODUCTION

The role of a diagnostic laboratory is to complement the work of the field officer; to assist him in arriving at a diagnosis or to confirm a tentative diagnosis made in the field so that appropriate action can be taken to prevent losses and thus increase productivity. Certain general principles and recommendations should be followed if specimens submitted are to be of any value for laboratory investigation.

VISION OF DEPARTMENT OF VETERINARY SERVICES

The animal industry becomes a fully integrated commercial sector producing quality food from animal products towards national self-sufficiency and export.

MISSION OF DEPARTMENT OF VETERINARY SERVICES

We are committed to the growth of a sound animal industry to supply quality and safe food from animal products for national consumption and export through the provision of quality veterinary services from motivated, skilled and creative individuals.

OBJECTIVES OF DEPARTMENT OF VETERINARY SERVICES

1. To prevent, control and eradicate animal and zoonotic diseases
2. To promote the growth and development of a sound animal industry.
3. To ensure that food of animal origin are clean, wholesome and fit for human
    consumption
4. To promote the growth and development of animal feed industry
5. To ensure the welfare and wellbeing of all animals.

MISSION OF REGIONAL VETERINARY LABORATORY SERVICES

The mission of the Regional Veterinary Laboratory (RVL) is to promote healthy livestock and companion animals and to ensure safe animal products for the consumer by assisting farmers, veterinarians, their clients, and others responsible for animal health in the detection and prevention of disease by providing accessible, accountable, timely and accurate diagnostic services.

FUNCTIONS OF THE LABORATORIES

1. Diagnosis of animal diseases
2. Monitoring of animal health status
3. Monitoring of pathogens
4. Provide disease information
5. Provide support for training to entrepreneurs in animal industry and
    departmental staffs in animal health.

PURPOSE OF THIS MANUAL

The purpose of this manual is to guide veterinary practitioners and assistants in selections, preservation, and delivery of specimens to the RVL for more timely and accurate evaluations and tests. We hope that this guide will be useful to you in your daily routine of attending to animals in good health, but much more importantly when diseased or deceased. On our part we can only assess its value by an improvement in the quality of future submissions and by your response in the form of criticisms which will help us to improve this guide.

LOCATIONS OF REGIONAL VETERINARY LABORATORIES IN MALAYSIA

The laboratory services are available in six strategic locations in Peninsular Malaysia. The locations of the RVLs are as follows:

(1) RVL Bukit Tengah - serving the northern states of the Peninsular Malaysia
     such as Perlis, Kedah, Penang and north Perak.
(2) RVL Petaling Jaya - serving the central states of the Peninsular Malaysia
     such as south Perak, Selangor, Negeri Sembilan, Malacca, east Pahang
     and Kuala Lumpur.
(3) RVL Johor Baharu - serving the southern states of the Peninsular Malaysia
     such as Johore.
(4) RVL Kuantan - serving the east coast states of the Peninsular Malaysia
     such as Pahang, Kelantan and south Terengganu.
(5) RVL Ipoh (attached to the Veterinary Research Institute) - serving the state
     of Perak
(6) RVL Kota Bharu - serving the east coast states of the Peninsular Malaysia
     such as Kelantan and north Terengganu.

In addition to the six RVLs in Peninsular Malaysia, there are also one Animal Disease Research Centre (ADRC) in Sabah and one State Veterinary Diagnostic Laboratory (SVDL) in Sarawak. RVL Kota Bharu is a reference laboratory for FMD monitoring and diagnosis using ELISA to detect antigen and antibody. Veterinary Research Institute (VRI) also acts as a reference laboratory for RVL for further laboratory test and confirmation. Certain tests such as Brucella CFT, Johne's CFT, Leptospira MAT, Nipah virus ELISA and Salmonella serotyping are carried out in VRI.

