Laboratory Diagnosis and Test Protocols

Richard B Ford; Elisa M Mazzaferro

doi:10.1016/B0-72-160138-3/50006-0

. 2009 May 21:573–642. doi: 10.1016/B0-72-160138-3/50006-0

Laboratory Diagnosis and Test Protocols

Richard B Ford ^1,^2,³, Elisa M Mazzaferro ^4,⁵

PMCID: PMC7182103

COMMON REFERENCE RANGE VALUES*

SAMPLE HANDLING

SAMPLE IDENTIFICATION

Identification of specimens is critical if the right result on a given patient is to get back to the right clinician in a timely manner. The following steps are recommended:

1.
Write the animal/client name on each specimen container.
2.
Write the animal and client name, species, breed, gender, and date on the test requisition form.
3.
Make sure that the originating clinic name and account number are clearly identified on the form.
4.
Clearly mark or write down the needed tests on the form. (NOTE: Commercial laboratories receive hundreds of samples each day with no test marked!)
5.
Indicate the source, if other than a blood sample, on the form.
6.
Identify the tissue or fluid source and clinic ID on all slides submitted for cytology (use a lead pencil to write on the frosted side).

SAMPLE COLLECTION TUBES

Most practices utilize a variety of glass vacuum tubes (Vacutainer)† to collect and submit blood, serum, or plasma from individual patients. The tubes are actually designed for collecting blood samples from humans. A variety of tube sizes, each of which maintains a pre-determined negative pressure (vacuum) inside, are available. The vacuum facilitates collection of an appropriate volume of the patient's blood to nearly fill the tube. Additionally, most of the blood collection tubes contain an additive that will either accelerate or prevent clot formation.

Adult (human) tubes are available in 5-mL, 7-mL, 10-mL, and 15-mL sizes. Pediatric (human) tubes, appropriate for use in companion animal patients, are available in 2-mL, 3-mL, and 4-mL sizes. For tubes containing an additive, filling the tube with an appropriate volume of blood is important. Underfilling any tube that contains an additive may sufficiently alter the sample such that the test results are adversely affected and may not accurately represent the patient's status.

The color of the stopper in the top of the tube indicates the type of additive, if any, and the specific type of tests that can be performed with that sample. For example, do not send serum when plasma is required!

Reference range values listed for TABLE 5-1, TABLE 5-2, TABLE 5-3, TABLE 5-4, TABLE 5-5 are representative values only and will vary among individual laboratories.

TABLE 5-1.

Hematology Reference Range Values

Test	Adult canine	Adult feline	Units
Red Blood Cell (total)	5.32–7.75	6.68–11.8	×10⁶ cells/mm³
Hemoglobin (Hgb)	13.5–19.5	11.0–15.8	grams
Hematocrit (Hct)	39.4–56.2	33.6–50.2	%
Mean Corpuscular Volume (MCV)	65.7–75.7	42.6–55.5	fL
Mean Corpuscular Hemoglobin (MCH)	22.57–27.0	13.4–18.6	pg
Mean Corpuscular Hemoglobin	34.3–36.0	31.3–33.5	g/dL
Concentration (MCHC)
Platelet Count	194–419	198–405	×10³ cells/mm³
Mean Platelet Volume (MPV)	8.8–14.3	11.3–21.3	fL
White Blood Cell (Total)	4.36–14.8	4.79–12.52	×10³ cells/mm³
Segmented Neutrophils (Segs)	3.4–9.8	1.6–15.6	×10³ cells/mm³
Non-Segmented Neutrophils (Bands or Non-Segs)	0–0.01	0–0.01	×10³ cells/mm³
Lymphocytes (Lymphs)	0.8–3.5	1.0–7.4	×10³ cells/mm³
Monocytes (Monos)	0.2–1.1	0–0.7	×10³ cells/mm³
Eosinophils (Eos)	0–1.9	0.1–2.3	×10³ cells/mm³
Basophils (Basos)	0	0	×10³ cells/mm³

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TABLE 5-2.

Biochemistry Reference Range Values

Test	Adult canine	Adult feline	Units
Glucose	73–116	63–150	mg/dL
Blood Urea Nitrogen (BUN)	8–27	15–35	mg/dL
Creatinine (Cr)	0.5–1.6	0.5–2.3	mg/dL
Phosphorus (P)	2.0–6.7	2.7–7.6	mg/dL
Calcium (Ca)	9.2–11.6	7.5–11.5	mg/dL
Ionized Calcium (iCa)	1.15–1.39	—	mg/dL
Total Protein (TP)	5.5–7.2	5.4–8.9	g/dL
Albumin (Alb)	2.8–4.0	3.0–4.2	g/dL
Globulin (Glob)	2.0–4.1	2.8–5.3	g/dL
Cholesterol (Ch)	138–317	42–265	mg/dL
Bilirubin (Total)	0–0.2	0.1–0.5	mg/dL
Alkaline Phosphatase (SAP or Alk Phos)	15–146	0–96	IU/L
Alanine Aminotransferase (ALT)	16–73	5–134	IU/L
Gamma Glutamyltransferase (GGT)	3–8	0–10	IU/L
Creatine Kinase (CK; formerly CPK))	48–380	72–481	IU/L
Sodium (Na)	147–154	147–165	mEq/L
Potassium (K)	3.9–5.2	3.3–5.7	mEq/L
A:G ratio	0.6–2.0	0.4–1.5	—
Na/K ratio	27.4–38.4	30–43	—
Chloride (Cl)	104–117	113–122	mEq/L
Bicarbonate (Venous)	20–29	22–24	mEq/L
Anion Gap	16.3–28.6	15–32	—
Osmolality (Calculated)	292–310	290–320	mOsm/kg
Amylase	347–1104	489–2100	IU/L
Lipase	22–216	0–222	IU/L
Triglyceride (TG)	19–133	24–206	mg/dL

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TABLE 5-3.

Urinalysis (Voided Sample) Reference Range Values

Test	Canine	Feline
Specific Gravity (SpGr)	variable	variable
Color	pale to dark yellow	pale to dark yellow
pH	5.0 to 8.5	5.0 to 8.5
Protein	negative to + 1	negative to + 1
Glucose	negative	negative
Ketones	negative	negative
Bilirubin	negative to trace	negative
Blood	negative	negative
*Microscopic:*
Red Blood Cell (RBC) Count	<5 RBCs/hpf	<5 RBCs/hpf
White Blood Cell (WBC) Count	<3 WBCs/hpf	<3 WBCs/hpf
Epithelial Cells	negative	negative
Casts	negative	negative
Bacteria	negative	negative
Special: Urine Protein:Creatinine	<0.3	<0.6

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TABLE 5-4.

Hemostasis Reference Range Values

Test	Canine	Feline
Platelet Count	166–600 × 10³/μL	230–680 ×10³/μL
Prothrombin Time (PT)	5.1–7.9 sec	8.4–10.8 sec
Activated Partial Thromboplastin Time (APTT)	8.6–12.9 sec	13.7–30.2 sec
Fibrin Degradation Products (FDP)	<10 μg/mL	<10 μg/mL
Fibrinogen	100–245 mg/dL	110–370 mg/dL
Activated Clotting Time (ACT)	60–110 sec	50–75 sec

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TABLE 5-5.

Blood Gas Analysis—Arterial Reference Range Values

Test	Canine	Feline	Units
pH	7.36–7.44	7.36–7.44	-
PO₂	90–100	90–100	mmHg
PCO₂	36–44	28–32	mmHg
HCO₃⁻	24–26	20–22	mEq/L
TCO₂	25–27	21–23	mEq/L

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Note: Most commercial laboratories recommend collecting a minimum volume of 2.0 whole blood for routine biochemical analyses; 2.0 mL of whole blood will yield close to 1.0 mL of serum. Dehydrated patients are expected to have a higher hematocrit, and therefore a larger volume of whole blood may be required in order to obtain a 1.0-mL sample of serum.

Note: When collecting blood from a patient, it is critical to use:

1.
The appropriate sized tubes
2.
Tubes that contain the proper additive for the test(s) being requested

Types of commercial blood collection tubes used in veterinary medicine are discussed in TABLE 5-6, TABLE 5-7, TABLE 5-8, TABLE 5-9, TABLE 5-10, TABLE 5-11, TABLE 5-12, TABLE 5-13, TABLE 5-14 . Interpretation of the in-office coagulation screen is discussed in Table 5-15 .

TABLE 5-6.

Red-Topped Tube

Additive	None
Effect	Allows blood to clot naturally; centrifugation is required to separate serum
Tests	Routine biochemistry, serology; cross-match; most liquid samples collected by centesis or aspiration for biochemistry or cytopathology— e.g., CSF, abdominal fluid

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TABLE 5-7.

Red and Gray Mottled Top (“Tiger-Topped Tube”); Also Called Serum Separator Tube (SST)

Additive	Serum separator gel with clot activator
Effect	Gel at the bottom of tube acts to separate cells (lower fraction) and serum (upper fraction); sample should be centrifuged to stabilize
Tests	Routine biochemistry, serology

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TABLE 5-8.

Lavender-Topped Tube; Also Called “Purple-Topped Tube”

Additive	EDTA (liquid)
Effect	Anticoagulant; removes calcium
Tests	Routine hematology; cross-match. NOTE: Invert eight times to prevent clotting and platelet clumping

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TABLE 5-9.

Dark Green-Topped Tube

Additive	Sodium heparin or lithium heparin
Effect	Anticoagulant; inactivates thrombin and thromboplastin, allowing isolation of plasma; sample must be centrifuged to isolate plasma
Tests	Ammonia, lactate, and other tests requiring plasma (see under Sample and Submit for individual tests listed in this section)

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TABLE 5-10.

Light Blue-Topped Tube

Additive	Sodium citrate
Effect	Anticoagulant; removes calcium; NOTE: EDTA anticoagulated samples do NOT substitute for samples requiring citrated plasma
Tests	Most coagulation profiles; NOTE: Tube must be filled to the capacity allowed by the vacuum

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TABLE 5-11.

Dark Blue-Topped Tube

Additive	Sodium heparin or Na EDTA
Effect	Anticoagulant; tube is designed to contain no contaminating metals
Tests	Toxicology and trace element testing (e.g., zinc, copper, lead, mercury) and certain drug level testing (consult with laboratory prior to submitting)

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TABLE 5-12.

Gray-Topped Tube

Additive	Sodium fluoride and potassium oxalate
Effect	Antiglycolytic agent that serves to preserve glucose for up to 5 days
Tests	Glucose and 12- hour glucose curves; NOTE: Inadequate volume may result in sample hemolysis

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TABLE 5-13.

Yellow-Topped Tube

Additive	Acid-Citrate-Dextrose (ACD)
Effect	Inactivates complement
Tests	DNA testing

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TABLE 5-14.

Brown-Topped Tube

Additive	Sodium heparin
Effect	Inactivates thrombin and thromboplastin
Tests	Lead determination (consult with individual laboratory regarding alternative handling)

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TABLE 5-15.

Interpretation of the In-Office (or Point-of-Care) Coagulation Screen

Platelet (estimate)	Low	Thrombocytopenia
ACT	Rapid, prolonged	Intrinsic or common clotting pathway defect
APTT	Rapid, prolonged	Intrinsic or common clotting pathway defect
BMBT	Prolonged	Thrombocytopenia, thrombocytopathia

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SAMPLE STORAGE AND TRANSPORT

Several types of sample collection/sample submission storage tubes are available. It is critical that the type of blood collection and/or storage tube used meets the requirements of the test as defined by the laboratory.

To prepare a sample for storage and transport:

1.
Stabilize serum from serum separator tubes (SSTs) by centrifuging the specimen before submission. If being mailed, it is preferable to transfer the separated serum to a labeled plain red-topped tube (RTT).

Note: Depending on the test requested, the tube used to COLLECT the sample is frequently NOT the same tube used to SUBMIT the sample. Sample collection and sample submission requirements are provided for ALL tests listed in this section.
2.
Centrifuge the blood samples in a plain RTT and transfer the serum to another RTT.
3.
Refrigerate and transport all blood specimens, cytology fluids, tissues, viral cultures, and urines for urinalysis or culture with ice packs.
4.
Keep all routine microbial cultures (except urine) and blood cultures at room temperature.
5.
If a specimen must remain frozen for transport, dry ice is required. It is usually the responsibility of the individual practice to package frozen samples correctly. Most laboratories do not provide dry ice for shipping.

PATIENT PREPARATION

Fasting the patient for 8-12 hours (an overnight fast with free access to water) is often helpful to reduce the likelihood of lipemia, which may interfere with several tests by falsely increasing or decreasing the results. When applicable, comments about the presence and influence of lipemia and/or hemolysis should appear on the laboratory reports. For special tests, patient preparation may include restriction of food as well as water and certain drugs. It is important to follow the guidance provided in this section regarding patient preparation or to contact the laboratory for specific instructions.

MINIMIZING HEMOLYSIS

Hemolysis during blood drawing can be minimized by adhering to the following recommendations.

Procure a nonlipemic (fasted) sample, because lipemia can increase red cell fragility. During phlebotomy, negative pressure created by the vacuum tube or syringe may collapse the lumen of the vein against the needle, thereby crushing numerous red cells. The flutter of the lumen against the needle can be stopped by reducing the negative pressure exerted during collection and by repositioning the needle with slight rotation or deeper insertion.

Excessive negative pressure exerted as the blood enters the vacuum tube or syringe can create hemolysis. This occurs during a slow or difficult collection, because the natural tendency is to use more negative force to enhance blood flow. More patience and “milking” the vein by alternating gentle negative pressure with a short release of all pressure usually solves the problem.

Hemolysis often occurs during the transfer of blood from a syringe into vacuum or other tubes. If a small-gauge needle is used, transfer of blood to specimen tubes is slowed, especially if small clots are present. Forcing the blood through a small-bore needle contributes to hemolysis. This problem can be avoided by removing the needle and top of the specimen tube, and transferring the blood directly into the open tube. Recapping the tube and aspirating a small amount of air to reestablish negative pressure helps to avoid having caps coming off in transit.

AVOIDING CLOTS AND PLATELET CLUMPS

The presence of clots and clumped platelets in anticoagulated blood is most commonly caused by a slow blood draw and the resulting delay in mixing it with the appropriate anticoagulant. If the venipuncture was traumatic, tissue fluid (thromboplastin), activated clotting factors, and hemolysis will quickly promote clot formation. The slight transfer delay when using a syringe for collection can also contribute to this problem. To avoid the formation of clots:

1.
Select a vein with good blood flow—the larger the better.
2.
Minimize the trauma of venipuncture.
3.
Collect blood directly into anticoagulated vacuum tubes (e.g., blue-topped tube [citrated] or lavender-topped tube [EDTA]).
4.
Mix the tube well by inverting several times immediately after filling.

If the syringe method is selected and a difficult draw is anticipated, the potential for clotting can be minimized by first rinsing the needle and syringe with a small quantity of liquid citrate (blue-topped tube) or EDTA (lavender-topped tube). However, the anticoagulant must be emptied from the syringe before proceeding, and care must be taken to match the anticoagulant chosen with the tests to be performed. Even trace amounts of heparin or EDTA will invalidate coagulation testing, whereas EDTA or citrate will alter the accuracy of several chemistry assays. A small amount of heparin contamination is acceptable in most chemistry assays and complete blood count parameters.

Platelet clumping in samples from cats is very common and is caused by contact aggregation. An effective method to prevent this clumping has not been found. Applying fresh blood directly to the slide from the syringe and making the blood smear immediately after collection is an effective method of assessing platelet numbers in cats.

SUBMISSION REQUIREMENTS FOR RABIES SUSPECTS

Guidelines for submitting tissue from dead dogs or cats for rabies diagnostic testing vary somewhat from state to state. It is always important to contact your State Veterinary Diagnostic Laboratory or Dept of Public Health prior to shipping any samples. Most public health authorities require advance notification about impending submission of samples for rabies testing. Veterinarians should verify the address, paper work requirements and shipping requirements prior to submitting any samples for rabies testing.

Note: CAUTION: Care must be taken during sample preparation to avoid direct personal contact with specimens. Pre-exposure rabies vaccination is recommended for persons preparing rabies specimens.

SAMPLE SUBMISSION FOR RABIES TESTING

1.
Laboratories may limit acceptance of tissue from dead animals for rabies testing to those for which there is a documented reason for considering that animal a rabies-suspect mammal. Generally this includes animals for which there has been a reported bite, scratch, or other possible saliva or nervous tissue exposure to a human.
2.
Most laboratories will accept any bat as long as there is reasonable likelihood that a human was exposed.
3.
Brain tissue from a rabies-suspect mammal reported to have bitten (or otherwise had “intimate” contact with) a domestic animal will likely be acceptable (e.g., brain tissue from a stray dog or cat that bit a pet dog or cat).
4.
Highly suspect surveillance specimens (with no reported contacts) include
- a.
  A rabies vector species (e.g., skunk or raccoon) showing clear signs of rabies infection
- b.
  A mammal not commonly recognized as a rabies vector, but showing clear signs of rabies infection
- c.
  A domestic animal that dies or is euthanized under the care of a veterinarian for which rabies is part of the differential diagnosis of a neurologic disorder.
5.
Most laboratories will not accept live animals as rabies suspects. The intact head only of authorized specimens will be accepted. Exceptions include bats, which should be submitted whole, and livestock, for which a cross-section of the brainstem and representative sections of brain (as defined by the laboratory) may be removed by a veterinarian and submitted. Special livestock instructions may apply.

