- Email: [email protected]
Assay of the biologic activity of allergen skin test preparations
Original
articles
Assay of the biologic test preparations
activity
of allergen
Fred BjiirkstBn, Ph.D., Tari Haahtela, M.D., Alf Backman, llpo Suoniemi, M.Sc. Helsinki and Tiuruniemi, Finland
skin
M.D., and
We studied the biologic activity of allergen preparations using a method involving skin prick tests in humans and the use of HEP (histamine equivalent prick) units. Results were found to be dependent on the population groups used in assays. If populations are not carefully standardized, results may vary by I power of IO. Accuracy can also be improved by the use of suitable allergen standard reference preparations, but such were not available to us. Conjidence ranges for the biologic activities were relatively wide and varied with the allergen preparation and the population group. Typically, the 95% confidence range included values from one-fifth to jive times the estimated HEP value when the number of subjects in the assay was 30 to 50 persons. When the preparations representing the same source material (e.g., timothy pollen) were assayed simultaneously in one population group of this size, a twofold or larger dtference in HEP values generally proved significant. An examination of 43 commercial products showed that allergen preparations with biologic activities declared in HEP units had a more uniform biologic activity than those assayed with traditional methods and units (PNUIml or weightlvolume). (.I ALLERGY CLIN IMMUNOL 73:324 -331, I984 .)
The potency of allergen preparations (extracts) intended for diagnostic or therapeutic use can be declared in biologic units, which describe the biologic activity in humans. The histamine equivalent prick (HEP) unit described by Aas et al. l has been endorsed by Nordic (Danish, Finnish, Norwegian, and Swedish) regulatory authorities, and the unit is now in use.* For the establishment of a HEP value, it is necessary to perform skin prick tests in a population of subjects with immediate hypersensitivity to the allergen in question. Each subject’s skin-reactivity to an allergen preparation is compared to the reactivity to a histamine
From the Hospital for Allergic Diseases, Helsinki University Central Hospital, Helsinki, and Department of Pulmonary Diseases, Tiuru Hospital, Tiuruniemi, Finland. The study was supported by a grant fmm the Finnish AntiTuberculosis Association. Received for publication Feb. 9, 1983. Accepted for publication Aug. 3 1, 1983, Reprint requests: Fred Bjkltsten, Hospital for Allergic Diseases, Meilahdentie 2, SF-00250 Helsinki 25, Finland.
324
1
0.1
1 INDIVIDUAL
100
10
1000
HEP
Fig. 1. Distribution
of individual HEP values in a group of 50 patients skin prick-tested with the allergen preparation “Cat 3” (cf. Table I). In nine patients the result was 0 HEP, and these have been omitted from the figure.
Histamine equivalent RAST:
Radioallergosorbent
prick (unit) test
VOLUME hUMRER
?3 3
Biologic
TABLE I. Allergen
preparations
-_.- _----
Source
studied
material
Cat epithelium
Cocksfoot pollen (Ductylis glomeruta) Dog epithelium 4
Fish Horse epithelium House dust
House dust mite
Mugwort pollen vulgaris)
Timothy pollen (Phleum pratense)
White oxeye daisy pollen (Chrysanthemum leucanthemum) ALK
< Allergologisk
Laboratorium,
_~._. Allergen
Alder pollen (A/nu.s sp.) Birch pollen (Be/u/a sp.)
(Artemisia
activity of skin test preoarations
Copenhagen,
code
Alder I Birch I Birch 2 Birch 3 Birch 4 Birch 5 Birch 6 Birch 7 cat I Cat 2 Cat 3 Cat 4 Cat 5 Cat 6 Cocksfoot I Cocksfoot 2 Cocksfoot 3 Dog I Dog 2 Dog 3 Dog 4 Dog 5 Fish 1 Horse 1 House dust 1 House dust 2 House dust 3 Mite I Mite 2 Mite 3 Mugwort I Mugwort 2 Mugwort 3 Mugwort 4 Mugwort 5 Timothy I Timothy 2 Timothy 3 Timothy 4 Timothy 5 Timothy 6 Timothy 7 White oxeye 1
Manufacturer
-_
i.nt
325
._~. - ..__..
#lo.
Dome .4LK 4LK ALK Dome Dome Phannacia Phannacia ALK ALK ALK Bencard Bencard Dome ALK ALK Dome ALK ALK Bencard Bencard Dome Bencard Bencard ALK Bencard Dome ALK Bencard Dome ALK ALK ALK Bencard Dome ALK ALK ALK Bencard Dome Pharmacia Pharmacia Dome
Denmark.
standard. This histamine standard is used to decrease interindividual variability, since the skin response to an allergen is dependent on the response to histamine.3~ 4 In the present study we have investigated the usefulness of a HEP method. We will discuss the difficulties involved and present some accuracy and confidence range data. We also report HEP values for
43 commercial allergen preparations intended for skin prick testing. In the future it will become possible to declare the concentration of individual allergens-at least main allergens-in mass units, and this is very desimble. Even then, biologic units will also be needed because the biologic activity of allergen preparutions and purified allergens will remain of interest. It will, for
326
Bjiirksten
et al.
J. ALLERGY CLIN. IMMUNOL. MARCH 1984
TABLE II. Effect of the lower (HEP value)
of allergen
limit chosen for wheals to be measured on the apparent preparations in subjects from the patient population Smallest
Allergen code (from Table I)
measured
whea12 Biologic
No. of subjects
Birch 2 Birch 3 Birch 5 Cat 2 Cat 3 Cat 5 Cocksfoot 1 Cocksfoot 2 Dog I Dog 2 Dog 3 Mugwort 2 Mugwort 3 Mugwort 4 Timothy 1 Timothy 2 Timothy 4
42 42 42 50 50 50 36 36 51 51 51 45 45 45 42 42 42
mm*
All wheals
activity WEP)
MATERIALS,
Biologic
AND METHODS
Subjects came from the following three population groups. Conscripts were 220 unselected young men, ages 18 to 19, from Imatra, a small industrial town. Skin testing took place in April and May 1979 in conjunction with a compulsory health examination for military service. Patienrs were persons referred to the outpatient department of the Hospital for Allergic Diseases, University Central Hospital, Helsinki, because of a variety of respiratory and/or dermal complaints related to type I allergy. The mean age of the patients was 25.7 years (range 5 to 70). Skin tests were performed in 92 to 112 patients (64% females, on the average); the number varied with the allergen source material. Testing took place in May and June 1979. Students were 708 unselected ninth grade students (55% girls) in Imatra schools. The study was carried out in conjunction with compulsory tuberculosis screening. Of those studied, 99% were 15 to 17 yr old. Skin testing was carried out in March to May 1978.
