Heather L. Wamsley, BS, DVM, PhD, DACVP-Clinical Short bio Dr. Wamsley is from Bradenton, FL. She is a graduate of the University of Wisconsin-Madison, where she completed her BS in Bacteriology and DVM. She did a one-year internship in small animal medicine and surgery at The Animal Medical Center NYC. Then she completed a residency in clinical pathology and PhD in molecular biology, tissue culture, and arthropod-borne infections at the University of Florida. Longer bio Heather is board-certified in veterinary clinical pathology with 18 years’ experience in hematology, marrow evaluation, clinical biochemistry, urinalysis, cytology, and pathology. She is a passionate educator and has received national and college teaching awards recognizing her abilities. Since 2004, she has been active in continuing education online and at local, national, and international conferences for all levels of veterinary professionals, preveterinary students, and K-12 students. She has published multiple book chapters in areas of urinalysis, clinical pathology as it relates to neurology, and other topics. Her other publications are in the areas of in situ molecular diagnostics, mammalian and insect cell culture, arthropod-borne disease pathogenesis, Parvo virus, preclinical evaluation of prospective therapeutics, histomorphometry, and clinical pathology. She is currently a clinical pathologist for Mars Petcare VCA Antech Diagnostics. Saturday 3pm-7pm Cytology Submission, Review of Interpretation, and Interactive Case Session Brush up on cytology! We will discuss multiple techniques for optimal collection, handling, and submission of cytology and histology samples along with tips that will help yield the most diagnostic information for you and your patients. Comparison with surgical biopsies and a systematic method for cytology interpretation will also be presented. We will continue with an interactive case session, during which we will evaluate photomicrograph cases and discuss the cytodiagnosis of skin lesions, effusion fluid, and internal lesions. Sunday 8am to 2:30pm 8-8:50 CBC Innovations We will discuss technology advances and artificial intelligence used to limit error, increase standardization, add value, shorten turn-around-time for CBC results. 9-9:50 CBC - RBC, PLT, and WBC Values CBC data and peripheral blood film evaluation are rapidly available and critical during evaluation of most patients. During these interactive sessions, clinical cases that focus on improved understanding of erythrocyte, platelet, and leukocyte data from CBCs will be presented and correlated with blood film and patient abnormalities. 10-10:50 All You Need to Node - Tips for Lymph Node Cytology Submission, Interpretation Guidelines, and Advanced Diagnostics for Lymphoid Neoplasia This presentation will review collection, preparation, and staining of samples for cytology followed by an overview of common abnormalities in lymph nodes. Special considerations for cats and advanced diagnostics will be discussed with case correlates. 11-11:50 Urinalysis Tips and Tricks This presentation will review urinalysis values that change post-collection, aspects of sample collection that affect urinalysis results, tips for optimal analysis in-house, methods for sediment evaluation by wet mount and cytology with examples, DNA testing for transitional cell carcinoma, bacteriuria, and other common urine sediment abnormalities. 12:40-1:30 Urinalysis Sediment Microscopy Review Designed for both novices and pros, this presentation is a case-based, rapid, interactive review of both common and unique urine sediment abnormalities. 1:40-2:30 Proteinuria Tests Tests used to diagnose proteinuria will be reviewed, including dipstick, sulfasalicylic acid precipitation, microalbuminuria test, and urine protein to creatinine ratio. We will also review causes of proteinuria, use of microalbuminuria test and creatinine to boost sensitivity of routine testing, brief discussion of SDMA, and case examples.

Cytology Submission, Review of Interpretation, and Interactive Case Session Heather L. Wamsley, BS, DVM, PhD, Dipl. ACVP-Clinical

Tampa, Florida, USA

OVERVIEW OBJECTIVES OF THE PRESENTATION Cytology is a useful aid for clinical decision-making. With practice and knowledge of its

limitations, cytology can be used as a rapid and inexpensive diagnostic test to improve patient

care.

OBJECTIVES OF THE PRESENTATION Tips for cytology and fluid slide preparation and staining

Tips for evaluating and interpreting cytology

Brief discussion of surgical biopsies

Important minimum information for cytology submission to pathologist

KEY CLINICAL DIAGNOSTIC POINTS Proper slide preparation is critical. Getting cellular material onto the slide in a thin-prep

monolayer that is also not lysed requires some practice, but is achievable.

Improve staining quality by placing slides in the stain fixative for at least two minutes. No

heat-fixation is needed for cytology. Heat fixation is for Gram stain, not cytology. If material

tends to “fall off” in the quick stain, it is very likely because preps are too thick – too much

material on the slide. Adipose tissue does actually adhere. Close the microscope condenser

partially to see the free lipid.

In-house quick stains create morphology artifacts usually due to inadequate fixation or

under-staining. If future submission to a pathologist is likely, try to reserve unstained at least

one slide that is likely to be highly cellular. Do also submit the in-house quick-stained slides.

Cells with intracytoplasmic granules, e.g., certain canine eosinophils, certain types of

lymphocytes, and some mast cells, may not stain well with in-house quick stains.

Approximately 20% of canine mast cell tumor cells do not stain with quick stain.

For fluid preps, hunt for microorganisms at the feathered edges.

For fluid preps, prepare slides soon after collection. Use a microhematocrit tube to transfer a

small drop of fluid to the slide. Sediment pellet cytology is also useful.

For tissue aspirates, use a systematic approach and attempt to classify the lesion into one

of the five diagnostic categories.

Basic signalment – species, breed, age, and sex, is essential minimum information to

include with submission to pathologist.

Label individual cytology slides somehow – either indicate the mass sampled and/or the

animal’s name/ID. If non-frosted slides are used, label the slide somehow to indicate which

side is up and, thereby, what part of the slide is safe to cover with an identifying sticker in

the lab. Simply writing Top or Up or the animal’s name/ID is extremely useful.

A brief description of the gross appearance of the lesion – size, shape, color, texture,

superficial appearance, etc. is very helpful.

If intra-cavitary (abdomen, thorax, etc.) lesions/organs are aspirated, it is valuable to include

information about what was sampled – one lesion, two lesions, four lesions/nodes vs.

general parenchyma etc., AND label the slides!! Is the organ enlarged, was a mass

sampled, were masses sampled, was a node sampled, were nodes sampled, was random

parenchyma sampled, staging for metastasis – what neoplasm? If the ultrasound was

performed outpatient by a specialist, then include the consult/ultrasound report with the

cytology slides.

“Don’t give any history – don’t want to bias the pathologist” is rotten advice. There is

precedence in human medicine. The attending physician, not the pathologist, was held

legally liable for misdiagnosis attributed to lack of appropriate history.

ADDITIONAL DETAILS Cytology slide preparation – aspirates, fluids, biopsy imprints

A 22- or 23-gauge needle is suitable for most subcutaneous aspirates. Imprinting an

ulcerated mass does not yield the same diagnostic information as an aspirate. Aspirates may be

obtained using a needle alone or with the needle attached to a syringe with negative pressure.

Either method usually results in a diagnostic sample. Using the needle alone without a syringe

attached allows for better needle control, decreases hemorrhage, and allows a needle core-type

sample.

When spreading an aspirate into thin film prep, quickly place the spreader slide over the

aspirated material without additional downward pressure. This will usually cause the aspirated

material to diffuse into a thin layer between the two slides. Then, gently slide the two glass

slides apart to form a monolayer of aspirated cells. In some situations, surgical biopsies can

also be imprinted for cytology.

Allow the slides to air-dry. Do not heat-fix or formalin-fix the slides. As an aside, also do

not ship in the same bag or box cytology slides and surgical biopsies that are in formalin. If

material seems to fall off of slides, the slide preps are very likely too thick. When using quick

stain in-house, let slides site in fixative for at least two minutes. Try to reserve at least one

cellular slide unstained for submission to a pathologist if desired. If using an outside lab, include

basic signalment – species, breed, age, sex, and try to include a gross description of the lesion

or diagnostic imaging results on the submission form. It may be faster to include a digital image

of the lesion or the ultrasound/consult report with the request form.

For fluid cytology, EDTA tubes are preferred. Plain, red top tubes should be used for

microbial culture. Thin film preparations of fluid samples should be made as soon as possible

after collection and submitted along with the fluid if an outside lab will be used. It is helpful to

use a microhematocrit tube to control the size of the fluid drop used to prepare the slide. The

drop should be small, similar to preparing a blood film. Preps made using a transfer pipet

usually have too much fluid, are too thick, partially detach in the stain, and limit ability to detect

bacteria and other small structures. Cytology of the fluid sediment pellet is particularly useful to

screen for microorganisms and to concentrate transudates/modified transudates. If this method

is used, it is important to note it on the submission form and helpful to also include a smear of

unconcentrated fluid.

Surgical biopsies can be used to prepare both cytology and histopathology samples. If

the biopsy is sufficiently large, a portion of the biopsy can be imprinted onto glass slides before

the sample is placed in formalin. The internal cut-surface of the biopsy should be imprinted;

avoid imprinting the serosal surface or outer epithelium of tissues. Use a clean gauze or paper

towel to gently dab away excess fluid prior to touching the tissue sample to the slide. As the

tissue is repeatedly imprinted onto the slide, additional fluid may appear. Dab away excess fluid

as it accumulates on the gauze or other absorbent material.

Very small pieces of soft tissue, e.g., liver, can simply be smeared into a thin film on the slide

using a blade or a second spreader slide. Firm tissues, e.g., very firm tumors, like a sarcoma,

may not exfoliate well. To promote exfoliation of cells, the surface area of the cut-surface of the

tissue can be increased by scoring it using the point of an 11 blade or a needle, similar to what

is done during a grid keratectomy. This gridding procedure and stronger pressure may be

required to encourage exfoliation.

Surgical biopsies – small and large Biopsies or portions of biopsies that are intended for histopathology should not be

macerated as above described. Small biopsy pieces are easily lost in jars of formalin. They

should be placed in plastic cassettes or wrapped in formalin-soaked gauze and indicate such on

the submission form. An adequate volume of formalin is 10 parts formalin to 1 part tissue for

proper tissue preservation. To promote adequate fixation, organs with a lumen can be flushed

with formalin, and large organs can be “loafed”. If necessary, very large samples can be

formalin-fixed in-house and then shipped with a small volume of formalin in double-bagged zip

closure bags. Inking of tissue edges can aid in measurement of surgical margins around

neoplasms. Though it may seem obvious at the time of biopsy, along with a gross description of

the lesion, it is helpful to indicate on the submission form exactly what has been submitted from

a mass – specifically, if the excised tissue contains the whole mass (e.g., a skin biopsy with a

small mass) or if the excised tissue is an incisional biopsy of the mass. It can be hard for

technical staff in the laboratory to discern once the tissue is formalin-fixed.

