Veterinary Dermatology 2004, 15, 304 308

Blackwell Publishing, Ltd.

Determination of threshold concentrations of allergens and

evaluation of two different histamine concentrations
in canine intradermal testing
PATRICK HENSEL*, MICHAELA AUSTEL*, LINDA MEDLEAU*, YING ZHAO
and ANAND VIDYASHANKAR
*Department of Small Animal Medicine, College of Veterinary Medicine and
Department of Statistics, The University of Georgia, Athens, GA 30602, USA
(Received 9 December 2003; accepted 11 February 2004)

Abstract The purpose of this study was to determine the optimal histamine concentration and allergen threshold
concentrations for canine intradermal testing. Thirty healthy dogs were tested using two different concentrations
of histamine and four different concentrations of each allergen. The optimal histamine concentration was determined to be 1:10 000 w/v. The threshold concentration was at least 1750 PNU/mL for all tested grasses, weeds,
trees, moulds and insects, except for fleas which was as least 1:500 w/v. For Dermatophagoides pteronyssinus, the
optimal threshold concentration was 250 PNU/mL, whereas for Dermatophagoides farinae and Tyrophagus
putrescentiae, it was 100 PNU/mL. Threshold concentration for all epidermals except human dander was at least
1250 PNU/mL. The optimal threshold concentration for human dander was 300 PNU/mL. Our results suggest
that the currently used 1:100 000 w/v concentration of histamine and the 1000 PNU/mL concentration for most
grasses, weeds, trees, moulds, epidermals and insects may not be appropriate for canine intradermal testing.
Keywords: allergen, atopy, dog, histamine, intradermal testing, threshold concentration.

I NTRO D U CTI ON
Canine atopic dermatitis (AD) is a genetically predisposed inflammatory and pruritic allergic skin disease
with characteristic clinical features, which is associated
most commonly with immunoglobulin (Ig)E antibodies
to environmental allergens.1 To confirm a diagnosis of
canine atopy and to identify the offending allergens,
intradermal testing (IDT) is a valuable and useful
diagnostic tool.25 In IDT, histamine phosphate
solution is used as the positive control. However, a
standard concentration for its use in veterinary medicine has not been established. In the USA, a 1:100 000
(0.01 mg/mL) concentration of histamine is used,
whereas in Europe, a 1:10 000 (0.1 mg/mL) concentration is often employed.2,3,6,7
Because there is no standardized method for
measuring the biological potency of allergens, their
concentrations may be expressed in protein nitrogen
units (PNU), weight to volume (w/v), Noon units (NU)
or as a percentage.3,6 One PNU is equal to 0.00001 mg
of phosphotungstic acid-precipitated protein nitrogen,
whereas w/v is determined by the ratio of dry defatted
allergenic material to diluent. The definition of 1 NU is
This project was funded by the Companion Animal Research Fund
of the University of Georgia (project number: CA-115).
Correspondence: P. Hensel, The University of Georgia, College of
Veterinary Medicine, Veterinary Teaching Hospital, Athens,
GA 30602, USA. E-mail: phensel@vet.uga.edu
304

1 mL of an extract made from 1 mg of pollen in 1 L of

extracting fluid and allergen concentrations in percentages represent the amount of raw allergenic material in
g per 100 g extraction volume.3,6 Today, most manufacturers sell the allergens either in PNUs or w/v.
To ensure reliable IDT results, an allergen should
be tested at its threshold or optimal concentration. The
optimal concentration of an allergen is the highest
concentration that does not produce irritant (false
positive) reactions in at least 90% of normal individuals.3
Although optimal allergen concentrations for canine
IDT have not been well studied, the following allergen concentrations are routinely used in veterinary
medicine: 1000 PNU/mL for grass, tree and weed
pollens, moulds and insects; 250500 PNU/mL for
feathers and epithelia; and 250 PNU/mL or 1:1000
1:5000 w/v for individual house dust mites. The flea
allergen is usually tested at 1:1000 w/v.24
The purpose of this study was to determine the
optimal histamine concentration and threshold concentrations for allergens in canine IDT.

