History of rubber latex

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1.0 Introduction

1.1 History of rubber latex

     Brazil provided the world with the rubber tree, Hevea Brasiliensis, but now she is no longer playing any significant part in the world natural rubber trade. Seeds were exported from the lower Amazon area of Brazil to London UK by Henry Wickham, a local planter of British Government in 1876.

The seeds were germinated at the Tropical Herbarium in Kew Gardens, London later that year. From there seedlings were exported to Ceylon (Sri Lanka). In 1877, 22 seedlings were sent from Ceylon to Singapore, where they grew strongly, and the technique of tapping was developed.

Prior to this, the trees had to be felled before the latex could be extracted. By 1900, most of the techniques and agricultural practices required to establish large plantations had been developed. One key technique was bud grafting. This is essentially a cloning technique which ensures that genetically identical trees can be produced in unlimited numbers. The rubber industry often talks about high-yielding clones, or other types of clone; and this is the basis of that terminology.

Over the next 40 years or so, the British in Malaysia and the Dutch in Indonesia cleared large areas of rainforest to create rubber plantations. Simultaneously, local farmers saw the opportunities of rubber cultivation, and planted small groves of trees to supplement their own income. This gives rise to two types of rubber plantations in most producing countries: the estates, or plantations and the smallholdings. Smallholdings tend to produce solid rubber while estates are essentially large-scale farms, with professional management. Most latex comes from professionally managed estates.

1.2 Latex production

     Latex is an aqueous elastomeric emulsion that represents the cytoplasm of highly specialized cells of several plants (Fahn, 1979) The latex of the tropical Hevea brasiliensis tree containing rubber particles is known as natural rubber latex (NRL) (Niggemann and Breiteneder, 2000). The rubber particles actually can be specifically known as 1-4 cis-polyisoprene molecules and it belongs to a very important family called the isoprenoids which constituted of more than 200,000 different molecules. It can be found not only in plant kingdom but also animal kingdom. The isoprenoid, which is cholesterol, is very important in building the cells animal and plant cells membranes, and another group which is ubiquinones can act as electron carrier. 

The latex produced by Hevea brasiliensis contains up to 50% cis-polyisoprene (C5H8)n. Although this monomerous isoprene can be produced by many plants and animals, nobody knows the reasons for latex production in a lot of plant species, especially in the rubber tree. According to some research, the latex secretion is a form of defence against wounding or predators such as insects and microorganisms while other state that rubber and other hydrocarbon compounds act as carbon traps to maintain the balance between biomass and atmospheric CO2 (Dickenson, 1969). Another uses of the latex could be that isoprene emissions by numerous tropical plant species constitute an antidote against the toxicity of atmospheric ozone.

All the part of the Hevea brasiliensis rubber tree is capable to produce latex but so far only the bark of the trunk is exploited (Dickenson, 1969). There is a productive latex bark between the external bark and the cambium. The cambium is very important as it act as a lateral meristem. It generates wood and xylem vessels' growth to the inside and the soft bark containing the phloem and the latex vessels' growth to the outside. Laticiferous vessels were organized in concentric layers around the cambium and these organized layers are called latex mantles. In each mantle, the tubes are connected by anastomoses, which act as the interconnection between vessels, constitute an advantage in the collection of latex. The laticiferous vessels are constitute of a kind of specialized plant cell called laticifer and the isoprene, 1-4 cis-polyisoprene is synthesized in the milky cytoplasm of this cell. All the laticifer cells contain the genetic information (DNA) and the machinery needed for protein biosynthesis (RNA, ribosomes) which is indispensable to allow the regeneration and restoration of the latex lost by the previous tapping.

To produce the latex, the cell must be supplied with raw material. Water and mineral absorbed in roots go up into the trunk using xylem vessels located in the wood near the cambium. Sugar, amino acids, hormones and more generally elaborate molecules are produced and distributed by the source organs, for example the leaves through photosynthesis, and are carried by the sieve tubes which located in the bark very near to the cambium. Cambium and sieve tubes are very fragile and they must not be wounded during tapping because these are the most important part for molecule transportation and survival of the plant. The latex vessels are not in direct contact with these two sources of nutrition supply but there is a horizontal circulatory system, constituted of rays, connects the wood and the fiber to the latex tubes. In the tapped trees, the laticiferous vessels show intensive metabolic activity. Hence, the cells do not look like normal sieve tube cell that do not contain any organelles. They contain the usual organelles of plant cells, such as nucleus, mitochondria, vacuoles, ribosomes, golgi apparatus and endoplasmic reticulum.

The rubber particles are the more typical latex component. The rubber particles usually ranging from 50 Što about 30.000 Š(0.003µm) in size and compose up to 50% of the latex by weight. These particles are wrapped in a single membrane made of proteins and lipid. The rubber particles are also associated with triglycerides, sterols, sterol esters, tocotrienols and other lipids. Dupont et al., (1976) have confirmed the presence of phosphatidylcholine and small amounts of phosphatidyl ethanolamine in the lipids associated with rubber particles (Dupont et al., 1976). The protein envelope of rubber particles is visible in sections of osmium stained rubber particles and is approximately 100 Šthick (Andrews and Dickenson, 1961). The envelope is negatively-charged and these negative charges on the membrane increased the stability of the latex colloidal suspension. A recent hypothesis implies that this membrane protects the cis-polyisoprene molecules against oxidative degradation. It is responsible, after latex maturation, for decreases in some technological properties such as Plasticity Retention Index (PRI).

The latex particles can be clearly seen after the latex was process by the Talalay process that the latex would be cooled to a temperature of -30°C. As the water crystallized, the rubber particles are forced into a boundary around the ice.

Particle size of greater proportion is far too small to be resolve by power of light microscope and reliable information in proportion could be obtained only with electron microscopic observations. Tempel recorded 1000 Å size at the maximum frequency of electron microscope (Tempel, 1952). This was confirmed later by Gomez and Moir (Gomez and Moir, 1979). Schoon and van der Bie, observed a multi-modal distribution of rubber particles in latex of mature Hevea trees (Schoon and van der Bie, 1955). Then they come out with a postulation that proposed larger particles are formed by the association of smaller particles. Gomez found a multi-modal distribution in latices from young potted plants. In the laticifers of very young plants, small osmiophilic (readily stained with osmic acid) particles can be seen freely move in the cytoplasm (Gomez, 1966). The structure and colloidal properties of Hevea latex has been well studied throughout the year (Cockbain and Philpott, 1963; Ho et al., 1976).

