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What type of cystic fibrosis testing platform is right for your laboratory?
By Christine Tubb, CLS(NCA), CLSp(MB)
Cystic fibrosis (CF), a disorder of the exocrine glands, results from mutations in the cystic fibrosis gene, commonly known as CFTR (cystic fibrosis transmembrane conductance regulator).1 It is a chronic pulmonary disease that causes rapidly progressive, life-threatening infections of the respiratory system.2,3 The disease is characterized by dry, thick, sticky secretions in airways and ducts, eventually causing their blockage and subsequent inflammation and infection. This leads to failure of multiple organs, such as the pancreas, lungs, and sweat glands.1,4,5 Patients with cystic fibrosis also will often suffer from intestinal obstruction, diabetes, biliary cirrhosis, pancreatic insufficiency, growth retardation, and dehydration because of the amount of salt they lose in their sweat. It is the most lethal inherited disease of childhood, even though carrier frequency and incidence of CF vary with race and ethnic group.
Cystic fibrosis is inherited recessively; therefore, most parents who have a child with CF are carriers, having no signs or symptoms of the disease in their family history. The autosomal-recessive disease itself has a frequency of 1 in 3,300, and approximately 1 in 30 Caucasians in the United States is a carrier of ΔF508, the most common gene mutation that can produce CF. CF is far less common among African- and Asian-Americans.
This first gene mutation linked to CF was discovered in 1988. The CTFR gene codes for a ion channel protein that allows ions such as chloride to move from the inside to the outside of cells. Transport of chloride ions helps control the movement of water into mucus and other secretions, thereby affecting their viscosity. Loss (delta indicates deletion) of the phenylalanine (F) amino acid residue from the CFTR protein at position 508 alters its function. Approximately 70% of CF cases among Caucasians are due to the presence of two ΔF508 genes.3,56
Several testing platforms have become available for CF testing in clinical laboratories. Laboratories are looking for an assay’s cost, hands-on time, start-to-finish time, ease of use, and efficiency, along with several other criteria.2 There are four CF platforms that seem to be at the top of laboratories’ lists when it comes to testing for the CFTR. This review will look at the similarities and differences among these four testing platforms in order to help determine which platform will work best for which size laboratory. The four CFTR testing platforms examined and compared are as follows:
The eSensor Cystic Fibrosis Carrier Detection System (commonly known as CFCD) is an in vitro clinical multiplex genetic test system. Its prime functions are genotyping and detection of the 24 ACMG/ACOG (American College of Medical Genetics/American College of Obstetrics and Gynecology) cystic fibrosis mutations to determine whether or not the patient blood specimen is a carrier.2 Each blood specimen must be converted into genomic DNA. The genomic DNA is then converted to single-stranded targets by using multiplex polymerase chain reaction (PCR) amplification and exonuclease digestion. The single-stranded targets are combined with appropriate buffers that have allele-specific signaling probes, which are labeled with signaling molecules called ferrocenes.2,7 Once this mixture is complete, it is loaded into cartridges. These cartridges are unique because they have an array of electrodes that are bound to single-stranded capture probes. Each electrode contains capture probes for a single, specific mutation.
Once the cartridges are placed in the eSensor 4800 instrument, the single-stranded targets will hybridize to their appropriate complementary sequences of the signal and capture probes. In order for the target and probe complex to be detected, an alternation current voltammetry that gives off specific electrical signals from the hybridized signaling probes is used.2 After all of this occurs, the eSensor software then sorts out the signals from each mutation and reports them in a fashion that is easy for the user to understand. Each specimen’s report will state either the carrier or non-carrier status of that particular patient for each of the 24 cystic fibrosis panel mutations for which the eSensor can test.7
The Tag-It Cystic Fibrosis kit (commonly known as the Tag-It mutation detection kit for CFTR 40+4) uses multiplex PCR and multiplex allele-specific primer extension (ASPE).2 The kit comes with a bead mix consisting of 86 different beads that each have their own fluorescent signature and an oligonucleotide "tag" for specific ASPE hybridization of product.1,8 Each of the 5’-tailed ASPE primers has its own antitag oligonucleotide that is complementary to a "tag" oligonucleotide on a particular bead. Genomic DNA is amplified by multiplex PCR. The PCR products are treated with Exonuclease I to digest any unincorporated primers and shrink alkaline phosphatase to remove the 5’ phosphates of any nucleotides that are not incorporated. The treated PCR product is used in the ASPE reaction. This reaction contains biotin-labeled deoxycytidine triphosphate (dCTP) and 86 primers that have sequences specific for each allele assayed. The ASPE reaction also has a specific 3’ "tag" sequence to allow further bead attachment.
