Serum allergy testing of normal dogs

Do you wonder about the specificity of allergen-specific IgE tests in dogs (the percentage of dogs not allergic to an allergen that are correctly identified as non-allergic)?  Hopefully, you are using clinical criteria to make the diagnosis of canine atopic dermatitis (CAD), not serum allergy test results.  Addressing this issue definitively is not as simple as it may seem, since there is no true gold standard to determine allergen-specific sensitivity. 

 Several studies have reported results, however, that should give you a healthy skepticism about the specificity of serum allergy tests, even when considering the tests for the sole purpose of selecting allergens for immunotherapy, if not for diagnosing CAD.  These false positive results may occur due to non-specific binding in assays or cut-off values that are set too low.

 Codner submitted serum samples from five normal greyhound dogs (http://www.vetrespit.com/resources/codner.pdf).  Four of the five dogs had multiple highly positive reactions.

In a recent report, 18 non-atopic West Highland White Terrier dogs were allergy tested.  Multiple highly positive reactions were reported by the laboratory for 45 of the 48 allergens tested with the Allercept® test, leading the authors to conclude that the test was not specific for this population of dogs.

 Today, at the World Congress of Veterinary Dermatology in Vancouver, there was a nice presentation describing why horseradish peroxidase conjugated monoclonal IgE antibody should not be used in assays, due to the high level of non-specific binding which results with certain allergens.  Some laboratories disclose the conjugate that they use in their assays, but others do not.

 The bottom line:  do not use allergy test results to make a diagnosis of atopic dermatitis and don’t rely upon them exclusively for allergen selection for immunotherapy.

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Intradermal testing reliability in dogs

This month I will review the reliability of “allergy” testing in dogs.  If you are going to recommend allergy testing to clients, you should understand their limitations.  The topics I have planned for the coming weeks are:

  1. Intradermal testing reliability
  2. Serological testing  of normal dogs
  3. Serological testing repeatability
  4. Serological testing reproducibility

Intradermal testing (IDT), sometimes referred to by the redundant term intradermal skin test, involves injecting numerous allergen extracts into the dermis.  Ccanine intradermal testommonly, between 50 and 70 individual injections are performed.  A positive control (usually histamine) and a negative control (saline) are also injected.  After some period of time, usually 15 to 20 minutes, the sites are graded for wheal formation.  This is usually done with a subjective 0 to 4 ordinal scale, although some also measure the average wheal diameter.

The test results can be influenced by a number of factors, including medications, skin pliability, and patient stress.   The optimal testing concentration of aqueous allergens is also of paramount importance and varies with different allergens.  The ideal concentration causes few wheal reactions in normal dogs, but does so in dogs truly allergic to the allergen.  Despite the importance of the testing concentration, guidelines are often not followed by those performing IDT.

Even when performed under ideal conditions, IDT causes false positive reactions to occur. These may represent irritant reactions as described by Koebrich et al to house dust mites (HDM).  Seventeen clinically normal beagles were tested with IDT for sensitivity to HDM.  Seven of 17 reacted to HDM on IDT at the standard testing concentration.  Mueller et al found that clinically normal dogs and atopic dogs reacted to storage mites at the same rate (about 1/3 of each group).

I participated in a study that looked at the fundamental questions of IDT repeatability and reproducibility in dogs.  To examine repeatability, duplicate intradermal injections were evaluated in a blinded study and the agreement of the two readings by the same observer examined.  To examine reproducibility, the agreement of three independent observers, each scoring the same dogs, were examined.  The results were instructive, but not surprising to someone who has done a lot of intradermal testing.  The negative (0) and strongly positive (4+) reactions were generally repeatable and reproducible, but those values in the mid-range were less reliable than desired of a health measurement scale that has been considered the “gold” standard.

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Variability of allergen-specific immunotherapy formulation

Allergen-specific immunotherapy (ASIT) is often discussed as though it represents a uniform treatment option.  In fact, the process of customizing an allergen prescription for a pet is not standardized and there is a great deal of subjectivity involved, even when veterinarians are presented with identical test results. One veterinarian’s ASIT is not the same as the next’s ASIT.