Cytology interpretation overview When evaluating cytology, identify an area of the preparation that contains intact cells

that are not lysed, distorted, or otherwise damaged. This is one of the hardest parts of cytology.

If an area of intact cells within readable monolayer is found, then attempt to classify the lesion

into one of five general categories. In many cases, more than one pathologic process may be

present in a single lesion. Real life cytology interpretation is typically not straightforward. Five

general lesion categories:

Inflammatory – leukocytes are increased above that which is subjectively expected to be

caused by blood contamination

Cystic – large number of mature, keratinized, squamous epithelial cells or abundant,

amorphous, proteinaceous material

Hemorrhagic – bloody and pink proteinaceous fluid with macrophages that contain

phagocytosed erythrocytes or erythrocyte breakdown pigments

Neoplastic – a monotypic cell population of one tissue origin

Mixed cell population – both inflammation and atypical-appearing monotypic cell population

ADDITIONAL READING

Burton AG. Clinical Atlas of Small Animal Cytology. 1st ed. Hoboken, NJ: Wiley-

Blackwell, 2017.

Valenciano AC, Cowell RL, eds. Cowell and Tyler's Diagnostic Cytology and Hematology

of the Dog and Cat. 5th ed. New York City, NY: Elsevier, 2019.

CBC - RBC, PLT, and WBC Values and Blood film preparation and evaluation. Heather L. Wamsley, BS, DVM, PhD, Dipl. ACVP-Clinical

Tampa, Florida, USA

OVERVIEW CBC data and peripheral blood film evaluation are critical during evaluation and management of

most patients. Abundant information is rapidly provided and may yield diagnosis of the case or,

more commonly, guide decisions about future diagnostic testing and medical management.

OBJECTIVES OF THE PRESENTATION This presentation will review what the numeric parameters reported in a CBC mean.

The discussion will be case-based and will include examples of how review of a blood film is

needed for full interpretation. KEY CLINICAL DIAGNOSTIC POINTS CBC numeric data include multiple RBC and PLT parameters that may give clues about the

cause of anemia, conditions associated with thrombocytopenia, and response to treatment of

anemia and thrombocytopenia. Blood film evaluation enhances the value of these data and

will detect abnormalities that hematology analyzers cannot, such as parasites, abnormal cell

shapes, other.

CBC data include information about WBC quantity and differential WBC count. All nucleated

cells in the sample are counted and reported as WBC. Cells that are not actually WBCs, such

as nucleated red blood cells (nRBC) or neoplastic cells, are included in this value. In order to

fully interpret WBC data, a blood film must also be evaluated. A WBC correction formula may

be needed if high nRBC is present.

Important, clinically germane information may be missed if a well-prepared peripheral blood

film is not evaluated as part of a complete CBC. This may lead to delayed case diagnosis or

misdiagnosis with delayed therapy or inappropriate therapy and diagnostic evaluation.

Automated hematology analyzers may produce erroneous data; it is important to verify

information from automated analyzers by peripheral blood film evaluation.

Automated hematology analyzers are incapable of detecting clinically important changes, for

example, neutrophil toxic change, blood-borne parasites, neoplastic cells, and others.

ADDITIONAL DETAIL RBC values Parameters in the CBC that provide information about red blood cell quantity include,

RBC count (RBC), hemoglobin concentration (Hgb), and hematocrit (HCT). Some laboratories

may also report a spun packed cell volume (PCV) based on the centrifugation of blood in a

microhematocrit tube. The analyzer actually measures the RBC and the Hgb. The analyzer

calculates the HCT based on the measured mean cell volume (MCV) and measured RBC.

There are multiple parameters in the CBC that give information about red blood cell quantity.

This is useful to help verify the information, especially since some conditions, such as,

agglutination, can affect multiple parameters and cause false results. One way to verify the data

is to multiply the Hgb value by 3. The product should be approximately equal to the calculated

HCT or spun PCV.

Parameters in the CBC that provide information about red blood cell quality include,

mean cell volume (MCV), red cell distribution width (RDW), mean cellular hemoglobin

concentration (MCHC), mean cell hemoglobin (MCH). The analyzer actually measures the

MCV. The RDW, MCHC, and MCH are calculated based on other data. The MCV indicates the

average size of the red blood cells. The RDW describes variation in red blood cell size.

Anisocytosis (unequal cell size) is a term used to describe variation in red blood cell size based

on peripheral blood film evaluation. RDW conceptually is an electronic version of anisocytosis.

The MCHC and MCH indicate how much hemoglobin there is in the red blood cells. Of the two

values, MCHC is more useful. MCH is often ignored, particularly since other red blood cell

features, not just the quantity of hemoglobin in the red blood cells, may affect it. The MCHC is a

ratio of hemoglobin to the number of red blood cells. High values are typically false. There is no

condition that causes the red blood cells to make extra hemoglobin. Conditions that cause

elevated MCHC include hemolysis, lipemia, Heinz bodies, and extremely increased WBC. Low

values may be seen in anemia with very strong, marked regenerative response or with

hemoglobin deficient RBCs – iron deficiency. Some laboratories may also report cellular

hemoglobin concentration mean (CHCM). This value is conceptually similar to the MCHC - ratio

of hemoglobin to the number of red blood cells; however, it is based on an actual measurement,

rather than a calculation. The CHCM is typically not affected by the conditions that cause false

increases in calculated MCHC.

PLT values Parameters in the CBC that provide information about platelets include, platelet count

(PLT), mean platelet volume (MPV), platelet distribution width (PDW), and plateletcrit (PCT).

The analyzer counts the platelets and determines the average size, which are reported as the

platelet count, PLT, and mean platelet volume, MPV, respectively. Clumped platelets are not

counted and included in the reported platelet count. Clumped platelets are common, particularly

when the Vacutainer collection system is not use. Cats often form platelet clumps even with

excellent venipuncture. A low automated platelet count should be confirmed by looking at a

blood film since platelet clumping is very common. A normal platelet count should also be

confirmed by blood film review since hemolysis and lipemia can both cause falsely increased

automated counts.

Active platelet production can cause large platelets and high MPV. A mix of giant and

large platelets with high MPV can also be seen with hereditary macrothrombocytopenia, which

is production of large platelets in quantity that is lower than the general canine reference

interval. This condition has been documented in the following breeds/mixes, Cavalier King

Charles Spaniel, Chihuahua, English Toy Spaniel, Havanese, Parson Russell Terrier,

Labradoodle, Labrador Retriever, Maltese, Poodle, Shih tzu. In the United States, 30-50% of

CKCS are affected. Clinically normal dogs with this condition may have platelet counts between

30,000 to 200,000/uL. Affected dogs are sometimes misdiagnosed with immune-mediated

thrombocytopenia. DNA testing is available to confirm this condition via Auburn, UPenn, and

others.

The MPV may not be included with the reported CBC values from some laboratories.

The platelet distribution width, PDW, describes variation in platelet size. It is conceptually similar

to the red blood cell RDW. This value may not be included with the CBC from some

laboratories. The plateletcrit, PCT, is a calculation based on the platelet count and the size of

the platelets. It is conceptually similar to the red blood cell HCT. It gives an idea of overall

platelet mass and accounts for both quantity and size of platelets. During treatment of immune-

mediated thrombocytopenia, the PCT may become normal before the platelet count. The PCT

can be used as an early indicator of positive response to treatment. The PCT may become

normal before the platelet count normalizes because larger than normal platelets are released

into circulation during active platelet production. With hereditary macrothrombocytopenia, the

mean platelet volume (MPV) is typically very high with normal plateletcrit (PCT), though the

platelet count is low. In affected dogs, the plateletcrit may be more useful than the traditional

platelet count to evaluate the patient’s platelet mass.

WBC values

CBC data include information about WBC quantity and differential WBC count. All

nucleated cells in the sample are counted and reported as WBC. Cells that are not actually

WBCs, such as nucleated red blood cells (nRBC) or neoplastic cells, are included in this value.

A WBC correction formula may be needed with high nRBC:

uncorrected WBC count X 100 = corrected WBC count /µL

number of nRBCs per 100 WBCs + 100

Blood film microscopy is required for full WBC evaluation. For example, when

neutrophilia is present, considerations may include stress leukogram (glucocorticoid response),

physiologic leukogram (epinephrine/excitement response), inflammation, or other less common

causes. To distinguish between these possibilities, information about neutrophil morphology

from the blood film is required – whether or not toxic change is present. When lymphocytosis is

present, considerations may include physiologic leukogram (epinephrine/excitement response),

response to antigenic stimulation, or lymphoid neoplasia. To help distinguish between these

possibilities, information about lymphocyte morphology from the blood film is required, for

example, whether or not large immature cells (blasts) or atypical lymphocytes are present. Most

hematology analyzers provide a WBC differential. The automated WBC differential may not be

accurate. Examples follow. When there is marked toxic change, the neutrophils may be counted

as monocytes. Neutrophil left shift is not detected at all or is not definitively characterized by

hematology analyzers, depending on the analyzer used. Eosinophils of some breeds are

incorrectly counted as monocytes. Circulating neoplastic cells may be miscounted as any one of

the WBCs, depending on the condition, for example, a dog with marked mastocythemia had

60K WBCs reported by the automated analyzer. The hematology analyzer reported the WBCs

as neutrophils. The clinical diagnosis was inflammation and infection. The dog arrested during

biopsy of skin masses presumed to be infected. The skin masses were actually mast cell

tumors. The diagnosis was missed prior to invasive testing because a properly made blood film

was not examined.

