Allergy 1986, 41, 526-531

Histamine-Dependent Allergy Blood TestClinical Evaluation

B. A. FAR-'̂ J, V. M. CAMP and P. LOLIES

Department of Radiology (Division of Nuclear Medicme), Emory University School of Medicine Atlanta, Georgia, U.S.A.

Allergen-mediated histamine release from human leukocytes represents an importanimodel for m viSro studies of allergic reactions. The purpose of this study was to determinewhether the measurement of histamine released in allergic patients by radioenzymaticassay following mixing of their blood with common allergens represents a reliable indexfor diagnosis of atopic allergy. Three categories of allergens were used: 1) house dust andraite; 2) cat and dog dander; 3) trees, grasses and ragweed mixture. The presence ofallergy w3s established by clinical history and intradermal skin testing in tlie study groupof 150 patients. A significant allergen-mediated histamine release ranging from 4 to 65%of the total blood histamine content was observed in 96 % of the patients with skin testsensitivity of > 3 + . There was a significant correlation between skin testing andhistamine release in terms of the allergens causing the response. Thus, the measurementof histamine by radioenzymatic technique following its release in blood in response toallergen challenge represents a clinically useful in vttro test for the diagnosis of atopicdisease.

Key words: Eillergy; asthma; liistamine; radioenzymatic; rhinitis.

Accepted far pubiication 25 April 1986

CLINICAL ASPECTSA liistamine-dependent aUergy blood test is developed. The test was used to measurethe released histamine of 150 patients witli allergic disorders following the incuba-tion of their blood with common allergens in three major categories. An excellentcorrelation was found between histamine released from whole blood and theresponse of skin mast cells to intradermal antigen administration. The availabilityof this test should he useful to various physicians to assist them in the diagnosis andmanagement of their patients witti allcTgic diseases.

AUergy is a major public health concern as large Skin tests and provocational tests have beennumbers of the American public, approximately widely used for many years in providing useful35 million, suffer from all sorts of allergic disor- confirmatory evidence for a diagnosis of specificders. The cost involved in caring for the allergic allergy which has been made on clinical groundssufferer is close to one billion dollars in the Unit- (3, 8, 11). Recent reports by Imber (5), Reddy eted States alone. More than two-thirds of the al. (14) and Pascual et al. (13) challenged theschooltime lost is a direct consequence of upper reliability of various skin test techniques. Therespiratory tract insults, including asthma and reasons for this are numerous and include theother respiratory tract allergies (7). These promi- following: a) it is expensive, b) it requires a greatnent concerns accentuated the need for specific deal of time on the part of the patient, c) there is aand clinically reliable biochemical tests for the risk of anaphylaxis (it cannot be used to evaluateappropriate recognition and prediction of the potent antigens that might produce anaphylaxisseverity of atopic disease. with even the minor exposure of skin testing), d)

HISTAMINE-DEPENDENT ALLERGY BLOOD TEST 527

dermographism and widespread skin diseaseinterfere with testing, and e) it is a qualitativebiological testing, as the interpretation of resultsdepends entirely on the experience and the dex-terity of the skin testerhimself. These observationshave stimulated others to seek far less invasive,safe and convenient methods for the identificationof l̂n inciting factor in an allergic patient.

For the most part, allergic reactions are a phys-iological response to the release of histamine (6).In 1966, Lichtenstein et al. (10) carefully assessedin patients with ragweed-sensitive allergic rhi-nitis, the release of histamine from isolated bloodleukocytes when challenged with allergen. Thetest was reproducible and correlated with clinicalsymptoms. Even though this method is represen-tative of a cellular IgE-mediated reaction com-parable to the wheal and flare reactions followingthe release of the histamine from skin mast cells, ithas not been widely accepted in clinical practicemainly because of the requirements of large bloodvolumes and inheritable complexities associatedwith the measurement of histamine.

In view of the importance of histamine inallergic disorders, we took the initiative to developa radioenzymatic assay for histamine (4). Theassay was used to study the released histamine of alimited number of atopic subjects following theincubation of their blood with a group of commonallergens. A significant correlation was foundbetween histamine released from whole blood,clinical history, and the response of the skin mastcells to intradermal antigen administration. Stim-ulated by this preliminary observation, we modi-fied this assay in order to develop an in citro his-tamine-dependent allergy test suitable for clinicalroutine application. This test was evaluated bythe measurement of histamine released fromwhole blood of a diversified population of allergicpatients as a result of the incubation of theirbloodwith a mixture of common extrinsic allergens inthree nniajor categories.

