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How to Improve Urine Culture Results

A recent study shows that refrigeration of urine at 5 degrees C for 24 hours does not significantly decrease the colony forming unit (CFU) counts in dogs with signs of urinary tract infection (UTI).  Dr Mark Acierno and colleagues at LSU found that bacterial growth was 35.7% in immediately processed samples and 33.7% in refrigerated samples, but that refrigeration in tryptic soy broth (TSB) resulted in bacterial growth in only 31.7% of the samples.  Thus, the sensitivity of culture of refrigerated samples compared to non-refrigerated samples was 95% but only 89% in refrigerated samples in TSB.  For best results, we recommend that urine samples be submitted as undiluted urine or on a culture swab, and not in broth.

Urine sediment may deteriorate with time irrespective of refrigeration as crystal may form or dissolve depending on the pH of the urine.  Refrigeration will tend to preserve cells and possibly slow bacteria overgrowth in free catch samples.  Erythrocytes may lyse due to osmolal stress especially in dilute samples.

If a cytologic exam is desired, please make an air-dried sediment slide, since this will preserve cell integrity and prohibit bacterial overgrowth.

 

Abstract: JAVMA, Acierno, MJ et al, 7/15/2018:Vol 253, No. 2, pgs 177-180.

 

Hemolysis and Lipemia Effects on Sample Results

Serum chemistry values are often derived by spectrophotometrically measuring change in the amount of light transmitted through serum after a chemical reaction changes the color density of the sample by an amount relative to the concentration of the target substance (glucose, creatinine, or ALT for example).  If the serum is hemolysed it changes the baseline color and there may be fragments of RBCs in the serum as well, interfering with the measurement of light in unpredictable ways.  If the serum is lipemic, globules of fat may interfere with the measurement of light in unpredictable ways.  With hemolysis, typically our lab sees marked elevations in total protein, albumen and phosphorus with variable increases in ALT, AST. T Bili, glucose, calcium and occasionally decreased creatinine and ALP.  Lipemic specimens can have variably increased creatinine, ALT, AST. ALP. T Bili, glucose, calcium, phosphorus, and total protein.

 

CBC results can be affected by hemolysis due to decreased RBCs, and both hemolysis and lipemia can affect turbidometric values such as hemoglobin and the indices calculated from hemoglobin.

 

It is suggested that serum be centrifuged and separated from the blood to decrease in-vitro hemolysis and document lipemia.  If the sample is lipemic, it might be helpful to wait a 4-8 hours and redraw the sample.  Remind owners that feeding before a blood draw can cause inaccurate results.

Maintain a Stable Sample in Winter

When it is cold outside, samples shipped via postal or commercial carriers could be exposed to very low temperatures that destroy intact cells. Consider the following, to minimize the risk of damaged samples:

 

  • Serum samples should be spun and separated, since frozen RBCs can lyse and result in hemolysis in the serum.
  • Separator gel may prove an inadequate barrier in very cold weather. For those who have 3-step quick stains, Cytology slides can be fixed with a few dips in methanol fixative prior to shipping.
  • Liquid samples are best preserved by packing them in insulating material. Styrofoam packs, silver mylar baggies and bubble wrap all help with insulation.

 

If you have questions about ensuring the integrity of your samples, just give us a call.

Cytology Sample Submission

Fine needle aspirates (FNA) for cytologic examination can be submitted several ways. FNA material from a mass should be gently expressed from the syringe onto a clean, frosted edge glass slide after which a slide should be placed over the first slide, then slid across it quickly (not pulled apart), producing a monolayer that will stain well. 

 

Fluid can be submitted in a tube, (EDTA is acceptable, if culture is not desired), but a slide should be made at the hospital before shipping, to insure intact cell morphology and minimal bacterial overgrowth in the event of delay in transit or damage by heat or cold.

 

All slides should be labeled with the name of patient, the site of collection, and the date. When sending multiple sites, labeling the slide holder without labeling the slides can result in lab error; once the slides are unpacked, the location will not be obvious on the appropriate slide.

 

Samples can be submitted air dried or, after drying, fixed in methanol.  Fixation prior to shipping, especially if formalinized biopsies are shipped in the same box, will preserve the integrity of the cells and eliminate formalin damage that causes stain failure.

 

It is always a good idea to stain a slide to confirm that the sample is adequate.

Diagnosing Suspected Lymphoma

Clinical signs that cause suspicion of lymphoma include lymphadenopathy, hepatomegaly, splenomegaly, and chronic GI disorder such as vomiting or diarrhea.  Fine needle aspirate (FNA) of the area of interest is a good first step.  A monomorphic population of lymphocytes, especially in an area where lymphocytes do not usually reside, can be diagnostic.  Antigen receptor site rearrangement PCR (PARR) of stained or unstained cytology slides can be performed at a reference lab if additional information is needed.  PARR is helpful to identify the lymphocyte type (T or B) and confirm clonality (which supports lymphoid neoplasia when positive).  In cases where a presurgical FNA is impractical, formalin fixed biopsy can be diagnostic of lymphoma. Immunohistochemistry (to determine the lymphocyte type) or PARR (for lymphocyte type and confirmation of clonality) can be performed on the fixed tissue for additional information.

Mast Cell Tumor in Cats

Mast cell tumor in cats typically presents as a skin mass composed of slightly pleomorphic mast cells with 0-1 mitotic figures/10 HPF (400x) and vague granulation.  There can be variably dense eosinophilic infiltrates.  Unlike canine mast cell tumors which have not just one but 2 prognostically significant grading systems (the Patnaik and the Kiupel/MSU systems), there is not a widely recognized grading system for feline mast cell tumors. Molecular parameters such as Kit receptor analysis are also not applicable at this time.  A recent study however, reported by Sabattini and Bettini  in Veterinary Pathology 56(1), 43-49, 2019 finds that tumors with >5 mitotic figures/ 10 HPF and 2 of the 3 criteria such as tumor diameter over 1.5 cm, irregular nuclear shape, and nucleolar prominence, exhibited high grade behavior with reduced survival time (median 349 days). 

 

This tumor should always be considered to have potential for local invasion and distant metastasis. Tumors may arise in the skin or in the internal organs, usually the spleen. Upon diagnosing this tumor in the cat, check the local lymph nodes, watch adjacent skin for other masses, palpate (gently) the liver and spleen for organomegaly that might indicate visceral mast cell tumor, and check the CBC for circulating mast cells. Aggressive palpation may result in mast cell degranulation, release of vasoactive amines, and hypotension, so care is warranted. It is important to determine if the margins are clean with histologic evaluation as complete excision of this type of tumor is often curative.