M AT E R IA L S A N D M E T H O D S
Dogs
Thirty privately owned, clinically healthy dogs were
used in this study. They included 2 intact males, 12
neutered males and 16 spayed females; their ages ranged
from 2 to 8 years (mean age 4.1 years) and weights
2004 European Society of Veterinary Dermatology

Threshold concentrations in canine IDT

305

Table 1. Allergens used for IDT in this study

1. Bahia grass
2. Bermuda grass
3. Bluegrass Kentucky/June
4. Fescue grass, meadow
5. Johnson grass
6. Redtop grass
7. Ryegrass, Perennial
8. Timothy grass
9. Cocklebur
10. Dock Yellow
11. Dog Fennel
12. Lambs Quarter
13. Marsh Elder, True/Rough
14. Pigweed Rough/Redroot
15. Plaintain, English
16. Sorrel, Sheep/Red

17. Ragweed, Southern

18. Ash Red /Green
19. Birch, Red / River
20. Cottonwood, Eastern
21. Elm, American
22. Hickory, Shagbark
23. Maple, Red
24. Mulberry, Red
25. Oak, Virginia live
26. Sycamore, American, Eastern
27. Alternaria tenuis
28. Aspergillus niger
29. Curvularia spicifera
30. Fusarium moniliforme
31. Helminthosporum sativum
32. Hormodendrum hordei

ranged from 11.5 to 39 kg (mean weight 25.6 kg).

There were 12 mix-breed dogs, 7 Labrador retrievers,
3 German shepherd dogs, 2 rottweilers and 1 each of
pitbull, boxer, Doberman pinscher, border collie, Jack
Russell terrier and golden retriever. None of the dogs
had any history of pruritus or skin and ear diseases. No
abnormalities were found on physical examination.
Haemograms, serum chemistry profiles and urinalysis
were within normal limits in all 30 dogs.

Histamine phosphate control solution

Each dog was intradermally tested with two different
concentrations of histamine phosphate solution (Greer
Laboratories Inc, Lenoir, NC, USA) at 1:100 000
(0.01 mg/mL) [H1] and 1:10 000 (0.1 mg/mL) [H2].
The test histamine solutions were stored in glass
vials at 4 C. Because of the stability of the histamine
phosphate solution and the short duration of the study
the same solution was used in all 30 dogs.

Allergens
Forty-eight commercially available aqueous allergens
were used for IDT. The allergens included eight grasses
(18), six weeds (914), twelve trees (1526), eight
moulds (2734), three mites (3537), five epithelia (38
42) and six insects (4348) (Table 1). All allergens were
obtained from Greer Laboratories Inc., except for the
storage mite (Tyrophagus putrescentiae), which was
obtained from Center Laboratories (Port Washington,
NY, USA). Each allergen was diluted with phosphatebuffered saline to obtain four different test concentrations. Pollens, moulds and insects (except flea) were
tested at the following concentrations: 1000, 1250, 1500
and 1750 PNU/mL. The flea allergen was tested at 1:500,
1:750, 1:1000 and 1:1250 w/v. The house dust mites and
storage mite were tested at 100, 250, 500 and 750 PNU/
mL. All epithelia were tested at 500, 750, 1000 and
1250 PNU/mL except for human dander. Human
dander was tested at 50, 100, 300 and 500 PNU/mL.
The reason for different serial dilution range of human
dander is due to its maximal available strength of
700 PNU/mL. In addition, test strength recommendations were not available at the beginning of this study

33. Penicillium notatum

34. Pullularia pullulans
35. Dermatophagoides farinae
36. Dermatophagoides pteronyssinus
37. Tyrophagus putrescentiae
38. Human epithelia
39. Cat epithelia
40. Sheep epithelia
41. Duck feathers
42. Goose feathers
43. Flea
44. Cockroach American
45. Cockroach German
46. House fly
47. Mosquito
48. Moth

for this new allergen. The diluted allergens were stored

in glass vials at 4 C and made up fresh every 3 weeks.