A few years ago, I took a stab and finding out just how much variability could be expected.  I invited veterinary dermatologists on the vetderm and dipderm list serves to participate.  In this informal survey, 11 veterinary dermatologists responded.  All were given the same patient history and allergy test results and asked for their immunotherapy recommendations.  Among the findings:

  • each dermatologist prescribed a unique allergen formulation
  • only 2 allergens were prescribed by every participant
  • the number of allergens included ranged from 10 to 20
  • 28/50 allergens were prescribed by at least one dermatologist
  • 16/50 allergens were prescribed by >50% of dermatologists

Now, this certainly is affected by the number of positive reactions on the test.  Had there been fewer positive reactions in my hypothetical case, there would have been better agreement.  But, I often see intradermal test results on second opinion cases for which 40 or more allergens are scored as positive.

When we consider the variability in test results as well as the variability in formulation recommendations, I suspect that if the same patient saw ten different veterinarians for allergy testing/ASIT, the client would get ten unique allergen prescriptions, some quite different from one another.  Which is why I now refer to this treatment as subcutaneous immunotherapy rather than allergen-specific immunotherapy.

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Getting the most from your skin biopsies

It’s frustrating for you and your clients if a skin biopsy result comes back non-diagnostic. Here are some of the things I do to get the most information that I can from dermatopathology.

1. For inflammatory skin diseases, do not do a surgical scrub or remove crusts at the site. You can clip the hair short, if there is any in the area, but try not to alter the skin surface. Valuable information will be thrown in the trash if you do!

2. Select a range of lesions if there is an obvious progression evident. If there are papules, pustules, and crusts, take samples of each. Most pathologists will charge the same if you send in multiple samples from the same disease process.

3. Rotate the biopsy punch in one direction to minimize shearing artifact. This is especially important for delicate vesicles and pustules.

4. Find a good dermatopathologist. A pathologist with a special interest in skin is more likely to give your unusual skin biopsies that extra attention that can make all the difference.

5. Once you’ve found a dermatopathologist, help them out! The goal should not be to withhold a good history and your differential diagnosis in hopes that they will reach an unbiased conclusion. Providing them with a history and accurate lesion descriptions are crucial to getting the most useful information back.

Even with these precautions, there is a chance that you won’t be able to give the client a definitive diagnosis, so it’s best to set realistic expectations, as in “even if it is not 100% definitive, it will likely point us in the right direction.”

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Skin cytology: let the lesion morphology guide your technique

When it comes to taking samples for skin cytology, there is no one technique that is best in every situation.  Here are my favorite techniques, and when I find them most useful:

1.      Cellophane tape stripping is the technique that I now use most often.  It is great for relatively dry, scaly, seborrheic skin and for getting into tight corners, like the interdigital spaces.  Inexpensive clear cellophane tape, a glass slide, a modified Wright’s stain kit, and a good microscope are all that you need.  Most veterinarians have the first three; if you don’t have a good microscope that allows you to see clearly with the oil immersion lens, invest in getting it cleaned up or replaced.

cellophane tape stripping for cytology

cellophane tape stripping for cytology

 To make a sample, tear off a 2-inch piece of tape and press it against the affected area(s) several times.  If the body regions are clinically distinct, make multiple samples.  Place the tape loosely on the slide to transport it to your laboratory, peel of the tape and dip it directly into the dark blue stain for 5-10 seconds, then place it on the slide.  Blot it with bibulous paper and you are ready to examine it in less than a minute.

2.      Direct impression with a glass slide works great if you are interested in a greasy or moist area, which will transfer sufficient material to the slide.  Again, press it 2-3 times in the affected area(s), let it air dry, then follow the staining technique above.

3.      Cotton swabs are the least used technique, but may be helpful when you need to reach into the crevice of a Bulldog’s tail fold, or some similar situation.  Roll out the swab as you would an ear swab, then stain it as above.

4.      Pustule cytology samples are taken by pricking a pustule with a 25g needle and smearing the contents on a glass slide.  These are best stained with the full complement of 3 stains in your modified Wright’s stain kit.

That’s it!  Skin cytology is one of the most widely used and informative tests performed by veterinary dermatologists.  Make it part of your diagnostic work up of skin cases too!