PACKAGING REQUIREMENTS FOR AUTHORIZED SAMPLES

In the case of a suspect dog or cat, the entire brain must be properly packaged in a standard rabies shipping container (these are often provided at County Health Departments). Specimens must be accompanied by a completed rabies specimen history form. Forms can often be downloaded from a website designated by the State Public Health authorities or the diagnostic laboratory.

INFORMATION REQUESTED ON THE RABIES SPECIMEN HISTORY FORM

1.
Name and address of veterinarian submitting the specimen.
2.
Name and address of owner (if known).
3.
Indicate whether or not human exposure occurred and the type of exposure (e.g., bite, scratch). Also note whether exposure to a rabid animal is known or highly suspect.
4.
Specimen:
- a.
  Type of specimen.
- b.
  Age/breed/gender/pet versus stray versus wildlife.
- c.
  Cause of death (euthanasia, killed, natural causes).
- d.
  Medical history of the animal (if known), including date of last rabies inoculation.
- e.
  Health status of the animal at the time of death.
5.
Location: describe the geographic location (exact address) of the animal when the specimen was collected.

SUBMISSION GUIDELINES

1.
Diagnostic testing of the specimen is generally performed by a designated laboratory within the state. Prior authorization to submit a rabies-suspect specimen is generally required; it is always recommended.
2.
If the submission is an emergency, or made over a weekend/holiday, most laboratories will provide specific instructions to accommodate a veterinarian's request.
3.
Do not submit live animals.
4.
If the suspect animal is alive, it should be humanely euthanized without damaging the head. The head must then be removed from the body and submitted intact for examination. Brain tissue that is damaged may not be accepted by the laboratory. Dead suspect bats can usually be submitted with the head intact.
5.
Specimens must be preserved by refrigeration. Freezing should be avoided. Only if refrigeration is not available can the tissue be submitted frozen.
6.
Tissues must not be fixed with chemical preservatives.
7.
Tools, cages, and other surfaces potentially contaminated with infectious saliva or blood can be disinfected with a solution of sodium hypochlorite (1 part household bleach to 10 parts water) in water.
8.
Properly packaged specimens may be shipped directly to the rabies laboratory (verify correct address) by parcel post or commercial mail carrier. Special arrangements are likely to be required for samples arriving over weekends or holidays.

PACKING AND SHIPPING DIRECTIONS

An acceptable rabies suspect shipping set may include any of the following:

1.
One pre-assembled shipping container, including outer cardboard box, insulated cooler, and two gallon-sized cans with lid-locking plastic seal. Packing instructions for package are printed on top inner flaps.
2.
Two gel packs of refrigerant (store the pack—not the specimen—frozen until needed).
3.
Two plastic bags (13 × 20 inches × 4 mil) in which the animal head, brain of livestock or other large animal, or intact bat is to be sealed before placing in can.
4.
Two plastic bags (13 × 20 inches × 4 mil) in which to place the cans.
5.
One large plastic bag that surrounds the closed insulated cooler.
6.
Two absorbent pads to be placed in the cans, surrounding the specimen.
7.
Two blank rabies history forms and directions for collection and submission of specimens.

To prepare the specimen for shipping:

1.
Remove the head from the body of the animal (except bats) and place the head in a small plastic bag. Cool specimen in a refrigerator or freezer before packaging, to enhance preservation.
2.
When shipping samples consisting of only cerebellum and brainstem, first place the brain tissue in a small plastic container, then place the container in the small plastic bag. If sharp objects protrude from the specimen (e.g., bone fragments, porcupine quills) wrap specimen in several layers of newspaper before putting head in the plastic bag. Wrap bagged specimen in provided absorbent material and place inside the metal can.
3.
Place the lid on the metal can and secure with a mallet. Place a plastic pressure ring (provided) on the can and secure with a mallet. The plastic ring will be seated more easily if a hard surface is placed on top of the ring before using the mallet. This will allow even pressure to be applied to the ring. CAUTION! Infectious splashes can occur when hammering the lid in place if the groove is contaminated with blood or body fluids in the specimen.
4.
Wash hands well with soap and water. Disinfect or burn all materials contaminated in specimen preparation.
5.
Complete the rabies specimen history form provided with the package. Answer all questions as accurately as possible; the history form will be used to report results to the local health authority. Place form on the outside of the plastic bag that surrounds the EPS cooler. When shipping more than one specimen in the container (e.g., bats), be certain that: each specimen is individually bagged to prevent cross contamination; each is clearly identified, and a separate history included for each specimen.
6.
CAUTION: Do not use glass, wire, or other packaging materials capable of causing wounds or injuring skin.

HISTOPATHOLOGY AND CYTOPATHOLOGY

Histopathology and cytopathology are among the most important diagnostic tools available for use in clinical practice. Generally, diagnostic specimens are submitted to a commercial laboratory or university where specially trained technologists can prepare and stain the cells/tissue to be interpreted by a pathologist. One critical limiting factor in obtaining diagnostic cytology or histopathology is the quality of the specimen submitted. It is the responsibility of the practice to not only obtain, but also prepare, specimens properly before submission and interpretation. This part of Section 5 describes standards for preparing and submitting specimens for cytologic or histopathologic interpretation. Sample collection techniques are described in Section 4.

HISTOPATHOLOGY

Biopsy Tissue

Tissue specimens for histology must be preserved and transported in formalin (10 parts formalin to 1 part tissue). The ideal tissue specimen is less than an inch thick. OSHA and transportation safety regulations limit the size and quantity of formalin containers that can be shipped. It is strongly recommended to use containers supplied by the laboratory or the FAA-approved airline, place the container in a ziplock plastic bag, and then in a second outer bag that contains the requisition. Samples packaged inappropriately may not be picked up by the courier. CAUTION: Do not enclose cytology samples in bags containing formalin-fixed tissues because this may alter the cytologic appearance and staining of the cells of interest.

Very Large Specimens

Several (preferably three or more) representative sections of large tissues or organs should be selected, preserved, and transported for histology. The remainder should be placed in a large plastic container of formalin, refrigerated, and retained in case additional samples are needed.

Tissue Orientation and Information

Knowing the orientation and other facts about the tissue mass is critical for the pathologist. A diagram may be included on the requisition form. Borders and areas of interest on the mass can be marked with colored or numbered sutures. State whether the entire mass has been excised, if all is being submitted, or if the tissue had to be divided into sections before submission.

Very Small Specimens

Tiny samples, such as endoscopic biopsy specimens, are best preserved if they are first placed in a labeled tissue cassette holder (usually available from the laboratory) and then dropped into formalin. Small biopsies should not be placed in a container with large tissue, because they are easily lost.

CYTOPATHOLOGY

Used alone, as a diagnostic screening test for underlying disease, or used in conjunction with the surgical biopsy to facilitate rapid assessment of a potentially serious lesion, cytopathology is among the most fundamental and important diagnostic tools used in clinical practice. Cytopathology is not a clinical discipline restricted to the realm of board-certified clinical pathologists. Several continuing education short courses and laboratories on diagnostic cytopathology are offered at major conferences throughout the U.S. In addition, excellent textbooks, with abundant color plates, are available to facilitate cytologic interpretation of specimens collected from dogs and cats.

Cytologic preparations are perhaps most useful for distinguishing details between cell types (e.g., mesenchymal vs. epithelial) and cellular activity (e.g., inflammation vs. neoplasia). Detection of intracellular vs. extracellular organisms can provide immediate clues, without waiting for organisms to be cultured, about the nature of the disease. Noninflammatory lesions can generally be distinguished as benign or neoplastic (Box 5-1).

Although it is the responsibility of the individual clinician to understand personal limitations, there is one special advantage that the clinician does have over the pathologist—familiarity with the patient's health status and the nature of the lesion/disease under consideration. Described here are guidelines for preparing and submitting samples for cytologic interpretation (see Section 4 for sample collection techniques). Whether samples are sent to a commercial laboratory or a university, or are interpreted within the practice, the following recommendations are important when preparing a high-quality specimen.

Note: The accuracy of interpreting cytopathologic specimens is dependent on four key variables:

•
Experience and training of the clinician
•
Selection of the appropriate case/lesion
•
Cellular quality of the specimen selected
•
Techniques used to collect, prepare, and stain the sample

Fine Needle Aspiration (FNA)

Indications

FNA Involves the use of a syringe and needle to extract cells from a palpable lesion. Most commonly, FNA is performed on cutaneous and subcutaneous lesions. However, with the increasing use of ultrasound in private practice, it may not be necessary to actually “palpate” a lesion in order to extract diagnostic cytology (e.g., ultrasound-guided hepatic or splenic aspirates). Additional experience and training are essential when attempting to perform ultrasound-guided FNA.

Sample preparation

Because sample size (“harvest”) typically is small, the cells collected are discharged directly onto a dry, clean slide and allowed to rapidly (within 5 to 10 seconds) air-dry. It is recommended that the needle tip actually contact the slide as the aspirate is discharged rather than blowing the sample over the slide. If fluid is inadvertently recovered, the FNA should be reattempted from the peripheral limits of the lesion.

Extremely small harvest of cells can be sprayed directly on the slide, remain untouched, and allowed to air-dry. If the volume recovered allows placement of a formed drop onto the slide, the sample should be spread over the surface of the slide, prior to air-drying, in the same way that a peripheral blood smear is prepared.

Staining options

Once the sample has air-dried (rapidly), use of a quick Romanowsky-type (Wright's) stain is appropriate. Alcohol fixation is not recommended if the specimen is to be reviewed/interpreted immediately. Alternative stains, such as new methylene blue (wet mount), Gram stain, Giemsa stain, or Wright-Giemsa stain, can be used in practice as dictated by cytologic objectives.

FNA specimens mailed to an outside laboratory typically are air-dried and left unstained. Some laboratories recommend that the specimen be immersed in methyl alcohol for a few minutes prior to sending, although this additional step seems to be optional.

Common mistakes

Low cell harvest, high cellular density on the slide (e.g., the result of making a “bad” slide or failing to adequately disperse the sample), and obtaining nondiagnostic material are the three most common mistakes when obtaining samples for diagnostic cytopathology. Contamination of the “wet” (not yet air-dried) sample with water, alcohol, or stain can create artifacts that will compromise the diagnostic value of the specimen. Excessive blood or tissue fluids may profoundly dilute the diagnostic sample, making interpretation difficult or impossible.

Exfoliative Cytology (“Impression Smear”)

Indications

Exfoliative cytology is made from a clean surface of exposed lesions or from the surface of tissue collected during biopsy. Preparations made from the cut surface of fresh biopsy specimens or post-mortem tissues provide the greatest diagnostic yield.

Sample preparation

To avoid one of the most common mistakes, excessive tissue fluid or blood is absorbed from the cut surface (using a scalpel blade) of the specimen before attempting to exfoliate cells on a slide. Clean, high-quality absorptive paper (such as filter paper) works well, and fragments of paper will not be left on the specimen.

Once excess fluid has been absorbed from the surface, the specimen is gently grasped and allowed to make gentle contact with a clean slide. The actual weight of the specimen is usually sufficient; it is usually not necessary to press the specimen onto the slide. After multiple contacts with the slide have been made, the sample is rapidly air-dried.

Staining options

Once the sample has air-dried (rapidly), use of a quick Romanowsky-type (Wright's) stain is appropriate. Alcohol fixation is not recommended if the specimen is to be reviewed/interpreted immediately. Alternative stains, such as new methylene blue and Gram stain (wet mounts), Giemsa stain, or Wright-Giemsa stain, can be used in practice as dictated by cytologic objectives.

Common mistakes

Excessive or rough handling of the specimen before attempting exfoliation will compromise the quality of the specimen. In addition, excessive blood or tissue fluid on the cut surface of the tissue may effectively “dilute” the diagnostic cells in the specimen, making interpretation difficult. When additional pressure is used to exfoliate cells or specimen is rubbed across the slide, individual cells are likely to rupture and smear, rendering the sample nondiagnostic. Failure to obtain adequate numbers of diagnostic cells is more likely to be the consequence of the type of tissue being examined than poor technique. Epithelial tissues (liver/spleen/adenoma/carcinoma) tend to exfoliate abundant numbers of cells when applied to a slide. In contrast, mesenchymal cell tissues (fibrosarcoma/chondrosarcoma) tend not to exfoliate well. Diagnostic yield of cells from mesenchymal tissue may be so low as to warrant submission of fixed tissue for histopathologic examination.

Swabs, Scrapings, Washings, or Brushings

Indications

A variety of techniques are available to collect cytologic specimens from the upper and lower respiratory tract, conjunctiva, ear canals, and vaginal mucosa. In most cases, cytologic objectives focus on the recovery and identification of infectious organisms (mites, bacteria, etc). Section 4 describes the various techniques of sample collection from these locations.

Sample preparation

Skin scrapings and ear swabs for diagnosis of infectious agents, and occasionally neoplasia, are perhaps the most common samples used in practice to collect diagnostic specimens. Gentle handling of the specimen once collected is the rule when attempting to exfoliate diagnostic cells or organisms. In addition, it may not be necessary to air-dry or apply a stain depending on the samples collected (e.g., skin scrapings or ear swabs for mites).

Samples collected from washings vary considerably in the cell harvest, the consistency of the fluid recovered, and the quality of the diagnostic specimen. In some cases, fluid recovered from washings (e.g., bronchoalveolar lavage, transtracheal aspiration) will require centrifugation to acquire sufficient numbers of diagnostic cells. The supernatant (fluid portion) of the sample is discarded. The cells recovered may be re-suspended in 1 or 2 drops of sterile saline or a volume of saline equal to the volume of specimen remaining in the centrifuge tube. A pipette is used to apply a sample of the fluid to a slide. The sample is distributed over the slide in the same way that a peripheral blood smear is prepared. The slide is air-dried and stained. In other cases, the sample collected from cytologic washings will be highly cellular and may be applied directly to a slide, air-dried, and stained.

Samples collected from brushings normally are obtained with specially made cytology brushes designed for use during endoscopy. Although small “pinch” biopsies are preferred, occasionally the use of a brush may be the only practical option. Cytologic specimens collected by brushing tend to be especially low in yield. Furthermore, the additional manipulation required to extract cells from the brush and onto a slide for examination tends to yield specimens of poorer quality. Cells obtained during brushing may be applied directly to a clean slide, air-dried, and stained. In other cases, it may be preferable to wash the brush in a centrifuge tube containing a small volume (<1.0 mL) of sterile saline. The suspended cells may be applied directly to a slide, distributed, and then air-dried and stained. It may be necessary to centrifuge the sample (as described for washings previously) before preparing the sample.

Staining options

Generally, the same staining options previously described apply to specimens collected from washings or brushings. Samples collected from skin scrapings typically are suspended in oil or hydrogen peroxide on the slide and examined “wet” without the use of additional stain. Swabs, especially from ears, may be stained with a quick Romanowsky-type stain or a Gram stain (wet mount) to facilitate identification of organisms.

Common mistakes

Samples collected from skin scrapings and swabs tend to be relatively high in yield when diagnostic cells or organisms are present. Cells collected from washings and brushings are usually collected during endoscopic procedures; the yield of diagnostic cells can vary, depending on the extent of the lesion as well as the skill of the individual performing the procedure.

Body Fluids

Indications

The accumulation of fluid in either the pleural space or the abdomen, or in both, justifies attempts to remove fluid for diagnostic cytology. The volume of sample can be difficult to determine, but ideally would be 2-3 mL of fluid collected by needle and syringe (centesis) under sterile conditions. Smaller samples of joint fluid and CSF are also collected for chemical and cytologic analysis. Any fluid recovered should be examined for color, consistency, total nucleated cell count, and protein concentration as well as for morphology of the cells recovered. Other chemistries (creatinine, amylase, etc.) can be determined depending on the nature of the fluid recovered and the patient's condition.

Sample preparation

Because the volume of fluid obtained may be large and the concentration of cells in the fluid recovered may be low, centrifugation is indicated to concentrate cells in small aliquots of fluid. After centrifugation and removal of the supernatant, cells can be resuspended in 1-2 drops of sterile saline/supernatant. Suspended cells should be distributed directly on a slide, allowed to air-dry, and stained.

Note: It is important not to delay processing of cytologic samples recovered from body fluid. The longer cells are allowed to remain in suspension, the greater is the opportunity for morphologic changes of cells to occur.

Spinal fluid must be processed within 30 minutes of collection because of the fragility of cells in CSF. Furthermore, conventional centrifuges may damage any cells collected. Because of the complexities associated with processing of CSF for cytopathology, most samples are evaluated within specialty or referral hospitals.

Staining options

Air-dried cytologic preparations can be stained in the same manner described previously.

Common mistakes

Attempting to evaluate uncentrifuged cytologic specimens collected from body fluids can result in a low yield of diagnostic cells and may compromise the study. Allowing the cells to remain in the fluid for an extended period of time before making the cytologic preparations may significantly alter the morphology of individual cells, making interpretation difficult or impossible. Furthermore, the presence of peripheral blood in any sample collected from a body cavity must be distinguished from contamination associated with the sampling technique versus a primary bleeding disorder.

Bone Marrow

Indications

Cytologic examination of a bone marrow aspirate is an especially valuable tool in the assessment of patients with persistent anemia, particularly nonregenerative anemia, abnormal numbers (either high or low) of leukocytes, thrombocytopenia, any blood dyscrasia detected in peripheral blood, and any combination of these findings. Bone marrow specimens will yield the most information if both a core biopsy and aspirate slides are submitted. The biopsy should be cut first, and the core placed in a tissue-processing cassette, labeled, and dropped into a formalin container. The aspirate needle can then be placed into the same puncture site as the biopsy needle. (Bone marrow biopsy and aspiration collection techniques are described in Section 4.)

