Introduction

Loxosceles reclusa (L. reclusa), commonly referred to as the Brown Recluse, is a venomous spider endemic to the southeastern and Midwestern United States [1-5]. The active component of L. reclusa venom is a broadly reactive, 305 amino acid enzyme [6,7]. Envenomation can result in disfiguring dermatonecrosis and/or a fatal hemolytic anemia [8-11]. The severity of injury has led to the consideration of a number of possible treatments such as dapsone, glucocorticoids and hyperbaric oxygen; but, none of these has been effective and severely ill patients generally receive only supportive therapy [2,8,9]. Although an ELISA for L. reclusa venom exposure has been described, it is not available for general use [12,13]. There are relatively few case reports and recommendations for the management of Brown Recluse spider bite (BRSB) mediated hemolysis [9,14,15].

Our center, located in the endemic region of L. reclusa, encounters several cases of severe envenomation per year. The victims we treat are frequently children who follow an unpredictable clinical course requiring inpatient management. In our experience, once BRSB mediated hemolysis becomes clinically apparent it can accelerate rapidly and can be difficult to manage in the absence of a diagnostic test and definitive treatment. Here, we report the salient clinical and laboratory features of 17 cases of severe BRSB mediated hemolysis – the majority of which were in children – as determined by a 10-year retrospective chart review.

Previous investigators have shown that BRSB mediated hemolysis is complement dependent [6,16-19]. However, until eculizumab was approved by the United States Food and Drug Administration (FDA) in 2007, there was no viable complement inhibitor available for clinical use [16,20]. Eculizumab carries FDA labels for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) and has been used off-label to successfully treat other complement-mediated hemolytic processes such as refractory cold agglutinin disease and catastrophic antiphospholipid antibody syndrome in the setting of renal transplantation [21-23]. Here, we investigate whether eculizumab could curtail BRSB associated hemolysis in an in vitro model.

Previous investigators have also determined that levels of a highly expressed RBC membrane protein, glycophorin A, may be altered by exposure to the venom of a different Loxosceles species [17]. In order to clarify the mechanism of BRSB mediated hemolysis, and to move towards the goal of developing an easily accessible laboratory test for L. reclusa venom exposure, we determined the effect of venom exposure on glycophorin A as well as two membrane bound complement regulatory proteins (CD 55 and CD 59) which are associated with PNH and drug induced immune hemolytic anemia.

Materials and Methods

Results

Clinical Features of Severe BRSB Hemolysis

Our 10-year retrospective chart review identified 17 cases of life-threatening BRSB mediated hemolysis at our institution over the past 10 years. The laboratory findings in each of the 17 cases are summarized in Figure 1 and listed in Table S1. The median age of patients was 9 years, and the majority (71%) of cases were age 18 or younger (Figure 1A). Envenomation was associated with a decreased hematocrit (median nadir value = 16%; adult reference range = 42-50%), elevated LDH (median maximum value = 878 U/L; adult reference range = <225 U/L), elevated total bilirubin (median maximum value = 5 mg/dL; adult reference range 0.2-1.2 mg/dL) and a median of 3.5 blood transfusions (Figure 1B-E). On average, patients presented approximately 4 days after envenomation and were admitted to the hospital for 5 days (Figure 1 F-G). Most victims were female (Figure 1H). Two patients died despite supportive therapy, including one toddler (Figure 1I). The case fatalities were ages 4 and 54, were Caucasian versus African American, were male and female, with neck and torso bites, respectively. Typically, young children (under age 12) presented with symptoms sooner after envenomation compared to older patients (median of 2 vs. 7 days until presentation, P =.0037; Figure 1J). All patients were otherwise healthy and had no evident co-morbidities predisposing them to hemolysis. Patients presented with a wound at the site of envenomation, at least one sign of (e.g., hemoglobinuria or jaundice) or symptom (e.g., abdominal pain) of hemolysis.

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Figure 1. Characteristics of victims of severe Brown Recluse spider bite mediated hemolysis from 2002 through 2012 at Vanderbilt University Medical Center.

Horizontal line identifies the median value.

https://doi.org/10.1371/journal.pone.0076558.g001

The direct antiglobulin test (DAT) was not consistently positive for either C3 or IgG on the samples for which data were available, although a previous report demonstrated consistent deposition of complement in envenomed adolescents [9]. In our study, patients who did have a positive DAT for IgG were found to have a negative eluate when tested with panel cells (n=2), indicating that the IgG attached to RBCs in vivo did not demonstrate reactivity with common RBC antigens.

