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Studies on the hymenoptera
Studies on the Hymenoptera IV. Correlation of passive transfer in Hymenoptera allergy with culprit insect and direct skin tests IIalka
Brown,
M.D.,
and Harry
S. Bernton,
M.D.,
Washington,
D. C.
Direct &in tests and passive transfer tests with the sera of 16 patients who had suffered a generalized reaction to the sting of a hymenopterous insect were found to be positive. In contrast, these tests in &in and sera of 18 patients who had szcffered only a local reaction tended to be negative. Passively transferable antibodies correlated more nearly with the species of the culprit insect than did direct skin tests, and these antibodies seemed on occasion to result from former stings rather than from the most recent sting. No significant difference was fownd in family history and past history of allergy between the 2 groups.
T
he purpose of this study was to determine whether antibodies concomitant with anaphylaxis in patients sensitive to stings of the Hymenoptera could be demonstrated by passive transfer and to classify them, if present, as groupspecific or species-specific antibodies. The reliability of skin tests in Hymenoptera allergy has been questioned by allergists. Although skin sensitivity usually parallels clinical sensitirity,l there are many exceptions.2, 7r 8 Occasionally, a patient, despite a plausible history of anaphylaxis, gives negative skin tests to insects even when retested 2 months after a sting. Equally perplexing to the allergist is a patient who reacts with large skin test reactions to all common stinging Hymenoptera, but who insists tllat he has been stung by only a single species” throughout his life. In the latter case, skin tests do not necessarily differentiate between irritating qualities of the extracts and an antigen common to the honeybee, hornet, wasp, and yellow jacket. In neither case will skin testing guide the allergist in his choice of the correct therapy. A few studies3-5, 8 have shown that certain normal persons who have never From the Department of Medicine (Allergy), George Washington University Supported by a grant from the John A. Hartford Foundation, Inc. Received for publication Oct. 25, 1967. *In this paper, for reasons of simplicity, the word species is used to designate types of insects. Entomologically, this is correct for honeybee, hornet, and yellow the wasp is a genus.
146
Hospital.
the different jacket, but
Volume Number
Studies on the Hymcvoptwa
44 3
1.17
experienced anaphylaxis from a sting may give positive skin tests. The percentage of patients giving positire tests cannot be accurately compared because the extracts used in the different studies were not of the same concentration.” Bernton and Browq5 with the use of honeybee extracts containing 200 and 20 protein nitrogen units (PNU) per milliliter? noted positive tests in 38 per cent of 380 normal volunteers. Apparently, a stronger extract products a grcat,er incidence of positive t,ests, and one must agree with Bensol? tllat honeyhcc venom “is of moderate toxicity to the human species, nonsp,ccific, ;111(1 therefore tending to cause somewhat of a reaction in every one.!’ Because of the discrepancy between clinical symptoms and skin teats ;rlrcad!’ noted and because of apparent inconsistency in direct skin testing, the sera oi IIymenoptcra-sensitive patients were studied hy passive transfer. Evcq patient had suffered generalized itching and swelling, asthma, weakness, or anaphy1ac.t in shock following a single sting. METHODS Passive transfer
tests
The usual precautions were taken when sera were obtained from 16 patients who had suffered anaphylaxis from a Hymenoptera sting and from 12 patients who had suffered only a large local reaction. The passive transfer tests were performed as follows: Sites mere sensitized on the arms of volunteers with the use of 0.05 ml. of t,he patient’s undiluttcl serum. Forty-eight hours later the sites were challenged with 0.025 ml. of one of the antigenic extracts. These were commercial extracts,* each made from whole bodies of a single species of Hymenoptera-bees, hornets, wasps, or yellow jackets. Tn our preliminary testing, it had been found that 38 per cent of “normal” subjects reacted to an antigen containing 200 PNU per milliliter. Therefore, in the present study 100 PNU per milliliter was used as the standard strength .for challenging. This extract was diluted in sterile normal saline from a 50 per cent glycerinated extract containing 10,000 to 20,000 PNU per milliliter. Tests wer’c perforrned in triplicate and read as follows: t, wheal greater than 0.5 cm. but less than 0.75 cm. in diameter and surrounded by flare; +, wheal 0.75 cm. i~t diameter, surrounded by flare; st, wheal 1.5 cm. in diameter with pseudopods and flare; tt+, wheal greater than 2.0 cm. in diameter with pseudopods ant/ flare. Controls were as follows: (1) The serum of a pollen-allergic patient was challenged with honeybee antigen. (2) The serum of a honeybee-allergic patient was challenged with pollen antigen. As a positive control, the serum from ;I ragweed-sensitive patient was injected into each recipient and challenged wit11 ragweed antigen. A positive reaction indicated that the subject was capable 01 passive transfer. Every recipient tested negatively to the hymenopterous cxtracr, at the time of challenge of the sites. Since dilutions of antigen or of serum in our preliminary testing had in general proved unrewarding, dilutions in most cask were climinnt~cd. “Hollister-Stier
Laboratories,
Spokane,
Wash.