Thrombocytopenia is not necessarily a contraindication to performing bone marrow aspiration. Assuming normal platelet function, bone marrow aspiration is indicated even when platelet counts are extremely low (e.g., 5000 platelets/mm³). The author has observed persistent bleeding and large hematoma formation at the site of aspiration in dogs with platelets counts <3000 platelets/mm³ in peripheral blood.

Sample preparation

In most patients undergoing bone marrow aspiration, sufficient numbers of platelets will be present in the sample to justify routine use of an anticoagulant. Before collecting the sample, a few drops of 4% EDTA are placed in the center of a watch glass. The same 12-cc syringe used to draw the EDTA is used to collect the sample. This syringe will contain a small amount of EDTA. Collection of marrow is typically limited to 0.5 mL. Larger volumes may cause hemodilution of the sample, making interpretation difficult. On withdrawing the appropriate volume, the sample is immediately added directly to the EDTA and mixed thoroughly. A glass pipette can be used to transfer the aspirated marrow onto a clean, dry slide. Other techniques are described in Section 4. Using the same technique to distribute peripheral blood for a differential count will suffice. The sample is allowed to air-dry.

Staining options

Bone marrow staining routinely entails use of a quick Romanowsky-type stain. Special staining, usually performed by a commercial or university laboratory, may be indicated when looking for the presence of iron stores or specific types of organisms.

Common mistakes

If the bone marrow contains functional platelets, failing to quickly transfer the aspirate into the EDTA can result in clot formation. The presence of clots is likely to entrap diagnostic cells, making interpretation difficult or impossible. Hemodilution and an excessive volume of EDTA are also common mistakes that can compromise the quality of the smears. Other complications usually are caused by errors in the technique of making the slide. For example, failing to adequately distribute the sample across the slide can result in a unusually thick preparation. Bone marrow aspirates taken from the head of the humerus can become contaminated with joint fluid, making the sample completely unusable.

BIOCHEMISTRY—ROUTINE

The ability to obtain a comprehensive biochemical profile, and to do so quickly and inexpensively, has become a routine part of the clinical work-up for the companion animal patient. Clearly, the biochemistry profile greatly expands the clinician's ability to assess the patient presenting with a history of clinical illness. Additionally, it is now feasible to obtain a biochemical profile on seemingly healthy patients as part of a routine “wellness examination.”

This section discusses those analytes offered by most clinical laboratories performing companion animal (dog and cat) biochemistry profiles. Although specific analytes included on panels vary among laboratories, any individual test not discussed here can probably be found in the following section entitled Special Diagnostic Tests and Test Protocols.

The following criteria are applicable to all samples in which blood/serum/plasma is collected for which a routine biochemisty profile or special laboratory test is requested.

ANALYTE OR TEST NAME (SYNONYMS)

The name of the individual chemical analyte being measured (e.g., alkaline phosphatase) is followed in parentheses by common abbreviations used by laboratories when reporting results (e.g., SAP or Alk Phos). In some cases the name of the test is presented rather than the actual chemical being tested for (e.g., ACTH stimulation, in which cortisol is the actual analyte measured).

NORMAL

Representative reference range values for normal adult dogs and cats are listed with each analyte. In addition, TABLE 5-1, TABLE 5-2, TABLE 5-3, TABLE 5-4, TABLE 5-5, TABLE 5-6 summarize reference ranges for dogs and cats.

Note: Reference range values listed throughout this section are for general reference only. Test results from individual patients must be compared with the reference range values of the laboratory that performs the test.

PATIENT PREPARATION

Any unique patient preparation parameters should be followed before collecting the sample. For routine biochemistry profiles, an 8- to 10-hour fasting period is recommended when feasible. When performing routine profiles on normal patients, it is preferable to collect samples in the morning. Owners are instructed to withhold food and water after midnight on the day the blood sample is to be collected.

COLLECT

This section stipulates the type and volume of sample to be collected, as well as the type of collection tube to be used. For routine biochemistry, collecting at least 2.0 mL of whole blood in a red-topped tube (or serum separator tube) is required to obtain the minimum 1.0 mL of serum required for sample analysis. Dehydrated patients have a higher hematocrit, and a larger volume of whole blood may be required to obtain 1.0 mL of serum.

SUBMIT

This section stipulates the type and volume of sample that is to be submitted for analysis. Also, the type of vial/container in which the sample should be shipped is specified. Unless specified in the protocol, do not store or ship samples as whole blood; instead, separate whole blood from serum before shipping unless using an appropriate serum separator tube. Serum samples should be shipped in a sterile red-topped tube. Freezing of the sample is not required for routine biochemistry profiles.

INTERPRETATION

Each analyte and test procedure is described separately.

INTERFERENCE

This section stipulates common interfering substances/factors and indicates, when known, if the interference will falsely elevate or lower test results. Samples that are lipemic, icteric, and/or hemolyzed may cause test interference with individual analyte assays, resulting in unreliable test results. Interference may be positive (false increased test results) or negative (false decreased test results). The degree and type of interference vary depending on the test methodology used. Most laboratories provide details in final reports pertaining to known or potential interfering factors.

PROTOCOL

Normal:

3.9-5.2 mEq/L (dog); 3.3-5.7 mEq/L (cat).

Interpretation:

Increased value (hyperkalemia) may indicate mineralocorticoid deficiency (Addison's disease or hypoadrenocorticism) but must be interpreted with serum sodium and an ACTH stimulation test. Numerous causes of decreased potassium are recognized. GI and renal losses are the most common and most significant. Persistent hypokalemia warrants significant efforts to determine the underlying cause(s).

WARNING: Potassium levels >7.5 mEq/L may cause cardiac arrhythmias (profound bradycardia) and death. Potassium levels <2.5 mEq/L may cause profound weakness.

SODIUM (Na⁺)

Normal:

147-154 mEq/L (dog); 147-165 mEq/L (cat).

Interpretation:

Increased value (hypernatremia) may result from excess dietary consumption or severe dehydration. Decreased value (hyponatremia) may indicate mineralocorticoid deficiency (Addison's disease or hypoadrenocorticism) but must be interpreted in light of other tests (e.g., serum osmolality, potassium, ACTH stimulation test). Persistent diuresis caused by drugs (furosemide) or an inherent medical disorder (nephrotic syndrome) can deplete serum sodium to significantly low levels. Depending on the laboratory methodology, pseudohyponatremia may occur in patients with profoundly lipemic serum.

TOTAL PROTEIN (TP)

Normal:

5.5-7.2 g/dL (dog); 5.4-8.9 g/dL (cat).

Interpretation:

TP must be evaluated with constituent proteins albumin and globulin. Increased TP (hyperproteinemia) may indicate dehydration (elevated albumin and globulin) or extreme elevations in globulin (chronic inflammation, infection, neoplasia, especially myeloma). Decreased TP may indicate increased protein loss (especially albumin), chronic malassimilation/maldigestion, starvation, or chronic illnesses (tumor cachexia).

TRIGLYCERIDE (TG)

Normal:

19-133 mg/dL (dog); 24-206 mg/dL (cat).

Interpretation:

Increased TG is normally increased in any animal during the postprandial state (with 6 hours following a meal). TG is the cause of gross lipemia when concentrations exceed ~500 mg/dL. Increased TG (in the fasted patient) is associated with familial hypertriglyceridemia, a condition most often reported in Miniature Schnauzers (other breeds and mixed breeds may be affected) born in the U.S. (the condition has not been described in Miniature Schnauzers in Europe or the United Kingdom) and certain lines of mixed-breed cats. There is no clinical significance associated with decreased TG in either the dog or cat.

SPECIAL DIAGNOSTIC TESTS AND TEST PROTOCOLS

This section includes advanced biochemical laboratory tests not typically included in routine companion animal medicine laboratory profiles. These tests are selected on the basis of abnormal findings revealed during routine physical examination and laboratory profiling. Additional special laboratory tests and test procedures can be found in the organ system-specific sections that follow (see Chapter Outline).

Note: Throughout the Special Diagnostic Test section, the following information is provided, where appropriate, for each laboratory test described:

•
Test or analyte name (abbreviations or common names)
•
Normal (representative reference range value for normal adult dogs and cats)
•
Patient preparation (includes any special requirements before sample collection)
•
Sample (type of sample and recommended minimum volume to be collected)
•
Submit (component of sample to submit for analysis, store, or mail)
•
Interpretation (basic interpretation of test results that are outside the reference range)
•
Interference (variables that may falsely elevate or decrease test results)
•
Protocol (as applicable, accepted procedures for performing the test are outlined)

ACTH STIMULATION TEST: See ENDOCRINOLOGY

ADRENOCORTICOTROPIC HORMONE (ACTH) Endogenous: See ENDOCRINOLOGY

ALDOSTERONE: See ENDOCRINOLOGY

AMMONIA (NH₃) (FASTING AMMONIA)

Normal:

45-120 μg/dL (dog); 30-100 μg/dL (cat).

Patient Preparation:

Overnight fast.

Collect:

Whole blood, 2.0 mL minimum, in EDTA (purple-topped tube) or in heparin.

Submit:

Plasma, 1.0 mL minimum.

Interpretation:

Decreased levels of ammonia are not considered clinically significant. Elevated ammonia levels support the diagnosis of underlying, significant liver disease. This test generally is considered a liver function test and usually is performed to support a diagnosis of hepatic encephalopathy. Fasting ammonia and ammonia tolerance tests are uncommonly performed today because of sample instability and specimen handling requirements. These tests have largely been replaced by pre– and post–bile acid assay.

Interference:

Hemolysis; elevated BUN; glucose values >600 mg/dL. NH₃ is unstable if not frozen at −20° C. LIMITING FACTORS: ideally, blood should be collected in a sealed, cold glass collection tube, centrifuged immediately, and plasma analyzed within 20 minutes of collection. Alternatively, plasma can be stored for up to 48 hours if frozen immediately after collection and kept frozen until time of analysis.

AMMONIA TOLERANCE TEST

Normal Resting Values:

45-120 μg/dL (dog); 30-100 μg/dL (cat).

NOTE: Minimal change should be detected following oral challenge because clearance is nearly 100% following a single pass through the liver.

Patient Preparation:

Overnight fast.

Collect:

Whole blood, 2.0 mL minimum, in EDTA (purple-topped tube) or in heparin.

Submit:

Plasma, 1.0 mL minimum, for each pre- and post challenge sample.

Interpretation:

Elevated ammonia levels support the diagnosis of underlying, significant liver disease. This generally is considered a liver function test and is usually performed to support a diagnosis of hepatic encephalopathy. Fasting ammonia and ammonia tolerance tests are uncommonly performed today because of sample instability and specimen handling requirements. These tests have largely been replaced by pre– and post–bile acid assay.

Interference:

Hemolysis; elevated BUN; glucose values >600 mg/dL. NH₃ is unstable if not frozen at −20° C. LIMITING FACTORS: Ideally, blood should be collected in a sealed, cold glass collection tube, centrifuged immediately, and plasma analyzed within 20 minutes of collection. Alternatively, plasma can be stored for up to 48 hours if frozen immediately after collection and kept frozen until time of analysis.

Protocol:

Two plasma samples are required. The first is a baseline sample. The second sample is collected 30 to 45 minutes following administration of ammonium chloride (NH₄Cl) at 100 mg/kg body weight as an oral 5% solution in approximately 20-50 mL of saline. NH₄Cl is also available as a powder that can be administered orally, at the same dose, which lowers the risk of vomiting/aspiration.

ANTINUCLEAR ANTIBODY (ANA): SEE IMMUNOLOGY

BILE ACIDS

Normal (dog and cat):

Pre-feeding, ≤7 μmol/L; Post-feeding, ≤15 μmol/L.

Patient Preparation:

12-hour or overnight fast before collecting the pre-feeding sample.

Collect:

Whole blood, 2.0 mL minimum, in red-topped tube for each sample collected.

Submit:

Serum, 1.0 mL minimum, for each sample submitted.

Interpretation:

Bile acids are indicated for the assessment of hepatobiliary disease in non-icteric patients. There is no value in performing this test in patients that are icteric. Hepatobiliary disease (e.g., portosystemic shunt) is supported with either a pre-feeding sample >7 μmol/L or a post-feeding sample >15 μmol/L. NOTE: Reference range values may vary among different laboratories.

Interference:

Lipemia; icterus; hemolysis. Results in patients that vomit the meal prior to collecting the 2-hour post-feeding sample cannot be expected to be reliable. Individual variations in gastric emptying and absorption can result in discordant results (e.g., the pre-feeding sample is higher than the post-feeding sample). Such results are not reliable and the test should be repeated.

Protocol:

1.
The pre-feeding (or fasting) blood sample is collected following a 12-hour fast. Label the tube accordingly.
2.
Feed a relatively high-fat meal (to stimulate gallbladder contraction). A protein-restricted diet with corn oil added is appropriate for those patients with protein intolerance and signs of hepatic encephalopathy.
3.
Two hours following consumption of the meal, collect the post-feeding sample. Label the tube accordingly.

BLOOD GASES (ARTERIAL AND VENOUS)

NOTE: values represented below are expected from patients breathing room air.

Normal:

	Arterial		Venous
Value	Dog	Cat	Dog	Cat
pH	7.36–7.44	7.36–7.44	7.34–7.46	7.33–7.41
PCO₂	36–44	28–32	32–49	34–38
PO₂	90–100	90–100	24–48	35–45
TCO₂	25–27	21–23	21–31	27–31
HCO₃⁻	24–26	20–22	20–29	22–24

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Patient Preparation:

Patients breathing 100% oxygen at the time of sample collection are expected to have different results than those of patients that are breathing room air during sample collection. Note the conditions under which the sample was collected.

Collect:

Whole blood, either arterial or venous, depending on the assessment required.

Submit:

Sample cannot be stored. Immediate testing is required to obtain reliable results.

Interpretation:

Tco ₂ is synonymous with HCO₃ ⁻ in patients breathing room air. The overall interpretation of venous and/or arterial blood gas results will vary considerably depending on the patient's health status. Several variations in test results are possible. The clinician should consult appropriate references to interpret results of individual patients (see Section 1).

Interference:

The test should be performed immediately on collecting the sample. Delays could cause significant abnormalities in actual results. Exposure of the sample to room air (bubbles within the sample) may cause Pco ₂ to decrease, whereas pH and Po ₂ may increase.

BODY FLUIDS (SUBMITTED FOR CHEMISTRY ANALYSIS)

Normal:

Not applicable.

Patient Preparation:

When feasible, the skin over the site selected for centesis should be shaved and surgically prepared prior to attempting sample collection to avoid contamination of either the sample or the body cavity from which the sample is collected.

Collect:

1-2 mL, minimum, by direct centesis of body cavity or fluid-filled compartment.

Submit:

Centrifugation of whole blood contamination is indicated to remove particulate material (e.g., blood cells, cellular debris) when prompt evaluation of specimen is not possible.

Interpretation:

Any biochemical analyte determined in serum or plasma may be assayed in body fluid—e.g., amylase, lipase (pancreatitis), urea nitrogen, creatinine (ruptured bladder), glucose, lactate

Interference:

Interference from blood and blood components, bilirubin, bile, and urine may significantly interfere with test results. Centrifugation of the sample (blood contamination) may be necessary before performing any biochemistry test.

CALCIUM, IONIZED (iCa)

Normal:

1.12-1.42 mmol/L (dog); 1.12-1.42 mmol/L (cat)

Patient Preparation:

None.

Collect:

Whole blood, 2.0 mL.

Submit:

Serum, 1.0 mL.

Interpretation:

Results reflect the concentration of the biologically active, ionized fraction of calcium without the influence of plasma proteins (e.g., albumin).

Interference:

The reported values of iCa can vary with patient's blood pH; iCa decreases as pH increases.

CEREBROSPINAL FLUID (CSF)

Normal:

Value	Dog	Cat
WBCs (× 10³/L)	≤3	≤2
RBCs (×10⁶/L)	≤30	≤30
Protein (mg/dL)	≤33	≤36
Cytology (%)
Monocytes	87	69–100
Lymphocytes	4	0–27
Neutrophils	3	0–9
Eosinophils	0	0
Macrophages	6	0–3

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Patient Preparation:

General anesthesia is required. For a description of the technique for collecting spinal fluid from dogs and cats, see section 4, Advanced Procedures. Specific training and/or experience is strongly recommended before collecting CSF from the cisterna magna (between the head and C1). Fatalities can result from improper technique.

Collect:

Usually, two 0.5- to 1.0-mL samples are collected in red-topped tube (no additives).

Submit:

Samples collected.

Interpretation:

If one sample contains excessive numbers of neutrophils, the second sample is submitted for culture and sensitivity; treatment recommendations should include use of an antibiotic (preferably intravenous) that will penetrate the blood-brain barrier.

Interference:

Blood contamination is the most common interfering factor. An RBC count greater than 30 × 10⁶/L is consistent with peripheral blood contamination. Immediate analysis is recommended. It is not recommended to submit CSF via mail for assessment.

Protocol:

Proper patient preparation and collection technique is critical (see Section 4).

COBALAMIN (VITAMIN B₁₂)

Normal:

Results vary considerably among laboratories; consult individual laboratory.

Patient Preparation:

Fasted.

Collect:

Whole blood, 2.0 mL minimum (red-topped tube).

Submit:

Serum, 1.0 mL.