After this, the ASPE products are hybridized to the bead mixture and are then filtered in order to remove any free biotin-labeled dCTP and any unhybridized primers.8,10 The bead-captured ASPE products are incubated with reporter dye and the samples are read. Tag-It Data Analysis Software analyzes the fluorescence values of each sample in order to determine if any wild type or mutant alleles for each of the variations has been detected. In essence, the Tag-It Cystic Fibrosis kit is used to determine if a patient has mutation in his CFTR gene. This is important because it can confirm whether or not a patient has cystic fibrosis and also can help couples know whether one or both of them are carriers of the CFTR gene.1,8
Commonly known as the OLA, the Oligonucleotide Ligation Assay is an analyte specific reagent (ASR) that is centered around the hybridization of a PCR primer with an exact match to a target sequence. One oligonucleotide probe specific to the genotype and one common oligonucleotide probe both hybridize to the resulting amplicon. The ligation products are separated electrophoretically because the common probe has a fluorescent dye marker, and the genotype probe has a modifying 5’ tail. Finally, the signal is detected by using a genetic analyzer.8,9,10 OLA can detect up to 32 mutations in the cystic fibrosis gene.2,9
The CFTR InPlex ASR designed by Third Wave technology incorporates Third Wave Invader DNA chemistry. This chemistry uses enzymes called cleavases. These will recognize and cleave certain specific structures once two oligonucleotides are added to a nucleic-acid target at the same time. A second reaction will occur in which fluorescent resonance energy transfer (FRET) generates a detectable fluorescent signal. InPlex cards, which are microfluidics cards that contain dried Invader oligos/FRET cassettes, are used for both of these reactions. The end result is production of a signal that is detected using a fluorometer. InPlex can test for a total of 42 CF mutations.2
The following tables contain a brief synopsis of each platform’s procedure:
The InPlex assay seems to be easiest to use because it does not require many steps or transfers.2 OLA and eSensor follow closely with a few more transfers and steps in their processes.2,10 Tag-It’s protocol is not as easy but has advantages in other areas.1 InPlex requires the shortest amount of hands-on time, about 45 minutes.2 OLA comes in second at about 1.5 hours, followed by Tag-It ranging from 1.5 hours to 2.5 hours.6 The assay requiring the most hands-on time is eSensor’s.2 Shortest start-to-finish time is InPlex at about 3.5 hours. eSensor and OLA both take around 6.5 hours; the Tag- It assay takes 8 hours from start to finish.1,2,10
Currently, only eSensor and Tag-It are FDA cleared, and the eSensor is only cleared for carrier testing.1,7 The Tag-It, eSensor, OLA, and InPlex all successfully detect the ACMG/ACOG panel, which consists of 24 mutations; all have excellent specificity and sensitivity.1,2,10 InPlex, Tag-It, and OLA test for additional mutations, with Tag-It and InPlex tests for unique mutations for which OLA cannot test. The following chart shows total mutations tested for each:
Tag-It, InPlex, and OLA are all capable of using extracted DNA from blood spots. eSensor is not capable of using DNA extracted from blood spots.2 Tag-It and eSensor have not been fully tested using DNA extracted from buccal swabs.1,2 InPlex and OLA, however, have successfully used DNA extracted from buccal swabs in their platform testing.2,9
InPlex is the cheapest at $39; Tag-It is around $50 per year;2 OLA is a bit more expensive at around $64,2,8 and eSensor costs anywhere from $65 to $100 per 250 samples because Osmotech only offers a reagent rental agreement.2,7
Three of the four platforms are available for direct purchase. The InPlex direct-purchase instrumentation costs, which include the fluorometer, card bucket and clips, and card sealer are the least expensive of these four at roughly $13,000.2 The Tag-It Luminex 100xMAP system costs $45,000,1,2 and the OLA ABI Prism 3100/3130 genetic analyzer with 16 capillaries costs anywhere from $100,000 to $145,000.2,9 Carefully consider these and other aspects when choosing which platform for CF testing is right for your laboratory, weighing the advantages and disadvantages of each.
Christine Tubb, CLS(NCA), CLSp(MB), is a clinical laboratory scientist at the School of Allied Health Sciences, Department of Laboratory Science and Primary Care, Texas Tech University Health Sciences Center, Lubbock, TX.
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By John F. Halsey, PhD, and Michelle Altrich, PhD, HCLD(ABB)
Adverse drug reactions (ADRs) are defined as any unintended noxious or deleterious effect from the administration of a therapeutic drug at doses appropriate for standard therapy. These adverse reactions are common and often have an important impact on determining the safety and efficacy of pharmacologic agents.1,2,3 Such reactions account for 5% of hospital admissions and occur in 10% to 20% of hospitalized patients.4 This relatively high incident rate, coupled with current limited clinical-laboratory testing available for ADRs, has spawned significant interest and focus in actively researching new testing approaches.