DIRECTORY OF VETERINARY LABORATORIES

It is important that all specimens collected should arrive at the laboratory in a state as similar as possible to that in which it existed in the animal body just before death. All forms of distortion should be prevented and include such things as bacterial contamination, autolytic changes and putrefaction.

The specimen submitted should always be correctly labeled. The labeling should not come off in the presence of water or some other liquids.

Packing of the specimens must be done in a manner to prevent leakage so that dissemination of infective agent does not occur. Plastic bags, wax paper and glass container may be used. These should then be suitably packed in boxes/thermos flasks to prevent breakage while in transit. Non-disposable containers will be returned; so make sure that your own address is on them.

The specimens should be sent by the fastest means of transports to the laboratory nearest to you. Specimens should preferably arrive during office hours. Where specimens are due to arrive outside office hours or on public holidays, the laboratory concerned should be notified in advance so that arrangement can be made to collect them.

The appropriate submission form, duly filled in must accompany all specimen submission.

1.  MAKVET 01 Mammalian submission form
2.  MAKVET 02 Avian submission form
3.  MAKVET 03 Serological submission form
4.  MAKVET 04 Feed samples submission form
5.  MAKVET 05 Inter laboratory submission form
6.  MAKVET 05 Rabies sample submission form
7.  MAKVET 06 Submission form for toxicology
8.  MAKVET 07 Laboratory report for all samples
9.  MAKVET 08 Laboratory report for biochemistry
10. MAKVET 09 Submission form for National Pullorum Programme samples
11. MAKVET 10 Aquatic animal submission form
 

CARCASS SUBMISSION

1. General Information

Whenever possible, submit the entire carcass to the laboratory. Ideally, the carcass should be submitted to the laboratory as soon as possible after death. If submission is unavoidably delayed, the carcass should be kept cool but not frozen. Please, DO NOT FREEZE CARCASSES FOR POST-MORTEM EXAMINATION. Freezing produces severe artifacts, which make interpretation difficult or impossible. Please include a complete clinical history with all submissions. It will be most helpful if the attending veterinarian writes the history.

1. Place in fixative as soon as possible.
2. Fix tissue slices (0.5 cm thick) 24 - 48 hours. Formalin volume must be 10 times or more than the volume of tissues.
3. Use wide-mouthed, leak proof containers. DO NOT USE NARROW-NECKED CONTAINERS: A fresh and pliable tissue can easily be put into a narrow-mouthed container, but it becomes hard when fixed, and cannot be readily removed.
4. Alternatively, tissues can be fixed in formalin solution for 24 - 48 hours, removed from solution, wrapped in formalin-soaked gauze sponge, placed in plastic bag, and sealed for transportation. This technique decreases the possibility of spillage and leakage of formalin during transportation.
5. Improper handling or fixation of tissues can induce artifacts that may result in non-diagnostic or unsuitable specimens. A few examples that cannot be corrected by processing are:

a. Chemical dehydration - disinfectants and medications applied
    to the skin may damage (burn) tissues
b. Freezing - intracellular and extra cellular ice formation causes
    cell lysis and disrupts tissues
c. Pressure - excessive pressure from forceps or digits can
    rupture cells and compress tissues
d. Delayed fixation - un-refrigerated specimens are subjected to
    dehydration, autolysis and proliferation of saprophytic
    bacteria
e. Inadequate fixation - using inadequate volume of fixative or
    trying to fix large specimens (e.g. whole kidney or testis) will
    result in incomplete fixation and autolysis.

3. Writing a Post-mortem Report

The post-mortem report should record accurately and thoroughly all observations made during the examination of the carcass. A summary of the available history, clinical observations and results of field tests conducted must be included.

OBSERVE carefully and then DESCRIBE completely. Do not interpret! Theoretically, a person with a reasonably good command of a language will be able to describe well what that person has seen while performing the postmortem examination even though he/she does not know the significance of what he/she has seen and described! To help remember points that need to be described, remember this phrase: TeN DiSC SPaCeS

Use extra paper if the space in the standard form is insufficient and clip it together with the form.