Interpretation:

Test usually is performed with folate and trypsin-like immunoreactivity (TLI). It is used in the assessment of chronic small bowel diarrhea with associated weight loss. Significantly decreased cobalamine supports the need to measure TLI (exocrine insufficiency) and also supports mucosal disease, and may be indicative (in cats) of hepatic disease (hepatic lipidosis).

Interference:

Hemolysis; lipemia.

ETHYLENE GLYCOL*

Normal:

Negative. “Trace” amounts may be detected in normal patients.

Patient Preparation:

None.

Normal:

225-375 μmol/L (dog and cat).

NOTE: consult individual laboratory because test results may vary.

Patient Preparation:

None.

Collect:

Whole blood, 2.0 mL (red-topped tube).

Submit:

Serum, 1.0 mL; sample must be frozen and shipped on cold packs for overnight delivery.

Interpretation:

This is a single-sample test representing mean blood glucose over the last 1 to 3 weeks. Increased fructosamine indicates poor glycemic control (hyperglycemia); declining fructosamine indicates improved or adequate glycemic control. Values >500 μmol/L suggest inadequate glycemic control over the past 1 to 3 weeks. Values less than the lowest reference range value suggest that the patient has sustained significant periods of hypoglycemia over the past 1 to 3 weeks. Values <400 μmol/L and clinical signs of polyuria/polydipsia (PU/PD) and polyphagia are suggestive of a Somogyi phenomenon. Fructosamine levels should not be used to make specific adjustments in daily insulin therapy.

Interference:

The assay is a colorimetric procedure; therefore, significant hemolysis or icterus could affect results. Hypoproteinemia and/or hypoalbuminemia will cause falsely low values. Hyperlipidemia and azotemia may also alter results similarly.

GLYCOSYLATED HEMOGLOBIN (GLYCATED HEMOGLOBIN; Gly Hb)

Normal:

1.7% to 4.9% (dog and cat).

NOTE: consult individual laboratory because test results may vary.

Patient Preparation:

None.

Collect:

Whole blood, 2.0 mL in EDTA (purple-topped tube).

Submit:

Plasma, 1.0 mL; separate plasma and refrigerate until assayed.

Interpretation:

This is a single-sample test representing mean blood glucose over the lifespan of red blood cells (~3-4 months). This test is used less in veterinary medicine than the fructosamine assay. In dogs, values consistently between 4% and 6% are associated with adequate glycemic control and owner satisfaction.

Interference:

Storage at room temperature and for longer than 7 days will decrease values; patients with a hematocrit (Hct) <35% may have lower than expected values. NOTE: laboratories must use an assay that has been validated for dogs and for cats. Human assays performed on animal plasma may not be valid.

IRON

Normal:

NOTE: Consult individual laboratory because test results may vary.

Patient Preparation:

None.

Collect:

Whole blood, 2.0 mL (red-topped tube).

Submit:

Serum, 1.0 mL.

Interpretation:

Results should be interpreted with total iron-binding capacity (TIBC) and ferritin. Decreased values reflect chronic, not acute, blood loss (e.g., hookworms, intestinal ulceration, bleeding from neoplasia). In cases of iron deficiency, expect TIBC to be normal or high, whereas serum ferritin will be low. Patients with anemia associated with chronic inflammatory disease are expected to have normal to low TIBC, whereas serum ferritin will be normal to high.

Interference:

Hemolysis; lipemia.

LACTIC ACID (LACTATE)

Normal:

2-13 mg/dL (0.22-1.44 mmol/L) (dog); results not reported for cats.

Patient Preparation:

Avoid venous stasis when collecting sample. Clean venipuncture and rapid draw of sample is important.

Collect:

Whole blood, 2.0 mL, in lithium heparin plasma or in iodoacetate tubes. Some laboratories will accept samples collected in fluoride tubes.

Submit:

Plasma, which should be rapidly separated from blood. If this is not possible, the sample may be refrigerated immediately at 4° C, but only for 2 hours, at which time the plasma must be separated from blood.

Interpretation:

Resting values >6.0 mmol/L indicate severe acidosis and a poor prognosis. Test is also used to assess metabolic myopathies, especially in Labrador Retrievers.

Interference:

Aspirin, phenobarbital, and epinephrine may alter lactate values. Also, allowing the sample to sit at room temperature will result in increased level of lactate.

Protocol:

To diagnose metabolic myopathy in Labrador Retrievers: two samples are recommended; the first blood sample is collected at rest. A second sample is collected following 10-15 minutes of brisk walking/running.

LEAD, BLOOD

Normal:

Results vary considerably among laboratories; consult individual laboratory. Usually, values <0.05 ppm in whole blood (or <3 ppm in liver or kidney) are within the range of normal.

Patient Preparation:

None.

Collect:

Whole blood, 2.0 mL, in EDTA (lavender-topped tube) or heparin.

Submit:

Entire sample.

Interpretation:

Refer to the individual laboratory for specific interpretation of the values reported. Values >0.3 ppm suggest exposure. Values >0.4 ppm are generally considered diagnostic of toxicosis.

Interference:

Incorrect tube used for collection/storage of whole blood.

LIPOPROTEIN ELECTROPHORESIS

Normal:

Normal values have not been established for the dog and cat.

Patient preparation:

12-hour fast.

Collect:

Whole blood, 1.0 mL (red-topped tube).

Submit:

Serum, 0.5 mL.

Interpretation:

Test consists of electrophoretic separation of various lipoprotein categories in serum. It may qualitatively identify various categories of lipoproteins, including chylomicrons, very-low-density lipoproteins (VLDLs), low-density lipoproteins (LDLs), and high-density lipoproteins (HDLs). Standards have not been established for the dog or cat.

Interference:

Lipemia is not an interfering factor because electrophoresis will separate various lipid fractions.

MAGNESIUM (Mg)

Normal:

1.5-2.5 mg/dL (dog and cat).

Patient Preparation:

None.

Collect:

Whole blood, 2.0 mL, in red-topped tube.

Submit:

Serum, 1.0 mL.

Interpretation:

Increased Mg may reflect renal failure or insufficiency. Decreased Mg is observed in many gastrointestinal disorders (malabsorption, pancreatitis, chronic diarrhea), renal disease (glomerulonephritis, diuresis, tubular necrosis), and multiple endocrine diseases, as well as with sepsis, blood transfusion, and parenteral nutrition.

Interference:

Mg-containing drugs (oral antacids and laxatives) will falsely elevate test results. Some intravenous fluids contain Mg, which also may falsely elevate test results. Falsely decreased values may result from diuretic therapy or intravenous fluid therapy–induced diuresis.

OSMOLALITY, ESTIMATED (SERUM)

Normal:

290-310 mOsm/kg (dog); 308-335 mOsm//kg (cat).

Patient Preparation:

None.

Collect:

Whole venous blood, 2.0 mL, in a red-topped tube or serum separator tube.

Submit:

Serum, 1.0 mL.

Interpretation:

Osmolality of extracellular fluid (ECF) is determined predominantly by electrolytes, especially sodium, and small molecules (glucose and urea) and is reflective of fluid shifts between the vascular space and the interstitium. Increased ECF osmolality (>350 mOsm/L), or hyperosmolality, is likely to be associated with clinical signs (especially neurologic) because of the shift of water from the interstitial space into the vascular space.

NOTE: Direct laboratory measurement of serum osmolality can be performed but is expensive. Serum osmolality is usually calculated according to the following formula:

mOsm / kg = 1.86 ({Na}^{+} + K^{+}) + (glucose \div 18) + (BUN \div 2.8) + 9

PANCREATIC LIPASE IMMUNOREACTIVITY (PLI)

Normal:

2.2-102.1 μg/L (dog); 2.0-6.8 μg/L (cat)

Patient Preparation:

Fasted for 12 hours before collecting blood.

Collect:

Whole blood, 3.0 mL minimum in red-topped tube or serum separator tube.

Submit:

Serum,1.0 mL minimum. Immediately s eparate serum from clot. Ship serum only. Do not ship whole blood.

Interpretation:

NOTE: PLI is species-specific; samples must be labeled “DOG” (cPLI) or “CAT” (fPLI). Interference:

Anticoagulant, hemolysis; moderate or greater lipemia.

Protocol:

Serum should be separated immediately following clot formation and retraction.

PROTEIN ELECTOPHORESIS (SERUM)

Normal:

Value	Dog	Cat
Total protein (g/dL)	6.0–7.6	7.3–7.8
Albumin (g/dL)	2.7–3.7	2.8–4.2
α1-Globulin (g/dL)	0.25–0.60	0.3–0.65
α2-Globulin (g/dL)	0.72–1.40	0.40–0.68
β1-Globulin (g/dL)	0.63–0.89	0.77–1.25
β2-Globulin (g/dL)	0.60–1.0	0.35–0.50
γ1-Globulin (g/dL)	0.50–0.83	1.39–2.22
A:G ratio	0.8–1.0	0.63–1.15

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Patient Preparation:

Fasted for 12 hours (overnight) to prevent postprandial lipemia.

Collect:

Whole blood, 2.0 mL (red-topped tube).

Submit:

Serum, 1.0 mL (most laboratories will accept a volume of serum from 0.5 to 1.0 mL).

Interpretation:

Multiple interpretations are possible, depending on the patient's condition. Test usually is performed to assess degree of loss of albumin or increases in one or more globulin fractions (e.g., hypergammaglobulinemia associated with FIP, canine ehrlichiosis, multiple myeloma). The test is NOT used to confirm a diagnosis.

NOTE: Most clinical assessments are made from the shape of the curve in a densitometer tracing of the electrophoresis rather than specific numbers. When requesting serum protein electrophoresis, it is important to request a copy of the curve as well as the quantitated results for each protein fraction.

Interference:

Lipemia; hemolysis.

TRYPSIN-LIKE IMMUNOREACTIVITY, CANINE (CANINE TLI)

Normal:

5.0-35.0 μg/L.

Patient Preparation:

Fasted.

Collect:

Whole blood, 2.0 mL in red-topped tube.

Submit:

Serum, 1.0 mL. Separate serum from clot. Ship serum only. Do not ship whole blood.

Interpretation:

NOTE: TLI is species-specific; samples must be labeled “DOG”. It is a sensitive and specific test for the diagnosis of exocrine pancreatic insufficiency in dogs and cats. Values <2.5 μg/L, in the presence of clinical signs, support the diagnosis. Values >50 μg/L have been used to diagnose pancreatitis in dogs. However, TLI has been replaced by the canine pancreatic lipase immunoreactivity (cPLI) assay to diagnose pancreatitis in dogs.

Interference:

Hemolysis; moderate or greater lipemia.

TRYPSIN-LIKE IMMUNOREACTIVITY, FELINE (FELINE TLI)

Normal:

12-82 μg/L.

Patient Preparation:

Fasted.

Collect:

Whole blood, 2.0 mL (red-topped tube).

Submit:

Serum, 1.0 mL. Separate serum from clot. Ship serum only. Do not ship whole blood.

Interpretation:

NOTE: TLI is species-specific; samples must be labeled “CAT”. This is a sensitive and specific test for the diagnosis of exocrine pancreatic insufficiency in dogs and cats. Values <2.5 μg/L in the presence of clinical signs supports the diagnosis. Values >100 μg/L have been used to diagnose pancreatitis in cats. However, TLI has been replaced by the feline pancreatic lipase immunoreactivity (fPLI) assay to diagnose pancreatitis in cats.

Interference:

Hemolysis; moderate or greater lipemia.

Note: Serum for PLI and TLI assays may be submitted to the Gastrointestinal Laboratory Department of Small Animal Medicine and Surgery, Texas A &M University, 4474 TAMU, College Station, Texas, 77843-4474.

HEMOSTASIS AND COAGULATION

Tests of hemostasis are directed at determining platelet numbers and function, activation and abnormalities of the intrinsic and extrinsic clotting cascade, and quantitation of breakdown products of thrombosis and fibrinolysis. Obtain blood samples for evaluation of coagulation abnormalities by careful venipuncture, and insert samples into plastic or silicone-coated glass syringes.

Because tissue thromboplastin can activate the clotting cascade, some authors advocate using two syringes and two needles to obtain blood for coagulation tests. First, carefully insert the needle into the vein and withdraw 1 mL of blood. Leave the needle in the vessel, and remove the first syringe. Attach a second syringe and obtain the appropriate volume of blood; then remove the needle from the vessel. Rapidly replace the needle on the second syringe with a fresh needle and then inject the blood sample into the appropriate tubes for later analyses.

Platelet tests should be performed on fresh samples within 2 hours of collection. Plasma samples can be spun down and frozen at −20° C for several days, and at −40° C for several months to a year for later analyses.

INITIAL IN-OFFICE SCREENING TESTS

The initial in-office screening tests for coagulation defects include a hematocrit (Hct), peripheral blood smear, activated coagulation test (ACT) or activated partial thromboplastin time (APTT), prothrombin time (PT), and, if indicated, buccal mucosal bleeding time assay.

Hematocrit

The patient's hematocrit and total protein should be evaluated to determine whether anemia is present. The color of the plasma in the spun-down microhematocrit tube can aid in making a diagnosis if intravascular hemolysis (red) or icterus (yellow) is present. The buffy coat from a microhematocrit tube can be evaluated microscopically for the presence of microfilaria in heartworm disease or mast cells in systemic mastocytosis.

Peripheral Blood Smear

The peripheral blood smear should be evaluated for RBC morphology, RBC fragments (schizocytes), platelet count, large platelets, WBC count and morphology, and blood parasites.

Platelet Count

One of the most simple cage-side tests when determining the cause of a coagulopathy is the platelet count. To perform this test:

1.
Obtain an anticoagulated (trisodium citrate or sodium oxalate are the anticoagulants of choice for platelet and coagulation testing) sample of peripheral blood, and make a stained blood smear.
2.
Scan the slide, including the peripheral edge, for platelets and platelet clumps. If platelet clumps are present, the platelet estimate cannot be accurately measured; also, it is unlikely that thrombocytopenia is the cause of the patient's hemorrhage.
3.
If no platelet clumps are present in the feathered edge of the blood smear, scan multiple areas of the slide on 100× (oil) magnification. Count the number of platelets per high-power field (hpf) and then multiply the value by 15,000 to give an approximate estimation of platelet number.

Hemorrhage secondary to thrombocytopenia occurs when platelet numbers decrease to <40,000/uL (<2-3 platelets/hpf). If there are signs of superficial hemorrhage and more than 4-5 platelets/hpf, a thrombocytopathia (platelet function problem) such as von Willebrand's disease, DIC, or aspirin-induced coagulopathy may be present.

Activated Coagulation (Clotting) Time (ACT)

The ACT measures the function of the intrinsic and common coagulation pathway (factors II, V, VIII, IX, X, XI, and XII). The ACT can be used reliably to screen for disorders of secondary hemostasis. Severe thrombocytopenia (<10,000-20,000 platelets/μL) and decreased fibrinogen, in addition to decreases in activated clotting factors listed previously, can cause prolongation in the ACT. An ACT tube contains diatomaceous earth that stimulates blood clotting on contact.

To perform the ACT:

1.
Warm the ACT tube to 37° C in a heating block or water bath.
2.
Use a 3-mL syringe without any anticoagulant to obtain 3.0 mL of blood. The venipuncture should be atraumatic. Because tissue factor stimulates the clotting cascade, quickly change the needle and push 2.0 mL of the blood sample into the ACT tube, inverting the tube several times to mix the contents, and then place the tube in the water bath or heat source. Start counting the time at the moment you inject the blood into the tube. (The remaining blood can be used to fill microhematocrit tubes and make peripheral blood smears.)
3.
To check the tube for clots, quickly invert the tube and then return it to the heat source at 60 seconds and then every 5 seconds thereafter. Record the time that the first signs of a clot (gel) is observed.

Normal ACT time is 90-120 seconds for dogs, and 80-100 seconds for cats.

Activated partial thromboplastin time (APTT) is another, more sensitive test to detect defects in the intrinsic clotting cascade. It is more sensitive than the ACT in that it will become prolonged earlier than the ACT. Point-of-care coagulation analyzers (SCA-2000, Symbiotics, Inc., San Diego) are available that require less blood than an ACT, and thus may be the preferred test.

Prothrombin Time (PT)

PT is a test to determine abnormalities in the extrinsic (factor VII) coagulation pathway. Because factor VII is the most labile clotting factor and has the shortest half-life, PT will become prolonged before any changes in ACT or APTT (intrinsic pathway). The prothrombin-complex clotting factors are II, VII, and X; these factors interact with factor V and fibrinogen in the presence of tissue thromboplastin and calcium chloride.

Buccal Mucosal Bleeding Time (BMBT)

The BMBT measures the time required for platelets to become activated and interact with damaged vascular endothelium to form a primary platelet plug. It is a test of primary hemostasis. The BMBT becomes prolonged with thrombocytopenia (<100,000/μL) and platelet dysfunction syndromes such as von Willebrand's disease. The BMBT is usually performed without any sedation in dogs and with ketamine in cats.

To perform the BMBT:

1.
When performing the BMBT in dogs, place a loose tie of gauze around the dog's muzzle to lift the lip so that the buccal mucosa is exposed and the veins slightly engorged. It is important to not tie the gauze too tightly, as vasoconstriction can artifactually change test results.
2.
Use a BMBT template (Simplate R) to make two small nick incisions in the buccal mucosa. Gently wick the blood away from the site with a piece of filter paper (if you don't have filter paper, a coffee filter works well). Allow the blood to wick into the filter paper without touching the incisions or the clot.
3.
Note the time from making the initial incision to the time that hemorrhage stops (i.e., a platelet plug has formed). Normal BMBT is less than 3 minutes in dogs and cats.