According to Dickenson there are rubber particles with different stained regions. The inner osmiophilic region surrounded by a weakly stained periphery is attributed to lack of uniformity when newly synthesize rubber particles are deposited on existing particles during biosynthesis (Dickenson, 1969). He has also suggested that the inner particulate inclusion, having 50-80 Å thickness, might be molecules of rubber of molecular weight about 100,000 (Dickenson, 1969).

Among the proteins located in the membrane of rubber particles, two enzymes implicated in the elongation of the cis-polyisoprene chain have been discovered: the Rubber Elongation Factor (REF, 14.6kD), cis-prenyl transferase (76kD) and also a glycoprotein (22kD) which is involve in latex coagulation by its glucidic moiety.

The latex vacuoles, called lutoids, are single-membrane particles that work as polydispersed lysosomal vacuomes. Lutoids is the second major component of Hevea latex and they are membrane-bound bodies that are mostly larger in size than the rubber particles. Usually they are 2-5 µm in diameter and bounded by a unit membrane of about 80 Šthick. Their membrane is negatively charged on its external face, the internal part is acidic (pH 5) and positively charged due to Mg++, Ca++, proteins with either + and - charges. It was Wiresum  who first suggested that the lutoids behave like cell vacuoles due to stainability with neutral red (Wiresum, 1957). Though some arguments existed in this postulation, the work of Ribaillier et al. provided strong evidence for the vacuolar properties of lutoids (Ribaillier et al.,1971).

The content of lutoids which is the B-serum has a very rapid flocculating action on the aqueous suspension of rubber particles in latex, resulting in the formation of microfloccs (Southorn and Edwin, 1968). This activity is apparently moderated by the surrounding C-serum and is much reduced if B-serum is boiled. Southorn and Yip demonstrated that this fast initial flocculating action of B-serum is an electrostatic one involving the interaction between the cationic contents of B-serum and the anionic rubber particle surface (Southorn and Yip, 1968).

By using phase contrast microscopy observation and application of suitable staining agents and procedures, the structure of lutoid particles have been studied in detail. They can be divided into mainly three types of fibrillar structures. The first type, known as microfibrils, are the characteristic of latex vessels in young tissues (Dickenson, 1965; Dickenson, 1969; Audley, 1965; Audley, 1966). As seen by phase contrast microscopy of tapped latex from young tissue, the microfibrils are found freely suspended in the fluid content of the lutoid B-serum. The microfibrils are usually seen grouped together in bundles. Each bundle has a diameter range from 450-500 Å. Individual microfibrils are several micron long and 70-80 Å in diameter.

The microfibrils can be isolated from the sediments of latex from young tissues which on negative staining with phosphotungstic acid that enable to shows further details. Each microfibril is a tightly coiled continuous helix with a hollow axis where the diameter of the helix is about 125 Å and that of the hollow axis 30 Å. The microfibrils consist of an acidic protein while DNA seems to be absent. Microfibrils however are not found in tissue or latex collected from the mature bark. It is commonly believed that they disintegrate or degraded as the particles mature or else the young lutoids containing microfibrils themselves disintegrate as the tissue grow old and are replaced by a population of lutoids without microfibrils. However, the microfibrils do not seem to have vital role in the rubber latex biosynthesis.

The second type of fibrillar structures, is observed in lutoids of latex collected from mature bark of stimulated trees, and they are known as 'microhelices' (Gomez and Yip, 1975). They are named as 'microhelices' (Gomez and Yip, 1975) because of their spring like shape. These structures were first observed by Dickenson (Dickenson, 1965; Dickenson, 1969). Sometimes they are found in unstimulated trees and their number increases base on dilution. However, microhelices are more frequently found in lutoids of tapped latex than in situ latex (Southorn and Edwin, 1968; Gomez and Yip, 1975) and are sometimes observed in latex collected from young tissue also.

As reviewed by Gomez and Moir (Gomez and Moir, 1979) the microhelices are approximately 1 µ in length with a diameter of 200 Å, having a fibre width of about 50 Å and an open hollow helix having a 300 Å wide pitch. Dickenson suggested the formation of microhelices from microfibrils but is not widely accepted and has been questioned by Gomez and Yip (Dickenson, 1965; Gomez and Yip, 1975).

A third type of lutoid inclusion is minute spherical particles in Brownian movement and was observed by Schoon and Phoa (Schoon and Phoa, 1956). Later Southorn found such particles in large numbers in the bottom fraction of ultra-centrifuged latex of long rested trees and this was confirmed by Dickenson (Southorn, 1960; Southorn, 1961; Dickenson, P.B., 1969). But so far the role of such particles in latex is unknown.

Intralutoidic proteins and enzymes play an important role in latex production physiology. Among the enzymes, all ranges of acidic hydrolases typical of animal and plant lysosomes, are found present. These enzymes play role in the recycling of worn molecules and by function of their lutoid contents like animal lysosomes, are the suicide bags of the latex cells, and their bursting leads to clamping of the cytoplasmic metabolism and to latex coagulation. Some defence proteins (Pathogenesis Related Proteins) against pathogenic agents such as chitinase, β- 1-3 glucanase and lysozymes, are accumulated in this compartment. Hevein is a very important protein used for latex coagulation. Other proteins, mostly be considered as a form of storage for reduced nitrogen (Vegetative Storage Protein), or stress proteins have also been found.

Other types of particle were discovered in Bogor by Frey-Wyssling in the 1930s which possess a double layer membrane. It shows a complex internal structure which assimilates them into chromoplasts. They are characterized by having the almost equal importance as carotenoids content, which gives a slight yellow colour to the latex. After the discoveries of latex organelles at Bogor and after optical and electron microscopy observation done by researchers in England and Malaysia, the cytoplasmic nature of Hevea latex was now clearly reviewed and proved.

1.3 Latex allergy

The first recorded immediate-type reactions to natural rubber appear to be two cases in the German literature from 1927 (Fuchs, 1994). The first case was reported by Stern, and it involved urticaria and laryngeal edema after dental exposure to natural rubber (Stern, 1927). The second report involved asthma that was provoked by the fumes given off by a rubber-coated electrical cable when the wire warmed from current passing through it (Grimm, 1927) Although these cases do not meet current standards for a diagnosis of latex allergy, they are certainly suggestive. The concept of immediate-type allergic reactions to natural rubber latex was then dormant until 1979, with the exception of a mention of rubber allergy in an allergy textbook by K. Hansen in 1957.7

Downing reported delayed-type reactions to rubber gloves in 1933, not in medical personnel but in electrical company linemen (Downing, 1933). Downing's report is interesting and frustrating. Seven cases of skin reactions are described in linemen who were wearing heavy rubber gloves to insulate them from the electrical wires they worked around. Three of the cases were related to a particular brand of "chocolate-colored" gloves. In these 3 cases, the symptoms could have been due to chemical irritation or toxicity, judging from the relatively rapid onset of symptoms. In other cases, a 24-hour patch test produced no reaction at 24 hours but a marked reaction at 72 hours, a finding highly suggestive of delayed-contact reactions. This report also contains an interesting quotation, attributed to the chief chemist at a rubber glove company.