Example: The liver is tan to brown and there are multiple 2 mm to 1 cm black foci on the surface and randomly distributed in the parenchyma.

Report negative findings only if relevant eg: absence of gross lesions in animal which had shown neurological signs

REMEMBER: For every diagnosis missed because you do not know about it, you will miss nine diagnoses just because you do not observe carefully!
 

Organs such as liver, spleen and kidney should be cut into slices perpendicular to the surface to demonstrate their anatomic structure, whenever possible, one surface should consist of the natural boundary of the organ. Sliced tissue should be between 0.5 - 1.0cm thick to allow proper fixation. Please submit adequate samples and avoid small minute samples.

If lesion is small and localized, please include adjacent normal tissue as well as the lesion. This will help in identifying the tissue as well as defining the nature of spread of the lesions. In the case of large lesion, submit the entire mass with transverse cuts to allow formalin penetration, or if this is not feasible, submit appropriate sized sections from several different areas.

Submit sections of abnormal tissue and organs. Clinical history and gross pathological findings may help in the selection of tissue samples for histopathological examination. One of the most common problem for the pathologist involves the receipt of a history and/or gross description that indicates the probability of lesions in specific organ or organ system, along with several tissues, none of which originate from the involved organ! This happens often with animals showing CNS signs, as there seem to be some reluctance to remove the brain and/or spinal cord. Another common example is a case of diarrhea with no submission of faecal or gastrointestinal tract samples. In cases where there are no clinical history, no gross lesions or sudden death, a complete set of organs should be collected. The recommended set of organs include:

Tissues with lesions
Brain
Heart
Lungs
Liver
Spleen
Kidney
Stomach
Small intestines
Large intestines
Lymph nodes
Urinary bladder
Thymus (young animals)
Bone marrow
Muscles
Adrenal glands
Thyroid glands
Other tissues in certain cases

REMEMBER: WE LOSE NOTHING IF WE TOOK MORE TISSUE SAMPLES
                    THAN WE ACTUALLY NEED, BUT WE MAY LOSE EVERY
                    THING IF THE TISSUES REQUIRED FOR FURTHER TESTS
                    HAVE BEEN THROWN AWAY!!!

Brains submitted for pathological examination may be submitted intact and immersed in 10 times or more volume of 10% formalin. Better fixation and therefore examination can be obtained by prefixing the entire brain for two days in a large container in 10% formalin and then shipping the entire brain in a smaller container with a small volume of 10% formalin. This method is preferred.

Skin biopsy samples require careful selection and handling. Do not shave biopsy sites as the hairs are useful guidelines for proper plane of section. Select recent active lesions and margins of lesions incorporating normal skin. If a bullous skin disease is suspected, the active lesion i.e. an actual blister is required for definitive histological diagnosis.

The heart may be submitted in its entirety, particularly if a malformation is suspected. Rinse the chambers with water and fill with 10% formalin.

It is always wise for the pathologist to freeze and hold portions of organs, so that further tests such as virology, bacteriology, toxicology, etc. may be carried out later if necessary. So, please submit bigger portions of organs in ice to the laboratory.

In order to provide you with the most reliable test results, we have established criteria for specimens submitted to the Regional Veterinary Laboratories. We request your assistance in providing quality specimens.
 

Submit at least 15 sera/group. A group = a house, flock. Do not pool samples or make up a group with sera from more than 1 house or source.

1. Whole blood specimens should be allowed to clot, the serum micro centrifuge tubes.
2. Each group of specimens should be put into a plastic bag and labeled appropriately.
3. Pack the samples in an insulated container on dry ice or freezer packs and ship to the laboratory within one day. About 4.5 kg of dry ice or 6 - 10 freezer packs are required. These are minimum quantities to adequately chill the specimens. The specimens must arrive at the laboratory within 36 hours of collection.
4. Do not ship whole blood specimens, please.
5. Sera to be tested for Mycoplasma are not to be frozen. Put these specimens with freezer packs only in the Styrofoam container. Do not use dry ice.
6. Serum to be tested for antibodies to agents other than Mycoplasma may be frozen.

