March 2005

Pathology

Judith A. Taylor DVM, DVSc, DACVP
LabVet Consultations Inc.



Harvesting The Hemogram:
Maximize Your Diagnostic Return

Introduction

In practice, the hemogram is an integral part of the minimum data base. It may provide information regarding the etiology, severity, and duration of the disease process (es) affecting the patient. It is also used to monitor response to treatment and/or progression of disease.

There are four basic components to the hemogram:
  1. The erythron
  2. The leukon
  3. The platelets
  4. Plasma protein
Each part should be evaluated individually, and then a collective interpretative summary should be established. The emphasis placed on any individual constituent will be dictated by the individual case. The evaluation of the hemogram should not be done in isolation, but rather interpreted along with the clinical history, physical examination, clinicopathologic data, treatment history, and ancillary tests.

Hematological evaluation of the veterinary patient often requires sequential blood sampling. Consideration should be given to concurrent bone marrow, intravascular and peripheral tissue dynamics as each compartment ultimately will influence the others.

Smear Preparation and Interpretation


Improperly prepared smears impede accurate assessment of the blood film. A small drop of blood should be placed near the end of a clean, dry glass slide. A second slide angled at 30 should be placed just ahead of the drop and quickly backed into the blood. AS THE DROP IS SPREADING along the contact surface, a quick, smooth forward motion with flat contact of the spreader slide results in a perfect feathered edge. The slide should be immediately air-dried by rapid waving to preserve the cells properly.

A well-made blood film has a thin, flat, uninterrupted surface with a translucent sheen. It should cover of the length, and of the width of the slide. The feathered edge is thin, symmetrical, and elliptical in shape, and the depth of the smear should be consistent from tip to the butt.

Initial low-power scan should cover the entire smear and will enable the identification of any hemoparasites (i.e. microfilaria), platelet clumping, atypical cells (usually large and at the feathered edge), and should confirm adequacy of smear quality and staining.

Blood smears should be routinely examined in-house irrespective of whether the CBC is evaluated within the clinic or sent to an outside laboratory. Every clinic using a QBC or other in-house analyzer should be performing smear evaluations on each patient, if only to verify the reported values. Duplicate blood smears should be prepared from every patient and one smear can be sent off, while the other is held for further review, especially if serial sampling is anticipated.

The Erythron


The erythrocytes are the most numerous cells on the blood smear in health. Evaluation of the size, shape, maturation, color, saturation, density, distribution, and inclusions provides insight into the overall health and tissue oxygen delivery of the patient.

Numerical and morphologic red cell indices aid in the assessment of the adequacy of the red cell mass. Interpretation of the red cell count, hemoglobin, hematocrit, polychromasia, reticulocytosis, and indices such as MCV, MCH, MCHC, and RDW will assist in the characterization of the red cell disorder, and may narrow the possible clinical diagnoses under consideration.

MCV or mean corpuscular volume is measured directly by automatic cell counters or can be calculated by (PCV x 10) RBC count = MCV (femtolitres).

Macrocytosis is most often caused by reticulocytosis, but can also be an artifact if agglutination is present or if the blood is old as red cells will imbibe fluid and swell in-vitro. Macrocytosis may also be seen in Poodles (congenital), in Greyhounds (perhaps due to a significantly shorter red cell lifespan), in malabsorption of vitamin B12 in Giant Schnauzers, and in dogs with hereditary stomatocytosis (Alaskan Malamutes, Drentse-Partrijshond, and Miniature Schnauzers).

Microcytosis is most often seen with iron deficiency, portosystemic shunts, and in certain breeds in health (Akita, Shiba Inu, Chow Chow, and Shar Pei). It can also be an artifact of osmotic fluid loss when the ratio of anticoagulant to blood is altered. This results from placing a small amount of blood in large collection tubes.

MCH or mean corpuscular hemoglobin is the amount of hemoglobin per red cell in pg, and is an indication of corpuscular saturation. It can be calculated by Hb concentration RBC count = MCH (pg). Factors should affect both the MCH and MCHC in a similar fashion. If divergent values are noted, it usually indicates an artifact within the hemoglobin, hematocrit or red cell measurements

MCHC or mean corpuscular hemoglobin concentration is the most accurate of the indices as its calculation is independent of the RBC count. It represents the amount of hemoglobin over the red cell mass and is calculated by Hb concentration Hct (L/L). It is usually low in iron deficiency or reticulocytosis. Any increased value is an artifact of hemolysis or interference with the hemoglobin measurement.

Hct (L/L) represents the proportion of blood composed of erythrocytes. It is a calculated parameter using the formula Hct % = (RBC/ l) x MCV (fl).

PCV (%) is derived by the centrifugation of blood and provides similar information as does the Hct. In comparison, the PCV is often slightly higher as cell packing is not as efficient as direct measurement.

Hemoglobin (g/L) is determined by a colorimetric technique in automated cell counters and is the most direct assessment of oxygen carrying capacity. It is approximately one-third of the hematocrit if normocytosis if present. It may be falsely increased by lipemia, heinz bodies, hemolysis, and treatment with oxyhemoglobin.

RDW or red cell distribution width is an index of the degree of variation of the erythrocytes, and is usually increased with reticulocytosis, and in some myeloproliferative diseases (erythremic myelosis in cats).

These parameters must be compared to species, age, and in some cases, breed-specific reference intervals. For example, regenerative anemias are most often due to blood loss or hemolysis. IHA, heinz body hemolytic anemia, infectious causes (Hemobartonella, Babesia), microangiopathies, and neoplasia may be underlying causes. Poorly regenerative anemias often accompany chronic inflammatory, metabolic, endocrine, toxic, some immune hemolytic anemias (aplastic anemia), and myelophthisic diseases.

Disorders of the red cells include anemia, polycythemia, dysmaturation, enzyme abnormalities, and abnormal inclusions.

The Leukon


The leukocytes are the least numerous cell component in health, with a typical ratio of 1:500 to 1:1000 of white cells to red cells in most species. Evaluation of the size, distribution, maturation, inclusions and any atypical cells should be done in each cell line and may provide an indication of underlying disorders such as infectious, inflammatory, immune, neoplastic, and toxic (i.e. drug-induced) myelosuppression.

Neutrophils are carefully evaluated for toxic changes. These morphologic abnormalities represent disruption of cell maturation and give an overall picture of "phagocyte homeostasis". These changes can occur in the bone marrow or peripheral circulation. Specifically, toxic changes include alterations in the cytoplasm (retention of RNA (basophilia), vacuolation, or rarely in domestic animals, disruption of primary granules (aberrant granulation). Dohle bodies are intracytoplasmic aggregates of RER, the significance of which is very species dependent (cats, horses, minor compared to major significance in the dog). Nuclear changes are rarer and include karyolysis or swelling of the nuclear chromatin. Karyopyknosis may be a toxic change or a part of the normal aging process. Abnormal cell size and altered nuclear shape, number and segmentation are other indications of dysmaturation.