If the BMBT is prolonged, von Willebrand's disease, NSAID influence, congenital thrombopathies (Bassett and Otter Hounds), and systemic illness (azotemia, hepatic failure, malignancy) should be ruled out. If the BMBT is normal in the face of a normal platelet count and clinical bleeding, tests of the coagulation cascade (APTT, PT, ACT) should be considered.

ANCILLARY TESTS OF HEMOSTASIS

Thrombin Time

The thrombin time is a measure of the amount of functional fibrinogen in plasma. The test is used in the diagnosis of DIC when fibrinogen levels are low. Fibrinogen levels may also be normal in DIC, but thrombin time will still be altered because of in vivo fibrinolysis. This test is now rarely used, because since more sensitive and specific tests such as D-dimer concentration are available for the diagnosis of DIC.

Fibrinogen

Fibrinogen levels are used in the detection of DIC. In DIC, fibrinogen levels can decrease as a result of the activation of thrombin and fibrin formation and the activation of plasmin, which causes degradation of fibrin and fibrinogen. Fibrinogen levels can be decreased, normal, or increased in cases of chronic DIC due to a compensatory overproduction. Because of the variability in fibrinogen levels, this test alone is not conclusive to make a diagnosis of DIC.

Fibrin(ogen) Degradation Products (FDPs)

FDPs (also called fibrin split products [FSPs]) are formed when the enzyme plasmin acts on fibrin monomers, cross-linked fibrin, and fibrinogen. Because fibrinogen can increase during periods of inflammation without DIC, the presence of FDPs alone does not allow a diagnosis of DIC. FDPs are cleared by the hepatic reticuloendothelial system. In cases of hepatic insufficiency or hepatic failure, FDPs can be elevated without concurrent DIC.

D-Dimers

D-dimers are used in the diagnosis of DIC. D-dimers are released as the result of the breakdown of cross-linked fibrin by plasmin. Because D-dimers occur as a result of a stable fibrin clot, elevated levels are more sensitive and specific for a diagnosis of DIC.

The PIVKA Test

The PIVKA (proteins induced by vitamin K absence or antagonism) test is most useful in diagnosing vitamin K deficiencies. Moderate deficiencies in Vitamin K–dependent coagulation factors (II, VII, IX, and X) will cause abnormal PIVKA test results. The PIVKA test becomes prolonged 12-24 hours after the PT test becomes prolonged.

Saline Agglutination

The saline agglutination test is simple to perform in-house and aids in the diagnosis of immune-mediated hemolytic anemia. To perform a saline agglutination test:

1.
Place one drop of 0.9% saline on a microscope slide. Mix the drop of saline with one drop of the patient's anticoagulated blood and observe for the presence of agglutination under the microscope.
2.
If agglutination is present, mix a second drop of saline with the blood-saline mixture on the slide and review under the microscope a second time.

If the “agglutination” disperses, it is likely caused by rouleaux secondary to inflammation. If the agglutination remains, autoagglutination of red blood cells is occurring due to interaction with antibodies directed against glycoprotein moieties on the surface of the RBC membranes.

Note: Management of patients with a confirmed coagulopathy involves correcting any underlying cause, replenishing oxygen-carrying capacity in the form of red blood cells or purified hemoglobin, replacing clotting factors and antithrombin in the form of fresh frozen plasma, and maintaining end-organ perfusion. The management of specific conditions and coagulopathies is listed under their subheadings. A more thorough approach to transfusion management is listed in Section 1.

SUBMISSION OF SAMPLES FOR COAGULATION TESTING

1.
Draw blood sample into a blue-top tube (BTT) that contains sodium citrate. Fill the BTT to at least 75%, but preferably 90% or more, because results will be affected by excess citrate anticoagulant.
2.
Centrifugation and separation of plasma from cells is strongly recommended if transportation to the laboratory may require more than 12 hours.
3.
Use a plastic pipette or small syringe to transfer the plasma to a clean plastic tube. Cap the tube and keep cold or freeze at −20° C or lower. Freezing the plasma is not necessary unless testing will be delayed for more than 24 hours, but it should always stay cold. Repeated freezing and thawing of plasma denatures coagulation proteins.
4.
If samples will be mailed, ship overnight with frozen cold packs.

ACTIVATED COAGULATION (CLOTTING) TIME (ACT)

Normal:

90-120 seconds (dog); 80-100 seconds (cat).

Patient Preparation:

Direct penetration of the vein is important.

Collect:

Venous blood in an ACT Vacutainer tube. Fill to maximum allowed by vacuum.

Submit:

Blood sample in collection tube per protocol.

Interpretation:

ACT is a convenient in-hospital screening test that evaluates both the intrinsic and common coagulation pathways. Prolonged clotting time implies coagulation factor deficiency. A specific coagulation factor deficiency must be less than 5% to increase the ACT. NOTE: hemophiliac patients may have factor VIII or IX activity only 40% to 60% of normal and yet would have a normal ACT (and a normal APTT).

Interference:

The presence of tissue thromboplastin in sample (e.g., failing to obtain blood from a “clean” venipuncture) will activate the extrinsic pathway.

Protocol:

A two-tube technique is recommended to eliminate any chance of tissue thromboplastin contaminating the sample. Fill two tubes from the same draw. Use the second tube only. Pre-warm the tubes in a water bath or heating block (37° C). Place the filled sample tubes in the water bath or heating block and begin timing. Incubate the sample in the collection tube for 60 seconds for dogs, and 45 seconds for cats. Invert sample every 5 seconds to assess for evidence of clot formation. Stop procedure at first sign of clot formation.

ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT)

Normal:

8.6-12.9 seconds (dog); 13.7-30.2 seconds (cat).

Patient Preparation:

None (atraumatic venipuncture is recommended).

Collect:

Normal:

2.6 ± 0.48 minutes (dog); results not reported in cats.

Patient Preparation:

None.

Collect:

Not applicable.

Submit:

Not applicable. This is an in-hospital screening test for platelet function.

Interpretation:

BMBT is a sensitive and specific test of platelet function. Prolonged BMBT is expected in patients with von Willebrand's disease and uremia. Test is NOT generally recommended for thrombocytopenic patients.

Interference:

Improper technique thrombocytopenia.

Protocol:

The test entails a standardized cut into the buccal mucosa with subsequent “capture” of blood onto filter paper until bleeding ceases.

CLOT RETRACTION TEST

Not generally recommended.

NOTE: Because of the insensitivity of this test, the clot retraction test is NOT recommended for the assessment of patients with suspected disorders of hemostasis.

COAGULATION FACTOR ACTIVITY (FACTOR ASSAY)

The following inherited coagulation factor deficiencies have been reported in dogs and cats:

Hemophilia A (factor VIII deficiency)—the most common factor deficiency
Hemophilia B (factor IX deficiency)
Factor XII deficiency (Hageman trait)—of minor significance in affected cats
Vitamin K–dependent factor deficiency— occurs in Devon Rex cats, with severe bleeding

Other, rare deficiencies have been reported.

Diagnosis of Coagulation Factor Deficiency

Coagulation factor deficiency usually is suggested in the individual dog or cat on the basis of initial test results from routine coagulation profiles (see ACT, APTT, and PT in this section). occasionally, It is possible to measure activity of specific factors in individual patients. Specialized laboratories experienced in performing these assays should be consulted regarding sample, sample size, submission requirements, and interpretation.

CROSS-MATCH: MAJOR AND MINOR

Normal:

Results (in dogs and cats) are reported as “compatible” (“no agglutination”) or “incompatible” (“agglutination and/or hemolysis”) in either major or minor cross-match tubes.

Patient Preparation:

None.

Donor Preparation:

None.

Collect (Patient):

Venous blood, 2 mL, in red-topped tube PLUS anticoagulated venous blood, 2.0 mL, in lavender-topped tube.

Collect (Donor):

The same (this is where it's important to label the tubes!).

Submit (Patient):

Serum, 1 mL, PLUS anticoagulated whole blood, 1.0 mL.

Submit (Donor):

The same.

Interpretation:

No agglutination and/or hemolysis in either tube indicates that the match is compatible and the donor's blood may be used.
The presence of agglutination and/or hemolysis in the major cross-match tube indicates that the donor's blood should not be used.
The presence of agglutination and/or hemolysis in the minor cross-match tube suggests that the compatibility is not ideal; if another donor cannot be found, the blood can be used—although with caution.
The presence of agglutination and/or hemolysis in the donor control (donor cells mixed with donor serum) suggests incompatibility; the donor's blood should not be used.
The presence of agglutination and/or hemolysis in the patient control (patient cells mixed with patient serum) likely reflects the patient's diagnosis. Transfusion is indicated.

Interference:

In vitro hemolysis associated with difficulty collecting a sample or inappropriate handling of the blood; profound lipemia (lactescence).

Protocol:

1.
Wash RBCs from patient and donor in 0.9% SALINE solution three times; add 4.8 mL of saline to 0.2 mL of RBCs from patient and donor. Mix accordingly:
- a.
  Major cross-match: Mix 0.1 mL (2 drops) of donor RBCs + 0.1 mL (2 drops) of patient serum
- b.
  Minor cross-match: Mix 0.1 mL (2 drops) of patient RBCs + 0.1 mL (2 drops) of donor serum
- c.
  Patient control: Mix 0.1 mL (2 drops) of patient RBCs + 0.1 mL (2 drops) of patient serum
- d.
  Donor control: Mix 0.1 mL (2 drops) of donor RBCs + 0.1 mL (2 drops) of donor serum
2.
Incubate for 15 minutes at 37° C. Centrifuge for 1 minute.
3.
Observe the supernatant in all tubes for evidence of hemolysis in the test samples. Examine the suspension of RBCs for agglutination (macroscopically and microscopically).

D-DIMER (FRAGMENT D-DIMER; FIBRIN DEGRADATION FRAGMENT)

Normal:

Consult laboratory reference range (dog); studies in cats are lacking.

Patient Preparation:

None.

Collect:

Anticoagulated venous blood, 2 mL, in EDTA or heparin.

Submit:

Plasma, 1.0 mL.

Interpretation:

D-dimer is the proteolytic fragment of fibrinogen degradation. D-dimer concentration is used in the assessment of DIC in dogs. Elevated levels represent a marker of clot lysis and therefore support a diagnosis of DIC; a negative test result has a high negative predictive value and reliably rules out a diagnosis of DIC. The test also has the potential to identify patients with pulmonary thromboembolic disease, although results are not reliably predictive.

Interference:

None reported.

FIBRINOGEN, QUALITATIVE (ESTIMATED)

Normal:

Refer to laboratory reference range (dog and cat).

Patient Preparation:

None.

Collect:

Whole venous blood, 2.0 mL, in EDTA (lavender-topped tube).

Submit:

Plasma, 1.0 mL.

Interpretation:

Fibrinogen levels can be estimated as the difference between plasma protein concentrations before and after heating. An increased value correlates with clot lysis and supports the diagnosis of DIC.

Interference:

Clots in sample.

Protocol:

Invert tube several times to assure adequate mixing of venous blood and anticoagulant.

FIBRINOGEN, QUANTITATIVE

Normal:

100-245 mg/dL (dog); 110-370 mg/dL (cat).

Patient Preparation:

None.

Collect:

Completely fill a citrated (blue-topped) tube with whole blood. Mix thoroughly. Centrifuge immediately. Transfer plasma to a red-topped tube.

Submit:

Plasma, 1.0 mL, in a red-topped tube; label as CITRATED PLASMA.

Interpretation:

Increased concentration is associated with DIC. However, there is no single test for the diagnosis of DIC. The clinician must also assess fibrin degradation products (increased), APTT (prolonged), PT (prolonged), and platelet count (decreased).

Interference:

Incorrect ratio of citrate (anticoagulant) to whole blood; clots in sample; use of anticoagulants other than citrate.

Protocol:

On collection of blood, invert tube several times to assure adequate mixing of sample and anticoagulant. Centrifuge immediately. Transfer plasma to a red-topped tube; (label as CITRATED PLASMA). NOTE: Sample is stable for only 24 hours if held at 2° to 8° C; for extended storage, sample must be frozen.

FIBRIN DEGRADATION PRODUCTS (FDPs; FIBRIN SPLIT PRODUCTS [FSPs])

Normal:

<10 μg/mL (dog); <10 μg/mL (cat).

Patient Preparation:

None.

Collect:

Venous blood, 2.0 mL, in EDTA or in a red-topped tube.

Submit:

1.0 mL serum or plasma, 1.0 mL.

Interpretation:

Assay is used to document breakdown of fibrin clots. Increased concentration is associated with DIC (see also D-Dimer). However, there is no single test for DIC diagnosis. The clinician must also assess fibrinogen (increased), APTT (prolonged), PT (prolonged), and platelet count (decreased).

Interference:

Clots in sample.

PARTIAL THROMBOPLASTIN TIME (PTT): SEE ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT)

PIVKA TEST (PROTEINS INDUCED BY VITAMIN K ANTAGONISM TEST; “THROMBOTEST”)

Normal:

Refer to laboratory reference range (dog and cat).

Patient Preparation:

Atraumatic venipuncture is recommended.

Collect:

Completely fill a citrated (blue-topped) tube with whole blood. Mix thoroughly. Centrifuge immediately. Transfer plasma to a red-topped tube.

Submit:

Plasma, 1.0 mL, in red-topped tube; label as CITRATED PLASMA.

Interpretation:

Test is used in conjunction with prothrombin time in the assessment of patients suspected of warfarin toxicosis.

Interference:

Incorrect ratio of citrate (anticoagulant) to whole blood; clots in sample; use of anticoagulants other than citrate.

Protocol:

On collection of blood, invert tube several times to assure adequate mixing of sample and anticoagulant. Centrifuge immediately. Transfer plasma to a red-topped tube; label as CITRATED PLASMA. NOTE: Sample is stable for only 24 hours if held at 2° to 8° C; for extended storage, sample must be frozen.

PLATELET COUNT

Normal:

166-600 × 10³/μL (dog); 230-680 × 10³/μL (cat).

Patient Preparation:

Atraumatic collection is recommended.

Collect:

Venous blood, 1.0 mL, in EDTA (lavender-topped tube).

Submit:

Entire sample.

Interpretation:

Decreased platelet count is indicative of many disorders, including immune-mediated thrombocytopenia (extremely low platelet count), infection, sepsis, and DIC, and therefore must be assessed in light of other physical, hematologic, and biochemical parameters. Elevated platelet counts (up to 1 million cells/μL) can be normal for some patients. Cats with extreme thrombocytosis should be tested for feline leukemia virus.

Interference:

Slow draw of blood from the vein, transfer of blood from syringe to tube, and traumatic venipuncture may falsely decrease platelet count.

PROTHROMBIN TIME (PT)

Normal:

5.1-7.9 seconds (dog); 8.4-10.8 seconds (cat).

Patient Preparation:

None.

Collect:

Collect whole blood in a citrated (blue–topped) tube). Fill the tube to the capacity allowed by the vacuum. Invert immediately to mix. Centrifuge immediately and collect plasma. Transfer to a sterile plastic tube, using plastic pipette. Freeze. NOTE: label as CITRATED PLASMA.

Submit:

Citrated plasma, 1.0 mL. Ship sample with dry ice. Store frozen.

Interpretation:

PT is used to assess extrinsic and common coagulation pathways. Prolonged PT is used to assess patients with suspected vitamin K antagonism (warfarin toxicosis).

Interference:

Incorrect ratio of citrate to whole blood; clots in specimen; use of a non-citrated anticoagulant (e.g., EDTA in lavender-topped tube). NOTE: If the citrated plasma sample contacts glass, clotting factor activation may occur.

Protocol:

On collection of blood, invert tube several times to assure adequate mixing of sample and anticoagulant. Centrifuge immediately. Transfer plasma to a red-topped tube. (Label as CITRATED PLASMA). NOTE: sample is stable for only 24 hours if held at 2° to 8° C; for extended storage, sample must be frozen.

VON WILLEBRAND FACTOR (vWD FACTOR)

Normal:

Results reported are specific for the laboratory performing the test.

Patient Preparation:

None.

Collect:

Collect whole blood in a citrated (blue-topped) tube. Fill the tube to the capacity allowed by the vacuum. Invert immediately to mix. Centrifuge immediately and collect plasma. Transfer to a sterile plastic tube, using a plastic pipette. Freeze. Label tube as CITRATED PLASMA. Although EDTA (lavender-topped tube) may be accepted by some laboratories, sodium citrated samples are preferred when submitting samples for vWD factor.

Submit:

Citrated plasma, 1.0 mL. Ship sample with dry ice. If storing longer than 24 hours, store frozen.

Interpretation:

vWD is the most common inherited hemostatic disorder reported in dogs. This test is usually performed to confirm the diagnosis of vWD, in conjunction with the BMBT. Although the condition is inherited, variable degrees of expression are recognized. Dogs with vWD levels ≤30% have a tendency to bleed spontaneously (e.g., epistaxis).

Interference:

Recent transfusion may falsely elevate vWD levels. Incorrect ratio of citrate to whole blood, a clotted specimen, and use of a non-citrated anticoagulant (e.g., EDTA in lavender-topped tube) can also affect results. Do NOT use a glass pipette or glass tube. If the citrated plasma sample contacts glass, clotting factor activation may occur.

ENDOCRINOLOGY

ADRENOCORTICOTROPIC HORMONE (ACTH), ENDOGENOUS

Normal:

10 to 70 pg/mL (dog); results not reported for cat.

