Diagnostic and therapeutic applications of a modified radioallergosorbent test and comparison with the conventional radioallergosorbent test

D@~osGc and thermutic ap@wW~ of a modi#ed radioMer~ test and comparison with the conlsentian~al radioa#ergosorbent test P. J. Santrach, B.A., J. L. Parker, D.O., I?. 1. Jonas, B.S., and J. W. Yunginger, M.D.* Rochester. Minn.

A mod$cd rutliouIler~o.sorhettt test (RAST) assuy and scoring system fFuduliNu/ehuj’ RAST) hu~v been purported to increuJe the diagnostic accurucy und .sen.sitivit~ of rhe RAST. In order to t~s.w.~.~ such cluim.s, WC compared this modified RAST with the conventional Phudebas RAST in 40 putients with .sea.so,lul ullergic rhinitis. Phadebus und modified RAST scores to nine trller,qvn\ I~I’W highly correluted (r = l .93) and results from either RAST correlated with skin-test titrutiori end point.\. Clinical sensitivity to test allergens 11’1i.siissessed u.rinLq nasul prin~ncutron titrution: po.siti).e RAST determinutions by both method.5 \cvre ussociuted with ne~uti~v pro~‘ocution tests in fewer than 107~ of cuse.s. HoM,e\,er. po.siti\,e pro~vcation tests hvre noted in o\‘cr htrlf the putients echo bud negative Phudehas RAST results: the inclu.tion of an udditronul reference poinr ut the lok,er end of the standard c‘rtrve mu! increase the diugno.stic trc~c~rrtrc~~ of the test. .~o,l-I~E-c.o,ltciininK control preparations produced equivocul results in JIM to .stww 01 21 mod$ed RAST assuy. but produced negutirv results in all Phudebu.s RAST u.s.suy. Buck~qround binding produced b! negatil*e control prepururions luried c.onsidertrbly umoq d@,rent ullergen discs, indicuting that the cur&’ point between po.siti\ic und negurivi~ mq! \urv f?om uller~en to ullergen. The modified RAST NIJ.S ulso rtsed to c~ult~tlt~re initiul irn,?lrt,lottlc~rcii~~ doses. Preliminary skin tests ut these predicted allergen (,oll(.elitr(lliO))5 proa’ucccl It~r;ty whetrl.s I >I6 mm) in I5 of 50 triuls (30%). und in this sirltution immunotherup~ do.se.s were withheld. The remuining 35 patients toleruted the initial immunotherup! do.se.s withortt incident. A.5 presently performed. the modiJed RAST c~.ssu~ does not u&d ~i@ficunt diugnostic~ ullergen extruct.s are trdvuntuge.s o\,er the Phudebus RAST; until such u time (IS stundurdted tr\Gluble. potential therupeutic upplicutions of the mod$ed RAST remuin .specultrtil.e.

Pbadebas RAST (Ph RAST; Pharmacia) kits for the measurement of allergen-specific IgE antibody levels

have been marketed commercially for several years by Pharmacia Diagnostics (Piscataway. N. J.). With these kits RAST results are expressed either on a semiquantitative basis (0 to 4) or in arbitrary Phadebas RAST units. More recently, “modif$zd” RAST (FadaVNalebuff) scoring systems have been proposed by other commercial and noncommercial groups.‘-” Proponents of the modified RAST scoring system allege that the diagnostic sensitivity of the test is increased, thus permitting the identification of patients with lower but clinically significant 1gE antibody levels. The proponents have also used these modified RAST scores to predict optimal starting doses for allergen immunotherapy. However, these

From the Departments of Pediatrics and Internal Medicine (Aller#y), the Allergic Diseases Research Laboratory, and the Mayo Grtiuate School of Medicine, Mayo Clinic and Foundation. This -tudy was supported in part by grants from Pharmacia Diagnosks. Inc.. from the National Institutes of Health (AI-1 1483). and from the Mayo Foundation. Recejged for publication Aug. 15. 1980. Accepted for publication Oct. 13. 1980. Reprint requests to: Dr. J. W. Yunginger. Allergic Diseases Research Laboratory. Mayo Clinic, Rochester, MN 55901. *Dr. ‘k’unginger - - is the recipient of an Allergic Diseases Academic Arard (AI-00107). 0091.6749/81/020097+08$00.80/0

0

1981

The

C. V. Mosby

Co.

