Donald Beezhold, PhD, William C. Beck, MD
Latex surgical gloves have recently been identified as a potential source of allergens. Much of the current information suggests that the soluble proteins in latex may cause significant reactions in sensitive individuals. The starch powders used as a lubricant on some latex gloves have also been identified as potential allergens in some patients. In this study, we determined these powders to act as potential carriers of latex allergens. We have produced a polyclonal antiserum to be used as a reagent to study latex proteins. By Western blot analysis, we identified a significant interaction between latex proteins and starch powders. The binding of latex proteins to starch particles results in a glove particle that may have an increased potential to act as an allergen.The latex protein\p=n-\starchparticles represent a potential mechanism for exposure and sensitization of health care workers to latex allergens. Elimination of these particles from the operating room should reduce the route of sensitization and the potential for adverse reactions to latex.(Arch Surg. 1992;127:1354-1357)
The recent emergence of allergy to latex is a serious concern to surgeons, operating room personnel, health care workers, and dental practitioners. While latex aller¬gies have long been noted, the mandatory implementation of universal precautions in 1988 dramatically increased the incidence of reported allergies to latex. Recent estimates report that 7.4% of surgeons and 5.6% of operating room nurses are sensitive to latex.1 While the dermatitis and ur¬ ticaria associated with latex allergy are not life threatening,severe reactivity to latex, including IgE-mediated anaphy- laxis and death,2 has been reported in sensitized workersand patients. The danger of anaphylactic shock appears to arise when sensitized individuals undergo medical pro¬cedures (surgery, barium enema, and dental work) in which they are exposed to latex internally or on mucousmembranes.
The causative agents that induce reactivity to latex products have not been determined. Latex products con¬tain many chemical ingredients in addition to the latex.Several of these components have been identified as pro¬ducing allergies, including the accelerators used in vulca¬nization,3 absorbable dusting powders,4 and water-soluble proteins derived from the rubber tree, Hevea brasiliensis.5 However, little is known about the route of sensitizationinvolved in latex allergy.The number of reports that surgical gloves induce res¬piratory symptoms (rhinitis and asthma) and conjunctivi¬tis in operating room personnel has been on the increase.6"8 Baur and Jäger" suggested that latex antigens may be ab¬sorbed by the powders on surgical gloves and become air¬borne allergens. These airborne allergens then have the potential of affecting not only the operating room personnel but also the patient. Turjanmaa et al9 demonstrated thatIgE antibodies from a patient sensitive to latex recognized protein antigens in raw latex, glove extracts, and on glove powders. In this study, we confirmed and extended these results. We have produced a rabbit antiserum as an immunoreagent to study soluble latex proteins. Using this antiserum, we compiled data characterizing extractable latex proteins and demonstrating that the proteins adsorbed to the glove powders are derived from latex.
Ammonified latex (Guthrie Latex Inc, Phoenix, Ariz) was obtained through Vanguard Research Inc (Mamaroneck, NY). The latex slurry (20 mL) was poured onto a plastic plate and the
emulsion was allowed to collapse and air dry for 18 hours. The resulting latex film was cut into small pieces (1 cm2) and placedin a polypropylene bottle. The latex pieces were extracted over night (37°C) in 20 mL of distilled water. The latex was removed and the supernatant centrifuged at 1000g to remove particulate matter and then dialyzed in phosphate-buffered saline (PBS). The protein concentration of the extract was determined using the bicinchoninic acid (BCA) method (Pierce, Rockford, 111).
An antiserum was made that would specifically recognize all soluble latex proteins. TiterMax Adjuvant (CytRx Corp, Norcross,Ga) was chosen as a vehicle for immunization because it contains
no bacterial or plant components. Such components potentially induce antibodies that could cross-react with latex proteins. Inaddition, TiterMax Adjuvant produces high titer IgG antiserum
that is ideal for enzyme-linked immunosorbent assay and West¬ern blot assay, usually after a single injection.A 50-50 mixture (volume-volume ratio) of latex proteins (10 mg/mL) and TiterMax Adjuvant was prepared. A New Zealand white rabbit was injected with a total of 0.8 mL of the mixture infour sites on the back. The rabbit was bled at 3, 5, and 7 weeks.
