American Society for Veterinary Clinical Pathology
2424 American Lane
Madison, WI 53704

Telephone: +1-608-443-2479
Fax: +1-608-443-2474
email: info@asvcp.org

1. Preanalytical Factors Important in Veterinary Clinical Pathology

 

1.1. General, including Hematology, Endocrinology, Chemistry and Serology

 

1.1.1.   Specimen Collection, Handling and Transport to the Laboratory.

Information concerning sample requirements, proper collection, handling, and delivery or shipping procedures for any assay performed in the laboratory should be available to clients electronically, in written materials (such as a laboratory services manual, special information sheets, journal or newsletter articles), or by personal telephone conversation.  Samples should be collected according to standard practices.  Instrument manufacturer's package inserts have detailed descriptions of appropriate samples, including collection tubes and handling conditions.  The specifications for sample submission should be provided to the client by each laboratory. The specimens should be handled carefully and transported to the laboratory in a timely manner under conditions appropriate for the type of sample and its stability.  Type of specimen (e.g., whole blood, serum, plasma, urine) should be clearly stated on the specimen label. Deviation from recommended protocols can adversely affect test results. Contact manufacturers for specific details.

a. Hematology

i. Blood films made in the clinic should not be refrigerated and should be protected from condensation and freezing during transport to the laboratory.

ii. Anticoagulated samples for hematology that are found to have macroclots that can be found upon visual inspection will produce variably erroneous results.  The clinic should be contacted either in writing or by phone and informed that the sample will produce erroneous results. Because the varying degree of inaccuracy can't be predicted, clotted samples are unsuitable for analysis and it is not recommended that these samples be analyzed.  If samples of questionable or substandard quality are analyzed, any procedures and possible inaccuracies should be documented in writing by the laboratory.  Additionally, any possibly inaccurate results should have easily seen comments on the report to the clinician that clearly state those values may be inaccurate and misleading.

 

1.1.2. Specimen Identification.

Specimens should be identified with pertinent information as determined by the laboratory, such as owner, species, animal signalment, name of clinic or doctor, address, telephone and fax numbers, e-mail address, location from which the specimen was collected, etc. Unique and matching identifiers should be located on both the submission form and container.

 

1.1.3. Test Identification.

The requested test(s) should be clearly marked or stated on the submission form.

 

1.1.4. Specimen Accessioning.

The specimen information, identification and requested tests should be correctly entered into the laboratory information system (LIS).  Information entered into the LIS may be used to track the location and appropriate storage of the sample, e.g., immunology vs. hematology section or frozen vs. refrigerated.  Specimen aliquotting and delivery to the appropriate section within the laboratory or between several departments should be coordinated. Any problems with sample quality, including but not limited to hemolysis, lipemia, gelling of the sample, or other analytically significant problems should be recorded and reported to the clients and laboratory staff.  If the degree of inaccuracy associated with sample quality is likely to be significant, testing should not be performed on the sample in question and/or results should not be reported. Problems with the reported or unreported results should be communicated to the client and, if possible, a new sample obtained.

 

1.1.5. Client Communication and Education.

Communication between laboratory personnel and clients (internal and external) should be timely and courteous regarding pre-analytical factors influencing laboratory test results (e.g., incomplete submission forms, inappropriate sample or sample handling, or poor sample quality). Clients should be informed of the expected time for receipt of preliminary and final reports.  Similarly, feedback from clients to laboratory should be encouraged.  All verbal or written complaints/feedback/suggestions should be documented and forwarded to the appropriate level of management.  Management meetings, etc. must be documented and organizational reviews conducted to ensure timely and appropriate follow up on corrective actions.

 

1.1.6. Personnel Safety.

Conditions should be comfortable and appropriate for computer entry, data transcription, handling of specimens, specimen disposal, and all other tasks.  Special consideration should be given for repetitive work.  Personal protective equipment (PPE) should be appropriate for handling specimens and operating equipment in all areas of the clinical laboratory.  Safety procedures for the disposal of all samples, waste, and other supplies should be appropriate for the type of material.  Personnel should receive safety and biohazard training regarding exposure to potentially hazardous chemicals or infectious pathogens present in biological materials.  Documentation of environmental, health and safety training should be available and readily accessible for each staff member. Training should include basic prevention of bacterial contamination as well as information on zoonotic diseases.  All training should be documented.

 

1.1.7. Laboratory Environment.

The laboratory environment should meet standard requirements necessary for safe, rapid, efficient and effective performance.  The workspace should be well-lit and organized in order to promote efficiency and safety.   Equipment and instrumentation should be in working order. Up-to-date procedure protocols should be easily accessible for reference when needed. Laboratory facilities and operation should be in compliance with appropriate government agencies.