Vol.

67, No. 2. pp. 97-104

J. ALLERGY

98 Santrach et al.

CLIN. IMMUNOL. FEBRUARY

A/hw~icrtio~r~s uwd RAST: Radioallergosorbent test PRU: Phadebas RAST units FCS: Fetal calf serum HSA: Human serum albumin HCS: Human cord serum PRIST: Paper radioimmunosorbent test TA: Total activity TC: Time control

reports have been anecdotal, have not presented comparative data using Ph RAST scoring in the same patient groups, and have not included provocation tests to document the diagnoses of IgE-mediated disease. The aims of the present study were to measure serum IgE antibodies by both the Ph and the modified RAST, to compare the Ph and modified RAST scores with those obtained using a background-based scoring method, to compare these RAST data with results of both skin testing and nasal provocation testing, and to

document the safety of initial immunotherapy doses predicted by the modified RAST. MATERIALS Patients

AND METHODS

We studied 40 patients (10 male, 30 female) aged 14 to 46 yr, with a positive history of seasonal allergic rhinitis or perennial rhinitis with a definite seasonal flare who responded to an advertisement soliciting volunteers. By clinical history there was a reasonable suggestion of pollen sensitivity in all patients. Pregnant or lactating females and patients who had received immunosuppressive or systemic glucocorticoid therapy in the preceding year were excluded. Thirty patients had never received immunotherapy for inhalent allergy, while IO had discontinued such immunotherapy at least 3 yr before the study. Patients omitted antihistamine and bronchodilator medications for at least 24 hr before all in vivo studies.

1981

skin tests were reconstituted to 1: 100 and I : 1,000 (w :v) with Sorenson’s phosphate buffer” containing 0.5% HSA (S-HSA). Sufficient allergen was injected to produce an initial 5-mm wheal; subsequent wheal-and-flare diameters were recorded 20 min later. The negative control wax S-HSA, while histamine HCI (0.0275 mgiml) was the positive control. Subsequently, we performed intradermal skintest end-point titrations with a single allergen (timothy. oak, birch, maple, or ragweed) that had been selected earlier on the basis of a positive clinical history and a positive or equivocal modified RAST result. Extracts were reconstituted in S-HSA to I : 100 (No. 1 dilution), from which serial fivefold dilutions were prepared in S-HSA to 1 : 39,062,500 (No. 9 dilution). End-point titration skin tests were read at both 10 and 20 min. The larger values were reported and were graded according to the system of Norman.’ The skin tests were serially administered beginning with the lowest concentration until a 2f reaction was observed or the I : 100 concentration was attained. The allergen concentration producing the first 2+ reaction in a series of progressively increasing wheal reactions (at least 2 mm from concentration to concentration) was designated as the end point.

Nasal provocation

tests

Nasal provocation tests were performed in a modified double-blind manner with the same 1 : 100, I :500, and I : 1,000 (w: v) extracts in S-HSA used for skin testing. Initially, a control test was administered by instilling S-HSA (0. I ml) into each nostril. The allergen solution was then instilled into one nostril, while S-HSA was placed into the opposite nostril. Provocation tests were administered by one investigator and were scored by a second investigator who, along with the patient, was unaware of which nostril had been challenged with allergen. Allergens were administered serially beginning with the I : 1,000 concentration until a positive response was noted or the 1 : 100 solution was given. Patients were observed for 20 min after instillation; tests were considered positive if sneezing, pruritus, rhinorrhea, congestion, or wheezing was induced. Challenges to different allergens were performed on different days.

Ph RAST Skin tests Lyophilized pollen extracts were provided by Pharmacia Diagnostics; these included short ragweed, English plantain, orchard grass, timothy grass, alder, elm, oak, birch, and maple. By RAST inhibition the manufacturer compared the allergenic potency of the short ragweed, birch, oak, timothy grass, and orchard grass pollen extracts with reference extracts provided either by the Bureau of Biologics, Food and Drug Administration (Bethesda, Md.) or by the National Institute for Biological Standards and Control (London, England). For preliminary puncture skin tests we reconstituted each extract with 50% glycerin to I : 20 (w : v) concentration. Positive and negative control tests included histamine HCI (I mg/ml in 50% glycerin) and 50% glycerin alone, respectively. Extracts for preliminary intradermal

