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What are the ways to test for protein in the canine urine?

What are the ways to test for protein in the canine urine?


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I know that one way is to use the reagent sticks and the refractometer, but this is inaccurate. Is protein distinguishable when using a microscope? Is it time sensitive(i.e. have to test it right away)?


After some research, I could primarily find two tests for testing the protein content in dog urine.

1) The Urinalysis dipstick test (reference) which has a chance of giving false positive results (reference). Here is the link to a lab test done using the dipstick method and the results that the researchers got.

A sample Dipstick (reference)

2) The second test would be the benzethonium chloride test (reference).

This test precipitates the protein and increases the turbidity of the sample. The turbidity is proportional to the protein concentration. This technique is very sensitive and can yield accurate results in samples with very low protein concentrations (< 20 mg/dL).

A lab study done using this procedure to precipitate protein (reference).

The urine sample is best read within 30 minutes of collection otherwise certain factors (like crystals, bacteria and cells) may alter their composition (dissolve or multiply) over time. If you collect a urine sample at home, please drop it off at your veterinary clinic as soon as possible (reference).

Reasons for excess protein if present in the urine of your pet is listed in this article.


What are the ways to test for protein in the canine urine? - Biology

Post-glomerular causes of renal protein loss such as hemorrhage and inflammation also contribute to urine protein quantification.

Traditionally, urine protein loss has been detected either through a qualitative test such as a urine dipstick or via a semi-quantitative test such as a urine protein creatinine ratio.

A urine protein creatinine ratio greater than 0.5-1 is considered abnormal.
However, both the dipstick method and the urine protein creatinine ratio can be inaccurate and can yield false positive results due to contamination of urine with red blood cells, white blood cells and bacterial protein and therefore must be measured on urine sample with an inactive sediment and a negative culture.

A 24-hour urine protein quantification is more accurate but is technically more difficult to obtain, requiring hospitalization and 24-hour urinary catheterization with a closed collection system.

However, a new alternative test is now available to detect proteinuria in dogs and is currently being released for cats. The Heska E.R.D. HealthScreen Canine Urine test is a semi-quantitative test for microalbuminuria in dogs.

The advantages of this test are that it is a point-of-care test which detects small amounts of albumin in the urine prior to the detection of protein via a urine protein creatinine ratio. Microalbuminuria is defined as the presence of albumin in the urine between the concentrations of 1.0 and 30.0 mg/dl (below the detection ability of a urine dipstick test or urine protein creatinine ratio).

Thus this provides an earlier indicator of renal damage and protein loss. This test utilizes an anti-canine albumin antibody and therefore is specific for canine albumin in the urine.

A secondary advantage is that there is little cross reaction between urine albumin and red blood cells. If macroscopic hematuria is present (i.e. the urine is obviously red in color), then cross-reactivity with microalbuminuria can occur, but this does not occur with microscopic hematuria (Proceedings of the 21st ACVIM Forum, SL Vaden, Microalbuminuria: What is it, and how do I interpret it?).

The cross-reactivity of pyuria is somewhat more variable. In one study, 67 percent of dogs with pyuria had urine albumin concentrations less than 1 mg/dl. [Proceedings of the 21st ACVIM Forum, SL Vaden, Microalbuminuria: What is it, and how do I interpret it? Vaden SL et al. J Vet Intern Med 2002 16:378 (abstract)].

In a large scale study of 3,041 dogs owned by employees of veterinary hospitals, the overall prevalence of microalbuminuria in dogs was 24.7 percent (S. Radecki et al. Proceedings of the 21st ACVIM Forum, abstract #110).

In the same study, the prevalence of microalbuminuria was significantly higher in the older population of dogs (4 percent at one year and 55 percent at 15 years).

This finding also correlates with the fact that proteinuria occurs in dogs with underlying diseases, which are more common in geriatric dogs. A follow-up study was conducted to survey for underlying diseases in the population of dogs with positive tests.