B. Serum samples - To be brought in to the laboratory

1. The specimens may be prepared as in para A1 and A2. These may be refrigerated up to 24 hours before transporting to the laboratory.
2. Freshly drawn whole blood may be submitted if they are submitted on the same day as drawn. Older specimens tend to hemolyze or contaminate.
   
   a. These need to be collected in covered vials, snap-cap culture tubes,
       or vacutainer tubes.
   b. Please do not collect in open tubes.
   c. The blood should be allowed to clot on a 5 degrees slant. This
       allows maximum yield of serum. Do not allow blood to clot forming
       a 'plug' in the bottom of the tubes.
   d. If there is any delay in getting the samples to the lab, the clotted
       tubes may be refrigerated temporarily. Please do not put them in car
       trunks or anywhere that may overheat.

C. Tissue specimens for virus isolation - To be shipped

1. Tissues submitted for virus isolation should be collected as aseptically as possible and put in plastic bags. All the air is expressed from the bag and the bag is closed securely.
2. The plastic bags are placed into a small light-weight cardboard box which is in turn packed on dry ice in a Styrofoam shipping container. This prevents the tissues from being 'burned' direct contact with dry ice. This method requires 4.5 kg dry ice for transit times of 36 hours or less. Cool packs may be used in place of dry ice. Use at least 6 packs for overnight transit.
3. We must insist on shipment by overnight express (e.g. Federal express, Nationwide or any other courier services).

D. Tissue specimens for virus isolation - To be brought in to the laboratory

1. Tissue are collected and placed in plastic bags as in para C1 and labeled.
2. Place the labeled plastic bags on ice and transport to the laboratory.

E. Bacteriological specimens - Shipped or brought in

1. Obtain specimen using a 'culturette'. This is a pre-packed sterile swab which has a small ampoule of liquid media at the bottom. Be sure to contact only the material to be tested. Be sure to crush the ampoule immediately.
2. Ship labeled culturettes at room temperature. Please do not refrigerate or freeze.
3. Send culturettes by overnight express or any appropriate method so as to arrive at the laboratory in less than 24 hours from the time the specimen was obtained. Swabs in transit longer than 24 hours are almost always overgrown disproportionately or fail to yield the desired isolate.

F. Tissues for histopathological examination - To be shipped or brought in

1. Tissues should be collected and put promptly into 10% buffered formalin. The tissues must not constitute more than 10% of the combined tissue/formalin volume. Pieces of tissue larger than 1 cubic centimeter should be partially cut to permit rapid penetration of formalin to the interior portions.
2. Permit tissues to fix for 24 hours, drain excess formalin and reseal the container. This will save on the shipping charges; or
3. The formalin need not be poured off and the tissues shipped as soon as they can be put into the formalin in suitable containers.
4. Do not freeze any tissue intended for histopathological examination whether they are fresh or fixed.
5. Pack the specimens in a leak-proof container with absorbent material and seal.
6. Ship as expeditiously as possible.
7. Be sure to label all specimens.

G. Necropsy

1. Domestic poultry - Necropsy. When birds are submitted for necropsy, it is advantageous for you to:

a. Submit 4 - 5 freshly dead birds, that have died of the
    condition, preferably refrigerated and kept cool until they
    arrive to the laboratory. Time is important as small carcasses
    decompose quickly.

b. Submit 4 - 5 live, sick birds, that are clinically affected with
    the condition. Take care not to select cull birds from the flock
    which do not represent the disease condition. These
    specimens should be shipped by express in containers
    suitable for live birds.

c. If dead birds have to be submitted then try to bring the
    freshest specimens possible.

d. Please supply flock history, medication, vaccination, or any
    other information that may be helpful.