Venom Exposure Results in IgG and Complement Fixation to the RBC surface, resulting in Complement Mediated Hemolysis

We found that venom naïve erythrocytes had no detectable IgG or complement bound to surface membranes. However, venom sensitization resulted in deposition of both IgG and complement on the RBC membrane after incubation with ABO-identical fresh frozen plasma (Table 1). In addition, venom sensitized erythrocytes - but not venom naïve erythrocytes - hemolyzed in the presence of ABO-identical fresh frozen plasma (Figure 2A). When complement-poor (heat inactivated) plasma was substituted for fresh frozen plasma from the same donor, hemolysis was almost completely prevented (Figure 2C).

Donor	IgG		Complement
	Untreated	Venom Treated	Untreated	Venom Treated*
1	0	4+	0	1+/4+
2	0	2+	0	w+/2+
3	0	2+	0	1+/2+
4	0	3+	0	0/3+
5	0	3+	0	0/3+

Table 1. Venom exposure and incubation with a source of IgG and complement (fresh frozen plasma) leads to IgG and complement deposition on RBCs that initially were negative for complement and IgG.

Although venom exposed, plasma incubated washed cells from some donors demonstrates mild autoagglutination, exposure to complement antibody increased the strength of agglutination in all instances. Grading is on a scale of 0 (no agglutination) to 4+ (strong agglutination). w = weak.

*Pre/Postincubation with anti-C3b/d.

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Figure 2. Effect of eculizumab on L. reclusa associated hemolysis in vitro.

(A) L. reclusa venom sensitized cells are hemolyzed over time when incubated with ABO identical plasma, compared to venom naïve RBCs. (B) This hemolysis is reduced by an average of 79.2% (SD = 18.8%) when eculizumab is added to the assay. (C) Eculizumab was statistically indistinguishable from heat inactivation at all timepoints. Results are averages of 3-4 experiments. Error bar shows results +/- 1 standard deviation. Asterisk indicates statistical significance, p<.001.

https://doi.org/10.1371/journal.pone.0076558.g002

Eculizumab Inhibits L. Reclusa-Mediated Hemolysis

The addition of eculizumab to experiments containing venom exposed RBCs and ABO-identical fresh frozen plasma reduced hemolysis at 24 hours of incubation, from 10.9% hemolysis without eculizumab to none detectable with eculizumab (p<.001). At 48 hours, there was 16.0% hemolysis without eculizumab vs. 3.3% with eculizumab (p<.001). At 72 hours, there was 22.4% hemolysis without eculizumab vs. 6.9% with eculizumab (p<.001). At 96 hours, there was 37.6% without eculizumab vs. 16.1% with eculizumab (p<0.001; see Figure 2B and Figure 3). Eculizumab did not reduce hemolysis to a statistically significant degree 4 hours after exposure: 6% hemolysis without eculizumab vs. none detectable with eculizumab. On average at all time points, eculizumab decreased venom-mediated hemolysis by 79.2% (SD 18.8%). The extent of hemolysis in experiments containing eculizumab was statistically indistinguishable from negative controls consisting of complement-poor (heat inactivated) plasma (p>.05 at all time points, see Figure 2C).

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Figure 3. Photographs of plasma under various experimental conditions after 72 hours of incubation, compared to initial plasma (right).

https://doi.org/10.1371/journal.pone.0076558.g003

Venom Exposure Results in Reduced RBC Glycophorin A Expression

Flow cytometry was employed to compare the levels of glycophorin A, CD55 and CD59 in venom naïve versus venom sensitized cells. These experiments showed that exposure to L. reclusa venom does not significantly change erythrocyte expression of CD 55 or CD 59, compared to venom naive (PBS) controls (P=.079 and P=.714, respectively, see Figure 4A and 4B). In contrast, exposure to L. reclusa venom dramatically reduced the mean fluorescence intensity (MFI) of glycophorin A on the surface of erythrocytes, compared to incubation with PBS (P<0.001, see Figure 4C).

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Figure 4. Top Row: Mean data from 5 experiments show that venom exposure results in a statistically insignificant alteration in the expression of CD 55 (A) and CD 59 (B), but a statistically significant reduction of glycophorin A (C). Error bars indicate +/- 1 standard deviation. Bottom Row: Representative histograms from a single experiment (Red = untreated cells; Blue = PBS treated cells; Orange = venom treated cells; Green = isotype control).

https://doi.org/10.1371/journal.pone.0076558.g004

Discussion

The majority (71%) of patients with severe BRSB mediated hemolysis in our case series were children under age 18. Our experience and the results of our 10-year retrospective chart review indicate that it is not easy to reliably identify which children (or adults) will develop profound hemolysis (vs. mild to no hemolysis) in the immediate aftermath of L. reclusa envenomation. This is due the fact that there is no commercially available laboratory test for L. reclusa venom exposure, nor are there known parameters which will predict whether or not venom-induced hemolysis is likely to occur after envenomation [12,13].