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The quick and dirty on ear canal cytology

Continuing with my focus on diagnostic tips, today I’ll share my approach to ear canal cytology in dogs and cats.  As you know already, this is a simple, but valuable test that should be recommended whenever otitis externa is suspected, and on follow up examinations for those pets. I like to take samples from the junction of the vertical and horizontal canals.  You may venture into the deeper horizontal canal if the pet is immobilized and you are confident that you are not jamming more debris deeper into the canal. This means a light touch on the cotton-tipped swab.

Once you’ve collected your swabs, roll each on opposite sides of the slide. I insist on frosted slides so that I can easily keep track of where I’ve placed what.  It also avoids the frustration (most of the time!) of trying to focus with your oil-immersion lens on the wrong side of the slide.

The next point is where I might save you or your technician a few minutes a day. When staining ear cytology slides, you can skip heat fixing and the first two stain jars.  That’s right, go straight to the blue/purple stain of your modified Wright’s stain kit.  A blinded study found that the interpretation of ear cytology samples did not change when you bypass these steps.  If your practice does 20 ear cytology stains per day and this saves you or your technician 1-2 minutes, it adds up over the course of a year.

Scan the slide and find representative areas to hone in on.  Using oil immersion so you can get a better look at any bacteria, scan at least 10 fields then score the exudate with this scale, which is derived from some recent research papers:

Scoring ear cytology

Score Yeast per oil field Bacteria per oil field WBCs per oil field
1 (normal) 0 <1 none
2 (gray zone) 0-1 1-4 rare
3 (abnormal) >1 ≥5 >0.5
Malassezia on ear canal cytology

Malassezia on ear canal cytology

Always interpret the samples in light of your clinical examination of the ear canal. For example, yeast can be found in normal ears, but it would be unlikely that an ear with abnormal numbers of white blood cells is clinically normal.

I hope this helps you get the most out of your ear cytology samples!

Jon Plant, DVM, DACVD

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Tips for interpreting your DTM cultures

One of the topics that veterinarians ask me about most frequently is how to interpret fungal cultures grown on dermatophyte test medium (DTM).  The fungal culture is an important early step in working up many presentations, including folliculitis, patchy alopecia, and even facial crusting that resembles pemphigus foliaceus. As a zoonotic disease, dermatophytosis is not one you want to overlook on your differential diagnosis list.

M. canis on DTM/Sabaroud's agar

M. canis on DTM/Sabaroud's agar

Understanding what makes a DTM “tick” will help your interpretation. The DTM incorporates antibiotics (gentamicin and chlortetracycline) to suppress bacterial growth, cycloheximide to suppress saprophytic fungal growth, and phenol red as a color indicator. It provides both carbohydrate and protein nutrients. Dermatophytes (fungi belonging to the genera Microsporum, Trichophyton, and Epidermophyton) preferentially use protein as an energy source, whereas most saprophytic fungi use carbohydrates first. This characteristic is the key to interpreting the color change that may be seen with DTMs. The early growth of dermatophytes will usually (but not always!) cause the DTM to change from yellow to red due to the production of alkaline byproducts of protein metabolism. Saprophytes may do the same, but usually only when the colony is much larger. Check your cultures daily to make sure the red color change precedes the growth of a large colony. In addition to observing for a red color change, the interpretation of DTM growth should include an assessment of gross and microscopic colony morphology.  Dermatophytes grown on DTM are generally lightly pigmented (white, buff, tan or cinnamon-colored), whereas common saprophytes are often darkly pigmented. The colony surfaces of the dermatophytes of veterinary importance are often described as powdery, granular or cottony.

tape prep

tape prep

I prepare a slide for microscopic examination of suspect colonies by lightly touching a piece of clear cellophane tape to the surface of the colony, then placing it on the slide with a drop of the blue stain from a modified Wright’s kit. Macroconidia of M. canis and M. gypseum are usually easy to find, but T. mentagrophytes often doesn’t produce macroconidia on DTM. M. canismacroconidia typically have

M. canis

M. canis

six or more cells, thick cell walls and knobby ends. M. gypseum macroconidia tend to be numerous, ellipsoid, have thinner cell walls, and four to six cells. Some microscopic characteristics of T. mentagrophytes that can be seen are spiral hyphae and grape-like clusters of one-celled microconidia.