Allergens We studied altogether 43 allergen preparation lots from 12 allergen source materials and four manufacturers (Table I). All preparations were intended for skin prick testing. Different manufacturers used different solvents: extracts
material
activity
rejectedt
37 37 37 28 31 31 26 29 18 29 34 29 35 38 24 29 32
*Wheals under the limit were given the diameter 0 mm. If all lots from the same source patient’s results for the source material in question were omitted from analysis. ?A11 wheals smaller than the limit were omitted from analysis.
SUBJECTS, Subjects
3 mm
No. of subjects
IO 43 31 1.3 3.2 1.8 2.8 IO 0.1 1.3 0.8 2.3 IO IO I.8 IO 54
instance, be necessary to measure the biologic activity of standard reference preparations.
under
biologic
activity (HEI’)
IO 77 77 10 31 10 10 25 3.2 IO 2.8 IO 55 37 IO 31 330 produced
0 mm wheals
in a patient,
this
from Allergologist Laboratorium (Copenhagen, Denmark) contained 50% glycerol; 0.45% sodium chloride; extracts from Bencard, Ltd. (Betchworth, Surrey, U.K.) contained 50% glycerol; extracts from Dome Laboratories, Div. Miles Laboratories (Stoke Poges, Slough, U.K.) contained 50% glycerol and 0.4% phenol; Pharmacia Diagnostics (Uppsala, Sweden) supplied freeze-dried allergen preparations and a solvent containing 0.9% sodium chloride and 0.4% phenol. In addition, we used a negative reference solution containing 50% glycerol and a positive reference solution containing 1 mg/ml (5.43 mmol/L) histamine dihydrochloride in 50% glycerol.
Skin prick tests Skin prick tests were performed with undiluted allergen solutions as they were supplied by the manufacturers. A droplet of each solution to be tried was applied to the volar aspect of the forearm. A disposable needle was then pushed through the droplet, the skin was punctured, and the epidermis lifted without causing bleeding. The largest diameter of the wheal (D) and the diameter vertical to this (d) were measured after 15 min. At this time the histamine wheal is already decreasing in size, but change from the maximum is slight. As the final result, we used the mean allergen wheal diameter D, = (D + d)/2. The mean histamine wheal diameter, D,,, was obtained in an analogous manner. The different population groups were skin-tested by different persons, and minor variations in technique may have occurred. In the conscripts, a coefficient of variation of 15%
VOLUME 73 N;JMB::R :i
Biologic
TABLE 111.Effect of the choice
of assay
population Biologic
Allergen code (from Table 1)”
on the biologic activity (HEPIt
activity
(HEP) observed in ;~ssays ___ ___-.-__...” I-..--_-.-. -..-
found
Biologic activity or concentration reported by manufacturer
Patients
Conscripts
Birch I Birch 7 cat I cat 2 Dog I Mugwort I Mugwort 2 Timothy 3 Timothy 1
activity of skin test aret*~%‘a!ior$s 327
I .o 10 0.4 10 3 .2
I.0 1 .o
IO 3.1 IO
* 411 allergen lots came from Allergologisk Laboratorium. t For each allergen source material, those patients were included in whom at least one of the allergen lots in question gave ‘8 when1 equal to or larger than 2 mm
TABLE IV. Variation (patients)
included
of the biologic activity in the assay population
and its confidence
range
with Biologic
Akgen (from
code Table I)
No.
of subjects*
cat 3
Assay
the number activity
result
(HEPIt
---_.
.-
950/e confiience
IO
IO
of subjects
il.!)-54
20
I .o
tJ. I- 11:
30
I.0 2.8
fl.1, 1%) :I. i- 1i! C!..Glli
40 50
range
3.2
*.U results are from the same assay in a group of 50 patients. Results were randomly removed to obtain groups with fewer members. tFor the allergen source material, those patients were included in whom at least one of the allergen lots in question gave a wheal equal to or larger than 2 mm
to 20% in wheal diameters was observed when multiple skin tests with the same allergen preparation were performed in the same subject.
tions). Inserting values for u and b in equation i, and solving for C. we obtain the following (in HEP unit.sr.
Calculation
When the diameters of the wheals caused in a subject by skin tests with an allergen preparation (D;,) and the histamine reference (D,,) are known, an “individual HEP’~ can be calculated from equation 3. The individual HEP is a function of the biologic activity of the prepanttion and the subject’s sensitivity to the allergen in question. The biologic activities (HEP values) that we report for allergen preparations are medians of the individual HEP values obtained in studies of sensitive population groups. Use ot medians eliminates any error due to the inclusion of hughly sensitive patients with large whealing reactions far whom the linear relationship characterized by equation 1 may not hold.
of HEP values
The dose-response relationship to borh histamine and allergens in skin tests can be approximated by the following equation’. ,‘: log Da,h = N log C + h
(1)
where Da,h is the mean wheal diameter, C is the allergen concentration, and a and b are constants. When HEP units are used, then by definition the allergen concentration (C) is 1 HEP when the allergen wheal diameter (D,) is equal to the histamine wheal diameter (D,,). As a result, we obtain the following from equation 1: log Dh = b
(2)
For the constant u, we have obtained the same empirical value 0.198 in the case of both allergens and histamine (Backman, BjiirkstCn, and Suoniemi, unpublished observa-
C = (D,/D,,y.“”
Significant
diffwenme
(3)
in f4EP w&es
We examined the significance of difference?, in (median) HEP values obtained when several lots of allergen from similar source material were assayed with simultaneous skin tests in the same subjects. In this case, interindividual and
328
Bjiirksth
J. ALLERGY CLIN. IMMUNOL. MARCH 1984
et al.
