S U M M E R
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The Saline Agglutination Test—why you
might reconsider performing it
Jennifer L. Brazzell, DVM, MVetSc, MRCVS, Diplomate ACVD;
Veterinary Pathologist;
Marshfield Labs Veterinary Services
BACKGROUND
Inside This Issue
The Saline Agglutination
Test—why you might
reconsider performing it.....1
How and why you should read
all of your pathologist’s
report.......................................3
Did you know? --surgical
biopsy submissions. ................5
A few times a year I receive a phone call from a veterinarian wanting
to discuss a patient with suspected immune-mediated hemolytic
anemia. During the discussion, there is mention of a “positive
saline agglutination test” which prompts a discussion about why
the test was performed to begin with and what is meant by the
statement “positive saline agglutination test.” Does this statement
mean that saline was added to a peripheral blood sample and
agglutination was noted or that suspected agglutination was noted
macroscopically and persisted after addition of saline? I find that
more often than not, the test does not provide any additional
information that could not be gleaned from a CBC, peripheral blood
smear review, and biochemical analysis; that it is often performed
without standardization; and that interpretation is equivocal at best.
TEST USE
This test is purported to be a clinical method to differentiate
erythrocyte agglutination from erythrocyte rouleaux. This is a
problem most often encountered in cats that are prone to low-level
rouleaux normally but can also be noted less frequently in dogs.
TEST PROTOCOL
To perform the test, whole blood is diluted with saline and a
wet preparation of the blood is made on a microscope slide,
coverslipped, and microscopically examined. A quick literature and
internet search on this topic led me to multiple non-standardized
protocols recommending the use of anywhere from a 1:1 dilution
blood:saline all the way up to a 1:9 dilution blood:saline; single or
multiple washes; immediate examination or rocking the slide and
waiting 30 seconds to 1 minute before evaluation; macroscopic
examination only versus macroscopic and microscopic examination.
You can see how the usefulness of the test is limited when there is
no standardized method for performing or interpreting the test.
BEYOND numbers
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RFPT0616
TEST INTERPRETATION
In theory, saline should disrupt the protein-protein interactions between plasma proteins
and the erythrocyte membranes responsible for rouleaux but will not disrupt true antibodymediated erythrocyte agglutination. You can see now how the name “saline agglutination
test” is not appropriate as it implies that the saline causes agglutination. Historically, a
positive saline agglutination test is one where agglutination is NOT dispersed—very confusing
indeed! A preferred name for this test is the “saline dispersion test” or “saline dilution test”
and a positive test would be a test where the saline does in fact disperse the erythrocyte
aggregation.
PROBLEMS WITH THE TEST
This is a test that is only used in veterinary medicine with no correlate in human laboratory
medicine. This test lacks standardization with regard to: the ratio of blood to saline; the
time to wait before reading results; degree of mixing; temperature at which to perform the
test; and whether results can be seen macroscopically or require microscopic examination.
There is also lack of standardization of result interpretation and reporting--some published
literature implies that agglutination will form when saline is added to whole blood and
that this is a positive test. In addition, there is remarkable biologic variability and multiple
etiologies responsible for rouleaux and erythrocyte autoagglutination that even with test
standardization, results would be difficult to interpret. It is recognized that some rouleaux
will not disperse with typical 1:1 or 1:2 dilutions with saline; that overdilution can disperse some
low affinity antibody-mediated erythrocyte binding; and that some cases of immune-mediated
hemolytic anemia will not have obvious macroscopic or microscopic agglutination. Therefore,
the lack of agglutination and/or dispersion of rouleaux does not rule out the possibility of
immune-mediated hemolytic anemia and likewise the continued presence of erythrocyte
aggregation following saline dilution does not rule in agglutination or rule out rouleaux!
THE BOTTOM LINE
The saline dilution/dispersion test is not recommended by most clinical pathologists due
to lack of standardization, misconceptions about performing and interpreting the test,
and inability to produce accurate, reproducible results. A diagnosis of rouleaux versus
agglutination should rely on combined interpretation of the history, biochemical profile,
complete blood count, peripheral blood smear review, +/- Coombs’ testing. Classic cases of
immune-mediated hemolytic anemia will involve a strongly regenerative anemia, an elevated
serum bilirubin concentration, spherocytosis (in dogs) and/or erythrocyte agglutination.
CLINICAL PEARL
Rouleaux is unlikely to be the cause of erythrocyte aggregation if it incorporates
polychromatophilic erythrocytes.
Rouleaux (Figure 1A, page 3) tends to form variable length linear, sometimes branching,
chains of rowed erythrocytes and is typically associated with normal erythrocyte mass
and morphology. Rouleaux WILL NOT incorporate polychromatophilic erythrocytes
(reticulocytes). Agglutination (Figures 1B and 1C, page 3) tends to accompany a regenerative
anemia with anisocytosis, polychromatic spherocytosis (in dogs), and will form varisized
clusters of erythrocytes that may incorporate polychromatophilic erythrocytes.
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Figure 1: Rouleaux vs. Agglutination.
Note the presence and incorporation of polychromatophilic erythrocytes (arrows)
into the erythrocyte aggregates associated with agglutination.
Figure 1 (A):
Rouleaux in a Cat.
