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.

References
1. Gisbert JP, Gomollon F. Common misconceptions in the diagnosis and management of anemia in inflammatory bowel disease. Am J Gastroenterol 2008; 103(5): 1299–1307.
2. Goodhand JR, Kamperidis N, Rao A, Laskaratos F, McDermott A, Wahed M, Naik S, Croft NM, Lindsay JO, Sanderson IR and others. Prevalence and management of anemia in children, adolescents, and adults with inflammatory bowel disease. Inflamm Bowel Dis 2012; 18(3): 513–519.
3. Cartwright GE, Lauritsen MA, Humphreys S, Jones PJ, Merrill I M, Wintrobe MM. The anemia associated with chronic infection. Science 1946; 103(2664): 72–73.
4. Stein J, Hartmann F, Dignass AU. Diagnosis and management of iron deficiency anemia in patients with IBD. Nat Rev Gastroenterol Hepatol 2010; 7: 599–610.
5. Stein J, Dignass A. Management of Iron Deficiency Anemia in Inflammatory Bowel Disease with Special Emphasis on Intravenous Iron. Practical Gastroenterol 2011; 35: 17–30.
6. Stein J, Dignass A. Management of iron deficiency anemia in inflammatory bowel disease – a practical approach. Ann Gastroenterol 2012, in press.

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