TABLE V. Biologic
activities
(HEP) found Subjects
Allergen code (from Table I)
Alder I Birch 1 Birch 2 Birch 3 Birch 4 Birch 5 Birch 6 Birch 7 Cat 1 Cat 2 Cat 3 Cat 4 Cat 5 Cat 6 Cocksfoot I Cocksfoot 2 Cocksfoot 3 Dog 1 Dog 1 Dog 2 Dog 3 Dog 4 Dog 5 Fish 1 Horse I House dust I House dust 2 House dust 3 Mite I Mite 2 Mite 3 Mugwort I Mugwort 2 Mugwort 3 Mugwort 4 Mugwort 5 Timothy I Timothy 2 Timothy 3 Timothy 4 Timothy 5 Timothy 6 Timothy 7 White oxeye I
Type
of population
Students Conscripts Patients Patients Students Patients Conscripts Conscripts Conscripts Patients Patients Students Patients Students Patients Patients Students Conscripts Patients Patients Students Patients Students Students Students Conscripts Students Students Conscripts Students Students Conscripts Patients Patients Patients Students Patients Patients Conscripts Patients Students Conscripts Conscripts Students
and activities
or concentrations
reported
Biologic activity or concentration reported by manufacturer
tested Biologic activity (HEP)*
No.
80
22 1.0 IO 77 19 77 1.0 7.7 0.4 IO 31 I.8 IO I.1 IO 25 IO 1.0 3.2 IO 0.9 2.8 1.1 0.8 0.6 0.3 1.3 0.9 1.0 1.3 0.6 1.0 IO 55 37 3.6 IO 31 3.1 330 7.5 4.3 130 0.9
37 37 39 91 37 27 41 59 28 31 221 31 loo 26 29 128 43 18 29 100 34 34 66 143 81 273 145 24 137 98 69 29 35 38 103 24 29 45 32 124 44 61 66
by manufacturers
5000 PNU/mI I .O HEP I .O HEP 5.0 HEP 5000 PNU/ml 3000 PNU/ml 1.0 HEP IO HEP I .O HEP I .O HEP 5.0 HEP 150% 150% 5000 PNU/mI 1 .O HEP 5.0 HEP 5000 PNU/ml 1: 100 I:100 I:20 150% 150% 5000 PNU/ml 10% 150% 1:IOO 150% 5000 PNU/ml 1.0 HEP 1.2% 1000 PNU/ml 1.O HEP I .O HEP 5.0 HEP 2.5% 5000 PNU/ml I .O HEP 5.0 HEP 1 .O HEP 2.5% 5000 PNU/ml I .O HEP 10 HEP 5000 PNU/ml
*All wheals smaller than 3 mm in diameter were rejected from analysis. interpopulation variability can be ignored. When evaluating the two medians HEPb and HEP,, we counted the number of subjects in whom the wheal diameter D,, (or the individual HEP value C,) was larger than wheal D, (or C,). This number is k and the total number of subjects is n. If k > n/2, we used instead k = n - k. The binomial probability for the zero hypothesis is as follows:
s=‘i, 2” i=,i!(n
n! - l)!
(4)
A significant difference between HEP, and HEP, was taken to exist if 2s < 0.05. This nonparametric procedure is robust with respect to the distribution of diameters D (and individual HEP values C).
\/OLIJMt 73 Y JMBER 3
Biologic
TABLE VI. Biologic activities and the significance of the differences assayed by simultaneous skin tests in one patient group
activity of
observed
skin
when
lest
pr’vxvations
ailergen
329
lots were
Biologic activity (HEPI* Allergen code (from Table I)
No. of subjects
Reported manufacturer
Observed
Birch 2
42
I0
I
Birch
3
42
43
5
Birch 5
42
31
Cat
2
50
1.3
I
cat
3
50
3.2
s
cat
5
50
1.8
Cocksfoot 1 Cocksfoot 2 Dog I Dog 2 Dog 4 Mugwort 2 Mugwort 3
36
45
10
i\4U&WOrt
4
45
10
Timothy 1 Timothy 2 Timothy 4
42
5
10
Bir.
Cat
3 =
3 =
Bir
Cat
7 :-
(p C 0LJ5) between pairwise -.-HO
.I
5 :* c-ar
Cocksf. 2 > Cocksf.
’
0.1
51 51
1.3
51
0.8
45
Dog 2 = Dog 4 1;. Do:: 1
2.3
I 5
1.8
42
10
42
54
*For each allergen source material, to or larger than 2 mm.
Confidence
2.8
36
Significant differences lots compared
by
those
patients
were
included
1 5
in whom
limits for HEP values
The HEP values reported are medians. A nonparametric confidence interval for quantile q is the interval [Trj, XJ, where X refers to observations, and r and s are the ranks of the observations. Ranks r and s are calculated by the following formulas: r=nq-wsnq(l-q)
(5)
s=nq+wsnq(l-q)
(‘5)
where q is the quantile for which the confidence limits being calculated (in the present case q = 0.5, since q is median), and ws is the upper lOO(t)th percentile of standard normal distribution. These confidence limits based on the sign test.
Mugw. 3 = Mugw. 4 a Mugw. 2
are the the are
RESULTS
When the biologic activity of allergen preparations is assayed by using skin prick tests in humans, a choice has to be made as to which persons should be regarded as sensitive and suitable for inclusion in the assay population. When the choice is based on skin test results, the apparent biologic activity of the preparation to be assayed will increase as the limit for acceptable sensitivity (wheal size) is raised. When we raised the limit for an acceptable wheal size from 2 to 3 mm, the HEP value for 17 different allergen preparations rose on the average by a factor of 3. When, in addition, it was required that the minimum wheal size
Tim. 4 > Tim. 2 :, ‘PIN. i
at least one of the allergen
lots in question
gave a wheal
equal
(3 mm) had to be obtained with the prepNation being assayed and not with any preparation from similar allergen source material, there was an additional twofold rise in the HEP value. Data in Table II demonstrate the phenomenon. Another circumstance that will influence the accuracy is the choice of basic population group from which those considered sensitive to an allergen are to be selected. When the patients at the Hospital for Allergic Diseases were the basic pop&ion, HEP values on the average 10 times higher were obtained than when conscripts were tested with preparations ascribed the same biologic activity (Table HI). Conscripts in assay populations were selected for skin reactivity only, not for any disease. Human skin responsiveness varies widely: in sensitive populations usually over a range of 4 powers of 10 or more (Fig. 1). As a consequence, confidence ranges for biologic activities obtained with skin tests will be wide when population groups of a reasonable size are studied. In an example, we decreased the number of subjects from 50 to 10 in steps of 10. The apparent biologic activity then rose from 3.2 to 10 HEP, and the confidence range widened from 0.3- 10 to 0.0-55 HEP (Table IV). We have assayed the biologic activity of 43 allergen preparations, using similar criteria for the selection of sensitive subjects but three types of popula-
330 Bjorksten et al. tion groups (conscripts, patients, and students). The observed biologic activities varied over the range 0.4 to 330 HEP. For preparations that, according to the manufacturer, had the biologic activity 1 HEP, the range was narrower, from 0.4 to 10 HEP (Table V). A part of the variability was due to the use of different population groups. With a single group and somewhat different selection criteria, the observed range was 0.1 to 54 HEP for 17 allergen preparations. For each source material, significant differences in the biologic activity of the preparations studied could be detected (Table VI).