MATERIAL AND METHODS

Patient population

One hundred and fifty patients ranging in agefrom 9 to 60 years admitted to an outpatient

allergy clinic were evaluated by history andintradermal skin tests. These patients weredivided into four major categories: Group 1consisted of 78 subjects with rhinitis. Group 2consisted of 43 patients with asthma. Group 3consisted of 27 patients with upper respiratorytract infection, and Group 4 was made up of 30patients with combined symptoms of asthmaand rhinitis.

Skin Testing:

The skin tests were performed with the sameantigens used for m vitro histamine release. Theconcentrations used for skin testing were 1,000PNU/ml for house dust, dog dander, and treepollen, and 100 PNU/ml for house dust mite,cat dander, ragweed, and grass pollen. For skintests, 0.02 ml of allergen was injected at eachsite on the upper arm with a disposable syringeand reactions read at 15 min. Skin-test gradingwas based on measurements of the greatestdiameter of the wheal following the usual crite-ria; negative, (site no different from diluentcontrol), 3 + (8-12 mm of wheal) and 4 + (>12 mm wheal).

Histamine release from whole blood:

A schematic representation of the assay isshown in Fig. 1. Histamine release reactionswere carried out in 12 by 75 mm plastictubes. To each of the tubes were added 0.5 mlof Tris-albumin buffer, allergens (in PNU/ml:category I; house dust/house dust mite, 200:5;category II; cat/dog dander, 60:1000; and cat-egory III; trees/grasses/ragweed, 150:150:100)and 0.5 m! of heparinized blood. Controltubes were prepared by mixing buffer withblood alone. The tubes were incubated in ashaking water bath for 15 niin at 37°C. Thiswas followed by centrifugation at 500 x g for10 min. The plasma was separated and storedat -70°C until analysis. Samples for deter-mination of total blood histamine wereachieved by boiling 0.5 ml of whole bloodwith 1.5 ml of 0.05 mol/1 phosphate buffer(pH 7.9) in a heating boiling water bath for20 min. After centrifligation at 15,000 x g for

528 B. A. FARAJ ET AL.

2 Efixymm R*«ctkon 3, Extractlofi siid Counting

Fig. 1. Scheme of assay profile of histamine-dependent allerg\' blood test. C represents an allergen-free tube as a controland S denotes an allergen-containing tube.

30 min, the supernatant was removed andstored at -70° C until analysis.

Plasma histamine assay

The concentration of histamine released fromblood was measured by radioenzymatic tech-nique according to Faraj et al. (4). The methodis based on the use of purified enzyme his-tamine-N-methyltransferase (HNMT) isolatedfrom rat kidney, to transfer a radioactive meth-yl group from the cofactor [3H-methy]-S-ade-nosyl-L-methionine (SAM) to an endogenoushist£miine acceptor molecule in the sample toform the radioactive metabolite [3H-methy]-N-histatnine. This was followed by selectiveextraction of the liberated metabolite and itssubsequent quantification by liquid scintillationcounting (Fig. 1). Lower level of sensitivity is65 picograms of histamine per sample; intra-and interassay coefficients of variation were 5 %and 7%, respectively.

Percentage of histamine release

The percentage of histamine released from 1 mlof blood by the allergen tnixture in each of thethree categories was determined from the fol-lowing formula: 100 x A/B = % histaminerelease, where A = concentration of histaminein plasma following the incubation of 1 ml of

whole blood with allergens, minus con-centration of histatnine in plasma followingincubation of 1 ml of blood alone; B = con-centration of total histamine obtained from 1 mlof boiled blood minus blank sample.

RESULTS

Clinical observation

Of the 78 patients with rhinitis only 28 ex-hibited 4-^ (n = 10) and 3 -H (n = 18) skin testreactivity as compared to 50 patients with vas-omotor rhinitis having negative skin test reac-tion when evaluated against a series of commonallergens. In contrast, 30 of the asthmaticpatients had extrinsic asthma since they demon-strated positive skin test reactivity against avariety of conimon inhalants ( + i, n = 10; + 3,n = 20) as compared to only 13 with negativeskin test reactions (i.e. intrinsic asthma). Asimilar pattern was observed among thosepatients with recurrent upper respiratory infec-tion (n = 27) and the asthma-rhinitis group (n= 30).

