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Year : 2009  |  Volume : 75  |  Issue : 5  |  Page : 545-548

Autologous serum skin test: Methodology, interpretation and clinical applications

Department of Dermatology, Venereology and Leprosy, Indira Gandhi Medical College, Shimla, India

Date of Web Publication3-Sep-2009

Correspondence Address:
Nand Lal Sharma
Department of Dermatology, Venereology and Leprosy, Indira Gandhi Medical College, Shimla 171001 (H.P.)
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0378-6323.55424

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How to cite this article:
Vohra S, Sharma NL, Mahajan VK. Autologous serum skin test: Methodology, interpretation and clinical applications. Indian J Dermatol Venereol Leprol 2009;75:545-8

How to cite this URL:
Vohra S, Sharma NL, Mahajan VK. Autologous serum skin test: Methodology, interpretation and clinical applications. Indian J Dermatol Venereol Leprol [serial online] 2009 [cited 2020 Sep 21];75:545-8. Available from:

  Introduction Top

Autoimmune urticaria, a recently described subset of chronic urticaria, has often severe and continuous whealing associated with malaise, indigestion and sensation of hot and cold. A personal or family history of other autoimmune diseases or urticaria in family members is suggestive. It is associated with antithyroid antibodies in 27% of patients or with other autoimmune conditions such as vitiligo, rheumatoid arthritis, pernicious anemia and insulin-dependent diabetes mellitus. [1],[2] Diagnosing these patients becomes important as they need high doses of antihistamines and systemic corticosteroids during acute exacerbations. Immunomodulatory drugs, while their use is not justified in chronic idiopathic urticaria (except in antihistamine refractory chronic urticaria cases), are therapeutic benefit in recalcitrant to therapy autoimmune urticaria patients having significantly impaired quality of life. [3] Immunologically, it is characterized by the presence of functional autoantibodies that activate mast cells and basophils by crosslinking the high-affinity IgE receptor (FceRI). About 50% patients of chronic urticaria have functional autoantibodies to FceRI and 9% to the IgE antibody itself. [4],[5] Autoimmune urticaria is thus diagnosed in ASST-positive chronic urticaria patients who exhibit functional autoantibodies against IgE and/or its high-affinity receptor FceRI. However, it is often clinically difficult to distinguish chronic urticaria from autoimmune urticaria. Its diagnosis is practically relied upon clinical suspicion and autologous serum skin test (ASST) as facilities for assaying functional histamine release from basophils or mast cells, a suitable diagnostic laboratory investigation, are largely confined to research centers. Unfortunately, the immunoblot assays, for binding of autoantibody to autoantigen, have not shown any correlation with basophil histamine release and attempts to develop a suitable enzyme-linked immunosorbent assay (ELISA) for clinical use to differentiate between functional and nonfunctional autoantibodies too has remained unsuccessful. Therefore, it becomes imperative for all practising dermatologists to know the precise methodology, accurate interpretation and clinical applications of ASST for effective management of autoimmune urticaria patients.

  Methodology Top

Historically, in 1986, Grattan et al. [6] were the first to use ASST to differentiate autoimmune urticaria from chronic idiopathic urticaria. They injected intradermally 0.1 ml of autologous serum and normal saline as control in 12 patients of chronic idiopathic urticaria. The positive results were arbitrarily defined as formation of a wheal by serum within 2 h of injection that is at least 5 mm larger than that resulting from saline control and had a difference of 10 mm in the diameter of surrounding erythema. They observed positive responses in 7 patients within 30 min which attained their zenith in 90-120 min and remained positive for an average of 8 h. Subsequently, Sabroe et al. [7] standardized its methodology and defined the parameters which provide optimum sensitivity and specificity for detecting patients of chronic urticaria with autoantibodies [Table 1]. [4] There have been numerous modifications in the methodology and interpretation of ASST since then in various studies. O'Donnell et al. [8],[9] from their two separate studies interpreted positive ASST as the mean diameter of serum-induced wheal being at least 2 mm larger than that of saline-induced wheal at 30 min. Bakos and Hillander [10] used 0.1 ml instead of 0.05 ml each of serum, histamine and saline. Toubi et al. [11] also used 0.1 ml of sterile autologous serum and determined the wheal/flare size at 30 min and followed up for 60 min and graded the responsed from 0 to +3 of ASST by measuring wheal/flare diameter as 0 = negative control; +1 = wheal 1.5 mm > negative control, flare 15 mm; +2 = wheal 3-5 mm > negative control, flare > 15 mm; +3 = wheal 6-10 mm > negative control.

As ASST results tend to get modified with treatment, the patients should be off antihistamines for at least 2-3 days (long-acting antihistamine for 7 days) and doxepin for 2 weeks prior to the test to avoid false negative results. [7],[12],[13] Most studies also exclude patients taking corticosteroids or immunosuppressive agents during the foregoing 6 weeks to 3 months of ASST.[7],[14] In other words, the patient should have active disease at the time of ASST. However, the test is not performed over areas involved by wheals in the last 24 h.