DISCUSSION Several authors have proposed that the biologic activity of allergen preparations should be determined with the aid of skin tests performed in humans.‘*fi-‘n Recently the use of skin prick tests and HEP units has been endorsed by Nordic regulatory authorities.2 In another recent approach, intracutaneous skin tests and measurement of erythema reactions have been used.Y Since intracutaneous tests can cause anaphylactic reactions, we find it difficult to justify their use for assay purposes. Erythema reactions caused by skin prick tests are too vague for measurement. Previous workers have performed skin titrations with several concentrations of the allergen preparation to be assayed. In this paper we described a modification of the HEP method in which a single concentration is used. Our method was developed to enable studies on large numbers of allergen preparations in large population groups. Titration methods are useful-and more precise-in small studies. We find that the major difficulty encountered in biologic skin test assays is the great variability in the skin response of subjects considered sensitive to the allergen under study and suitable for inclusion in an assay population (Fig. l).“* ‘I As a consequence, confidence limits for HEP values will span a wide range; typically the 95% confidence range included values from one-fifth to five times the estimated HEP value when the number of subjects in the assay group was 30 to 50 (Table IV). Confidence ranges can be narrowed by increasing the number of subjects or decreasing their variability through careful selection. Use of the histamine standard, as done when HEP units are used, also serves to reduce variability. Concerning biologic assays, Aas et al.’ stated that “. . . lo-20 individuals are adequate for this purpose. The patients should have a positive history, a positive skin prick test to routine testing and a positive RAST (RAST class 2 or more) for the allergen in question . . . . ” We still agree with this except that 20 must now be considered as a defi-
J. ALLERGY CLIN. IMMUNOL. MARCH 1984
nite minimum for the number of individuals, and preferably assay groups should include many more (cf. Table IV). The accuracy is affected by the choice of basic population group from which those considered sensitive are picked. When the basic population consisted of hospital patients, 10 times higher HEP values were obtained than with an essentially unselected (but allergen-sensitive) population of conscripts (Table III). When we used conscripts to assay seven allergen preparations, which according to the manufacturers had the biologic activity 1 HEP, we obtained values in the range of 0.4 to 4.3 I-IEP. When five similar preparations were assayed in hospital patients, the result in all cases was 10 HEP (Tables III and V). As might be expected, skin sensitivity was different in the different basic populations. Skin sensitivity is also known to vary with sex,” age,‘* and, at least in the case of pollen allergens, with the time of the year.‘” When several population groups are used, accuracy can be improved through the use of allergen standard reference preparations.” Unfortunately such standards were not available to us. When allergen preparations representing one source material were analyzed in a single group of subjects, a twofold difference in HEP values generally proved significant (p < 0.05) (Table VI). The biologic activity of the skin test preparations studied by us varied over an approximately lOOO-fold range (Tables V and VI). Some readers may wonder how it has been possible at all to use so variable and poorly defined test materials. The explanation is that the slope of the dose-response curve is low; equation 3 contains the information that a lo-fold increase in the concentration of an allergen preparation will, on the average, result in an only 1.6-fold increase in the diameter of skin-test wheals. An analysis of the results in Tables V and VI suggests that manufacturers who used the HEP unit had allergen preparations of a more uniform biologic activity than others included in the study. Not unexpectedly, we found no relationship between biologic activity and concentrations declared in PNU per milliliter or as weight/volume ratios. There is a great need for good, specific, chemical and immunochemical methods for the determination of allergen mass concentrations. Generally, the accuracy and precision of such methods are much better than those obtainable in biologic assays. For the direct determination of biologic activity, however, skin prick tests in conjunction with HEP units is a useful method. We thank Dr. Hannu Jokela, who assistedus in the study of conscripts.
‘IOLVME 73 ‘J:JMREF 3
REFERENCES 1. .& K, Backman A, Belin L, Weeke B: Standardization of ,lllergen extracts using appropriate methods. The combined use (tf >km prick testing and radio-allergosorbent tests. Allergy t?: iio, 1078 1. Nor&c Council on Medicines: Registration of allergen prepi!rations. Nordic guidelines. NLN Publication No. 7, Uppsala, j 982. Nordiska L&emedelsn&mden, pp 1-16 3. Barbee RA. Brown WG, KaItenbom W, Halonen M: Allergen \kir!-test reactivity in a community population sample: correlation with age, histamine skin reactions, and total serum immunoglobulin E. J ALLERGY CLIN IMMUNOL 68: 15, 1981 4. Swain HH, Becker LH: Quantitative studies in skin-testing. V. The whealing reactions of histamine and ragweed pollen extract. J ALLERGY 23441, 1952 5. Haahtela T, Bjijrkstln F. Heiskala M, Suoniemi I: Skin prick test reactivity to common allergens in Finnish adolescents. Allergy 35:425. 1980 6. Becker EL: Quantitative studies in skin testing. I(A). The assay of ragweed extracts by means of scratch test utilizing an “all or none” response J ALLERGY 19:108, 1948
Biologic
activity
of skip
rest
~reoarationc
331
7. Brighton WD, Topping MD. Henocy I:. A TUG, *r\ III:I!\ !irr allergen extracts. Clin Allerg) 9:591_ igl-rehpon\c curve and effect of wheal. erythema, and pallent irt’lect!on on asray results. J AI LEKGY cl IN ~~IMC!N~I ?O .+la, i.28: IO. Voorhorst R. Nikkels AH: Atoplc skin te\l me e\dluated VII. How to make allergen-test extracts blolo~icali\ equrvalent and keep them at constant strengths. Ann .Alierg!. ,.L; liiCt 1970 11. BJtirkstCn F. Haahtela T. Hannuksela M .$-,a) cli allergen preparations using direct RAST tilratiorlt, an!i laid tw:~. Allergy 35:233, 1980 12. Barbec RA. Lebowitz MD, Thompson WC. Hunow\ H: Inmediate skin-test reactivity in a general poptrlatlon \amplr. Ann Intern Med 84: 129. 1976 13. Haahtela T, Jokela H. Influence ol’ rhe pollen ir:rson on immediate skin test reactivity 10 comm\>r ali~~r~:en\. Allerg! 35.15. 1980
articles
Assay of the biologic test preparations
activity
of allergen
Fred BjiirkstBn, Ph.D., Tari Haahtela, M.D., Alf Backman, llpo Suoniemi, M.Sc. Helsinki and Tiuruniemi, Finland
skin
M.D., and
We studied the biologic activity of allergen preparations using a method involving skin prick tests in humans and the use of HEP (histamine equivalent prick) units. Results were found to be dependent on the population groups used in assays. If populations are not carefully standardized, results may vary by I power of IO. Accuracy can also be improved by the use of suitable allergen standard reference preparations, but such were not available to us. Conjidence ranges for the biologic activities were relatively wide and varied with the allergen preparation and the population group. Typically, the 95% confidence range included values from one-fifth to jive times the estimated HEP value when the number of subjects in the assay was 30 to 50 persons. When the preparations representing the same source material (e.g., timothy pollen) were assayed simultaneously in one population group of this size, a twofold or larger dtference in HEP values generally proved significant. An examination of 43 commercial products showed that allergen preparations with biologic activities declared in HEP units had a more uniform biologic activity than those assayed with traditional methods and units (PNUIml or weightlvolume). (.I ALLERGY CLIN IMMUNOL 73:324 -331, I984 .)
