Chronic Urticaria - Pooled Donor Basophil Activation Testing (PD-BAT)

Chronic spontaneous urticaria (CSU) is a debilitating mast cell-driven disease characterized by recurrent wheals and/or and/or angioedema.[2,3] Chronic spontaneous urticaria (CSU) is a disorder where symptoms of urticaria occur without an identifiable provoking factor and persist for more than six weeks.[2] CSU is thought to result from the pathogenic activation of mast cells and basophils causing them to release histamine and other proinflammatory mediators.[2,4] CSU frequently occurs in patients with autoimmune conditions including autoimmune hypothyroidism, rheumatoid arthritis, type I diabetes mellitus, Sjogren syndrome, celiac disease and systemic lupus erythematosus.[5-8] The exact mechanisms leading to activation of mast cells in CSU are, as of yet, not fully characterized, but autoimmunity is thought to play a key role.[2,9-15] There are two identified autoimmune endotypes of CSU with different types of autoantibodies that have been associated with the activation of skin mast cells.[9,16-19] In both type I and type IIb endotypes, mast cells are the primary drivers and basophils are important secondary contributors. Type I and type IIb CSU patients share the same phenotype but may differ in disease course, clinical features, and response to treatment.[3] In contrast to classical type I hypersensitivity and allergy, which involve the development of IgE to exogenous allergens, type I CSU is characterized by IgE antibodies directed to self-antigens (also called autoallergens).[7,11,20] Autoallergic chronic spontaneous urticaria is present in more than 50% of patients with chronic spontaneous urticaria.[21] The beneficial effects of the anti-IgE drug omalizumab in many CSU patients further points to a role of type I autoimmunity in CSU.[9,22,23] Type IIb autoimmunity, related to the presence of IgG autoantibodies against the high affinity Fc epsilon receptor I (FceRI) or, less frequently, to IgE itself, also has been implicated as a causative mechanism for CSU.[2,8,9,11-13] The type IIb endotype is present in 8% to 40% of patients with CSU and is characterized by higher disease severity, concomitant autoimmune diseases, low levels of total IgE, elevated levels of IgG-anti-thyroid peroxidase, basopenia, eosinopenia, poor response to antihistamines and to omalizumab, and a good response to cyclosporine.[3] A recent study found that the majority of patients with a type IIb profile also had a type I profile, suggesting there is greater overlap and heterogeneity in these "endotypes" than previously recognized.[21] Another study suggest that patients who have IgG and IgE autoantibodies to the same autoantigen might more often be late or insufficient responders to omalizumab, as compared with patients with only one class of autoantibodies.[24] In CSU, dermal mast cells and basophils express IgE receptors on their surface. Cross-linking of these receptors by an IgE/antigen complex or by an autoantibody can result in cell activation and degranulation. [3,4,22] As is the case with many autoimmune conditions, the presence of antibodies to autoallergens or to FcepsilonRIalpha and/or IgE does not necessarily result in a disease phenotype. Antibodies to these proteins can be found in a number of other autoimmune disorders including dermatomyositis, systemic lupus erythematosus, pemphigus vulgaris, bullous pemphigoid and in health controls.[26] Consequently, only functional antibodies (i.e., those that cause mast/basophil degranulation) are considered diagnostic of autoimmune CSU.[27,28] The autoimmune nature of CSU is further supported by the autologous serum skin test (ASST), an in vivo assay of mast cell activation induced by intradermal injection of a patient's serum into self.[2,29-34] Nearly 50% of patients with CSU will develop a wheal-and-flare response at the point of injection when inoculated with their own serum. A positive ASST reaction in CSU patients has been found to be associated with the presence of autoantibodies against FcepsilonRIalpha in 30% to 40% of patients and/or against IgE itself in an additional 5% to 10% of patients.[8,11,30- 32,34-39] Recent guidelines recommended that the ASST test be performed in the diagnostic workup of CSU to determine the subtype of autoimmune urticaria.[2] Unfortunately, positive ASST results are not unique to patients with CSU and have been noted in a substantial proportion of patients with allergic or non-allergic rhinitis, multiple drug allergy syndrome and even in healthy control subjects.[40, 41] Moreover, the positivity of the test has been shown to persist even when CSU patients are in clinical remission, particularly in subjects with autoimmune thyroiditis.[42] The utility of the ASST is further limited by the fact that it cannot be used in patients treated with antihistamines, a status that is quite common for patients with CSU in clinical practice.