Table 1. Adverse drug reactions (ADRs)
It is important to recognize that not all ADRs are allergy related. The spectrum of adverse reactions to drugs includes both immune and non-immune pathological mechanisms as indicated in Table 1. The term drug allergy should be limited to those ADRs where an immune mechanism has been clearly demonstrated. Immune-based ADRs also are variable with respect to symptom and mechanism of action. The Gell-Coombs classification system describes these types of immune mechanisms of tissue injury (see Table 3).
Table 3. Gell-Coombs classification system
Type I (IgE mediated) allergic reactions are of primary concern and most studied, since these reactions may lead to anaphylaxis, a life-threatening condition. One of the best-studied examples is penicillin allergy, which causes significant morbidity and mortality. Penicillin molecules are capable of combining directly with proteins in the body and stimulating immune cells. Once the person has been sensitized and has had time to produce IgE, the next administration of penicillin may activate the primed mast cells and basophils, and lead to the systemic release of histamine and other mediators (e.g., mast cell tryptase, leukotrienes — see Figure 1). To help confirm a presumptive diagnosis of allergy, clinical laboratories can provide tests for drug-specific IgE, including penicillin.
Figure 1. Schematic illustrating a drug allergen (Drug) interacting with IgE specific for the drug (IgE). This interaction results in mediator release (Histamine) by the basophil or mast cell.
Penicillin allergy is an interesting case since it is capable of eliciting both Type I and IV Gell-Coombs hypersensitivities.5 Type IV hypersensitivities are delayed and may occur after the patient has been taking the drug for multiple days. Type IV reactions are mediated by drug-specific T cells. Symptoms of Type IV reactions are generally cutaneous or skin-related in nature, with eczema and rashes as the typical clinical presentation. These patients may not demonstrate any Type I reactions.
Although many patients will report to their physicians that they have a drug allergy, this diagnosis will be incorrect in many cases. It is problematic to rely solely on the patient’s clinical history, since some ADRs may mimic allergic type reactions. These pseudoallergic (i.e., "anaphalactoid") reactions may be caused by direct effects of the drug on immune cells but are not the result of immune sensitization. Causes of pseudoallergic reactions commonly include opiates, aspirin, nonsteriodal anti-inflammatory drugs, and radiocontrast media. In addition, patients may have a rash or other inflammatory symptom caused by a coexisting medical condition, such as a viral infection, that may incorrectly be attributed to an adverse drug reaction or drug allergy. Of course, any ADR is clinically important and should be evaluated, whatever the actual mechanism.
Correctly making a drug allergy diagnosis is further complicated if the drug must undergo some bioactive transformation before it can be immunogenic and stimulate the immune system. Most small molecule drugs are not immunogenic in their native state and must be coupled in vivo to a protein in the body before they can activate the immune system. For example, the sulfa drugs are believed to require the formation of a sulfonamide-protein complex before sensitization can occur. In this case, the sulfa drug must be converted by liver enzymes to a reactive molecule that can link to proteins in the body to be of sufficient size to effectively stimulate immune cells. Finally, it also is possible that the patient’s adverse reaction is due to an additive or excipient in the final drug formulation, such as gelatin, rather than the drug itself. Such sensitivities can be difficult to sort out.
Allergic reactions to new biotherapeutics, such as monoclonal antibody drugs, are being increasingly reported.6 These biologically-derived drugs, especially monoclonal antibodies, are an important class of new drugs that have provided many new options for cancer treatment and other difficult-to-treat diseases. While the incidence of ADRs is low in most new biotherapeutic drugs, these monoclonal antibody drugs consist of large glycoprotein molecules; therefore, they have the potential for stimulating the immune system. The table "Examples of some biotherapeutic drugs with reported ADRS" lists examples of several important biological drugs for which the U.S. Food and Drug Administration (FDA) has required the manufacturers to provide a specific warning in the labeling because of reported ADRs.
Table 2. Examples of some biotherapeutic drugs with reported ADRs
*Giezen TJ, Mantel-Teeuwisse AK, et al. Safety-Related Regulatory Actions for Biologicals Approved in the
United States and the European Union. JAMA, 2008; 300(16):1891.
It is important to recognize that most new biotherapeutic drugs provide great benefit to the patient. The incidence of ADRs is low but the severity of the reaction can be life threatening. Therefore, drug developers continue to develop improved methods to reduce the immunogenicity of this important new class of drugs.