The antilatex titer was determined using an enzyme-linkedimmunosorbent assay and reached a titer of 1 / 5000 after 7 weeks.
Three common glove powders were secured for testing: Biosorb(Johnson & Johnson Co, Indianapolis, Ind), Keoflo 7136-USP(Hubinger Inc, Keokuk, Iowa), and calcium carbonate, United States Pharmacopeia, Extra Light (Pfizer Inc, New York, NY).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) electrophoretically separates proteins according to molecular size based on their ability to sieve through a polyacrylamide gel. The SDS-PAGE was performed as previously described1" using a mini-PROTEAN II gel apparatus (BIO-RAD Laboratories, Rockville Centre, NY). Samples containing the latex
proteins were placed in reducing sample buffer, heated at 100°C for 2 minutes, and electrophoresed on 15% polyacrylamide gels.The proteins were visualized by 0.1% Coomassie blue R250 (BIORADLaboratories) staining of the gels or by Western blot analysis. For Western blot analysis, the latex proteins that were separated on 15% SDS-PAGE were transferred to nitrocellulose
(Schleicher and Schuell, Keene, NH) using the buffer system described by Towbin et al.11 The membranes were blocked for 1 hour at ambient temperature with 100-mg/mL bovine serum albumen
and incubated with 1/5000 dilution of rabbit antilatex antiserum (37°C, overnight). The blots were washed three times and reacted for 1 hour at 37°C with an alkaline-phosphatase-labeled antirabbitIgG. Following five washes to remove unreacted second antibody, the latex protein bands were visualized using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Promega,Madison, Wis) as a substrate.
To determine if latex proteins are adsorbed by glove powders,we used powders that had not previously been in contact with latex. A 10% (weight-volume ratio) suspension of the powders
was prepared in PBS. Latex proteins (100 u,L, 1 mg/mL) were in¬cubated with 100 u.L of powder suspension for 1 hour. The particles were pelleted by centrifugation and the supernatant was
removed. The pellet was washed three times in PBS, resuspended in electrophoresis sample buffer, and analyzed by SDS-PAGE and Western blot.In other experiments, starch particles were removed from surgical gloves by cutting the glove into 1-cm2 pieces and washingin PBS. The particulate matter was recovered by centrifugation after removal of the latex pieces. To test for latex proteins, the glove powder pellet was suspended as a 50% slurry (volumevolume ratio) in PBS, and 100 u.L of the glove powder slurry was washed three times in PBS and suspended in SDS sample buffer.The samples were analyzed for the presence of latex proteins by SDS-PAGE and Western blot analysis.
Antilatex Antiserum Recognizes the Entire Range of Latex Proteins
Latex proteins were separated by SDS-PAGE on 15% gels and stained with Coomassie blue to visualize the proteins (Fig 1). In lane 1, the extract from crude latex contained a large range of proteins. The bulk of the protein had a molecular weight above 30 000 d, with less protein in the 17000- to 30000-d range. Significant amounts of proteins migrated below 17000 d.Not surprisingly, proteins from ammoniated latex failedto migrate as distinct bands. This is presumably because proteolytic degradation has occurred. The crude latex slurry used by glove manufacturers is stored in ammonia(pH 11.4) to prevent putrefaction. Therefore, hydrolysis and degradation of the proteins would be expected to occur. Although the latex proteins tend to smear, distinct protein bands were observed at 14 to 17, 30, 43, and 70 kd.Next, the preparation of total latex proteins was used for the production of a polyclonal antiserum. A polyclonal antiserum was desirable as an immunoreagent with which to specifically identify proteins by Western blot. In theWestern blot technique, proteins separated by SDS-PAGE are transferred to nitrocellulose paper and then identified by reactivity with an appropriate specific antiserum. When Western blot analysis was performed using the antilatex antiserum, we found a similar profile of proteins that were recognized by our antiserum (lane 2) to that observed following total protein staining with Coomassie blue R250 (lane 1). These data demonstrate that the antiserum is reactive with the complete range of latex proteins. In contrast, no reactivity was observed in control Western blots using nonimmune rabbit serum.