 

1.1.8. Personnel Requirements.

Personnel should meet training requirements as indicated for specific areas of the laboratory.  Training, continuing education, and recertification for specialized tasks should be regularly scheduled and documented.  The laboratory should be staffed appropriately to meet the workload.

 

1.1.9. Laboratory Information Systems (LIS).

LIS are intended to improve work-flow and efficiency of the laboratory. Prior to implementation, a LIS should be thoroughly evaluated, and the ability to maintain accurate records verified.  Inefficient and unwieldy LIS should be updated or enhanced based on the needs of the laboratory.  LIS should meet all governing legal regulations for medical record archives.  Problems with sample accessioning or archival storage should be corrected immediately.

 

1.1.10. Identification of out-sourced tests ("send-outs").

Clients should be informed of those tests that are referred to other laboratories. 

 

1.2. Manual Hematology of Non-mammalian Species

 

1.2.1. Specimen Collection, Handling and Transport to the Laboratory.

Acceptable transport times for avian blood are shorter than that for mammalian and reptilian blood.  Controlled studies have shown that refrigerated avian blood deteriorates within twelve hours regardless of anticoagulant (Harr et al, 2005). Acceptable transport time for avian blood smears on glass slides is similar to reptilian and mammalian blood smears.  Evidence of leukocyte/thrombocyte aggregation in the hemacytometer should be reported to indicate erroneous total WBC and differential cell counts.  Hematology samples for shark species should be processed within 5 hours (Arnold, 2005).  EDTA (7.5% or 1-2 mg/mL of blood) is acceptable for most animal species, but is not suitable for all.  Blood from stingrays, some bony fishes and some avian species reacts atypically in commercially prepared EDTA tubes. Elasmobranch blood (shark, skates and rays) should be placed in a dry anticoagulant due to the high plasma osmolality values (~ 1000mmol/kg).  Liquid anticoagulants may be used if adjusted for osmolality.

 

1.2.2. Personnel Requirements.

Laboratory personnel should have specific training in specimen handling and sample preparation for exotic species. Training should include basic prevention of bacterial contamination as well as information on zoonotic diseases including Chlamydophila, West Nile Virus, Salmonella, Avian Influenza and Giardia. Documentation of training, continuing education and periodic proficiency assessment should be at the discretion of the laboratory director.

 

1.3. Urinalysis

 

1.3.1. Specimen Collection, Handling and Transport to the Laboratory.

Identification of the urine collection method is important when interpreting the presence and concentration of potential contaminants including blood and bacteria.  The submitter should clearly state the method by which the urine was obtained, such as free flow (midstream, early, or late), catheterization, cystocentesis, or from the floor or metabolism cage.  Clear specimen containers can be used to facilitate gross examination if urine will be examined within 30 minutes.  However, if urinalysis will be delayed, urine should be protected from exposure to UV light to prevent degradation of urine constituents (eg, bilirubin).  Lids should be secure to prevent evaporation and/or volatilization of urine constituents (eg, ketones).   

 

1.3.2. Urine Storage.

Optimally, urine should be examined within 30 minutes of collection.  If immediate examination is not possible, urine should be stored at refrigerated temperatures to minimize changes in urine physical and chemical make up and to inhibit bacterial growth.  Strict recommendations for duration of refrigerated storage cannot be made, because this depends on specific urine components. (Rabinovitch, 2009) Storage for a maximum of 24 hours in the refrigerator is generally recommended (Osborne cautiously suggests 6-8 hours), but urine may be stable for shorter or longer periods depending on its initial make up.  Chemical constituents that are particularly unstable include bilirubin and glucose, and pH if bacteria are present.  (Rabinovitch, 2009; Osborne, 1999)  Stability of formed elements depends on urine pH and concentration.  Crystals may form in vitro during storage at either room temperature or under refrigeration. (Albasan, 2003; Sturgess, 2001) If crystalluria is a clinical concern, freshly collected urine should be examined immediately. Refrigerated samples should be brought to room temperature prior to analysis.  Because urinalysis results may be affected by storage duration and temperature, the time the urine was collected, the time it arrived in the laboratory, and method of storage should be recorded.  Alternative methods of preservation are available for stabilization of urine chemistry, inhibition of bacterial growth, and preservation of formed elements.  Manufacturer's claims should be followed regarding intended use of particular preservative and duration of storage.

 

1.3.3. Microbiological Culture.  Quantitative microbiologic culture techniques are recommended for determining presence of significant bacteriuria.  Urine specimens collected by cystocentesis are preferred, but specimens properly collected by catheterization and free-catch are acceptable if quantitative culturing methods are employed. Urine should be submitted for the microbiological culture prior to urinalysis procedure to avoid contamination of the specimen.  Alternatively, a sterile aliquot can be set aside for possible microbiological culture subsequent to urinalysis procedure.  Refrigerated urine samples are acceptable for microbiological culture for at least 6 hours and often up to 24 hours.  Refrigeration of urine specimens for 24 hours may result in false negative culture results. (Padilla, 1981) If bacteriostatic transport media is used, urine samples do not need to be refrigerated.