The potency of allergen preparations (extracts) intended for diagnostic or therapeutic use can be declared in biologic units, which describe the biologic activity in humans. The histamine equivalent prick (HEP) unit described by Aas et al. l has been endorsed by Nordic (Danish, Finnish, Norwegian, and Swedish) regulatory authorities, and the unit is now in use.* For the establishment of a HEP value, it is necessary to perform skin prick tests in a population of subjects with immediate hypersensitivity to the allergen in question. Each subject’s skin-reactivity to an allergen preparation is compared to the reactivity to a histamine
From the Hospital for Allergic Diseases, Helsinki University Central Hospital, Helsinki, and Department of Pulmonary Diseases, Tiuru Hospital, Tiuruniemi, Finland. The study was supported by a grant fmm the Finnish AntiTuberculosis Association. Received for publication Feb. 9, 1983. Accepted for publication Aug. 3 1, 1983, Reprint requests: Fred Bjkltsten, Hospital for Allergic Diseases, Meilahdentie 2, SF-00250 Helsinki 25, Finland.
324
1
0.1
1 INDIVIDUAL
100
10
1000
HEP
Fig. 1. Distribution
of individual HEP values in a group of 50 patients skin prick-tested with the allergen preparation “Cat 3” (cf. Table I). In nine patients the result was 0 HEP, and these have been omitted from the figure.
Histamine equivalent RAST:
Radioallergosorbent
prick (unit) test
VOLUME hUMRER
?3 3
Biologic
TABLE I. Allergen
preparations
-_.- _----
Source
studied
material
Cat epithelium
Cocksfoot pollen (Ductylis glomeruta) Dog epithelium 4
Fish Horse epithelium House dust
House dust mite
Mugwort pollen vulgaris)
Timothy pollen (Phleum pratense)
White oxeye daisy pollen (Chrysanthemum leucanthemum) ALK
< Allergologisk
Laboratorium,
_~._. Allergen
Alder pollen (A/nu.s sp.) Birch pollen (Be/u/a sp.)
(Artemisia
activity of skin test preoarations
Copenhagen,
code
Alder I Birch I Birch 2 Birch 3 Birch 4 Birch 5 Birch 6 Birch 7 cat I Cat 2 Cat 3 Cat 4 Cat 5 Cat 6 Cocksfoot I Cocksfoot 2 Cocksfoot 3 Dog I Dog 2 Dog 3 Dog 4 Dog 5 Fish 1 Horse 1 House dust 1 House dust 2 House dust 3 Mite I Mite 2 Mite 3 Mugwort I Mugwort 2 Mugwort 3 Mugwort 4 Mugwort 5 Timothy I Timothy 2 Timothy 3 Timothy 4 Timothy 5 Timothy 6 Timothy 7 White oxeye 1
Manufacturer
-_
i.nt
325
._~. - ..__..
#lo.
Dome .4LK 4LK ALK Dome Dome Phannacia Phannacia ALK ALK ALK Bencard Bencard Dome ALK ALK Dome ALK ALK Bencard Bencard Dome Bencard Bencard ALK Bencard Dome ALK Bencard Dome ALK ALK ALK Bencard Dome ALK ALK ALK Bencard Dome Pharmacia Pharmacia Dome
Denmark.
standard. This histamine standard is used to decrease interindividual variability, since the skin response to an allergen is dependent on the response to histamine.3~ 4 In the present study we have investigated the usefulness of a HEP method. We will discuss the difficulties involved and present some accuracy and confidence range data. We also report HEP values for
43 commercial allergen preparations intended for skin prick testing. In the future it will become possible to declare the concentration of individual allergens-at least main allergens-in mass units, and this is very desimble. Even then, biologic units will also be needed because the biologic activity of allergen preparutions and purified allergens will remain of interest. It will, for
326
Bjiirksten
et al.