Fig 1.—Analysis of latex protein extracted from ammonified crude latex.
Proteins extracted from latex were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis on 15% acrylamide gels. Lane 1 demonstrates total latex proteins stained with 0.1% Coomassie blue R250; lane 2, Western blot analysis of total latex proteins.
Latex Proteins Bind to Starch Particles
To test if latex proteins interact with glove powders,glove powders were treated with soluble latex proteins, followed by three washes in PBS to remove any nonbound protein. Latex proteins that bound to the starch particles were eluted in electrophoresis sample buffer and analyzed by Western blot. As shown in Fig 2, latex proteins bound to the cornstarch powders (lanes 1 and 2) but not to thecalcium carbonate powder (lane 3). This interaction appears to be nonspecific in that the entire range of proteins observed in the latex protein preparation (lane 4) were bound to the particles. In controls, no immunoreactive proteins could be detected in the powders (lanes 5 to 7) that were not treated with the latex proteins.
Latex Proteins Are Present on Starch Particles Isolated From Surgical Gloves
We tested We tested particles removed from eight brands of surgical gloves. Of the eight brands tested, one was powderfree,one was non-latex but powdered, and the remaining six brands were powdered. We found variable levels of protein on the particles removed from the gloves (Fig 3).Although insoluble material was obtained from the powder-free glove, no latex protein could be detected (lane 1). Likewise, no latex protein could be detected on the powder removed from the latex-free glove (lane 7). Variable levels of protein were observed on the powder
removed from the remaining six brands of surgical gloves.Levels of latex proteins ranged from significant (lane 4) to nearly undetectable (lane 5). Interestingly, the protein profiles differed among the various gloves. Most notably,glove powders in lanes 2 and 4 demonstrated very similar protein patterns; most of the proteins were 70 kd or lower, with a prominent band at 17 kd. Glove powders in lanes3 and 8 demonstrated a different pattern in which numerous high-molecular-weight proteins were visible and much of the protein was above 30 kd.
gnized with increasing frequency. While soluble proteins in the latex have been implicated as one causative agent, it is not clear which of these proteins are allergens and/or which route of exposure triggers an allergic response. Inthis study, we described a polyclonal antibody that recognizes soluble latex proteins. The antilatex antiserum was developed as a tool by which to quantitate levels of proteins on latex products14 and to identify latex proteins byWestern blot technique. We characterized the extractable proteins in crude latex and observed a broad range of polypeptides with evidence for breakdown of proteins. Crude latex used for manufacture of latex medical devices typically is stored in 0.7% ammonia at pH 11.4. This harsh treatment causes extensive hydrolysis of the proteins as evidenced by the electrophoretic profiles being smeared rather than a distinct banding pattern. Nonetheless, some distinct polypeptides could be identified in our preparation. Similarly, extensive hydrolysis of proteins isolated from ammoniated latex was reported by Slater and Chhabra.15 They found, however,that distinct bands are observed when proteins are isolated from nonammoniated latex (latex collected directly from the tree without the addition of ammonia). It should be
noted, however, that the glove wearer is exposed and sensitized to the hydrolyzed protein and/or polypeptides.We have demonstrated that the extractable proteins in latex interact with the powders used as lubricants on surgical gloves. We found a substantial interaction between the latex proteins and cornstarch powders; however, no interaction was observed with the calcium carbonate powder. Calcium carbonate is often used as a mold release agent in the manufacture of surgical gloves. The possibility of glove powders as carriers of latex allergens was first suggested by Baur and Jäger.6 This was further substantiated by Turjanmaa et al,9 who showed that patients with latex sensitivity had serum IgE that reacted with glove extracts, crude latex extracts,and glove powder extracts. We have extended these observations and now show that surgical glove powders contain substantial amounts of latex proteins. Using specific antiserum for latex proteins, we demonstrated that the proteins found on the glove powders originated from the latex. In