Immature neutrophils signaling a "left shift" (note whether it is "regenerative" or "degenerative") are noted and interpreted with the overall leukocyte count and the clinical history of the patient. This is a non-specific change, and while it is seen most commonly with peracute sepsis or endotoxemia, it may also accompany any disorder where there is significant tissue lysis (i.e. immune, toxic, ischemic, neoplastic and degenerative disorders). The degree of toxicity in the immature cells should be noted and compared to the mature cells as this may reflect on the chronicity or resolution of the disease.

A stress leukogram is commonly encountered in practice and is non-specific (neutrophilic leukocytosis with lymphopenia, monocytosis and eosinopenia is the classic scenario, but not all changes are always present, and are somewhat species-dependent, i.e. dog neutrophilia (without left shift and toxicity), cat lymphopenia). Physiologic lymphocytosis is common in young, excited, healthy animals, and counts up to 20 x 109 /L have been observed in healthy cats. Lymphocytes in these cases are usually uniformly mature and well differentiated.
Atypical blast cells in circulation may represent asynchrony of maturation due to intra- or extramarrow disease (i.e. cats with Mycoplasma haemofelis, formerly H. felis, FeLV, or panleukopenia), and may be present in animals with myeloproliferative disorders. Rarely animals with non-hematogenous malignancies will have cancer cells in circulation. Mast cells are infrequently seen in the peripheral blood but may be noted in cases of inflammatory disease (parvovirus infection, acute hemorrhagic pancreatitis, gastric torsion, pericarditis, and peritonitis). This must be kept in mind when evaluating buffy coat smears from patients with a mast cell tumor.

Disorders of the leukocytes include leukopenia, leukocytosis, leukemia, dysmaturation and abnormal inclusions.

The Platelets


Platelets are the second most numerous cell in health, with most domestic species having counts in the 100 to 1000 x 109/L range. The role of platelets in the coagulation process is intuitive, but there is an increasing interest and awareness of their role in the inflammatory process as they are "microtransporters" of many inflammatory cytokines such as platelet derived growth factor and transforming growth factor. Evaluation of the size, distribution, maturation, and any inclusions (i.e. Ehrlichia platys) may provide an insight into underlying immune, infectious, neoplastic and thrombopathic disease.Parameters such as PCT (platelet crit) and PDW (platelet distribution width) are interpreted similar to the equivalent measures for erythrocytes. An idiopathic asymptomatic macrothrombocytopenia in Cavalier King Charles Spaniels has been determined to have an autosomal recessive pattern of inheritance. A microthrombocytosis is often seen with some anemias ( possibly due to colony stimulating factors released in the marrow) and also in hypothyroid dogs. The pathogenesis of the latter mechanism is poorly understood.

Disorders of platelets include thrombocytopenia, thrombocytosis, leukemia, dysmaturation and abnormal function or inclusions.

Plasma Proteins


Serum proteins include albumin (which accounts for 75% of the oncotic pressure of the blood), alpha and beta globulins (most of which are synthesized by the liver), and the immunoglobulins, which are secreted by B lymphocytes and plasma cells.

Evaluation of proteins may be through direct measurement in the plasma or serum, or by breakdown into the respective components via serum protein electrophoresis and immunoelectrophoresis. Evaluation of the plasma proteins is critical in anemic patients when distinguishing between hemolysis and hemorrhage, or in monitoring the patient with ongoing hemorrhage. Hyperproteinemia may be relative as in dehydration, or may be absolute as with inflammation or neoplasia, especially lymphoproliferative disease or multiple myeloma. Hypoproteinemia may be seen with hemorrhage, protein loss through nephropathy, enteropathy, diffuse dermatopathy, third space effusions or rarely through decreased synthesis as in end stage liver disease. Maldigestion, malabsorption, starvation or cachexia of neoplasia may result in lower serum proteins. Estimation of the albumin, globulins, and the A/G ratio may guide further investigation. For example, selective albumin loss suggests early glomerular leakage of the lower molecular weight protein (albumin has a molecular weight of about 69,000 D compared with 150,000 D and higher for globulins). Panhypoproteinemia is more consistent with gastrointestinal losses or hemorrhage. Inflammatory intestinal disease may present with an apparent selective albumin loss as the increased production of acute phase reactant proteins may mask ongoing globulin losses.



Cytology and Telecytology:
Practical Tips and Pitfalls To Avoid

Introduction: The Basic Rules

Cytology is a powerful, inexpensive and relatively non-invasive diagnostic tool. The utility of this procedure lies in answering the following questions: "What is the lesion and how do I treat it?" In order to maximize the benefit derived from fine needle aspiration (FNA), two things must be inherent to the technique:
  1. Access to the lesion to harvest a sufficient number of intact, representative cells.
  2. Knowledge to recognize and evaluate the cells independent of the architecture of the lesion, and put it into the context of the overall disease process(es) affecting the patient.
There are certain principles, which if adhered to, will ensure a satisfactory diagnostic yield to assist the practitioner. In my opinion, adherence to the following three guidelines will maximize the return on in-house procedures:
  1. Know how and when to use, and not to use cytology.
  2. Stain and preview 1 or 2 slides after smears are prepared.
  3. Use a simple algorithm each and every time you look at a specimen.
After following these guidelines, it may be decided to send the sample to a pathologist for assistance in interpretation. However, by following these steps, it will ensure the harvest of a representative, adequate specimen upon which a conclusive diagnosis may be rendered.

How to Use It


Cytology can be used on any mass that can be seen or touched, immobilized, brought to the surface and aspirated. Specimens may be obtained from solid lesions that are superficial, or from diffuse or focal lesions within body cavities or individual organs, providing they are accessible and can be immobilized. Diagnostic imaging improves the accuracy of yield from internal lesions, usually with less effort, risk and expense relative to surgical biopsies. Cytology is also suitable for the diagnosis of lavage and body cavity fluids.

Cytological detail may be superior to histopathology in some instances. For example, lymph node or bone marrow evaluations in patients with lymphoma, and liver aspirates from cats with hepatic lipidosis may provide superb detail for morphologic classification and clinical staging. Aspirates may be more desirable due to the ease of technique, minimal risk to the patient, and the high sensitivity of a diagnostic sample, especially in diffuse lesions.

Cytological specimens may be obtained by fine needle aspiration (FNA), impression smears, scrapings or swabs. FNA is the most commonly used technique. Lesions that do not allow for the accurate placement of the needle and withdrawal of a representative sample may be better sampled by impression smears, scrapings, swabs, or excisional biopsies.

Fine needle aspiration (FNA):


For routine aspirations of superficial cutaneous lesions, surgical preparation of the overlying skin is optional. For aspirations obtained from deeper structures, or those requiring entry through body cavities, the skin should be clipped and aseptically prepared. The lesion should be demarcated and immobilized. A 22 gauge needle attached to a 12 cc syringe is inserted into the area of interest. A pistol grip aspiration gun facilitates smooth, one-handed aspiration with minimal assistance, but is not necessary. Once the needle is correctly placed within the lesion, the barrel of the syringe is withdrawn to about the 8-10 cc mark, and this negative pressure is held steady while the tip of the needle is gently redirected a few millimeters around the point of entry, taking care to remain inside the mass. Pumping of the syringe must be avoided as this may increase the likelihood of iatrogenic hemorrhage and tissue trauma at the site of aspiration. Material should be aspirated only as far as the hub of the needle; more material usually denotes contamination, especially if it is blood tinged. The negative pressure is released first, and then the needle is withdrawn from the tissue.