  Preparation of Autologous Serum and Controls Top

Two milliliters of patient's venous blood is collected in a sterile glass tube and allowed to clot for 30 min at room temperature. The serum is then separated by centrifugation at 500× g for 15 min and used immediately for ASST. [12],[13]

Histamine diphosphate 10 µg/ml for positive control and sterile physiological saline (0.9%) for negative control are used. [12],[13]

  Procedure and Interpretation Top

Approximately 0.05 ml (equivalent to 2 units on insulin syringe that has 1 ml marked as 40 units) each of autologous serum, histamine diphosphate and sterile physiological saline is injected separately intradermally over volar aspect of the left forearm.[1],[4],[12],[15] Every time a separate syringe should be used for each solution. Autologous serum is injected most proximally and histamine more distally with normal saline in the middle keeping a gap of at least 5 cm between the two injection sites. After 30 min (15 min for histamine), the wheal formed at each injection site is measured at two perpendicular diameters (d 1 and d 2 ) and the average of the two is calculated. [1],[4],[12],[15] Wheal area can also be calculated according to the formula ð [(d1 +d 2 ) 2 /4].[2] If needed the wheal volume can be calculated by multiplying the wheal area by half the change in skin-fold thickness measured with a low-tension spring-loaded thickness measuring gauge. The surrounding flare is often ignored but the redness response is measured as ' redness score ' [Table 2]. [4]

Positive ASST is the one with serum-induced wheal which has both redness score = 2 and a diameter (average of d 1 and d 2 ) of ≥1.5 mm as compared to the saline-induced wheal at 30 min [Figure 1]. Using this criterion, the sensitivity and specificity of the ASST for detecting autoantibodies is 70 and 80%, respectively, and false positive results in healthy subjects and controls are minimal.

  Clinical Relevance Top

The reported prevalence of ASST positivity in patients of chronic urticaria varies from 35 to 58% in various studies. [6],[8],[9],[10],[16] Autologous serum skin test (ASST) positivity has been found to correlate well with the severity and duration of attacks of urticaria. [11],[17] However, it needs to be kept in mind that positive ASST does not specifically imply mast cell degranulation by autoimmune stimulation as a cause of the wheal response. It rather helps to define a subgroup of patients with chronic urticaria who are most likely to have endogenous cause and the results must be interpreted in the context of clinical relevance.

  Comments Top

Despite being the most accessible and useful test for demonstrating endogenous vasoactive factors in the patient's blood with chronic idiopathic urticaria, a positive ASST is not synonymous to autoimmune urticaria. The significance of a negative ASST also remains less clear. For instance, the reported prevalence of positive ASST in chronic urticaria is about 60% while IgG antibodies specific for the high-affinity IgE receptor FceRI or IgE are detected in not more than 30−40% of ASST-positive patients. [18] This signifies that different histamine-releasing factors, some chemokine-like, may be involved in vivo, and that such factors may also be clinically relevant in ASST-negative patients. Validity of ASST also remains questionable in view of the findings of a recent study by Bajaj et al ., [19] wherein they used autologous serum therapy in ASST-positive and ASST-negative chronic urticaria patients and could prevent relapse almost equally well in both the groups for as long as 2 years suggesting existence of a subset of ASST-negative autoimmune urticaria patients. They suggested that the ASST positivity perhaps reflects an autoreactive state to multiple and yet to be identified circulating factors in the patient's own blood. It is probable that the ASST-negative patients possess a different IgG subclass with decreased ability to release histamine from basophil activation. Although all these may partly be explained on the basis of results being false negative in ASST-negative autoimmune urticaria patients, future studies may unravel these aspects.

Autologous serum skin test (ASST) is also not bereft of limitations. Although it may not hold true in all cases, a positive ASST has been demonstrated to be associated with the presence of H. pylori antibodies. [14],[20] Interestingly its reactivity also correlates strongly in patients who have multiple intolerances to nonsteroidal antiinflammatory drugs. [21] It is time-consuming, needs expertise for reproducibility, stopping antihistamines prior to the test in severe cases may be difficult, testing over the site of recent wheals may alter the results and the results may sometimes need to be correlated with in vitro basophil histamine-release assays. [4],[5] There are also health and safety issues involved in relation to the preparation of autologous serum. Nevertheless, ASST offers a simple screening test for potentially relevant biological activity and may help to define a subgroup of patients with urticaria who are more likely to have an endogenous cause for their disease than do patients without a positive test. It has good sensitivity and even better sensitivity in detecting autoantibodies in children as well. [22] Therefore, it can reasonably be used as a predictive clinical test to diagnose autoimmune urticaria, especially in situations where the basophil histamine-releasing test is not available.

