Recent advances in the understanding of the compositions and structures of allergens now make it possible to use allergenic components instead of allergenic extracts in allergy testing.
In this interview, Jean-Charles Clouet, Director of Assay Business Development & Scientific Marketing at Siemens Healthcare Diagnostics, discusses the role of these molecular allergens in allergy diagnosis.

Q. What is the prevalence of allergies, and are there any geographical disparities?
Allergic diseases and asthma represent a growing and major healthcare challenge worldwide, as reported by the recent World Allergy Organization (WAO) White Book. [1] The authors confirm the steady increase of allergic diseases during the last decades that now affect approximately 30–40% of the industrialized world’s population, with an especially high percentage among the youngest subjects (40–50% of school-aged children are sensitized to one or more common allergens).

For example, allergic rhinitis is one of the most common allergic conditions, impacting roughly 500 million people of all social classes and ages globally. [2] In Europe, a study showed allergic rhinitis prevalence at approximately 25%. [3,4] It is important to note that, although direct costs induced by allergic rhinitis are limited, the condition affects subjects’ quality of life and has significant impact on performance at work or school. Therefore, its overall economic impact is probably underestimated. [2]

Another recent study, this one surveying more than 38,000 children (up to 18 years old) in the United States, reported that 8% had food allergy, including a rate of about 6% for those aged 0-2 years, and more than 8.5% for those aged 14-18 years. [5] Worldwide, it is estimated that 220–520 million people may suffer from food allergy. [1]

These statistics illustrate the high prevalence of allergy worldwide and why the World Health Organization (WHO) ranks allergy as the fourth most common global chronic disease. [6]

Q. What are the most common clinical manifestations of allergy that demand further testing?
Subjects suffering from allergic diseases tend to develop IgE-mediated immune reactions to normally harmless substances called allergens. These can include tree pollens, grasses and weeds; foods; mites; animal danders; molds; insects; and drugs. Associated clinical manifestations range from mild to severe and affect the upper and lower airways, gastrointestinal tract and skin. The consequent allergic diseases may include rhinitis, asthma, allergic conjunctivitis, atopic eczema, food allergy, insect allergy, drug allergy and anaphylaxis. Some can even be fatal, in the cases of allergic reactions to certain foods, insect venoms or drugs.

The model for the “Allergy March” published in the late 1990s emphasized that the most common forms of allergic diseases in early infancy are gastrointestinal symptoms and skin conditions (e.g., atopic dermatitis) caused by food proteins, such as hen’s egg and cow’s milk. [7] Additionally, IgE reactivity to food allergens in early infancy is a strong predictor for reactivity to respiratory allergens later in childhood. Other forms, such as allergic rhinitis and reactions to aeroallergens, happen later in life (1–10 years).

In 2003, the European Academy Of Allergy and Clinical Immunology (EAACI) published a position paper on allergy in children recommending testing for all subjects with severe, persistent or recurrent “allergic symptoms” (irrespective of age), along with those requiring a prophylactic treatment,. Proposals to select relevant allergens based on the subject’s age were provided. [8] Additional position papers are available for other forms of allergic reactions, such as drug allergy (causing 20% of deaths due to anaphylaxis) or insect allergy (fatal reactions in up to 50% of individuals with no documented history of reaction). [3]

Q. What are the current testing methods?
The objectives of allergy diagnosis are to identify both the symptoms’ origin (i.e., is the reaction IgE-mediated?) and the offending allergen(s). Allergy diagnosis is multi-factorial and includes a detailed case history and in vivo (i.e., skin tests) and/or in vitro (i.e., allergen-specific IgE measurements) testing. For some allergens (e.g., foods), oral challenges may also be performed to support diagnosis of food allergy.

Skin tests and blood tests, performed by allergists and laboratories respectively, present their own advantages and limitations. Skin tests are highly sensitive, with results immediately available for the patient. However, patients must discontinue medications (e.g., antihistamine) prior to testing, and interpretations of skin-test results are highly subjective and depend largely on operator skills. In vitro tests have the advantage of providing precise, quantitative results for each allergen, validated through extensive internal and external quality-control procedures and programs.

It is important to note that “allergens” used for in vivo and in vitro testing procedures are still primarily allergenic extracts. Obtained by extraction of proteins from crude allergenic sources, these extracts consist of a mixture of known and unknown proteins. Due to molecular-biology techniques and research begun in the 1980s, it is now possible to better understand compositions and structures of allergens, to classify them into families of proteins and to obtain for a significant number of them more qualified and standardized materials called “molecular allergens” or “allergenic components.” Whether highly purified in native form from the allergenic source or produced via recombinant protein expression techniques, molecular allergens have ushered in a new era in allergy diagnosis.