Patient Preparation:

Patient should be hospitalized overnight.

Collect:

Whole blood, 2.0 mL, in EDTA (chilled, lavender-topped tube); Immediately transfer plasma to plastic tube (ACTH adheres to glass) and freeze. Samples should be stored frozen until assayed. Maximum storage time: 1 month at −20° C.

NOTE: Contact laboratory directly before collecting samples for ACTH. Some laboratories request plasma samples be submitted in aprotonin and will provide specially prepared tubes for this purpose. Aprotonin (protease inhibitor) is added to a lavender-topped tube to stabilize ACTH. Freezing the sample is not necessary. The treated plasma should be separated immediately by centrifugation, transferred to a plastic tube, capped, and refrigerated. Transport sample to the lab with cold packs.

Submit:

Plasma, 1.0 mL; sample should NOT be allowed to sit at room temperature even for a short period.

Interpretation:

Adrenal tumors and iatrogenic Cushing's syndrome are expected to suppress ACTH secretion; pituitary-dependent Cushing's syndrome is characterized by excessive plasma concentration of ACTH.

Interference:

Recent or current corticosteroid administration; “stress” at or around the time of blood collection. NOTE: samples must be handled quickly because ACTH disappears quickly from whole fresh blood.

Protocol:

Following overnight hospitalization, the sample is collected between 8 and 9 am the following morning.

ACTH STIMULATION TEST (ACTH “Stim”)

Normal:

	Dog	Cat
Pre-test:	0.5 to 6.0 μg/dL	0.5 to 5.0 μg/dL
Post-test:	6 to 17 μg/dL	≤13 μg/dL

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NOTE: 17-22 μg/dL is considered “borderline” for dogs; 13-16 μg/dL is considered “borderline” for cats.

Patient Preparation:

No prior treatment with corticosteroids for at least 5-7 days before testing.

Collect:

Heparinized whole blood, 2.0 mL (green-topped tube). NOTE: Do NOT submit blood collected in EDTA.

Submit:

Plasma, 0.5 mL minimum for each sample submitted; sample should be refrigerated if shipped. Sample is assayed for cortisol.

Interpretation:

This is the most commonly used screening test for hyperadrenocorticism in dogs and cats. Patients with pituitary-dependent hyperadrenocorticism or adrenal tumor are expected to have an exaggerated cortisol response following stimulation with ACTH, assuming that the adrenal glands have retained ACTH responsiveness. Post-stimulation values ≥22 μg/dL are considered diagnostic for hyperadrenocorticism in dogs (>16 μg/dL in cats) in the presence of clinical signs (especially polydipsia) and supporting laboratory data and abdominal ultrasound. NOTE: ACTH stimulation does NOT differentiate between pituitary-dependent hyperadrenocorticism and adrenal tumor. An alternative test to use when screening for canine Cushing's syndrome is the low-dose dexamethasone test (see following entry).

The ACTH stimulation test is the only reliable test for monitoring patients undergoing o, p′-DDD (Lysodren) treatment of pituitary-dependent hyperadrenocorticism. Dogs with adequate pharmacologic suppression of adrenal function should have unchanged pre- and post-stimulation values (typically <2.0 μg/dL for both).

Interference:

Concurrent or recent treatment with corticosteroids. Anticonvulsant medications may adversely affect test results.

Protocol:

Several protocols are available; the following are representative.

Collect pre-test sample; then administer ACTH gel at 2.2 IU/kg IM (dog). Collect post-test sample 2 hours after ACTH administration;

or

Collect pre-test sample; then administer synthetic (expensive) ACTH (tetracosactrin, cosyntropin [Cortrosyn]) at 0.25 mg (dog) or 0.125 mg (cat) IM or IV. Collect post-test sample 1 hour after ACTH administration (dogs).

or

Collect pre-test sample; then administer ACTH at 125 μg IM (cat). Collect two post-test samples 30 minutes and 60 minutes after ACTH administration (cats).

ALDOSTERONE, SERUM

Normal:

Pre-test, 49 pg/mL (mean); post-test, 306 pg/mL (mean); reported range, 146 to 519 pg/mL (dogs). Results not reported for cats.

Patient Preparation:

None.

Collect:

Venous blood, 2.0 mL, in EDTA (lavender-topped tube) as baseline (pre-test); and repeat in 1 hour.

Submit:

Plasma, 1.0 mL, for each of the two samples collected.

Interpretation:

Low baseline and minimal or no increase in aldosterone levels support a diagnosis of hypoaldosteronism. The test is designed to distinguish dogs with primary hypoadrenocorticism from those with secondary hypoadrenocorticism. However, the sensitivity of the test in dogs is such that the positive predictive value is relatively low.

Interference:

Clots in sample.

Protocol:

In dogs, the pre-test and post-test samples are collected at 1-hour intervals following administration of ACTH. Follow the same protocol used to test for hyperadrenocorticism (see ACTH Stimulation).

COBALAMIN (VITAMIN B₁₂)

See Also TLI and Folate.

Normal:

249-733 ng/L (dog); 290-1500 ng/L (cat).

Patient Preparation:

Overnight fast.

Collect:

Whole blood, 4.0 mL (red-topped tube); separate serum from cells immediately.

Submit:

Serum, 2.0 mL.

Interpretation:

The test usually is performed in conjunction with TLI and serum folate. Decreased levels of cobalamin (vitamin B₁₂) support the diagnosis of small intestinal mucosal disease, small intestinal bacterial overgrowth, and exocrine pancreatic insufficiency. There is no significance attached to levels above the reported reference range.

Interference:

Hemolysis; lipemia.

CORTISOL, RESTING (BASAL): See ACTH STIMULATION TEST

Not Generally Recommended.

Normal:

0.5-6.0 μg/dL (dog); 0.5-5.0 μg/dL (cat)

Resting plasma cortisol in dogs is not routinely recommended because of the wide range of values in healthy animals. Although dogs with hyperadrenocorticism are expected to have increased values, reported values may still be within the limits of the reference range listed for normal dogs.

DEXAMETHASONE SUPPRESSION TEST, LOW-DOSE (LDDS TEST; DEXAMETHASONE SCREENING TEST)

Normal:

	Dog	Cat
Pre-test:	0.5 to 6.0 μ g/dL	0.5 to 5.0 g/dL
4-hr Post-test:	Usually <1.0 μg/dL	Same
8-hr Post-test:	Usually < 1.0μ g/dL	Same

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Patient Preparation:

No prior treatment with corticosteroids for at least 5-7 days before testing.

Collect:

Heparinized whole blood, 1.0 mL to 2.0 mL at each collection (green-topped tube). NOTE: Do NOT collect blood in EDTA.

Submit:

Plasma, 0.5 mL minimum for each sample submitted; sample should be refrigerated if shipped. Sample is assayed for cortisol.

Interpretation:

This a screening test for hyperadrenocorticism in dogs and cats. Administration of dexamethasone decreases plasma cortisol to <1.0 or 1.4 μg/dL (depending on the laboratory) within 2-3 hours in normal dogs. An 8-hr post-test sample >1.4 μg/dL is consistent with Cushing's syndrome in dogs and cats with clinical signs (especially polydipsia) and supporting laboratory data and abdominal ultrasound. NOTE: LDDS does NOT differentiate between pituitary-dependent hyperadrenocorticism and adrenal tumor.

The 4-hr post-test sample is not interpreted as part of the screening test but is considered an aid in differentiating pituitary-dependent hyperadrenocorticism from adrenal-dependent disease. Demonstrating a transient cortisol suppression (4-hour post-tests sample) supports pituitary-dependent disease and rules out adrenal-dependent disease.

Interference:

Recent or concurrent corticosteroid administration. Anticonvulsant medications may adversely affect test results.

Protocol:

Collect pre-test sample of plasma; administer dexamethasone (either in sodium phosphate or polyethylene glycol) at 0.01 mg/kg IV in dogs, or 0.1 mg/kg IV in cats; then collect a 4-hr post-test plasma sample, followed by an 8-hr post-test plasma sample. Submit the three plasma samples. (NOTE the higher dose of dexamethasone used in cats vs. dogs.)

DEXAMETHASONE SUPPRESSION TEST, HIGH-DOSE (HDDS TEST)

Normal:

	Dog	Cat
Pre-test:	0.5 to 6.0 μg/dL	Same
4-hr Post-test:	Usually <1.0 μg/dL	Same
8-hr Post-test:	Usually <1.0 μg/dL	Same

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Patient Preparation:

No prior treatment with corticosteroids for at least 5-7 days before to testing.

Collect:

Heparinized whole blood, 1.0 mL to 2.0 mL at each collection (green-topped tube). NOTE: Do NOT collect blood in EDTA.

Submit:

Plasma, 0.5 mL minimum for each sample submitted; sample should be refrigerated if shipped; sample is assayed for cortisol.

Interpretation:

The HDDS test is used in dogs with abnormal ACTH stimulation or LDDS test results to distinguish between pituitary-dependent disease and adrenal tumor. Administration of dexamethasone decreases plasma cortisol to <1.0 or 1.4 μg/dL (depending on the laboratory) within 2-3 hours in normal dogs. Dogs with an adrenal tumor or pituitary-dependent hyperadrenocorticism are not expected to demonstrate suppression of cortisol following administration of dexamethasone at the dose prescribed.

NOTE: “Suppression” is defined as:

Plasma cortisol concentration <50% of baseline at 4 hours or at 8 hours following dexamethasone administration.

or

Plasma cortisol concentration <1.4 μg/dL at 4 hours or at 8 hours following dexamethasone administration.

Interference:

Recent or concurrent corticosteroid administration. Anticonvulsant medications may adversely affect test results.

Protocol:

Collect pre-test sample of plasma; administer dexamethasone (either in sodium phosphate or in polyethylene glycol) at 0.1 mg/kg IV in dogs, or 1.0 mg/kg IV in cats; then collect a 4-hour post-test plasma sample, followed by an 8-hour post-test plasma sample. Submit the three plasma samples. (NOTE the higher dose of dexamethasone used in cats vs. dogs.)

ESTRADIOL (BASELINE)

Normal:

Not normally detectable (dog and cat).

Patient Preparation:

None.

Collect:

Venous blood, 2.0 mL, in a red-topped tube.

Submit:

Serum, 1.0 mL.

Interpretation:

This assay is not commonly requested for dogs and cats. Elevated levels have been used to detect testicular tumors and ovarian remnant syndrome. However, better tests are available.

Interference:

Variations in results occur with different methodologies used.

FOLATE: See also TLI AND COBALAMIN

Normal:

6.5-11.5 μg/L (dog); 9.7-21.6 μg/L (cat).

Patient Preparation:

Overnight fast.

Collect:

Whole blood, 4.0 mL (red-topped tube); separate serum from cells immediately.

Submit:

Serum, 2.0 mL.

Interpretation:

Assay usually is performed in conjunction with TLI and serum cobalamin. Elevated levels of folate support the diagnosis of small intestinal bacterial overgrowth in the upper small intestine. Values below the reference range support the diagnosis of proximal small intestinal disease.

Interference:

Hemolysis; lipemia.

FRUCTOSAMINE

Normal:

225-375 μmol/L (dog and cat).

NOTE: refer to laboratory reference range; ranges vary depending on methodology used.

Patient Preparation:

Fasted.

Collect:

Whole blood, 2.0 mL, in a red-topped tube (serum), lavender-topped tube (plasma in), or green-topped tube (plasma in heparin). CAUTION: sample must be non-hemolyzed.

Submit:

Serum or plasma, 1.0 mL.

Interpretation:

Test reflects glycemic levels over the preceding 1 to 3 weeks; it generally is used in assessing quality of glycemic control in patients with diabetes mellitus.

Interference:

Hemolysis; icterus.

GASTRIN

Normal:

Varies according to individual laboratory (dog); results not established for cat.

Patient Preparation:

None.

Collect:

Venous blood, 2.0 mL, in red-topped tube.

Submit:

Serum, 1.0 mL; sample should be kept frozen until assayed.

Interpretation:

Test is not commonly performed. Levels will be elevated in patients with functional gastrinoma, pyloric obstruction, renal failure, and gastric ulcers. There is no significance associated with decreased values.

Interference:

Concurrent administration of H₂ antagonist drugs (e.g., cimetidine).

GLUCAGON STIMULATION (IVGS TEST)

This is a complex test protocol to perform and yields results that are not generally reliable in distinguishing patients with type 1 diabetes from those with type 2 diabetes. It has been used to diagnose patients with insulin-secreting tumor. However, risk is associated with performing this test in patients with insulin-secreting tumor. Administration of glucagon will elevate serum glucose, which, in turn, promotes secretion of excessive amount of insulin; subsequent hypoglycemic crisis is a potential consequence.

GLUCOSE CURVE, 12-HOUR

Normal:

Glucose concentration ranges between 100 mg/dL and 250 mg/dL for the entire sampling period (dog and cat).

Patient Preparation:

Ideally, a venous catheter should be placed 1 hour before starting serial collections. Patient attitude during testing is important, because stressed or unusually aggressive animals may not be appropriate subjects for this test.

Collect:

Venous blood, approximately 1 mL per collection; plan on drawing as many as seven samples.

Submit:

Serum from each sample for routine glucose determination.

Interpretation:

This test is used to evaluate glucose levels in diabetic patients receiving insulin, particularly those that may experience recurrence of clinical signs as a result of under-treatment. Objectively, sufficient numbers of samples should be collected to establish a true nadir (lowest point) during the day. For example, if the nadir is >450 mg/dL, each dose of insulin might be increased by 1 to 2 units. When increasing insulin dose, it is appropriate to increase the dose by the same units for each administration throughout the day.

Interference:

Stress. Also, use of portable glucose meters to measure serial glucose levels in individual patients tends to result in lower values than actually are occurring.

Protocol:

The patient is given the usual dose of insulin and fed at home in the morning. On arrival at the hospital, a short intravenous catheter is placed in a suitable vein and secured appropriately. Serial samples are collected at 2-hour intervals over a 10-12 hour period. At the conclusion of the sampling period, the patient is usually fed and given a second daily dose of insulin, as appropriate. Then you, and the patient, can go home.

GLUCOSE TOLERANCE TEST, INTRAVENOUS (IVGT TEST)

Not Generally Recommended.

Normal:

By 60 minutes post-injection, serum insulin should be within 1 standard deviation of the baseline, and serum glucose should be within normal reference range for both dogs and cats.

Patient Preparation:

24-hour fast. Pre-placement of an intravenous catheter is recommended.

Collect:

Whole blood, 2.0 mL in a red-topped tube, for each sample submitted.

Submit:

Serum, 1.0 mL, for each sample submitted.

Interpretation:

Uncommonly performed, the IVGT test is an “insulin secretagogue test” used to distinguish type 1 diabetes from type 2 in cats. (NOTE: It is appropriate to consider all diabetic dogs as having type 1 [insulin-dependent] diabetes.) Patients with a mean serum insulin level >15 μg/mL by 60 minutes following injection are likely to have type 2 (non–insulin-dependent) diabetes. However, in cats, results are inconsistent and rarely diagnostic—another reason why this is NOT a popular test.

Interference:

Hemolysis. Prolonged contact of serum with RBCs will cause a false decrease in glucose concentration. (NOTE: Do NOT use gray-topped tubes to collect samples.) The IVGT test can be adversely influenced by diet, certain drugs (steroids, insulin), stage of estrus, underlying illness or infection (sepsis), and stress.

Protocol:

1.
Fast the patient overnight.
2.
Place an intravenous catheter.
3.
Collect venous blood in a red-topped tube. Submit 1.0 mL serum for a baseline glucose.
4.
Administer 0.5 g/kg of 50% glucose solution IV over 30 seconds.
5.
Collect approximately 2.0 mL of whole blood each: 1 minute, 5 minutes, 15 minutes, 25 minutes, 35 minutes, 45 minutes, 1 hour, and 2 hours following administration of glucose. (times may vary slightly depending on author and/or reference used).
6.
Submit 0.5-1.0 mL of serum for each sample. NOTE: Centrifuge and separate each serum sample as soon as practical after clot formation.

Each sample is submitted for both insulin and glucose determination.

GLUCOSE TOLERANCE TEST, ORAL (OGT TEST)

Not Generally Recommended.

The OGT test, although commonly performed in humans, is rarely performed in dogs and cats because of the difficulty associated with reliably administering the required volume of glucose orally.

An oral glucose absorption test has previously been described in the literature as a means of assessing patients with malabsorptive gastrointestinal disorders. Today, considering that superior tests are available, this test is no longer recommended for the assessment of malassimilation in dogs and cats.

INSULIN

Normal:

5-20 μU/mL (dog and cat).

Patient Preparation:

Overnight fast.

Collect:

Whole blood, 2.0 mL, in red-topped tube.

Submit:

Serum, 1.0 mL.

Interpretation:

Test is indicated for the diagnostic assessment of patients suspected of having an insulin-secreting tumor (e.g., insulinoma). If the patient has profound hypoglycemia at the time the test sample is collected, test results for insulin may be reported as normal. Simultaneous testing of serum glucose is recommended. Low glucose (<60 mg/dL) and an insulin level >20 μU/mL are consistent with insulin-secreting tumor.

Interference:

Hemolysis; blood collected in EDTA (plasma).

Protocol:

Most laboratories recommend that the patient's insulin level be determined in conjunction with blood glucose. Profound hypoglycemia may result in a normal insulin level being reported. A recent meal as well as several drugs can influence insulin concentrations.

