Collection and Handling of Specimens for Laboratory Diagnosis by Dr M Mananggit

1. Collection and Handling
of Specimens for
Laboratory Diagnosis
Dr. Milagros R. Mananggit
Chief, Regional Animal Disease Diagnostic Laboratory
Department of Agriculture, Regional Field Unit 3

2. Purpose of collecting samples
1. Direct examination thru microscopy
Blood parasite examination
Fecalysis
Impression smears for Fluorescent Antibody Test
eg. Rabies and Hog choleSra
2. Isolation of causative microorganisms
Bacterial Isolation and identification
Tissue culture
Egg inoculation
Mice Inoculation

3. Purpose of collecting samples
3. Serological Investigation
 ELISA Test
 AGPT
 HA-HI Test
 Rapid plate test
4. Molecular Test
 LAMP
 Polymerase Chain Reaction (PCR)
 Conventional PCR
 Real Time PCR (quantitative PCR)
5. Diagnosis of the disease

4. Purpose of testing samples
1. Confirmatory diagnosis of suspect or
clinical case
2. Demonstration of freedom from
infection in a defined population
3. Eradication of disease or elimination of
infection from a defined population
4. Estimation of prevalence of infection or
exposure to facilitate risk analysis

5. Purpose of testing samples
5. Certification of freedom from infection
or presence of the agent in
individual animals or their products
for trade or shipment.
6. Determination of immune status of
individual animals or populations
7. For specific therapeutic medication

6. Basics in collecting samples
1. Always protect yourself, other people and
the environment
2. Use clean, preferably sterilized implements
3. Collect samples before treatment is initiated
4. Sample volume or quantity must be sufficient
for all necessary tests with enough residual
specimen for archival purposes
5. Practice proper restraint of animals during
sample collection

7. Basics in collecting samples
6. Packaging containers for specimens should
be leak-proof and sterile
7. Label all specimen containers and account
for all of them in a sample information sheet
8. Maintain cold chain during transport of
samples
9. Provide sufficient epidemiological information
on the case
10. Notify the receiving laboratory

8. Checklist for Field Sample Collection
1. Personel Protective Equipment (PPE)
 Scrub suit
 Face mask
 Gloves
 Boots
 Goggles
 Apron

9. Checklist for Field Sample Collection
2. Necropsy Kit
 Knife
 Scissors
 Bone cutter
 Forceps
 Scalpel with
blade

10. Checklist for Field Sample Collection
3. Sample Containers
a. Blood/Serum
1) Vacutainer tubes
Violet or green top –whole
blood
Red top – serum samples
Glass slides –blood smears
2) Disposable Syringes
3) Cryotubes for serum (2 ml)
4) Vacutainer needle and holder

11. Checklist for Field Sample Collection
b. Organs/Tissues – each organ
must have separate
container
 Screw cap sterile
containers
 Zip lock plastic bags

12. Checklist for Field Sample Collection
c. Swabs - nasal, tracheal,
oro-pharyngeal, cloacal
with viral or bacterial
transport media

13. Checklist for Field Sample Collection
4. Labelling of Samples
 Permanent water proof pen
 Masking tape
5. Preserving samples
 Ice chest/Styropor box
 Gel coolant
 10 % buffered formalin
– for histopathology
 Slide box
 Methanol to fix blood smears

14. Checklist for Field Sample Collection
6. Recording Forms
 Surveillance or disease investigation forms
 Sample submission forms
7. Restrainer – rope, nose grip, pig restrainer
8. Disinfectant (hand sprayer)
 70 % alcohol
 10 % lysol or bleaching agent
9. Documentation –camera, tablet, cell phone
10. Garbage bag

15. Sample Collection During Necropsy
 Tissue samples should be collected
aseptically using rat tooth forceps and
scalpel blade dip in a container with
alcohol.
 Cut tissue samples with demarcation
between normal and abnormal with enough
quantity to perform differential diagnosis.
 Each tissue must have a separate sterile
container. Intestines should be tied with yarn
at both ends.

16. Samples that Should Always be Collected
Tissue/Sampl
e
Fresh (Chilled-not frozen) Fixed (10% buffered formalin)
Serum 5 ml
Whole Blood 3 ml in EDTA
Swabs Brain, epicardium, joint
Brain
Lungs 6x6x6 cm, 2 sections 2x2x1 cm
Heart 4x4x4 cm piece 2x2x1 cm w L & R ventricles and
septum
Liver 4x4x4 cm piece 2x2x0.5 cm
Kidney Half of a kidney 0.5 cm slice thru center
Spleen 5 cm piece
Lymph nodes Mandibular, sternal,
tracheobronchial, mesenteric,
inguinal
Mandibular, sternal,
tracheobronchial, mesenteric,
inguinal
Ileum 10 cm segment 2 cm segment

17. Sample Collection in Live Animals
1. Blood:
 jugular vein - horses, and small ruminants
 tail vein - big ruminants,
 wing vein - avian species
 lateral saphenous or middle cephalic in dog/cat
Serum: Let stand at room temperature for 12 to 24 hours
then transfer into cryotubes.
BPE: Use violet or green top cryotubes, blood smears
can be prepared, air dry and fix in methanol for 3
minutes.