In addition to the risk of anaphylaxis from drug-specific IgE, the patient’s immune response to a biological drug or biotherapeutic can have other undesired consequences. Drug-specific IgG may be a significant problem in the management of the patient for two primary reasons:
The clinical laboratory of the future will likely be asked to measure the patient’s drug-specific IgG for many of these new biotherapeutics. These tests are already an important part of FDA-required testing being performed during clinical trials for many biotherapeutics.
When an ADR occurs during the initial exposure to a biotherapeutic drug, one usually predicts that the reaction was not an immune mediated reaction, since there was not sufficient time for the immune system to generate IgE. In the case of the important cancer biotherapeutic drug cetuximab (trade name, Erbitux), however, a serious, immediate anaphylactic reaction (IgE -mediated) following the initial infusion of a drug was actually observed in small percentage of the patients. Since these patients had not previously been exposed to the drug, the basis for the reaction was at first problematic. This unique medical mystery was eventually solved, however, by Thomas A. E. Platts-Mills, MD, and colleagues at the University of Virginia.7,8 They found an interesting link between beef allergy and the anaphylactic responses to cetuximab. When they tested the serum of all the patients who had reacted to cetuximab, they found these individuals had pre-existing IgE to cetuximab and beef allergen. The researchers also were able to detect specific IgE in a relatively high proportion of control subjects who had never been exposed to the drug. These individuals would possibly be at risk for an ADR if infused with cetuximab.
Cetuximab is a chimeric (i.e., mouse-human) monoclonal IgG antibody molecule and a large glycoprotein with many possible immunogenic structures or epitopes. The unique part on this monoclonal antibody drug responsible for these cross-reactions was determined to be the carbohydrate structure galactose-alpha-1,3-galactose; found on both the monoclonal antibody protein and beef proteins. Thus, patients who had acquired an allergy to beef, lamb, or pork, were also likely to have an allergic response to cetuximab.9
Few FDA-cleared test kits are available to investigate drug allergy so testing options are limited. Currently, the most widely available tests are to confirm a Type I drug hypersensitivity by measuring drug-specific IgE. Skin and patch testing also can be done to evaluate other immune mechanisms, including tests for Type IV or T cell-mediated sensitivities.
The use of ex-vivo live blood-cell testing is being evaluated by a number of laboratories and has the potential to confirm an immune-mediated mechanism for many drugs. Such tests can be used to assess immediate reactions when drug-specific IgE tests are not available.
These tests involve the challenge of basophils and/or leukocytes with the drug of interest and the measurement of biomarkers released on the cell surface. Multiple markers of activation can be measured (e.g., histamine, sulfidoleukotrienes, CD63, or CD203c). Drug-specific T cells that may be responsible for delayed drug reactions can be evaluated by T-cell proliferation, CD69 upregulation, or cytokine production.9 Currently, ex vivo tests with live blood cells are not available in an FDA-cleared kit; they are only available at specialized reference labs.
John F. Halsey, PhD, founder and former CEO of IBT Laboratories, is currently a clinical associate professor at the University of Kansas School of Medicine’s Department of Internal Medicine, Division of Allergy, Immunology, and Rheumotology. Michelle Altrich, PhD, HCLD(ABB), is clinical laboratory director at IBT Laboratories, and formerly held an appointment at the University of Virginia where she specialized in molecular and cellular immunology.
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By Narayan Nayak, PhD; Mark Van Cleve, PhD; and Nelson Thune
A child suddenly develops severe respiratory symptoms, and her concerned parents take her to their family physician. The doctor needs to determine quickly if the child has the 2009 H1N1 flu strain or the common cold. The doctor believes that the child has a cold and sends her home after taking blood to rule out H1N1, but the symptoms recur two weeks later. Is it another cold or a more serious chronic respiratory disease, such as allergic rhinitis or asthma? Specific IgE testing can help the physician determine if there is an allergic component to the illness and help to guide effective treatment. Patients who suffer from asthma and other chronic respiratory diseases may experience more severe and frequent attacks during a bout of the flu, putting them at greater health risk. In fact, patients with asthma and other chronic respiratory diseases are priority recipients for both the seasonal and the 2009 H1N1 flu vaccinations.
It is estimated that 400 million people worldwide suffer from allergic rhinitis, and 300 million suffer from asthma, including 6.3 million children in the United States. The incidence of asthma is also increasing rapidly. The most common form among children is allergic asthma; and if the disease is not treated in time, it can even be fatal. Early detection through allergy testing is a key to identification of the cause of respiratory symptoms, and all treatment strategies significantly improve when the identity and degree of allergen sensitivity are known as early as possible.