Intradermal testing
For this procedure, all dogs were sedated with medetomidine 2040 g/kg IV (Domitor, Pfizer, Exton, PA,
USA). The skin over the left ventro-lateral thorax wall
was gently clipped. The skin test sites were marked with
a waterproof marker and injected intradermally with
0.05 mL of each histamine solution (H1 and H2), each
allergen solution and a negative control solution (0.9%
buffered saline) using 0.5-mL syringes and 27-G (0.5 mm)
needles. Each skin test site was evaluated subjectively by
the same investigator, 15 and 30 min post injection for
erythema and induration and objectively by measuring
the diameter (average of the vertical and horizontal
diameter) of any wheal formation.
A reaction to an allergen test concentration was considered positive if it was erythematous and /or indurated
and its wheal diameter was equal to or greater than the
mean diameter between the negative control and the
positive histamine phosphate control (H1 or H2) being
measured. The allergen threshold concentration was
defined as the highest concentration to which 10% or
fewer of the dogs ( 3/30 dogs) reacted positive.

Statistical analysis
The threshold concentration of each allergen was analysed
in reference to the two different histamine concentrations
using the chi-square test. The differences between the two
histamine concentrations and the equality of the threshold concentrations between the two histamine concentrations were evaluated by analysis of variance (),
including Tukeys studentized range (HSD) and Duncans
multiple range (HSD) test. All statistical comparisons
were evaluated at a 5% level of significance (P < 0.05).

R E S U LT S
Histamine phosphate control solution
The two histamine concentrations resulted in positive
reactions in all 30 dogs. The reactions were characterized

2004 European Society of Veterinary Dermatology, Veterinary Dermatology, 15, 304308

306

P Hensel et al.

Table 2. Thresholds using the H1 concentration as positive control

Table 3. Thresholds using the H2 concentration as positive control

Allergen

Threshold

P-value

Allergen

Threshold

P-value

14, 634, 4447

5
35
36
37
38
3942
43
48

1750 PNU
1500 PNU
100 PNU ( 1:12 000 w/v)
100 PNU (1:9600 w/v)
100 PNU ( 1:10 000 w/v)
300 PNU (1 : 233 w/v)
1250 PNU
1:500 w/v (0 PNU)
1000 PNU

0.05
0.5428
< 0.0001*
0.5428
0.0003*
0.5428
0.05
0.05
0.0679

134, 44 48
35
36
37
38
39 42
43

1750 PNU
100 PNU (1:12 000 w/v)
250 PNU (1:3840 w/v)
100 PNU (1:10 000 w/v)
300 PNU (1:233 w/v)
1250 PNU
1:500 w/v (0 PNU)

0.05*
1.00
0.0679
0.5428
0.6753
0.05*
0.05*

*Suggest even lower threshold concentrations.

Suggest even higher threshold concentrations.

by round to oval wheals with the maximum magnitude

of wheal formation occurring between 15 and 30 min
post injection. Although all reaction sites were erythematous and indurated, the H2 reactions were slightly redder
and firmer than the H1 reactions. The mean diameters
of the H1 and H2 reactions were 11.6 mm (range, 9
14 mm) and 14 mm (range, 1118 mm), respectively.
Pairwise comparison between the two histamine
concentrations revealed significant differences in
wheal diameter (P < 0.0001 between H1 and H2).

Allergen threshold concentrations

When H1 was used as the positive control, the threshold
concentration for all tested moulds, weeds and trees
was at least 1750 PNU/mL (Table 2). The threshold
concentration was also at least 1750 PNU/mL for all
grasses (except Johnson) and for the insects (except
moth and flea). The threshold concentration for
Johnson grass was 1500 PNU/mL, for the moth it was
1000 PNU/mL and for the flea it was 1:500 w/v. For
all epithelia besides human dander, the threshold
concentration was at least 1250 PNU/mL. For human
dander, the threshold concentration was determined to
be 300 PNU/mL. The optimal threshold concentration
for Dermatophagoides farinae and Tyrophagus putrescentiae were not determined because > 10% of the dogs
reacted positive to all test concentrations including the
lowest tested concentration of 100 PNU/mL. However,
the threshold concentration for D. pteronyssinus was
found to be 100 PNU/mL (Table 2). Thus, using the
0.01 mg/mL histamine phosphate solution (H1) as
positive control, the optimal threshold concentrations
could only be determined for Johnson grass, moth, human
dander and D. pteronyssinus. For all other allergens,
threshold concentrations still need to be determined.
Using the H2 concentration as the positive control,
the threshold concentration for all grasses, weeds, trees,
moulds and insects (except for the flea) was at least
1750 PNU/mL (Table 3). The threshold concentration
for the flea was 1:500 w/v. For all epithelia, except
human dander, the threshold concentration was at
least 1250 PNU/mL. For human dander, the threshold
concentration was determined to be 300 PNU/mL
(Table 3). The optimal threshold concentration for
D. farinae and T. putrescentiae was determined to be
100 PNU/mL; whereas, for D. pteronyssinus it was

*Suggest even higher threshold concentrations.

