Collection and Handling of Specimens for Laboratory Diagnosis

Dr. Milagros R. MananggitChief, Regional Animal Disease Diagnostic LaboratoryDepartment of Agriculture, Regional Field Unit 3

Purpose of collecting samples1. Direct examination thru microscopyBlood parasite examinationFecalysisImpression smears for Fluorescent Antibody Test eg. Rabies and Hog choleSra2. Isolation of causative microorganismsBacterial Isolation and identificationTissue cultureEgg inoculationMice Inoculation

Purpose of collecting samples3. 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

Purpose of testing samples1. Confirmatory diagnosis of suspect or clinical case 2. Demonstration of freedom from

infection in a defined population3. Eradication of disease or elimination of infection from a defined population 4. Estimation of prevalence of infection or

exposure to facilitate risk analysis

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 populations7. For specific therapeutic medication

Basics in collecting samples1. Always protect yourself, other people and the environment2. Use clean, preferably sterilized implements3. Collect samples before treatment is initiated4. Sample volume or quantity must be sufficient for all necessary tests with enough residual specimen for archival purposes5. Practice proper restraint of animals during sample collection

Basics in collecting samples6. Packaging containers for specimens should be leak-proof and sterile7. Label all specimen containers and account for all of them in a sample information sheet8. Maintain cold chain during transport of samples9. Provide sufficient epidemiological information on the case10. Notify the receiving laboratory

Checklist for Field Sample Collection 1. Personel Protective Equipment (PPE)

Scrub suit Face mask Gloves Boots Goggles Apron

Checklist for Field Sample Collection 2. Necropsy Kit

Knife Scissors Bone cutter Forceps Scalpel with

blade

Checklist for Field Sample Collection 3. Sample Containers

a. Blood/Serum 1) Vacutainer tubes

Violet or green top –whole blood

Red top – serum samplesGlass slides –blood smears

2) Disposable Syringes3) Cryotubes for serum (2 ml)4) Vacutainer needle and holder

Checklist for Field Sample Collection

b. Organs/Tissues – each organ must have separate container Screw cap sterile

containers Zip lock plastic bags

Checklist for Field Sample Collection c. Swabs - nasal, tracheal,

oro-pharyngeal, cloacal with viral or bacterial transport media

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

Checklist for Field Sample Collection 6. Recording Forms

Surveillance or disease investigation forms Sample submission forms

7. Restrainer – rope, nose grip, pig restrainer8. Disinfectant (hand sprayer)

70 % alcohol 10 % lysol or bleaching agent

9. Documentation –camera, tablet, cell phone10. Garbage bag

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.

Samples that Should Always be CollectedTissue/

SampleFresh (Chilled-not frozen) Fixed (10% buffered formalin)

Serum 5 mlWhole Blood 3 ml in EDTASwabs Brain, epicardium, jointBrainLungs 6x6x6 cm, 2 sections 2x2x1 cmHeart 4x4x4 cm piece 2x2x1 cm w L & R ventricles and

septumLiver 4x4x4 cm piece 2x2x0.5 cmKidney Half of a kidney 0.5 cm slice thru centerSpleen 5 cm pieceLymph nodes Mandibular, sternal,

tracheobronchial, mesenteric, inguinal

Mandibular, sternal, tracheobronchial, mesenteric, inguinal

Ileum 10 cm segment 2 cm segment

Sample Collection in Live Animals1. 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.

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

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 oil6. Tissue impression smears in slide7. Milk – 5 to 10 ml in sterile container

Sample Collection for Toxicology1. Body fluids –blood and urine2. 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

Samples for Specific DiseasesAbortion

mid portion of kidney, placenta, adrenal gland, liver, spleen, tied-off stomach, brain and serum

Store at 2-4 °C and submit under refrigerationAbscesses

Purulent exudates on a sterile swab or in a sterile tube collected from the margin of the abscesses

Store and submit under refrigeration

Samples for Specific DiseasesArthritis

Swab or fluid from affected joint or unopened joint

Send in bacterial transport medium, fluid in syringe

Anthraxcotton swab soaked in exuded blood taken from

superficial ear vein or cut surface of hemorrhagic lymph node placed in tube media.

Submit under refrigeration

Samples for Specific Diseases

Blackleg and Malignant Edema2 inches cube of affected muscle Packed in sterile container and submit under

refrigerationBrucellosis

Blood and serum sample collected 10 to 20 days after abortion, aborted fetus, tied off stomach

Shipped in sealed container under refrigeration

Samples for Specific DiseasesCaseous Lymphadenitis

affected nymph nodes place in sealed container and submit under

refrigeration, preserve 2x2 cm in buffered formalin

Colibacillosispackage section of small intestine, mesenteric

lymph node, spleen, liver, kidney ship under refrigeration

Samples for Specific DiseasesHaemophilus

large portion of the lung or pleural fluid from untreated sick animal with respiratory infection

Keep under refrigerationLeptospirosis

blood collected at time of clinical signs then 10 to 20 days after,

submit under refrigeration

Samples for Specific Diseases

MastitisMilk, 5 to 10 ml in sterile container Keep under refrigeration

Pasteurellosis3 inches square of affected lung and

mediastinal lymph node, deep nasal or tracheal swab

Keep under refrigeration

Samples for Specific DiseasesSalmonellosis

Sections of liver, spleen, kidney and tied off section of intestine

Submit under refrigerationAvian Pest

Brain, lung, trachea, spleen serum collected on the 4th and 14th days

following onset of the disease Freeze and submit under refrigeration

Samples for Specific Diseases

PseudorabiesBrain, tonsil, lung, lymph node and serum Freeze and submit under refrigeration

RabiesUnopened head of the animal Freeze and send under refrigeration, don’t

preserve

Samples for Specific DiseasesSwine Flu

Lung, trachea, mediastinal lymph node Freeze and submit under refrigeration

AflatoxicosisSuspected feed sample Keep dry and cool

TumorPortion of tumor mass and adjacent normal tissue Preserve in 10% buffered formalin

Write the ReportNo 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 number2. Description of the animal: species, breed, age,

sex3. Duration and condition of the outbreak4. Mortality and morbidity rate5. Animal population and nearby farms

Write the Report6. Clinical signs observed7. Treatment given and vaccination8. Feeds and water given9. Possible contact with other animals10.Tentative diagnosis11.Necropsy report12.Samples collected and submitted13.Veterinarian’s name, address and contact

number

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.

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.

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.

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.

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

Storage and Archives

System of documentation and storage conditions

Biosecurity measures Back-up plan in case of compromises

to the storage environment

Thank You andGod Bless…

RADDL3 (045)961 2934