addition, we have observed that powders removed from gloves of various manufacturers are contaminated with differing amounts of the latex proteins and exhibit different electrophoretic profiles. Thus,we have confirmed starch powder as a potential route of exposure to latex protein allergens. The latex protein-starch particles found on latex glovesrepresent a potentially reactive antigen. Immunologically,complex antigens have proven to be more immunogenic than more highly purified or biochemically simple antigens.16 Furthermore, antigens composed of a proteinpolysaccharide complex are known to be strongly immunogenic.16 Thus, the addition of starch powders to latex gloves during the glove manufacturing process may create a new set of antigens with the potential to be highly immunogenic. The protein-polysaccharide nature of thesenew antigens may favor the development of an IgE response.It has been well documented that a significant proportion of patients with latex allergy have a detectable IgE response by radioallergosorbent test assay.17 The protein antigens have not been well characterized, but multiple antigens appear to be involved. Protein antigens to which the IgE response is directed have been reported as having a molecular weight higher than 30000 d18 and in another report, as having molecular weights of 2000, 5000, and 30000 d.5 Likewise, Slater and Chhabra15 demonstrated that IgE in serum of patients sensitive to latex recognizes multiple proteins in the 6- to 45-kd range and suggested that the major antigen is a 14-kd peptide found in extracts from nonammoniated latex. Polypeptides with these molecular weights as well as additional proteins are present in our protein preparation from ammoniated latex and
were found in variable amounts on surgical glove starch powders.
The route of exposure to an antigen is considered to be crucial in determining what type of immunologie response(IgG vs IgE) is induced. Starch powders easily become aerosolized when donning gloves and can remain airborne for many hours. Airborne starch particles may be inhaled by operating room personnel and have been reported to incite asthma and rhinitis.6 Thus, airborne starch particlesmay represent a major route of sensitization to latex protein allergens in health care workers. In preliminary studies, we observed that a large number of patients with latex allergy also have a history of previous surgery. Exposure to latex proteins via inhalation of protein-coated starch particles or via contamination of a surgical incision may adversely influence the outcome of the immune response to latex proteins. Another issue related to the use of starch powders is the complication of starch peritonitis or granulomatous reactions. Cornstarch powders were originally introduced as an absorbable alternative to the use of talc powder.19 Initially, the powder proved inert and caused few problems;
however, since the adoption of gamma irradiation for sterilization of surgical gloves, the reported incidence of starch granuloma has increased.20 Autoclaving the starch reduces adhesion and granuloma formation21 and alters the ability of starch to influence lymphocyte reactivity.22 Thus, the finding that latex proteins are adsorbed to the starch powders may further help to explain the increasedincidence of starch-induced granulomatous reaction. Before gamma irradiation, sterilization of the gloves by autoclaving may have caused significant degradation of the
proteins, thereby reducing the antigenicity of the proteins. The presence of foreign proteins on the starch particlessignificantly increases the likelihood that the particles will induce inflammatory reactions in susceptible individuals.
Our data demonstrate that the starch particles used to lubricate some surgical gloves are carriers of latex protein antigens. Starch particlesfrom surgical gloves contaminate surgical incisions; adhere to instruments, sutures, and equipment; and will become airborne during donning. Exposure to latex protein-starch particles during surgery or by breathing latex protein-starch particles may predispose the development of IgE antibody to latex proteins. Thus, for the benefit of the patient and the health care worker, prepowdered gloves should be eliminated from the operating room.This work was supported in part by a grant from Regent Hospital Products Inc, Greenville, SC.The authors thank Christine Personius, Zong-Lu Shen, and TerrieZimmer for excellent technical assistance and Margaret Fay, PhD, forcritical reading of the manuscript.
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