Q. What is the role of molecular allergens in allergy testing?
Allergenic extracts allow the detection of specific IgE directed against an allergenic source. In contrast, molecular allergens permit detection of precisely specific IgE directed against the disease-eliciting component(s) of the allergenic source. Therefore, measurements of specific IgE against molecular allergens yield additional key information that cannot be obtained by testing allergenic extracts. In particular, use of molecular allergens can help allergists define a more personalized and relevant sensitization profile for each patient.

For example, testing with molecular allergens makes it possible to determine if a patient’s sensitization is genuine (i.e., specific to one allergenic source) or comes from a cross-reactivity to proteins that have similar structures and are present in different sources. This is an important consideration when assessing a patient’s risk of reaction to some allergic sources and recommending appropriate avoidance measures. Allergenic molecules can also help clinicians assess the severity of a patient’s allergic reaction and, in the case of food allergy, decide whether or not to perform an oral food challenge. Finally, allergenic molecules can help clinicians identify patients who will benefit from immunotherapy treatment and decide which allergens should be used for treatment.

Q. Is this new technology likely to shift the burden of allergy testing towards the lab and away from in vivo (i.e., skin-based) testing?
In vitro testing is an accurate complement or alternative to skin testing for most allergens. However, despite their growing number, molecular allergens are not yet available for all types of allergens. Using allergenic extracts in conjunction with in vitro and/or skin testing is still the only option for a large number of
allergenic sources.

Q. In your view, what is the ideal sequence of tests to optimize the early diagnosis and treatment of allergy?
First of all, it’s important to raise awareness among the public and in the physician community of the importance of early diagnosis to better prevent and treat allergic diseases. Equally important is educating both clinicians and the public on the availability of new diagnostic tools, such as molecular allergens, along with the best way to leverage them to improve patient care.

An “ideal sequence” should begin with a careful and detailed case history taken by an allergist. This is critical in deciding whether further testing is necessary. Also, the results of specific IgE measurements should always be analysed in conjunction with the patient’s clinical history, since an allergen sensitization does not necessarily imply a clinical responsiveness.

If the clinical history suggests an allergic reaction, the clinician generally will request detection of specific IgE against the suspected allergens. Testing will then be performed using allergenic extracts in most cases. The reason is that allergen extracts are complex heterogeneous mixtures made of major and minor allergenic determinants. In some instances, a few molecular allergens will be enough to replace the corresponding allergenic extract. Unfortunately, this is not the case for sources with more complex compositions and in situations in which as-yet unidentified determinants (even minor ones) could be of clinical importance for particular patients. Also, as already mentioned, allergenic components have not yet been developed for a large number of allergenic extracts.

Therefore, in the majority of cases, testing allergenic extracts as a first step is the best or perhaps the only option to detect all sensitized patients. If specific IgE against the allergenic extract are found, relevant molecular allergens should then be tested to provide more information, as explained above.

References
1. World Health Organization. White Book on Allergy 2011-2012 Executive Summary. By Prof. Ruby Pawankar, MD, PhD, Prof. Giorgio Walkter Canonica, MD, Prof. Stephen T. Holgate, BSc, MD, DSc, FMed Sci, and Prof. Richard F. Lockey, MD.
2. Bousquet J, et al. Allergic rhinitis and its impact on asthma (ARIA) 2008 update (in collaboration with the World Health Organization, GA(2)LEN and AllerGen). Allergy. 2008;63 Suppl 86:8-160.
3. Bauchau V, Durham SR. Prevalence and rate of diagnosis of allergic rhinitis in Europe. Eur Respir J. 2004;24:758–764.
4. Bauchau V, Durham SR. Epidemiological characterization of the intermittent and persistent types of allergic rhinitis. Allergy. 2005;60:350–353.
5. Gupta, R, et al. The prevalence, severity and distribution of childhood food allergy in the United States. Pediatrics. 2011;10.1542/ped.2011-0204.
6.I nternational classification of diseases (ICD). http://www.who.int/classifications/icd/en/.
7. Kulig M, Bergmann R, Klettke U, Wahn V, Tacke U, Wahn U. Natural course of sensitization to food and inhalant allergens during the first 6 years of life. J Allergy Clin Immunol. 1999;103:1173–1179.
8. Host, et al. Allergy testing in children: why, who, when and how? Allergy. 2003:58:1-11.