The Ph RAST utilizes a standard curve produced by reacting birch pollen RAST discs with four concentrations of a reference serum pool containing IgE antibodies to birch. These four references (A, B, C, D) contain 17.5,3.5, 0.7, and 0.35 PRU of birch IgE antibody. We attempted to increase the sensitivity of the Ph RAST by adding a fifth reference point (E) containing 0.14 PRU birch IgE antibody. Otherwise, the Ph RAST was performed using all reagents supplied with the kit, according to the manufacturer’s instructions. Patient sera (0.05 ml) were tested in duplicate against the same nine pollen allergens used for skin testing; results were reported in both PRU and Ph RAST class (0 to 4). IgE antibody levels were considered elevated if the Ph RAST result was greater than 0.35 PRU (class I or greater). We examined three negative control

VOLUME NUMBER

Modified radioallergosorbent

67 2

preparations in each assay. including FCS, a 5% HSA solution, and pooled HCS. These controls produced results ranging from 0.01 to 0.18 PRU (class 0) for all 21 assays performed. Each assay also contained a positive control preparation, either a single patient serum (for birch, oak, aider, English plantain, and maple) or a serum pool from individuals known to have elevated specific IgE antibodies (for timothy grass, orchard grass, and short ragweed). No positive control was available for the elm assays. The mean levels for these controls ranged from 2.3 to 30.2 PRU with coefficients of variation between assays (2 to 3 assays/ allergen) ranging from 2% to 62% depending on the particular allergen.

Modified

RAST (FadallNalebuff

test

99

TABLE I. Modified RAST scoring system (Condensed from Fadal RG, Nalebuff DJ: Modified RAST procedure. New Jersey, 1979) Modified RAST class

Range expressed as % time control

0

<3.0% 3.0%-6.7% 6.7%- 14.8% 14.8%-32.5% 32.5%-72% 72%- 160% > 160%

I 2 3 4 5 6

RASTI

The modified RAST utilizes “time control” tubes for scoring RAST results.x A Phadebas IgE PRIST kit (Pharmacia) standard serum that contained 100 U IgE/ml (Pharmacia Diagnostics) and that was diluted 1: 4 with 0.9% NaCl was tested (0.1 ml) in duplicate with PRIST discs (containing anti-IgE) to serve as the time control. Patient sera (0.1 ml) were tested in duplicate using Ph RAST allergen discs. We used negative and positive controls for the modified RAST that were identical to those in the Ph RAST. All tubes were covered with plastic film and allowed to stand overnight at room temperature. The discs were subsequently washed three times by adding 2.5 ml of 0.9% NaCl, allowing them to stand for 10 min, and removing the saline by aspiration. In the second stage of the assay ‘21-labeled anti-IgE (Pharmacia Diagnostics, 0.05 ml) was added, after which the tubes were capped and incubated overnight at room temperature. The washing procedure was then repeated, and the discs were transferred to clean test tubes and capped. The duplicate time control tubes were first counted in a gamma scintillation counter until 40,000 counts were obtained. The entire assay was then counted for the average time necessary for the time control tubes to reach 40,000 counts.* Results from control and patient sera were calculated as a percent of the time control and assigned a modified RAST class according to Table I.’ Modified RAST class 0 was considered negative, class 1 was considered equivocal, and classes 2 through 6 were increasingly positive. Using the nine previously designated allergen discs, the FCS binding ranged from 0.7% to 3.3% of the time control, while the ranges for the HSA and the HCS were from 0.9% to 4.7% and 0.9% to 3.2% of the time control, respectively. The radioactive binding produced by FCS and HCS was sufficient to score as class 1 in one of 2 1 modified RAST assays, whereas HSA scored as class 1 in seven of 21 assays. For the positive control sera, the mean binding in terms of the percent of the time control ranged from 26.8% to 116.7% with interassay coefficients of variation from 0.6% to 39.4% depending on the allergen. The *The originators of the modified RAST advocate 25,000 counts as the desired level of activity for the time control.’ Because of the design of the gamma scintillation counter used in this study, we could set the machine to count for 40,000 but not for 25,000 counts. The subsequent use of a percent of the time control to assign RAST class makes the two methods comparable.