The syringe is separated from the needle, filled with air and reattached. Small drops of material are gently expelled onto several clean, dry glass slides (an average aspirate will usually yield 3-6 slides). The material is spread thinly using the spreader technique as for a blood smear (crush techniques should be reserved for select tissues such as bone marrow). The advancing edge of the spreader slide should not go beyond the edge of the bottom slide. This produces a "feathered edge" effect as seen in a blood smear, where larger, possibly diagnostic cells may be preserved. Slides must be waved rapidly by hand (rather than placed on the bench top or in front of a fan) to avoid slow drying and pyknosis of cells which obscures nuclear and cytoplasmic details. Air drying is adequate fixation for most tissues, although wet-fixed slides will allow for specialized stains such as Papanicolaou's which may be required in isolated cases of epithelial neoplasia where improved nuclear detail is required. Special cytofixative sprays are available, but must be applied immediately after slides are prepared. Any air-drying of the sample will compromise the quality of the wet-fixed smear.

If blood, fat, or fluid, is aspirated and starts to fill the barrel of the syringe, smears may be made, or the material may be saved in an EDTA tube if deemed significant (for example from a cyst). This fluid may be processed further (for culture and sensitivity, biochemistry, or concentrated for cytologic examination). If peripheral blood contamination is suspected, the needle should be withdrawn, replaced with a clean one, and inserted in a different site to try to avoid iatrogenic hemorrhage. In very vascular structures, (i.e. thyroid, hemangiopericytomas), a smaller gauge needle (23-25 gauge) may be substituted.

Impression Smears


Impression smears may be made from surface lesions where size or shape precludes adequate placement of a needle for aspiration, from poorly exfoliating lesions, or from cut surfaces of excision biopsies. In the latter case, cytology has the advantage of providing a better turnaround time while a histologic sample is pending. Any superfluous exudate or debris is removed with a saline-moistened sponge, and the exposed surface is blotted dry. For biopsies, a scalpel should be used to provide a fresh cut surface which is then blotted dry. A clean dry slide is laid onto the lesion so that contact results with minimal pressure. This avoids negative suction and smearing of the tissue which cause cell disruption. Several thin imprints can be made on a slide which is then air-dried. Wet-fixation may be used, but imprints must be made and fixed quickly. If the surface of the tissue has been adequately prepared, the sample should dry quickly with a minimum of waving. Slides may be stained in the usual manner.

Scrapings & Swabs


Scrapings are indicated for the same reasons as impression smears. The surface of the lesion or biopsy is prepared similarly, and a clean scalpel blade is used to gently scrape the exposed site. The material is spread thinly and evenly across a clean glass slide which may be air-dried or wet-fixed as above.

Swabs may be required to evaluate invaginated, or deep surfaces (for example, vaginal or conjunctival cytology). The tip should be moistened with saline and applied to the surface of interest. The swab is then gently rolled across the surface of the slide which is fixed and stained appropriately. Dragging or simply wiping the swab across the slide will result in rupture artifact of cells.

Cytology of Effusions, Lavages and Cavity Fluids.


The equipment required for fluid cytology will depend upon the collection procedure and specimen of interest. In general, glass slides, routine stains, various sizes of syringes (3 cc for synovial fluid, to 35 cc with stopcock for some chest and abdominal effusions), and needles (most frequently used is 22 gauge) along with appropriate vacutainer tubes (usually red top and lavender), cell counting apparatus (ie.hemocytometer), refractometer, and centrifuge (low speed, 1500 RPM) are the basic essentials. Over-the-needle catheters, endoscopes, sterile physiologic saline, pH paper or meter, sedimenting chambers (for CSF), and microbiology may also be useful. Specialized centrifuges such as the cytocentrifuge (Cytospin, Shandon-Southern Products, Cheshire, England), provide for superior cell preservation and may be available in large referral hospitals or diagnostic laboratories.

Fluids should be collected into EDTA to prevent clotting, which will invalidate cell counts (a clean CSF or synovial fluid collection may be the exception to this rule). Serum tubes (red-top) should be used if microbiology is required (EDTA is bacteriostatic).

Most samples of fluids contain few cells and some concentration technique is required to make smears which are diagnostic. In most cases, this involves concentrating the sample by low speed centrifugation (1500 rpm for 5-10 minutes) and making smears of the resuspended sediment. If the fluid is very cellular (usually counts greater than 15 x 10 9/L), direct smears of fluid are prepared using a spreader technique, again taking care to produce a "feathered edge". For centrifuged samples, the supernatant can be removed and either saved for further biochemical analysis, or discarded. Once the supernatant is removed, it is important to gently mix the sediment by flicking the bottom of the centrifuge tube. A pasteur pipette should not be used, as suction of the specimen results in disruption of cells. Fluid smears are air-dried quickly, labeled as to site (this is especially important where multiple sites are sampled, i.e. multiple joints, left and right thoracocentesis, etc.), patient ID, and whether they are direct or sedimented. They can then be examined or submitted to a diagnostic laboratory. If slides are sent out, any excess, unspun fluid should be submitted at the same time for further evaluation if warranted.

Alternative concentration techniques include gravity sedimentation for CSF and lavage fluids, use of a cytospin centrifuge (excellent cell morphology, but cost prohibitive for most practices), or membrane filtration techniques. If a centrifuge is not available, direct smears should be made immediately after collection. Cell morphology will be better preserved for direct smears than from slides prepared from a specimen concentrated several hours, or days, post-collection. Fluids should be processed as soon as possible after collection (preferably within one hour or less). Cell degeneration and lysis, in-vitro erythrophagocytosis and bacterial overgrowth can all be noted as artifactual changes in fluids left for prolonged periods of time. This is especially true with fluids left at room temperature, or exposed to extreme temperature fluctuations during shipment. In- vitro phagocytosis is inhibited by anticoagulants such as EDTA. CSF slides must be prepared within one hour of collection to avoid cell degeneration.

Synovial fluids usually have increased viscosity and present some difficulty in the preparation of a monolayer that is readily air-dried without cell shrinkage. Usually, an assessment of joint fluid will require direct smears (and if enough sample, cell counts and protein). Mucin clot test is done to allow a qualitative evaluation of the hyaluronic acid of the glycosaminoglycan protein. Care must be taken to dry the thin smears quickly to allow for accurate identification of cell types such as segmented neutrophils which, when pyknotic, can be difficult to distinguish from the mononuclear cells. If intra-articular neoplasia is suspected, concentration techniques may be helpful.