Table 4. Variation in threshold concentrations using different

histamine solutions
Allergen

H1

H2

Johnson grass
D. farinae
D. pteronyssinus
T. putrescentiae
Moth

1500 PNU
100 PNU
100 PNU
100 PNU
1000 PNU

1750 PNU
100 PNU
250 PNU
100 PNU
1750 PNU

H = Histamine concentration.

250 PNU/mL (Table 3). Using a 10-fold higher histamine phosphate solution (H2) as positive control, the
optimal threshold concentration could be determined
for D. farinae, T. putrescentiae, D. pteronyssinus and
human dander. For all other allergens, a higher
optimal threshold concentration is suggested.
Although the threshold concentrations for Johnson
grass, D. farinae, D. pteronyssinus, T. putrescentiae and
moth varied depending on which histamine concentration was used as a positive control (Table 4), these
differences were not statistically significant (P > 0.05).

D ISC U S S IO N
A standard concentration for histamine solution in
canine IDT is not used in veterinary dermatology. In
the USA, histamine is typically used at a concentration
of 1:100 000 w/v and in Europe it is used at both
1:100 000 and 1:10 000 w/v. However, when we
compared these two concentrations, we found that the
1:10 000 w/v concentration resulted in reactions that
were subjectively more erythematous and indurated
than those of the 1:100 000 w/v concentration. By
objective evaluation, the 1:10 000 w/v concentration
resulted in significantly larger reactions than did the
1:100 000 w/v concentration. Mean wheal diameter for
the 1:10 000 w/v histamine concentration was 14 mm,
with the smallest wheal measuring 11 mm and the
largest measuring 18 mm in diameter. With the
1:100 000 w/v histamine concentration, mean wheal
diameter was only 11.6 mm, which was barely larger
than the smallest 1:10 000 w/v histamine wheal. Also,
4 of the 30 dogs had weak reactions with 1:100 000 w/v
that ranged from 8 to 10 mM in diameter. According to
Scott et al.2 positive control reactions with wheal
diameters < 10 mm should not be used for skin test
evaluation. Our findings are similar to those of an earlier

2004 European Society of Veterinary Dermatology, Veterinary Dermatology, 15, 304 308

Threshold concentrations in canine IDT

study performed by Willemse & Van Den Brom in
1982.6 In that study, the investigators found that mean
wheal diameter for the 1:100 000 w/v histamine concentration was 10 mm, whereas 12.5 mm was the mean
diameter for the 1:10 000 w/v histamine concentration.6 Because the 1:100 000 w/v histamine concentration
occasionally resulted in positive reactions that were too
weak compared with the 1:10 000 w/v concentration, we
believe it should not be used in canine IDT. If a weak
positive control is used there is a risk of misinterpreting
a weak reaction to an allergen as positive, especially if
the reactions are evaluated subjectively.2,8
In IDT, an allergen should be tested at its threshold
concentration, the highest concentration that does not
produce irritant reactions (false positive) in at least
90% of normal individuals.3 If the allergen concentration used is above its threshold concentration, falsepositive irritant reactions may occur. However, if the
allergen concentration is below its threshold concentration, allergic individuals may fail to react, resulting
in false-negative results.3
The best method to establish the threshold concentration of an allergen is to intradermally inject serial
dilutions of the allergen extract in clinically healthy
nonallergic dogs to determine the highest concentration
that does not produce positive reactions in > 10% of
the dogs.3 To the authors knowledge, only two such
studies have been performed in dogs to evaluate the
threshold concentrations for pollens, moulds and
epithelia.6,9 However, the results of these studies, both
published more than 20 years ago, are not valid today
because either the allergens tested are no longer in use
or they were tested as allergenic mixes. In addition, in
one of these studies allergen units such as NU and percentage were used which cannot be compared directly
with the allergen units PNU and w/v used here. In our
study, we determined that the threshold concentration
for human dander was 300 PNU/mL. We also found
that the threshold concentrations for all other tested
epithelia, pollens and moulds were > 1000 PNU/mL,
the concentration used by most veterinary dermatologists. However, we were unable to identify their exact
threshold values, because fewer than 10% of the dogs
reacted positive to any of the concentrations we evaluated, including the highest tested concentrations of
1250 PNU/mL for epithelia and 1750 PNU/mL for
pollens and moulds. Thus, further studies are needed
evaluating higher allergen concentrations in order to
establish the threshold concentrations for pollens,
moulds and epithelia other than human dander.
In our study, the threshold concentrations for the
tested insects were at least 1750 PNU/mL, whereas, the
threshold concentration for flea allergen was at least
1:500 w/v. Our results differ from those of an earlier
study in which skin test threshold concentrations were
determined for 13 insects and arachnid allergens.10 The
investigators in that study defined the threshold concentration as the highest concentration that produced
a positive reaction in < 25% of dogs. As opposed to the
10% cut-off level in our study, their threshold concen-