Follow-up information was provided for 572 dogs. Fifty-six percent of dogs were subsequently diagnosed with inflammatory, infectious, metabolic or other disease processes (www.heska.com/erdscreen/erd_data572.asp). These results are also consistent with those found by Whittemore et al., in which 56 percent of clinically ill dogs with microalbuminuria were found to have infectious, inflammatory or neoplastic disease (JC Whittemore et al. Proceedings of the 21st ACVIM Forum, abstract #234).

This is an important finding because proteinuria is often associated with underlying disease processes and thus the ERD test can be used as a screening test in older patients as a marker for these underlying processes.

Whittemore et al. also demonstrated that this ERD test was more sensitive in detecting proteinuria than the urine dipstick or the urine protein creatinine ratio. In a population of clinically ill dogs, 55 dogs with proteinuria were diagnosed positive using the ERD test but were not detected using a urine dipstick or urine protein creatinine ratio.

Current recommendations for clinical practice are to use the ERD test as a screening test in young dogs of breeds with hereditary predisposition to glomerulopathy. Since there is a marked increase in the prevalence of microalbuminuria in dogs greater than 6 years of age, it may be prudent to begin screening dogs beginning at that age (Proceedings of the 21st ACVIM Forum, S. Vaden `Microalbuminuria: What is it and how do I interpret it?).

If a positive test is found (and is a repeatable finding), then additional testing can be pursued to assess for underlying diseases. Further quantification can be done, if desired, using an ELISA for canine albumin but must be submitted to a reference laboratory. Progression to overt proteinuria can be monitored closely.

Overt proteinuria is currently managed with a low protein diet and administration of an ACE inhibitor such as Enalapril. Addition of an anti-thrombotic such as aspirin may be used in advanced cases.

Further information is required in order to determine at which point therapeutic intervention would be most beneficial, although theoretically early detection does allow for earlier intervention where appropriate.

Source: Johanna Frank (2003): An update on the diagnosis of proteinuria in dogs. In: DVM Newsmagazine Oct 1, 2003 www.dvmnewsmagazine.com/dvm/

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Urinalysis Protocols

Collection protocol

Instruments

Samples are analyzed using Multistix urine dipsticks (Siemens Healthcare Diagnostics Inc., Deerfield, IL), and refractometry and light microscopy.

Sample Requirements

Urine collected by free catch, catheterization or cystocentesis may be submitted. Sterile/aseptic collection technique, when possible, is recommended to prevent fecal and environmental contamination.

Preferred sample volume*

* Occasionally, smaller volume samples may be accommodated. Please contact the lab regarding submission of these samples and the testing that is available.

Appropriate Tubes

Urine may be submitted in any sterile, additive-free, plastic or glass container of appropriate volume, including syringes, Eppendorf tubes, and plain red top tubes.

Sample Handling

Label samples using permanent marker with a legible, simplified numbering system. For dipstick, specific gravity and sediment examination, samples should be refrigerated as soon as possible following collection and must be analyzed within 24 hours for valid results. They should not be frozen or transported directly on ice. Transport with cold packs is recommended. Chemistry tests on urine have prolonged stability. Please contact the lab directly if you wish to submit frozen or archived urine samples for chemistry tests.

Results

Parameters generated from the urine dipstick include semi-quantitative measurement of urobilinogen**, protein, pH, blood***, ketones****, bilirubin and glucose. Confirmatory tests for the presence of bilirubin (Ictotest), ketones (Acetest, acetoacetic acid) and protein (sulfasalicylic acid precipitation test, SSA) may be performed if there is a positive result on the dipstick. Urine specific gravity is measured via refractometry. Sediment examination evaluates for the presence of red blood cells, white blood cells, epithelial cells, crystals, casts, sperm, lipid/fat and bacteria or fungus.