Figure 1 (B):
Agglutination in a Cat.
Figure 1 (C):
Agglutination in a Dog.
How and why you should read all of your pathologist’s
report
COMPONENTS OF A PATHOLOGY REPORT
A typical pathology (cytology or histology) report will consist of the following seven
components:
1. Patient Identifiers and Clinic Information
2. Specimen
3. Clinical History
4. Gross Description
5. Microscopic Description
6. Diagnosis
7. Comments
1. Patient and Clinic Information
To ensure that the report represents the patient in question, each report contains patient and
clinic identifiers. Make sure that the report you receive matches the patient signalment and
your clinic name. Is “Mrs. Fluffypants” really an intact male 5 year old German Shepherd cross
dog? Possibly but this may be questioned. This section will also contain a unique accession
number that can be cross-referenced with your in-clinic or on-line requisition forms/requests.
2. Specimen
This section will describe the sample received. For example: “Four unstained slides” for a
cytologic specimen or “One container” for a histologic specimen. Reading this section will
ensure that all slides or tissues collected were received and processed.
3. Clinical History
The clinical history you provide on the submission form is transcribed into this section. This
should be read to ensure accurate transcription since interpretation of microscopic findings is
often made with the clinical history in mind. On that same note, be sure to provide pertinent
clinical history to aid with interpretation.
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4. Gross Description
This section is present only in histology reports. It will describe the macroscopic (gross)
appearance of the tissues received including the number of pieces and their physical
appearance (whole and upon sectioning). It will also describe how the sample was trimmed
in order to paraffin embed the tissue, including how many pieces of tissue and the number
of cassettes collected. For example, if you see 4c8p in your report, this indicates that 8
pieces of tissue were collected into 4 cassettes.
5. Microscopic Description
This section is often overlooked but contains important information even to the practicing
veterinarian. This section describes how the submitted specimen looks under the
microscope. The pathologist will indicate what tissue is being examined and this should
correlate with the sample submitted. Likewise, the description of the lesion should
correlate with the gross description and your clinical impression of the lesion. Incidental
findings that may or may not be related to the primary diagnosis will also be described.
For example, there may be low level chronic (cholangio) hepatitis adjacent to the focus of
nodular hyperplasia in a liver. While this finding is incidental, it may impact the treatment
and monitoring of the patient going forward. If the sample represents a tumor, this section
is where surgical margins are reported which tells you whether the tumor was completely,
narrowly, or incompletely excised.
6. Diagnosis
The pathologist will interpret the macroscopic and microscopic findings in light of the
clinical history provided in order to make a diagnosis. If incidental findings are thought to
be clinically relevant, they will also be reported in this section. The diagnosis is occasionally
not definitive in which case the cytological or histologic microscopic appearance
(morphologic diagnosis) may be recorded and differential diagnoses listed.
7. Comments
The comments section is used to provide information regarding the cause, behavior, and/
or typical prognosis of the diagnosed lesion. This section is also used to discuss why the
submitted sample may not be definitively diagnostic. Additional tests that might help
determine the etiology, expected clinical behavior or prognosis, or that might help make a
more definitive diagnosis may also be recommended in this section.
It is important to read all of the report and not just the diagnosis! Reading the entire
report is a way for you to ensure that no sample submission, processing, or reporting
mistakes have been made. Nowadays, it is also common for clients to request and read
copies of their pet’s pathology reports and I can assure you that they read the report front
to back and will ask you to explain parts they don’t understand. Familiarizing yourself
with pathology descriptors will help with client communications regarding the report, and
discussion of the report with your pathologist should you have any questions.
Marshfield Labs prides itself on superior customer service and any of our pathologists
would be happy to discuss any questions or concerns you might have regarding a
pathology report.
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Did You Know?...
Did you know that there are six steps involved in processing surgical biopsy submissions?
Or that on average, it takes approximately 18-24 hours to take your submitted surgical
biopsy sample and turn it into a glass slide that can be evaluated by a pathologist?!
Before a pathologist can even look at a slide from your submission, the tissue must be:
1. Entirely fixed—this may take an additional 24-48 hours after arrival in the lab if the
sample is large.
2. Examined macroscopically and trimmed to fit into plastic tissue cassettes (“grossing”).
Once tissues are examined and trimmed to fit in the tissue cassettes, the macroscopic
examination is dictated into the “Gross Examination” portion of the histology report.
3. Processed—this involves sequential dehydration of the tissue and replacement of fluids
with paraffin. This step usually occurs overnight and takes 8-12 hours.
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4. Paraffin embedded—the plastic cassettes containing pieces of paraffinized tissue are
removed from the cassette and placed in a mold which is then filled with liquid paraffin.
After cooling, the cassette top and the paraffin embedded tissue become one unit known
as a paraffin block.
5. Cut into thin sections and placed on a slide (microtomy)—a microtome is used to cut
sequential 4-5 um sections of the paraffin block producing a long ribbon which is floated
on a bath of warm water to smooth wrinkles and allow placement on a glass slide.
6. Stained and coverslipped—the slide is dried and passed through another series of
chemical reagents to remove the paraffin and rehydrate the tissue in order to stain with
hematoxylin and eosin (H&E) stain. Once stained, slides are coverslipped and are finally
ready to be evaluated by a pathologist.
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