Histamine blood release

Allergen-mediated histamine release (in % oftotal blood histamine) from whole blood of the150 patients is shown in Fig. 2. The results of

HISTAMINE-DEPENDENT ALLERGY BLOOD TEST 529

CHtegofy tilCategory I Category II (Trees/GrMMS

{Housedust/MKal ICat/Dog» RagMsed]

70-

60-

50-

40

30-

20-

to-

5-

5

L̂ Non.Moplc

Fig. 2. Allergen-mediated histamine release (in % of totalblood histamine) from whole blood of 150 patients withdifferent allergic disorders following the incubation of theirblood with common allergens in three major categories.

100-

I- 75-

- 50-

25-

Negative 3-*- 4-i-

Skin Test (Wheal Diameter)

Fig. 3. Percent agreetnent between histamine blood releaseand skin test results.

this study showed 90-100 % agreement betweenhistamine blood release and skin testing (Fig.3). Further, a significant correlation was notedbetween in vitro histamine release from wholeblood and response of skin tests in thesepatients (Fig. 4). This was demonstrated by anaverage of 12-17% of histamine blood releasefollowing antigenic challenge with allergen mix-ture in each of three major categories when theskin test was 3 + , compared with an averagehistamine blood release of 27-30% in patientswith skin test results of -f 4 or greater. Non-allergic blood from donors with negative skintest released no histamine following incubationwith the above allergens (Fig. 4).

DISCUSSION

In an earlier report (4) we demonstrated thedevelopment of a simple and sensitive radio-enzymatic assay for the direct measurement ofhistamine in plasma. This assay was used toformulate a histamine allergy blood test amen-able to clinical routine use. We measured theamount of histamine released from whole bloodof atopic patients after in vitro provocations with

suspected allergens in three major categories.We found that this method can be routinelyapplied and easily performed: a) The sensitivityof the system allows in vitro quantitative testingwith allergens in three major categories withonly 2.5 ml of blood; with 5 ml of blood the testcan be expanded to include an additional fiveother categories (i.e., penicillin, insect venom,mold mixture, albumin and altemaria). b)Freshly prepared assay reagents includingenzyme (histamine-N-methyltransferase), radio-labeled cofactor ('H-SAM), buffers, and stop-ping solution are stable for at least 6 months ifstored appropriately (4° to -70°C). c) The stor-ing of a blood sample for 48 h at 4°G had noeffect on the allergen-induced release of his-tamine from whole blood. This is a dear-cutadvantage to both patient ziid physician, d)Many samples can be assayed per day andreporting of the results can be achieved within24 h of receiving the sample, e) The test isinexpensive and easy to perform.

In agreement with previous observations (9,32, 15, 16), an excellent correlation was notedbetween histamine released from whole blood ofpatients with allergic asthma, rhinitis and upper

530 B. A. FARAJ ET AL.

Negative 3 * 4+

Skin Test (Wheal Diameter)

Fig. 4. Relationship between aliergen-mediated Iiistaminerelease and skin test results in patients with allergic disor-ders.

respiratory infection and the response of theskin mast cells to intradermal antigen admin-istration. A significant amount of histamine wasreleased, ranging from 25-30% of the totalhistamine content following incubation of theblood with allergens in any of the three catego-ries. This effect was observed in 83-100% ofthe patients with skin test sensitivity of 4 -i- orgreater to grasses, trees, house dust, ragweedand animal dander. There was good agreementbetween the intensity of the skin reaction andmagnitude of histamine release, since patientswith skin test sensitivity of 3 -(- had a histamineblood release upon antigen challenge in vitro ofonly 12 to 17%. This in vitro histamine-depen-dent allergy test is highly specific, since patientswith vasomotor rhinitis and those with intrinsicasthma who were clinically non-atopic did notrelease histamine from blood.

The histamine-release results from this studycompare favorably with published reports of theuse of RAST technique in allergy diagnosis (1,2). Several reports have shown that the RASThas an overall accuracy of about 70% whencorrelated with a patient's clinicEil history (12).

The present results showed that the histaminerelease was positive in over 90 % of the patientswho were allergic by clinical evaluation. Fur-ther, histamine release from whole blood prob-ably is a biologically more sensitive index of apatient's sensitivity than RAST test because itis a cell-attached IgE that reacts with allergen,resulting in the release of allergic mediator his-tamine. The basophil histamine release testseems to be superior to RAST because noallergen coupling procedure is needed, and thesame allergens can be used as for skin testing.

