- Top of page
- Materials and methods
Background: To study the mechanisms of passive sensitization of patients receiving plasma containing IgE antibodies to a defined allergen.
Methods: When required for medical reasons, regular donor plasma with IgE antibodies to timothy grass allergen (8–205 kUA/l), was given. Kinetics of IgE antibodies in the recipients’ serum and his/her basophil allergen threshold sensitivity, CD-sens, was monitored up to 2–3 weeks after transfusion. The IgE antibodies were quantitated by ImmunoCAP. The CD-sens in plasma recipients, determined by CD63 up-regulation, was measured by flow cytometry and compared to CD-sens of patients with allergic asthma and/or rhinitis.
Results: There was a significant correlation (r = 0.98; P < 0.001) between amount of IgE antibody given and recipient serum peak concentration. The T1/2 for IgE antibody in circulation was 1.13 days (95% confidence limit 0.35–1.91 days). The recipients became CD-sens positive already 3 h after transfusion. The CD-sens peak was observed after 3.4 days and the value were correlated (r = 0.68; P < 0.02) to the amount of IgE antibody transfused and were of the same magnitude as found in allergic patients. The T1/2 of CD-sens indicated two populations of basophils; one with a CD-sens decrease T1/2 of 4 days and one of 10 days.
Conclusion: Transfused IgE antibodies will sensitize mast cells and basophils to CD-sens levels similar to those of allergic patients. The recipients expressed ‘slow’ or ‘rapid’ CD-sens decline, indicating two different basophil populations. After transfusion of plasma with >10 kUA/l IgE antibody the recipient could have allergen reactive basophils for up to 7 weeks.
In 1919 Ramirez reported (1) the first case of allergy passively transferred by a blood transfusion. The patient was suffering from aplastic anemia and received blood regularly, and usually without any reactions. However, this particular time, when riding a horse carriage in Central Park a few days after the transfusion, the patient developed asthma. Dr Ramirez noticed the significance of this special event and when tracing the blood donor found that he was suffering from asthma due to horse allergy. Since this first description of transfer of an allergy, a few reports have followed. In 1941 Loveless reported skin and mucosal sensitivity to ragweed after transfer of blood from ragweed allergic patients shown to have circulating ‘reagins’ to ragweed (2). The nonallergic recipients became skin test positive for quite long times, in two of the five cases for 7 and 20 weeks, respectively. A couple of other cases have also been reported (3, 4).
The blood transfusion specialty has, through questionnaires and by simple case histories taken by blood center personnel, tried to identify and eliminate allergic, potentially dangerous blood donors. However, this procedure has not been very successful; we found that 23% of the donors were IgE-sensitized and had significant levels of IgE antibodies to common allergens (5). Thus, the risk of passive sensitization by blood and plasma transfusion is a reality. However, it also opens an opportunity to study serological and inflammatory aspects of human IgE-sensitization in vivo.
In the 1960s, before the discovery of the central role of IgE (6) in allergic inflammation, extensive studies were performed on basophil granulocytes and their role in allergic reactions (7). Cells, sensitized in vivo or in vitro with ‘reagin’, were challenged with allergen and the basophil response was measured as histamine release. In the early 90s, it was reported that when the basophils empty their histamine containing granulae a structure on the granulae vesicle, CD63, becomes exposed on the cell surface (8). Appearance of CD63 on the surface of basophils, measured with flow cytometry, is now an established procedure to detect allergen or anti-IgE triggering of basophils, and the accessibility of the basophil specific marker CD203c (9) has made it possible to more easily identify basophils.
The objective of this study was to transfuse defined amounts of IgE antibodies to an allergen, and to examine the degree and duration of IgE-reactivity in the recipient, measured by skin prick tests (SPT) and basophil allergen threshold activation (CD-sens). The IgE sensitization characteristics of the basophils were also studied.