[43] Basophil activation testing has been shown to better identify patients with autoimmune CSU, than ASST or individual antibody serology.[13,44] An indirect assessment of functional antibodies in patients with CSU can been accomplished by in vitro measurement of the activation of donor basophils on exposure to patient serum.[13] Historically, the measurement of the of inflammatory mediators (usually histamine) released by donor basophils has been employed as an indicator or basophil activating potential of the patient's serum in the CSU-Basophil Histamine Release Test (CSU-BHRT).[45] A quantitative assessment of donor basophil activation by patient serum also can be accomplished by flow cytometric analysis of CD63 expression on the donor basophils in the CU-Basophil Activation Test (CSU-BAT).[12,13,33,34,43,44,46-56] Incubation of donor basophils with serum from patients with CSU leads to increased CD63 expression in approximately 50% of cases. Basophil activation tests are considered the best single tests for diagnosing type IIb CSU and showed better correlation with other markers of type IIb CSU and response to treatment than the ASST or immunoassays for autoantibodies.[3,13,44,57] A recent systematic review of 42 studies by Monino-Romero and colleagues identified the basophil activation testing to show a strong association with higher disease activity in patients with CSU.[58] There is a significantly higher prevalence of CSU-BAT positive results in patients with positive ASST results than in those that are ASST negative.[34,44,54,59] Patients with positive CSU-BAT results tend to have a higher prevalence of other autoantibodies, [34,44,46,47,59] more severe urticaria,[34, 59] and are more likely to have angioedema [34,44] than CSU-BAT negative patients. CSU-BAT positive children have been shown to be more likely to exhibit an earlier spontaneous resolution of urticaria than CSU-BAT negative children.[48] A positive basophil test has been linked to long disease duration, higher disease activity, a poor response to antihistamines and omalizumab, and a better response to cyclosporine and fenebrutinib.[44,23,59-61] Omalizumab, a humanized monoclonal antibody against the Fc portion of the IgE antibody, prevents free IgE from binding to high-affinity FceR1 receptors on mast cells and basophils. This drug has been approved by the U.S. Food and Drug Administration for the treatment of CSU that is unresponsive to H1 antagonists.[2] Total serum IgE levels are significantly lower in CSU-BAT positive patients than CSU-BAT negative patients as well as in ASST-positive vs ASST-negative CSU patients. [34,59,62] Low IgE levels have been linked to lower rates of response to anti-IgE treatment in CSU patients, [22,63-65] suggesting that type IIb CSU patients respond differently to omalizumab. Studies have segregated CSU patients treated with omalizumab into groups referred to as "fast responders" (subjects who show a complete response as short as three to seven days after the first administration of the drug; these represent about 60% to 70% of patients), "slow responders" (subjects who need three to five monthly administrations before showing a significant clinical response; about 10% to 20% of cases), and "non-responders" (about 10% to 20% of cases).[23,66] Positive results in basophil activation tests and/or ASST have been linked to slower responses to omalizumab.[23,59- 61,67] The results of the basophil activation tests, and autologous serum skin test are significantly correlated with the time to symptom relief with omalizumab.[23] CSU-BAT is more specific for the detection of histamine-releasing autoantibodies in CSU patients than the ASST and may thus be a useful test in assessing patient responsiveness to omalizumab.[23] As such, patients with refractory CSU may benefit from longer, more "personalized" courses of omalizumab.[68] Results of CSU-BAT tests may help clinicians to set patients' expectations for therapy. One of the challenges in performing indirect BAT is the intra-individual variability in the response of donor basophils in the assays. While some donor basophils are highly reactive to pathologic serum samples, others are not. Conversely, some donor basophils are non-specifically activated by healthy control serum. In common laboratory practice, this variability in donor responsiveness is accounted for by testing patient samples with multiple donors in separate experiments.[50] Arbitrary rules are established to assess the results when activation for individual donors varies.[44-45] We have developed a procedure for prescreening individual donors and creating a pool of 20 selected blood donors to optimize the pooled donor basophil activation test (PD-BAT) assay performance and ensure similar results for samples tested from run to run.[1]