TABLE II. Modified RAST-based initial immunotherapy dose (Condensed from Fadal RG, Nalebuff DJ: Modified RAST procedure. New Jersey, 1979) Modified RAST class

0 1 2 3 4 5 6

RAST-1 concentration

No therapy 1:500 1:2,500 1: 12,500 1:62,500 1:312,500 1: 1,562,500

RAST-0 concentration

No therapy 1: 100 1:500 1:2,5OO 1:12,500 1:62,500 1:312,500

positive control sera scored as either class 3, 4, or 5 in all assays.

Background-based

RAST scoring

Our laboratory has previously utilized a backgroundbased method of scoring the RAST .9 The Ph RAST binding values were recalculated and expressed in terms of a percent of the HCS negative control for each allergen. Results of 200% to 250% were considered equivocal while those above 250% were considered positive.

Study

protocol

Initial serum samples were obtained from all patients between May and July, 1979 (visit l), and were tested for allergen-specific IgE antibody by both the Ph and modified RAST. During a second visit, from August to November, a detailed clinical history was obtained from each patient, and screening puncture and intradermal skin tests were performed with all allergens. Based on the clinical history and an elevated or borderline modified RAST class, we initially chose to study one pollen in each patient. lntradermal endpoint skin-test titrations and nasal challenges were performed with this allergen. Data from these two visits were used to compare the diagnostic efficacy of the Ph and modified RASTs. We also determined whether the modified RAST results could be used to predict a safe immunotherapy starting dose, as proposed by Fadal and Nalebuff.‘. ”

100

Santrach et al.

J. ALLERGY

Phadrbas

RAST

Modified

r-to.70

r*+O.SO

(n-50)

. .

__

. . .

It if)t .. .

.

W” . .

.

tion was given subcutaneously as the initial immunotherapy dose. If either skin test produced a 16-mm or greater wheal diameter, the initial immunotherapy dose was withheld; a serum sample was obtained for a repeat Ph and modified RAST determination, and the skin-test end point was retitrated. Ten of the 40 patients returned for two additional visits to undergo the same in vivo tests (skin-test end-point titration, nasal challenge, and initial immunotherapy dose) for a different allergen, giving a total of 50 allergen trials.

(n.50)

RAST

.

.

.

”

.

RESULTS Diagnostic

studies

The fifty allergen trials were distributed among five pollens: short ragweed (n = 23), birch (n = 7), maple (n = 7), oak (n = 6), and timothy (n = 7).

.

.”

3.0

All patients showed a 2f Skin

Test

Endpoint

Dilution

FIG. 1. Correlation between skin-test end points and the two RAST procedures.

TABLE III. Correlation between nasal provocation tests

RAST class and

Ph RAST class

Modified

01234

0

Nasal provocation 8 3 13 14 4 Positive Negative 5 1 2 0 0 13 4 15 14 4 Total

ND ND 0

RAST class

123456

3 7 8 14 10 0 5 2 1 0 0 0 8 9 9 14 10 0

ND = Not done. TABLE IV. Correlation between expanded classes and nasal provocation tests

CLIN. IMMUNOL. FEBRUARY 1981

Ph RAST

Ph RAST class Nasal provocation

0

%

1

2

3

4

Positive Negative Total

2 3 5

6 2 8

3 1 4

13 2 15

14 0 14

4 0 4

This recommended starting dose is 0.1 ml of the “RAST-0” concentration calculated from Table II.’ A more conservative alternative approach is to begin immunotherapy with 0.1 ml of the “RAST-1” concentration listed in Table II; RAST-1 concentrations are fivefold more dilute than RAST-0 concentrations. Patients returned for a third visit between 1 and 4 wk later and were skin tested intradermally as described previously with the RAST-1 and RAST-0 concentrations of the allergen in question. If a wheal of less than 16-mm diameter was elicited with the RAST-1 and RAST-0 concentrations, 0.1 ml of the RAST-0 concentra-