148 Direcf
Brown skin
J. Allergy September, 1969
and BerntolL testing
Direct skin tests were performed on the lateral aspect of the arm by the intracutaneous injection of 0.02 ml. of extracts of honeybee, hornet, wasp, and yellow jacket in varying dilutions so that a negative, a threshold, and sometimes a strongly positive test was obtained with each extract. The strongest extract used for direct tests contained 1,000 PNU per milliliter and 2.5 per cent glycerine. The wea.kest extract giving a positive skin test among these patients contained 0.01 PNU per milliliter. Direct skin tests were read as follows: +, wheal slightly larger than the intradermal injection, mild flare; +t, wheal 0.5 cm. in diameter, definite flare; +tt, wheal 0.75 cm. in diameter, large flare, often with pseudopods and itching ; +++t-, wheal 1.0 cm. in diameter, large flare, pseudopods, and itching. Identification
of insect
The diagnosis of the species of Hymenoptera was determined by the shape of the insect and by the type and site of the nest. An insect was considered identified upon presentation by the patient. An insect was considered presumptively identified if it was obtained from the nest of the culprit or if the patient brought in the nest from which the culprit was seen to fly. If neither of these conditions prevailed, the insect was not considered identified. RESULTS Passive transfer
Table I shows in 12 cases of local reactions to a sting the relation between passive transfer and direct skin tests with the use of the same antigen in the same dilutions for both types of test.
Table 1. Relation between passive transfer and intradermal patie?ats who had suffered only a large local reaction Hornet
Honeybee Culprit
Patient Ala. Ale. Gib. Gol. Has. Hoc. Kir. Kre. Pre. Pri. Rea. sto.
insect*
Passive transfer+
Skirt
Slcin
transfer
transfer
testt.
testi
0 0 0 0
0 0 ttt 0
0 0
0 0 +t+ 0
0
0
0 0 0 0 i
FO)
0
“y
0
0
0
0
0 0 0 0 fl
tt 0 0 0 t 0 0
0 0 0 0 0 0 0
++ 0 0 0 0 0 0
0
Passive
test$
0 0 +tt 0
“W” W “\V” TV w HB HB
Wasp Passive Skin
Passive transfer
L‘HB” HB ‘TJ” (Tes” J”
0 :
Skin
*HB = honeybee; W = wasp; YJ = yellow not positively identified are written within quotation tPassive transfer technique explained in text. *Direct skin tests performed with 0.02 ml. of Other concentrations are noted in parentheses.
skin tests in 12
test$
i
Yellow jacket
0 :
0 0 tt+ 0
0
0 0 + :
0
0
O 0 0 0 0 :
+OfP) 0 0 + 0 0
0 jacket; marks.
Ves
=
extract
containing
unidentified
100
vespid.
PNU
per
Insects
milliliter.
Tables II and III show in 16 patients who had suffered a generalized reaction the relation between passive transfer and direct skin tests with the same antigSc>ns as those in Table I. The culprit insect was the honeybee in 4 patients ant1 ;I, vespid in 8 patients with local reactions. Among patients who had snffcut~l ;I generalized reaction, the honeybee was incriminated in 5 cases and a vcspi(l ilt 11 cases. The scra of the patients reacting only IocalIy to a sting did not t,ransfcr scllsitivity with any of the Ilymenoptera antigens tested. T,iken;isc, the sera 01 \: Table II. Relation of passive transfer sufferecl a generalized reaction*
to direct skin tests in 7 pntieafs
Honeybee
Patient Cav. Nit. Hap. writ. Gre. coo. Flo.
Cdprit insect UYJ,’ HB “Hym” “HB” “FIB” YJ YJ
Passive tmnst
Hornet Passive tmns-
Direct test$
fert
skin
-t + 0 IO 0 0 0
fer 0 0 0 0 0 0 0
(! O(l0, ++t+ 0 i-++ ?-c +
0 ++++(0.01)ll ++++ ++(O.l) ++ II
Passive tmnsfw
Direct
Two species Mau. YJ&HB “H” Dau. Har. HB
skin
Passive
test
transfer
WOl.
YJ IIT3 II
i + 0
Bia. Car.
II Y’J
0 t++t
YJ
+
Three species C:ES. Ber.
Four speoies Trn. the
Passive
0 t++(lO) +t+t
“Hera exhibited positive same as those for Table
t
j
Wasp ;K?$
‘d 0 +t i-
:I.+.. + 1 i?+ ++ unidcntifird
per millilitt~r. (0.01 :z i).llt
who hut1
Pellow
jacket
~~~l~~~~I~~~~~~rI~~~'
i
0 + +
L+(l) +t
Zt(lO, 0
0 0 +
0 iCk 0
+ + +
0 +++ +++
++ t++
+ -I-
tt
t 0
t
0
++t II.
100 PNU parentheses
++ t++(l) t+
transfer
0 oi I I
Direct
transfer
+ ttt ++
0 0
one species only. yellow jacket, Hym = within quotation marks.
Hornet
Baneybee
Culprit insect
0 Cl(l0) +c++ i-c ttt ++t +
containing noted in
jazz_- ._ / Dir{ cl I skt~i i ttstj
Passive tramfer
to direct skin tests i,rt 9 patieds
Table
Patient
Birect skin I testj
0 0 i& i-+ 1r 0
“Sera demonstrated passive transfer with extract of tHB = honeybee, H = hornet, W = wasp, YJ = member of Hymenoptera. Insects not identified are written STechnique explained in text. $Direct skin tests performed with 0.02 ml. of extract If a weaker concentration was used, the concentration is PNU per milliliter). IlConstitutional reaction from single skin test.
111.Relation of passive transfer sujfered a geraeralixed reaction*
Ycllotu
m-asp Direct skin test$
who l1w1
with
extracts
+cL
iti of
2 or more
0 0 0
*-++ i----t ( 1 i +-
t
0 *L -3+
+
-+
1i r-I-r
+++
t+
if?+
species.