18. Sample Collection in Live Animals
2. Swabs – collect exudates from abscess and
lesions, oropharyngeal, tracheal and cloacal
swabs for avian influenza
3. Feces – take directly from rectum or just
after defecation
4. Urine collection thru catheterization

19. Sample Collection in Live Animals
5. External parasites
 large parasites can be picked off and place in
container with 70% alcohol for identification
 smaller parasites such as mites can be scraped
with blade and placed in slide with mineral oil
6. Tissue impression smears in slide
7. Milk – 5 to 10 ml in sterile container

20. Sample Collection for Toxicology
1. Body fluids –blood and urine
2. Tissues to collect at necropsy – stomach or
rumen contents, feces, brain, liver, kidney,
body fat, skin, and urine
3. Suspected source – feeds and water,
poisonous plants, soil
4. Test for nitrates – best sample is aqueous
fluid from the eye of dead animal

21. Samples for Specific Diseases
Abortion
mid portion of kidney, placenta, adrenal gland,
liver, spleen, tied-off stomach, brain and serum
 Store at 2-4 °C and submit under refrigeration
Abscesses
Purulent exudates on a sterile swab or in a sterile
tube collected from the margin of the abscesses
 Store and submit under refrigeration

22. Samples for Specific Diseases
Arthritis
Swab or fluid from affected joint or unopened
joint
 Send in bacterial transport medium, fluid in
syringe
Anthrax
cotton swab soaked in exuded blood taken from
superficial ear vein or cut surface of hemorrhagic
lymph node placed in tube media.
 Submit under refrigeration

23. Samples for Specific Diseases
Blackleg and Malignant Edema
2 inches cube of affected muscle
 Packed in sterile container and submit under
refrigeration
Brucellosis
Blood and serum sample collected 10 to 20 days
after abortion, aborted fetus, tied off stomach
 Shipped in sealed container under refrigeration

24. Samples for Specific Diseases
Caseous Lymphadenitis
affected nymph nodes
 place in sealed container and submit under
refrigeration, preserve 2x2 cm in buffered
formalin
Colibacillosis
package section of small intestine, mesenteric
lymph node, spleen, liver, kidney
 ship under refrigeration

25. Samples for Specific Diseases
Haemophilus
large portion of the lung or pleural fluid from
untreated sick animal with respiratory
infection
 Keep under refrigeration
Leptospirosis
blood collected at time of clinical signs then
10 to 20 days after,
 submit under refrigeration

26. Samples for Specific Diseases
Mastitis
Milk, 5 to 10 ml in sterile container
 Keep under refrigeration
Pasteurellosis
3 inches square of affected lung and
mediastinal lymph node, deep nasal or
tracheal swab
 Keep under refrigeration

27. Samples for Specific Diseases
Salmonellosis
Sections of liver, spleen, kidney and tied off
section of intestine
 Submit under refrigeration
Avian Pest
Brain, lung, trachea, spleen
serum collected on the 4th and 14th days
following onset of the disease
 Freeze and submit under refrigeration

28. Samples for Specific Diseases
Pseudorabies
Brain, tonsil, lung, lymph node and serum
 Freeze and submit under refrigeration
Rabies
Unopened head of the animal
 Freeze and send under refrigeration, don’t
preserve

29. Samples for Specific Diseases
Swine Flu
Lung, trachea, mediastinal lymph node
 Freeze and submit under refrigeration
Aflatoxicosis
Suspected feed sample
 Keep dry and cool
Tumor
Portion of tumor mass and adjacent normal tissue
 Preserve in 10% buffered formalin

30. Write the Report
No necropsy is complete until all findings have
been recorded in written form. The report should
include the following information:
1. Owner’s name, address and contact number
2. Description of the animal: species, breed, age,
sex
3. Duration and condition of the outbreak
4. Mortality and morbidity rate
5. Animal population and nearby farms

31. Write the Report
6. Clinical signs observed
7. Treatment given and vaccination
8. Feeds and water given
9. Possible contact with other animals
10.Tentative diagnosis
11.Necropsy report
12.Samples collected and submitted
13.Veterinarian’s name, address and contact
number

32. Handling and Transport of Samples
1. Keep tissue samples cool to prevent
decomposition and growth of nonspecific
bacteria
2. Keep various tissues separated from one
another using ziplock or whirlpack bags, or
sterile screw cap containers.
3. Swabs should be kept moist by using sterile
saline/water or transport media and keep at 4
degrees Centigrade till reach the lab.

33. Handling and Transport of Samples
4. Blood samples should be kept at room
temperature for 12 to 24 hours before
separating the serum to avoid hemolysis.
Freeze if can’t reach the lab for a week.
5. Fecal samples should be kept cool but don’t
freeze.
6. Samples for bacteriology should be kept
under 2-4 degrees Centigrade. Those for
virology can be frozen.

34. Key Concepts in Sending Specimens to
the Laboratory
1. Speed in getting the materials to the
laboratory.
2. Keep the samples cool on the way to the
laboratory.
3. Use packaging that will prevent leakage
and crushing.

35. Key Concepts in Sending Specimens to
the Laboratory
4. Be sure that all your samples are well-
labeled.
5. Be sure that appropriate paperwork is
included with all of the samples such as
necropsy report, sample submission form,
disease investigation form.
6. Alert the laboratory on the arrival of your
samples.

36. Storage and Archives
 Future research and development efforts
 Retrospective studies
 Epidemiological studies
 Providing critical reference materials
used in assay standardization, validation
and proficiency testing programs

37. Storage and Archives
 System of documentation and storage
conditions
 Biosecurity measures
 Back-up plan in case of compromises
to the storage environment

38. Thank You and
God Bless…
RADDL3 (045)961 2934