J. ALLERGY CLIN. IMMUNOL. MARCH 1984
TABLE II. Effect of the lower (HEP value)
of allergen
limit chosen for wheals to be measured on the apparent preparations in subjects from the patient population Smallest
Allergen code (from Table I)
measured
whea12 Biologic
No. of subjects
Birch 2 Birch 3 Birch 5 Cat 2 Cat 3 Cat 5 Cocksfoot 1 Cocksfoot 2 Dog I Dog 2 Dog 3 Mugwort 2 Mugwort 3 Mugwort 4 Timothy 1 Timothy 2 Timothy 4
42 42 42 50 50 50 36 36 51 51 51 45 45 45 42 42 42
mm*
All wheals
activity WEP)
MATERIALS,
Biologic
AND METHODS
Subjects came from the following three population groups. Conscripts were 220 unselected young men, ages 18 to 19, from Imatra, a small industrial town. Skin testing took place in April and May 1979 in conjunction with a compulsory health examination for military service. Patienrs were persons referred to the outpatient department of the Hospital for Allergic Diseases, University Central Hospital, Helsinki, because of a variety of respiratory and/or dermal complaints related to type I allergy. The mean age of the patients was 25.7 years (range 5 to 70). Skin tests were performed in 92 to 112 patients (64% females, on the average); the number varied with the allergen source material. Testing took place in May and June 1979. Students were 708 unselected ninth grade students (55% girls) in Imatra schools. The study was carried out in conjunction with compulsory tuberculosis screening. Of those studied, 99% were 15 to 17 yr old. Skin testing was carried out in March to May 1978.
Allergens We studied altogether 43 allergen preparation lots from 12 allergen source materials and four manufacturers (Table I). All preparations were intended for skin prick testing. Different manufacturers used different solvents: extracts
material
activity
rejectedt
37 37 37 28 31 31 26 29 18 29 34 29 35 38 24 29 32
*Wheals under the limit were given the diameter 0 mm. If all lots from the same source patient’s results for the source material in question were omitted from analysis. ?A11 wheals smaller than the limit were omitted from analysis.
SUBJECTS, Subjects
3 mm
No. of subjects
IO 43 31 1.3 3.2 1.8 2.8 IO 0.1 1.3 0.8 2.3 IO IO I.8 IO 54
instance, be necessary to measure the biologic activity of standard reference preparations.
under
biologic
activity (HEI’)
IO 77 77 10 31 10 10 25 3.2 IO 2.8 IO 55 37 IO 31 330 produced
0 mm wheals
in a patient,
this
from Allergologist Laboratorium (Copenhagen, Denmark) contained 50% glycerol; 0.45% sodium chloride; extracts from Bencard, Ltd. (Betchworth, Surrey, U.K.) contained 50% glycerol; extracts from Dome Laboratories, Div. Miles Laboratories (Stoke Poges, Slough, U.K.) contained 50% glycerol and 0.4% phenol; Pharmacia Diagnostics (Uppsala, Sweden) supplied freeze-dried allergen preparations and a solvent containing 0.9% sodium chloride and 0.4% phenol. In addition, we used a negative reference solution containing 50% glycerol and a positive reference solution containing 1 mg/ml (5.43 mmol/L) histamine dihydrochloride in 50% glycerol.
Skin prick tests Skin prick tests were performed with undiluted allergen solutions as they were supplied by the manufacturers. A droplet of each solution to be tried was applied to the volar aspect of the forearm. A disposable needle was then pushed through the droplet, the skin was punctured, and the epidermis lifted without causing bleeding. The largest diameter of the wheal (D) and the diameter vertical to this (d) were measured after 15 min. At this time the histamine wheal is already decreasing in size, but change from the maximum is slight. As the final result, we used the mean allergen wheal diameter D, = (D + d)/2. The mean histamine wheal diameter, D,,, was obtained in an analogous manner. The different population groups were skin-tested by different persons, and minor variations in technique may have occurred. In the conscripts, a coefficient of variation of 15%
VOLUME 73 N;JMB::R :i
Biologic
TABLE 111.Effect of the choice
of assay
population Biologic
Allergen code (from Table 1)”
on the biologic activity (HEPIt
activity
(HEP) observed in ;~ssays ___ ___-.-__...” I-..--_-.-. -..-
found
Biologic activity or concentration reported by manufacturer
Patients
Conscripts
Birch I Birch 7 cat I cat 2 Dog I Mugwort I Mugwort 2 Timothy 3 Timothy 1
activity of skin test aret*~%‘a!ior$s 327
I .o 10 0.4 10 3 .2
I.0 1 .o
IO 3.1 IO
* 411 allergen lots came from Allergologisk Laboratorium. t For each allergen source material, those patients were included in whom at least one of the allergen lots in question gave ‘8 when1 equal to or larger than 2 mm
TABLE IV. Variation (patients)
included
of the biologic activity in the assay population
and its confidence
range
with Biologic
Akgen (from
code Table I)
No.
of subjects*
cat 3
Assay
the number activity
result
(HEPIt
---_.
.-
950/e confiience
IO
IO
of subjects
il.!)-54
20
I .o
tJ. I- 11:
30
I.0 2.8
fl.1, 1%) :I. i- 1i! C!..Glli
40 50
range
3.2
*.U results are from the same assay in a group of 50 patients. Results were randomly removed to obtain groups with fewer members. tFor the allergen source material, those patients were included in whom at least one of the allergen lots in question gave a wheal equal to or larger than 2 mm
to 20% in wheal diameters was observed when multiple skin tests with the same allergen preparation were performed in the same subject.
tions). Inserting values for u and b in equation i, and solving for C. we obtain the following (in HEP unit.sr.
Calculation
When the diameters of the wheals caused in a subject by skin tests with an allergen preparation (D;,) and the histamine reference (D,,) are known, an “individual HEP’~ can be calculated from equation 3. The individual HEP is a function of the biologic activity of the prepanttion and the subject’s sensitivity to the allergen in question. The biologic activities (HEP values) that we report for allergen preparations are medians of the individual HEP values obtained in studies of sensitive population groups. Use ot medians eliminates any error due to the inclusion of hughly sensitive patients with large whealing reactions far whom the linear relationship characterized by equation 1 may not hold.
of HEP values
The dose-response relationship to borh histamine and allergens in skin tests can be approximated by the following equation’. ,‘: log Da,h = N log C + h
(1)
where Da,h is the mean wheal diameter, C is the allergen concentration, and a and b are constants. When HEP units are used, then by definition the allergen concentration (C) is 1 HEP when the allergen wheal diameter (D,) is equal to the histamine wheal diameter (D,,). As a result, we obtain the following from equation 1: log Dh = b
(2)
For the constant u, we have obtained the same empirical value 0.198 in the case of both allergens and histamine (Backman, BjiirkstCn, and Suoniemi, unpublished observa-
C = (D,/D,,y.“”
Significant
diffwenme
(3)
in f4EP w&es
We examined the significance of difference?, in (median) HEP values obtained when several lots of allergen from similar source material were assayed with simultaneous skin tests in the same subjects. In this case, interindividual and
328
Bjiirksth
J. ALLERGY CLIN. IMMUNOL. MARCH 1984
et al.