Common Interferences

Samples with marked blood contamination/hematuria, hemoglobinuria and bilirubinuria may result in color interference with dipstick parameters. Oral ascorbic acid supplementation may result in false negative urine glucose. Bleach, peroxide and other strong oxidizing agents may result in false positive urine glucose. Quarternary ammonium compounds and highly alkaline urine may result in false positive urine glucose.

* Occasionally, smaller volume samples may be accommodated. Please contact the lab regarding submission of these samples and the testing that is available.

** Urobilinogen is only reported out in non-human primate samples, as it is not considered to be reliable or valid in most veterinary species.

*** Intact red blood cells, myoglobin and hemoglobin may each result in a positive dipstick reaction for blood.

**** Acetoacetic acid is the only ketone measured by the urine dipstick. Other undetected ketones (beta-hydroxybutyrate, acetone) may be present in the urine of veterinary species.


Large Volume Protocols

Large volume biochemical profile: Collection protocols for large volume specimens (dogs, pigs, camelids, horses, etc.)

Instrument

Beckman AU480, Beckman Coulter, Brea, CA

Sample Requirements

Blood should be collected into plain red top tubes with no additive or gel separator. Following collection, blood should be allowed to clot, centrifuged at 1700 rcf* for 10 minutes, the serum removed and placed into another plain red top tube or other, sterile, additive-free, plastic tube. To prevent analytical error, serum should be removed from the clot within 1 hour of sampling. Required volumes are listed in the table below.

*RCF = relative centrifugal force = 0.00001118 x r (rotational radius in cm) x RPM2

Required serum volume
(Note: not whole blood volume)

**If the specimen is short volume, the lab will attempt to run as many tests as possible.

Appropriate Tubes

The following tubes are suitable for blood collection and analysis using the Beckman Coulter AU480. Please contact the lab if you have questions regarding the appropriate tube to utilize for your samples.

Name Sample volume Mfr #
BD Vacutainer Blood Tubes (glass tubes, no additive) 3 mL 366397
BD Vacutainer Blood Tubes (glass tubes, no additive) 7 mL 366397
BD Vacutainer Blood Tubes (glass tubes, no additive) 10 mL

Sample Handling

Label tubes using permanent marker with a legible, simplified numbering system. Samples should be refrigerated as soon as possible following collection. Transport with cold packs is recommended do not transport directly on ice. Please contact the lab for further instruction if you would like to submit archived, frozen samples for analysis.

Results

Results are available in Excel format.

Common Interferences

Certain degrees of lipemia, hemolysis, and icterus (LIH) may interfere with analysis of some analytes. LIH indices and the expected interferences are reported for basic and full profile samples. Potassium levels may be artifactually increased and glucose artifactually decreased in samples where serum has sat on the blood clot for an extended period of time.


When is crystalluria a concern?

Crystalluria is often an incidental finding, and in many cases, no further investigation or treatment is needed your veterinarian can advise you if this is the case with your dog. However, if your dog is showing signs of a urinary tract infection — having difficulty urinating, urinating frequently, urinating in unusual locations, or has blood in the urine — it’s important to have a veterinarian examine your dog and perform further diagnostic tests. The signs could be caused by bladder stones, and the earlier the stones are diagnosed, the more options may be available for treatment other than proceeding straight to surgical removal.


Urine Testing: Why Test Your Cat's Urine?

Having regular veterinary examinations performed for your cat is the best way to keep your cat healthy. In most cases, your veterinarian will likely recommend blood and urine testing as part of a thorough examination. If your cat is not feeling well, blood and urine testing may be necessary to diagnose your cat’s illness.

We’ve talked about common blood tests and what we can learn from them in a previous post. Today, I’d like to talk about urine tests and explain what your veterinarian may be looking for in your cat’s urine.