Abbreviations
i’
nrt’
150
Brown
J. Allergy September, 1969
and Bernton
beekeepers stung during the season (100 times or more) failed to give a positive reaction with the use of the passive transfer technique with honeybee extract. In contrast, each of the sera of 16 generalized reactors was capable of transfer when challenged with allergens of the Hymenoptera. Seven sera reacted positively with the extract of one species, 3 with the extracts of 2 species, 5 with the extracts of 3 species, and one with all 4 antigenic extracts tested. Controls in every case were negative. Direct
skin
test
reactions
It is apparent from Table I that skin tests were negative in a dilution of 100 PNU per milliliter in 9 of the 12 cases of local reaction. In one patient, the intradermal reaction was +tt when challenged with an extract containing 100 PNU per milliliter. In only 2 tests of the 48 performed was the skin test positive with the use of a concentration of 10 PNU per milliliter. In sharp contrast were the intradermal tests of the 16 generalized reactors, all of whom had shown positive passive transfer tests. Eight patients reacted positively with honeybee antigen at a concentration of 100 PNU per milliliter, 3 at a concentration of 10 PNU per milliliter, one at a concentration of 0.01 PNU per milliliter. Nine patients reacted positively when tested with hornet extract at 100 PNU per milliliter and one patient with a concentration of 1 PNU per milliliter. Eleven patients showed positive direct tests with wasp extract at 100 PNU per milliliter and one with 1 PNU per milliliter. Twelve patients reacted when challenged intracutaneously with yellow jacket extract, 100 PNU per milliliter, and one patient with 1 PNU per milliliter. Family
history
More than half of the family members of the 12 patients suffering local reactions had experienced some type of allergy, 2 to stings of Hymenoptera (Table IV). Fifty per cent of the family members of the 16 patients suffering a generalized reaction had experienced typical symptoms of allergy. Past
history
Fewer patients than family members in both groups had experienced a personal history of allergy : 4 of 12 local reactors and 6 of 16 generalized reactors to a sting (Table IV). In both groups, hay fever was the most frequent allergy noted. The number of cases, 28 in all, is too small for further interpretation. Accuracy
of identification
of insects
The insect was identified accurately in 11 of the 16 cases of generalized reaction. In one of these, the sting may have been intravenous since a hematoma occurred at the site of sting, and symptoms occurred within 5 minutes. One patient had suffered anaphylaxis 5 times, once caused by a honeybee, once by a yellow jacket, and 3 times by a hornet. In 5 cases the insect was not identified. In several the insect was not seen.
Volume Number
44 3
IV. Family and past history of Hymenoptera patients reaction compared with those suffering a genera,lized reaction
Table
Large
reaction
Generalized
___.-.--. Past histol-y
history
Patient
Ah. Ale. Gib. Gol. HM. Hoc. Kir. Jirc.
i t t 0 t + +
0 0 0 + + 0 0 0
Cav. Sit. Hap. wat. Gre. C!oo. No.
0 0 0 0 0 t% 0
1\hU.
t
Pro.
0
0
Pri. Krx.
t t
t 0
Ihu. Ihr. CM.
t +
+ -c
sto.
0
t
CJ 1
0 .L 0
Bia. CX. TIX.
il i i
0 II t
14
x
ti
s
-i
1
reaction
Family history
13
Past
a 10~1
Family history
Patient
Totals
local
suffering
Lh. Wol.
t 1) Ii 0 IJ II 0
-
In one cast, a small boy (Hap.) who had been playing out-of-doors rushed into the house screa.ming, became cyanotic and clammy, and collapsed. ITe was hospitalized for 5 days for complete examination. All tests were negative. ~1 similar experience was repeated the next summer, again with hospitalization and a negative work-up. The child then was rrferrecl for testing with insect allergens. Exceptional entomological situations must not be overlooked in searching for accurate identification. Patient Coo., a 6-year-old boy who had been playing near a hedge which contained a 12 inch hornets’ nest, appeared at the kitchen door “covered with yellow jackets,” only one of which stung him. Because the mother’s description was at rariance with the hornets known to exist in the hedge, an entomologist examined the nest. To his surprise, attached to thtl hornets’ nest at the farthest point from the opening was a smaller >--ellov jackets’ nest. (He surmised that the 2 nests had been started independently ant1 with increase in size had become contiguous.) Hence, the mother’s description oi’ the insect was correct. The insect was definitely identified in 6 of the 12 C~SCSof local reactions. DISCUSSION
In this series of patients, anaphylactic hypersensitivity to IIymenoptcAra stings was associated in every instance with the presence of circulating skinsensitizing antibodies to the specific insect. The circulating antibodies possiblymay not be etiologically related to the serious reactions suffered by the patients. The presence of these antibodies may simply be an index of abundant antihod:of this type being fixed to tissues. In this regard, passire transfer reactions