- Top of page
- Materials and methods
Since the prevalence of IgE-sensitization among blood donors, at least in Sweden, is very high (5), a follow-up of routine plasma transfusions offers a unique way to study passive sensitization. The plasma donors at the Karolinska University Hospital, having high concentrations of IgE antibodies to timothy grass pollen allergens were identified. When their plasma was used as a complement to erythrocytes and electrolyte solutions in cases of severe blood loss, the transfused IgE antibodies to timothy grass as well as their basophil sensitizing capacity was monitored for days to weeks after the transfusion.
The IgE from transfused plasma could be detected in the circulation of the recipient already in the first sample, i.e. 3 h after transfusion. As expected, there was a relation between the amount of IgE antibodies transferred and the concentration observed in the serum of the patient. Studies of the metabolism of IgE, based on injections of radio-labeled monoclonal IgE, has indicated a half life for IgE in circulation in the order of 0.7–4.4 days (11, 12), with 2.3 days being a representative and commonly used figure. Our finding in this study of a mean half-life for IgE of 1.13 days is a bit low compared to previous reports; however, it was obtained using native, antibody-active, IgE.
Different approaches have been used to document the allergen sensitivity of a patient. Provocation challenge tests using the offending substance in food, drug and occupational allergy and allergen extract inhalation tests in nasal and bronchial allergy have been applied (13, 14), but could for ethical and medical reasons not be used in this study. The most commonly used test is the SPT, which is easy to perform, but has a low precision (15). In this study the CV for wheal area duplicates was 52%.
In a few patients in this study a SPT to timothy was performed and found to change from negative before to positive after transfusion. Obviously, the risk of a clinically significant IgE-sensitization of mast cells, through transfusion of plasma containing IgE antibodies, is a reality.
The approach of in vitro challenge of tissue sensitized in vivo is an attractive alternative to in vivo provocation tests. Basophils as target organ were extensively explored before IgE was discovered (7) and allergen sensitivity, calculated by using allergen threshold measurements, was found to give the best information of the patient's allergen sensitivity, although a relation to cell reactivity has occasionally been found (16). The CD63 up-regulation after stimulation at one, high dose of allergen, i.e. basophil cell reactivity, has during recent years been used as a measure of both allergic (17) and nonallergic (18) cell responses. In our study CD-sens, i.e. the allergen concentration giving 50% of maximal up-regulation of CD63 was used as a measure of general allergen sensitivity after transfusion and, in contrast to CD63 up-regulation, it was found to correlate significantly with SPT.
When the recipient's CD-sens was monitored during the first 2 weeks after transfusion, it was found that even transfusion of plasma with a moderate concentration of IgE antibody, e.g. 8 kUA/l, resulted in allergen reactive basophils. The peak CD-sens of the transfused patients had a geometric mean of 7.6, which is comparable to that of patients with asthma and/or rhinoconjunctivitis due to timothy induced, IgE-mediated allergy (geometric mean 20.4; 95% confidence interval 0.9–438). Thus, passive sensitization of basophils results in recipients as IgE-sensitized as allergic patients.
The allergen sensitivity of the recipient's basophils increased rapidly after the transfusion and the CD-sens peaked at 3.4 days. This time-point is interesting since it might mirror the time-span for basophils to fully mature in the bone marrow before they are released into the circulation. During this period the genes for FcɛRI are turned on, leading to accumulation of receptors on the cell surface in a linear fashion (19), and the density of surface FcɛRI receptors is suggested to be a function of the current IgE concentration. Thus, we propose that the increase in IgE concentration caused by the passive transfer of IgE, leads to a higher FcɛRI expression on the maturing basophils in the bone marrow. Moreover, the composition of surface IgE, as well as IgE antibody specificity, on the newly released basophils mirrors the blend of the IgE from the recipient and the plasma donor, thus giving these basophils the capacity to react to allergen in a similar fashion as basophils from the allergic donor. Since it has previously been reported that basophils circulate in the blood for approximately 1 day (19), one can assume that the main part of the circulating basophils at day 3.4 have matured in a bone marrow environment consisting of a blend of IgE from recipient and donor.