Urinalysis is, by far, the most commonly performed urine test. A urinalysis (or UA as it is often called) is actually made up of many different tests. A typical urinalysis tests for the following:

  • Visual Evaluation: If your cat’s urine is discolored or has an abnormal clarity (i.e. cloudy urine for instance), these findings will be noted here. Normal urine should be yellow and clear.
  • Urine Specific Gravity (USG): This is a measure of the concentration of your cat’s urine. Urine passed through the kidneys with no change in concentration has a specific gravity of 1.008 to 1.012. This urine is termed isosthenuric. Healthy cats should be able to produce relatively concentrated urine, often with a USG of 1.050 or higher. If the urine is too dilute, measured as an abnormally low urine specific gravity, your cat may be suffering from a disease condition that affects his ability to produce concentrated urine. This may result from diseases such as diabetes, kidney disease, and many others. It is important to remember that USG can change drastically between one urine sample and the next. In some cases, multiple urine samples may need to be tested to determine whether a cat is producing consistently dilute urine. Evaluating USG in conjunction with clinical signs, physical examination findings, and blood test results is also useful and will help your veterinarian determine the significance of the USG result or other abnormal laboratory results.
  • Urine pH: pH is a measurement of acidity, in this case the acidity of your cat’s urine. The lower the pH number, the more acidic the urine. The pH of urine will impact which types of stones and/or crystals can form in your cat’s urine. Some types of stones form in urine with lower pH values and others are more likely to be found at higher pH values. Some types of bacteria also prefer specific pH ranges. Manipulating the pH value can be useful for handling some urinary tract issues.
  • Glucose: Commonly referred to as “sugar”, glucose in the urine is most frequently an indication of diabetes although stress can cause glucose to show up in the urine in some cases also.
  • Ketones: Ketones are most often found in the urine of diabetic animals. Ketosis occurs when glucose cannot be utilized for energy production. Body fat is then broken down into ketones that can pass through the kidneys into the urine. Ketones in the urine quite often indicate a crisis situation.
  • Bilirubin: Bilirubin, a product of red blood cell breakdown, is normally removed in the liver and becomes part of the bile. When it is found in the urine, it can be an indication of liver disease or other illnesses, such as bleeding disorders.
  • Blood: Blood may be found in the urine for a number of different reasons. Referred to as hematuria, blood in the urine can be an indication of urinary tract infection (UTI), cystitis, kidney or bladder stones, kidney disease, cancer of the urinary tract, or bleeding disorders.
  • Protein: Protein in the urine can be caused by kidney disease as well as other illnesses.
  • Urine Sediment: Examining the urine sediment involves separating the cells and other solid matter from the fluid portion of the urine through centrifugation. The sediment is examined for red blood cells, white blood cells, bacteria, casts, crystals, mucous, or other cells. Essentially, this portion of the urinalysis looks at the cellular and solid component of the urine, searching for abnormal numbers of cells or other materials which should not normally be present in the urine. It can provide additional clues as to the state of your cat’s health.

In some cases, your veterinarian may also perform even more specialized urine testing:


How to keep your dog from urinating within the few hours prior to their veterinary visit

  • If they’re small enough to comfortably do so, carry your dog from the car into your vet’s office.
  • If they’re too heavy to carry, run with your leashed dog from the car into your vet’s office, don’t give them a chance to sniff and pee.
  • Don’t let your dog out into your yard unattended prior to their vet visit. If they must go out, go with them, have them on a leash and collect their urine (see tricks and tips below).
  • The veterinary team will collect a fresh sample during your appointment, so having your dog’s bladder full is helpful.


The Review:

I was sent the PawCheck Urinary Tract Infection kit which included:

  • An adjustable ladle to catch the urine
  • Easy to read instructions
  • Urinary Tract Infection Paw Test Strip
  • A color chart to refer to with the test strip

The adjustable ladle is an excellent idea and kept me from getting any urine on my hands, which is a first. The packaging was great and gave me peace of mind that I was using sterile materials.

I took Leroy out and caught a urine sample using the adjustable ladle.

I carefully poured the urine into a small cup.