After years and years research and studies, eventually, we are able to found out that the latex allergy is an immune response. It can also be specifically known as IgE-mediated hypersensitivity to the the allergen found in natural rubber latex product. The most common way people become sensitized to latex allergens is through direct or indirect contact in health care setting or daily use product (Alwilda et al., 2003). In recent years, this allergy response has become a worldwide major clinical problem. Those children with spinal bifida or urogenital malformation, those undergo repeated surgery or those who work as a health care workers and also rubber industry workers are the high risk group to develop hypersensitivity symptom due to repeat and unavoidable exposure to latex products (Sanz et al., 2003; Frankland, 1999; Alwilda et al., 2003).

So far there are more than forty thousand different products made of latex and frequently used both in our daily life and in the hospital and it can cause allergy for example such as gloves, injection port, balloon, condom and many other more (Sanz et al., 2003; Alwilda et al., 2003). This potent allergen not only present in those products but it can be a potential allergen from the tyre fragments. In a volumetric sampling of the air 48m from the nearest street level had shown 3800-6900 particles per m³ and over 90% were inhalable (Frankland, 1999). These inhalable particles gave latex-specific IgE reactions. The abundance of latex particles in the urban air may be considered as another reason for the increase in latex allergy too (Frankland, 1999).

This incidence of latex allergy seems to be less than 1% in the general population (Rendeli et al., 2005). The increase of this latex sensitizations is due to the frequent use of latex gloves since 1980 as a protection against AIDS and also other infectious diseases (Chardiu et al., 1999). The prevalence of sensitization to natural rubber latex is about 2-17% among exposed health care workers, up to 30-50% in childhood with spina bifida, a disease that needed repeated surgical procedures (Pires et al., 2002), and less than 1% in general childhood (Roberts et al., 2005 ) and adult (Liss and Sussman, 2005) population. But even in none risk groups in the sera of unselected healthy blood donors it was found that 6.4% had latex-specific immunoglobulin (IgE) antibodies (Frankland, 1999). Sub-clinical sensitization with a positive skin test or latex-specific IgE without clinical manifestations was found in 24% of the same cohort (Frankland, 1999).

1.5 The Increased Prevalence of Latex Allergy

Although it is a general fact, but it is too easy to be explained as owing to the increased use of gloves, due to the risk of hepatitis, AIDS and other blood transmitted diseases, and new manufacturing methods (Frankland, 1999). The temporal relationship of latex sensitization agrees poorly with the initiation of universal precautions towards those diseases (Charous et al., 1997). In 1940, mineral talc has been replaced by cornstarch to serve as donning powder in surgical gloves (Ellis H., 1990) because it was found that the silica talc powder can cause peritoneal granulomata. In term of transfer, mineral talc is heavy and only temporarily airborne, while the cornstarch remains airborne when used as a glove powder and it had also been shown that it used to be a latex antigen carrier (Tomazic et al., 1994; Beezhold, 1992).

Latex contaminated glove powder provokes allergic reactions in sensitized subjects (Bragnami et al., 1995) and acts as a "latex aeroallergen" in operating theatres (Heilman et al., 1996). It was shown (Lundberg et al., 1997), however, in immuno-clinical investigations that mineral talc has a high capability to bind strongly to latex allergens in contrast to cornstarch which captured the latex allergens, but the binding was weak and unstable. Hence, the silica talc powder actually able to acts as an efficient latex allergen eliminator (Frankland, 1999). The cornstarch powder consists of a mixture of amylose and amylopeptose sugar and it was chemically treated to improve its lubricant properties. Besides that, it was found that lower molecular weight latex allergens show a higher tendency to bind than the high molecular weight allergens (Turjanmaa et al., 1996).

1.6 Physiological response to latex

Hypersensitivity to latex may result in two types of immuno-response, Type I immunoglobulin E (IgE) or Type IV cell-mediated response (Alwilda Scholler-Jaquish, et al., 2003). Type I latex immunoglobulin E response are triggered by IgE antibodies produced in response to water-soluble proteins present in natural rubber latex products. The most severe Type I reaction is an anaphylactic shock, which may occur within minutes after exposure to the latex allergen. This immediate response is mediated primarily by the release of histamine, which causes temporary, rapid constriction of bronchial smooth muscles, increased vascular permeability, and dilation of postcapillary venules. Clinical manifestations may include symptom such as contact uritcaria, pruritus erythema, angioedema, thinorrhea, sneezing, coughing, apprehension, anxiety, dizziness, dyspnea, stridor, wheezing, tachycardia, hypotension, fainting, loss of consciousness, and cardiopulmonary arrest (Cormio, et al., 1993; Douglas et al., 1997; Field, Longman, Al- Sharkawi, King, 1997; McCance, Huether, 1998; Thurlow, 2001).

Type IV latex reactions occur in response to those chemicals that act as accelerants and antioxidants during the manufacturing process. This immune response is delayed and typically occurring 24 to 72 hours after the exposure to the allergen. Specialized lymphocytes cause pruritis, erythema, and vesicles or blisters at the point of contact. Because latex is found in many products, the greatest risk for people to have a Type IV reaction comes from breaks in the skin. It is important to remember that during the first exposure, no allergic response occurs, but antigens are formed. Through continual exposures, a hypersensitivity reaction may occur. Its treatment varies with the severity of the symptoms but typically involves removal of the offending agent, application of cool compresses and topical steroid or antihistamine creams, and oral administration of antihistamines (Cormio et al., 1993; Douglas et al., 1997; Field et al., 1997; Lewis et al., 2000; McCance & Huether, 1998; Thurlow, 2001).