Chronic spontaneous urticaria (CSU) is a debilitating mast cell-driven disease characterized by recurrent wheals and/or and/or angioedema.^2,3 Chronic spontaneous urticaria (CSU) is a disorder where symptoms of urticaria occur without an identifiable provoking factor and persist for more than six weeks.² CSU is thought to result from the pathogenic activation of mast cells and basophils causing them to release histamine and other proinflammatory mediators.^2,4 CSU frequently occurs in patients with autoimmune conditions including autoimmune hypothyroidism, rheumatoid arthritis, type I diabetes mellitus, Sjogren syndrome, celiac disease and systemic lupus erythematosus.^5-8 The exact mechanisms leading to activation of mast cells in CSU are, as of yet, not fully characterized, but autoimmunity is thought to play a key role.^2,9-15 

There are two identified autoimmune endotypes of CSU with different types of autoantibodies that have been associated with the activation of skin mast cells.^9,16-19 In both type I and type IIb endotypes, mast cells are the primary drivers and basophils are important secondary contributors. Type I and type IIb CSU patients share the same phenotype but may differ in disease course, clinical features and response to treatment.³ In contrast to classical type I hypersensitivity and allergy, which involve the development of IgE to exogenous allergens, type I CSU is characterized by IgE antibodies directed to self-antigens (also called autoallergens).^7,11,20 Autoallergic chronic spontaneous urticaria is present in more than 50% of patients with chronic spontaneous urticaria.²¹ The beneficial effects of the anti-IgE drug omalizumab in many CSU patients further points to a role of type I autoimmunity in CSU.^9,22,23 

Type IIb autoimmunity, related to the presence of IgG autoantibodies against the high affinity Fc epsilon receptor I (FceRI) or, less frequently, to IgE itself, also has been implicated as a causative mechanism for CSU.^2,8,9,11-13 The type IIb endotype is present in 8% to 40% of patients with CSU and is characterized by higher disease severity, concomitant autoimmune diseases, low levels of total IgE, elevated levels of IgG-anti-thyroid peroxidase, basopenia, eosinopenia, poor response to antihistamines and to omalizumab, and a good response to cyclosporine.³ 

A recent study found that the majority of patients with a type IIb profile also had a type I profile, suggesting there is greater overlap and heterogeneity in these "endotypes" than previously recognized.²¹ Another study suggest that patients who have IgG and IgE autoantibodies to the same autoantigen might more often be late or insufficient responders to omalizumab, as compared with patients with only one class of autoantibodies.²⁴ In CSU, dermal mast cells and basophils express IgE receptors on their surface. Cross-linking of these receptors by an IgE/antigen complex or by an autoantibody can result in cell activation and degranulation.^3,4,22

As is the case with many autoimmune conditions, the presence of antibodies to autoallergens or to FcepsilonRIalpha and/or IgE does not necessarily result in a disease phenotype. Antibodies to these proteins can be found in a number of other autoimmune disorders including dermatomyositis, systemic lupus erythematosus, pemphigus vulgaris, bullous pemphigoid and in health controls.²⁶ Consequently, only functional antibodies (i.e., those that cause mast/basophil degranulation) are considered diagnostic of autoimmune CSU.^27,28 The autoimmune nature of CSU is further supported by the autologous serum skin test (ASST), an in vivo assay of mast cell activation induced by intradermal injection of a patient's serum into self.^2,29-34 Nearly 50% of patients with CSU will develop a wheal-and-flare response at the point of injection when inoculated with their own serum. A positive ASST reaction in CSU patients has been found to be associated with the presence of autoantibodies against FcepsilonRIalpha in 30% to 40% of patients and/or against IgE itself in an additional 5% to 10% of patients.^{8,11,30-32,34-39} 

Recent guidelines recommended that the ASST test be performed in the diagnostic workup of CSU to determine the subtype of autoimmune urticaria.² Unfortunately, positive ASST results are not unique to patients with CSU and have been noted in a substantial proportion of patients with allergic or nonallergic rhinitis, multiple drug allergy syndrome and even in healthy control subjects.^40,41 Moreover, the positivity of the test has been shown to persist even when CSU patients are in clinical remission, particularly in subjects with autoimmune thyroiditis.⁴² The utility of the ASST is further limited by the fact that it cannot be used in patients treated with antihistamines, a status that is quite common for patients with CSU in clinical practice.⁴³