Because transient periods of profound anemia are associated with serious complications such as ischemic stroke [26] and patients may die despite supportive measures, eculizumab (or other systemically active inhibitors of the complement pathway) may have a role in management of severe cases of BRSB mediated hemolysis. The efficacy of eculizumab, which reduced hemolysis by an average of 79.2% at the time points evaluated in our in vitro model, establishes a rational basis for the further study of complement inhibitor therapy for the treatment of severe BRSB mediated hemolysis. The risks of profound anemia and exposure to multiple blood donors should be weighed against the possibility of complications – particularly meningitis due to Neisseria meningitis – which has been reported to result from treatment with eculizumab [21]. We note that recent changes to pediatric meningococcal vaccination schedules [27] indicate that children who are over age 11 should be vaccinated against Neisseria meningitidis, which may reduce the likelihood of an adverse outcome after eculizumab therapy for a large proportion of victims of envenomation. However, eculizumab does not carry an FDA approval for the treatment of BRSB mediated hemolysis and the clinical utility of eculizumab therapy in this population has not been established in vivo. It may also be useful to compare the efficacy of eculizumab against loxosceles anti-venoms, which are not currently FDA approved or available in the United States [28].

Towards the goal of developing a test that could determine whether a patient with an insect bite had been exposed to L. reclusa venom, we compared the expression of three red cell membrane surface proteins - CD 55, CD 59, and Glycophorin A (CD235a) – in venom exposed versus venom naïve human RBCs. We studied CD 55 and CD 59, which are key complement regulatory proteins, because it is known that levels are reduced in paroxysmal nocturnal hemoglobinuria. In addition, CD55 is altered following exposure of RBCs to cephalosporins [29]. We found no significant alterations in CD 55 and CD 59 levels that would make these assays useful in diagnosis or monitoring victims of BRSB (Figure 4). Our results were consistent with previous investigators studying a different species of Loxosceles spiders [17].

We also evaluated glycophorin A, which is a highly glycosylated structure that carries the M and N blood groups and is associated with the Wright B (Wr^b), Pr, and high incidence En^a blood group antigens [30,31]. It is expressed on erythrocytes in the circulation as well as erythroid precursors in the bone marrow, and the average RBC expresses approximately 1 million glycophorin A structures on its surface [32,33]. Similar to previous investigators studying Loxosceles intermedia [17], we found that levels of glycophorin A are reduced to a statistically significant extent in Loxosceles reclusa venom-exposed erythrocytes compared to venom naïve erythrocytes.

We considered several possible explanations for the observed reduction in erythrocytic glycophorin A following in vitro venom exposure. One possibility is that venom exposure leads to the production of an autoantibody associated with glycophorin A. Support for this theory comes from the observation that some patients with BRSB mediated hemolysis demonstrate IgG deposition on their red cells in vivo (positive DAT) [9]. Moreover, cases of severe autoimmune hemolytic anemia with a similar clinical presentation as BRSB mediated hemolysis are known to be caused by autoantibodies directed against minor blood group antigens associated with glycophorin A [30,34,35]. However, we believe that the fact that BRSB mediated hemolysis can be modeled in vitro rules out an active immune component (such as autoantibody formation) to the hemolysis. In addition, whereas autoantibodies tend to agglutinate normal cells, in our experience antibodies eluted from the RBCs of victims of BRSB mediated hemolysis do not react with normal cells. Given the ubiquitous expression of glycophorin A on RBCs, we would expect the eluate from affected patients to react with all normal cells if these patients were developing glycophorin A specific autoantibodies. Finally, the timing of hemolysis – within a few days after envenomation - seems too short for the generation of an autoantibody.

A second possibility is that L. reclusa venom directly hemolyzes RBCs. We believe that this hypothesis is very unlikely, given our observation that venom sensitized RBCs fail to hemolyze when incubated with complement depleted plasma in vitro.