tended more to be obtained with sera from patients who gave strongly positive direct skin tests. There were csceptions to this general trend. Combining the data from Tables T, II, and III, passive transfer was achieved with scra of 4 of 8 patients showing 4t- direct. skin test reactions, with sera of 11 of 21 patients showing 3+ direct react,ions, vith the scra of 10 of 23 subjects 2+ direct reactions, with 0 of 5 patients showing It reactions, and with 0 of 2 subjects showing & reactions. In 11 of the 12 cases in which the insect W’BS identified, passive transfer was nchievd to the specific ins&. In contrast, passive transfer reactions were positive in 32 tests with only 14 h>-mcnopterous allergens other than the one t,hought to hare caused anaphplaxis. On direct skin testing of the patients who had identified the culprit ins&, the specific insect gave the largest reaction in 5 of 12 cases; another insect showccl the largest reaction in 3 of 12 patients, and in 3 patients specific and nonspecific insects showed about equally marked reaction. In one case (Kit.) the culprit insect gave the only positive direct skin reaction, and it occurred with the use of an extract of high dilution. The sera of 8 beekeepers who had suffered many rcvcnt stings without urltoward symptoms tcstcd negatively with the use of the passive transfer tcchniyuc. In the 3 instances in which passive transfer responses were positive in the presence of negative direct tests to the same antigcnic extract (Car., May., and Gas.), every serum showed the posit,ive reaction in triplicate, i.c., in each of’ 3 different recipients. In some cases t.he apparent specificity of the passive transfer test was striking. Patient Nit. had been stung onl,v by a honeybee and had suff&~~tl armphylaxis only from the stin, 0’ of a honeybee. IIcr serum, when testctl 1)~ the passire transfer technique, gave a positive result only ~hcn honcybec cstract was used. Patient F’lo. had been stung only h- ~cllow jackets, and his serun~ transferred sensitirity only when challenged with yellow jackrt, extract. Similarly, Patient Har., the wife of a bcekecpcr~ xhose son, aged 9, had diet1 oi anaphylaxis shortl)- after a hone~hcc sting, hat1 cqericnccd se~crc gcncralixctl reactions with a honcyber and a wasp sting only. 11~ s~uin transferred sensitivity when tested with these 2 antigenie cstracts. It (lid not transfer sensitivity when hornet or ;veIlow jacket extract was nscd. Some of the inconsistencies in skin testing may bc esplainctl hy tlio fact that most patients had been stung se\-era1 times previously by diff’ercnt members of the Hymmoptera. All the antibodies found at the time the patient was examined may not necessarily bc the result of the last, sting. This is clearly shown in the case of Patient Bia., who had suffcrcd anaph~lasis from the sting of a honeybee, in 1964, and of a pcllow jacket, in 1962. In 1960, during a single month he received 3 separate hornet stings. each causin g mow severe anapli,~ laxis than the last. The duration of the lift of transferable antibotlics is not known. It is possible that the titer of antibodies to one species of insect may 1~ higher because of more freqncnt stings by that insect. There appears to be a tendency for sensitivity to the H.vmcnoptcra, as measured by direct skin testing, to decrease with the passage of time.” The erolntion of the passire transfer has not yet hen studied in a similar manner. It ma-
1)~ that lesser titers of antibody following a sting nli\y (lisdppvar ~OI*O ~il1>i(lI~ from the circulation than higher titers of antibody. Whether the patients who had sufYcrrt1 only lov;11 tv;r&~ns will ~WOIIII. more sensitive in timc and develop more st14011glypositi\.ca skin tests j)ositi\~c, passive trdnsfcr t,rsts remains to bc seen. ilncl
CONCLUSIONS In 16 I-lpmcnoptera-sensitive patients who had suffcretl a generalized I*c~a(~tio~~ to a single sting, antibodies concomitant with anaphylaxis conl(l btl passircily transferred. In 12 patients who had suff%r~~l only a local reaction, passive* transfer was not, achieved. Direct skin tests to l~~nlrnopteronn untigerls ill I ho patients who had suffered a generalized rcac*tion n-erc larger nncl positii-rx ;11 higher dilutions than were Qose of patients who had suffered only local reactions. There was a tendency for larger tlirect, skin tests to be corrclatrd with I)osit ivc passive transfer. Half, or more, of the family memb~s of both groups 1x111t~sl)~lrit~xctl symptoms of allergy. Passi\-r transfer (W~rPlilt(Y~ II101’e c~lcarly wit11 till’ spccics of culprit insect than did direct skin tests. In 2 vases the insect ;tppc~a~v~l to be identified by passive transfer. The prc~ncc of passiveJ. tramsFvi2hlt~ antibodies may result from former stings or from the most vrccnt sting. The
:~uthors
wish
to thank
Dr.
Philip
J. Torsnq
for
contrilnlting
on,’ wrum
to thi5
surt)..
REFERENCES
1. Mueller, If. L.: Insect allergy, Prdiat. Clin. Sorth Amcric:~ 6: 91T, 19SH. 2. Htirr, H. A., and Foubert, E. L.: Antigenic relationships betnwn hon~~~l~ws, wvas[,s. vc~l IOU jwkrts, and yellow and black hornets, J. AI,I,&RGY 27: 98, 1956. (Abst.) 3. Wmson, R. L.: Diagnosis of bypersensitirity to tllv bw anti to mosquito, A\r(,l~ I nt. M~,II. 64: 1306, 1939. 4. Mnc~Lnrcw, 11’. R., Eisenberg, B. C., Frank, J). E., xnd Kesslw, .l. : Rractionr to inr;wi nllrrgen~. The incidence of response to testing among :kIlt+c am11 non:rllcrgi(, pcrsou~, (%lifomin Med. 93: 224, 1960. 3. Ht%lton. 11. H., and Hrown, H. : Studies on the Ilymenoptwa. I. Skin wavtions of norn!:t I persons to honeybee (Apis mellifern) extract,, J. A&I.IiER(iY 36: 315, 1963. 6. Brown, II., and Rernton, II. 8.: Evolution of the skin tf,st in 100 II~mrnopter:1-s~r~siti~-~~ patients, prwentcd at the meeting of the American Acr~Iwny of Allvrgy, Iiostou, Ffltj.. Id@. i. Mueller, H. L.: Aerixl intracutaneous testing for Irwc ;lrltl wrsp sclllsitivit)-. .T. .\I.I.F‘lifi\ 30’ I”? 195Y. 8. S~‘il\v\-a;,i~,‘H: .J.: Skin sensitivity in insect allergy, ,1. A. 31. h. 194: 119, l!Wj.