307

trations for insects were lower than ours. The reason for
this discrepancy is unclear. One possible explanation is
that we measured the skin test reactions objectively,
whereas in the earlier study, skin test reactions were
evaluated subjectively. Also, the extracts may have been
inherently more potent in the earlier study, which
might be problematic in veterinary medicine because
of the use of nonstandardized allergen extracts.
Currently, most veterinary dermatologists test for
house dust mite hypersensitivity using individual house
dust mite extracts at 250 PNU/mL or, as recommended
by the manufacturer, at 1:10001:5000 w/v.3 However,
when this concentration is used in mixed house dust
mite extracts, it may be too high, causing false-positive
irritant reactions in clinically healthy, nonallergic dogs.
For example, in one study, when normal, healthy dogs
were intradermally tested with a mixed house dust mite
extract at 1:5000 v/w, 58% of the dogs had false-positive
reactions.11 In another study, concentrations of a mixed
house dust mite extract > 31.25 PNU/mL resulted in a
high number of false-positive reactions.12 In our study,
we used single house dust mite allergen extracts.
The threshold concentration for D. pteronyssinus was
250 PNU/mL but for D. farinae it was 100 PNU/mL.
The threshold concentration for the storage mite and
T. putrescentiae was also 100 PNU/mL.
Ideally, this study should have been designed using a
blinded controlled protocol. However, limited funding
precluded that option. The investigator did not know,
except for the control solutions, which allergen he was
injecting during the skin test until the results were
transferred to a table. For this reason we believe that
the results of our study are scientifically valid.
In conclusion, we found that a 1:10 000 w/v concentration of histamine phosphate solution is preferred
to a 1:100 000 w/v concentration as a positive control.
By using a concentration of 1:10 000 w/v we were able
to identify the optimal threshold concentrations for
human dander and all three mite allergens (D. pteronyssinus, D. farinae and T. putrescentiae). Further studies
are needed to confirm our threshold concentration
results for these allergens as well as to elucidate the
exact threshold concentrations for the pollens, moulds,
nonhuman epithelia and insects.

AC K N OW L E D G E M E N T S
The authors want to thank Mrs Barbara Vignola (Greer
Laboratories Inc.) for material support (allergen
extracts, diluent vials and syringes) and Dr Harvey L.
Crumm (Pfizer Inc., Animal Health Group) for donating the drugs (Domitor and Antisedan) used in
this study.