Non-invasive prenatal testing (NIPT) based on cell free fetal DNA (cffDNA) circulating in maternal blood is moving rapidly forward. With promises of improved safety, earlier detection and easier access to tests, NIPT has the potential to bring many positive benefits to prenatal care. Here we discuss the recent developments in this area.

by Dr Melissa Hill, Dr Angela Barrett, Dr Helen White and Professor Lyn Chitty

Non-invasive testing using cell free fetal DNA
Prenatal diagnosis of genetic conditions or aneuploidy has traditionally required invasive diagnostic tests [chorionic villus sampling (CVS) and amniocentesis] which carry a small but significant risk of miscarriage of around 1% and can only be safely conducted after 11 weeks in pregnancy. In 1997 Lo and colleagues identified the presence of cell free fetal DNA (cffDNA) in maternal plasma and in doing so opened the door to a safer approach to prenatal diagnosis whereby non-invasive prenatal testing (NIPT) of fetal genetic material could be performed using a maternal blood test [1].

The cffDNA is an attractive target for prenatal testing. In addition to avoiding the risk of miscarriage it is anticipated that NIPT will be available early in pregnancy as cffDNA can be detected from 4 to 5 weeks with sufficient levels for analysis by 7 to 9 weeks. The cffDNA emanates from trophoblast cells in the placenta and is pregnancy specific as it is cleared from the circulation within 30 minutes of delivery. It is now also evident that the whole fetal genome is represented in the maternal plasma, suggesting that tests for many genetic conditions will be possible [2].

The major barrier to developing specific prenatal tests based on cffDNA has been the relative concentration of the fetal material. The cffDNA represents only a small proportion (around 10%) of the cell free DNA that is present in the maternal circulation, as the vast majority is maternal in origin. As a result, it is difficult to determine what genetic information is specific to the fetus against the large background of maternal cell free DNA.
For this reason NIPT was initially limited to the identification of alleles present in the fetus but not in the mother because they were inherited from the father or because they arose de novo. These tests include fetal sex determination, which uses targets on the Y chromosome, fetal rhesus genotyping in Rhesus D (RhD) negative mothers, and paternally inherited or de novo autosomal dominant single gene disorders. All of which can be conducted using relatively straightforward molecular techniques. More recently, new technologies such as digital PCR and massively parallel sequencing (MPS) have allowed researchers to develop NIPT for single gene disorders where parents have the same mutations and for aneuploidies, both of which need to take into account the presence of the mother’s allele or chromosomes.

Early clinical successes with non-invasive testing
The two early success stories for NIPT have been fetal rhesus genotyping and fetal sex determination, which are performed using real-time quantitative PCR. Analysis of cffDNA in the plasma of RhD-negative pregnant women who have a past history of haemolytic disease of the newborn or have elevated levels of Anti-D antibodies has been used clinically to determine the fetal RHD status for almost a decade. Large scale validation studies demonstrate high specificity and sensitivity and fetal RHD typing of all RhD-negative pregnant women has the potential to become routine clinical practice in the next few years.

Similarly, NIPT for fetal sex determination is increasingly offered as standard genetic care for women with pregnancies at risk of genetic conditions that primarily affect a particular sex. Fetal sex determination informs the need for genetic diagnosis, allowing up to 50% of carriers to avoid unnecessary invasive testing, and is important for guiding pregnancy management in some conditions. The test has been shown to be reliable when performed after 7 weeks gestation [3], and clinical utility was demonstrated in a recent UK audit as only 32.9% of women subsequently underwent invasive testing [4]. Importantly for implementation, NIPT is viewed positively by women who have had the test and has been shown to be cost neutral compared to invasive testing, which means women can have the clinical benefits of the test at no extra cost to health services [4].

NIPT for single gene disorders
The first use of cffDNA for the diagnosis of a single gene disorder was for the autosomal dominant condition myotonic dystrophy [5]. NIPT has also been possible for other paternally inherited autosomal dominant disorders such as early onset primary dystonia and Huntington’s disease. NIPT for the autosomal dominant condition achondroplasia, which commonly occurs as a de novo mutation, has also been successful. NIPT for autosomal recessive or maternally transmitted autosomal dominant disorders is more difficult due to the need to distinguish between the maternal and fetal free DNA. Exclusion of the paternal mutation is possible in autosomal recessive conditions where the parents carry different mutations. If the paternal allele is detected, there is a 50% risk that the fetus will inherit the disorder, and invasive testing would be recommended. If the paternal allele is not detected invasive testing is not required.