TABLE V. Biologic
activities
(HEP) found Subjects
Allergen code (from Table I)
Alder I Birch 1 Birch 2 Birch 3 Birch 4 Birch 5 Birch 6 Birch 7 Cat 1 Cat 2 Cat 3 Cat 4 Cat 5 Cat 6 Cocksfoot I Cocksfoot 2 Cocksfoot 3 Dog 1 Dog 1 Dog 2 Dog 3 Dog 4 Dog 5 Fish 1 Horse I House dust I House dust 2 House dust 3 Mite I Mite 2 Mite 3 Mugwort I Mugwort 2 Mugwort 3 Mugwort 4 Mugwort 5 Timothy I Timothy 2 Timothy 3 Timothy 4 Timothy 5 Timothy 6 Timothy 7 White oxeye I
Type
of population
Students Conscripts Patients Patients Students Patients Conscripts Conscripts Conscripts Patients Patients Students Patients Students Patients Patients Students Conscripts Patients Patients Students Patients Students Students Students Conscripts Students Students Conscripts Students Students Conscripts Patients Patients Patients Students Patients Patients Conscripts Patients Students Conscripts Conscripts Students
and activities
or concentrations
reported
Biologic activity or concentration reported by manufacturer
tested Biologic activity (HEP)*
No.
80
22 1.0 IO 77 19 77 1.0 7.7 0.4 IO 31 I.8 IO I.1 IO 25 IO 1.0 3.2 IO 0.9 2.8 1.1 0.8 0.6 0.3 1.3 0.9 1.0 1.3 0.6 1.0 IO 55 37 3.6 IO 31 3.1 330 7.5 4.3 130 0.9
37 37 39 91 37 27 41 59 28 31 221 31 loo 26 29 128 43 18 29 100 34 34 66 143 81 273 145 24 137 98 69 29 35 38 103 24 29 45 32 124 44 61 66
by manufacturers
5000 PNU/mI I .O HEP I .O HEP 5.0 HEP 5000 PNU/ml 3000 PNU/ml 1.0 HEP IO HEP I .O HEP I .O HEP 5.0 HEP 150% 150% 5000 PNU/mI 1 .O HEP 5.0 HEP 5000 PNU/ml 1: 100 I:100 I:20 150% 150% 5000 PNU/ml 10% 150% 1:IOO 150% 5000 PNU/ml 1.0 HEP 1.2% 1000 PNU/ml 1.O HEP I .O HEP 5.0 HEP 2.5% 5000 PNU/ml I .O HEP 5.0 HEP 1 .O HEP 2.5% 5000 PNU/ml I .O HEP 10 HEP 5000 PNU/ml
*All wheals smaller than 3 mm in diameter were rejected from analysis. interpopulation variability can be ignored. When evaluating the two medians HEPb and HEP,, we counted the number of subjects in whom the wheal diameter D,, (or the individual HEP value C,) was larger than wheal D, (or C,). This number is k and the total number of subjects is n. If k > n/2, we used instead k = n - k. The binomial probability for the zero hypothesis is as follows:
s=‘i, 2” i=,i!(n
n! - l)!
(4)
A significant difference between HEP, and HEP, was taken to exist if 2s < 0.05. This nonparametric procedure is robust with respect to the distribution of diameters D (and individual HEP values C).
\/OLIJMt 73 Y JMBER 3
Biologic
TABLE VI. Biologic activities and the significance of the differences assayed by simultaneous skin tests in one patient group
activity of
observed
skin
when
lest
pr’vxvations
ailergen
329
lots were
Biologic activity (HEPI* Allergen code (from Table I)
No. of subjects
Reported manufacturer
Observed
Birch 2
42
I0
I
Birch
3
42
43
5
Birch 5
42
31
Cat
2
50
1.3
I
cat
3
50
3.2
s
cat
5
50
1.8
Cocksfoot 1 Cocksfoot 2 Dog I Dog 2 Dog 4 Mugwort 2 Mugwort 3
36
45
10
i\4U&WOrt
4
45
10
Timothy 1 Timothy 2 Timothy 4
42
5
10
Bir.
Cat
3 =
3 =
Bir
Cat
7 :-
(p C 0LJ5) between pairwise -.-HO
.I
5 :* c-ar
Cocksf. 2 > Cocksf.
’
0.1
51 51
1.3
51
0.8
45
Dog 2 = Dog 4 1;. Do:: 1
2.3
I 5
1.8
42
10
42
54
*For each allergen source material, to or larger than 2 mm.
Confidence
2.8
36
Significant differences lots compared
by
those
patients
were
included
1 5
in whom
limits for HEP values
The HEP values reported are medians. A nonparametric confidence interval for quantile q is the interval [Trj, XJ, where X refers to observations, and r and s are the ranks of the observations. Ranks r and s are calculated by the following formulas: r=nq-wsnq(l-q)
(5)
s=nq+wsnq(l-q)
(‘5)
where q is the quantile for which the confidence limits being calculated (in the present case q = 0.5, since q is median), and ws is the upper lOO(t)th percentile of standard normal distribution. These confidence limits based on the sign test.
Mugw. 3 = Mugw. 4 a Mugw. 2
are the the are
RESULTS
When the biologic activity of allergen preparations is assayed by using skin prick tests in humans, a choice has to be made as to which persons should be regarded as sensitive and suitable for inclusion in the assay population. When the choice is based on skin test results, the apparent biologic activity of the preparation to be assayed will increase as the limit for acceptable sensitivity (wheal size) is raised. When we raised the limit for an acceptable wheal size from 2 to 3 mm, the HEP value for 17 different allergen preparations rose on the average by a factor of 3. When, in addition, it was required that the minimum wheal size
Tim. 4 > Tim. 2 :, ‘PIN. i
at least one of the allergen
lots in question
gave a wheal
equal
(3 mm) had to be obtained with the prepNation being assayed and not with any preparation from similar allergen source material, there was an additional twofold rise in the HEP value. Data in Table II demonstrate the phenomenon. Another circumstance that will influence the accuracy is the choice of basic population group from which those considered sensitive to an allergen are to be selected. When the patients at the Hospital for Allergic Diseases were the basic pop&ion, HEP values on the average 10 times higher were obtained than when conscripts were tested with preparations ascribed the same biologic activity (Table HI). Conscripts in assay populations were selected for skin reactivity only, not for any disease. Human skin responsiveness varies widely: in sensitive populations usually over a range of 4 powers of 10 or more (Fig. 1). As a consequence, confidence ranges for biologic activities obtained with skin tests will be wide when population groups of a reasonable size are studied. In an example, we decreased the number of subjects from 50 to 10 in steps of 10. The apparent biologic activity then rose from 3.2 to 10 HEP, and the confidence range widened from 0.3- 10 to 0.0-55 HEP (Table IV). We have assayed the biologic activity of 43 allergen preparations, using similar criteria for the selection of sensitive subjects but three types of popula-
330 Bjorksten et al. tion groups (conscripts, patients, and students). The observed biologic activities varied over the range 0.4 to 330 HEP. For preparations that, according to the manufacturer, had the biologic activity 1 HEP, the range was narrower, from 0.4 to 10 HEP (Table V). A part of the variability was due to the use of different population groups. With a single group and somewhat different selection criteria, the observed range was 0.1 to 54 HEP for 17 allergen preparations. For each source material, significant differences in the biologic activity of the preparations studied could be detected (Table VI).
