sample collection

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. Purposeof collecting samples
1.Direct examination thru microscopy
Bloodparasite examination
Fecalysis
Impressionsmears for Fluorescent Antibody Test
eg. Rabies and Hog choleSra
2. Isolationofcausative microorganisms
Bacterial Isolation and identification
Tissueculture
Egginoculation
Mice Inoculation

3. Purposeof 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 (quantitativePCR)
5.Diagnosis of the disease

4. Purposeof testing samples
1. Confirmatory diagnosis of suspector
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 riskanalysis

5. Purposeof testing samples
5.Certification of freedom from infection
or presence of the agent in
individual animals or theirproducts
for trade orshipment.
6. Determination of immunestatus of
individual animals orpopulations
7. For specific therapeutic medication

6. Basics incollecting samples
1.Always protect yourself, other people and
the environment
2. Use clean, preferably sterilizedimplements
3.Collect samples beforetreatment isinitiated
4.Sample volume or quantity must be sufficient
for all necessary tests with enough residual
specimen for archivalpurposes
5.Practice proper restraint of animals during
sample collection

7. Basics incollecting samples
6.Packagingcontainers for specimens should
be leak-proof andsterile
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 SampleCollection
1. Personel Protective Equipment(PPE)
Scrub suit
Face mask
Gloves
Boots
Goggles
Apron

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

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

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

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

13. Checklist for Field SampleCollection
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 SampleCollection
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 differentialdiagnosis.
Each tissue must have a separate sterile
container. Intestines should be tied with yarn
at both ends.

16. Samplesthat ShouldAlways beCollected
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. SampleCollection in LiveAnimals
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. SampleCollection in LiveAnimals
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. SampleCollection in LiveAnimals
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. SampleCollection 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. Samplesfor 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. Samplesfor 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. Samplesfor Specific Diseases
Blackleg and Malignant Edema
2inches 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. Samplesfor Specific Diseases
Caseous Lymphadenitis
affected nymph nodes
place in sealed container and submit under
preserve 2x2 cm in buffered
refrigeration,
formalin
Colibacillosis
package section of small intestine, mesenteric
lymph node, spleen, liver, kidney
ship under refrigeration

25. Samplesfor 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. Samplesfor 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. Samplesfor 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. Samplesfor 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. Samplesfor 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 theReport
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 theReport
contact
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.V
eterinarian’s name, address and
number

32. Handling and Transport ofSamples
samples cool to prevent
and growth of nonspecific
1. Keep tissue
decomposition
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 ofSamples
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 Conceptsin SendingSpecimensto
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 Conceptsin SendingSpecimensto
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 anddevelopment efforts
Retrospective studies
Epidemiological studies
Providing critical reference materials
used in assay standardization,validation
and proficiencytesting programs

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

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