A third possibility, and the explanation which we favor, is that exposure to venom enzymatically alters the RBC membrane, making it vulnerable to non-specific attachment of immune globulin and complement, eventually resulting in hemolysis. This hypothesis is supported by two observations: 1) the hemolysis can be modeled in vitro and thus does not require an active (specific) immune response; 2) the antibodies eluted from the surface of RBCs from affected patients do not demonstrate reactivity with commonly tested, venom naive RBCs.

This third hypothesis would also suggest that reduction in glycophorin A levels is indicative of venom exposure and not directly causative of RBC lysis. A previous study found that RBCs exposed to venom from a species of Loxosceles found only in South America also results in the reduction of erythrocyte glycophorins [17,36]. This may indicate that glycophorin A expression may be used to detect exposure to the venom from various Loxosceles species.

The fact that so many of the patients in our case study were children may also lend a clue to the factors that may predispose patients to severe hemolysis. It has been previously reported that L. reclusa venom attachment to red cells in vitro is dose dependent [37]. Perhaps the smaller blood volume of children, in combination with a particularly venomous bite in certain situations, leads to a predilection towards severe disease in the pediatric population. Because so many individuals in the endemic region anecdotally report a history of L. reclusa spider bite, but very few develop severe hemolysis, other factors such as variations in serum complement and immunoglobulin concentrations or as yet undiscovered genetic factors may also play a role in the immune response to envenomation. When we compared the extent of hemolysis between several combinations of donor RBCs and donor plasma, we found no obvious evidence of individual-specific predisposition to BRSB mediated hemolysis (Figure S1). Further investigation is needed to firmly establish the pathogenesis of this phenomenon.

There are several limitations to our study. First of all, the retrospective chart review was limited by the fact that BRSB mediated hemolysis is a clinical diagnosis that cannot be confirmed by a “gold standard” laboratory assay. We had very stringent case definition criteria – the patients had to have laboratory evidence of severe hemolysis, as well as a clinical presentation and history that led the primary treating team of physicians to have a high suspicion for spider bite hemolysis. This strategy was intended to limit the number of “false positives” in our case series but may have led us to underestimate the true prevalence of this disease at our medical center. Along those lines, many of the victims of envenomation were young children who may not have been able to give a reliable history of the envenomation. If an insect bite was not noted on physical exam, and the clinical team did not suspect a spider bite pathogenesis, then the case would have been missed by our chart review. Second of all, our in vitro model was designed to test the efficacy of eculizumab, and therefore used doses of spider venom that are much greater than what would be observed clinically. This approach was intended to help to clarify the mechanism underlying this hemolysis, not as a close approximation of in vivo proportions. Further studies are needed to address the optimal timing of eculizumab administration after venom exposure. Third of all, it is difficult to definitively establish the significance of the reduction of glycophorin A levels. Although we are not aware of any disease that reduces expression of glycophorin A, it is possible that the reduction that we observed is fairly non-specific in terms of venom effect on RBCs. Therefore, additional work is needed to determine whether venom from harmless spiders also results in a significant reduction in glycophorin A.

Two of the pediatric patients in our case series were not initially recognized as victims of L. reclusa envenomation. One patient, a 7 year-old girl, presented one day after suffering a spider bite. She was discharged home with a normal direct serum bilirubin, only to present 4 days later with symptoms and laboratory findings consistent with severe BRSB mediated hemolysis. She was hospitalized for 4 days before recovering. A second patient, a 4 year-old girl, was initially sent home from an outside facility after evaluation for an insect bite. Her symptoms worsened, and she presented to our institution the next day. She eventually died as a result of the BRSB mediated hemolysis, despite supportive measures.

At our institution, we now counsel our clinical colleagues to admit patients with suspected BRSB mediated hemolysis and to obtain frequent hematocrit measurements (every 1-6 hours, depending on the patient’s age and ability to tolerate blood draws) to monitor for hemolysis, which can be sudden in onset. We also recommend frequent monitoring of urine for free hemoglobin, which is especially useful for very young children who are unable to tolerate frequent blood draws. RBC transfusions should be considered when the hematocrit drops below 25-30% or when otherwise clinically indicated.

Conclusion

In conclusion, L. reclusa envenomation can result in a life threatening hemolytic anemia with mortality despite supportive measures. Our studies indicate that this hemolytic process is complement mediated and is substantially reduced by eculizumab in vitro. Because venom from L. reclusa results in a reduction of detectable glycophorin A, glycophorin A levels should be further investigated as a supportive test for management of Loxosceles venom exposure. A clinical trial to establish the utility of glycophorin A expression and eculizumab therapy in the diagnosis and treatment of L. reclusa-mediated hemolysis is needed.

Supporting Information