Urine cytology warrants special mention. The two most common diagnoses are urinary tract inflammation (UTI), and neoplasia, and these may be very difficult to substantiate if the specimen is not handled correctly. Routine wet mounts should be examined in-house, preferably soon after specimen collection. If there is a concern, unstained, air-dried smears should be made of the sediment, AT THE SAME TIME, for the best preservation of cell morphology. Formalin should not be added to the specimen as this interferes with cell morphology, and may invalidate some of the chemical tests. Wet mounts should not be submitted to external labs unless the coverslips are permanently mounted to prevent drying of the sample. Any unspun, unfixed urine, taken at the same time, should be submitted with the slides, if they are sent out.

Cell counts can be estimated by examination of direct smears of most fluids. Ten oil fields are counted, an average number of cells/field is obtained, and multiplied by 10 to give a rough indication of the number of cells x 10 9/L. CSF and synovial fluid cell counts can be done using a hemocytometer. The CSF should be loaded into the hemocytometer chamber undiluted. Both sides of the chamber are counted, and an average number of cells is obtained and multiplied by 10/9 to give the number of cells/cu. mm. This is then divided by 1000, and equals the number of cells x 10 9/L within the specimen. Synovial fluid should be diluted in normal saline, (do not use the unopette for WBCs), and then counted and calculated as above, taking the dilution factor into account.

Protein estimation of fluids may be done using a refractometer (read directly off the total protein scale, or may have to use specific gravity and conversion table to obtain in g/L, depending on the model). For fluids with very low protein such as CSF, urine dipsticks may be used to estimate an increase in protein. These observations should be confirmed using precipitation or microprotein methods which are more accurate at the lower ranges (<1 g/L).

Bone marrow aspiration and biopsy.


The indications for bone marrow biopsy include the following:
  • any unexplained cytopenias or increased peripheral cell counts
  • any unexplained, non-regenerative anemia
  • diagnosis of hematopoietic neoplasia or tumors metastatic to the marrow
  • clinical staging of certain diseases
  • assessment of adequacy of a therapeutic protocol
  • evaluation of certain immunofluorescent techniques (FeLV, anti-megakaryocyte antibodies)
  • assessment of erythropoiesis in the horse
Aspiration and core biopsies may be performed using local anesthesia over the proposed site. Bone marrow aspiration may be sufficient, but a core biopsy should be taken at the same time if an adequate cellular specimen cannot be obtained due to technique, in suspected cases of marrow architectural abnormalities (ie. myelofibrosis) or for diseases with focal hematopoietic involvement (neoplasia such as lymphoma, mast cell tumor). In small animals, the dorsal wing of the ilium, ischium in obese patients, neck of the femur or proximal humerus are appropriate sites. In young ruminants and horses, the rib or dorsal crest of the ilium are preferred, while in mature large animals, the sternum is the site of choice.

The skin over the proposed site should be clipped and surgically prepared. A skin bleb of local anesthetic is injected and then the needle is advanced to the surface of the bone which is infiltrated ahead and behind the aspiration site. A Rosenthal needle (Dynamedical, London, ON) may be used for aspiration (14-16-gauge, 1.5 inch needle in large dogs, and a 20-22-gauge, 0.5-1.0 inch needle in cats). Presterilized EDTA preserves cellular detail better than heparin and will avoid clotting in samples that are aspirated slowly. The inner surface of the barrel of a 12 cc syringe should be coated with the anticoagulant, and about 0.2 cc of fluid should be left in the syringe to be mixed with the marrow sample. The skin may be directly penetrated by the bone marrow needle with the stylet in place, or a small stab incision with a scalpel blade may be made. The needle is advanced to the bone surface where it is seated and then penetration of the cortex may be completed by manual pressure and rotation, or with the aid of a small hammer. There is usually a sense of "popping" through the inner cortex with a slight decrease in resistance. At this point the stylet is removed and the syringe is attached. The marrow must be aspirated vigorously. Usually after several pumps against some resistance, the thick, bloody marrow with fat globules will be observed entering the barrel of the syringe. Only a small amount of marrow (0.5-1.0 ml) should be aspirated. The syringe may be removed from the needle, the stylet replaced and the needle may be left seated in the bone while smears are made. The fluid should be flooded onto 4-6 glass slides which are then tipped up on their side and placed on an absorbent surface to soak up the excess blood. Tiny pinpoint bone spicules should be evident on the surface of the angled slide. In some species, platelet clumps and fat may mimick the granules. The bone marrow is transferred to other slides by using a combination of spread and crush techniques in order to adequately spread the spicules. If a definite grittiness is felt when smears are made, bone marrow granules have likely been obtained. The needle may then be removed, and if necessary, a skin suture placed.

If a core biopsy is obtained at the same time, a single needle (Jamshidi, Trudell Medical, London, ON) may be used for both aspiration and core samples. The aspiration procedure is carried out as outlined above. Once cytologic specimens have been prepared, the needle may be redirected ahead or behind the aspiration site, (this avoids an acellular sample due to previous aspiration of site). It is advanced through the cortex, and then the stylet is removed. The needle is advanced downwards with a manual clockwise rotation to a depth equal to the length of the needle. The needle is then retracted while redirecting the tip to cut the base of the core. The needle is removed by counterclockwise rotation and retraction.The stylet is gently inserted into the beveled end of the needle to remove the biopsy, which is then placed into 10% formalin. Impression smears of the core may be made just before it is fixed. The smears should be stained using routine stains but due to thickness of the samples, the length of time of staining must be modified (usually takes twice as long, at least). Assessment of bone marrow biopsies is best done by an experienced cyto/histopathologist, and a current blood film and history should accompany all specimens for appropriate interpretation. Smears for special staining (i.e. reticulocyte stains in horses, immunofluorescence, etc.) should be clearly marked.

Telecytology


Telecytology is the electronic submission of digital images of cytology specimens. It accelerates the diagnostic procedure by avoiding the submission and handling of glass slides. It is particularly suited to facilitating intra-operative diagnosis and decision-making.

Requirements


A digital still camera with good resolution (3 Mpixels or higher), preferably mounted , and a good quality, well-maintained microscope are essential to producing images of optimal diagnostic quality. Internet access, a mid- to high-end computer with the most RAM and the fastest CPU you can afford, a good quality monitor and imaging software are required to complete the process. Finally, an essential requirement is a trained cytopathologist with experience and interest in digital imaging.

As with glass slides, the quality of the diagnosis is dependent upon the quality of the material submitted. It is essential to scan all smears and capture representative, well-focused and bright images since there is no opportunity for the pathologist to go back and review alternative fields in the smears. Ideal images are derived from monolayer areas containing well-illuminated, well-stained, intact cells that are representative of the lesion, and that are found within a minimum of background contamination. This requires more images in the case of a mixed lesion. Low-power scans of representative fields should be included to illustrate overall cellularity and cell arrangement. High-dry and oil fields are most critical for nuclear and cytoplasmic details, and to define etiologic agents. It is important to include a neutrophil or red cell in the background for size reference. In general, submission of 1-3 images at low power (10X), 5-10 at intermediate and high dry (20X and 40X), and 3-5 images under oil (100X) are recommended.