Brown,
M.D.,
and Harry
S. Bernton,
M.D.,
Washington,
D. C.
Direct &in tests and passive transfer tests with the sera of 16 patients who had suffered a generalized reaction to the sting of a hymenopterous insect were found to be positive. In contrast, these tests in &in and sera of 18 patients who had szcffered only a local reaction tended to be negative. Passively transferable antibodies correlated more nearly with the species of the culprit insect than did direct skin tests, and these antibodies seemed on occasion to result from former stings rather than from the most recent sting. No significant difference was fownd in family history and past history of allergy between the 2 groups.
T
he purpose of this study was to determine whether antibodies concomitant with anaphylaxis in patients sensitive to stings of the Hymenoptera could be demonstrated by passive transfer and to classify them, if present, as groupspecific or species-specific antibodies. The reliability of skin tests in Hymenoptera allergy has been questioned by allergists. Although skin sensitivity usually parallels clinical sensitirity,l there are many exceptions.2, 7r 8 Occasionally, a patient, despite a plausible history of anaphylaxis, gives negative skin tests to insects even when retested 2 months after a sting. Equally perplexing to the allergist is a patient who reacts with large skin test reactions to all common stinging Hymenoptera, but who insists tllat he has been stung by only a single species” throughout his life. In the latter case, skin tests do not necessarily differentiate between irritating qualities of the extracts and an antigen common to the honeybee, hornet, wasp, and yellow jacket. In neither case will skin testing guide the allergist in his choice of the correct therapy. A few studies3-5, 8 have shown that certain normal persons who have never From the Department of Medicine (Allergy), George Washington University Supported by a grant from the John A. Hartford Foundation, Inc. Received for publication Oct. 25, 1967. *In this paper, for reasons of simplicity, the word species is used to designate types of insects. Entomologically, this is correct for honeybee, hornet, and yellow the wasp is a genus.
146
Hospital.
the different jacket, but
Volume Number
Studies on the Hymcvoptwa
44 3
1.17
experienced anaphylaxis from a sting may give positive skin tests. The percentage of patients giving positire tests cannot be accurately compared because the extracts used in the different studies were not of the same concentration.” Bernton and Browq5 with the use of honeybee extracts containing 200 and 20 protein nitrogen units (PNU) per milliliter? noted positive tests in 38 per cent of 380 normal volunteers. Apparently, a stronger extract products a grcat,er incidence of positive t,ests, and one must agree with Bensol? tllat honeyhcc venom “is of moderate toxicity to the human species, nonsp,ccific, ;111(1 therefore tending to cause somewhat of a reaction in every one.!’ Because of the discrepancy between clinical symptoms and skin teats ;rlrcad!’ noted and because of apparent inconsistency in direct skin testing, the sera oi IIymenoptcra-sensitive patients were studied hy passive transfer. Evcq patient had suffered generalized itching and swelling, asthma, weakness, or anaphy1ac.t in shock following a single sting. METHODS Passive transfer
tests
The usual precautions were taken when sera were obtained from 16 patients who had suffered anaphylaxis from a Hymenoptera sting and from 12 patients who had suffered only a large local reaction. The passive transfer tests were performed as follows: Sites mere sensitized on the arms of volunteers with the use of 0.05 ml. of t,he patient’s undiluttcl serum. Forty-eight hours later the sites were challenged with 0.025 ml. of one of the antigenic extracts. These were commercial extracts,* each made from whole bodies of a single species of Hymenoptera-bees, hornets, wasps, or yellow jackets. Tn our preliminary testing, it had been found that 38 per cent of “normal” subjects reacted to an antigen containing 200 PNU per milliliter. Therefore, in the present study 100 PNU per milliliter was used as the standard strength .for challenging. This extract was diluted in sterile normal saline from a 50 per cent glycerinated extract containing 10,000 to 20,000 PNU per milliliter. Tests wer’c perforrned in triplicate and read as follows: t, wheal greater than 0.5 cm. but less than 0.75 cm. in diameter and surrounded by flare; +, wheal 0.75 cm. i~t diameter, surrounded by flare; st, wheal 1.5 cm. in diameter with pseudopods and flare; tt+, wheal greater than 2.0 cm. in diameter with pseudopods ant/ flare. Controls were as follows: (1) The serum of a pollen-allergic patient was challenged with honeybee antigen. (2) The serum of a honeybee-allergic patient was challenged with pollen antigen. As a positive control, the serum from ;I ragweed-sensitive patient was injected into each recipient and challenged wit11 ragweed antigen. A positive reaction indicated that the subject was capable 01 passive transfer. Every recipient tested negatively to the hymenopterous cxtracr, at the time of challenge of the sites. Since dilutions of antigen or of serum in our preliminary testing had in general proved unrewarding, dilutions in most cask were climinnt~cd. “Hollister-Stier
Laboratories,
Spokane,
Wash.