1. Exotic or pet birds - necropsy. More often than not these birds have already died. Dead birds should be wetted and refrigerated immediately in a plastic bag. Package the wet bird in a plastic bag and ship to the laboratory on ice so as to arrive in less than 24 hours if possible. If this is impossible, freeze the bird and ship as suggested. However, we cannot perform histopathological examination on previously frozen tissue, only gross pathology, virus isolation, and bacteriology are possible.

Respiratory problems

1. Histopath: Fix 0-5 - 1.0 cm thick portions of tissue in 10 volumes 10% formalin per volume tissue. Do not freeze, if at all possible.

2. Bacteriology: Submit tissues in plastic bags labeled "BACT", put placenta in separate bag: fluids should be submitted in labeled plain (red top) tubes; these specimens should be refrigerated but not frozen.

3. Serology: Collect serum at the same time of abortion and 2-3 weeks later; remove serum from clot and store frozen at your office; paired sera can then be submitted as indicated. Never freeze clotted whole blood with the expectation of using it for serology later as this will interfere with serological tests.

Suggested Standard Submissions for Neonatal Scours

1. HISTOPATH: Fix 0.5 - 1 cm thick portions of tissues in 10 volumes of 10% formalin per volume of tissue; do not freeze; either open intestine longitudinally or fill lumen with formalin and ligate both ends.

2. BACTERIOLOGY: Submit tissues in separate, labeled plastic bags. Smears for Gram stain may be made from a composite ileal/jejunal swab (not the sections for culture) and air-dried.

3. VIROLOGY: Tissues may be submitted frozen, clearly labeled "VIROLOGY".

4. PARASITOLOGY: To make ileal mucosal smears, transfer ileal mucosal scrapings to a glass slide and air dry. Fecal material should arrive at the laboratory in refrigerated condition (not frozen) as soon as possible after the sample is taken. Alternatively, feces may be preserved in 2.5% potassium dichromate solution (1 volume feces to 1 volume potassium dichromate solution).

2) Blood (Heparin/EDTA)

Hematology is the first step in the diagnosis of a disease or condition so blood samples should be labeled with the animal identification and sent to the laboratory as soon as possible (if travel time to the laboratory takes more than 6 hours it should be packed in ice) for hematology and blood protozoa examination. On collection, blood should be gently mixed to prevent clotting. For the preparation of thin/thick blood smear refer to page ___. Blood samples for determining trypanosomiasis (Surra) MUST be packed in ice and sent to laboratory within 24 hours of collection otherwise the organism will die and cannot be identified.
Tests: CBC, Blood protozoa e.g trypanosomes, Babesia.

b) Nematodes such as round/hook worms from the GIT, heart, should be washed in normal saline gently and dropped into hot 70% methylated spirit. The worms may be left in this solution and submitted in a leak proof container for identification.

c) Tapeworms for identification should be submitted with their heads and mature segments intact. To collect the head, leave the worms that are still attached to the intestine in normal saline and carefully tease out their heads, then place worms in water bath at about 40-50^oC for 10-30 mins. to kill the worm in the extended state. Transfer to 70% ethylated spirit and pack in leak proof bottle to be sent to laboratory.

d) Flukes should be washed in normal saline and gently pressed between 2 glass slides bound together with a rubber band. Leave in 10% formal-saline for 24 hours, then remove the slides and return the flukes to the 1% saline, put in a leak proof bottle and send to laboratory for identification.
 

PREPARATION OF THIN AND THICK BLOOD SMEARS

Collection of blood for preparation of films

It may be necessary to first clip the hair. Puncture the vein with a needle or pricker. The first few drop of blood should not be used as they are always contaminated and rich with platelets. Only a small drop of blood is required as it is almost impossible to prepare a film that is too thin. A very common fault is to use too much blood. A drop the size of the head of a pin is sufficient. From carcasses, blood from the cut end of an ear may be used.