Allergic respiratory conditions account for more than 10% of per capita healthcare spending in the United States, and the allergy testing market is large and growing as a result.
Today’s automated in vitro-specific IgE laboratory tests provide accurate, comprehensive allergy-testing results to physicians without the need to perform traditional skin testing. In vitro testing can provide multiple results from one small blood draw, making them ideal for pediatric patients, and do not require a patient to discontinue medications prior to testing. Traditional methods are at best semiquantitative, while in vitro tests are quantitative, precise, and highly specific. For the patient, in vitro testing requires less time and discomfort and avoids the possibility of inducing a dangerous systemic allergic reaction.
An allergic reaction is triggered when allergens and allergen-specific IgE antibodies bind to receptors on basophils and mast cells, inducing the release of inflammatory mediators and resulting in the allergic response. In vitro serum-based allergy tests quantitatively measure the concentration of IgE directed against a variety of allergens, thus determining the potential for an allergic reaction to each allergen.
Advancements in enzyme immunoassay (EIA) technology have significantly improved the sensitivity and reliability of in vitro allergy testing. These tests are available for a wide variety of allergens such as foods, insect, or environmental contaminants. Current EIA tests provide a quantitative result for IgE, referenced to a World Health Organization standard, and expressed in kilounits per liter (kU/L). Today’s tests have an assay range of zero to 100 kU/L, providing results covering the range of non-symptomatic sensitivity to extreme sensitivity. The most recently introduced EIA tests can deliver a limit of detection, or LOD, as low as 0.043 kU/L and a limit of quantitation, or LOQ, of 0.07 kU/L. While these values are below the typical cutoff level for negative sensitivity, they indicate the ability of the test to reliably detect extremely low levels of specific IgE antibodies. They may also allow the physician to detect an emerging sensitivity and to treat the patient early enough to halt the "allergy march," whereby allergic sensitivity continues to increase and, ultimately, can initiate more severe symptoms or, potentially, asthma.
With a typical intra-assay coefficient of variation (CV) of 5% to 10%, precision is considered excellent. Most importantly, results obtained on different in vitro assay systems are quite comparable. Robert G. Hamilton, MD, director of the Johns Hopkins University Dermatology, Allergy, and Clinical Immunology Reference Laboratory, showed data at the 2009 American Association of Clinical Chemistry meeting demonstrating superb dilution linearity in results obtained on the three major systems used for in vitro allergy testing, and excellent correlation of quantitative results among systems. This data is indicative of a much better alignment among these leading manufacturers’ systems than previously reported in other publications.
Another important aspect of EIA-based allergy testing is that it is highly automated. Much of the sensitivity and accuracy of the testing is due to automation that allows operators with lower levels of training to produce more reliable results faster, and with less hands-on time.
In vitro allergy testing can also offer cost advantages versus traditional testing, an important attribute given current economic conditions and pending healthcare reform in the United States. Traditional testing often involves patient exposure to a large number of allergens (as many as 150) to screen for the patient’s specific allergy. This testing is then billed as an office procedure. In contrast, in vitro testing is often ordered by an ear-nose-throat physician or pediatrician in a targeted way involving a small number of core allergens to determine if the patient is sensitive (atopic). In some cases, the physician will be able to determine from this initial round of testing that the patient does not suffer from allergy-related disease, resulting in an effective diagnosis with substantial healthcare cost savings. If initial testing reveals that the patient is atopic, further targeted testing can then be performed to determine additional allergens to which he may be sensitive.
The key advantages of in vitro, serum-based testing will result in broader utilization worldwide as more allergy testing is performed by primary-care physicians, and more allergists use specific IgE testing to supplement skin testing. Current research includes investigations into the benefit of using recombinant allergens in testing and treatment. Work is also underway to evaluate the potential for using multiplex techniques to survey more allergens in each test, thus further driving down cost.
The demand for in vitro allergy testing is expected to continue to grow as the health threat posed by chronic respiratory diseases such as asthma and allergic rhinitis grows, and more patients gain access to this convenient form of testing. State-of-the-art, automated in vitro testing systems from multiple suppliers, delivering highly correlated results at very affordable cost are now available to laboratories to enable them to meet the rapidly growing need by physicians for sensitive, accurate, and reliable allergy testing.
Nayak Narayan, PhD, director, Systems Development; Mark Van Cleve, PhD, applications development manager; and Nelson Thune, general manager, are all employed at Hycor, an Agilent Technologies Division, Garden Grove, CA, where they have a combined total of more than 50 years of experience in the development of innovative allergy-testing systems.
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