Summary of WBC leukogram patterns Stress leukogram “Stress response” “Steroid response”

The classic response is very common in dogs

– Leukocytosis

– Mature neutrophilia

– Lymphopenia **most consistent change**

– Eosinopenia

– Monocytosis (dogs)

Frequently observed; need to recognize and distinguish from inflammation

– Hypercortisolemia (endogenous or exogenous): shipping, trauma, pain,

hyperadrenocorticism, chronic illness

– But, often CONCURRENT with inflammation: animals have both “stress” and

inflammation

Changes in neutrophil pools due to glucocorticoids

– Decrease the rate at which neutrophils leave blood and enter tissues

– Mature neutrophils stay in circulation longer; hypersegmentation common

– Cause neutrophils in circulation to demarginate from vessel walls

– The number of mature neutrophils in the circulating neutrophil pool rises

– Cause marrow to release segmented neutrophils at a faster rate

– The number of new mature neutrophils entering circulation is increased

Leukocytosis: due to neutrophilia, lymphopenia, eosinopenia, monocytosis

Neutrophilia without left shift & without hyperfibrinogenemia

– Occurs 4-8 hours after single episode of hypercortisolemia

– Returns to normal within 24 hours after single episode

– After long term hypercortisolemia, takes 2 to 3 days to return to normal

Lymphopenia**most consistent change**

– Due to sequestration in lymphoid tissue and cell death

– Long term can cause lymphoid involution

– Stress OFTEN accompanies inflammatory disease

– Lymphopenia may be the only indication of steroid release

– The clinical importance is to recognize the pattern

Eosinopenia is often seen with stress leukogram, but eosinophils are already typically

low in health

– With hypoadrenocorticism, can have eosinophilia “reverse stress leukogram”

Species differences: dogs and cats more responsive to this process than other domestic

species

Physiologic leukogram “Physiologic neutrophilia” “Physiologic lymphocytosis” “Excitement

response”

The classic response is common in young cats and horses

– Transient leukocytosis with or without concurrent erythrocytosis and

thrombocytosis

– Transient mature neutrophilia

– Transient lymphocytosis

– No bands, normal fibrinogen

Endogenous epinephrine release

– Increased capillary blood flow due to increased HR and BP

Changes in neutrophils and lymphocytes due to epinephrine

– High capillary blood flow demarginates mature neutrophils

– The number of mature neutrophils in the circulating neutrophil pool rises

– Mature lymphocytes are flushed out of lymphoid organs and channels

– The number of mature lymphocytes in circulation is also increased

Occurrence

– Young, healthy animals

– Occurs rapidly after epinephrine release and is transient

– Fear, strenuous exercise, restraint for blood draw, sudden seizures

– Returns to normal within ~30 minutes

– Species differences: more common in cats and horses

– Cat: Mild neutrophilia and marked lymphocytosis (lymphocytes ≤ 20,000/µL)

– Horse: Neutrophilia and lymphocytosis (lymphocytes 6000-14000/µL)

– Bovine: Mild to mod. leukocytosis due to neutrophilia and lymphocytosis

– Dog: Uncommon in dogs; mild leukocytosis due to mature neutrophilia +/-

lymphs

Neutrophilia with regenerative left shift There is a left shift

There is a neutrophilia

There are more mature segmented neutrophils than immature cells (e.g., band

neutrophils, metamyelocytes)

At that time, marrow production is adequate

Neutropenia or normal neutrophil count with degenerative left shift There is a left shift

The total neutrophil count is normal or neutropenic

There are more immature cells (e.g., band neutrophils, metamyelocytes) than mature

segmented neutrophils

At that time, tissue demands exceed marrow capacity to produce neutrophils

Close monitoring and intervention warranted

Leukemoid response

A regenerative left shift with very high leukocyte counts, ~100K +/- 20K

Concurrent toxic change common.

Marked, established inflammation

Rarely, paraneoplastic; neoplasm makes compound that stimulates WBC production

Lymphocytosis

The total lymphocyte count and the lymphocyte morphology are used to categorize

lymphocytosis.

In dogs and cats, counts higher than 30 K/uL are usually considered neoplastic

regardless of cell morphology.

When counts are lower than 30 K/uL, the following are typically used to help classify a

lymphocytosis - cell morphology, concurrent disease and medications, whether or not

the lymphocytosis is persistent, and other diagnostics tests, e.g., vector-borne infection

screening, diagnostic imaging, advanced tests to characterize the lymphocytes (flow

cytometry or PCR for antigen receptor rearrangement - PARR).

Blood collection & blood film preparation and evaluation Vacutainer tubes containing EDTA should be filled with the designated amount of blood.

Partial filling of vacutainer tubes may cause false changes in cell morphology and numerical

data. Over-filling of tubes may result in clotted blood samples. Blood films should be prepared

soon after collection, preferably within 4 hours of collection, to minimize post-collection changes

in erythrocytes and leukocytes such as echinocyte formation, leukocyte degradation, which can

cause false differential counts and decrease accuracy of interpretation. In addition, prolonged

exposure to EDTA may make it more difficult to identify blood-borne parasites, like

haemoplasmas (formerly, hemobartonella), which may detach from the surfaces of erythrocytes

due to the EDTA, or some Borrelia.

There are two commonly used techniques for blood film preparation, the push smear

and the coverslip methods. Each has advantages and disadvantages. The push smear

technique is easier to learn, and the slides that are produced are easier to stain in the Diff Quik

jars (Coplin jars) used in most practices. The coverslip technique produces blood films that have

a more even distribution of cells in the smear – there is no feathered edge with clumps of

platelets and leukocytes, and the cells are less likely to lyse when blood films are prepared in

this way. Using the coverslip technique permits more accurate estimation of leukocyte and

platelet numbers and a better opportunity to evaluate cell morphology given the more even

distribution of erythrocytes and fewer lysed cells. Blood films should be rapidly dried with a blow

dryer to preserve optimal erythrocyte morphology. Instead, when blood films are permitted to

air-dry on the lab bench top, refractile air-drying artifacts may occur and are more pronounced in

humid environments. Refractile air-drying artifacts should be avoided since they severely

impede one’s ability to identify red blood cell parasites and to evaluate red blood cell shape

changes (poikilocytes). Tips for quick staining and a summary of one systematic approach to

blood film evaluation are listed below here.

Tips for improved blood film staining

Control the amount of blood or fluid put on the slide. o Use hematocrit tube to put blood on glass slide to control drop size. o Avoid applying too large of a drop or too much material to the slide. True for

cytology and hematology. Too much material will decrease ability to make

diagnosis. Rapidly air-dry blood film with heated air to avoid air-drying artifacts. Fixative for 2 minutes

o Set slides in the fixative for at least 2 minutes. Improves staining quality of blood

and cytology. Rinse

o Distilled or deionized water is preferred. Tap water is usually used, though it may

alter cell staining due to pH differences. Low magnification examination (10X or 20X objective)

Resist the urge to jump immediately to high magnification.

Find an area of the blood film where the erythrocytes are evenly distributed, neither too

thick, nor too thin. In this area, the erythrocytes are typically individual or only touching

one or two other erythrocytes.

Form initial impressions about the leukocyte and erythrocyte counts.

Determine the predominant leukocyte, which should be the neutrophil in dogs and cats.

Screen for relatively large structures, like, clumped platelets, clumped leukocytes, large

atypical cells, microfilariae.

High magnification examination (50X or 100X objective)

Leukocytes

o Estimate the leukocyte count to validate automated data (detail how to is below).

o Estimate the leukocyte differential to validate automated data, or perform a 100-

or 200-cell differential count.

o Evaluate leukocyte morphology: look for left shifting, toxicity, altered

morphology, infectious organisms, other cellular inclusions, and atypical or

neoplastic cells.

Erythrocytes

o Assess whether the density of the erythrocytes corresponds appropriately with

RBC count data.

o Evaluate the arrangement of erythrocytes: look for rouleaux, agglutination.

o Evaluate erythrocyte morphology: look for changes in RBC size, shape, color,

and inclusions.

Platelets

o Assess whether or not platelet clumps are present, which would cause

automated hematology analyzers to falsely report a low platelet count.

o Estimate the platelet count to validate automated data (detail how to is below).

o Evaluate platelet morphology: look for changes in platelet size and inclusions.

To estimate the total leukocyte count, determine the average number of leukocytes in at least

ten microscopic fields in the monolayer portion of the blood film. Then, multiply the average

number of leukocytes by the square of the objective that was used to perform the estimate. If

the 50X objective was used, then multiply by 2,500. If the 100x objective was used, then

multiply by 10,000. The product provides a rough estimate of the total leukocyte count per

microliter. The 50X or 100X objective is typically preferred when performing leukocyte

estimates, except when the patient is leukopenic – it may be better to use the 20X objective.

In dogs and cats, there should be a minimum of seven to ten platelets per 100X field. To

estimate the total platelet count, determine the average number of platelets in at least ten

representative 100X objective fields. For thrombocytopenic patients, determining the average

number of platelets in thirty 100X objective fields may be necessary to obtain a more accurate

estimate. Multiply the average number of platelets by 15,000. The product provides an estimate

of the total platelet count per microliter. Some authors recommend multiplying by 20,000 for

cats. Also, feline platelets are prone to clumping even with excellent venipuncture technique.

When platelets are clumped, automated counts are unreliable, but a subjective assessment of

whether or not the total number of platelets is adequate is typically possible.

All You Need to Node - Tips for Lymph Node Cytology Submission, Interpretation Heather L. Wamsley, BS, DVM, PhD, Dipl. ACVP-Clinical

Tampa, Florida, USA

OBJECTIVES OF THE PRESENTATION Review collection, preparation, and staining of samples for cytology

Overview of common abnormalities in lymph nodes

Special considerations for cats will be discussed

Advanced diagnostics will be discussed

KEY CLINICAL DIAGNOSTIC POINTS If using in-house quick stain, make sure to adequately fix the slides. Use fixative reagent

that has not been diluted by topping-off old fixative with new fixative. Leave slides at least

two minutes in proper fixative reagent.

If multiple lymph nodes are enlarged, do not only sample the largest one, and do not only

sample submandibular nodes.

It is helpful to get a small amount of blood with lymph node aspirates, especially in

generalized peripheral lymphadenomegaly.

Immunocytochemistry is now routinely available at a practical price for previously stained

cytology slides. This can be used to determine B-cell or T-cell lymphoma and should be

more accurate than PCR for antigen receptor rearrangement (PARR).

Other advanced testing to characterize lymphocytes include flow cytometry, PCR for

antigen receptor rearrangement (PARR), and immunohistochemistry. Each has strengths

and weaknesses.

Flow cytometry can be helpful to characterize peripheral blood lymphocytoses, but results

may be equivocal. Serial monitoring or follow-up testing with PARR may be needed.

ADDITIONAL DETAILS When to aspirate lymph nodes and which ones to sample

Indications for lymph node FNA include lymph node enlargement, staging or diagnosing

neoplasia, screening for a cause of unexplained hypercalcemia, and screening for certain

infectious diseases.

When multiple lymph nodes are enlarged, include samples from the prescapular and/or

popliteal nodes if possible. Picking only the biggest lymph node can backfire. Very large

lymph nodes may be necrotic and may not yield the specific cause of lymph node

enlargement by FNA.

When screening for metastatic neoplasia, nodes are selected based on anatomy drained

by the lymph node. Submandibular - Head, most of it; Oral cavity, rostral

Prescapular - Caudal head: pharynx, pinna; Thoracic limb, most of it; Thoracic wall, part

of it

Axillary - Thoracic limb, deep structures; Thoracic wall, most of it; Neck, deep structures;

Mammary glands: thoracic & cranial abdominal

Inguinal - Mammary glands: caudal abdominal & inguinal; Abdominal wall, ventral half;

Penis, prepuce, scrotum; Pelvis, ventral; Thigh and stifle, medial; Tail

Popliteal - Distal to stifle; Perineum

How to aspirate and what next FNA is performed similar to other subcutaneous masses. It is helpful to have a small

amount of blood in the FNA, especially with generalized lymphadenomegaly. Use a 22/23-

gauge needle and the needle only, non-suction technique. Negative pressure FNA with

syringe attached is not necessary. Spread FNA material into a thin monolayer prep, as for

other cytology samples. Squash preps may help, but these almost always are too thick

because too much aspirated material is placed on the slide. Do not use the blood film smear

technique for node FNAs.