In summary, the data presented here outlinethe availability of a safe, quantitative, reliableand easy to perform histamine-dependent aller-gy blood test. This test should be useful tophysicians with a wide range of specialities,including allergists, pediatricians, internists,anesthesiologists, ophthalmologists, and oto-laryngologists, to assist them in the manage-ment of their patients with allergic symptoms.When acute symptoms are due to extrinsicagents, the histamine aliergy blood test can beutilized to identify the offending allergens. Themanagement of the patient could includeallergen avoidance and pharmacological block-ade of effector mechanisms.

ACKNOWLEDGEMENTS

We thank Dr. George Gottlieb of Asthma and Diseases ofAllergy, Decatur, Georgia, for providing us with bloodspecimens and skin test data of the padents evaluated in thismanuscript. We greatly appreciate his clinical assessmentand advice ttiroughout the study.

REFERENCES

1. Aas, K. & Johansson, S. G. O,: The radioailergosor-bent test in the in vitro diagnosis of muitiple reaginicallergy. A comparison of diagnostic approaches. J.Allergy Clin. Immunoh 48, 134-142, 1971.

2. Berg, T.. Bennich, H. & Johansson, S. G. O.: In vitrodiagnosis of atopit allergy. I. A comparison betweenprovocation tests and the radioallergosorbent test. Int.Arch. Allergy Appl. Immunol, 40, 770-778, 1971.

3. Davies, R. J. & Corrado, O. J.: Diagnostic tests -challenge tests. Oral and nasal and bronchial. In Lessof,M. H. (ed): Allergy: immunological and ciinicaiaspects, pp. 83-105. John Wiley, New York, 1984,

4. Faraj, B. A,, Gotdieb, G. R., Camp, V. M., et al.;Development of a sensitive radioassay of histamine

HISTAMINE-DEPENDENT ALLERGY BLOOD TEST 531

for in vitro allergy testing. J., Nucl. Med. 25, 56-63.1984.

5. Imber, W. E.: Allergic skin testing. A dinical investiga-tion. J. AUergy Clin. Immunol. 60, 47-55, 1977.

6. Ishizaka, T.: Molecular and biochemical mechanisms ofIgE-mediated histamine release from mast cells. InGanellin, R. & Schwarz, J. C. {eds.): Frontiers inhistamine research, pp. 401-409. Pergamon Press, NewYork. 1985.

7. Lee, D. A., Winslow. N. R. & Speight, A. N. P.:Prevalence and spectrum of asthma in childhood. Br.Med. J. 286, 1256-1258, 1983.

8. Lessoff, M. H. & Bametson, R. C ; Diagnostic meth-ods - skin tests. In Les&of, M. H. (ed): Allergy, immu-nologicaJ and clinical aspects, pp, 73-82. John Wiley,New York, 1984.

9. Lichtenstein, L. M., Marone, G. & Thomas, L- L,:The roie of basophils in inflammatory reactions. J,Invest. Dermatol. 71, 65-69, 1978.

10. Lichtenstein, L. M., Norman, P. S., Osier, A. G., etal,: In vitro studies of human ragweed allergy. Changesin cellular and humoral activity associated with specificdesensitisation. J. Clin Invest. 45. 1126-1229, 1966.

11. Morley, J., Hanson, J. M. & Youlton, L. J. F.: Media-tors of allergy. In Lessof, M. H. (ed): Allergy: immu-nological and clinical aspects, pp. 45-72. John VVUey,New York, 1984.

12. Norman, P. S., Lichtenstein, L. M. & Ishizaka. K.:Diagnostic tests in ragweed hay fever. J. Allergy Clin.Immunol. 52, 210-224, 1973.

13. Pascual, H. C . Reddy, P. M., Nagaya, H., et al.Agreement between radioaliergosorbent test and skintest. Ann. Allergy 39, 325-327, 1977.

14. Reddy, P. M., Nagaya, H., Pascual, H. C , et al.Reappraisal of intracutaneous tests in the diagnosis ofreaginic allergy. J. .Miergy Clin. Immunol 61, 36-41,1978.

15. Siraganian, R. P.: Automated histamine analysis for inaitro allergy testing. J. Aliergy Clin. Immunol. 59, 214-222, 1977.

16. Taylor, K. M., Krillis, S. & Baldo, B. A.: An enzym-atic-isotopic microassay for measuring allergic release ofhistamine from blood and mast cells in vitro. Int. Arch.Allerg>'Appl. Immunol. 61, 19-27, 1980.

Dr. B. A. Faraj

Department of RadiologyEmory University467 WoodrufT Memorial BuildmgAtlanta, GeorgiaUSA