Harvesting The Gold:
Interpretation and Techniques of Urinalysis

Introduction

A complete urinalysis should be included as an integral part of the minimum data base of any veterinary patient presented for laboratory evaluation. This includes wellness testing, preoperative evaluations of healthy patients, and any patient with well-defined or undetermined illness. Detection of abnormal findings in any urine sample may direct further evaluation of the patient, as the results often pinpoint the body system or organ affected, and may specify further necessary diagnostic or clinical procedures.

All too often, urinalysis is overlooked as a routine part of a minimum data base. In one study of 2,000 routine canine and feline urine evaluations, results indicated that failure to examine urine sediment of macroscopically normal samples would have missed abnormalities in 16. 5% of canine patients (pyruia and bacteriuria), and 5.7% of feline patients (hematuria and bacteriuria). The underlying reasons for failure to include routine urinalysis usually include inappropriate resource allocation (time, training, labor-intensive, tools), and a technical aversion (mostly practitioners!).

Technique


The most important rule is to STANDARDIZE your procedure. Urine should be evaluated within 60 minutes of collection to minimize temperature- and time-dependent in-vitro changes. If the urine is preserved by refrigeration, it should be warmed to room temperature before analysis. Note that a recent study found that increased storage time and decreased temperature were associated with a significant increase in the number of CaOx crystals found within specimens. This study concluded that refrigeration may enhance in-vitro crystal formation.

Read all manufacturers recommendations for storage, handling and use of test strips. Never touch the reagent pads with your fingers.
  1. Identify the method of collection and record the time of collection and of analysis along with the signalment of patient. Urine obtained by cystocentesis normally is aseptic until it reaches the mid-urethra.

  2. Macroscopic (physical & chemical) and microscopic evaluations should be done on all samples.

  3. Record physical findings: volume, color, odor, viscosity, turbidity, debris.

  4. Chemical findings should include pH, protein, glucose, ketones, blood and bilirubin. The most accurate results are obtained on fresh, well mixed, uncentrifuged urine. Dip the stick thoroughly and briefly into the urine, remove excess fluid to avoid "wicking", and hold the stick horizontally, reading tests at the appropriate recommended times.

  5. Urine sediment should be obtained by centrifugation of 5 ml of urine for 5 minutes at 1,500 rpm (or RCF of 400g). Duration of centrifugation may vary between hospitals depending on equipment used. A range of 4-10 minutes is recommended. However, the most important factor is consistency. Supernatant should be decanted by tipping the tube (preferably save it to repeat any suspicious test results such as positive protein). Resuspend sediment in 1-2 retained drops of urine by holding the tube at the rim and flicking the distal end several times with your finger. DO NOT PIPETTE TO MIX.

  6. Make 2 air dried smears of the sediment and set aside. These may be submitted to a cytopathologist if atypical or neoplastic cells are suspected on wet-mount examination.

  7. Transfer a large drop of unstained sediment to a clean glass slide and "float" a coverslip over the drop.

  8. If preferred, add 1-2 drops of sedistain to the remaining sediment and mix by flicking. Transfer a drop of stained sediment to the slide and apply a coverslip in a similar fashion.

  9. Evaluate the wet mounts at low power (x100, LPF), and high-dry (400x, HPF) magnifications. Reduce the light intensity on the microscope by lowering the condenser and close the iris diaphragm.

    Examine the urine for the 5 "C"s: cells, crystals, casts, creatures and crud. The latter is a rather "un-scientific" term, but is appropriately descriptive of many samples with "dirty" sediment.

    Scan the entire specimen with the low power objective and record the average number of casts per LPF (you may want to count the number observed in 10 fields). Leukocytes, erythrocytes, and epithelial cells are reported per HPF. Crystals and creatures should be reported as present, and semi-quantitated.

What is Abnormal?


Macroscopic: Physical


Volume Amount of urine produced may be important in the interpretive process. In dogs and cats normal urine production is approximately 20-40 ml/kg/day. Less or more than this daily volume may represent oliguria or polyuria, respectively. This must be interpreted with regards to many environmental and physiologic factors such as body weight, size, diet, temperature, humidity, exercise, health and current therapeutics.

Color Any color other than clear or yellow. Urochromes and urobilin impart the yellow or amber color which is influenced by the volume and concentration of urine. May be altered by current diet, therapy, or metabolic disease. It is a crude index of the degree of urine concentration and dilution.

Turbidity Any degree of turbidity. Note species differences (i.e. rabbit, horse, birds).

pH <5.0 or > 7.5. Note carnivorous diets may result in pH as low as 4.5, while vegetarian diets will be associated with alkaline urine. Urine pH is not necessarily a good indicator of blood pH or renal function as it is affected by factors such as diet, diurnal variation, and bacterial contamination. Urine pH is stable in sterile containers stored at room temperature for several hours.

Specific Gravity Urine specific gravity and osmolality are indices of renal function, namely the ability of the kidneys to respond to the ionic balance of the body and conserve or excrete water appropriately. SG is the ratio of the weight of urine to the weight of water and is based on the number, weight and size of dissolved solids. It provides an estimate of the osmolality of urine but is easier to measure. Most commonly in practice it is measured by refractive index, that is the transmission of light through the urine. A higher solute load alters (bends) the transmission of light, resulting in a higher reading. Osmolality is dependent only on the number of dissolved solutes in urine. The osmolality of the urine can be estimated from the SG by multiplying the last two digits of the reading by 36 (i.e. urine with a specific gravity of 1.012 has an osmolality of roughly 432 mOsm per kg (12 x 36=432)). Osmolality of the urine is interpreted similarly to SG, that is it is an index of renal function relative to the water balance and concentration of plasma.

1.007-1.0030 in a dog, and 1.007-1.0035 in a cat may be normal under conditions of normovolemia. Maximal specific gravity in healthy dogs ranges from 1.050-1.076, and in healthy cats it can reach 1.080. Glucosuria may increase urine SG at a rate of 0.004/g/dl, while proteinuria will increase SG 0.003/g/dl.

Isosthenuria means that the osmolality of the urine is equal to that of plasma and includes readings of 1.007-1.012. Essentially the kidneys are filtering and excreting the urine in an unaltered state. This must be interpreted in light of the animals water balance and metabolic health. Hyposthenuria indicates urine with an osmolality less than that of plasma. (note: beware of owners collecting urine in the snow!). Abnormalities in concentrating ability arise when two-thirds of the filtering capacity of the kidneys is damaged. Note however, in the cat, and in the early stages of primary glomerular disease in any species, increases in urea and creatinine (azotemia) may precede concentration abnormalities

Reagent strips detecting specific gravity have not been adequately validated in veterinary medicine, so refractometry should be relied on. It is important to periodically calibrate the instrument by obtaining a zero reading for distilled water.

Macroscopic: Chemical


Protein Positive results are usually abnormal, but must be interpreted in light of specific gravity and species. Positive samples should be repeated on supernatant after centrifugation to eliminate false positives from blood cells and casts.

Reagent strips are detecting albumin (smaller MW of 65,000) and are not reliable for globulins or Bence Jones paraproteins. An alternative test is the sulfosalicylic acid test whereby equal amounts of 5% sulfosalicylic acid and the supernatant are mixed and the level of turbidity is determined against a dark background.