Air-dry the slides completely and stain or place in slide carriers for delivery to outside lab.

In general for all cytology, do not heat-fix, formalin-fix, or expose the slides to formalin

fumes. Adequate fixation is very important for all cytology. Cell damage due to improper

fixation is common when lymph node aspirates are stained in-house. Make sure the aspirate

is completely dry before staining. Make sure that the in-house quick stain fixative is

completely changed periodically. Do not top it off with fresh fixative. Topping off dilutes the

active fixative ingredient and creates difficulties reading slides. Let slides sit in the fixative for

at least two minutes before staining routinely.

Try to avoid staining all of the slides with in-house quick stain. There are certain cell types

that may not stain properly with in-house quick stain, such as, mast cells and a certain form

of lymphocytes/lymphoma – granular lymphocytes. Approximately 20% of mast cell

neoplasia cases do not stain with in-house quick stain. When screening for mast cell tumor

metastasis, quick-staining lymph nodes reduces sensitivity and is not recommended (Vet

Comp Oncol. 2018 Dec;16(4):511-517).

Advanced tests for lymphoid neoplasia – why and how When lymphoid neoplasia – lymphoma or leukemia, has been diagnosed, advanced

testing may be performed to further characterize the neoplastic cells beyond their

cytomorphology. Cytology should report cell size – small, intermediate, or large; a subjective

estimate of mitotic frequency; and whether or not intracytoplasmic magenta granules –

granular lymphocytes, are present.

Information about whether or not a lymphoma is B-cell or T-cell may be used to aid

prognostication along with the information about neoplastic cell size – small, intermediate, or

large, and clinical presentation at time of diagnosis – no clinical signs or presence of clinical

illness. B-cell versus T-cell information may also be useful in treated pets that subsequently

come out of remission or if novel treatments may be pursued.

Another scenario when advanced testing may be performed is when a lymphoid

population is present, but it is uncertain from cytomorphology/histopathology whether or not

the cell population is a neoplastic proliferation or an inflammatory infiltrate.

Test options include immunocytochemistry, flow cytometry, PCR for antigen receptor

rearrangement (PARR), and immunohistochemistry. No one test is perfect. There are

advantages and disadvantages to each test. When practically possible, these tests are often

used in tandem for diagnosis.

Immunocytochemical staining on slides that have been previously stained for routine

cytology is now readily available for clinical samples at a practical price. For example, this

test can be used to characterize lymphoma as B-cell lymphoma or T-cell lymphoma from

routine cytology samples.

Flow cytometry and PARR use different methods to categorize the lymphoid cells either

as polyclonal – a mixed population of lymphocytes that likely is not lymphoid neoplasia, or as

monoclonal – a population of one lymphocyte (or very few lymphocytes - oligoclonal) that

likely is lymphoid neoplasia. Flow cytometry uses antibodies to label the cells. PARR uses

DNA from the cells. Either of these tests may yield equivocal results or interpretive

comments from the laboratory that do not match with the clinical presentation and

progression of clinical disease. Flow cytometry provides more information about the cells

than PARR, but PARR may be more practical to perform in most situations.

Flow cytometry test results indicate whether or not the cell population is likely

neoplastic or is not likely neoplastic, the size of the neoplastic cells, and whether or not the

cells are B-cells, T-cells, or other. The specimen for flow cytometry must be a fresh liquid

sample, e.g., peripheral blood, effusion fluid, or solid tissue FNA in saline-serum transport

medium. Rapid, overnight transport to the lab is required. Flow cytometry is often used for

peripheral blood lymphocytoses.

PARR test results indicate whether or not the cell population is likely neoplastic or is not

likely neoplastic and whether or not the cells have a DNA arrangement that indicates B-cell,

T-cell, or both B-cell and T-cell. The antibody-based tests – flow cytometry and

immunostaining of histology or cytology, are more accurate than PARR for telling B-cell

versus T-cell lymphoma. Despite this, PARR is often used instead of flow cytometry for

practical reasons. PARR can be performed on slides that have been previously stained for

cytology and have been in archival storage for months. No second pet visit, no second

sample, and no rapid, overnight transport are required for PARR. Now that

immunocytochemistry on previously stained cytology slides is routinely available,

immunocytochemistry may be a practical alternative to PARR that has higher diagnostic

accuracy.

There are three additional caveats for PARR. (1) When one intends to do PARR, it is

suboptimal to collect two sets of aspirates – one set for cytology and one set for PARR.

Cytology is needed prior to PARR to confirm that lymphoid cells are actually present on the

slides that are submitted for PARR. (2) Chronic ehrlichiosis may cause a false-positive result

by PARR. This is due to an oligoclonal population of lymphocytes – just a few lymphocyte

clones, in chronic proliferation due to Ehrlichia infection. (3) It is important to only use PARR

when a lymphoid population is known to be present based on microscopy. Uncommonly,

other hemolymphatic cancer types, like, acute myeloid leukemia, can appear to be

“lymphoma” – a false-positive result from PARR.

Diagnostic categories for lymph node cytology Nondiagnostic

Normal cells

Hyperplastic/reactive lymph node

Lymphadenitis

Neoplasia – primary or metastatic

Lymphedema

The normal cell population is composed of greater than 80 to 90% small lymphocytes.

The small lymphocyte nucleus is about 1 to 1.5 times the size of a canine erythrocyte or 1.5

to 2 times the size of a feline erythrocyte. Small lymphocytes are smaller than neutrophils.

Other cell types are present in much lower numbers.

The hyperplastic lymphoid population is usually due to response to antigenic stimulation.

Small cells predominate. Immature cells are increased, but are less than 50% of the total cell

population. Plasma cells are variably increased – mild to marked. Mild increases in other

leukocytes - granulocytes, macrophages, mast cells, are expected. Feline hyperplastic

nodes may only have few plasma cells with moderately increased large immature

lymphocytes and mildly increased mast cells.

Inflamed lymph nodes – lymphadenitis, are characterized based on the type of

inflammation, which helps rank differential diagnoses. Lymphoid hyperplasia is often

concurrent.

When screening for metastatic neoplasia in lymph nodes, sample lymph nodes if they

are palpable, even if they are not enlarged. Note that early metastasis can be missed by

microscopy. Advanced metastasis may only yield neoplastic cells, inflammation, and

necrosis without lymphoid cells. FNA of normal-sized nodes, prominent nodes, or slightly

enlarged nodes may only yield perinodal adipose tissue.

There are many morphologic types of lymphoma. The different forms of lymphoma are

associated with varied prognosis and treatment approaches. Some forms can be diagnosed

cytologically. Other forms may require advanced testing, like, PARR or flow cytometry, or

histology. In the lecture presentation, I will show cytology examples of multiple lymphoma

types.

Urinalysis Tips and Tricks & Sediment Microscopy Review Heather L. Wamsley, BS, DVM, PhD, Dipl. ACVP-Clinical

Tampa, Florida, USA

OVERVIEW Urine samples are labile; components of the urine sample may change or may be lost during the

delay between sample collection and sample analysis. Ideally, a trained individual should

examine urine samples soon after collection. Routine urinalysis is a test that may be improved

by performing it in-house.

OBJECTIVES OF THE PRESENTATION Brief review of ways to maximize the potential of urinalysis.

INTRODUCTION Complete urinalysis includes assessment of several physical and chemical characteristics of

urine. It is a simple, economical test that requires minimal specialized equipment and can be

easily performed by trained staff in general veterinary practice. With proper sample handling

and testing, data generated by urinalysis rapidly disclose vital information about the urinary tract

and also provide a general screen of other body systems (e.g. endocrine, hepatic). Taking

advantage of the full potential of in-house urinalysis can rapidly provide you with information that

will benefit you and your patients.

ADDITIONAL DETAIL

Urine Sample Collection Method, Timing, and Handling Prior to Analysis In addition to biologic variability of the patient, urinalysis results are influenced by the

urine collection method, the timing of urine collection, administration of therapeutic or diagnostic

agents prior to collection, and how the sample is handled prior to analysis. There are several

methods of urine collection each with their own advantages and disadvantages. Ideally at least

6 mL of urine should be collected prior to the administration of therapeutic or diagnostic agents

so that baseline information can be established. In most situations, either naturally voided urine,

collected midstream into a sterile container or urine obtained by cystocentesis is preferred.

Since urine collection method can have a significant effect upon urinalysis results, it is important

to record the collection method in medical records. It should also be indicated whether or not

therapeutic or diagnostic agents (e.g. parenteral fluids, antimicrobials, glucocorticoids, diuretics,

antihypertensives, radiographic contrast) have been administered prior to urine collection. If so,

the type of agent and timing and duration of administration relative to urine collection should be

recorded.

Timing of collection during the day also influences results. First morning urine is

maximally concentrated. This is good for USG monitoring. However, this urine is held in bladder

for prolonged period, which may reduce viability of fastidious organisms and alter cell

morphology due to cell degradation. First morning urine samples are also more likely to be

acidic. Time of collection relative to a meal may influence biochemical tests. The effect of a pH

modifying diet is maximal within 3-6 hours of a meal. Samples collected within 3-4 hours are

helpful to detect hyperglycemic glucosuria. Samples collected within 1 hour of a meail are more

likely to be alkaline due to postprandial alkaline tide. Randomly timed urine samples are held in

bladder for less time. This may be good for cell morphology and culture of fastidious organisms.

However, the USG value will not represent the maximal urine concentration ability of the renal

tubules.

Ideally, urine should be collected into a sterile, opaque, sealable, labeled container and

analyzed within 60 minutes of collection. If it is not possible to analyze the sample within 60

minutes, the sample should be preserved by refrigeration soon after collection for up to

approximately 12 hours (i.e. overnight). Samples intended for routine urinalysis should not be

frozen. Also, though numerous chemical preservatives of urine exist (e.g. boric acid, formalin,

Mucolexx™), routine use of these preservatives is not recommended, since each may affect

different components of the urinalysis. It is important that sealed containers are used for urine

sample storage since certain volatile compounds may evaporate (e.g. ketones), and samples

should be protected from light since certain compounds may degrade due to light-exposure (e.g.

bilirubin).

Refrigeration is considered the optimal preservation method since it prevents bacterial

overgrowth, preserves cellular and cast morphology, and does not affect chemical testing as

long as the sample is returned to room temperature prior to analysis. Cold urine may cause a

false increase in urine specific gravity, and cold urine may inhibit the chemical reactions on the

urine multitest dipstick. Refrigeration also promotes crystal formation (i.e. calcium oxalate

dihydrate, magnesium ammonium phosphate) that becomes worse as the duration of storage

increases (Albasan et al., 2003). To minimize refrigeration artifacts, samples should be allowed

to warm to room temperature prior to urinalysis. Also, if crystalluria is observed in a sample that

has been refrigerated or that has been stored for greater than 6 hours regardless of the storage

temperature, the finding should be confirmed with a freshly obtained, nonrefrigerated urine

sample that will be analyzed within 30 to 60 minutes of collection.