It is now clear that NIPT can be successfully applied to recessive conditions where parents carry the same mutation using digital PCR, which allows high copy number counting and quantification of alleles. Digital PCR requires the dilution of template DNA to an average concentration of less than one molecule per well, and hundreds to thousands of replicates of a PCR reaction are analysed. The mutation status of the fetus is predicted with an approach known as relative mutation dosage (RMD), which is based on the premise that if both the woman and the fetus are heterozygous there will be an allelic balance between the wild type and normal alleles; if the fetus is homozygous for either the mutant or the wild type allele there will be an over-representation of one or the other [6]. Another approach to identify single gene disorders non-invasively that we may see more of in the future is MPS, which has been used to determine the inheritance of two different β-thalassemia alleles [2].

NIPT for aneuploidies
Prenatal screening and diagnosis for fetal aneuploidy is offered routinely to all pregnant women in many countries to detect trisomy 21 (Down’s syndrome), trisomy 18 (Edwards syndrome) and trisomy 13 (Patau syndrome). The extensive scale of current prenatal screening programmes for these conditions means that success in developing accurate NIPT for aneuploidies will transform antenatal care. Several approaches have been explored including fetal specific epigenetic markers and SNP based methods. The most promising approach to date utilises MPS, which allows large scale single molecule counting to detect the increase in the number of sequences that result from the trisomic chromosome.

The first successful proof-of-principle studies utilising MPS to detect fetal aneuploidy from maternal plasma were published in 2008 [7,8]. Using this methodology, millions of short DNA sequences are generated from genomic locations. The sequences are then compared with the known human genome sequence, to establish how many sequences have been derived from each chromosome. For example, by comparing the total number of uniquely mapped chromosome 21 sequences obtained from a cfDNA sample with the number obtained from a normal genomic DNA sample, very small increases in the amount of chromosome 21 can be detected in the cfDNA sample if the fetus carries an additional chromosome 21.

Several large scale validation studies have subsequently demonstrated high levels of sensitivity (100%) and specificity (98–99%) using MPS. Efforts to decrease costs and increase throughput have seen many groups use multiplexing of patient samples into samples libraries that are run on one lane of the sequencing platform (2–12 patients per lane). Another strategy to decrease costs has been the use of targeted or ‘chromosome-selective’ MPS approaches where the sequencing assay is targeted to non-polymorphic loci on specific chromosomes such as 18 and 21 [10]. Studies with targeted MPS also show the potential for greater accuracy with the use of a novel bioinformatic algorithim (FORTE) that considers the proportion of specific cffDNA in the samples and accounts for the prior risk of trisomy (taken from published data on maternal and fetal gestational age related risks) to predict the likelihood of fetal trisomy for each patient [9].

Following the success of the validation studies, NIPT for aneuploidy is being offered through commercial providers in some countries (Sequenom, BGI, Berry Genomics, Aria, LifeCodexx). It is not yet clear, however, how these new tests will be introduced more widely into antenatal care. At present the small false positive rate means that the test is considered an “advanced screening test” that should be confirmed by invasive testing. Other considerations for implementation include the cost of the technology, the gestational limits of the test and the structure of existing screening programmes. All of these factors will impact on whether NIPT is introduced as a replacement for invasive testing or as an adjunct to current screening tests offered to all women.

Conclusions
NIPT is rapidly bringing about dramatic changes to antenatal care. Fetal sex determination and RhD genotyping are now available as clinical services in a number of countries. Testing is already possible for some single gene disorders, and new technologies such as digital PCR and MPS are allowing the challenges of testing for recessive conditions and aneuploidies to be met. Successful implementation, however, will require more than the development of laboratory tests and we must consider ethical issues, research stakeholder views and assess implementation strategies to ensure NIPT is offered in a way that best meets women’s needs. For this reason studies such as the RAPID programme in the UK (www.rapid.nhs.uk) that look at all aspects of test development and implementation are important.

Notification
This article summarises a recent review published in Best Practice in Clinical and Obstetric Gynaecology: Hill M, Barrett AN, White H, Chitty LS. Uses of cell free fetal DNA in maternal circulation. Best Pract Res Clin Obstet Gynaecol. 2012 Apr 27 [Epub ahead of print].