Proteinuria supports inflammation, hemorrhage, glomerular leakage, or decreased proximal tubular reabsorption. The degree of proteinuria may help to distinguish the source. Mild proteinuria may accompany some urinary tract infections or endocrine disorders such as hypercortisolemia (Cushings). Fever, exercise, orthostatic, (humans), and congestive heart failure may result in mild proteinuria. Trace protein in very concentrated urine from cats and dogs may not be significant. Tubular disease causes mild to moderate proteinuria, while primary glomerular dysfunction often results in profound proteinuria.

Clinically normal mice and rats routinely have proteinuria. Neonatal animals may have proteinuria derived from colostral proteins. False positive results may be obtained in alkaline urine due to cross contamination of buffer in the dipstick pad, or contamination of the sample with quaternary ammonium compounds (disinfectants).

Glucose Positive results reflect hyperglycemia which exceeds the renal threshold (reabsorption capacity) of the proximal renal tubules. This is species dependent, and may vary with the state of health (dog: 10 mmol/L, cat 16 mmol/L, and cattle 6 mmol/L).
Occasionally glucosuria may occur in the absence of hyperglycemia as a result of decreased tubular resorption such as in cases of canine Fanconi-like syndrome and leptospirosis.

Stress may rarely incite transient glucosuria in cats, and in horses, xylazine may result in detectable glucosuria. The reagent strips are enzyme-dependent (glucose oxidase) and the urine must be brought to room temperature before testing. Marked ketonuria, ascorbic acid, and very concentrated urine may cause false negatives. The most common cause of false negative readings is outdated test strips. Oxidizing cleaning agents (hydrochlorite, chlorine), and contamination with hydrogen peroxide may cause false positive results with Chemstrip . Pseudoglucose has been noted in the urine of cats with urethral obstruction, and is of undetermined origin.

Ketones Positive results indicative of altered lipid metabolism in a catabolic state. Most commonly in small animals ketonuria accompanies hyperglycemia and glucosuria and is indicative of diabetes mellitus with ketoacidotic state (a critical emergency). Starvation, especially in immature animals, may result in detectable ketones in the urine. Rarely false positive results are obtained in highly pigmented samples, and with certain drug therapies (tricyclic ring compounds, drugs with free sulfhydryl groups such as captopril). In some species like the rat, low levels are normal. Decreased sensitivity of ketones may occur in the presence of bacterial contamination.

Blood Any blood is abnormal. Detection of blood does not allow for identification of source (i.e. upper or lower urinary tract). Hematuria must be interpreted in light of collection technique as catheterization or cystocentesis may produce traumatic, not pathologic hemorrhage. The reagent strips are more sensitive to free hemoglobin than intact erythrocytes, and decreased sensitivity may occur in acid urines, and in urine preserved with formalin. Although the peroxidase test strip is much more sensitive for hemoglobin than are the urine protein tests, a strong positive for blood usually results in some positive protein detection as well.

Bilirubin Positive results are usually abnormal, and indicative of conjugated (water soluble) bilirubin. However, in concentrated urine, especially from male dogs (but also from females), trace to 2+ may be normal because of a low renal threshold or tubular conjugation as renal tubular cells contain glucuronyl transferase. Bilirubinuria is a common finding in healthy ferrets. Any bilirubinuria in cats is abnormal and should not be ignored. Bilirubinuria may precede bilirubinemia in early disease. Note that bilirubin is light-sensitive and false negative results may be obtained in urine kept at room temperature in transparent containers.

Microscopic


Blood >5 RBC/hpf. Microscopic detection of intact red cells will assist in the differentiation between hematuria versus hemolysis or myoglobinuria.

Leukocytes > 5 WBC/hpf. It is impossible to identify the level of the source of inflammation based on urine sediment alone. Other abnormalities such as excessive transitional or renal epithelial cells, casts may help to narrow down the possibilities.

Casts >2 hyaline casts, >1 granular cast, or > 1 waxy cast /lpf. Casts may disintegrate in dilute or alkaline urine. Accurate identification and quantitation of casts is essential as they imply renal tubular damage or stasis in an active disease state. They are not a reliable index of the severity of renal lesions, but may be one of the earliest indicators of tubular disease.

Creatures Bacteria, fungi, or parasites. Again, must be interpreted with the method of collection and possibility of contamination, and should be assessed in light of other findings, i.e. hemorrhage, inflammatory cells, casts etc. Parasitic eggs of Capillaria plica, Stephanurus dentatus or Dioctophyma renale are rarely encountered. Microfilaria of Dirofilaria immitis may occur in urine. Environmental contaminants of flea eggs, spores or mites are rarely retrieved by owners upon collection.

Cells Hyperplastic, metaplastic, or neoplastic epithelial cells. Usually very few squamous, transitional and renal tubular cells are encountered in normal urine. This will depend upon the method of collection. Because of the in vivo effects of various bladder disorders on cell morphology, urine cytology often requires the assistance of a pathologist.

Crystals Because of the in vitro effects of time and temperature, there is poor correlation between crystalluria and urolithiasis. Therefore, crystals may be normal or abnormal. They reflect saturation of urine with crystalloid material. If in doubt as to whether the crystals formed in vitro or in vivo, repeat analysis of freshly collected urine. Any cystine, tyrosine, leucine, urate, calcium oxalate monohydrate or unidentifiable crystals are abnormal. Amorphous crystals are difficult to identify accurately based on visual examination alone. If dissolution occurs with the addition of 1 drop of solution of 6.25M sodium hydroxide, but not acetic acid, they are likely amorphous urates. If dissolved by the addition of 1 drop of 10% acetic acid, crystals are likely amorphous phosphate.

Debris includes mucus, fat, sperm. Most of the time these compounds are not of pathologic significance. Fat is often found in urine from clinically healthy cats due to the high lipid content of feline renal epithelial cells.

Urine Cases


Case 1

8 year old Siberian Husky with a 2 day history of vomiting, diarrhea, depression, abdominal pain, irregular tachycardia and oliguria

Urinalysis:

Free flow, Clear, yellow  
Specific Gravity 1.008
pH 6.5
Protein 0.3g/L
Glucose +
Ketones -
Bile +
Blood 4+
Leukocytes 0-3/hpf
Erythrocytes TNTC
Epith transitional 0-3/hpf
Crystals 2+/hpf
Casts fine granular 4/lpf


Case 2


5 year old Golden Retriever F/S with history of pollakiuria, hematuria, dysuria.

Urinalysis:

Free flow, Cloudy red  
Specific Gravity 1.027
pH 7.5
Protein 0.3 g/L
Glucose -
Ketones -
Bile -
Blood 4+
Leukocytes 20-40/hpf
Erythrocytes TNTC/hpf
Bacteria 3+ gram negative rods
Culture E. coli heavy growth


Case 3


6 year old M Rottweiler with history of hematuria, stranguria.