1.7 Type of Latex Allergen

Latex allergen are the substance that causes the allergic response when it come into contact. There are 12 different natural rubber latex allergen have been identified, cloned and sequenced by the researchers and they are known as Hev b allergens (Yeang, 2004; Wagner and  Breiteneder, 2004). Hev b 1 and the Hev b 3 were the major allergens for children with multiple congenital anomalies (Yeang et al., 1996). Hev b 2 and  Hev b 4 are important sensitizing allergens for health care workers with latex allergy (Bernstein et al., 2003). Hev b 5 and Hev b 6 are recognized by IgE in a majority of both health care workers and natural rubber latex allergic children (Palosuo et al., 1996; Pamies et al., 2006; Sutherland et al, 2002). The major NRL allergens, Hev b 2 is glycosylated proteins. For both proteins, it has been demonstrated that IgE epitopes arise on the carbohydrate moiety (Yagami et al., 2002; Arif et al., 2004).

1.8 Food Allergies Associated with Pollen and Latex

The Silver birch (Betula) pollen allergy has been extensively studied, especially in the Scandinavian countries, because it is the pollen that causes the most seasonal allergic rhinitis and asthma. It is also the pollen that most commonly associated with IgE mediated symptoms related to vegetables and fresh fruits (Halmepuro et al., 1984 ; Calkhoven et al., 1987; Frankland & Aalbers, 1987). It has been shown (Pham & Baldo, 1995) that there may be allergenic relationships between taxonomically different pollens in a such way that they carried the same allergens. There is also evidence that shows a family of cytoskelous proteins called profilins, with a molecular weight of 14-18 kD, are present in a few different type of pollens and plant species (Frankland, 1995). Profilins are commonly known as thermolabile proteins. It means that patients who may be anaphylactically sensitive to many fresh fruits and vegetables can eat them without worries when they have been cooked, simply means that it denature at high temperature. Profilin is an allergen present in many plant species, so that we would consider that profilin may be an allergen in Hevea brasiliensis (Frankland, 1999). It has been found that  in a group of 19 subjects allergic to latex, only two of them had anti-profilin IgE antibodies. However, profilin is hardly detectable on glove extract immunoblots because it might be destroy in the process of sterilizing and heating in the making of gloves. But profilin is found present in banana extract. So it is more likely to be involved in cross-sensitivity to banana and latex and should be taken into account when interpreting the results of the latex IgE antibody assays (Frankland, 1999).

Other than profilin, lectin is another type of protein that might involved in the cross-sensitivity among fruits and latex (Freed, 1999). Lectins are carbohydrate binding proteins that bind to carbohydrate reversibly and noncovelently and it is present in most of the plants. These types of proteins are toxic, high resistance to heat and digestive enzymes, and present in many foods. Many lectins are powerful allergens and prohevein, which is one of the main allergens of rubber latex (Alenius et al., 1995). Recently, it has been genetically engineered into the transgenic tomatoes for its fungistic properties. According to Freed's hypothesis, we can expect an outbreak of tomato allergy in the near future among latex sensitive individuals (Freed, 1999 ).

The fresh fruits which usually cause allergic problems are banana, avocado pear and chestnut (Frankland, 1999). Other fruits less associated with latex allergy are peaches, kiwi, mango and walnut. The "latex fruit syndrome" is very common in the latex allergic individuals because 52% of latex allergic patients have allergic response to various fruits and systemic anaphylaxis occurred in 36% (Blanco et al., 1994; Tokuyama et al., 1997; Raihi et al., 1991; Levy et al., 1992; Bayerl et al., 1997; Holter et al., 1997) . It may be that the patient's reaction to fruit brings to light the latex allergic symptoms which may have been undiagnosed (Frankland, 1999).

The fruits that commonly cause allergic symptoms to pollen sensitive patients are not necessary the same as those in the latex allergic subject. Banana allergy can be found in young children, but this has not necessarily occurred subsequently with latex allergy (Bayerl et al., 1997). It is rare in infants but it has been much studied in relation to its cross-allegenicity with latex in adults and older children. In adults we have seen the latex allergic subject transform to become banana allergic after many years (Frankland, 1999). It can be shown by RAST inhibition that there is a dose-dependent inhibition with increasing concentrations of banana extracts (Raihi et al., 1991; Levy et al., 1992). There is an unusual way to have anaphylaxis from banana occurred in a latex allergic patient who was bitten by a monkey who had been eating banana (Bayerl et al., 1997). Lysozyme has been shown to be in detectable amounts in gloves, and this may be one of the cross-reacting allergens which account for the association of NRL and fresh fruit allergic responses (Holter et al., 1997).

To study the chemical and immunological properties of latex proteins, it is necessary to clone and express the dozen or more individual latex allergens. The proteins involved in rubber synthesis of H. brasiliensis are many, but profilin, the pan-allergen of fresh fruit and enzymes such as chitinases, glucanases and lysozymes, and papain, may be concern for causing the latex fruit syndrome (Breiteneder and Scheiner, 1998).

Chapter 2

2.0 Materials and Methods

2.1 Case Study 1 (Mari, et al., 2007)

2.1.1 Allergenic Extracts and Allergenic Molecules

All patients underwent a standardized SPT with a commercial NRL extract (Stallergènes, Antony, France), along with a standard panel of pollen and non-pollen allergen extracts (Allergopharma, Reinbeck, Germany; Stallergènes), as previously reported (Mari, 2001; Mari, 2002). NRL extract and NRL extract spiked with rHev b 5 (Lundberg et al., 2001) were used for IgE detection on a CAP System (Phadia, Uppsala, Sweden). Since extracts were commercially available at different times during the course of the study, it was not possible to avoid different batches. Escherichia coli -produced recombinant molecules Hev b 1, Hev b 2, Hev b 3, Hev b 5, Hev b 6.01, Hev b 6.02, Hev b 8, Hev b 9, Hev b 11, and native bromelain (nBRO) were obtained from Phadia and were used for IgE detection on the Phadia CAP System.

2.1.2 Patients, Skin Testing and Sera

A total of 6,126 subjects, aged 2-81 years, with a male/female ratio of 1.03, were enrolled in the study. All of them were unselected consecutive subjects referred to an outpatient allergy center of the Italian National Health Service for respiratory symptoms suggesting an allergic disease. Subjects reporting symptoms related to NRL exposure were excluded from the survey. Demographical (age and gender) and clinical (respiratory and food ingestion-related symptoms) data were recorded for all patients at history taking. Patients with a positive SPT to NRL were surveyed again for experiencing any allergy symptoms when exposed to NRL-containing products. For these asymptomatic NRL-sensitized subjects, a follow-up for checking any outbreaking symptoms was established for a period between 3 and 6 years. Patients underwent SPTs with a standardized procedure for allergenic extracts and molecules (Mari, 2001). A positive reaction was recorded for a wheal of at least 3 mm in diameter and greater than the negative control measured 15 min after performing the test. SPT reactivity was classified by comparative evaluation with histamine reactivity (Mari, 2001). Oral informed consent for skin testing and blood sampling was obtained from patients or caregivers during the allergy consult. Following SPTs, sera were collected from 475 patients, either NRL SPT-positive or control patients who had granted informed consent to blood drawing for an in vitro diagnostic procedure, and stored at -20 ° C.