References
1. Lo YM, Corbetta N, Chamberlain PF, et al. Presence of fetal DNA in maternal plasma and serum. Lancet. 1997; 350: 485–487.
2. Lo Y, Chan K, Sun H, et al. Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus. Sci Transl Med. 2010; 2: 61ra91.
3. Devaney SA, Palomaki GE, Scott JA, et al. Noninvasive fetal sex determination using cell-free fetal DNA: a systematic review and meta-analysis. JAMA. 2011; 306: 627–636.
4. Hill M, Lewis C, Jenkins L, Allen S, Elles R, Chitty LS. Implementing non-invasive prenatal fetal sex determination using cell free fetal DNA in the United Kingdom. Expet Opin Biol Ther. 2012; Suppl 1: S119–126.
5. Amicucci P, Gennarelli M, Novelli G, et al. Prenatal diagnosis of myotonic dystrophy using fetal DNA obtained from maternal plasma. Clin Chem. 2000; 46: 301–302.
6. Lun FM, Tsui NB, Chan KC, et al. Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma. Proc Natl Acad Sci U S A. 2008; 105: 19920–19925.
7. Fan HC, Blumenfeld YJ, Chitkara U, et al. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc Natl Acad Sci U S A. 2008; 105: 16266–16271.
8. Chiu RW, Chan KC, Gao Y, et al. Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. Proc Natl Acad Sci U S A. 2008; 105: 20458–20463.
9. Sparks AB, Struble CA, Wang ET, et al. Optimized Non-invasive evaluation of fetal aneuploidy risk using cell-free DNA from maternal blood. Am J Obstet Gynecol. 2012; 206: 319.

The authors
Melissa Hill PhD¹, Angela Barrett PhD¹,
Lyn Chitty MRCOG, PhD¹* and
Helen White PhD²

¹ Clinical and Molecular Genetics Unit, UCL Institute of Child Health and Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK
² National Genetics Reference Laboratory (Wessex), Salisbury District Hospital, Salisbury, UK

*Corresponding author
e-mail: l.chitty@ucl.ac.uk

Maternal screening is offered to all expectant women during the first or second trimester of pregnancy. The purpose of this screening is to test for fetal abnormalities including chromosomal abnormalities such as Down’s syndrome, Trisomy 18 and neural tube defects such as spina bifida. Testing is performed by taking a blood sample from the patient’s arm which is then tested for a combination of biomarkers. Clinical results in addition to the maternal age are considered and used to calculate the risk of Down’s syndrome.

by Leah Hoencamp and Lynsey Adams

Down’s syndrome is a genetic condition and occurs when an individual inherits an extra copy of one chromosome. This means that affected people have three copies of chromosome 21, where there should be only two. The extra chromosome causes characteristic physical and intellectual features. The reasons why an extra copy of chromosome 21 causes Down’s syndrome are not known, which is why screening is so essential.

A combination of tests is used to screen for Down’s syndrome. Two types of screening are available and which is used depends on the stage of pregnancy of the patient. These stages are divided into first and second trimester.

First trimester screening includes:
• Free beta-hCG
• Pregnancy associated plasma protein (PAPP-A)

Second trimester screening includes:
• Double test AFP and hCG)
• Triple Test (AFP, hCG and uE3)
• Quadruple Test (AFP, hCG, uE3 and inhibin A)

If the results generated from this screening appear within the ‘higher risk’ category, more definitive tests are needed to confirm a diagnosis, such as amniocentesis or a chorionic villus sample. These tests provide a definitive result and involve taking samples of fluid from around the unborn baby. However, it is a highly invasive procedure and carries a small risk of miscarriage.

Internal quality control in maternal screening
Quality control (QC) is a crucial part of any clinical testing programme to ensure the accuracy and reliability of patient test results. Quality control is designed to detect, reduce and correct deficiencies in the laboratory’s internal analytical process prior to the release of patient results and to improve the quality of the results reported by the laboratory. Quality controls are manufactured to mimic a patient sample and contain one or more analytes of known concentration. They are made using a base material normally human serum, bovine serum, urine or spinal fluid. A laboratory will use quality controls to validate the patient samples. If QC results are within their target range then patient results should also be accurate. Once validated, the patient results can be used for diagnosis, prognosis and treatment planning. If QC values are outside the target range, it may indicate a number of issues including inaccurate calibration, instrument failure, operator error or reagent issues. In the field of maternal screening, the main aim is to minimise the risk of false positive and false negative results, ultimately ensuring results obtained are accurate and reliable.

In any type of screening the majority of errors take the form of false positive or false negative results. In other areas false negative results are of more concern as the patient will be perceived as healthy and will therefore not receive the required treatment. However, in prenatal screening, false positive results are also of major concern. If a patient tests positive they may have to undergo an invasive amniocentesis procedure with risk to the fetus in order to confirm if a chromosomal disorder like Downs’s syndrome is present. It is clear that such screening requires a robust and reliable quality control procedure in order to avoid potential errors.