Urinalysis:

Free flow, red-tinged  
Specific Gravity 1.033
pH 6.0
Protein 0.3 g/L
Glucose -
Ketones -
Bile -
Blood 4+
Leukocytes 2-4/hpf
Erythrocytes TNTC/hpf


Case 4


7 year old M WHWT presented with dyspnea, weakness, pyrexia

Urinalysis:

Free flow sample, Clear, yellow  
Specific Gravity 1.018
pH 6
Protein 0.1 g/L
Glucose -
Ketones -
Bile -
Blood -
Leukocytes 0/hpf
Erythrocytes 0/hpf
Casts 1-2/hpf fine granular
Sperm 4+
Epithelial cells 2/hpf


Case 5


Dalmatian, 11 year old FS presented with urine dribbling, increased frequency of urination.

Urinalysis:

Free flow, light yellow, Clear  
Specific Gravity 1.035
pH 6.5
Protein -
Glucose 55 mmol/L
Ketones -
Bile -
Blood -
Leukocytes -


Case 6


Mixed breed canine, history of PU/PD and polyphagia.

Urinalysis:

Catheterized Sample, Clear, yellow  
Specific Gravity 1.006
pH 6.5
Protein 0.1 g/L
Glucose -
Ketones -
Bile -
Blood 2+
Leukocytes 10-20/hpf
Erythrocytes 25-30/hpf
Bact rare rods/hpf
Culture E coli moderate growth


Case 7


Feline, 8 year old acute onset anorexia, vomiting and neurologic signs.

Urinalysis:

Clear, pale yellow  
Specific Gravity 1.014
pH 6.0
Protein 1.0 g/L
Glucose trace
Ketones -
Bile -
Blood 2+
Leukocytes 20-25/hpf
Erythrocytes 20/hpf
Casts 2+ fine granular casts
Crystals 3+


Case 8


5 year old F/S GSHP with history of vomition, weight loss, and frank blood in stool.

Urinalysis:

Clear, pale yellow  
Specific Gravity 1.017
pH 7.0
Protein -
Glucose -
Ketones -
Bile -
Blood -
Leukocytes 3/hpf
Erythrocytes occ/hpf
Crystals -


Case 9


9 year old F Canine with history of PU/PD, vomiting.

Urea 85 mmol/L 3.0-8.0
Creat 565 umol/L 30-140


Urinalysis:

Specific Gravity 1.013
pH 6.5
SSA prot >1.0 g/L
Protein 10 g/L
Urine protein: creatinine 25.6


References


  1. Albasan H, Lulich JP, Osborne CA, Lekcharoensuk C, Ulrich LK, Carpenter KA. Effects of storage time and temperature on pH, specific gravity, and crystal formation in urine samples from dogs and cats. J Am Vet Med Assoc 2003; 222:176-179.

  2. Barlough JE, Osborne CA, Stevens JB. Canine and feline urinalysis: value of
    macroscopic and microscopic examinations. J Am Vet Med Assoc 1981; 178:61-63.

  3. Brobst D. Urinalysis and Associated Laboratory Procedures. Vet Clin North Am
    Small Anim Pract 19: 929-949, 1989.

  4. Duncan RJ, Prasse KW, Mahaffey EA, eds. Urinary System In: Veterinary Laboratory Medicine Clinical Pathology ed 2. Iowa State University Press, Ames. 1994; 162-183.

  5. Osborne CA, Stevens JB. Urine sediment: under the microscope In: Osborne CA, Stevens JB, eds. Urinalysis: guide to compassionate patient care. Shawnee Mission, Kan: Bayer Co. 1999; 125-150.

  6. Osborne CA, Stevens JB. "Prophet"-ing more from urinalysis:maximizing reproducible test results. CA, Stevens JB, eds. Urinalysis: guide to compassionate patient care. Shawnee Mission, Kan: Bayer Co. 1999; 51-63.



Update on the Use of PCR in Veterinary Practice


The availability and use of molecular biology tools such as the polymerase chain reaction (PCR) has been expanding rapidly in veterinary medicine. These assays, originally developed for use in research, have been accepted into the mainstream of diagnostic tools available for the diagnosis and investigation of many infectious, toxic, neoplastic and genetic diseases.

The basis of PCR is the detection of minute quantities of nucleic acids which would otherwise be undetectable in cells, tissues and fluids with conventional investigative methods. Specific segments of DNA unique to a particular species or even to strains of organisms have been identified and are targeted in the PCR. The precise nature of the isolated DNA can be determined by looking at the "code" or sequence of the individual base pairs and comparing it with a known sequence of the target organism of interest. The analogy has been made that PCR is like looking for a needle in a haystack, and then making a haystack of that needle.

The PCR process is brilliant in its simplicity, and extreme sensitivity. The double stranded DNA is separated by heating into its respective single strands. A "primer" or a synthetic sequence of base pairs that is complementary to small sections of the DNA of interest acts as an initial template for elongation of the new DNA molecule. These are inserted along with a polymerase enzyme "Taq" which drives the polymerization or elongation process after initial binding to the primers when the mixture is cooled. The resulting new single strands of DNA are joined together in the classic double helix configuration, expanding the original genetic material 2-fold each cycle. An analogy would be to take a single zipper, undo it, replicate the corresponding halves of each side and then do the zippers back up resulting in a pair from one original. This would represent one PCR cycle. With PCR, each cycle of heating, polymerization and cooling is repeated so that at the end of 20-40 cycles run on the original target DNA, up to 30 million or more copies (or "amplicons") may be produced.

In order to isolate the genetic material of interest, this amplified material is fragmented by the enzymatic digestion of the DNA at specific points, resulting in strips of various sizes. The digesting enzymes are known as "restriction endonucleases". The resulting mix of DNA fragments is then separated by size through electrophoresis, transferred to a special medium, and identified after the application of specific "probes" or complimentary sequences that bind to the separated DNA pieces. The probe is labeled with specific radioactive or enzymatic markers to highlight the sequences of interest.

PCR is the most widely used of the molecular diagnostic tools. With minor adjustments to the process, there are several different versions of PCR. PCR refers to the general detection of DNA which may be quite large in size. The RT-PCR is used for infectious agents such as viruses like FeLV and FIV that have RNA as their primary genetic material. With this technique, a DNA copy of the RNA genome (cDNA) is first produced using the reverse transcription process (transcription normally involves making a messenger RNA from a DNA template, hence the name "reverse transcription). Nested PCR refers to the search for even more specific sequences within the initial PCR by further limiting the scope of the primers. In-situ PCR refers to the detection of target DNA within intact cells.

Clinical applications of PCR are varied and include the detection of infectious agents, the diagnosis of neoplasia, and the identification of animals affected with inherited genetic disorders. These applications are expanding to include an elaboration of many facets of disease such as latent infections, antimicrobial resistance, the epidemiology of infectious agents as well as the emergence of new or mutated species. With regards to neoplasia, PCR is used to identify neoplasms and early tumor recurrence in patients in remission, as well as evaluating responses to chemotherapy and the development of drug resistance. The accurate identification of heterozygous, asymptomatic carrier animals among particular breeds at high risk of genetic diseases is a major benefit of PCR application.