Interestingly, two basophil populations with different CD-sens decline T1/2 were identified; one with a rapid and one with a slower decline. Earlier studies have shown that one or more mutations in the β-chain of FcɛRI (FcɛRI-β), leading to polymorphism in the receptor, could contribute to more elevated skin test responses to grass and house dust mite, higher concentrations of IgE antibodies to grass and bronchial reactivity to methacholine (20) in the tested individuals. Although not tested in the current study, this observation generates the hypothesis that a current FcɛRI-β polymorphism in individuals contributes to elevated reactions in vitro (basophil response) and in vivo (mast cell response), and to a sustained reactivity.
It has been suggested that the allergen reactivity of basophils is not representative of the tissue localized mast cells. If such a difference is of significant importance in a particular allergic disease, or in certain patients, the use of basophils as a surrogate marker for target organ allergen sensitivity must be questioned. However, much of our information of allergic inflammation in humans comes from studies of basophils, as these are more readily accessible. Limited studies of tissue mast cells have been done, and thus far some of the key features of the basophil translate well to the mast cell. In particular, mast cell and basophil allergen sensitivity appears to be very similar (21).
Obviously, transfusion of even moderate amounts of IgE antibodies represents a potential risk for an allergic reaction. In this study two standard units of plasma, with an IgE antibody concentration in the order of 10–20 kUA/l, could, as calculated from the T1/2 of 4 and 10 days, respectively, result in a positive CD-sens up to 2–3 weeks after transfusion in a patient with rapid and 5–7 weeks in one with a slow decline. Regular blood donors with IgE antibodies to common allergens at concentrations >10 kUA/l are not rare (5). In a recent study, as many as 7.5% of donors in Sweden and 5.6% in Norway had such high IgE antibody levels [unpublished observation], and a few of them to drugs (penicilloyl G), latex and foods (e.g. peanuts).
The mast cell allergen sensitivity seems to disappear even more slowly and studies based on Prausnitz–Küstner titration, indicate a half-life in the order of 2 weeks (22). This difference between mast cells (23) and basophils (24) has been observed also in recent studies on the function of omalizumab. Thus, a recipient will be at risk also when exposed to common inhalant and food allergens outside the hospital environment, many weeks after the transfusion.
In both allergic patients and transfusion recipients there was a relation between concentration of IgE antibodies and CD-sens. Such a relation between IgE antibodies, SPT and food challenge has been observed in other studies, e.g. recently in allergy to peanuts (13). Our findings of a significant correlation between the relative concentration of IgE antibodies and CD-sens are in agreement with studies using a rat mast cell line transfected with the α-chain of the human high-affinity IgE receptor and measuring of cell activation from release of a β-hexosaminidase (25). They reported a good correlation with the relative, but not with the absolute, concentration of IgE antibodies indicating that nonantibody active IgE molecules, when bound to Fcɛ-receptors on the mast cell or basophil surface, can interfere with the ability of the cell to respond to allergen stimulation.
The geometric mean of the total number of basophil surface IgE molecules per microlitre of blood was before transfusion 80 000 and at the peak after transfusion 1 100 000. This peak number of IgE molecules was comparable with that of allergic patients, 1 200 000, showing that passively transferred IgE molecules binds to the basophils to the same extent as in allergic patients. Interestingly, the two recipients (Nos. 3 and 4) with nonreactive basophils pretransfusion did reach peak values for IgE molecules, 1 900 000 and 2 000 000, respectively, and CD-sens, 49 and 7, respectively, as the other although both belonged to the ‘rapid’ decrease group.
In conclusion, transfusing plasma containing IgE antibodies results in a transient sensitization of basophils and mast cells; this reaches the same level as that of allergic patients. There seems to be two populations of basophils; those with a short allergen sensitivity half-life of 4 days and those with a longer half-life, 10 days. Their importance for the expression of allergic disease should be further studied. An IgE-sensitization has the potential to initiate severe transfusion complications during the first days postoperatively but also interfere with the recovery for weeks.