Demonstration of an aberrant mast-cell population with clonal markers in a subset of patients with “idiopathic” anaphylaxis

The cause of anaphylaxis remains unexplained in up to two-thirds of patients. The result is that the diagnosis of idiopathic anaphylaxis is assigned to the patient.¹  Because idiopathic hypotensive episodes are a feature of mastocytosis,^2,,–5  a clonal proliferative disorder of the mast-cell lineage, we asked the question of whether idiopathic anaphylaxis might in some cases be associated with a similar clonal disorder of mast cells

We thus determined clonal markers of mast-cell disease such as immunophenotypic or morphologic abnormalities and the 816D>V (D816V) c-kit mutation associated with systemic mastocytosis in this study, which enrolled patients with idiopathic anaphylaxis as well as those with a confirmed or probable diagnosis of mastocytosis. As will be shown, a group of patients with recurrent anaphylaxis without urticaria pigmentosa (UP) and multifocal bone marrow mast-cell aggregates exhibit an aberrant mast-cell population carrying the c-kit D816V mutation.

A total of 72 consecutive patients referred to the National Institutes of Health (NIH) Clinical Center in a 17-month period were enrolled in this prospective study after signing an informed consent in accordance with the Declaration of Helsinki. The study was approved by the National Institute of Allergy and Infectious Diseases (NIAID) intramural Institutional Review Board (IRB). The study population consisted of patients referred with the diagnosis of idiopathic anaphylaxis or confirmed or probable mastocytosis. Anaphylaxis was defined by self-limited syncopal or near-syncopal episodes accompanied by the presence of one or more symptoms suggestive of systemic mast cell degranulation, such as flushing, tachycardia, abdominal pain, and diarrhea. All patients underwent a medical evaluation including a bone marrow biopsy and aspirate to investigate presence of the World Health Organization (WHO) diagnostic criteria for mastocytosis.⁶ 

Mast cells in bone marrow biopsy sections were identified by immunohistochemical staining for tryptase.⁷  Mast cells in bone marrow aspirates were identified by flow cytometry as a CD117^high, IgE⁺ population as described.^8,9  Mutational analysis of the C-KIT gene was performed by reverse transcription–polymerase chain reaction (RT-PCR) and restriction fragment–length polymorphism analysis of the PCR products in bone marrow aspirate mononuclear cells as described.¹⁰  In some patients, bone marrow mast cells were enriched based on their surface CD25 expression using anti-human CD25 paramagnetic beads according to the manufacturer's instructions (Miltenyi Biotec, Auburn, CA).¹¹ 

Associations between variables were evaluated by Spearman correlation coefficient, and group differences by Wilcoxon rank-sum test.

We sought to examine the prevalence of clonal mast-cell disease in patients with the diagnosis of recurrent idiopathic anaphylaxis, and to determine laboratory or clinical parameters that would help to identify this patient group. We enrolled 72 consecutive patients (44 women and 28 men with a median age of 46 years [range, 22-69 years]) referred to the NIH Clinical Center with a diagnosis of confirmed or suspected systemic mastocytosis or idiopathic anaphylaxis (Table 1). Of those 72 patients, 36 (18 women, 18 men) had confirmed cutaneous or systemic mastocytosis and did not suffer from recurrent hypotensive episodes. Mutational analysis of the C-KIT gene was carried out in 22 patients in this group and was found to be positive for the 816D>V mutation in 18 (82%).

A total of 24 patients (18 women, 6 men) had a history of recurrent anaphylactic episodes. Among patients with anaphylaxis, 8 patients had the diffuse maculopapular hyperpigmented rash of UP, the characteristic skin finding of mastocytosis. Among 16 patients with recurrent anaphylactic episodes who did not have UP, 4 had characteristic multifocal mast-cell infiltrates in their bone marrow biopsy diagnostic of systemic mastocytosis. Thus, a total of 12 (50%) of 24 patients with recurrent syncopal episodes had evidence of cutaneous mastocytosis and/or multifocal bone marrow mast-cell aggregates, the histopathologic hallmark and the major diagnostic criterion of systemic mastocytosis. The 816D>V C-KIT mutation was detectable in bone marrow of 7 (88%) of 8 patients analyzed in this group, similar to the patients with mastocytosis without anaphylaxis. The frequency of detection of c-kit mutation in this study is consistent with a recent report that showed the mutation in 93% of patients,¹⁴  and suggests that 816D>V mutation alone is not a predictor of anaphylactic episodes in patients with mastocytosis.