Urinalysis: wet-mount versus dry-mount Urinalysis performed in-house by a trained staff member, soon after urine sample

collection is a great way to reduce or avoid artifacts that can arise during the delay between

sample collection and sample analysis. Keeping such analysis in-house may increase

productivity and potentially improve your urinalysis laboratory results. In addition to traditional

wet-mount urinalysis, dry-mount cytology of urine samples can be very useful in suspected

cases of urinary tract infection (Swenson et al., 2004) or urinary tract neoplasia. The method is

described below here. Slides prepared using this method can be evaluated in-house, or they

can be readily sent to an outside laboratory for review by a pathologist. The benefit is that

diagnostic material on slides prepared this way will not degrade the same way that it would in a

liquid urine sample during transport to an out-of-house, reference laboratory. Often when fluid

urine samples are sent to a reference laboratory for pathologist review to diagnose neoplasia,

cells deteriorate to the point where cytologic diagnosis no longer possible.

Method to prepare urine sediment for dry-mounting and routine cytologic examination 1) Centrifuge the urine as is done for wet-mounting.

2) Use a transfer pipette to aspirate the pellet from the bottom of the conical centrifuge tube.

3) Place a small drop of the aspirated material onto a clean, glass microscope slide.

4) Use a second clean, glass microscope slide to spread the material in a monolayer.

5) Allow the slide to air-dry. Heat fixation is not necessary and would alter cell morphology.

6) Stain as a routine cytology using Diff Quik® or other similar stain. Alternatively, the slide can

be stored in a covered container at room temperature and sent to a outside diagnostic

laboratory for evaluation by a pathologist.

REFERENCES AND FURTHER READING

1. Albasan H, Lulich JP, Osborne CA, Lekcharoensuk C, Ulrich LK and Carpenter KA

(2003) Effects of storage time and temperature on pH, specific gravity, and crystal

formation in urine samples from dogs and cats. Journal of the American Veterinary

Medical Association 222, 176-179

2. Osborne CA and Stevens JB (1999) Urinalysis: A Clinical Guide to Compassionate

Patient Care, 1st edn. Veterinary Learning Systems, Bayer Corporation, Shawnee

Mission, Kansas

3. Swenson CL, Boisvert AM, Kruger JM and Gibbons-Burgener SN (2004) Evaluation of

modified Wright-staining of urine sediment as a method for accurate detection of

bacteriuria in dogs. Journal of the American Veterinary Medical Association 224, 1282-

1289

4. Wamsley HL (2019) Examination of Urine Sediment. In Valenciano AC, Cowell RL (ed):

Cowell and Tyler’s Diagnostic Cytology and Hematology of the Dog and Cat, 5th ed–St.

Louis, MO: Elsevier, 2019, Chapter 23.

SEDIMENT MICROSCOPY - OVERVIEW OF URINE SEDIMENT FINDINGS Heather L. Wamsley, BS, DVM, PhD, Dipl. ACVP-Clinical

Tampa, Florida, USA

OVERVIEW Urine samples are labile; components of the urine sample may change or may be lost during the

delay between sample collection and sample analysis. Ideally, urine samples should be

examined soon after collection by a trained individual. Routine urinalysis is a test that may be

improved by performing it in-house.

INTRODUCTION Complete urinalysis includes assessment of several physical and chemical

characteristics of urine. It is a simple, economical test that requires minimal specialized

equipment and can be easily performed by trained staff in general veterinary practice. With

proper sample handling and testing, data generated by urinalysis rapidly disclose vital

information about the urinary tract and also provide a general screen of other body systems

(e.g. endocrine, hepatic). Taking advantage of the full potential of in-house urinalysis can rapidly

provide you with information that will benefit you and your patients.

ADDITIONAL DETAIL Urine Sample Collection Method, Timing, and Handling Prior to Analysis

In addition to biologic variability of the patient, urinalysis results are influenced by the

urine collection method, the timing of urine collection, administration of therapeutic or diagnostic

agents prior to collection, and how the sample is handled prior to analysis. There are several

methods of urine collection each with their own advantages and disadvantages. Ideally at least

6 mL of urine should be collected prior to the administration of therapeutic or diagnostic agents

so that baseline information can be established. In most situations, either naturally voided urine,

collected midstream into a sterile container or urine obtained by cystocentesis is preferred.

Since urine collection method can have a significant effect upon urinalysis results, it is important

to record the collection method in medical records. It should also be indicated whether or not

therapeutic or diagnostic agents (e.g. parenteral fluids, antimicrobials, glucocorticoids, diuretics,

antihypertensives, radiographic contrast) have been administered prior to urine collection. If so,

the type of agent and timing and duration of administration relative to urine collection should be

recorded.

Ideally, urine should be collected into a sterile, opaque, sealable, labeled container and

analyzed within 60 minutes of collection. If it is not possible to analyze the sample within 60

minutes, the sample should be preserved by refrigeration soon after collection for up to

approximately 12 hours (i.e. overnight). Samples intended for routine urinalysis should not be

frozen. Also, though numerous chemical preservatives of urine exist (e.g. boric acid, formalin,

Mucolexx™), routine use of these preservatives is not recommended, since each may affect

different components of the urinalysis. It is important that sealed containers are used for urine

sample storage since certain volatile compounds may evaporate (e.g. ketones), and samples

should be protected from light since certain compounds may degrade due to light-exposure (e.g.

bilirubin).

Refrigeration is considered the optimal preservation method since it prevents bacterial

overgrowth, preserves cellular and cast morphology, and does not affect chemical testing as

long as the sample is returned to room temperature prior to analysis. Cold urine may cause a

false increase in urine specific gravity, and cold urine may inhibit the chemical reactions on the

urine multitest dipstick. Refrigeration also promotes crystal formation (i.e. calcium oxalate

dihydrate, magnesium ammonium phosphate) that becomes worse as the duration of storage

increases (Albasan et al., 2003). To minimize refrigeration artifacts, samples should be allowed

to warm to room temperature prior to urinalysis. Also, if crystalluria is observed in a sample that

has been refrigerated or that has been stored for greater than 6 hours regardless of the storage

temperature, the finding should be confirmed with a freshly obtained, nonrefrigerated urine

sample that will be analyzed within 30 to 60 minutes of collection.

Urine Sediment Wet-Mount Findings

Crystalluria occurs when urine is saturated with dissolved minerals or other

crystallogenic substances that precipitate out of solution to form crystals. Crystals may form in

vivo for either pathologic or nonpathologic reasons, or crystals may precipitate in urine ex vivo

due to cold temperature or prolonged storage, postcollection alterations of urine pH, or

evaporation of water from the sample. To increase the likelihood that crystals present in the

urine sample actually represent those that may be present in the patient, fresh, nonrefrigerated

urine samples should be analyzed within approximately one hour of collection.

In most instances, crystalluria does not necessarily indicate the presence of uroliths or

even a predisposition to form uroliths. For example, a small number of magnesium ammonium

phosphate or amorphous phosphate crystals are frequently observed in clinically normal dogs

and cats. Detection of crystalluria may be diagnostically useful when abnormal crystal types are

identified (e.g. ammonium biurate, calcium oxalate monohydrate, cystine);

when large aggregates of magnesium ammonium phosphate or calcium

oxalate dihydrate crystals are found; or when crystalluria is observed in a

patient that has confirmed urolithiasis. Evaluation of the type of crystals present

may be useful to estimate the mineral component of the urolith(s), while

awaiting results of complete urolith analysis. Uroliths are often heterogenous;

therefore crystalluria is not a definitive indicator of urolith mineral content.

Sequential evaluation of crystalluria may aid in monitoring a patient’s response

to therapy for urolith dissolution. Specific types of common urine crystals are

discussed here. For a complete discussion including uncommon types of

crystalluria, consult a text devoted to urinalysis such as, Urinalysis: A Clinical

Guide to Compassionate Patient Care (Osborne and Stevens, 1999).

Magnesium ammonium phosphate crystals are referred to as

struvite crystals, triple phosphate crystals (a misnomer), or infection crystals

(an older term). They are colorless and frequently form variably sized casket

cover-shaped crystals. They also form three to eight sided prisms, needles, or

flat crystals with oblique ends. Magnesium ammonium phosphate crystals most

commonly form in alkaline urine, which often occurs in association with

bacterial infection. They may develop after collection in refrigerated, stored

urine samples (Albasan et al., 2003), or in those that become alkaline during

storage due to bacterial overgrowth or contamination of the sample with

cleanser residues, for example. When magnesium ammonium phosphate

crystals are detected in a stored urine sample, the finding should be verified by

prompt examination of a freshly obtained urine sample that has not been

refrigerated. Magnesium ammonium phosphate crystals are very commonly

seen in dogs and occasionally in cats. When found in significant number, they

are most frequently associated with bacterial infection by urease-producing

bacteria, such as Staphylococcus or Proteus. However, in cats they can occur

in the absence of infection, likely due to ammonia excretion by the renal

tubules. Magnesium ammonium phosphate crystals may also be seen in

clinically normal animals that have alkaline urine for reasons other than

infection (e.g. diet, recent meal), animals that have sterile or infection-

associated uroliths of potentially mixed mineral composition, or with urinary

tract disease in the absence of urolithiasis.

Struvite

Ca Ox Dihyd.

Ca Ox Mono

Ca Carb

Bilirubin

Amorphous

Uric

Biurate

Cystine

Calcium oxalate crystals occur in two forms, dihydrate and monohydrate. Calcium

oxalate dihydrate crystals occur much more commonly. They are colorless, variably sized,

octahedrons that resemble a small, gift card type envelope or a Maltese cross and most

commonly form in acidic urine. They may develop after collection in stored urine samples with or

without refrigeration (Albasan et al., 2003) or in those that become acidic during storage due to

bacterial overgrowth, for example. When calcium oxalate dihydrate crystals are detected in a

stored urine sample, the finding should be verified by prompt examination of freshly obtained

urine that has not been refrigerated. Calcium oxalate dihydrate crystals may be seen in clinically

normal animals. They also occur with calcium oxalate urolithiasis, hypercalciuria (e.g. due to

hypercalcemia or hypercortisolemia), or hyperoxaluria (e.g. ingestion of vegetation high in

oxalates [e.g. Brassica family], ethylene glycol, or chocolate). They have been reported with

increased frequency in cats as a complication of urine acidification to manage magnesium

ammonium phosphate formation.