PARATHYROID HORMONE (PTH)

Normal:

2-13 pmol/L (dog and cat). Results vary among laboratories.

Patient Preparation:

12-hour fast.

Collect:

Whole blood, 2.0 mL, in red-topped tube.

Submit:

Serum (FROZEN), 1.0 mL, in sterile plastic tube.

Interpretation:

PTH levels will be increased in patients with primary hyperparathyroidism, secondary renal or nutritional hyperparathyroidism, and other disorders causing hypocalcemia. No measurable PTH level is consistent with primary hypoparathyroidism. PTH testing should always include ionized calcium (iCa) assay.

Interference:

Hemolysis; thawing of sample for extended periods.

Protocol:

Serum should be separated from cells within 1 hour following collection; serum should be frozen and shipped on ice. Deliver to laboratory via overnight delivery. Keep frozen.

PARATHYROID HORMONE-RELATED PROTEIN (PTHRP)

Normal:

Refer to laboratory reference range values (dog and cat).

Patient Preparation:

12-hour fast.

Collect:

Whole blood, 2.0 mL, in red-topped tube.

Submit:

Serum (FROZEN), 1.0 mL, in sterile plastic tube.

Interpretation:

Interpretation of PTHrP entails simultaneous testing for calcium (or iCa) and PTH. PTHrP levels are low to undetectable in patients with primary hyperparathyroidism. Patients with hypercalcemia associated with lymphosarcoma or chronic renal insufficiency will have increased levels of PTHrP.

Test Interference:

Hemolysis; thawing of sample for extended periods.

Protocol:

Serum should be separated from cells within 1 hour following collection; serum should be frozen and shipped with ice in plastic tube. Deliver to laboratory via overnight delivery. Keep frozen.

T₃ (3,5,3′-TRIIODOTHYRONINE)

Normal:

0.8-1.5 mg/dL by radioimmunoassay (RIA) (dog); 0.8-1.5 ng/mL (by RIA) (cat).

NOTE: results will vary among different laboratories.

Patient Preparation:

None (patient should not be receiving exogenous thyroid hormone supplementation).

Collect:

Whole blood, 1-2 mL, in red-topped tube.

Submit:

Serum, 0.5 mL minimum. NOTE: Storage/shipment of samples in plastic, rather than glass, containers is recommended. Sample should be frozen and shipped with cold packs.

Interpretation:

T₃ is a poor indicator of thyroid function and generally provides little reliable diagnostic information pertaining to thyroid-related disease; baseline T₃ does NOT reliably distinguish between hypothyroid and euthyroid states. Test results for T₃ include both free T₃ (fT₃₎ and protein-bound T₃. RIA is the preferred test method.

Interference:

Patients receiving exogenous thyroid supplementation can have positive or negative test interference, depending on the dose of drug administered and the time the last dose was given. T₃ autoantibody, if present, may falsely lower test results. NOTE: Storage of serum or plasma in glass can cause a significant false increase in serum T₃ concentration.

REVERSE T₃ (RT_3; 3,3′, 5′-TRIIODOTHYRONINE)

There are currently no established diagnostic guidelines associated with baseline reverse T₃ values in dogs and cats.

T₃ SUPPRESSION

Normal:

Suppression of T₄ to 1.5 μg/dL following seven doses of synthetic T₃(in cats).

Patient Preparation:

None.

Collect:

Whole blood, 3.0 mL, in red-topped tube, for each sample (pre-test and post-test).

Submit:

Serum, 1.0 mL minimum for each sample.

Interpretation:

This test measures T₄and T₃ following sequential administration of seven doses of synthetic T_3; it may distinguish between euthyroid and slightly hyperthyroid cats. Hyperthyroid cats demonstrate minimal or no decrease in serum T₄, which remains at 2.0 μg/dL or more. T₄ values between 1.5 and 2.0 g/dL are nondiagnostic. T₃values should increase in all cats (normal as well as hyperthyroid). NOTE: If T₃ values do NOT increase, test results are considered invalid.

Interference:

Hemolysis; lipemia; icterus; blood collected in EDTA.

Protocol:

Pre-test sample is collected (to be submitted for T₃ and T₄).

FREE T₄ (fT₄)

Normal:

0.8-3.5 ng/dL (dog); 1.0-4.0 ng/dL (cat).

Patient Preparation:

None.

Collect:

Venous blood, 2.0 mL, in red-topped tube.

Submit:

Serum, 1.0 mL minimum; freeze and store in plastic tubes rather than glass; ship samples with cold packs to arrive for analysis within 5 days of collection.

Interpretation:

Free T₄ is used in preference to conventional T₄ to confirm hypothyroidism in dogs and hyperthyroidism in cats. Results <0.8 ng/dL (and especially <0.5 ng/dL) are consistent with a diagnosis of hypothyroidism in dogs. As with conventional T₄levels, free T₄ levels in cats that exceed 4.0 ng/dL are consistent with the diagnosis of hyperthyroidism.

Interference:

Storing/shipping serum in glass container may alter test results; circulating thyroid autoantibody does NOT interfere with test results. Results determined by RIA alone may be significantly lower than those determined by the MED method. Severe illness may cause low fT₄ values in dogs with normal thyroid function (sick, euthyroid). T₄ autoantibody does NOT interfere with the assay for free T₄.

TOTAL T₄, (THYROXINE OR TETRAIODOTHYRONINE)

Normal:

1.5-3.5 μg/dL (dog); 1.0-4.0 μg/dL (cat).

NOTE: A point-of-care ELISA test kit for in-hospital assessment of T₄is available. However, it is recommended that ELISA test results be confirmed by RIA.

Patient Preparation:

None (patient should not be receiving exogenous thyroid hormone supplementation).

Collect:

Whole blood, 1-2 mL, in red-topped tube.

Submit:

Serum, 0.5 mL minimum. NOTE: storage/shipment of samples in plastic, rather than glass, containers is recommended. Sample should be frozen and shipped with cold packs.

Interpretation:

T₄ is produced within the thyroid gland, and therefore the total T₄ is the preferred test of thyroid function. The test combines measurement of free T₄ (fT₄) plus protein-bound T₄. Dogs with T₄ levels <2.0 μg/dL are likely to have hypothyroidism (if associated clinical signs are present); cats with T₄ levels >4.0 μg/dL are likely to have hyperthyroidism.

Dogs: Decreased values suggest hypothyroidism (dogs); however, dogs with underlying illness NOT related to abnormal thyroid function may still have abnormally decreased T₄concentration (sick, euthyroid). A comprehensive physical examination and laboratory profile are indicated in establishing a diagnosis of hypothyroidism in dogs.

Cats: In middle-aged and old cats, hyperthyroidism becomes an important differential diagnosis when T₄ levels exceed 4.0 μg/dL in the presence of clinical signs. Most cases are caused by a functional multinodular adenoma. Less than 5% of cases are associated with thyroid adenocarcinoma.

Interference:

Patients receiving exogenous thyroid supplementation can have positive or negative test interference, depending on the dose of drug administered and the time the last dose was given. Underlying illness and T₄ autoantibody, if present, may falsely lower test results. NOTE: Storage of serum or plasma in glass can cause a significant false increase in serum T₄ concentration.

THYROTROPIN, CANINE (THYROID-STIMULATING HORMONE [TSH]; BASELINE TSH)

Normal:

Up to 0.6 ng/mL (dog); values not established for cats.

NOTE: Lower limits of normal (~ 0.1 ng/mL) for methodologies used are below the sensitivity of the assay.

Patient Preparation:

None, if patient is not receiving exogenous thyroid hormone supplementation.

Collect:

Venous blood, 1-2 mL, in red-topped tube.

Submit:

Serum, 0.5 mL minimum. NOTE: Storage/shipment of samples in plastic, rather than in glass, containers is recommended. Sample should be frozen and shipped with cold packs.

Interpretation:

This is a reasonable test for the assessment of hypothyroidism in dogs; however, TSH fluctuations can produce normal results in 20% to 40% of hypothyroid dogs. TSH should not be interpreted without having same-sample results for T₄ or fT₄. A low T₄ or fT₄ and increased TSH in a dog are consistent with the diagnosis of hypothyroidism. Normal T₄ or fT₄ and TSH effectively rule out hypothyroidism. Clinical signs and a routine laboratory profile must be part of the diagnostic assessment of any patient suspected of having thyroid disease.

Interference:

The same interfering factors that influence T₄ assays are likely to affect TSH.

THYROID-STIMULATING HORMONE, Canine: See THYROTROPIN, CANINE

THYROTROPIN RESPONSE (TSH RESPONSE TEST)

Not Generally Recommended.

Initially believed to be useful in diagnosing hyperthyroidism, the TSH response test has been shown in subsequent studies to be limited in the ability of abnormal thyroid tissue to respond to stimulation. Other test limitations, including the removal of bovine TSH from the market, have resulted in the current recommendation against its use.

IMMUNOLOGY

ALLERGEN-SPECIFIC IGE ANTIBODY TEST (RADIOALLERGOSORBENT TEST [RAST]; ALLERGY SCREEN)

Normal:

Refer to laboratory for interpretation of results reported.

Patient Preparation:

None.

Collect:

Venous blood, 2.0 mL, in red-topped tube,

Submit:

Serum, 1.0 mL.

Interpretation:

This in vitro assay is used to identify causative allergens in atopic animals. The RAST has also been suggested for evaluation of patients with suspected food-related hypersensitivity. At this time, results are inconclusive.

Interference:

Concurrent corticoid therapy.

ANTIBODY TITERS FOR INFECTIOUS DISEASE DIAGNOSIS: See INFECTIOUS DISEASE SEROLOGY and microbiology

ANTINUCLEAR ANTIBODY (ANA)

Normal:

Results are reported as a titer (ratio); refer to the laboratory reference range (dog and cat).

Patient Preparation:

ANAPLASMA PHAGOCYTOPHILA ANTIBODY (FORMERLY EHRLICHIA EQUI)

Normal:

Negative (dog)

Patient Preparation:

None.

Collect:

Venous blood, 2.0 mL, in red-topped tube.

Submit:

Serum, 1.0 mL.

Interpretation:

Limited studies are available about antibody responses to infection with A. phagocytophil a; refer to the laboratory test results for information on interpretation.

Interference:

Cross-reactivity with A. platys is expected.

ASPERGILLUS SPP. ANTIBODY TITER (NON-AVIAN)

Not Generally Recommended.

The high rate of false-positive and false-negative test results (depending on test methodology) limits the value of serology in establishing a diagnosis of aspergillosis in dogs without clinical signs.

Normal:

Negative (dog and cat).

Patient Preparation:

None.

Collect:

Venous blood, 2-3 mL, in red-topped tube.

Submit:

Serum, 1.0 mL minimum.

Interpretation:

It is recommended to concurrently request Penicillium spp. titer. A positive antibody titer in a dog that is not responsive to empirical antibiotic therapy and with persistent nasal discharge, masseter muscle atrophy, and erosions of the nasal planum is highly suspect for aspergillosis.

Interference:

A positive test result may simply denote exposure.

BABESIA ANTIBODY TITER, CANINE

Normal:

B. canis, <80; B. gibsoni, <320.

Patient Preparation:

None.

Collect:

Venous blood, 2-3 mL, in red-topped tube.

Submit:

Serum, 1.0 mL minimum.

Interpretation:

Titers >80 for B. canis or >320 for B. gibsoni are consistent with the diagnosis of infection in patients with corresponding clinical symptoms.

Interference:

There can be considerable cross-reactivity between serologic assays for B. canis and B. gibsoni. Negative results in patients suspected of being infected should be followed with a convalescent sample 4 weeks following the initial test.

BARTONELLA SPP. (BARTONELLA HENSELAE TITER)

Normal:

Negative (cat).

Patient Preparation:

None.

Collect:

Venous blood, 2.0 mL, in red-topped tube.

Submit:

Serum, 1.0 mL.

Interpretation:

Different test methodologies are used commercially, including IFA (immunofluorescent assay), ELISA (enzyme-linked immunosorbent assay), and Western blot analysis. Although there is cross-reactivity with other Bartonella species, the test is reported to be relatively sensitive and specific for infection in cats. At issue, however, is whether all cats that test positive are, in fact, clinically ill and whether treatment is indicated on the basis of one positive test result. Results on positive cats may be reported as Serum, +1 to +4.

Interference:

None reported.

BLASTOMYCOSIS ANTIBODY TITER

Normal:

Negative (dog and cat).

Patient Preparation:

None.

Collect:

Venous blood, 2.0 mL, in red-topped tube.

Submit:

Serum, 1.0 mL minimum.

Interpretation:

A positive serologic test in a dog with clinical signs consistent with blastomycosis does correlate with infection. Many cats with known blastomycosis infection, however, will have negative serologic results.

Interference:

None reported.

BLOOD CULTURE (BACTERIA)

Normal:

Negative at 10+ days following incubation (dog and cat).

Patient Preparation:

Ideally, sample should be collected while patient is febrile. The peripheral vein must be surgically prepared prior to venipuncture. Use at least two veins. Do not collect blood via a catheter.

Collect:

Venous blood, 6-10 mL, in a syringe (with no anticoagulant added).

Submit:

Transfer blood directly to a suitable (commercially prepared) vial containing a blood culture medium. NOTE: special media designed to remove certain antibiotics are available for patients that are concurrently receiving antibacterial therapy at the time of sample collection.

Interpretation:

The laboratory will report identification of any growth and minimum inhibitory concentration (MIC) susceptibility test results.

Interference:

Contaminating bacteria obtained during the collection process.

Protocol:

Samples from a separate vein, when feasible, should be collected. Generally, three samples are submitted from the same patient taken at approximately 1-hour blood intervals, collected by venipuncture (syringe and needle) from different sites.

BORRELIA BURGDORFERI: See LYME BORRELIOSIS

BRUCELLA CANIS ANTIBODY

Preliminary Assessment by RSAT OR TAT

(RSAT = rapid slide agglutination test; TAT = tube agglutination test)

Normal:

Negative (dog).

Patient Preparation:

None.

Collect:

Venous blood, 2.0 mL, in red-topped tube.

Submit:

Serum, 1.0 mL.

Interpretation:

Dogs with a negative test result are likely not to be infected. Follow-up testing is recommended for dogs with a negative test result but high likelihood of infection. Dogs with a positive test result should be retested by agar gel immunodiffusion (AGID) (see following entry) to confirm infection.

Interference:

Because of the nature of the screening tests, the frequency of false-positive test results can be high.

Protocol:

Both the RSAT and the TAT should be performed with 2-mercaptoethanol (2-ME) to eliminate interference caused by heterologous IgM (responsible for most false-positive reactions).

NOTE: optional testing by IFA is commercially available. Consider using IFA to compare with the RSAT and TAT.

Confirmatory Test by AGID (Agar Gel Immunodiffusion)

Normal:

Titers <50 are considered negative (dog).

Patient Preparation:

None.

Collect:

Venous blood, 2.0 mL, in red-topped tube.

Submit:

Serum, 1.0 mL.

Interpretation:

Titers >200 are generally consistent with positive results on blood culture.

Interference:

None reported.

CANINE DISTEMPER ANTIBODY

CEREBROSPINAL FLUID (CSF) (IgG or IgM)

Normal:

Negative (refer to laboratory reference range).

Patient Preparation:

Fluid should be collected from the cisterna magna; sample is collected with the patient under general anesthesia and an endotracheal tube placed; sterile technique is required.

Collect:

CSF.

Submit:

CSF, 1.0 mL.

Interpretation:

The presence of any titer for canine distempter virus antibody (IgG or IgM) in CSF is consistent with infection, provided there is no contamination of the sample with blood or plasma. CSF titers should be assessed in conjunction with serum antibody titer.

Interference:

Blood or plasma contamination of sample during collection may cause false-positive results in vaccinated dogs. Vaccine-induced antibody is not expected to cross into CSF.

NOTE: when assessing individual patients for distemper antibody, various laboratory methods are used to perform serology. The virus neutralization (VN) test method for CDV antibody is recommended.

SERUM (IgG or IgM)

Normal:

IgG (single sample titer is inconclusive). Uninfected dogs have no evidence of a rising titer when results of the acute and convalescent titers are compared. A single IgM titer will be negative. Vaccination will affect results. Refer to laboratory reference range.

Patient Preparation:

None.

Collect:

Whole blood, 2.0 mL.

Submit:

Serum, 1.0 mL.

Normal:

Refer to laboratory reference range (dog and cat).

A point-of-care test for in-hospital use in dogs will be negative in the non-exposed patient.

Patient Preparation:

None.

Collect:

Venous blood, 2.0 mL, in red-topped tube.

Submit:

Serum, 1.0 mL.

Interpretation:

Interpretation varies among laboratories and methodologies used to detect antibody. The clinician must consider test results with clinical signs and results of routine laboratory profiles when making the decision to treat a patient with a positive titer. Dogs with confirmed infections may continue to have a positive antibody titer for E. canis for several months following recovery.

NOTE: Correlation between antibody titer and active infection is poor with all available antibody tests on the market today.

Interference:

None reported.

EHRLICHIA EQUI ANTIBODY: See ANAPLASMA PHAGOCYTOPHILA ANTIBODY

EHRLICHIA SPP.: See POLYMERASE CHAIN REACTION (PCR) FOR TEST METHOD

FELINE CORONAVIRUS ANTIBODY (FeCoV Ab)

Inappropriately called the “FIP Ab test”; not generally recommended.