DISCUSSION Several authors have proposed that the biologic activity of allergen preparations should be determined with the aid of skin tests performed in humans.‘*fi-‘n Recently the use of skin prick tests and HEP units has been endorsed by Nordic regulatory authorities.2 In another recent approach, intracutaneous skin tests and measurement of erythema reactions have been used.Y Since intracutaneous tests can cause anaphylactic reactions, we find it difficult to justify their use for assay purposes. Erythema reactions caused by skin prick tests are too vague for measurement. Previous workers have performed skin titrations with several concentrations of the allergen preparation to be assayed. In this paper we described a modification of the HEP method in which a single concentration is used. Our method was developed to enable studies on large numbers of allergen preparations in large population groups. Titration methods are useful-and more precise-in small studies. We find that the major difficulty encountered in biologic skin test assays is the great variability in the skin response of subjects considered sensitive to the allergen under study and suitable for inclusion in an assay population (Fig. l).“* ‘I As a consequence, confidence limits for HEP values will span a wide range; typically the 95% confidence range included values from one-fifth to five times the estimated HEP value when the number of subjects in the assay group was 30 to 50 (Table IV). Confidence ranges can be narrowed by increasing the number of subjects or decreasing their variability through careful selection. Use of the histamine standard, as done when HEP units are used, also serves to reduce variability. Concerning biologic assays, Aas et al.’ stated that “. . . lo-20 individuals are adequate for this purpose. The patients should have a positive history, a positive skin prick test to routine testing and a positive RAST (RAST class 2 or more) for the allergen in question . . . . ” We still agree with this except that 20 must now be considered as a defi-
J. ALLERGY CLIN. IMMUNOL. MARCH 1984
nite minimum for the number of individuals, and preferably assay groups should include many more (cf. Table IV). The accuracy is affected by the choice of basic population group from which those considered sensitive are picked. When the basic population consisted of hospital patients, 10 times higher HEP values were obtained than with an essentially unselected (but allergen-sensitive) population of conscripts (Table III). When we used conscripts to assay seven allergen preparations, which according to the manufacturers had the biologic activity 1 HEP, we obtained values in the range of 0.4 to 4.3 I-IEP. When five similar preparations were assayed in hospital patients, the result in all cases was 10 HEP (Tables III and V). As might be expected, skin sensitivity was different in the different basic populations. Skin sensitivity is also known to vary with sex,” age,‘* and, at least in the case of pollen allergens, with the time of the year.‘” When several population groups are used, accuracy can be improved through the use of allergen standard reference preparations.” Unfortunately such standards were not available to us. When allergen preparations representing one source material were analyzed in a single group of subjects, a twofold difference in HEP values generally proved significant (p < 0.05) (Table VI). The biologic activity of the skin test preparations studied by us varied over an approximately lOOO-fold range (Tables V and VI). Some readers may wonder how it has been possible at all to use so variable and poorly defined test materials. The explanation is that the slope of the dose-response curve is low; equation 3 contains the information that a lo-fold increase in the concentration of an allergen preparation will, on the average, result in an only 1.6-fold increase in the diameter of skin-test wheals. An analysis of the results in Tables V and VI suggests that manufacturers who used the HEP unit had allergen preparations of a more uniform biologic activity than others included in the study. Not unexpectedly, we found no relationship between biologic activity and concentrations declared in PNU per milliliter or as weight/volume ratios. There is a great need for good, specific, chemical and immunochemical methods for the determination of allergen mass concentrations. Generally, the accuracy and precision of such methods are much better than those obtainable in biologic assays. For the direct determination of biologic activity, however, skin prick tests in conjunction with HEP units is a useful method. We thank Dr. Hannu Jokela, who assistedus in the study of conscripts.
‘IOLVME 73 ‘J:JMREF 3
REFERENCES 1. .& K, Backman A, Belin L, Weeke B: Standardization of ,lllergen extracts using appropriate methods. The combined use (tf >km prick testing and radio-allergosorbent tests. Allergy t?: iio, 1078 1. Nor&c Council on Medicines: Registration of allergen prepi!rations. Nordic guidelines. NLN Publication No. 7, Uppsala, j 982. Nordiska L&emedelsn&mden, pp 1-16 3. Barbee RA. Brown WG, KaItenbom W, Halonen M: Allergen \kir!-test reactivity in a community population sample: correlation with age, histamine skin reactions, and total serum immunoglobulin E. J ALLERGY CLIN IMMUNOL 68: 15, 1981 4. Swain HH, Becker LH: Quantitative studies in skin-testing. V. The whealing reactions of histamine and ragweed pollen extract. J ALLERGY 23441, 1952 5. Haahtela T, Bjijrkstln F. Heiskala M, Suoniemi I: Skin prick test reactivity to common allergens in Finnish adolescents. Allergy 35:425. 1980 6. Becker EL: Quantitative studies in skin testing. I(A). The assay of ragweed extracts by means of scratch test utilizing an “all or none” response J ALLERGY 19:108, 1948
Biologic
activity
of skip
rest
~reoarationc
331
7. Brighton WD, Topping MD. Henocy I:. A TUG, *r\ III:I!\ !irr allergen extracts. Clin Allerg) 9:591_ igl