148 Direcf
Brown skin
J. Allergy September, 1969
and BerntolL testing
Direct skin tests were performed on the lateral aspect of the arm by the intracutaneous injection of 0.02 ml. of extracts of honeybee, hornet, wasp, and yellow jacket in varying dilutions so that a negative, a threshold, and sometimes a strongly positive test was obtained with each extract. The strongest extract used for direct tests contained 1,000 PNU per milliliter and 2.5 per cent glycerine. The wea.kest extract giving a positive skin test among these patients contained 0.01 PNU per milliliter. Direct skin tests were read as follows: +, wheal slightly larger than the intradermal injection, mild flare; +t, wheal 0.5 cm. in diameter, definite flare; +tt, wheal 0.75 cm. in diameter, large flare, often with pseudopods and itching ; +++t-, wheal 1.0 cm. in diameter, large flare, pseudopods, and itching. Identification
of insect
The diagnosis of the species of Hymenoptera was determined by the shape of the insect and by the type and site of the nest. An insect was considered identified upon presentation by the patient. An insect was considered presumptively identified if it was obtained from the nest of the culprit or if the patient brought in the nest from which the culprit was seen to fly. If neither of these conditions prevailed, the insect was not considered identified. RESULTS Passive transfer
Table I shows in 12 cases of local reactions to a sting the relation between passive transfer and direct skin tests with the use of the same antigen in the same dilutions for both types of test.
Table 1. Relation between passive transfer and intradermal patie?ats who had suffered only a large local reaction Hornet
Honeybee Culprit
Patient Ala. Ale. Gib. Gol. Has. Hoc. Kir. Kre. Pre. Pri. Rea. sto.
insect*
Passive transfer+
Skirt
Slcin
transfer
transfer
testt.
testi
0 0 0 0
0 0 ttt 0
0 0
0 0 +t+ 0
0
0
0 0 0 0 i
FO)
0
“y
0
0
0
0
0 0 0 0 fl
tt 0 0 0 t 0 0
0 0 0 0 0 0 0
++ 0 0 0 0 0 0
0
Passive
test$
0 0 +tt 0
“W” W “\V” TV w HB HB
Wasp Passive Skin
Passive transfer
L‘HB” HB ‘TJ” (Tes” J”
0 :
Skin
*HB = honeybee; W = wasp; YJ = yellow not positively identified are written within quotation tPassive transfer technique explained in text. *Direct skin tests performed with 0.02 ml. of Other concentrations are noted in parentheses.
skin tests in 12
test$
i
Yellow jacket
0 :
0 0 tt+ 0
0
0 0 + :
0
0
O 0 0 0 0 :
+OfP) 0 0 + 0 0
0 jacket; marks.
Ves
=
extract
containing
unidentified
100
vespid.
PNU
per
Insects
milliliter.
Tables II and III show in 16 patients who had suffered a generalized reaction the relation between passive transfer and direct skin tests with the same antigSc>ns as those in Table I. The culprit insect was the honeybee in 4 patients ant1 ;I, vespid in 8 patients with local reactions. Among patients who had snffcut~l ;I generalized reaction, the honeybee was incriminated in 5 cases and a vcspi(l ilt 11 cases. The scra of the patients reacting only IocalIy to a sting did not t,ransfcr scllsitivity with any of the Ilymenoptera antigens tested. T,iken;isc, the sera 01 \: Table II. Relation of passive transfer sufferecl a generalized reaction*
to direct skin tests in 7 pntieafs
Honeybee
Patient Cav. Nit. Hap. writ. Gre. coo. Flo.
Cdprit insect UYJ,’ HB “Hym” “HB” “FIB” YJ YJ
Passive tmnst
Hornet Passive tmns-
Direct test$
fert
skin
-t + 0 IO 0 0 0
fer 0 0 0 0 0 0 0
(! O(l0, ++t+ 0 i-++ ?-c +
0 ++++(0.01)ll ++++ ++(O.l) ++ II
Passive tmnsfw
Direct
Two species Mau. YJ&HB “H” Dau. Har. HB
skin
Passive
test
transfer
WOl.
YJ IIT3 II
i + 0
Bia. Car.
II Y’J
0 t++t
YJ
+
Three species C:ES. Ber.
Four speoies Trn. the
Passive
0 t++(lO) +t+t
“Hera exhibited positive same as those for Table
t
j
Wasp ;K?$
‘d 0 +t i-
:I.+.. + 1 i?+ ++ unidcntifird
per millilitt~r. (0.01 :z i).llt
who hut1
Pellow
jacket
~~~l~~~~I~~~~~~rI~~~'
i
0 + +
L+(l) +t
Zt(lO, 0
0 0 +
0 iCk 0
+ + +
0 +++ +++
++ t++
+ -I-
tt
t 0
t
0
++t II.
100 PNU parentheses
++ t++(l) t+
transfer
0 oi I I
Direct
transfer
+ ttt ++
0 0
one species only. yellow jacket, Hym = within quotation marks.
Hornet
Baneybee
Culprit insect
0 Cl(l0) +c++ i-c ttt ++t +
containing noted in
jazz_- ._ / Dir{ cl I skt~i i ttstj
Passive tramfer
to direct skin tests i,rt 9 patieds
Table
Patient
Birect skin I testj
0 0 i& i-+ 1r 0
“Sera demonstrated passive transfer with extract of tHB = honeybee, H = hornet, W = wasp, YJ = member of Hymenoptera. Insects not identified are written STechnique explained in text. $Direct skin tests performed with 0.02 ml. of extract If a weaker concentration was used, the concentration is PNU per milliliter). IlConstitutional reaction from single skin test.
111.Relation of passive transfer sujfered a geraeralixed reaction*
Ycllotu
m-asp Direct skin test$
who l1w1
with
extracts
+cL
iti of
2 or more
0 0 0
*-++ i----t ( 1 i +-
t
0 *L -3+
+
-+
1i r-I-r
+++
t+
if?+
species.