or greater intradermal

skin

test reaction to either the 1: 1,000 or 1: 100 concentration of the study allergen; in 46/50 trials the puncture skin test was also 2+ or greater. The skintest end points ranged from dilution No. 9 to dilution No. 1. Fig. 1 shows the comparison of skin-test end points with both Ph and modified RAST results for all five pollens. For the 50 allergen trials there was good correlation between Ph RAST class and skin-test end point (r = +0.70, p < 0.001). For the separate allergens, the individual correlations ranged from +0.58 to +0.99, but were statistically significant only for timothy, maple, and short ragweed. Similarly, the correlation between modified RAST class and skin-test end point was good for all five allergens collectively (r = +0.80, p < 0.001). The individual correlations ranged from +0.66 to +0.99 and were statistically significant for timothy, oak, maple, and short ragweed. Fig. 2 shows the concordance between the two RAST assays. For all allergens collectively the correlation between Ph and modified RAST class was +0.93 (p < 0.001). Individually, the correlations ranged from +0.93 to +0.99 depending upon the allergen (p < 0.01). Clinical sensitivity to the test allergens was determined by nasal provocation tests. Table III shows the correlations between provocation tests and both the Ph and modified RAST classes. Provocation tests were negative in eight of 50 trials; the test allergens in these situations were oak (n = 4), maple (n = 2), birch and ragweed (n = 1 each). The skin-test end points in these cases were dilution No. 2 (n = 3), dilution No. 3 (n = 4), or dilution No. 4 (n = 1). Five of the eight cases were associated with a Ph RAST class of 0 (negative) and a modified RAST class of 1 (equivocal). The other three patients had positive Ph RAST classes of 1 or 2 and modified RAST classes of 2 or 3. Therefore, the association of negative challenge tests with positive RAST assays

VQLC:‘lE

67

NUMtl3H

2

Modified radioallergosorbent

test

101

l m

l ee

l

Modified RAST Ctors ffi. TAME -

V. Mean binding

2. Correlation between Ph and modified RAST results.

of negative

control

Mean % total

-

Wren Shon ragweed English plantain Timothy grass Orchard grass Alder Elm Oak Maple Birch

preparations activity

sera in the Ph RAST

Mean%~~t¶buf~t-~

FCS

HSA

tics

I .05 0.58 0.64 0.88 1.08 0.64 I.18 0.94 0.35

I .42 0.88 I.10 1.22 I .55 0.76 I .37 1.22 0.54

0.97 0.64 0.69 0.82 0.90 0.54 I.04 0.84 0.28

with nearly equal frequency in the Ph and nwdified RAST systems (3/37 or 8.1% and 3142 or 7. I %, respectively). Conversely, eight of 13 (62%) p&ents with a Ph RAST class of 0 had positive nasal provocation tests. This suggested that the cutoff point between negativity and positivity in the Ph RAST (0.35 PRU) was too high. The data from the 50 allergen trials in Table III were reexamined using the wu E reference (0.14 PRU) as the cutoff point bet&&en positivity and negativity, creating a new RAST category we called “class l/2” (Table IV). There were five patients who remained Ph RAST negative; t& 3 of these had positive nasal provocation tests. The occurred

and reference

Fkh-E 1.89(180)(195) I .42 (245) (222) I .65 (258) (239) I .87 (212) (228) 1.71 (158)(190) I .45 (227) (268) 1.64(139)(158) l.55(165)(184) I .70 (486) (607)

flewmocD 2.65 (252) I .% (338) 2.39 (373) 2.61(297) 2.42 (224) 2.27 (355) 2.41 (204) 2.35 (250) 2.41 (689)

(273) (306) (346) (318) (269) (420) (232) (280) (861)

remaining eight patients would now have positive Ph RAST results (class l/2) using the E reference as the cutoff point, and six of the eight exM&ed positive reactions to nasal provocation teesting. Thus the apparent incidence of negative Ph RAST-positive nasal challenge results would decrease (215, 40%), while the incidence of positive Ph RAST-negative nasal challenge results would increase only s#ightly (5/45, 11%). These analyses could not be a@ied to the modified RAST because no patients in modified RAST class 0 were enrolled in the My. Further comparisons were made berwam the RAST binding produced by the positive and negative control

102

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et al.