The advantages of using PCR are many. There is remarkable sensitivity as PCR can detect as few as 10 copies of the target DNA in clinical samples. The profound sensitivity enables PCR to accurately detect as few as a single affected cell or one viral particle within a tissue of interest. It is ideal for detecting latent infections such as FeLV, FIV, and Mycoplasma haemofelis (Hemobartonellosis). The technique is ideally suited for slow growing organisms such as Mycobacteria, and for infectious agents that are difficult or impossible to culture or identify from infected tissues (for example Borrelia burgdorferi or Toxoplasma gondii). It is ideal for detection of organisms which incorporate their own genetic material into the host genome such as Retroviruses.

In some cases it has been shown to be more sensitive than culture or enzyme immunoassays. Reports of detection of Chlamydia, adenovirus, mycoplasma, Campylobacter and feline and canine parvoviruses have supported this claim. In a recent study, PCR on brain tissue, feces, lymph node, thymus and ileum resulted in the detection of parvovirus in puppies and kittens with cerebellar hypoplasia, while immunohistochemistry was negative. The molecular assay may provide evidence that enables characterization of zoonotic infections, as in the cases tracing SARs, Bartonella and Chlamydia to similar or identical organisms in pet cats. PCR may define new causes of infectious diseases. For example, a novel species of Ehrlichia (E. ruminantium) has recently been found in dogs raising the possibility of a newly identified causative agent in the rickettsial family.

It enables earlier detection of the organism post-infection compared with serologic titres, and it eliminates the need for, and the time delay inherent with, acute and convalescent titres. For example in Leptospirosis, positive PCR results may be detected in urine as early as 3-4 days after the onset of illness, compared to 7-14 days when IgM is evaluated. A similar situation exists with Rocky Mountain spotted fever. PCR supplants the need for special media for transport of specimens, and in many laboratories offering PCR, it is cost-competitive.

PCR can be performed on formalin-fixed tissue, providing retrospective analysis in cases where blood or other tissues were not saved. PCR done on formalin-fixed tissues from cats with lymphoma diagnosed the presence of FeLV genetic material in several cases that were negative with immunohistochemical staining and enzyme-linked immunosorbent assay (ELISA). Similarly, PCR was successful in the detection of FIV DNA in the peripheral blood and bone marrow of many seronegative cats in an FIV-positive environment.

With increased demands for PCR, which has in itself spawned an entire new industry, specialized equipment, specific primers, and reagents are readily available over the internet and via other means, hence driving the development of newer assays as needs arise. PCR may enable distinction between active infection versus maternally derived or vaccine-induced antibodies in animals where seroconversion has been documented. Resistance to a particular class of antimicrobials may be supported by the finding of antimicrobial resistance proteins in infectious agents and may assist in the selection of appropriate antimicrobial therapy. Finally, with slight modifications, the PCR can provide quantitative information regarding the number of identical DNA segments in the sample which may be correlated with the actual number of organisms present. The RT-PCR may be adapted for quantitative purposes making it more useful in monitoring response to therapy. RT-PCR-based testing of viral load is standard for people infected with HIV-1.

The disadvantages of the technique are several. PCR requires specific reagents, equipment and trained personnel and is most suited to institutional diagnostic or research laboratories. However, this may change in the near future as bedside assays are already appearing in the human medical field. The quality assurance and regulatory aspect of the assay has been outpaced by the rapid spread and availability of the technique. Practitioners are at the mercy of the individual laboratory for quality assurance in the development and use of appropriate primers, and that the test is conducted in a responsible and reliable fashion including the use of appropriate controls. The target sequence of the organism of interest must be known before appropriate primers can be developed and applied in the assay. PCR results must be interpreted correctly and in light of the rest of the case data as detection of nucleic acid of an infectious organism does not prove the agent is the cause of the clinical disease.This is especially so with latent infections such as M. haemofelis (Haemobartonella) since many cats are asymptomatic carriers of the agent.

A negative result does not necessarily rule out a role for a particular agent, especially if a microbial toxin produced at a different site than one that is tested may underlie the disease occurrence. Appropriate selection of target tissues and timing of testing, especially in the face of treatment, is of paramount importance in obtaining accurate results. For example, false negative PCR results have been obtained when testing certain tissues in patients undergoing treatment for infections such as Ehrlichia or Leptospirosis. As agents mutate, the primers may not recognize a particular organism. Because of the explicit sensitivity, the PCR may not adequately assess response to therapy. Nucleic acids persist in the host after the organism has been killed, resulting in continued positive test results. Unfortunately, the duration of DNA persistence following appropriate therapy is unknown.

PCR, as with other assays, is susceptible to false-positive and false-negative results. DNA contamination of the laboratory equipment, reagents or in airborne droplets may result in false-positive results. DNA is indefinitely stable in the laboratory, and is not affected by standard decontamination procedures. Contamination may occur not only during processing of the sample, but also at the time of sample collection. Use of broad-spectrum or "universal" primers may result in identification of related organisms not of prime interest in particular cases.

False-negative results may occur in the presence of PCR inhibitors, including proteins such as hemoglobin. If there is extreme degradation of the sample, or technical problems with the assay, organisms of interest may fail to be detected. A restricted number of tissues tested, or an inappropriate tissue may result in negative findings. For example with canine distemper virus, the sensitivity was improved if whole blood, serum and cerebrospinal fluid were included in the assay. This is not unexpected, since the sensitivity of other conventional tests is similarly dependent upon tissue tropism of the virus at the time of sampling. Contamination of the RT-PCR with RNAses may also cause false negative results. Improperly prepared primers, inappropriate primers for the target organism, and lack of hybridization or binding of the probe may decrease the sensitivity of the test.

PCR testing is available for the diagnosis of genetic disorders such as pyruvate kinase deficiency in Basenjis, phosphofructokinase deficiency in English springer spaniels, renal dysplasia and canine von Willebrand's disease. In humans, over 50 different markers for cancer (known as oncogenes) have been identified and are under investigation. PCR has been used to evaluate feline neoplasms such as lymphoma and injection site sarcomas for the potential etiologic role of transforming viruses such as FeLV, FIV, papillomavirus and polyomavirus. It has also been applied to the study of monoclonality of B-cell and T-cell receptors in the diagnosis of canine lymphoma.

In summary, the use of the molecular diagnostic tool known as polymerase chain reaction (PCR) has been widespread in acceptance and availability in clinical veterinary medicine. Molecular diagnosis has been integrated into the mainstream of clinical applications such as the diagnosis of microbial infections, neoplasia and inherited genetic disorders. Nucleic acid probes are now available for almost every important viral, rickettsial, and bacterial pathogen in veterinary patients. PCR provides information that is not only useful in the diagnosis and treatment of cancer in domestic species, but may well play an important role in the study of human cancers through the development of animal models. PCR is also valuable in the diagnosis of genetic disorders through the identification of affected and carrier animals. Practicing veterinarians must have not only a basic understanding of the concepts underlying these molecular techniques, but must also possess the knowledge of how to accurately interpret and assimilate the information into the clinical caseload.




© 2005 - Judith A.Taylor DVM, DVSc, DACVP - All rights reserved