2.1.3 IgE Detection

Specific IgE to NRL extracts, NRL extracts plus rHev b 5 (latex+), to a panel of NRL recombinant allergenic molecules (rHev b 1, rHev b 2, rHev b 3, rHev b 5, rHev b 6.01, rHev b 6.02, rHev b 8, rHev b 9, and rHev b 11), and to native nBRO will be detected by using ImmunoCAP System following the manufacturer's instructions (Phadia) (Adriano Mari, et al., 2007). nBRO, a rarely allergenic glycoprotein, will be used to determine the levels of IgE to crossreactive carbohydrate determinants (CCDs) (Mari, 2002) . Values equal to or greater than 0.35kAU/l were considered positive for ImmunoCAP determinations (Maria et al., 2003).

2.1.4 Separation of B serum and C serum

Fresh Hevea latex will be prepared and the latex will be centrifuged at 44,000 g at 4 ° C for 1 h to obtain three main fractions: a top fraction containing rubber particles, an aqueous phase, the C serum, and a bottom fraction, the B serum, containing specialized organelles called 'lutoids'. The aqueous phase will be collected and centrifugation will be repeated. The clear aqueous C serum will be freeze dried and stored at -20 ° C. The bottom fraction containing the lutoids will be resuspended in 0.4 M mannitol, resedimented and subjected to repeat alternate freezing and thawing to rupture the lutoids. The fluid content of the lutoids, the B serum, will then recovered by centrifugation (Wagner et al., 2004).

2.1.5 Prepare the fractions of C serum

A total of two fractions of the B serum (B1, B2) and four fractions of the C serum (C1-C4) will used for immunoblotting. These fractions will be generated as follows. One hundred milligrams of the latex C serum will be dissolved in 20 m M Tris/HCl, pH 7.5. Proteins will be precipitated by adding (NH4)2 SO4 to a 25%, then to a 50%, and finally to a 75% saturation. Precipitation will be performed by stirring the samples for 30 min at 4 ° C with the (NH4)2 SO4 and subsequent centrifugation at 12,000 g for 20 min at 4 ° C. Precipitates will dissolved in 20 m M Tris/HCl, pH 7.5, and supernatants will be used for further precipitations. The resuspended precipitates will be obtained by addition of (NH4)2 SO4 to 50 and 75% saturation then will be desalted by a PD-10 column (Phadia) and applied to a MonoQ HR5/5 column (GE Healthcare, Little Chalfont, UK). Proteins will be eluted using a linear gradient of 0-1 M NaCl in 20 m M Tris/HCl, pH 7.5. Proteins derived from the 50% (NH4)2 SO4 precipitate was eluted with 150-200 m M NaCl will be pooled to fraction C1, proteins eluted with 300-500 m M NaCl to fraction C2. Fraction C3 contained proteins eluted with 300-450 m M NaCl, whereas fraction C4 contained proteins eluted with 450-550 m M NaCl; both C3 and C4 will be derived from the 75% (NH4)2 SO4 precipitate (Mari et al., 2007).

2.1.6 Prepare the fractions of B serum

Latex B serum (100 mg will be dissolved in 20 m M Tris/HCl, pH 7.5) will be directly applied to anion exchange chromatography yielding two fractions. Fraction B1 will contain unadsorbed proteins and fraction B2 proteins will be eluted with 150-250 m M NaCl. Protein extracts (50 µg per lane) will be separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE, 12%) under reducing conditions and will be transferred to Protran nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany) for immunodetection (Mari et al., 2007).

2.1.7 Immunoblotting

Immunoblots will be performed using 40 m M Na 2 HPO 4 , 7 m M NaH 2 PO 4 , 0.5% BSA, 0.05% w/v sodium azide, 0.5% w/v Tween-20, pH 7.5, for blocking, washing and antibody dilutions. Blocked membranes will be incubated with patient and control sera diluted 1: 5. Bound IgE will be detected using 125 I-labeled rabbit anti-human IgE (IBL, Hamburg, Germany), diluted 1: 20. Bound immunoglobulins will be visualized on Biomax MS film (Kodak, Rochester, N.Y., USA) (Mari et al., 2007).

2.1.8 Data Management and Statistical Analysis

Data were stored in customized databases and subsequently retrieved by means of study-tailored query programs. Statistical analysis was carried out by means of GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, Calif., USA).

2.2 Case Study 2 (Maria, et al, 2003)

2.2.1 Subject Selection

     43 patients that allergic to latex (consists of 40 women and 3 men) and they have a mean age of 38 years were selected for the study. All of them had a history of allergy to latex manifested by urticaria, rhinitis, bronchial asthma or anaphylaxis diagnosed by menas of clinical history and confirmed by positive skin tests to latex. Thirty subjects with a mean age of 33.2 years with a negative latex allergy history, skin test and negative serum-specific IgE to latex were selected as healthy controls. Subjects with infectious, conjucntival, cardiologic, hepatic and endocrinological diseases were excluded. All the allergy patients stopped from intake of antihistamines at least 5 days before blood sampling. Subject that took astemizole stopped 6 weeks before while corticosteroids stopped at least 1 month before (Maria et al., 2003).

2.2.2 Allergenic Extracts

     Aqueous sterile filtered standardized extracts of latex devoid of any preservatives (Bial Aristegui, Bilbao, Spain) at the initial concentrations of 10, 1, 0.25, 0.062 mg/ml, and the final concentrations of 5, 0.5, 0.125, 0.031 mg protein/ml were used, for Ag-specific sLT production detected with the cellular allergen stimulation test (CAST) and Ag-induced basophil activation test (FAST). The latex extract was analysed using SDS-PAGE and Hev b 5 allergen was identified (Maria et al., 2003).

2.2.3 In vivo Skin Tests

     Skin prick test were performed with latex extracts 10 mg/ml prepared earlier using ALK Abello lancets. Histamine hydrochloride (10mg/ml) was used as positive control and 0.9% NaCl was used as negative control. The results were read after 15 minutes. A wheal that appeared with a diameter equal to or greater than 3mm compared to negative control was considered positive (Maria et al., 2003).