Skin Test End poini

TABLE VI. Mean binding of negative preparations in the modified RAST

=9

control

Mean % time control

*8 Allergen

FCS

HSA

HCS

Short ragweed English plantain

2.28 1.94 1.41 1.38 1.60 I .20 2.56 1.80 0.81

2.80 2.92 1.46 2.22 4.49 1.76 3.22 2.41 1.02

2.19 2.06 1.79 1.47 2.43 1.35 2.57 1.84 1.10

Timothy grass Orchard grass Alder Elm Oak

Maple Birch

0 I

f s

CLIN. IMMUNOL. FEBRUARY 1981

I

5

-

4

-

3

-

2

-

I

-

Modified

O-

RAST

Vi&

I

visit

3

FIG. 3. Skin-test end points and RAST results in 15 patients who did not receive the calculated initial immunotherapy dose. A, Patients whose test results changed between either visit 1 or 2 and visit 3. These test results are connected by lines. ., Patients whose test results did not change between visits.

preparations. In Table V, the mean binding of the negative controls, reference E, and reference D in the Ph RAST are shown for each allergen. The mean binding is here expressed in terms of a percent of the TA to permit comparison of different assays. In most assays the radioactive counts bound by reference D (0.35 PRU of birch IgE antibody) were 2 to 4 times that bound by the negative controls; however, in the birch Ph RAST assay the counts bound by reference D were 6 to 8 times those of the negative controls. The addition of reference E (0.14 PRU of birch IgE antibody) reduced somewhat the gap between radioactive binding by positive and negative control sera. Binding by reference E ranged from 1.4 to 2.6 times that of the negative control preparations for all allergens except birch, where the mean binding was still over four

times that of the negative control preparations. The data in Table V also show the variation in RAST binding among different allergen discs. The three negative control preparations produced different binding for each of the allergens in the Ph RAST, with ranges from 0.35% to 1.18% TA for FCS, 0.54% to 1.55% TA for HSA, and 0.28% to 1.04% TA for HCS. Table VI shows simjlar variations when the negative control preparations were tested in the modified RAST. Here, mean binding was expressed as a percent of the TC; the ranges of percent of TC binding produced by FCS, HSA, and HCS were 0.81% to 2.56%, 1.02% to 4.49%, and 1.10% to 2.57%, respectively. Thus, in both types of RAST, marked variation in binding was noted among different allergens using the same negative control preparations. In addition, marked variation in binding occurred between different negative control preparations. For all allergens collectively, significantly greater binding was noted with the HSA preparation as compared with either FCS or HCS for both the Ph and the modified RAST (p < 0.001, analysis of variance). Table VII shows the comparison between the background-based RAST scores and nasal provocation test results. All seven patients who were considered RAST negative by this method were also negative by the usual Ph RAST criteria, while six had modified RAST classes of 1, and one had a class of 2. However, four of the seven had positive nasal challenges. The one equivocal result was from a patient with a Ph RAST class of 0 and a modified RAST class of 2. In the five patients with positive background-based RAST results and negative nasal challenges, the Ph RAST classes ranged from 0 to 2 and the modified RAST classes from 1 to 3. Initial

immunotherapy

dose studies

In 15 of 50 trials the patient reacted to the RAST-1 (n = 5) or RAST-0 (n = 10) skin-test concentration

VOLUME NUMBER

67 2

Modified

TABLE VII. Correlation between HCS as the negative control Nasal provocation Positive Negative

Total

background-based

RAST results and nasal provocation

Equivocal

RAST negative (~200% HCS)

(200% to 250% HCS)

4 3 I

0 I

with at least a 16-mm diameter wheal and thus were not given the initial immunotherapy injection. The challenge allergens in these cases were timothy (n = 3), birch (n = 5), and short ragweed (n = 7). The skin-test and RAST results in these 15 cases are shown in Fig. 3. The majority of these patients were highly sensitive in terms of skin-test reactivity; 11 of 15 patients had end points at dilution No. 9. On the other hand, the patients were fairly evenly distributed with respect to their RAST classes. Repeat skin-test end-point titrations and RAST determinations were performed to identify any changes that had occurred between the time of the original measurements and the immunotherapy visit. As seen in Fig. 3, skin-test end points changed significantly for only three of 15 patients. However, significant changes did occur in both the Ph and modified RAST classes between the first and third visit for some of the patients. There was an increase in Ph RAST class in six cases (two with timothy, two with birch, and two with ragweed), while for the modified RAST there was an increase in RAST class for five patients (three with timothy, one with birch, and one with ragweed) and a decrease for one patient (birch). The remaining 35 patients, who reacted to the preliminary skin tests with less than a 16-mm wheal, received the initial immunotherapy dose without incident; no systemic reactions were observed. The reactions to this injection ranged from no obvious skin reaction, or erythema only, to an g-mm wheal with surrounding erythema.