  References Top

1.Kaplan AP. Chronic urticaria: Pathogenesis and treatment. J Allergy Clin Immunol 2004;114:465-74.  Back to cited text no. 1  [PUBMED]  [FULLTEXT]
2.Grattan CE, Sabore RA, Greaves MA. Chronic urticaria. J Am Acad Dermatol 2002; 46:645-7.  Back to cited text no. 2    
3.Inamadar AC, Palit A. Management of autoimmune urticaria. Indian J Dermatol Venereol Leprol 2008;74:89-91.  Back to cited text no. 3  [PUBMED]  Medknow Journal
4.Grattan CEH. Autoimmune urticaria. Immunol Allergy Clin N Am 2004;24:163-81.  Back to cited text no. 4    
5.Sabroe RA, Greaves MW. Chronic idiopathic urticaria with functional autoantibodies: 12 years on. Br J Dermatol 2006;154:813-19.  Back to cited text no. 5  [PUBMED]  [FULLTEXT]
6.Grattan CEH, Wallington TB, Warin RP, Kennedy CTC, Bradfield JW. A serological mediator in chronic idiopathic urticaria: A clinical, immunological and histological evaluation. Br J Dermatol 1986;114:583-90.  Back to cited text no. 6    
7.Sabroe RA, Grattan CEH, Francis DM, Barr RM, Black AK, Greaves MW. The autologous serum skin test: A screening test for autoantibodies in chronic idiopathic urticaria. Br J Dermatol 1999;140:446-52.  Back to cited text no. 7    
8.O'Donnell BF, Nell CMO, Francis DM, Niimi N, Barr MR, Barlow RJ, et al . Human leucocyte antigen class II associations in chronic idiopathic urticaria. Br J Dermatol 1999; 140:853-58.  Back to cited text no. 8    
9.O'Donnell BF, Francis DM, Swana GT, Seed PT, Kobza BA, Greaves MW. Thyroid autoimmunity in chronic urticaria. Br J Dermatol 2005;153:331-35.  Back to cited text no. 9    
10.Bakos N, Hillander M. Comparison of chronic autoimmune urticaria with chronic idiopathic urticaria. Int J Dermatol 2003;42:613-15.  Back to cited text no. 10  [PUBMED]  [FULLTEXT]
11.Toubi E, Kessel E, Avshovich N, Bamberger, Sabo E, Nusem D, et al . Clinical and laboratory parameters in predicting chronic urticaria duration: A prospective study of 139 patients. Allergy 2004;59:869-73.   Back to cited text no. 11    
12.Kulthanan K, Jiamton S, Gorvanich T, Pinkaew S. Autologous Serum Skin Test in Chronic Idiopathic Urticaria: Prevalence, Correlation and Clinical Implications. Asian Pacific J Allergy Immunol 2006;24:201-6.  Back to cited text no. 12    
13.Godse KV. Autologous serum skin test in chronic urticaria, Indian J Dermatol Venereol Leprol 2004;70:283-4.   Back to cited text no. 13    
14.Baskan BE, Turker T, Gülten M, Tunali S. Lack of correlation between Helicobacter pylori infection and autologous serum skin test in chronic idiopathic urticaria. Int J Dermatol 2005;44:993-5.   Back to cited text no. 14    
15.Sabroe RA, Seed PT, Francis DM, Barr RM, Black AK, Greaves MW. Chronic idiopathic urticaria: Comparison of the clinical features of patients with and without anti-Fc ep-silon RI or anti-IgE autoantibodies. J Am Acad Dermatol 1999;40:443-50.   Back to cited text no. 15  [PUBMED]  [FULLTEXT]
16.Sabroe R, Fiebiger E, Francis D, Maurer D, Seed P, Grattan C, et al . Classification of anti FceRI and anti-IgE autoantibodies in chronic idiopathic urticaria and correlation with disease severity. J Allergy Clin Immunol 2002;110:492-99.   Back to cited text no. 16    
17.Fusari A, Colangelo C, Bonifazi F, Antonicelli L. The autologous serum skin test in the follow-up of patients with chronic urticaria. Allergy 2005;60:256-58.   Back to cited text no. 17  [PUBMED]  [FULLTEXT]
18.Riboldi P, Riccardo R, Tedeschi A, Gerosa M, Meroni PL. Chronic Urticaria: New Immunologic Aspects. Isr Med Assoc J 2002;4:872-3.   Back to cited text no. 18    
19.Bajaj AK, Saraswat A, Upadhyay A, Damisetty R, Dhar S. Autologous serum therapy in chronic urticaria: Old wine in a new bottle. Indian J Dermatol Venereol Leprol 2008;74: 109-13.   Back to cited text no. 19  [PUBMED]  Medknow Journal
20.Hizal M, Tuzun B, Wolf R, Tuzun Y. The relationship between Helicobacter pylori IgG antibody and autologous serum skin test. Int J Dermatol 2000;39:443-5.  Back to cited text no. 20    
21.Erbagci Z. Multiple NSAID intolerance in chronic idiopathic urticaria is correlated with delayed, pronounced and prolonged autoreactivity. J Dermatol 2004;31:376-82.  Back to cited text no. 21  [PUBMED]  [FULLTEXT]
22.Godse KV. Autologous serum skin test in children. Indian J Dermatol 2008;53:61-3.  Back to cited text no. 22    Medknow Journal


  [Figure 1]

  [Table 1], [Table 2]

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