Coronavirus titer is not a diagnostic test for feline infectious peritonitis (FIP). In fact, at this time there is no diagnostic test for FIP. The only value in submitting serum for a coronavirus titer is to identify cats that have a truly negative antibody titer (titered to zero). A negative antibody titer, although exceptionally rare among domestic cats, may denote no prior exposure to coronavirus (FIP).

EHRLICHIA spp (by Polymerase Chain Reaction [PCR])

Normal:

Negative (dog and cat).

Patient Preparation:

None.

Collect:

Venous blood, 2.0 mL, in EDTA (lavender-topped tube).

Submit:

Entire sample.

Interpretation:

Reported as positive or negative; positive samples should be subjected to further testing in order to confirm infection.

Interference:

PCR tests are subject to false-positive results because of minute traces of cross-reacting DNA in the sample.

FELINE INFECTIOUS ANEMIA: See HAEMOBARTONELLA (MYCOPLASMA HAEMOFELIS)

FELINE LEUKEMIA VIRUS ANTIGEN (FeLV Ag; p27 TEST)

NOTE: ALL commercial and in-hospital FeLV tests detect antigen, not antibody.

Normal:

Negative (denotes absence of virus).

Patient Preparation:

None.

Collect:

IFA: Whole blood, 1.0 mL, in EDTA (lavender-topped tube).

ELISA: Whole blood, 2.0 mL, in red-topped tube.

Submit:

IFA: buffy coat smear or 1.0 mL anticoagulated whole blood collected in EDTA; NOTE: IFA is the preferred method for assessing bone marrow aspiration samples for FeLV Ag.

ELISA: Serum, 1.0 mL.

Interpretation:

Both the IFA and the ELISA detect the presence of the core protein p27.

IFA: a positive test result identifies the presence of FeLV cell–associated antigen (in WBCs and/or platelets) and defines “persistent infection,” especially in cats with clinical and/or laboratory signs consistent with FeLV infection.

ELISA: a positive test result identifies the presence of soluble, circulating FeLV antigen; healthy cats with a positive test result should be retested 1 to 2 months later to reassess virus status OR be subjected to corroborative testing by IFA.

NOTE: The AAFP/AFM Task Force on Feline Retrovirus Testing stresses that healthy cats with positive test results may have false-positive test results (by either method). Corroborative testing is indicated using a different test method. A positive test result in a cat with clinical signs suggestive of chronic illness, lymphoid neoplasia, or significant hematologic abnormalities is highly indicative of infection. Negative test results are highly accurate.

Interference:

FeLV vaccination will not interfere with test results, regardless of test method used.

IFA: Thrombocytopenia and/or leukopenia may cause false-negative test results. Poor slide quality, eosinophilia, and hemolysis may influence ability to accurately read stained slides.

ELISA: Hemolysis.

NOTE: The AAFP/AFM Task Force on Feline Retrovirus Testing no longer stipulates that the IFA for FeLV Ag is the “confirmatory test” for cats with positive ELISA results. In fact, the ELISA is more sensitive than the IFA in detecting the presence of FeLV antigen.

FELINE IMMUNODEFICIENCY VIRUS ANTIBODY (FIV Ab)

Normal:

Negative (negative test results indicate no prior FIV exposure).

Patient Preparation:

None.

Collect:

Whole blood, 2.0 mL, in red-topped tube.

Submit:

Serum, 1.0 mL each for IFA (or ELISA) and Western blot analysis.

Interpretation:

Cats with a positive test result by IFA (or ELISA) should be subjected to confirmatory testing by Western blot analysis.

Interference:

Any cat having received at least 1 inoculation with the FIV vaccine (killed, adjuvanted product) will produce interfering antibody that is detected by all commercially available FIV Ab tests (IFA, ELISA, Western blot analysis). False-positive test results are expected to persist for at least 1 year following vaccination. Currently, there is NO test that will reliably and consistently distinguish between infected and vaccinated cats, including polymerase chain reaction (PCR). Also, kittens that have nursed from vaccinated queens are expected to have a false-positive test result for FIV Antibody associated with maternally derived antibody. Duration of the false-positive results is unknown.

Kittens less than 6 months of age are expected to have maternally derived antibody if the queen is infected and may have a false positive test result when tested by IFA or ELISA. Subsequent testing of positive kittens at 6 months of age or older is indicated to determine true infection status.

NOTE: The AAFP/AFM Task Force on Feline Retrovirus Testing stresses that healthy cats with a positive test result (by IFA or ELISA) may have false-positive test results, especially in populations in which the prevalence of infection is low. Confirmatory testing is indicated using the Western blot analysis. Negative test results are highly accurate.

GIARDIA ANTIGEN

Normal:

Negative for antigen (dog and cat).

Patient Preparation:

None.

Collect:

2 to 5 grams of fresh feces.

Submit:

Entire sample in a sterile container; sample can be stored for 24 hours at 2° to 8° C. Frozen feces may be stored for slightly longer periods.

Interpretation:

Test results are reported as either positive or negative for antigen; positive test results are expected in dogs or cats during active cyst and trophozoite shedding. The zoonotic potential of canine and feline giardiasis is controversial.

Interference:

Extended or improper storage of the sample could result in false-negative results.

HEARTWORM ANTIBODY, FELINE

NOTE: See also Heartworm Antigen, Feline.

Normal:

Negative.

Patient Preparation:

None.

Collect:

Whole blood, 1.0 mL, in red-topped tube.

Submit:

Serum, 1.0 mL.

Interpretation:

Test results should be interpreted in conjunction with a heartworm antigen test performed on the same sample. Because diagnostic confirmation of heartworm infection in cats is problematic (for several reasons), serologic test results must be considered in light of other laboratory and radiographic assessments.

A negative heartworm antibody (HW-Ab) test result suggests that there has been no exposure to Dirofilaria immitis. A negative result typically is used to rule out feline heartworm infection.

A positive HW-Ab test result only supports prior exposure. It does NOT confirm infection. Cats that are positive for HW-Ab should be subsequently tested for heartworm antigen (see below).

Interference:

Marked hemolysis or lipemia.

HEARTWORM ANTIGEN, FELINE

NOTE: See also Heartworm Antibody, Feline.

Normal:

Negative.

Patient Preparation:

None

Collect:

Whole blood, 2.0 mL, in a red-topped tube.

Submit:

Serum, 1.0 mL.

Interpretation:

Test results should be interpreted in conjunction with a heartworm antibody test performed on the same sample. Because diagnostic confirmation of heartworm infection in cats is problematic (for several reasons), serologic test results must be considered in light of other laboratory and radiographic assessments.

A negative heartworm antigen (HW-Ag) test result is not diagnostically useful; heartworm infection is still possible. A positive HW-Ag test result is highly specific; infection is likely.

Interference:

Marked hemolysis may cause a false-positive test result. A cat with male heartworm infection only will not have a positive result. Low worm burdens (common) may result in false-negative test results.

HEARTWORM ANTIGEN, CANINE

Normal:

Negative.

Patient Preparation:

None.

Collect:

Whole blood, 2.0 mL, in red-topped tube.

Submit:

Serum, 1.0 mL.

Interpretation:

A negative (HW-Ag) test result implies no infection; a positive HW-Ag test result strongly supports active infection.

Interference:

Marked hemolysis may cause a false-positive test result. A dog with a small worm burden may have a false-negative test result. NOTE: the canine HW-Ag test may remain positive for up to 16 weeks following adulticide therapy.

Protocol:

Sample may be submitted to a commercial laboratory or can be rapidly assessed in the hospital as a point-of care (SNAP) test; follow manufacturer's procedure outline.

Quantitative C6 Antibody Test

Normal:

Usually <30 antibody units (dog); refer to the laboratory reference range.

Patient Preparation:

None.

Collect:

RFFIT = rapid fluorescent focus inhibition test.

Normal:

Dog and cat: A titer of 0.5 IU/mL or higher is usually required for animals exported to most rabies-free areas. NOTE: it may to necessary to contact individual countries for current import requirements prior to shipping animals.

Patient Preparation:

None.

Collect:

Whole blood, 4.0 mL, in red-topped tube.

Submit:

Serum, 2.0 mL (minimum is 500 μL), in a leak-proof container (e.g., with screw-on cap). Place sample container should be placed inside a second container with gel packs or dry ice. Overnight shipping is recommended.

Interpretation:

As stipulated by the laboratory. NOTE: Values for a “protective titer” in animals have not been established.

Interference:

Gross hemolysis; lipemia; samples other than serum (e.g., plasma is NOT acceptable). NOTE: Other causes for sample to be rejected include insufficient quantity of serum, bacterial contamination of sample, and unlabeled sample.

Protocol:

Samples may be sent to: Rabies Laboratory/RFFIT, Mosier Hall, Kansas State University, 1800 Denison Avenue, Manhattan, KS 66506-5600 (Tel. 785-532-4483).

NOTE: For samples sent to Kansas State University:

1.
All specimen tubes must be labeled with the patient's name and/or identification number. Samples submitted without labels will not be accepted.
2.
All samples must have an accompanying RFFIT submission form.
3.
All animals being tested for export must have a microchip number (or tattoo) included.

Forms may be downloaded from the KSU Rabies Laboratory website: www.vet.ksu.edu/rabies

ROCKY MOUNTAIN SPOTTED FEVER (RMSF)

Normal:

Negative (dog and cat).

Patient Preparation:

None.

Collect:

Whole blood, 2.0 mL, in red-topped tube.

Submit:

Serum, 1.0 mL.

Interpretation:

Generally, two samples are recommended (“acute” and “convalescent”), obtained 2 to 3 weeks apart. Titers reported vary among laboratories. The laboratory performing the titer will provide recommendations for interpreting results.

Interference:

None.

TOXOPLASMOSIS TITERS (IgG AND IgM)

Normal:

See Interpretation.

Patient Preparation:

None.

Collect:

Whole blood, 2.0 mL, in red-topped tube.

Submit:

Serum, 1.0 mL.

Interpretation:

A positive titer denotes exposure, not active infection. IgG and IgM titers typically are reported individually. A titer >1:256 for IgM is consistent with active infection in patients with clinical signs (e.g., pneumonia in cats, myositis in dogs). It is recommended that two samples for IgG titer be submitted; samples should be collected 2 to 3 weeks apart. A fourfold or greater rise in titer within 2 to 3 weeks is supportive of the diagnosis of active infection. Cats seropositive on a single titer are unlikely to be shedding oocysts.

Interference:

Hemolysis; lipemia.

VACCINE TITERS: SEE LISTING UNDER SPECIFIC PATHOGEN

Laboratories providing vaccine titers typically limit these services to canine parvovirus, canine distemper, and feline panleukopenia. A limited number of laboratories offer titers for feline herpesvirus-1 and feline calicivirus.

Note: Several university and commercial laboratories now provide antibody titers for selected canine and feline viruses as a means of assessing immunity to prior vaccination.

Note: National laboratory standards for determining serum antibody titers against these pathogens have not been established. Because methods for performing titers vary among laboratories, titer results and ranges also can vary dramatically.

It is recommended that samples be analyzed by laboratories using the virus neutralization test (for canine distemper) and hemagglutination inhibition (for canine parvovirus and feline panleukopenia).

Note: A “positive” antibody titer usually will equate with “PROTECTIVE IMMUNITY”

A “negative” antibody titer does NOT necessarily equate to “SUSCEPTIBILITY”

URINE

A red-topped tube is the preferred collection tube for urinalysis. A Copan swab can be used for urine culture, but this precludes quantitation of bacteria, if present. Urine for culture is best collected by cystocentesis and transported with a cold pack to prevent bacterial overgrowth.

CORTISOL, URINE. SEE URINE CORTISOL:CREATININE RATIO (UC:CR; URINARY C:C RATIO)

MICROALBUMINURIA TEST (EARLY RENAL DISEASE (ERD) IN-HOSPITAL TEST KIT)

Normal:

Negative test strip indication (dog and cat).

Patient Preparation:

None.

Collect:

2-mL (minimum) aliquot of urine in a clean container.

Submit:

Same.

Interpretation:

Test strip indicator grades the approximate degree of microalbuminuria. The manufacturer of the test kit provides recommendations for interpreting test results. However, it should be noted that a positive test result in clinically normal dogs is NOT known to be predictive of impending renal disease. In various studies, it has been shown that a significant percentage of healthy dogs and certain breeds (soft-coated Wheaten Terriers) will have positive test results. Until more information is available about the clinical utility of this test, its use should be restricted to monitoring urine protein loss in patients with known or suspected glomerular disease.

Interference:

Blood contamination of urine sample.

URINE CORTISOL:CREATININE RATIO (UC:CR, URINARY C:C RATIO)

Normal:

Varies according to individual laboratory and test methodology used (dog and cat).

Patient Preparation:

Owner should collect urine at home on the day (morning is preferable) that the test is submitted, thereby reducing stress-induced artifact.

Collect:

3.0- to 5.0-mL aliquot of pooled urine in a sterile container.

Submit:

Same; sample should be refrigerated during transport to the laboratory.

Interpretation:

The UC:CR is reported to have high sensitivity (negative predictive value) and therefore has been recommended to rule out the diagnosis of hyperadrenocorticism in dogs.

Controversy exists regarding the diagnostic value of the UC:CR to diagnose canine Cushing's syndrome. Reference values for the cat are not reported. The test currently is NOT recommended as a single diagnostic test. In SERIAL UC:CR studies performed in hyperthyroid cats, elevated ratios were observed; successful treatment (medical and surgical) did result in a significant decrease in UC:CR in cats.

Interference:

The effect of urine collected from hospitalized dogs (stress) vs. urine collected from dogs at home remains an arguable variable. Owners should be advised to collect urine at home on the scheduled day of examination/testing.

Protocol:

Instruct the owner to collect urine in a single, clean container over 2 consecutive hours on the same day that the urine sample is to be submitted to the laboratory. A 3.0- to 5.0-mL aliquot of pooled urine is submitted for analysis. NOTE: Not all commercial laboratories offer this test. Check before submitting.

URINE PROTEIN-CREATININE RATIO (UPCr; P:Cr; UPC)

Normal:

Ratio <0.3 (dog); ratio <0.6 (cat).

Patient Preparation:

None.

Collect:

2- to 3-mL aliquot of randomly collected urine in a clean container.

Submit:

Same.

Interpretation:

UP:Cr titer >1.0 is consistent with the diagnosis of pathologic proteinuria. The ratio does not confirm the source of the protein loss. However, in patients with consistent hypoalbuminemia and significantly elevated urine P:Cr, loss of protein through the glomerulus is likely (e.g., glomerulonephritis).

Interference:

Blood contamination (cystitis, cystocentesis).

Footnotes

^*

NOTE: Reference Range values listed throughout this section are for general reference only. Test results from individual patients must be compared to the Reference Range Values for the laboratory that performs the test.

^†

Vacutainer® is the registered trademark of Becton, Dickenson and Company (B-D).

^*

Ethylene Glycol Test Kit, PRN Pharmacal Inc., Pensacola, Fla.

PERMALINK

Laboratory Diagnosis and Test Protocols

Richard B Ford, DVM, MS

Elisa M Mazzaferro, MS, DVM, PhD

COMMON REFERENCE RANGE VALUES*

SAMPLE HANDLING

SAMPLE IDENTIFICATION

SAMPLE COLLECTION TUBES

TABLE 5-1.

TABLE 5-2.

TABLE 5-3.

TABLE 5-4.

TABLE 5-5.

TABLE 5-6.

TABLE 5-7.

TABLE 5-8.

TABLE 5-9.

TABLE 5-10.

TABLE 5-11.

TABLE 5-12.

TABLE 5-13.

TABLE 5-14.

TABLE 5-15.

SAMPLE STORAGE AND TRANSPORT

PATIENT PREPARATION

MINIMIZING HEMOLYSIS

AVOIDING CLOTS AND PLATELET CLUMPS

SUBMISSION REQUIREMENTS FOR RABIES SUSPECTS

SAMPLE SUBMISSION FOR RABIES TESTING

PACKAGING REQUIREMENTS FOR AUTHORIZED SAMPLES

INFORMATION REQUESTED ON THE RABIES SPECIMEN HISTORY FORM

SUBMISSION GUIDELINES

PACKING AND SHIPPING DIRECTIONS

HISTOPATHOLOGY AND CYTOPATHOLOGY

HISTOPATHOLOGY

Biopsy Tissue

Very Large Specimens

Tissue Orientation and Information

Very Small Specimens

CYTOPATHOLOGY

Fine Needle Aspiration (FNA)

Indications

Sample preparation

Staining options

Common mistakes

Exfoliative Cytology (“Impression Smear”)

Indications

Sample preparation

Staining options

Common mistakes

Swabs, Scrapings, Washings, or Brushings

Indications

Sample preparation

Staining options

Common mistakes

Body Fluids

Indications

Sample preparation

Staining options

Common mistakes

Bone Marrow

Indications

Sample preparation

Staining options

Common mistakes

BIOCHEMISTRY—ROUTINE

ANALYTE OR TEST NAME (SYNONYMS)

NORMAL

PATIENT PREPARATION

COLLECT

SUBMIT

INTERPRETATION

INTERFERENCE

PROTOCOL

ROUTINE DIAGNOSTIC TESTS

ALANINE AMINOTRANSFERASE (ALT; FORMERLY SGPT)

ALBUMIN

ALBUMIN/GLOBULIN RATIO (A:G)

ALKALINE PHOSPHATASE (SAP OR ALK PHOS)

AMYLASE

BICARBONATE (HCO₃⁻)

POTASSIUM (K⁺)

SODIUM (Na⁺)

AMMONIA (NH₃) (FASTING AMMONIA)

COBALAMIN (VITAMIN B₁₂)