DISCUSSION The Ph and modified RAST results correlated highly in this study. Although the overall correlations between either RAST assay and the skin-test end points were quite good, there were instances in which negative or lower RAST class values were associated with skin-test end-point dilutions of No. 7 to No. 9. Perhaps the most critical area for comparison is in the lower Ph and modified RAST classes. Proponents of the modified RAST assert that their assay identifies a significant number of patients with low but definite levels of clinical sensitivity who would be considered negative with the Ph RAST. Of the 13 patients with a negative Ph RAST (CO.35 PRU) in this study, eight

radioallergosorbent

1

test

103

tests, using

RAST positive (>250% HCS)

37 5 42

had a positive nasal provocation test, suggesting that this claim may be warranted. If a new Ph RAST designation (class l/2) were defined by the D and E references, eight of the original 13 patients with a Ph RAST class of 0 would now have positive Ph RAST results. Of the remaining five patients whose Ph RAST class would still be 0, two had positive and three had negative nasal challenges. The addition of this fifth reference point to the Ph RAST standard curve would increase the sensitivity and accuracy of the assay, at least for this small group of patients. One of the dangers of lowering the cutoff point between RAST-positive and RAST-negative results is the possibility of increasing the number of falsepositive results. Accurate estimates of the frequency of false-positive and false-negative RAST results could not be made because our patients were selected by their RAST results and not by the results of nasal provocation testing. Negative nasal provocation tests occurred in eight of 50 allergen trials; of these, five were modified RAST class 1 (equivocal). In fact, negative nasal provocation tests were encountered in the majority (5/8) of modified RAST class 1 patients. Moreover, the high frequency with which the HSA negative control was designated as modified RAST class 1 (seven of 21 assays) should be emphasized. Collectively, these data suggest that the modified RAST class 1 designation is clinically meaningless. The variation in the RAST binding of negative control preparations among different allergens in both the Ph and modified RAST should also be noted. Because of this variation the degree of binding that is considered positive in one allergen system may be at the background level for another allergen. The optimal way to recognize this situation is to run a serum with low or absent IgE (negative control) with each solid-phase allergen being investigated. At present, the Ph RAST kit instructions do not recommend this approach; the use of a negative control preparation is not mentioned. In contrast, the modified RAST directions recommend that one or more negative controls be included in every assay. * The recommended negative controls include pooled HCS, pooled normal human serum (nonatopic), or serum from an atopic individual tested against an allergen to which the individual is not sensitive. The recommendation further

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specifies that the same negative control serum and a single allergen disc be tested in each assay, regardless of the allergens to which the patient is being tested, and lists a specific range of radioactive counts into which the binding of the negative control should fall. However, the recommendation does not take into account the variation in the binding of a single negative control serum among different allergen discs. Ideally, more than one negative control should also be employed, as we noted variations in binding among different negative control preparations. In the portion of the study dealing with initial immunotherapy doses, we performed preliminary skin tests using RAST-1 and RAST-0 allergen concentrations. Our cutoff point for positivity in this test was a wheal of 16-mm diameter or greater; this was higher than the 13-mm cutoff point recommended by other authors.x Nevertheless, skin-test wheals of greater than 16 mm were produced in 15 of 50 allergen trials (30%), and the patients thus did not receive the initial immunotherapy dose at the RAST-0 concentration. This was not particularly surprising, considering that the skin-test titers had ranged from 1:2,500 to 1: 39,062,500 in patients with a modified RAST class of 2. Had we performed preliminary skin tests and administered immunotherapy only with the RAST- 1 concentration, five patients (10%) would still have failed to receive the initial injection. The assumption was that patients exhibiting a skin-test reaction of greater than 16 mm would be at increased risk of systemic reaction if the calculated dose were administered. This assumption was not investigated further. The calculated modified RAST starting concentrations for the class 5 and 6 patients (1: 62,500 and 1 : 312,500 respectively) are actually less than the 1: 50,000 concentration recommended by some traditional allergists for very sensitive patientslO; thus, dosages recommended by the modified RAST system for class 5 and 6 patients may be more conservative than usual. However, proponents of the modified RAST overlook the fact that commercial allergen extracts of similar weight: volume labeling may vary lOO- to l,OOO-fold in terms of biologic activity.1i-13 Pending the availability of standardized allergen extracts, attempts to reduce the practice of immunotherapy to “cookbook” simplicity seem premature. When IgE antibody levels were remeasured in the 15 patients who did not receive the calculated initial immunotherapy dose, we noted an increase in RAST class in 30% to 40% of cases. This was probably due to the normal rise in IgE antibodies seen following the pollination seasons. I4 This assumption could have been tested by also remeasuring IgE antibody levels in the 35 patients who received the initial immunother-