2.2.4 In vitro Tests

2.2.4.1 IgE Determinations

     Specific and total serum IgE determinations were performed by enzyme immune analysis CAP-FEIA (Pharmacia-Upjohn, Uppsala, Sweden), following  the manufacturer's instructions. Specific IgE to latex equal to or higher than 0.35kU/l was considered positive (Maria et al., 2003).

2.2.4.2 Flow-Cytometric Cellular Allergen Stimulation Test

     50µl of patients' cell suspension were incubated with 50µl of aqueous extracts of latex (Bial Aristegui, Bilbao, Spain) at four different concentrations which is 5.0, 0.5, 0.125 and 0.031 mg protein/ml, in corresponding wells. A monoclonal anti-IgE antireceptor antibody (Buhlmann) at a final concentration of 1µl/ml at 37°C for 40 minutes was used as a positive control. 50µl of stimulation buffer was added to another well and the basal value without stimulation was evaluated (Maria et al., 2003).

The reaction was then stopped and plates were centrifuged for 5 minutes at 4°C, 1,000g. The basophils from the pellet were double-labelled by adding anti-CD63 PE- and anti-IgE FITC-labelled antibodies. The erythrocytes were incubated for 30minutes at 4°C and then were lysed. Flow-cytometric analysis of surface markers was performed at 488nm on a FACScan flow cytometer equipped with a 15-nW argon ion laser (Becton Dickinson) and analysed by CellQuest software. On the histogram defined by forward scatter and side scatter, the initial cell gate was defined by a bit map around lymphocytes. The second gate was defined around cells showing high-density anti-IgE label, identifying them as basophils. In each assay, at least 500 basophils were assessed. The other parameter analyzed on the identified basophils was CD63 and an example of such analysis is given in figure 2.1 (Maria et al., 2003).

Example of the FAST analysis in 1 patient as a scatter plot of the joint expression of the anti-IgE and CD63 of at least 500 unstimulated basophils (basal response), an equal number of basophils stimulated with 0.5, 0.125 and 0.031 mg/ml latex extract and basophils treated with monoclonal anti-IgE antibody as positive control.

     (Adapted from Maria et al., 2003)

     The cutoff points for anti-IgE FITC and anti-CD63 PE were established individually for each selected subject according to the start of the fluorescence peak in the positive control; threshold values are usually situated around a fluorescence intensity >102 (Maria et al., 2003).

2.2.4.3 Antigen-Specific sLT Production (CAST)

     For allergen-induced sLT ELISA quantification, instructions of the assay's manufacturer (CAST-ELISA, Buhlmann Laboratories) (Weck et al., 1993) were followed. The same concentrations as in FAST were used but the highest concentration (5mg/ml) was soon eliminated as it produced an unspecific leukotriene release. The result of latex at the lowest concentration was eliminated also as it produced a very low leukotriene release in 30% of the patients. Results that higher than 300Ñ€g/ml were considered as positive but only if they produced a stimulation index (SI) greater than 4 (SI=antigen-specific response/basal response). This condition was included in order to eliminate unspecific leukotriene. In order to avoid any bias, the three diagnostic tests were done by different people, none of them knew the results of the other tests (Maria et al., 2003).

2.2.5 Statistical Analysis

     The data were analyzed through the statistical program SPSS 10.0. The results of the descriptive study were expressed as median values, 25th to 75th percentile, since according to the results of the Kolmogorov-Smirnov test none of the variables were considered to stem from a normally distributed population. The comparison of means between both groups was done by means of the U-Mann-Whitney test for nonpaired groups and the Wilcoxon test for paired groups. For evaluating the correlation between FAST, serum-specific IgE, skin tests and sLT release in the patient group, Spearman rank correlation test was used. The reproducibility of FAST results was assessed by the intraclass correlation coefficient. This test measures the reproducibility between two diagnostic techniques with continuous results by creating two new variables: the difference between the determinations and the median of both of them. Results are considered acceptable when this coefficient shows values between 0.4 and 0.75, and excellent when the values are over 0.75. Values of p<0.05 were considered statistically significant (Maria et al., 2003).

     To obtain a specific cutoff point for the significant percentage of activated basophils, a receiver-operating characteristic (ROC) curve was used. The optimal cutoff point calculated by the ROC combines sensitivity and specificity in such way that the curve is remotest from the diagonal. However, the actual cutoff point chosen aimed at high specificity values instead of sensitivity since allergy to latex is a low prevalence pathology (Maria et al., 2003).

2.3 Case Study 3 (Marie-Laure et al., 2005)

2.3.1 Subjects Selection

     Natural rubber latex allergic patients  (n=46) were selected from Montpellier area for this study. All patients had a typical history of latex allergy with contact urticaria, rhinitis, asthma corroborated with positive skin prick tests and/or latex specific IgE (Marie-Laure et al., 2005).

     Allergic patients (n=33) without a history of latex allergy and with negative latex specific IgE and latex skin prick tests served as the control group.Subjects were asked not to take any medication at the time of analysis. The blood sampling was performed at the same time as the Basotest analysis (5ml were drawn into heparinised tubes, vacutainer, Becton-Dickinson) on as well as blood sample (5ml on dried tube, Becton-Dickinson) for specific latex IgE measurement (Marie-Laure et al., 2005).

2.3.2 In vivo Skin Tests

     Skin prick tests were performed with a latex standardized extract (100IR Stallergenes S.A., Antony, France) according to standard procedures. The extract contained 22µg of proteins per ml. Codeine phosphate was used as a positive control. The results were analyzed after 20minutes. The positivity was established when the size of wheal induced by the latex extract was 2/3 of that of the positive control (Marie-Laure et al., 2005).

2.3.3 Latex Specific IgE Determination

     Serum samples were stored at -20°C up until the measurement of latex-specific IgE. The CAP-FEIA K82 System® (Pharmacia Diagnostics, Uppsala, Sweden) was used and the instructions on the package insert were followed. Specific IgE to latex equal to or higher than 0.35kU/I were considered as positive (Marie-Laure et al., 2005).