It is important that all glass slides used are perfectly clean, free from scratches, grease, oil, thumb prints, etc. Grease can be removed with a clean cloth (or thoroughly-washed, clean, dry handkerchief) dipped in methylated spirit. The surface of the slide should not be touched with fingers but handled by the edges. The slides may be kept dust free by wrapping them in sheets of clean paper.

Technique for preparation of thin blood film

a) A clean slide is placed on a level surface and a very small drop of blood is deposited near the end of the slide leaving sufficient space for labeling. (see Fig.1 below)

b) Spread the blood in an even film by means of another slide (spreader). The spreading edge of the spreader must be perfectly smooth (free of chips)

c) i) The spreader is held between the thumb & index finger and drawn
       backwards towards the drop of blood. (See fig.2 below)

   ii) As it makes contact, the blood will spread along the edge of the
      spreader, which is then moved forward in a smooth gliding manner. (See
      Fig.3 below). The blood is therefore pulled behind the spreader and
      never pushed in front of it. The angle at which the spreader is held
      determines the thickness of the blood film. Generally a 30^o - 40^o angle is
      satisfactory.

d) Wave the slide in air to dry it.

e) The slide must then be identified by the name of the animal or its number on the same side as the smear.

A thin blood film should consist of a single layer of blood cells and its margins should not reach the edges of the slide. Thin blood films should be taken in the shade if possible or they may dry too quickly and the red blood cells may become cremated. For the same reason slides should not be left in the sun before use.

Some common faults in the preparation of blood smears and their causes are tabulated below:

• Tissues should be as fresh as possible
• Take samples from edge of lesion, and try to include tissue
• Be careful to collect in aseptic fashion
• Collect samples prior to antibiotic treatment
• Submit generous portions of tissue or several millimeters of pus, exudates or feces
• Maintain most samples at refrigeration temperatures (4⁰C) rather than frozen en route to laboratory.

• Good collection method is essential; exposure to air for more than 20 minutes can be detrimental

• Animals dead longer than 4 hours are usually unsuitable

• Tissues and liquid exudates are recommended (anaerobic bag or tube would be helpful)

• Swabs are not acceptable unless shipped in anaerobic containers (special collection devices with reduced oxygen environment)

• Collect milk in sterile snap cap or screw cap tubes
• Cool samples before submitting to the laboratory, and mail with ice packs. Samples may also be frozen without altering recoverability of pathogens.

• Not normally used for recovery of animal pathogens because bacteremia is intermittent. Call the laboratory for recommendations.

• Collect by cystocentesis, catheter or mid-stream catch.

• Pustules. Disinfect surface with alcohol, allow to dry and aspirate material with syringe and needle.
• If ringworm is suspected, pluck hair from lesion and scrape edge of lesion. Submit hair, skin scrapings, and scrap material.

• Clean anus/cloacae from feces with clean water and collect sample from the rectum or cloacae with sterile swabs or rectal pinch.

• Submit specimens collected early in the course of the disease as this enhances of isolating the virus
• Aseptic technique should be used in collecting materials
• Swabs should be placed in test tubes containing 50% glycerol saline
• All materials should be refrigerated (below 4⁰C) immediately
• Deep freezing is recommended for all materials without 50% glycerol saline
• Where vesicular disease (foot and mouth disease) is suspected, it is important to notify the laboratory nearest to you. Do not proceed to collect specimens. Proper authority will do specimen collection and submission.

With the large volume of serology cases, it is very important for samples to be submitted in such a way that they can efficiently be logged into the laboratory.

    a) Always use dry needles, containers, and tubes
    b) Do not freeze blood before collecting serum
    c) Do not force blood through needles
    d) Do not use outdated corvac tubes

2. Place serum in plastic snap cap tubes. This will aid efficient setup and
    storage, saving time in getting the results back to you. If you use blood
    tubes with silicone plugs, also put the serum in plastic snap tubes. This
    allows us to store the sera for up to 30 days following testing.