To facilitate the increased screening for Down’s syndrome, trisomy 18 and neural tube defects, Randox has developed the only commercially available multi-analyte; tri-level control specifically designed to cover both first and second trimester prenatal screening, with the following benefits:

• The unique combination of inhibin A and PAPP-A in addition to AFP, total hCG, free B-hCG and uE3 reduce the need to purchase separate controls thus saving money
• Manufactured from 100% human serum providing a matrix similar to the patient sample while reducing cross reactivity and ultimately shifts in QC values
• Three distinct levels of control are available, accurately covering the complete clinical range. The level one control contains suitably low levels of AFP whereas the level three control contains high levels of hCG. Moreover, the uE3 levels are in line with those typically found during the first twenty weeks of pregnancy
• True third party control providing an unbiased, independent assessment of performance. Highly accurate instrument specific target values and ranges are provided for the most popular analysers used in maternal screening
• Excellent reconstituted stability of seven days at +2–8 oC
• Excellent vial-to-vial homogeneity (%CV <1 %) • Suitable for first trimester double screen and second trimester triple and quad screens.
Internal quality control (IQC) will help ensure results are reliable. An inter-laboratory data management package such as Acusera 24.7 can be used to further ensure quality. An effective IQC and peer group reporting scheme will help improve your laboratory’s analytical performance, help meet regulatory requirements and most importantly ensure the accuracy and reliability of patient test results. Acusera 24.7 enables laboratories to monitor analytical performance, access peer group reports and compare results with other laboratories using the same quality controls, method and instrument.

External quality assessment in maternal screening
To further assess the performance of maternal screening tests, laboratories should also be involved in an external quality assessment (EQA)/proficiency testing (PT) scheme. External quality assessment (EQA) is an essential aspect of any laboratory operation. EQA measures a laboratory’s accuracy using ‘blind’ samples that are analysed as if they were patient samples. EQA provides a means of assessing the analytical performance of a laboratory compared to other laboratories utilising the same methods and instruments. Participation in an EQA scheme will help produce reliable and accurate reporting of patient results. Quality results will reduce time and labour costs, and most importantly provide accurate patient diagnosis and treatment. Such a scheme is of paramount importance during testing such as maternal screening.

Randox International Quality Assessment Scheme (RIQAS) offers a Maternal Screening Programme which is capable of monitoring all 6 parameters involved in first and second trimester screening. RIQAS is the world’s largest global EQA scheme with more than 20 000 participants in over 100 countries worldwide.

Effective screening is essential for the detection of fetal abnormalities including Down’s syndrome, trisomy 18 and spina bifida. However, equally important in this process for laboratories responsible for processing the results is quality control. Effective quality control will help reduce false positives and false negatives, thereby ensuring reliable results and improving care of the patient overall.

Abbreviations
AFP, alpha-fetoprotein; hCG, human chorionic gonadotropin; uE3, unconjugated estriol.

The authors
Leah Hoencamp BSc & Lynsey Adams BSc
Randox Laboratories
55 Diamond Road, Crumlin,
Co. Antrim, UK BT29 4QY
E-mail: marketing@randox.com

Iron deficiency and anemia are common side-effects of inflammatory bowel disease. Analysis of a number of components of the iron-metabolism pathway can aid a differential diagnosis.

by Professor Jürgen Stein

Iron deficiency occurs in about 60–80% of patients with inflammatory bowel disease (IBD), and anemia manifests in approximately one-third of patients. Anemia is thus by far the most common extraintestinal complication of IBD. In a recent review by Gisbert and Gomollón, study data showed the prevalence of anemia in patients with IBD to range from 16% to 74%, with a mean value of 16% in outpatients and 68% in hospitalised patients [1]. Goodhand et al. demonstrated in a more recently-published prospective trial that anemia and iron deficiency anemia (IDA) are particularly prevalent in children, the incidence of anemia being 70% in children, 42% in adolescents, and 40% in adults [2]. Iron deficiency was also found to occur more commonly in children (88%) and adolescents (83%) than in adults (55%).