Calcium oxalate monohydrate crystals are colorless and variably sized. They may be flat

with pointed ends and resemble picket fence boards. They may also form spindle or dumbbell-

shaped crystals. The same conditions that cause dihydrate formation can lead to monohydrate

formation, although the monohydrate form with picket fence board morphology is more

diagnostic of intoxication, since this form is usually only seen during acute ethylene glycol

toxicity. Formation of these crystals is time dependent and occurs only during the early phase of

intoxication. Crystalluria may be observed within 3 hours of ingestion in cats and within 6 hours

in dogs and may last up to 18 hours post-ingestion. Calcium oxalate monohydrate crystals with

spindle or dumbbell morphology are uncommonly observed with other causes of hyperoxaluria

(e.g. chocolate ingestion).

Calcium carbonate crystals are variably sized, yellow-brown or colorless, variably

shaped crystals (tic-tac-shaped, dumbbell-shaped, or spheres with radiant striations) that are

found individually or in clusters usually within alkaline urine. They are seen in clinically normal

horses, elephants, goats, rabbits, and guinea pigs. Anecdotally, they may very rarely be seen in

dogs. Sulfonamide crystals, which can be seen in dogs and cats after sulfa-containing antibiotic

administration, may form globules with radiant striations and could be mistaken for calcium

carbonate crystals. Calcium carbonate crystals look similar to the crystals observed in 2007

secondary to melamine-contaminated pet foods.

Bilirubin crystals may precipitate as orange to reddish-brown granules or needle-like

crystals. A low number of crystals are routinely observed in canine urine, especially in highly

concentrated samples from male dogs. When bilirubin crystals are found in other species or in

persistently large quantity in a canine patient, a disease associated with icterus (i.e. hemolytic or

hepatobiliary disease) may be present.

Amorphous phosphate and amorphous urate crystals are similar in shape and may

form amorphous debris or small spheroids. Amorphous phosphates are distinguished from

amorphous urates in two ways: phosphates are colorless or light yellow and form in alkaline

urine, while urates are yellow-brown to black and form in acidic urine. Amorphous phosphates

are commonly observed in alkaline urine of clinically normal animals, and they are not clinically

significant. Conversely, amorphous urates are an uncommon abnormal finding in most breeds.

They may be seen in animals with portovascular malformation, severe hepatic disease, or

ammonium biurate urolithiasis. Amorphous urates are routinely found in Dalmatians and English

Bulldogs and may represent a predisposition for urate urolithiasis in these breeds.

Compared to other breeds, Dalmatians excrete a larger amount of uric acid in their urine

and are therefore prone to form uric acid crystals. Uric acid crystals are colorless; flat;

variably, but often diamond-shaped; six sided crystals. Most other breeds convert uric acid to a

water soluble compound (i.e. allantoin) for excretion. Dalmatians have defective purine

metabolism, preventing this conversion, so that uric acid is excreted in its native form into the

urine. Also, Dalmatians have decreased tubular resorption of uric acid compared to other

breeds. Uric acid crystals can also occasionally be seen in English Bulldogs. They are rarely

seen in other dog breeds or cats and, when observed, have the same significance as

amorphous urate or ammonium biurate crystals.

Ammonium biurate crystals are golden-brown and spherical with irregular protrusions,

which engender a thorn-apple or sarcoptic mange-like appearance. In cats, they may form

smooth aggregates of spheroids. Ammonium biurate crystals are seen in animals with

portovascular malformation, severe hepatic disease, ammonium biurate urolithiasis, and

uncommonly in clinically normal Dalmatians and English Bulldogs.

Cystine crystals are colorless, flat hexagons that may have unequal sides. Cystine

crystalluria is an abnormal normal finding seen in animals that are cystinuric due to an inherited

defect in proximal renal tubular transport of several amino acids (i.e. arginine, cystine, lysine,

ornithine). Crystals are prone to develop in cystinuric patients that have concentrated, acidic

urine. Cystinuria is a predisposition for the development of cystine urolithiasis. Among dogs,

male French Bulldogs, Dachshunds, Basset Hounds, English Bulldogs, Yorkshire Terriers, Irish

Terriers, Chihuahuas, Mastiffs, Rottweilers, and Newfoundlands are affected with increased

frequency. Uroliths often lodge at the base of the os penis and may be missed on survey

radiographs since they are relatively radiolucent. Female dogs and other breeds also may be

affected. In cats, this disease has been recognized in male and female Siamese and American

Domestic Shorthairs.

Iatrogenic crystalluria can be seen with administration of some antibiotics, allopurinol,

and radiocontrast medium. Sulfonamide crystals are pale yellow crystals and may form

haystack-like bundles or globules with radiant striations. The latter morphology may be mistaken

for calcium carbonate crystals.

Renal tubular casts and pseudocasts

Renal tubular casts are formed by proteinaceous plugs of dense, mesh-like mucoprotein

(Tamm-Horsfall mucoprotein) that accumulate within the distal portion of the nephron. A low

number (<2 per low power field) of these proteinaceous hyaline casts can occasionally be

observed in urine of normal animals. Diuresis of dehydrated animals or proteinuria of

preglomerular or renal etiology can cause an increased number of hyaline

casts to be present in urine. Renal tubular epithelial cells that die and slough

into the tubular lumen can be entrapped within this dense mucoprotein matrix.

If present, inflammatory cells associated with renal tubulointerstitial

inflammation may also be entrapped. During microscopic sediment evaluation,

cellular casts are further classified as either epithelial, leukocyte, or erythrocyte

casts, if the constituent cells can be discerned. Once locked within the

proteinaceous matrix, cells continue to degenerate, progressing from intact

cells, to granular cellular remnants, and finally to a waxy cholesterol-rich end

product. A cast may dislodge from a given renal tubular lumen at any time

during this degenerative process and may be observed in the urine sediment.

However, in clinically normal animals only granular casts are rarely found (<2

per low power field). Other material can lodge within the proteinaceous matrix,

such as lipid from degenerated renal tubular epithelial cells, hemoglobin during

hemolytic disease, and bilirubin.

The number of casts observed in the sediment does not correlate

with the severity of renal disease or its reversibility; and the absence of casts

from urine sediment cannot be used to exclude the possibility of renal disease,

especially since casts are fragile and prone to degeneration, particularly in

alkaline urine. When hyaline or granular casts are present in increased

numbers or when other cast types are observed, one can only conclude that

the renal tubules are involved in an active disease process of unknown severity

Hyaline cast

Cellular cast

Granular cst

Waxy cast

Mucus

Fiber

or reversibility. When present, the type of cast observed may provide additional information.

Leukocyte casts indicate active renal tubulointerstitial inflammation. Waxy casts reflect a chronic

tubular lesion. To recognize the onset of nephrotoxicity in patients receiving aminoglycoside

antibiotic therapy, it is useful to monitor urine sediment for the appearance of tubular casts,

which should prompt withdrawal of the antibiotic. Other abnormalities seen with aminoglycoside-

induced nephrotoxicity include isosthenuria, proteinuria, glucosuria, aminoaciduria, all of which

may precede the onset of azotemia.

Structures such as mucus threads or fibers may resemble casts and should not be

mistaken for them during microscopic examination. Mucus threads are distinguished by their

variable width and tapered ends. Fibers are typically much larger than the surrounding cells and

may contain a repetitive internal structure, suggesting a synthetic origin.

Epithelial cells

Epithelial surfaces along the length of the genitourinary tract undergo constant turnover,

therefore it is routine to see a low number of epithelial cells (<5 per low power field) in normal

urine samples. A greater number of epithelial cells are seen in urine samples collected by

catheterization or in patients with inflamed, hyperplastic, or neoplastic mucosa. Using wet mount

preparations, it can be challenging to distinguish the different types of epithelial cells, since

transitional cells are highly pleomorphic and many types of epithelial cells will become rounded

once sloughed into fluid and degenerate when exposed to urine. Cell morphology is best

appreciated in freshly formed and collected urine that is promptly analyzed. When evaluation of

cell morphology is critical, the sediment pellet can be evaluated by Diff Quik-stained, dry-mount

cytology of the urine sediment pellet. Other methods to diagnose structural lesions within the

urinary tract (e.g. ultrasonography, catheter biopsy) are often more reliable and conclusive than

urinalysis.

Squamous epithelial cells line the distal third of the urethra, the vagina, and the

prepuce. They are large, flat, or rolled cells that have angular sides and usually a single small,

condensed nucleus or they may be anucleate. A variable number of squamous epithelial cells

are most commonly observed with lower urinary tract contamination of voided or catheterized

samples. Squamous epithelial cells should not be present in samples collected by

cystocentesis. A significant number of squamous epithelial cells are very rarely seen in

cystocentesis samples due to squamous cell carcinoma of the bladder or due to squamous

metaplasia of the bladder, which can occur with transitional cell carcinoma or chronic bladder

irritation. Squamous epithelial cells may also be found if the uterine body of an intact female

was unintentionally penetrated during urine sample collection.

Transitional epithelial cells line the renal pelves, ureters, bladder,

and proximal two-thirds of the urethra. They are highly pleomorphic, variably

sized cells that are smaller than squamous epithelial cells and two to four

times larger than leukocytes. They may be round, oval, pear-shaped,

polygonal, or caudate and often have granular cytoplasm with a single

nucleus that is larger than that of squamous epithelial cells. There should be

<5 transitional epithelial cells per low power field in normal urine sediments. A

greater number of transitional epithelial cells are seen in urine samples

collected by catheterization or in patients with inflamed, hyperplastic, or

neoplastic mucosa. Transitional epithelial cells with caudate morphology

specifically line the renal pelves. Caudate transitional epithelial cells are

rarely observed in urine sediments and are an abnormal finding that can

sometimes be seen in patients with pyelonephritis, renal pelvic calculi, or

other pathology involving the renal pelves.

Cuboidal-to-low columnar renal tubular epithelial cells often

become small round cells once they have exfoliated into urine and are not

always easily distinguished from leukocytes or small transitional epithelial

cells. Unless these cells are found within a tubular cast, observation of renal

tubular epithelial cells is not considered a dependable indicator of renal

disease, since a low number of tubular cells are sloughed normally and since

other similarly sized cells (e.g. small transitional epithelial cells, leukocytes)

may be mistakenly identified as renal tubular epithelial cells in wet mount

preparations. Though, observation of a very large number of these cells with

their cuboidal-to-low columnar morphology intact (not rounded) is a rare

abnormal finding that would indicate active renal tubular disease.