Took the cap off of the PawCheck urine device

Set the test strip in the urine for 2 minutes

As you can tell from the stick below, the test shows that Leroy has a trace of Leukocytes (white blood cells) in his urine, which means we are off to see the vet later this week.

I&rsquom not hugely concerned about this finding because it&rsquos a small amount, there&rsquos no blood and the nitrites are negative, but with his history, we&rsquoll have the vet check a sample of his urine under the microscopic just to make sure.

What do I think about this product?

You can tell so much from a pet&rsquos urine so I think that this a great product for pet owners who want to be proactive with their pet&rsquos health, or for pet owners who want to keep tabs on a specific condition in their pet.

Other posts you may be interested in:


What Helps or Hinders the pH Level of Your Dog’s Urine?

One of our customers recently told us how her dog’s urinary crystal issue cleared up after transitioning from a very high protein food to CARNA4 . Delighted with the results, she wanted to know exactly “what” urine pH level CARNA4 produced.

For answers, we consulted Dr. Scott Morton, President, Mortec Scientific Inc. (the independent research and laboratory testing firm that specializes in pet food, consumer food and natural health products and oversees CARNA4’s quality), as well as Maria Ringo, a classical, practising homeopath and one of our co-founders.

The answer is not a definitive number but speaks more to the holistic way a dog’s body functions, similar to humans. A pH level is defined as the concentration of Hydrogen ions or, more simply, the measure of acidity or alkalinity in the body. It is influenced by multiple factors. “It’s the sum of all the body’s metabolic processes, from digestion to elimination, that work together to determine the pH, not just the dog’s food input,” says Maria. “You need to consider the pH of both digested and absorbed food and drink, known as ash.”

“In practical terms, this means that eating too much of meats, fats and sugars compared to plant foods and whole grain foods with their alkaline, neutralizing nutrients can create an acidic pH body environment,” she explains. “And this acidic environment can cause inflammation and allow diseases to thrive.”

Dr. Morton goes further to outline the impact of various ingredients. “A diet containing high levels of poor quality ingredients, such as salt, protein, lactose, sucrose, fructose and magnesium, elevates and creates an unbalanced concentration of calcium in a dog’s urine. A low level or poor source of phosphorus also raises the calcium concentration – creating oxalate crystals and potentially kidney stones.”

He describes how vitamin imbalances and formulation errors, which can occur with synthetic premixes or sub-standard ingredients, can also wreak havoc. “A diet too low or too high in Vitamin D can increase urine calcium – leading to crystals or urinary disease over time. Similarly, a Vitamin B6 deficiency can increase blood oxalate levels and also form crystals,” explains Dr. Morton. “CARNA4 escapes these issues because it a balanced nutritional food that has all the required attributes from first class ingredients,” he adds.

In the end, we answered our customer’s query by explaining that we don’t test for, or guarantee specific urine pH levels. Why? Because we look at bladder health as being the result of a variety of dietary factors, including proper quality and balancing of certain vitamins, minerals and other nutrients, as explained above. We know other manufacturers will often add ingredients (which can cause irritating side effects) to acidify the pet’s urine and reduce crystal formation caused by poor quality ingredients. CARNA4 doesn’t need these urine acidifiers because the quality of its ingredients and the nutritional profile they create reduce the likelihood of crystal formations happening at all.

The bottom line: CARNA4 is a good balancer of pH of the blood of a dog accustomed to eating typical processed food.

Has your dog had urinary crystals? Have you changed their diet to solve this problem? Have you tried switching to CARNA4? Tell us your story and what helped your dog.



Comments:

  1. Rodel

    Yeah, got caught!

  2. Kerry

    You know that every effect has its causes. Everything happens, everything that happens is all for the best. If it were not for this, it is not a fact that it would be better.

  3. Gotzon

    curious, and the analog is?

  4. Win

    yes, yes, yes, let's see

  5. Winfrith

    Sorry, post deleted

  6. Manus

    You are not an expert, by any chance?



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