The cause of anemia in patients with IBD is multifactorial [Table 1]. The two most frequent etiological forms by far are IDA (resulting from iron deficiency secondary to blood loss through the ulcerations of the intestinal mucosa, reduced iron absorption and reduced intake) and anemia of chronic disease (ACD), described for the first time by Cartwright in 1946 [3]. ACD is characterised by normal or reduced mean corpuscular volume (MCV), reduced serum iron, reduced total iron binding capacity (TIBC), normal to elevated serum ferritin level, and reticuloendothelial system (RES) stores that are elevated relative to total body iron. While vitamin B12-folate deficiency and drug-induced anemia (sulfasalazine, thiopurines, methotrexate, calcineurin inhibitors) are less widespread, these possibilities should also be considered (for extended reviews
see Stein and Diagnass 2010, 2011, 2012 [4–6]).

Depending on severity, it is differentiated into three stages: (I) depleted iron stores, (II) functional iron deficiency with iron-deficient erythropoiesis, and (III) iron deficiency anemia.

Stage I depletion of iron stores is not associated with functional problems. It is only upon transition to stage II (iron-deficient erythropoiesis) that iron deficiency becomes a disorder because the cells can no longer be adequately supplied with iron. In stage III, the deficient iron supply to the body’s cells is already so pronounced that hemoglobin concentrations fall below the normal range.

In principle, all compartments of the body’s iron metabolism can be conveniently monitored with routine laboratory methods:
• Iron stores: serum ferritin
• Iron transport: transferrin saturation
• Iron utilisation with erythropoiesis: e.g. proportion of hypochromic erythrocytes or reticulocytes
Serum iron concentrations are governed by a circadian rhythm and can be low even in cases of anemia of chronic disease (ACD). Its role in the work-up of iron deficiency is, therefore, obsolete.

Serum ferritin
Serum ferritin is an indicator for the iron stores contained in the reticulohistiocytic system. Determining the serum ferritin concentration serves to identify disorders of the cellular iron stores (total body iron stores). The reference range for women is 15–100 μg/L and 30–200 μg/L for men; a serum ferritin concentration of 100 μg/L represents about 1000 mg of stored iron. Reduced concentrations are a sign of iron deficiency: a serum ferritin concentration <15 μg/L is considered a sign of an absolute iron deficiency. Because both ferritin and transferrin belong to the family of acute-phase proteins (APP), these reference ranges do not apply to patients with active inflammatory bowel disease.

In the context of inflammatory processes and the increased release of ferritin from damaged tissue, there may be an increase in serum ferritin levels. Hence, patients who actually suffer from iron deficiency will appear to have a normal iron status. In such cases, ferritin concentrations of 15 (30)–100 μg/L should be considered suspicious for iron deficiency. The differential diagnosis should, therefore, be based on serial measurements of inflammation parameters that are independent of iron metabolism [erythrocyte sedimentation rate (ESR), CRP].

Transferrin/transferrin saturation
Disorders of iron transport can be identified by determining the transferrin concentration. Iron deficiency is usually associated with a reduced transferrin saturation (TSAT). TSAT, expressed in per cent, is the quotient of the iron concentration (μmol/L) divided by the transferrin concentration (mg/dL) in serum or plasma multiplied by 70.9 (fasting blood sample).

Transferrin saturation is a measure for the iron load of circulating transferrin, the plasma protein responsible for transporting iron from its storage site to the bone marrow. Thus, determination of transferrin saturation does not provide any information regarding the status of the iron stores and provides only an indirect indication of the extent of iron utilisation in the bone marrow. Under physiological conditions, 16–45% of transferrin molecules in plasma are “loaded” with iron (3–4 mol of iron per mol of transferrin). Saturations <16% are considered to represent a suboptimum iron supply for the erythropoietic process. A reduced transferrin saturation (<20%) is associated with a relatively good sensitivity (90%) for recognising iron deficiency states, with, however, only a relatively low specificity (40–50%). Because the measurement of serum iron and serum transferrin are both subject to fairly significant circadian effects, blood samples should always be obtained at the same time of day and repeated frequently. Serum transferrin levels are increased in patients taking oral contraceptive steroids and reduced with inflammation (negative APP), meaning that, in patients with acute or chronic inflammatory disorders, TSAT may be reduced despite normal iron stores. Soluble transferrin receptor
While all cells in the body are supplied with transferrin receptors, the bulk of these (80%) are found in the bone marrow. The number of transferrin receptors on the cell surface is an indicator for that cell’s iron requirements. In cases of functional iron deficiency, i.e. inadequate availability of iron for normal erythropoiesis, the number of receptors on the cell membrane is up regulated. Because the transferrin receptors are continuously shed from the cell membrane and pass into the plasma as soluble transferrin receptors (sTfR), the serum concentration of sTfR serves as an indicator of iron supply for erythropoiesis. TfR is up regulated in iron deficiency. In contrast to ferritin and transferrin, neither chronic inflammation nor liver damage has any effect on TfR. Elevated concentrations of sTfR are found in iron deficiency as well as every other expansion of erythropoiesis, including haemolytic anemia, the thalassemias and polycytemias. Conversely, sTfR concentrations are reduced in aplastic anemia and other conditions characterised by hypoproliferative erythropoiesis, such as renal anemia.