Neoplastic epithelial cells are occasionally identified in urine

sediment. In a patient that has a bladder or urethral mass, the urine sediment

finding of atypical transitional epithelial cells in the absence of inflammation is

suggestive of transitional cell carcinoma. Neoplastic transitional epithelial

cells may exfoliate in cohesive sheets or individually. They are identified by

their malignant features, such as high nuclear-to-cytoplasmic ratio, variable cell and nuclear

size, clumped chromatin with prominent nucleoli, and mitotic activity. However when

Squamous

Transitional

Caudate

Neoplastic

Lipid

Erythrocytes

Erythrocytes

Leukocyte

inflammation is present, it is not possible to distinguish hyperplastic epithelial cells, which

develop similar cytologic features, from neoplastic epithelial cells. Since it is quite common for

bladder tumors to become secondarily inflamed, definitive diagnosis using urine cytology alone

is often not possible. In these instances, additional diagnostic information (e.g. imprint cytology

or histology of catheter biopsy material) may be helpful in making a definitive diagnosis. Other

less commonly observed tumors include rhabdomyosarcoma, urothelial papilloma, and

squamous cell carcinoma.

Blood cells, infectious organisms, and other sediment findings

Highly alkaline or dilute urine or improper sample storage may significantly reduce the

number of cells in the urine sediment. During microscopic examination care should be taken to

distinguish erythrocytes from lipid droplets. Lipid droplets are quite variably sized, refractile,

greenish discs that are usually smaller than erythrocytes, often float above the plane of focus,

and never exhibit the biconcave appearance of erythrocytes. They are frequently observed in

feline urine. Beyond their potential to be misidentified as erythrocytes, they are of little

significance.

Erythrocytes are quite translucent and may be pale orange due to their hemoglobin

content. Erythrocyte shape varies with the tonicity of the urine. They may maintain their

biconcave disc morphology; shrivel, becoming crenated in concentrated urine; or swell,

becoming rounded in dilute urine. There should be <5 erythrocytes per high power field.

However, the number observed can be influenced by collection method. Hematuria can be a

component of pathology seen with hemorrhagic diathesis (e.g. thrombocytopenia), infection,

inflammation, necrosis, neoplasia, toxicity (e.g. cyclophosphamide), or trauma.

In a sample collected by cystocentesis, there should be <3 leukocytes per high power

field. In a sample collected by catheterization or midstream voiding, there should be <8

leukocytes per high power field. Being larger than erythrocytes and smaller than epithelial cells,

leukocytes are intermediate in size compared to other cells that may be present in the sediment.

They are usually round with a stippled appearance and greyish internal structure that transmits

less light than erythrocytes; segmented nuclei are frequently visualized. Some leukocytes

contain granules that are occasionally visible as refractile structures within the leukocyte. These

cells may be referred to as glitter cells.

Observation of pyuria with concurrent bacteriuria indicates active urinary tract

inflammation with either primary or secondary bacterial infection. Urine culture is useful to

definitively identify microorganisms and to determine their antimicrobial sensitivities. Pyuria is

also seen with other causes of genitourinary tract inflammation, such as urolithiasis, neoplasms,

prostatitis, pyometra, and less common infections by viruses, mycoplasmas, or ureaplasmas.

Cystocentesis may avoid contamination of the urine sample by leukocytes from the genital tract

and aid in localizing the source of pyuria.

The absence of pyuria does not exclude the possibility of a urinary tract infection;

therefore urine sediment evaluation alone cannot be used to definitively exclude the possibility

of infectious urinary tract disease. Silent urinary tract infections (i.e. those lacking a detectable

inflammatory response) can be seen with hyperadrenocorticism/hypercortisolemia, diabetes

mellitus, and other immunosuppressed states. Also, leukocytes and bacteria may be diluted

below the detection limit of light microscopy in polyuric conditions when large volumes of dilute

urine are produced (e.g. pyelonephritis). At least 10,000 bacilli/mL or 100,000 cocci/mL are

required for detection by light microscopy.

Bacteria may be observed in urine sediment for reasons other than

urinary tract infection (e.g. normal finding in clinically normal animals,

overgrowth after collection). When microorganisms are observed in stained wet

mounts (e.g. sedi-stain, new methylene blue), it is necessary to distinguish

them from contaminants by confirming their presence in unstained wet mounts

or by cytologic examination of the dry-mount sediment pellet. The latter is a

more sensitive and specific method to detect bacteria than wet mounting and

permits more accurate identification of bacterial morphology (Swenson et al.,

2004). Urine cultures can occasionally be negative, even though bacteria were

presumptively detected during urinalysis. Reasons for this disparity include

antimicrobial administration prior to sample collection, prolonged urine storage

or extreme storage conditions prior to culture, improper culture technique,

bacterial contamination of the sample during urinalysis, misidentification of

nonbacterial structures as bacteria (common). Also, uncommon infections by

viruses or highly fastidious microorganisms (e.g. Mycoplasma, Ureaplasma)

may result in negative urine culture though a urinary tract infection is present.

Cystocentesis is the ideal collection method for urine culture, but take

care to avoid incidental enterocentesis. The likelihood of representative culture results may be

enhanced by collection of a randomly-timed urine sample (which will likely consist of freshly

formed urine that has not stagnated within the bladder) along with inoculation of a Culturette™

tube immediately after collection. Afterward, the Culturette™ should be refrigerated to prevent

overgrowth of robust bacteria.

Bacteria

Bacteria

Fungus, RBCs

Capillaria

With routine urine culture, results are reported that simply identify the bacteria present

and their respective antimicrobial sensitivities. Quantitative urine culture is an alternative may be

helpful to determine if the bacteria cultured from a urine sample likely represent a true infection

or if they are likely contaminants of the sample. Some labs offer quantitative urine culture as the

routine test. In addition to bacterial identification and elucidation of their antimicrobial

sensitivities, quantitative culture enumerates the bacteria as colony forming units (CFU)/mL.

Quantitative urine culture can be used even when the urine sample has been collected by

transurethral catheterization or by collection of midstream voided urine, instead of cytocentesis.

Quantitative urine culture results must be interpreted using published guidelines that are based

upon the urine collection method.

Other sediment findings Dependent on geographic distribution, other infectious organisms are occasionally

identified in urine sediment, such as fungi (e.g. Candida, Aspergillus, Blastomyces dermatitidis,

Cryptococcus); algae (e.g. Prototheca); and nematode ova, larvae, or adults (e.g. Capillaria,

Dirofilaria immitis, Dioctophyma renale). Trichuris (whipworm) parasite eggs appear very similar

to Capillaria parasite eggs. These two eggs can be distinguished by the positioning of their

bipolar caps and the texture of their outer shells. The bipolar caps of Capillaria ova are slightly

askew, rather than being perfectly bipolar as they are in Trichuris ova. And, the shells of

Capillaria ova have a granular appearance, rather than perfectly smooth as they are in Trichuris

ova. Capillaria are usually an incidental finding in the urine of asymptomatic cats. However,

Capillaria eggs are rarely identified in cats that present with hematuria, which resolves after

fenbendazole administration. Common contaminants of urine samples include sperm, talc, glass chips, plant pollen,

hair, and fibers. Aside from sperm, these contaminants can be mistaken for urine crystals (i.e.

talc, glass chips), transitional epithelial cells (i.e. plant pollen), or casts (i.e. hair, fibers).

Urinalysis: wet-mount versus dry-mount Urinalysis performed in-house by a trained staff member, soon after urine sample

collection is a great way to reduce or avoid artifacts that can arise during the delay between

sample collection and sample analysis. Keeping such analysis in-house may increase

productivity and potentially improve your urinalysis laboratory results. In addition to

traditional wet-mount urinalysis, dry-mount cytology of urine samples can be very useful in

suspected cases of urinary tract infection (Swenson et al., 2004) or urinary tract neoplasia. The

method is described below here. Slides prepared using this method can be evaluated in-house,

or they can be readily sent to an outside laboratory for review by a pathologist. The benefit is

that diagnostic material on slides prepared this way will not degrade the same way that it would

in a liquid urine sample during transport to an out-of-house, reference laboratory. Often when

fluid urine samples are sent to a reference laboratory for pathologist review to diagnose

neoplasia, cells deteriorate to the point where cytologic diagnosis no longer possible.

REFERENCES

1. Albasan H, Lulich JP, Osborne CA, Lekcharoensuk C, Ulrich LK and Carpenter KA (2003)

Effects of storage time and temperature on pH, specific gravity, and crystal formation in

urine samples from dogs and cats. Journal of the American Veterinary Medical Association

222, 176-179

2. Osborne CA and Stevens JB (1999) Urinalysis: A Clinical Guide to Compassionate Patient

Care, 1st edn, pp. 1-214. Veterinary Learning Systems, Bayer Corporation, Shawnee

Mission, Kansas

3. Swenson CL, Boisvert AM, Kruger JM and Gibbons-Burgener SN (2004) Evaluation of

modified Wright-staining of urine sediment as a method for accurate detection of bacteriuria

in dogs. Journal of the American Veterinary Medical Association 224, 1282-1289

PROTEINURIA TESTS Heather L. Wamsley, BS, DVM, PhD, Dipl. ACVP-Clinical

Tampa, Florida, USA

OBJECTIVES OF THE PRESENTATION Discuss diagnostic tests used to diagnose proteinuria

Provide information about guidelines for interpretation of urine protein to creatinine ratios and

microalbuminuria

To address questions that commonly arise during the presentation, SDMA will also be briefly

addressed.

KEY CLINICAL DIAGNOSTIC POINTS The duration and magnitude of proteinuria and whether or not there is concurrent azotemia

should guide the level of response to the proteinuria.

Microalbuminuria testing can be used to increase the sensitivity of UA for proteinuria in

samples with low USG.

Action values for UPC in azotemic animals are <0.5 for azotemic dogs and <0.4 for azotemic

cats.

Normotensive, non-hypercortisolemic, nonazotemic animals with persistent renal

microalbuminuria, UPC <1 should be monitored, particularly if they are breed-predisposed for

renal disease, have pre-existing renal disease, or have received treatments that may cause

renal damage.

Routine serum chemistry monitoring of blood creatinine by one laboratory with consistent

method is an excellent tool to assess renal function and diagnose early chronic kidney

disease. An increase of 0.3 mg/dL is significant even if creatinine value is still within the lab’s

reference interval.

In cats 15 years or older, creatinine >1.6 mg/dL should raise concern for renal disease.

Approximately 1 in 10 cats without chronic kidney disease can have elevated SDMA.

Feline diabetes mellitus and hypertrophic cardiomyopathy may affect SDMA reference

ranges. Cats with diabetes mellitus have significantly lower SDMA concentrations than

healthy animals. Values for cats with hypertrophic cardiomyopathy trended lower, but were

not statistically significant.

In humans, SDMA has been documented to increase with liver disease and multi-organ

failure. In trials that involved rabbits, diet and high cholesterol caused high SDMA.

As a test for pre-azotemic, “masked” chronic kidney disease in untreated hyperthyroid cats,

SDMA failed to predict the development of azotemia in most I131-treated hyperthyroid cats,

sensitivity 33%. Correlation between SDMA and GFR was poor in a population of ten I131-treated

hyperthyroid cats. Correlation between GFR and creatinine was moderate.