R E FE R E N C E S
1. Olivry T, DeBoer DJ, Griffin CE. The ACVD task
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Diseases of Dogs and Cats, 2nd edn. Philadelphia:
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injection of allergenic extracts. Journal of the American
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reactivity to various insect and arachnid allergens among
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Rsum Le but de cette tude tait de dterminer la concentration optimale en histamine et les concentrations
seuils pour les tests intradermiques chez le chien. Trente chiens sains ont t tests en utilisant deux concentrations
diffrentes dhistamine et 4 concentrations diffrentes de chaque allergne. La concentration optimale dhistamine tait de 1:10,000 w/v. La concentration seuil tait dau moins 1,750 PNU/ml pour toutes les gramines,
herbaces, arbres, moisissures et insectes, sauf pour lextrait de puce (1:500 w/v). Pour Dermatophagoides pteronyssinus, la concentration seuil optimale tait de 250 PNU/ml; tandis que pour Dermatophagoides farinae et
Tyrophagus putrescentiae, elle tait de 100 PNU/ml. La concentration suil pour tous les extraits de squames (sauf les
squames humaines) tait dau moins 1,250 PNU/ml. Pour les squames humaines, la concentration tait de 300
PNU/ml. Nos rsultats suggrent que la concentration actuellement recommande pour lhistamine (1:100,000
w/v) et celle de 1,000 PNU/ml pour la plupart des gramines, arbres, herbaces, moisissures, squames et insectes
pourraient tre trop faibles. A linverse, les concentrations de 250 PNU/ml utilises pour les acariens pourraient
tre trop leves.
Resumen El propsito de este estudio fue determinar la concentracin de histamina ptima y el umbral de concentracin de alrgeno para las pruebas intradrmicas caninas. Se aplicaron pruebas sobre treinta perros sanos
utilizando 2 concentraciones diferentes de histamina y 4 concentraciones diferentes de cada alrgeno. Se determin la concentracin ptima de histamina en 1:10,000 w/v. El umbral de concentracin fue como mnimo 1,750
PNU/ml para todos los cspedes, hierbas, rboles, mohos, e insectos excepto para las pulgas, que fue de 1:500
w/v. Para el Dermatophagoides pteronyssinus, el umbral de concentracin ptimo fue de 250 PNU/ml; mientras
que para Dermatophagoides farinae y Tyrophagus putrescentiae, fue de 100 PNU/ml. El umbral de concentracin
para todas las epidermis excepto la caspa humana fue al menos de 1,250 PNU/ml. La caspa humana fue de 300
PNU/ml. Nuestros resultados sugieren que la concentracin de histamina utilizada actualmente de 1:100,000 w/v
y la concentracin de la mayora de cspedes, hierbas, rboles, mohos, epidermis de 1,000 PNU/ml podra ser
demasiado baja. Contrariamente, las concentraciones utilizadas actualmente para caros del polvo domstico
de 250 PNU/ml podran ser demasiado altas.
Zusammenfassung Ziel dieser Studie war es, die optimale Histaminkonzentration und Allergengrenzwertkonzentrationen fr den Intradermaltest bei Hunden zu bestimmen. Dreiig gesunde Hunde wurden mit 2
verschiedenen Histaminkonzentrationen und 4 verschiedenen Konzentrationen jeden Allergens getestet. Die
optimale Histaminkonzentration wurde bei 1:10 000 w/v festgelegt. Fr alle getesteten Grser, Unkruter,
Bume, Schimmelpilze und Insekten lag die Grenzwertkonzentration bei mindestens 1750 PNU/ml, auer bei
Flhen, bei denen die Grenzwertkonzentration bei 1:500 w/v lag. Fr Dermatophagoides pteronyssinus war die
optimale Grenzwertkonzentration 250 PNU/ml, wogegen sie bei Dermatophagoides farinae und Tyrophagus
putrescentiae bei 100PNU/ml lag. Grenzwertkonzentration fr alle Epidermisarten auer menschlichen Schuppen war mindestens1250 PNU/ml. Bei menschlichen Schuppen lag sie bei 300 PNU/ml. Unsere Ergebnisse deuten
an, dass die augenblicklich bei Histamin verwandte 1:10 000 w/v Konzentration und die bei den meisten Grsern,
Unkrutern, Bumen, Schimmelpilzen, Epidermisarten und Insekten verwandte 1000 PNU/ml Konzentration
zu niedrig sein kann. Auf der anderen Seite kann die augenblicklich verwandte Konzentration von 250PNU/ml
bei Hausstaubmilben zu hoch sein.

2004 European Society of Veterinary Dermatology, Veterinary Dermatology, 15, 304 308