TfR-F index
The sensitivity and specificity of sTfR as a parameter for assessing iron-deficient erythropoiesis can be enhanced by the parallel determination of sTfR and ferritin, which can then be used to calculate the so-called TfR-F index. The TfR-F index is defined as the quotient of the concentration of sTfR (mg/L) and log serum ferritin (μg/L). This quotient represents a marker that is dependent on the status of the iron stores, the availability of iron for erythropoiesis as well as the erythropoietic activity. In individuals with a deficiency of iron stores, the TfR-F index is increased. Disadvantageous for the routine diagnostic use of the TfR-F index are its lack of uniform reference range (the reference ranges of the
individual components are assay-dependent) and the relatively high costs.

Hypochromic erythrocytes/reticulocyte hemoglobin
Determination of the cellular hemoglobin content of reticulocytes (CHr) and the proportion of hypochromic red cells (%HYPO) is a valuable marker in the temporal differential diagnosis of iron deficiency anemia. Because the maturation time for reticulocytes is 3–5 days in the bone marrow and 1 day in the peripheral blood, the drop in CHr represents a marker for current iron deficiency. By contrast, a decline in %HYPO, because it is dependent of the normal red cell life span of 120 days, reflects longer-standing iron deficiencies. Thus, CHr and %HYPO can be considered analogous to blood glucose and HgA1C determinations in diabetics.

Some blood count units (Adiva-120, Technicon H1, H2 und H3; Bayer, Leverkusen, Germany) have the capability, without significant additional expense, to measure the hemoglobin content of each individual erythrocyte and calculate the proportion of hypochromic red cells while at the same time assessing reticulocytes for their volume and hemoglobin content. In people without iron deficiency and in those in stage I, the proportion of hypochromic red cells (hemoglobin content <28 pg) is less than 2.5%. Values >10% are considered confirmatory for iron deficient erythropoiesis. The increase in %HYPO precedes microcytic changes in the blood count. CHr values <26 pg are also considered confirmatory for iron-deficient erythropoiesis.

Zinc protoporphyrin
A deficiency in available iron for erythropoiesis leads to a compensatory incorporation of zinc into the protoporphyrin complex [Figure 1], and the increased formation of zinc protoporphyrin (ZPP), because of its relatively strong fluorescence in whole blood, is easily measured using HPLC-coupled fluorescence detection. Individuals with iron store deficiency exhibit normal ZPP values as long as the erythropoietic process is adequately supplied with iron. The onset of iron-deficient erythropoiesis triggers continuously increasing ZPP concentrations. Concentrations <40 μmol/mol haeme are considered normal. Values of 40–80 μmol/mol haeme represent latent iron deficiency (hemoglobin normal); >80 μmol/mol haeme are associated with manifest iron deficiency. In severe cases, values up to 1000 μmol/mol haeme have been reported. Thus, ZPP determination not only recognises iron-deficient erythropoiesis but also quantifies it.

Hepcidin
The regulation of iron homeostasis in IDA, ACD and ACD/IDA involves the iron regulatory protein hepcidin, a type II acute phase protein. During inflammation, interleukin (IL) 6 induces hepcidin production which leads to a decrease in dietary iron absorption and macrophage iron release, leading to decreased circulating iron and impaired iron distribution within the body. Based on previous studies carried out in rats and humans showing elevated hepcidin-25 levels in ACD individuals and intermediate levels in ACD/IDA, it was assumed that measuring serum hepcidin levels could help differentiation between ACD and ACD/IDA.

Abbreviations
%HYPO, proportion of hypochromic red cells; APP, acute phase proteins; ACD, anemia of chronic disease; CHr, cellular hemoglobin content of reticulocytes; CRP, C-reactive protein; Hb, hemoglobin; IBD, inflammatory bowel disease; IDA, iron deficiency anemia; MCV, mean corpuscular volume; TSAT, transferrin saturation; (s)TfR, (soluble) transferrin receptor; ZPP;
zinc protoporphyrin.

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The author
Jürgen Stein MD, PhD
Crohn Colitis Clinical Research Center Rhein-Main
Frankfurt/Main, Germany
E-mail: J.Stein@em.uni-frankfurt.de