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.