 

1.4. Cytology/Microbiology

 

1.4.1. Specimen Collection, Handling and Transport to the Laboratory.

Information regarding cytologic/microbiologic submissions should be provided to the client in a laboratory service manual, special information sheet, journal or newsletter article, written material, or verbal instructions. Instructions should address issues such as collection techniques, appropriate containers (with or without anticoagulants), smear preparation and specimen fixation, if pertinent. Appropriate collection of cytologic/microbiologic specimens will increase the likelihood of a meaningful interpretation.

 

1.4.2. Unfixed cytologic specimens and air-dried, unstained cytology smears should be protected from exposure to formalin and formalin fumes, which interfere with subsequent staining, by shipping in tightly sealed containers or shipping separately from formalin-fixed biopsy specimens.

 

1.4.3. Identification of the site, method and time of collection is of great importance in determining optimal preparation and in interpretation.  The veterinary cytologist or cytopathologist should be knowledgeable about the effects of different collection methods, delayed preparation and improper handling of cytology specimens, especially fluid samples, with regard to expected cytologic features and interpretation.  For fluid samples, one or more direct smears should be made from the fluid sample before any concentration or fixation procedures are performed. The smear(s) can be stained or left unstained and should be submitted with the fluid sample.  This will allow estimation of the cell count and proportions of various cell types. This may provide valuable information that influences the cytologic interpretation and provides additional QC by allowing the cytologist/cytopathologist to ensure that the cell counts reasonably match estimates made from these smear(s).  This also avoids the situation in which extreme cell density of concentrated samples prevents optimal cytologic evaluation.

 

1.5. Hemostatic Testing (Coagulation)

 

1.5.1. Specimen Collection, Handling and Transport to the Laboratory.

Compliance with sample collection and storage requirements for coagulation is mandatory for accurate test results.  Whole blood should be collected in trisodium citrate anticoagulant in a 9:1 ratio and thoroughly mixed.  This typically is accomplished by filling to the indicated mark on the appropriate blood tube.  Specimens that do not conform to this dilution should be rejected. (Adcock, 1998) Citrate volume may need to be adjusted for samples from very anemic and polycythemic animals.(Stockham and Scott, 2008, p. 277) For tests requiring plasma, the citrated tube is centrifuged and chilled to 2-8ºC.  Plasma should be separated from blood cells and transferred to a plastic tube (not glass).(Fiebig, 2005, Kratz, 2006)  Specimen stability at room or refrigerated temperature (2-8ºC) is 4 hours and 24 hours for APTT and PT, respectively.  If testing is not performed within these time intervals, samples should be frozen at -20 ºC. (Adcock, 1998) Fresh citrated whole blood used for platelet function or other coagulation analyses should be ideally kept for <1 hour. (Giger, per communication).  If samples are mailed into a laboratory for testing versus direct transport, plasma should be placed in a plastic tube then frozen, then be packed on ice and shipped to arrive frozen within 24 hours.

 

1.5.2. Please see laboratory information bulletin 1 1.5.1.A.

Additional detail was added here as >90% of error in hemostatic assay results may be attributed to preanalytical factors of transport and handling.(Lippi, 2006; Valenstein, 2009; Bonini, 2002; Dale, 2002) 

 

1.6 Crossmatch

 

1.6.1 Specimen Collection, Handling and Transport to the Laboratory.

Sera or plasma may be used for crossmatching; however, the animal species and particular procedure may influence this selection.  Fresh sera could serve as a source of complement for use in detection of hemolysis in dogs and cats, but this is not typically performed.  Samples for major crossmatch include serum (non-additive tube) or plasma (EDTA or citrated) from recipient and anti-coagulated (EDTA, ACD, or citrated) whole blood or packed red blood cell sample from the donor(s).  Samples for minor crossmatch include anti-coagulated whole blood from recipient and sera or plasma from donor(s). Patient and donor samples should be less than 24 hours old when possible; donor samples may be as old as the unit of blood to be crossmatched.  If not used immediately, samples should be stored at 4ºC.  For some procedures, whole blood is used while others require a PBS-washed red cell suspension. General recommendations for sample collection, handling and transportation of hematology samples should be followed.

 

1.6.2. Specimen Identification.  Specimens from the patient and donor(s) should be clearly labeled as patient and donor(s) with date and time and for identification of the patient and each donor sample submitted.  Specific forms for submission should be considered to assure accurate assignment of patients and donors.

 

1.7. Radioimmunoassay (section left intentionally blank at this time)

Please see Hegstad-Davies, 2006 for a review of some of the literature.