ADDITIONAL DETAIL Urine dipstick protein testing is performed to screen for diseases that cause excess

protein loss by the urinary tract, e.g. glomerular diseases, or that cause excess systemic

production of protein with overflow into urine, e.g. multiple myeloma. However, since other

nonpathologic or pathologic causes of proteinuria occur more commonly, a positive protein

reaction should be interpreted judiciously.

The dipstick protein reaction is most sensitive to albumin. Other proteins, e.g.

hemoglobin, immunoglobulin light chains - Bence Jones proteins, must be present at very high

concentrations to react with dipstick reagents and cause a positive reaction. The sulphosalicylic

acid protein precipitation test is a facile method that detects all types of protein and permits of

verification dipstick results in urine samples that are not markedly alkaline. The test is very easy

to perform and the single reagent used in the test has a long shelf-life. All types of protein are

detected by this method, therefore Bence Jones proteinuria, which would most likely go

undiagnosed by urine dipstick, can be identified.

Additional data from the urinalysis, i.e. specific gravity, pH, sediment examination, are

needed to determine the significance of proteinuria detected by the dipstick reaction. A positive

protein reaction should be interpreted in the context of the urine specific gravity, since a small

amount of protein (trace to 1+, <0.30 g/L) can be a normal finding in a single well-concentrated

urine (specific gravity >1.030) sample. Persistent trace or 1+ proteinuria should prompt further

investigation (Lees et al., 2005). A similar protein concentration in dilute urine or in an animal

receiving potentially nephrotoxic drugs regardless of urine concentration would be abnormal.

Consideration of the urine pH is necessary, since urine that is either markedly alkaline (pH >9)

or moderately alkaline (pH 7.5) and well-concentrated (specific gravity >1.035) will likely cause

a false positive dipstick protein reaction. In one study, false positive dipstick protein reactions

were more common in cats than in dogs (Grauer et al., 2004). The specificity of the dipstick

protein reaction was 31% in cats and 69% in dogs when compared to a species-specific

albumin ELISA (microalbumin test). False positive reactions may have occurred due to the

presence of non-albumin proteins or other interfering substances. Contamination of the urine

sample with cleanser residues or improper dipstick usage or storage can also cause false

positive reactions.

Knowledge of the urine sediment is requisite for accurate interpretation of urine dipstick

protein. The most common pathologic causes of increased urine protein concentration are

urinary tract inflammation, infection, hemorrhage, or some combination of the three that most

often arises from the lower urinary or genital tracts. In these instances, microscopic examination

of the urine sediment will likely disclose pyuria, bacteriuria, or hematuria. In order to attribute

dipstick proteinuria entirely to hemorrhage, the heme reaction should be at least 3+ (marked)

and macroscopic hematuria should be present. If the heme reaction is less than 3+ and no other

causes of a persistently positive protein reaction (e.g. pyuria, bacteriuria, spurious) are

detected, then underlying disease (e.g. nephrotic syndrome, multiple myeloma, proximal renal

tubular defect) may be present.

When significant dipstick proteinuria has been verified as persistent based upon a

second urinalysis and when other sources of a positive protein reaction (e.g. pyuria with or

without bacteriuria) have been excluded, measurement of the urine protein-to-creatinine ratio

is necessary to more precisely determine the severity of proteinuria. The urine protein-to-

creatinine ratio should be <0.5 in nonazotemic animals, <0.5 for azotemic dogs, and <0.4 for

azotemic cats (Lees et al., 2005). A urine protein-to-creatinine ratio that is >1.0 in a sample

obtained by cystocentesis should rouse concern for glomerular disease (e.g. glomerulonephritis,

glomerulosclerosis, or canine amyloidosis), Bence Jones proteinuria, or much less commonly

tubular proteinuria. Serum biochemistry and serum or urine protein electrophoresis are useful

initial tests that will help localize the cause of proteinuria to preglomerular versus renal causes,

and these results may direct the course of future diagnostic testing (e.g. bone marrow biopsy

versus renal biopsy).

Serial measurement of the urine protein-to-creatinine ratio can be used to stage the

progression of renal disease and to evaluate the response to therapy. Determination of the urine

protein-to-creatinine ratio may also be performed to help establish the prognosis for newly

diagnosed cases of canine chronic renal failure (Jacob et al., 2005). A urine protein-to-

creatinine ratio that is >1.0 at the time of initial chronic renal failure diagnosis was a negative

prognostic indicator. Compared to patients with a urine protein-to-creatinine ratio that was <1.0,

canine chronic renal failure patients with a ratio that was >1.0 experienced more rapid

progression of renal disease, greater likelihood of uremic crisis, and greater risk of death due to

either renal or nonrenal causes. The rate of disease progression and risk of complications were

directly proportional to the magnitude of urine protein-to-creatinine ratio elevation. Similarly,

proteinuria predicts reduced survival times in healthy, nonazotemic cats (Walker et al., 2004)

and in cats with chronic renal failure (Syme and Elliot, 2003). Urine protein-to-creatinine ratios

>0.3 in healthy nonazotemic cats or >0.4 in cats with chronic renal failure were significant

predictors of reduced survival times in these two studies.

Microalbuminuria refers to increased urine albumin that remains beneath the detection

limit of the urine dipstick protein reaction (i.e. urine albumin >0.01 g/L but <0.30 g/L). If the urine

dipstick test is positive for protein, then the microalbuminuria test should also be positive. The

microalbuminuria test can be used to confirm the dipstick reaction. The prevalence of

microalbuminuria has been reported as 15 to 19% in clinically normal dogs (Gary et al., 2004;

Jensen et al., 2001) and 14% in clinically normal cats. Increased prevalence has been reported

with older age or the presence of nonrenal disease. In canine experimental models of

progressive glomerular disease, the prevalence of microalbuminuria was 75-100% and

persistent microalbuminuria preceded the development of overt proteinuria (Grauer et al., 2002;

Lees et al., 2002). These investigations suggest that, similar to humans, detection of persistent

microalbuminuria in dogs may aid in early diagnosis of occult glomerular disease prior to the

development of a positive dipstick protein reaction or increased urine protein-to-creatinine ratio.

The microalbuminuria test is a supplement to dipstick protein testing. It helps to detect protein in

the urine that is lower than what the dipstick can detect. It can be used to boost the sensitivity of

UA for protein detection in urine that is not maximally concentrated. The same rules for judicious

interpretation of proteinuria apply to the microalbuminuria test.

Algorithm for microalbuminuria / proteinuria interpretation:

Test for microalbuminuria---Negative---Rescreen in one year

Test for microalbuminuria---Positive---Condition that may contribute to protein in the

urine / invalidate albumin testing?----Yes----Treat or wait until resolved. Repeat

microalbumin test.

Test for microalbuminuria---Positive---Condition that may contribute to protein in the

urine / invalidate albumin testing?----No----Re-check in 2 weeks. Document 3 positive

tests. Monitor microalbumin or UPC. For monitoring, microalbuminuria testing may be

more economical and convenient than UPC testing.

o UPC >0.5 dog, >0.4 cat and nonazotemic, monitor

o UPC >1 and nonazotemic, investigate for conditions that cause renal proteinuria

o UPC >2, intervene

o UPC >0.5 dog, >0.4 cat and azotemic, intervene

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Symmetrical dimethylarginine: a new combined parameter for renal function and extent of coronary artery disease. Journal of the American Society of Nephrology 17, 1128-34

2. Buresova E, Stock E, Paepe D, Stammeleer L, Vandermeulen E, Smets P, Duchateau L, Lefebvre HP, and

Daminet S (2019) Assessment of symmetric dimethylarginine as a biomarker of renal function in hyperthyroid cats

treated with radioiodine. Journal of Veterinary Internal Medicine 33, 516-522. 3. Hall JA, Yerramilli M, Obare E, Yerramilli M and Jewell DE (2014) Comparison of serum concentrations of

symmetric dimethylarginine and creatinine as kidney function biomarkers in cats with chronic kidney disease.

Journal of Veterinary Internal Medicine 28, 1676-83 4. Gary AT, Cohn LA, Kerl ME and Jensen WA (2004) The effects of exercise on urinary albumin excretion in dogs.

Journal of Veterinary Internal Medicine 18, 52-55

5. Grauer GF, Oberhauser EB and Basaraba RJ (2002 Abstract) Development of microalbuminuria in dogs with heartworm disease. Journal of Veterinary Internal Medicine 16, 352 A103

6. Grauer GF, Moore LE, Smith AR and Jensen WA (2004 Abstract) Comparison of conventional urine protein test

strip method and quantitative ELISA for the detection of canine and feline albuminuria. Journal of Veterinary

Internal Medicine 18, 418-419 A127

7. Jacob F, Polzin DJ, Osborne CA, Neaton JD, Kirk CA, Allen TA and Swanson LL (2005) Evaluation of the

association between initial proteinuria and morbidity rate or death in dogs with naturally occurring chronic renal failure. Journal of the American Veterinary Medical Association 226, 393-400

8. Jensen WA, Grauer GF and Andrews J (2001 Abstract) Prevalence of microalbuminuria in dogs. Journal of

Veterinary Internal Medicine 15, 300 A113 9. Langhorn R, Kieler IN, Koch J, Christiansen LB and Jessen LR (2018) Symmetric dimethylarginine in cats with

hypertrophic cardiomyopathy and diabetes mellitus. Journal of Veterinary Internal Medicine 32, 57-63

10. Lees GE, Jensen WA and Simpson DF (2002 Abstract) Persistent albuminuria precedes onset of overt proteinuria in male dogs with X-linked hereditary nephropathy. Journal of Veterinary Internal Medicine 16, 353 A108

11. Lees GE, Brown SA, Elliot J, Grauer GF and Vaden SL (2005) Assessment and management of proteinuria in

dogs and cats: 2004 ACVIM forum consensus statement (small animal). Journal of Veterinary Internal Medicine

19, 377-385 12. Peterson ME, Varela FV, Rishniw M and Polzin DJ (2018) Evaluation of Serum Symmetric Dimethylarginine

Concentration as a Marker for Masked Chronic Kidney Disease in Cats With Hyperthyroidism. Journal of

Veterinary Internal Medicine 32, 295-304 13. Siroen MP, van der Sijp JR, Teerlink T, van Schaik C, Nijveldt RJ and van Leeuwen PA (2005) The human liver

clears both asymmetric and symmetric dimethylarginine. Hepatology 41, 559-65

14. Syme HM and Elliot J (2003 abstract) Relation of survival time and urinary protein excretion in cats with renal failure and/or hypertension. Journal of Veterinary Internal Medicine 17, 405 A106

15. Walker D, Syme HM, Markwell P and Elliot J (2004 Abstract) Predictors of survival in healthy, non-azotemic cats.

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