M. gypseum

M. gypseum

 Consider the occurrence of a color change, the macroscopic colony appearance, and the microscopic findings into your interpretation. The fungal culture is one of the most important laboratory procedures in veterinary dermatology. It should be used to rule out ringworm, as much as to confirm it. If most of their cultures come back positive, then you may not be recommending them often enough.

Jon Plant, DVM, DACVD

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Diagnosing Canine Atopic Dermatitis

How do you diagnose atopic dermatitis in a dog?  That’s right.  Based upon clinical features.  Serum IgE and intradermal testing can be used to identify potential allergens, but the tests are not sensitive or specific enough to rely upon for making the diagnosis.  Luckily, in 2010, Claude Favrot and colleagues published a nice paper on the clinical features of canine atopic dermatitis (Veterinary Dermatology 2010; 21: 23-31).  In the appendix, there is a very useful checklist that you can use to make a diagnosis.  There are various versions of it, but this one is 85% sensitive and 79% specific when dogs meet five out of eight of these criteria:

  • Age at onset < 3 years
  • Mostly indooratopic dog
  • Corticosteroid-responsive pruritus
  • Chronic or recurrent yeast infections
  • Affected front feet
  • Affected ear pinnae
  • Non-affected ear margins
  • Non-affected dorso-lumbar area

You’ll still want to rule out any other differentials that are suggested by the findings:

  • Food allergy
  • Sarcoptes
  • Flea allergy
  • Demodicosis

Next week’s post will be on interpreting fungal cultures.

Jon Plant, DVM, DACVD

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5 Tips for Better Skin Scrapings

What can I possibly teach you about that most basic test in veterinary dermatology, the skin scraping? Taking a skin scraping seems like a simple enough technique, yet at least once per month I am able to find a mite on a pet that previously had a “negative” scraping by their primary care veterinarian.  In this first installment of the SkinVet Blog, I’m going to share 5 tips for more productive skin scrapings.

Tip #1. Adjust your technique for the type of mite you suspect. Demodex canis, Demodex cati, and long-bodied Demodex injai all live in hair follicles and sebaceous ducts.  They require deep skin scrapings, forcing the mites to the surface by pinching and going deep enough to get capillary bleeding.

Demodex gatoi of cats and the mite tentatively named Demodex cornei of dogs are short, stubby mites that live superficially and may be more scarce.  You should use a superficial skin scraping technique, covering a large area of skin, but not going as deeply.  Similarly, Sarcoptes scabiei, Notoedres cati and Cheyletiella spp. live superficially and you need only scrape superficially, but over a larger area.

Tip #2. When suspicious of Sarcoptes, scrape the ear margins, even if they are minimally involved or unaffected.  The margin of the pinnae is the place Sarcoptes go to party. I’ve had more positive scrapings from ear margins than all other body sites.

Demodex canis mite and egg

Tip #3.  Learn to recognize Sarcoptes and Demodex eggs. Sarcoptes are hard enough to find on scraping, but finding even one oval egg is diagnostic, and enough to send me jumping for joy. Demodex eggs are described as fusiform, sort of like a deflated football. Finding a lot of Demodex eggs on follow up scrapings of a patient that you hoped and thought was improving is a downer.  It means the mites are still feeling well enough to be amorous.

Tip #4. Adjust the condenser of your microscope down to increase contrast.  This is especially important for mites with rather faint exoskeletons, like D. gatoi. They can be nearly transparent if the light is too bright and contrast is low. Recall, this cause of feline pruritus and alopecia is often rather scarce and hard enough to find. If suspected but not found, you’re going to have to do lime sulfur dips weekly to rule it out. Don’t stink up your clinic or your client’s home because you forgot to lower the condenser!

Tip #5. OK, this last one is more of a pet peeve of mine and has nothing to do with the results. If you’re still doing it, it’s time to ditch the old-school technique of re-using scalpel blades under the guise that it is better if they are dull. If your dermatologist came at you with the option of using a dull, used scalpel blade or a new one (that she could easily dull before using), which would you choose? If you are in need of something really dull, I recommend a stainless steel spatula. These work great around eyelids and other delicate areas, especially when scraping squirmy dogs.  Every pug should come equipped with one.

Do you have a skin scraping tip to share?

Next week’s blog will be on diagnosing atopic dermatitis.

Jon Plant, DVM, DACVD

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