Abbreviations
i’
nrt’
150
Brown
J. Allergy September, 1969
and Bernton
beekeepers stung during the season (100 times or more) failed to give a positive reaction with the use of the passive transfer technique with honeybee extract. In contrast, each of the sera of 16 generalized reactors was capable of transfer when challenged with allergens of the Hymenoptera. Seven sera reacted positively with the extract of one species, 3 with the extracts of 2 species, 5 with the extracts of 3 species, and one with all 4 antigenic extracts tested. Controls in every case were negative. Direct
skin
test
reactions
It is apparent from Table I that skin tests were negative in a dilution of 100 PNU per milliliter in 9 of the 12 cases of local reaction. In one patient, the intradermal reaction was +tt when challenged with an extract containing 100 PNU per milliliter. In only 2 tests of the 48 performed was the skin test positive with the use of a concentration of 10 PNU per milliliter. In sharp contrast were the intradermal tests of the 16 generalized reactors, all of whom had shown positive passive transfer tests. Eight patients reacted positively with honeybee antigen at a concentration of 100 PNU per milliliter, 3 at a concentration of 10 PNU per milliliter, one at a concentration of 0.01 PNU per milliliter. Nine patients reacted positively when tested with hornet extract at 100 PNU per milliliter and one patient with a concentration of 1 PNU per milliliter. Eleven patients showed positive direct tests with wasp extract at 100 PNU per milliliter and one with 1 PNU per milliliter. Twelve patients reacted when challenged intracutaneously with yellow jacket extract, 100 PNU per milliliter, and one patient with 1 PNU per milliliter. Family
history
More than half of the family members of the 12 patients suffering local reactions had experienced some type of allergy, 2 to stings of Hymenoptera (Table IV). Fifty per cent of the family members of the 16 patients suffering a generalized reaction had experienced typical symptoms of allergy. Past
history
Fewer patients than family members in both groups had experienced a personal history of allergy : 4 of 12 local reactors and 6 of 16 generalized reactors to a sting (Table IV). In both groups, hay fever was the most frequent allergy noted. The number of cases, 28 in all, is too small for further interpretation. Accuracy
of identification
of insects
The insect was identified accurately in 11 of the 16 cases of generalized reaction. In one of these, the sting may have been intravenous since a hematoma occurred at the site of sting, and symptoms occurred within 5 minutes. One patient had suffered anaphylaxis 5 times, once caused by a honeybee, once by a yellow jacket, and 3 times by a hornet. In 5 cases the insect was not identified. In several the insect was not seen.
Volume Number
44 3
IV. Family and past history of Hymenoptera patients reaction compared with those suffering a genera,lized reaction
Table
Large
reaction
Generalized
___.-.--. Past histol-y
history
Patient
Ah. Ale. Gib. Gol. HM. Hoc. Kir. Jirc.
i t t 0 t + +
0 0 0 + + 0 0 0
Cav. Sit. Hap. wat. Gre. C!oo. No.
0 0 0 0 0 t% 0
1\hU.
t
Pro.
0
0
Pri. Krx.
t t
t 0
Ihu. Ihr. CM.
t +
+ -c
sto.
0
t
CJ 1
0 .L 0
Bia. CX. TIX.
il i i
0 II t
14
x
ti
s
-i
1
reaction
Family history
13
Past
a 10~1
Family history
Patient
Totals
local
suffering
Lh. Wol.
t 1) Ii 0 IJ II 0
-
In one cast, a small boy (Hap.) who had been playing out-of-doors rushed into the house screa.ming, became cyanotic and clammy, and collapsed. ITe was hospitalized for 5 days for complete examination. All tests were negative. ~1 similar experience was repeated the next summer, again with hospitalization and a negative work-up. The child then was rrferrecl for testing with insect allergens. Exceptional entomological situations must not be overlooked in searching for accurate identification. Patient Coo., a 6-year-old boy who had been playing near a hedge which contained a 12 inch hornets’ nest, appeared at the kitchen door “covered with yellow jackets,” only one of which stung him. Because the mother’s description was at rariance with the hornets known to exist in the hedge, an entomologist examined the nest. To his surprise, attached to thtl hornets’ nest at the farthest point from the opening was a smaller >--ellov jackets’ nest. (He surmised that the 2 nests had been started independently ant1 with increase in size had become contiguous.) Hence, the mother’s description oi’ the insect was correct. The insect was definitely identified in 6 of the 12 C~SCSof local reactions. DISCUSSION
In this series of patients, anaphylactic hypersensitivity to IIymenoptcAra stings was associated in every instance with the presence of circulating skinsensitizing antibodies to the specific insect. The circulating antibodies possiblymay not be etiologically related to the serious reactions suffered by the patients. The presence of these antibodies may simply be an index of abundant antihod:of this type being fixed to tissues. In this regard, passire transfer reactions