3. Number the tubes consecutively, 1 through x, matching the actual tube
    number with the tube number written on the serology submission form.
    Use permanent marker to identify tubes. Grease pencil marks are easily
    rubbed off, and labels fall off during the heating process necessary for
    some tests.

4. Keep cases from different owners separate.

5. Make a copy of the paperwork submitted, and retain a portion of each
    serum sample in your freezer. This guards against un-forseen mishaps
    (lost or broken tubes) and may save you from having to re-bleed. Acute
    serum samples may also be retrieved from the freezer for pairing with
    convalescent samples. Paired sera should be tested together to
    maximize the diagnostic value of test results.
 

* Paired sera means acute & convalescent sera. The acute phase serum is collected at height of infection or at the time of clinical signs & the convalescent serum is collected 2-3 weeks later. Identify samples as A or 1 (for acute) & C or 2 (for convalescent). Preferably send both samples TOGETHER to the lab as paired sera. They may be sent separately but the reference no. of the acute or first serum must be stated when the convalescent or second serum is sent later.

A thorough interchange of information through a written or telephone history between clinician and diagnostician is important for confirming diagnoses suspected by the attending clinician. A complete account of history (including management and feed type), symptoms, and lesions submitted with specimens for laboratory evaluation is very important.

All specimens and exhibits for toxicological examination are to be sent to the Chemistry Department directly or via the regional laboratory. MAKVET 06 form should be used for this purpose.

There is no practical or affordable way to check for all possible poisons. The more information provided to the chemist, the more efficient and useful would be the selection process.

Selecting specimens for toxicology and chemical evaluation require three main criteria.

1. The correct specimens must be provided based on the type
    of poison involved
2. Adequate amount of specimen must be available
3. Preservatives should not be used unless absolutely
    necessary.

1. Specimens should be free of chemical contamination and debris. Contamination of samples with hair, vomitus, dust, dirt, etc. may produce erroneous results.
2. Freeze animal and tissue specimens for chemical analysis. Do not freeze whole blood samples, but keep them refrigerated.
3. Serum, if separated from the clot and not hemolysed, may be frozen.
4. Always package specimens from various organs and fluids separately.
5. Use clean glass or plastic containers that can be tightly sealed.
6. Label each specimen (container) for clear identification.
7. Never add preservative such as formalin unless there is a specific reason. If preservatives are added, include a sample of the preservative along with the specimen.
8. Submit samples for chemical analysis of low level organic contaminants, such as pesticide residues or PCB's, in glass containers, not plastic. Solid samples for this purpose may be wrapped in aluminium foil.
9. If ammonia, urea, or cyanide toxicosis is suspected, freeze rumen contents and blood or serum immediately after collection. Keep them frozen.
10. On all toxicology cases involving a dead animal, submit both fresh and formalin-fixed tissues.

Specimens that should be submitted from a live animal include:

Specimens that should be submitted from a dead animal include:
 

1. Sample size for hay and silage should be at least 1 quart. Cut all forages to a length of 3" or less.

2. Pack samples tightly to exclude air, and seal airtight in plastic bag. Very dry sample may be submitted in paper bags.

3. Take standing pasture and row crop samples for a minimum of 8-10 locations within a field. Remove forage from a four-foot area at grazing height. Mix all collected forage and take a representative sample (500 grams) for analysis.

1. Water samples should represent the water to be tested.

2. Submit water samples for inorganic analysis in clean containers of either glass or plastic. Container should be sterile if microbiologic testing is requested.

3. Submit all water samples for organic analysis in clean glass jars with clean aluminium foil placed over the mouth of the jar before attaching the lid.

QUICK GUIDE FOR TOXICOLOGY SPECIMENS

Specimens required for diagnosis of common toxicants are listed in Table 1.