2.3.4 Basotest Flow Cytometric Cellular Allergen Stimulation Test

     Basotest was used for the quantitative determination of in vitro basophil degranulation. Heparinized blood (100µl) was first incubated with a stimulation buffer for 10minutes at 37°C, and then with 100µl allergen solution diluted in a saline solution of the same extract of latex used for prick tests (without para-formaldehyde Stallergenes S.A., 100IR/ml) for 20minutes at 37°C. A dose-response curve was performed with 100, 50, 30, 10, 5 and 1µl of latex allergen (100IR/ml diluted in saline solution, 100µl final volume). The chemotactic peptide formylmethionylleucylphenylalanine (fMLP) was used as a positive control and the phosphate-buffered saline (PBS) solution served as a negative control. The activation process was stopped by incubating the blood samples at 4°C for 10minutes. The samples were then incubated for 20minutes at +4°C with 20µl of phycoerythrin (PE)-conjugated anti-IgE and fluorescein isothyocynate (FITC)-conjugated anti-gp53. Erythrocytes were subsequently removed by the addition of 2ml of lysing solution (Becton-Dickinson). Cells were washed twice with PBS solution and resuspended in 200µl of PBS solution and analyzed within 1hour by cytofluorimetry  (FASCalibur, Becton-Dickinson). The basophil population was gated on the PE anti-IgE positive cells and the expression of gp53 (CD63) was analyzed on this population. Acquisition was performed on 1,000 cells for each sample and results are given as the percentage of basophils (IgE-positive cells) expressing gp53 (CD63) (Marie-Laure et al., 2005). The theoretical cut-off of 15% of IgE-positive cells expressing CD63 in at least one of the concentrations was first used according to previous results and the notice of the kit (Paris-Kohler et al., 2000; Saporta et al., 2001).

2.3.5 Statistical Analysis

     The results were analyzed using the SAS software (version 6.1; SAS Institute Inc). p<0.05 was considered statistically significant. Results were expressed as median (25/75 percentiles) for quantitative items. χ2 analysis was used to compare frequencies. The Mann-Whitney U test was applied when appropriate. The sensitivity and specificity of the Basotest were calculated within the natural rubber latex allergic patients and the control group(Marie-Laure et al., 2005).

     ROC curves were established using mRoc software (Kramar et al., 2001) and the optimal cut-off point was determined by the Youde indice.

2.4 Case Study 4 (Hamilton et al., 1998)

2.4.1 Subjects Selection

     Recruitment of subjects was conducted over a 1 year period at 12 institutions across the United States (average 34 subjects per site). The investigators are members of the American Academy of Asthma, Allergy and Immunology's Latex Committee. Those that qualified for the study were classified into 3 groups on the basis of their clinical history: those not allergic to latex (n=180), those allergic to latex (n=124), or those with contact dermatitis (n=54). Skin test results from the contact dermatitis group, in which the primary clinical feature was a skin reaction confined to the area of glove contact, were not used in the data analyses for determining the diagnostic performance of the latex skin testing reagent. Because of requirements placed on the study by the FDA review panel to delay the enrollment of children for safety considerations, only 22 children (<18 years of age) were enrolled in the study. These included children with spina bifida who had a history consistent with latex allergy; 10 control subjects in the no latex allergy group with no known reactions to powdered latex rubber gloves, balloons, and catheters; and 2 subjects with contact dermatitis (Type IV) reactions (rash and itching) confined to the area of glove/skin contact (Hamilton et al., 1998).

2.4.2 Latex Skin Testing Reagent

Crude Malaysian Hevea brasiliensis (clone 600) tree latex was collected in sterile plastic bottles containing a nonhazardous (patented) Goodyear preservative (0.1 mol/L NaHCO3, 50% wt/vol glycerol, and 3 mmol/L cysteine with no azide). After 1 week of shipping on ice packs, the milky latex was ultracentrifuged for 1 hour at 4° C, and the yellowish latex "C-serum" was isolated. The Nonammoniated latex allergen reagent was filtered through a 0.22-mm Millipak 40 (Millipore), adjusted to 50% glycerin and 1 mg/mL total protein by the ninhydrin method, and stored at 2° to 8° C. General composition analyses were performed with a 12% nonreducing SDS-PAGE followed by a Western blot (Akasawa, et al., 1995) and a potency assessment in an ELISA inhibition assay analogous to the RAST inhibition assay was done (Bronzert, et al., 1994), with the E8 Nonammoniated latex allergen as a reference standard (CBER, FDA, Bureau of Biologics, Bethesda, Md) (Hamilton et al., 1998).

2.4.3 Study design

This project was conducted under Investigational New Drug application 6365 with a protocol approved by the Allergenic Products Committee of the FDA and by the institutional review board of each participating institution. The protocol was designed to blind the tester to the history taken by the investigator and to blind the subject as to the identity of the skin testing extracts. Initially, only individuals who had received no previous diagnostic skin or blood tests were included in the study (Hamilton et al., 1998).

However, this severely limited access to otherwise qualified subjects. This requirement was relaxed so that subjects with previous diagnostic tests could be enrolled but with the requirement that 2 investigators independently read the skin test results to minimize bias. After informed consent was obtained, blood was collected for serologic tests. Each subject completed a detailed questionnaire that examined their general atopic and latex allergy history status, the type and severity of their allergy symptoms after natural rubber latex examination and surgical glove use, and other known risk factors such as food allergies. It further examined the number of surgeries, frequency of glove use, and extent of exposure to common household rubber products, such as balloons and condoms (Hamilton et al., 1998).

A detailed description of the 2 most recent reactions to latex products was collected to identify the extent of exposure, rapidity of onset, and duration and severity of symptoms. The primary investigator assigned each subject to 1 of 3 groups (latex allergy, no latex allergy or contact dermatitis) on the basis of each subject's clinical history (Hamilton et al., 1998).

2.4.4 Skin Test

Baseline peak expiratory flow rate (PEFR) measurements were performed before skin testing. All skin testers were required to pass a validation test before participating in the study, which involved performing PSTs on (Kurtz, et al., 1998) nonatopic subjects with saline and histamine (1.8 mg/mL; Allermed, San Diego, Calif) in duplicate. In the validation process mean diameters of the wheal and erythema observed with histamine at 15 minutes were compared with population norms for the bifurcated needle. In the study PSTs were performed by applying glycerin-saline (negative control) and 1.8 mg/mL histamine (positive control) and NAL serially at 1, 100, and 1000 mg/mL to the volar aspect of the forearm, in duplicate, every 15 minutes.

All vials were coded "A" to "E" to blind the subject. A puncture was made through each drop by rocking the needle at 45° angles through 4 complete cycles, and the needle was wiped with alcohol between each skin test. A positive PST response was defined as one producing a greater than 2-mm wheal and a greater than 5-mm erythema above that caused by the saline control at 15 minutes after application. The mean diameters of the wheal and erythema were measured and, their perimeters were outlined with a fine tip rolling writer pen and transferred onto transparent tape (Transpore 3 inch, 3M Company) for a permanent record on the data forms. A repeat PEFR measurement was performed at any time during the study when it was deemed necessary and at the end of skin testing. A greater than 20% decrease in PEFR observed at any time during the study stopped all testing.