tended more to be obtained with sera from patients who gave strongly positive direct skin tests. There were csceptions to this general trend. Combining the data from Tables T, II, and III, passive transfer was achieved with scra of 4 of 8 patients showing 4t- direct. skin test reactions, with sera of 11 of 21 patients showing 3+ direct react,ions, vith the scra of 10 of 23 subjects 2+ direct reactions, with 0 of 5 patients showing It reactions, and with 0 of 2 subjects showing & reactions. In 11 of the 12 cases in which the insect W’BS identified, passive transfer was nchievd to the specific ins&. In contrast, passive transfer reactions were positive in 32 tests with only 14 h>-mcnopterous allergens other than the one t,hought to hare caused anaphplaxis. On direct skin testing of the patients who had identified the culprit ins&, the specific insect gave the largest reaction in 5 of 12 cases; another insect showccl the largest reaction in 3 of 12 patients, and in 3 patients specific and nonspecific insects showed about equally marked reaction. In one case (Kit.) the culprit insect gave the only positive direct skin reaction, and it occurred with the use of an extract of high dilution. The sera of 8 beekeepers who had suffered many rcvcnt stings without urltoward symptoms tcstcd negatively with the use of the passive transfer tcchniyuc. In the 3 instances in which passive transfer responses were positive in the presence of negative direct tests to the same antigcnic extract (Car., May., and Gas.), every serum showed the posit,ive reaction in triplicate, i.c., in each of’ 3 different recipients. In some cases t.he apparent specificity of the passive transfer test was striking. Patient Nit. had been stung onl,v by a honeybee and had suff&~~tl armphylaxis only from the stin, 0’ of a honeybee. IIcr serum, when testctl 1)~ the passire transfer technique, gave a positive result only ~hcn honcybec cstract was used. Patient F’lo. had been stung only h- ~cllow jackets, and his serun~ transferred sensitirity only when challenged with yellow jackrt, extract. Similarly, Patient Har., the wife of a bcekecpcr~ xhose son, aged 9, had diet1 oi anaphylaxis shortl)- after a hone~hcc sting, hat1 cqericnccd se~crc gcncralixctl reactions with a honcyber and a wasp sting only. 11~ s~uin transferred sensitivity when tested with these 2 antigenie cstracts. It (lid not transfer sensitivity when hornet or ;veIlow jacket extract was nscd. Some of the inconsistencies in skin testing may bc esplainctl hy tlio fact that most patients had been stung se\-era1 times previously by diff’ercnt members of the Hymmoptera. All the antibodies found at the time the patient was examined may not necessarily bc the result of the last, sting. This is clearly shown in the case of Patient Bia., who had suffcrcd anaph~lasis from the sting of a honeybee, in 1964, and of a pcllow jacket, in 1962. In 1960, during a single month he received 3 separate hornet stings. each causin g mow severe anapli,~ laxis than the last. The duration of the lift of transferable antibotlics is not known. It is possible that the titer of antibodies to one species of insect may 1~ higher because of more freqncnt stings by that insect. There appears to be a tendency for sensitivity to the H.vmcnoptcra, as measured by direct skin testing, to decrease with the passage of time.” The erolntion of the passire transfer has not yet hen studied in a similar manner. It ma-
1)~ that lesser titers of antibody following a sting nli\y (lisdppvar ~OI*O ~il1>i(lI~ from the circulation than higher titers of antibody. Whether the patients who had sufYcrrt1 only lov;11 tv;r&~ns will ~WOIIII. more sensitive in timc and develop more st14011glypositi\.ca skin tests j)ositi\~c, passive trdnsfcr t,rsts remains to bc seen. ilncl
CONCLUSIONS In 16 I-lpmcnoptera-sensitive patients who had suffcretl a generalized I*c~a(~tio~~ to a single sting, antibodies concomitant with anaphylaxis conl(l btl passircily transferred. In 12 patients who had suff%r~~l only a local reaction, passive* transfer was not, achieved. Direct skin tests to l~~nlrnopteronn untigerls ill I ho patients who had suffered a generalized rcac*tion n-erc larger nncl positii-rx ;11 higher dilutions than were Qose of patients who had suffered only local reactions. There was a tendency for larger tlirect, skin tests to be corrclatrd with I)osit ivc passive transfer. Half, or more, of the family memb~s of both groups 1x111t~sl)~lrit~xctl symptoms of allergy. Passi\-r transfer (W~rPlilt(Y~ II101’e c~lcarly wit11 till’ spccics of culprit insect than did direct skin tests. In 2 vases the insect ;tppc~a~v~l to be identified by passive transfer. The prc~ncc of passiveJ. tramsFvi2hlt~ antibodies may result from former stings or from the most vrccnt sting. The
:~uthors
wish
to thank
Dr.
Philip
J. Torsnq
for
contrilnlting
on,’ wrum
to thi5
surt)..
REFERENCES
1. Mueller, If. L.: Insect allergy, Prdiat. Clin. Sorth Amcric:~ 6: 91T, 19SH. 2. Htirr, H. A., and Foubert, E. L.: Antigenic relationships betnwn hon~~~l~ws, wvas[,s. vc~l IOU jwkrts, and yellow and black hornets, J. AI,I,&RGY 27: 98, 1956. (Abst.) 3. Wmson, R. L.: Diagnosis of bypersensitirity to tllv bw anti to mosquito, A\r(,l~ I nt. M~,II. 64: 1306, 1939. 4. Mnc~Lnrcw, 11’. R., Eisenberg, B. C., Frank, J). E., xnd Kesslw, .l. : Rractionr to inr;wi nllrrgen~. The incidence of response to testing among :kIlt+c am11 non:rllcrgi(, pcrsou~, (%lifomin Med. 93: 224, 1960. 3. Ht%lton. 11. H., and Hrown, H. : Studies on the Ilymenoptwa. I. Skin wavtions of norn!:t I persons to honeybee (Apis mellifern) extract,, J. A&I.IiER(iY 36: 315, 1963. 6. Brown, II., and Rernton, II. 8.: Evolution of the skin tf,st in 100 II~mrnopter:1-s~r~siti~-~~ patients, prwentcd at the meeting of the American Acr~Iwny of Allvrgy, Iiostou, Ffltj.. Id@. i. Mueller, H. L.: Aerixl intracutaneous testing for Irwc ;lrltl wrsp sclllsitivit)-. .T. .\I.I.F‘lifi\ 30’ I”? 195Y. 8. S~‘il\v\-a;,i~,‘H: .J.: Skin sensitivity in insect allergy, ,1. A. 31. h. 194: 119, l!Wj.