The remaining 12 of the 24 patients with recurrent anaphylaxis lacked both UP skin lesions and multifocal mast-cell aggregates in the bone marrow biopsy. On further analysis, 5 (42%) of these 12 patients (3 women, 2 men; 95% confidence interval, 0.15-0.72) had evidence of 1 or more minor criteria for mastocytosis (Table 2). All 5 patients had aberrant expression of CD25 on the surface of mast cells. In addition, inspection of the tryptase-stained bone marrow biopsy specimen or a Wright-Giemsa–stained aspirate smear showed evidence of morphologically aberrant mast cells, such as those with elongated shapes and hypogranulation, in all 5 patients. A total of 2 patients had serum tryptase levels greater than 20 ng/mL (Table 2). The 816D>V C-KIT mutation was detectable in bone marrow mononuclear cells enriched for CD25⁺ cells containing the aberrant mast-cell population in 3 of 3 patients examined. Of note, examination of unsorted bone marrow mononuclear cells in these 3 patients yielded a positive result in only 1 patient, suggesting that CD25 enrichment increased the sensitivity of the detection of the C-KIT mutation. CD25 subpopulation analysis for the C-KIT codon 816 mutation could not be carried out in the remaining 2 patients in whom only 2 diagnostic criteria were demonstrated due to sample limitations. These results show that an aberrant mast-cell population carrying clonal mast-cell markers is detectable in a subgroup of patients with idiopathic anaphylaxis. Some anaphylactic attacks followed yellowjacket hymenoptera stings in 1 patient (patient 5; Table 2). The findings in this patient were consistent with a recent report of subdiagnostic accumulation of mast cells in patients with recurrent anaphylaxis after hymenoptera stings.¹⁵  No stimuli that consistently brought on hypotensive episodes in other patients were identified.

A total of 7 patients with anaphylaxis (6 women, 1 man) had neither UP nor an aberrant bone marrow mast-cell population, and were diagnosed as having idiopathic anaphylaxis. An additional group of 12 patients of the 72 patients entered into this study had no syncopal or presyncopal events required to include them into the group with a history of recurrent anaphylactic episodes; however, they did have symptoms such as recurrent flushing, tachycardia, urticaria, or angioedema, which led their referral physicians to consider the diagnosis of mastocytosis. These patients had no diagnostic evidence of mastocytosis or of a clonal mast-cell disorder.

Serum IgE levels inversely correlated with serum tryptase¹⁶  in the full cohort (r = −0.46; P < .001), and patients with mastocytosis had significantly lower total serum IgE than those without mastocytosis (medians, 10 vs 84 IU/mL; P < .001). Patients with anaphylaxis as a group tended to have higher total IgE levels regardless of the etiology when compared with those without anaphylaxis, although this did not reach statistical significance (medians, 22.5 vs 12 IU/mL; P = .08). This may be pertinent to the pathogenesis of anaphylaxis, as IgE, even in its monomeric form, can potentiate mast-cell activation,^17,–19  and presence of a mutated c-kit molecule may further augment this process through convergent downstream signal-transduction pathways.^20,21 

In conclusion, an aberrant bone marrow mast-cell population carrying the clonal markers found in mastocytosis was detected in a high percentage of patients with recurrent anaphylaxis in this study. These findings provide a basis for the etiology of anaphylactic episodes in a significant subset of patients with “idiopathic” anaphylaxis.

This work was supported by the intramural National Institute for Allergy and Infectious Diseases/NIH funds.

National Institutes of Health

Contribution: All authors contributed significantly to the manuscript; C.A. designed the study and performed the research, collected data, and wrote the paper; L.M.S. collected data and performed research; C.N.K. performed research; N.K.S. collected and analyzed data; E.B. analyzed data; P.N. performed research; and D.D.M. performed research, provided funding for the study, critically reviewed the data, and wrote the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Cem Akin, University of Michigan, 5520-B MSRB-1, Box 0600, 1150 W Medical Center Dr, Ann Arbor, MI 48109-0600; e-mail:cemakin@umich.edu.