Only in cases where the history was discordant with the PST results (either a positive history with a negative PST result at 100 mg/mL or a negative history with a positive PST result), a 2-stage unblinded glove provocation test was performed as described previously to screen only for immediate upper and lower respiratory responses (Hamilton and Adkinson, 1997). In brief, the subject was equipped with plastic goggles and a silicone-based respirator mask equipped with 2 activated charcoal cartridges (mask model 72813, cartridge model 7251; 3M Company) to prevent ocular exposure or inhalation of latex allergen attached to glove cornstarch donning powder. The powdered latex examination glove used in the provocation studies at all (Kim, et al., 1998) study sites was shown to contain a high level of extractable latex allergen on the basis of RAST inhibition analysis (Bronzert et al., 1994) (mean latex allergen content ± SEM: 15,072 ± 1448 AU per glove; vinyl glove contains <1 AU/glove). Latex allergen was known to be attached to cornstarch donning powder by direct binding of latex-specific human IgE antibody to cornstarch particles16 collected from the glove used at all 12 study sites (data not shown).

In stage 1, 3 PSTs with saline were performed on the hand immediately before donning a high-allergen powdered latex examination glove on 1 hand and a synthetic (vinyl) glove on the opposite hand. Subjects were observed for skin symptoms over a 30-minute period. At stage 2, a provocational test was performed if no pruritus and no visually detectable erythema or swelling were observed by the investigator. The mask and goggles were removed and a new high-allergen powdered latex glove was blown up 3 times by the subject like a balloon and expelled gently each time into his own face. Each subject was then observed carefully over an hour period for objective evidence of any allergic symptoms. PEFR measurements were performed between the stage 1 and stage 2 provocation tests and at the end of the study to assess changes in lung function. Before leaving the site, each subject was queried for symptoms, given a diary card, and asked to contact the investigator at any time up to 48 hours to report any late asthmatic or delayed allergic reactions. A change in PEFR of greater than 20% from the subject's pretesting personal best was considered positive in this study, as well as in other studies (Vandenplas et al., 1995; Hamilton et al., 1998).

2.4.5 Serologic analyses

Total serum IgE level was measured by enzyme immunoassay (IMx; Abbott Laboratories, Abbott Park, Ill) and reported in nanograms per milliliter and as a percentile of the age adjusted nonatopic mean.18 IgE antibodies to common aeroallergens were measured in a single Phadiatop multiallergen screen (CAP System, Pharmacia-UpJohn, Kalamazoo, Mich) as a general marker for atopy. Natural rubber latex-specific IgE was measured by the 3 FDA-approved assays, and these results will be reported elsewhere (Hamilton et al., 1998).

2.4.6 Statistical analyses

All statistical analyses were performed by using SPSSx (SPSS Inc, Chicago, Ill). For all analyses, the NLA control group was used as a reference for comparison. Unpaired t tests were used to compare continuous variables, and a chi-squared test was used to compare categorical and binomial variables. All P values were 2-tailed, and those below .05 were considered significant. Sample size was chosen to allow an estimated sensitivity of greater than 95% to be established with a 95% confidence interval of ±3.5% (Hamilton et al., 1998).

2.5 Case Study 5

2.5.1 Subjects Selection

This study was conducted between May 2004 and April 2005 in a random sample of 23 out of 49 public health units in Florianopolis, Brazil. All 328 subjects who worked in the selected units were invited to participate in the study.

2.5.3 Questionnaire

After a brief explanation of the research project, they were invited to fill in a questionnaire on symptoms suggestive of latex allergy; the questionnaire took approximately 5 minutes to complete. There were no refusals to respond to the questionnaire.

After applying exclusion criteria (use of -blockers, antihistamines, tricyclic antidepressants, or corticosteroids in the 3 weeks prior the study, as well as pregnancy or breast feeding), 260 of the 328 individuals remained in the study and underwent SPT. The duration of SPT was 15 minutes on average for each test. The study was performed in compliance with the Declaration of Helsinki (1964) (World Health Organ. 2001) and the design was approved by the local ethics committee and included informed consent from all participants. The subjects were recruited from 2 groups with known differences in occupational exposure to latex: health care workers (nurses, pharmacists, physicians, dentists, and laboratory assistants) who had frequent contact with gloves or latex products and health care workers involved in administration (health unit director, accountant, financial director, and administrative assistant), who did not have frequent contact with gloves or latex products.

The questionnaire was adapted from the American Clinical Association of Allergy, Asthma and Immunology guidelines for latex allergy (Sussman and Gold, 1996). The questionnaire asked about age, sex, job category, current latex-glove-allergic symptoms, weekly frequency and daily hours of latex glove use, family and personal histories of allergic disorders (including asthma or rhinitis), symptoms of hand dermatitis (dryness, fi ssuring, swelling, pruritus, cutaneous rash), symptoms of fruit allergy (pruritus of the oral mucosa or local redness after eating avocados, bananas, kiwis, chestnuts, mango, melons, or peaches), multiple surgical interventions, sneezing or rhinorrhea associated with airborne glove powder or toy balloons containing latex allergen, immediate systemic, ocular, nasal, or pulmonary complaints, itching or redness associated with the use of condoms containing latex, direct mucosal or parenteral exposure to latex during medical procedures, or exposure to latex products such as diaphragms, balloons, shoe soles, rubber handles, rubber bands or elastics, or clothing (Table 1).

2.5.3 Skin Prick Test

SPT was performed with disposable sterile lancets on the volar surface of the forearm with latex antigen containing 2 mg of latex mix (Allergofar, Rio de Janeiro, Brazil). Histamine at 10 mg/mL (Allergofar, Rio de Janeiro, Brazil) and a sterile saline solution (NaCl, 0.95%) were used as positive and negative controls, respectively. SPT was considered positive when wheal diameters of at least 3 mm were obtained 15 minutes after puncture (Pepys, 1975). The tests were done in a room with access to emergency equipment and medical support.

2.5.4 Statistical Analysis

Statistical analysis included descriptive statistics of demographic and clinical characteristics of the population and logistic regression to predict SPT result from the questionnaire containing specifi c "yes/no" questions for past experiences with allergy. Statistical differences between variables were analysed by the 2 test. Various questionnaire items were tested, both separately and in combination, in order to identify those with best sensitivity, specifi city, and predictive values. A range of probability cutoff points from 0.1 to 0.5 was tested.

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