SAMPLE SUBMISSION AND OTHER REQUIREMENTS FOR DIAGNOSTICS

Snježana Zrnčić, PhD, DVMTCDC/TCCT Consultant No 2 – Diagnostics

zrncic@irb.hr

REQUIREMENTS FOR ACCURATE DIAGNOSIS

AT THE SAMPLING SPOT• Proper choice of fish/molluscs specimen at the cultivation

facility• Timing of sampling (for VHS/IHN water temperature should

not exceed 14oC, for KHV it should not be under 22oC)• Molluscs for bonamiosis in spring, for marteiliosis in the

autumn before decrease of sea temp. to 17oC• If lab personnel is not present during sampling all those

data together with location of sampling, farm registration, number of specimen in sample, date of sampling, and responsible person should be supplied on the document accompanied purcell

PACKING AND SHIPPING Veterinarians authorised for sampling on aquaculture

establishment should be well educated and trained for sampling , packing and mode of shipping of samples

All samples (whole fish, molluscs or organs in transport media) should be protected of contamination

Each pool of fish/molluscs should be packed in separate package (plastic bag) properly marked

liquids should not pour our of package Package (cooling box or styrofoam box) should maintain the

temperature (must not exceed 10oC) Purcell must be supplied with enough freezing blocks to secure

this temperature during the whole transportation Regular post must be avoid

If there is no possibility for vets to transport samples to diagnostic lab, reliable courier service with experience of transportation of cold purcells should be chosen

Some bad experience with some courier services during summer months

Purcell must be properly tagged (name, address and phone number of sender’s organisation, address, phone etc of laboratory, compulsory conditions, time of delivery)

Document describing the purcell must be properly protected of slubberness and wetting

UPON ARRIVAL Accompanied documentation must be checked to

register the sample Temperature inside the purcell must be measured be

check If those conditions are satisfactory, it is possible to

deliver samples to the diagnostic laboratory In the lab – registration of samples, opening of the

required protocols according to the sort of samples Determination of the priorities

PROCESSING OF SAMPLESAccording to the sample Organs in transport media (EMEM with 10% FBS and

pen/strep buffered with HEPES or TRIS) for virological examination shoul be subjected to the virological testing on the cell lines no later that 48 hours after sampling

Organs are homogenized in stomacher or mortar and pestle with sterile sand, diluted with transport media in ratio 1:10, centrifuged on 5000 rpm at 40C for 20 minutes, filtered through 450 μm and incubate for 4 hours at 150C or overnight at 40C

In the exceptional situation period between sampling and inoculation of cell lines should be prolonged to 72 hours

During some unpredictible situation exceptionally, a sample can be freeze at -800C and the inoçulation of cell line can be prolonged for 15 days

TARGET ORGANS FOR VIROLOGICAL EXAMINATION OF SALMONIDS

Anterior kidney

Heart

Spleen

Spleen

TARGET ORGANS FOR VIROLOGICAL EXAMINATION OF SALMONIDS

HEART

PROCESSING OF SAMPLES According to the sample Organs in transport media (EMEM with 10% FBS and pen/strep

buffered with HEPES or TRIS) for virological examination shoul be subjected to the virological testing on the cell lines no later that 48 hours after sampling

Organs are homogenized in stomacher or mortar and pestle with sterile sand, diluted with transport media in ratio 1:10, centrifuged on 5000 rpm at 40C for 20 minutes, filtered through 450 μm and incubate for 4 hours at 150C or overnight at 40C

In the exceptional situation period between sampling and inoculation of cell lines should be prolonged to 72 hours

During some unpredictible situation exceptionally, a sample can be freeze at -800C and the inoçulation of cell line can be prolonged for 15 days

PROCESSING OF ORGAN SAMPLES FOR CELL CULTURE

PROCESSING OF MUSSELS FOR CYTOLOGICAL AND MOLECULAR DIAGNOSTICS

STORAGE OF SAMPLES Samples submitted for laboratory diagnostics must be stored

until the written report has been issued According to lab protocols about service to customers all

negative samples are stored two weeks after completing the examination and positive samples are stored a month

Samples for virological testing on cell cultures are stored at -80oC

Samples submitted for cytologycal (stained tissue imprints )and histological testing for molluscan diseases are stored one year after completing testing

LABORATORY ORGANISATION AND EQUIPMENT Laboratory should be organised in the manner that there is no

possibility for cross contamination – it means that dissection should be 1st step, cell culture propagation should be performed in separate LAF with no possible contact with infectious material and work with fish viruses in another facility – IMPORTANT TO ENSURE ACCURATE RESULTS OF TESTING

Equipment must be calibrated (pipettes), termometers, incubators and they should operate in allowed range, centrifuges should be calibrated for speed and temperature, spectrophotometers should be calibrated in proper periods

MARKING AND TRACKING Traceability is the very important element during the whole

process The same mark should be traceable from submission of the

sample, during all step of laboratory procedure until reporting Used cell lines (number of passage, date of subcultivation)

should be marked with aim to give us possibility to go back through the whole process in the case of any suspicious of the correctness of the results

All used consumables (media, sera, chemicals etc. must be registered and marked

SECURITY MEASURESCleaning and disinfection protocols must be followed to avoid any possibility of contamination or spreading the pathogen from the labAll equipment should be properly cleaned and disinfected regularly, all disinfection procedures should be registered and efficacy should be controlled regularly (working surfaces, LAF, incubators, refrigerators etc.)Laboratory stuff should wear protective clothes, gloves, and shoes which are used only for lab workIt is not allowed to wear same coats during dissection and cell propagation,Material for testing should be disposed according in the manner which will not harm environment or allow spreading of infective material or possilitity of chemical contamination