Table 1: SPECIMENS REQUIRED FOR TOXICOLOGY TESTS

TABLE 2: SPECIMENS REQUIRED FOR SPECIFIC TOXICOLOGY TESTS

Several analyses grouped by clinical or chemical family are available as screening panels. See Table 3.

TABLE 3: CHEMISTRY SCREENING PANELS

Most clinical chemistry analyses require whole blood, plasma or serum. Always check the test protocol for the type of sample required.

For blood chemistry, enzymes, substrates and minerals are determined frequently.

Plasma samples can be refrigerated or frozen.

To avoid hemolysis, do not centrifuge clotted blood at speeds higher than 3000 rpm (700 x g) or for a prolonged time. Serum should be chilled immediately and transported in ice. Exposure to sunlight should be avoided.

Biochemical test combination kits e.g. wet chemistry kits from Boehringer Mannheim, Roche etc or dry chemistry slides from Idexx or Kodak are available commercially. Automated or semi-automated chemistry analyzers may be employed if the sample number is large.

Glucose concentration in blood can drop 10% an hour if kept at room temperature due to glycolysis. Use tubes with sodium fluoride (10mg/ml) or EDTA (2.5mg/ml) if plasma cannot be removed immediately by centrifugation.

Urine samples collected either by cystocentesis, catheterization or natural voiding should be chilled immediately after collection and kept chilled until arrival at the laboratory. Urine dipsticks which allow qualititative and semi-quantitative measurements may be purchased commercially.

Suggested profiles for tissue abnormalities and metabolic disorders is found in Table 1.

Sample handling before centrifugation and storage recommendations for serum or plasma after sampling is explained in Tables 2 and 3.

Table 2: SAMPLE HANDLING BEFORE CENTRIFUGATION FOR USE WITH VETTEST 8008

Table 3: STORAGE RECOMMENDATIONS FOR SERUM OR PLASMA AFTER SAMPLING

All carcasses of rabid animals must be handled with care so as to avoid exposure. Individuals involved with handling of rabid specimens should be adequately vaccinated against rabies and properly attired (lab coat or coveralls, plastic apron, thick rubber gloves, a mask, cap, rubber boots and goggles are a must). Ensure that any unauthorized personnel are not in the vicinity.

B. Submission Form

All details about the rabid animal must be entered into the submission form (MAKVET 5A). Ensure that the form is completely filled. All dog-bite cases must be clearly stated in the column provided to differentiate routine submissions. Each sample (head) should be properly labeled and accompanied by a form (one head one form).

C. Specimen Preparation

If the clinical history suggestive of rabies, extra precaution should be taken. The suspected rabid animal should be euthanised before decapitation. Avoid damaging the head if it has to be shot so that there is minimal brain damage. Extra precaution should be taken to prevent contact with saliva and other body fluids. The following procedures should then be followed:

(i)  The head should be decapitated at the atlanto-axial joint. Decapitation should be carried out on a solid surface or table so that it is easy for disinfection.

(ii)   The head should be cooled down promptly and kept cold (4⁰C). Freezing should be avoided. [Note: Although freezing the specimen in dry ice during transit or in liquid nitrogen flasks will preserve the virus, a prompt microscopic examination may be delayed because of the time taken to thaw the specimen].

(iii)  All openings that include the mouth, nostrils and ear orifices should be stuffed with formalinised cotton or cloth to prevent spillage of fluids.

(iv)  Each head should be put into a suitable watertight container and closed tightly. If plastic bag is used, choose the thicker one and make sure that it is sealed or tied properly with a rubber band. This in turn should be put into a larger watertight container, where cracked ice is packed between the inner and outer container. The package should be clearly labeled (given Bag No.) and accompanied by the completely filled MAKVET 5A form. The package is then put into a specially designed metal chest and shipped to the Veterinary Research Institute, Ipoh, Perak with the fastest possible courier. The metal chest should be labeled as below:

Poultry Cases

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