Basophil activation testing has been shown to better identify patients with autoimmune CSU than ASST or individual antibody serology.^13,44 An indirect assessment of functional antibodies in patients with CSU can been accomplished by in vitro measurement of the activation of donor basophils on exposure to patient serum.¹³ Historically, the measurement of the of inflammatory mediators (usually histamine) released by donor basophils has been employed as an indicator or basophil activating potential of the patient's serum in the CSU-Basophil Histamine Release Test (CSU-BHRT).⁴⁵ A quantitative assessment of donor basophil activation by patient serum also can be accomplished by flow cytometric analysis of CD63 expression on the donor basophils in the CU-Basophil Activation Test (CSU-BAT).^{12,13,33,34,43,44,46-56} Incubation of donor basophils with serum from patients with CSU leads to increased CD63 expression in approximately 50% of cases. 

Basophil activation tests are considered the best single tests for diagnosing type IIb CSU and showed better correlation with other markers of type IIb CSU and response to treatment than the ASST or immunoassays for autoantibodies.^3,13,44,57 A recent systematic review of 42 studies by Monino-Romero and colleagues identified the basophil activation testing to show a strong association with higher disease activity in patients with CSU.⁵⁸ There is a significantly higher prevalence of CSU-BAT positive results in patients with positive ASST results than in those that are ASST negative.^34,44,54,59 Patients with positive CSU-BAT results tend to have a higher prevalence of other autoantibodies,^{34,44,46,47,59} more severe urticaria,^34,59 and are more likely to have angioedema^34,44 than CSU-BAT negative patients. CSU-BAT positive children have been shown to be more likely to exhibit an earlier spontaneous resolution of urticaria than CSU-BAT negative children.⁴⁸ A positive basophil test has been linked to long disease duration, higher disease activity, a poor response to antihistamines and omalizumab, and a better response to cyclosporine and fenebrutinib.^44,23,59-61 

Omalizumab, a humanized monoclonal antibody against the Fc portion of the IgE antibody, prevents free IgE from binding to high-affinity FceR1 receptors on mast cells and basophils. This drug has been approved by the U.S. Food and Drug Administration for the treatment of CSU that is unresponsive to H1 antagonists.² 

Total serum IgE levels are significantly lower in CSU-BAT positive patients than CSU-BAT negative patients as well as in ASST-positive vs ASST-negative CSU patients.^34,59,62 Low IgE levels have been linked to lower rates of response to anti-IgE treatment in CSU patients,^22,63-65 suggesting that type IIb CSU patients respond differently to omalizumab. Studies have segregated CSU patients treated with omalizumab into groups referred to as "fast responders" (subjects who show a complete response as short as three to seven days after the first administration of the drug; these represent about 60% to 70% of patients), "slow responders" (subjects who need three to five monthly administrations before showing a significant clinical response; about 10% to 20% of cases) and "non-responders" (about 10% to 20% of cases).^23,66 Positive results in basophil activation tests and/or ASST have been linked to slower responses to omalizumab.^23,59-61,67 The results of the basophil activation tests and autologous serum skin test are significantly correlated with the time to symptom relief with omalizumab.²³ CSU-BAT is more specific for the detection of histamine-releasing autoantibodies in CSU patients than the ASST and may thus be a useful test in assessing patient responsiveness to omalizumab.²³ As such, patients with refractory CSU may benefit from longer, more "personalized" courses of omalizumab.⁶⁸ 

Results of CSU-BAT tests may help clinicians to set patients' expectations for therapy. One of the challenges in performing indirect BAT is the intra-individual variability in the response of donor basophils in the assays. While some donor basophils are highly reactive to pathologic serum samples, others are not. Conversely, some donor basophils are non-specifically activated by healthy control serum. In common laboratory practice, this variability in donor responsiveness is accounted for by testing patient samples with multiple donors in separate experiments.⁵⁰ Arbitrary rules are established to assess the results when activation for individual donors varies.^44-45 We have developed a procedure for prescreening individual donors and creating a pool of 20 selected blood donors to optimize the pooled donor basophil activation test (PD-BAT) assay performance and ensure similar results for samples tested from run to run.¹