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apy dose, but this was not done. However, such a rise may explain the patients’ intolerance to the modified RAST-based skin test. Although there were few significant changes in skin-test reactivity in the l- to 4-wk interval between the second and third visits, there was a lag of 2 to 6 mo between screening RAST assays (visit 1) and screening skin tests (visit 2). Ideally, future studies of this sort should be conducted outside of the pollination seasons, and the time interval between performance of the RAST assay and the institution of immunotherapy should be minimized. REFERENCES 1. Nalebuff DJ, Fadal RG, Ali M: IgE in investigation and management of atopic disorders: Recent advances. J Cont Educ ORL Allergy 40:47, 1978. 2. Nalebuff DJ, Fadal RG, Ali M: The study of IgE in the diagnosis of allergic disorders in an otolaryngology practice. Otolaryngol Head Neck Surg 87:351, 1979. 3. Fadal RG, Nalebuff DJ: Tools of the allergist: Old and new. Cont Educ (May), 1979, p. 37. 4. Nalebuff DJ, Fadal RG: The modified RAST assay: An aid in the diagnosis and management of allergic disorders. Cont Educ (May), 1979, p. 64. 5. Advertisement. J ALLERGY CLIN IMMUNOL 63:A35 (April), 1979. 6. Adolphson CR, Yunginger JW, Gleich GJ: Standardization of allergens, in Rose NR, Friedman H, editors: Manual of clinical immunology, ed. 2. Washington, D. C., 1980, American Society of Microbiology, p. 778. I. Norman PS: In vivo methods of study of allergy, in Middleton E Jr, Reed CE, Ellis EF, editors: Allergy: Principles and practice. St. Louis, 1978, The C. V. Mosby Co., vol. I, chap. 16, p. 256. 8. Fadal RG, Nalebuff DJ: Modified RAST procedure. New Jersey, 1979. (Pamphlet.) 9 Gleich GJ, Adolphson CR, Yunginger JW: The mini-RAST: Comparison with other varieties of the radioallergosorbent test for the measurement of immunoglobulin E antibodies. J ALLERGY CLIN IMMUNOL 65:20, 1980. 10 Patterson R, Lieberman P, Irons JS, Pruzansky JJ, Melam HL, Metzger WJ, Zeiss CR: Immunotherapy, in Middleton E Jr, Reed CE, Ellis EF, editors: Allergy: Principles and practice. St. Louis, 1978, The C. V. Mosby Co., vol. 2, chap. 49, p. 877. 11 Gleich GJ, Larson JB, Jones RT, Baer H: Measurement of the potency of allergy extracts by their inhibitory capacities in the radioallergosorbent test. J ALLERGY CLIN IMMUNOL 53:158, 1974. 12. Gleich GJ, Leiferman KM, Jones RT, Hooton ML, Baer H: Analysis of the potency of extracts of June grass pollen by their inhibitory capacities in the radioallergosorbent test. J ALLERGY CLIN IMMUNOL 58:3 1, 1976. 13. Yunginger JW, Jones RT, Gleich GJ: Studies on Alternaria allergens. II. Measurement of the relative potency of commercial Alfermria extracts by the direct RAST and by RAST inhibition. J ALLERGY CLIN IMMUNOL 58:405, 1976. 14. Henderson LL, Larson JB, Gleich GJ: Maximal rise in IgE antibody following ragweed pollination season. J ALLERGY CLIN IMMUNOL 55:10, 1975.