Guideline for the laboratory diagnosis of functional iron deficiency (2013)

Functional iron deficiency (FID) is a state in which there is insufficient iron incorporation into erythroid precursors in the face of apparently adequate body iron stores, as defined by the presence of stainable iron in the bone marrow together with a serum ferritin value within normal limits (Macdougall et al, 1989). In its broadest sense this definition encompasses the partial block in iron transport to the erythroid marrow seen in subjects with infectious, inflammatory and malignant diseases, and is a major component of the anaemia of chronic disease (ACD). One form of FID, found in some subjects treated with erythropoiesis-stimulating agents (ESAs), has been the subject of numerous studies following the widespread use of these agents, especially in subjects with chronic kidney disease (CKD). The clinical assessment of iron status has largely been focussed on the level of iron stores, as reflected in the serum ferritin concentration. However iron in stores is metabolically inactive and is not only unavailable for immediate use but may be difficult to bring into use at all. The real clinical issue lies in active iron metabolism, the movement of iron from effete red cells and into further generations of developing red cells. It is nevertheless true that replenishment of iron lost from the red cell pool will be compromised and iron supply to the erythroid marrow will be suboptimal as iron stores become depleted. Irrespective of cause, inadequate iron supply leads to impaired haemoglobin production and a reduction in the mean cell haemoglobin (MCH) value that becomes apparent after several weeks of impairment. In contrast, it has long been evident that the adequacy of iron supply might be estimated from the haemoglobin content of the reticulocyte within a time span of a few days. With the widespread introduction of automated cell counters capable of measuring the numbers, volume and haemoglobin content of reticulocytes, many laboratories are now in a position to detect the early indications of a failure of iron supply in this way. In 2006 the National Institute for Health and Clinical Excellence (NICE) published a guideline entitled, ‘Anaemia management in people with chronic kidney disease (CKD).’ (NICE, 2006). Among tests recommended for the assessment of iron status was the percentage of hypochromic red cells (%HRC). This variable, which continues to be recommended in the updated guideline (guideline 114, NICE, 2011), has limited availability, whilst the reticulocyte measures mentioned above have become more widely available. It is thus timely to review the use of these newer variables, together with more established measures of iron status, in the management of patients with FID. The guideline group was selected to be representative of UK-based experts in the clinical and laboratory fields of iron metabolism, CKD, quality control and method evaluation. MEDLINE was searched systematically for publications in English from 1966-2011 using key words: functional iron deficiency and each of the parameters discussed. The writing group produced the draft guideline, which was subsequently revised by consensus by members of the Task Force of the British Committee for Standards in Haematology (BCSH). The guideline was then reviewed by a sounding board of UK haematologists and members of both the BCSH and the British Society for Haematology. Comments were incorporated where appropriate. The ‘GRADE’ system was used to quote levels and grades of evidence, details of which can be found in Appendix 1. The object of this guideline is to provide healthcare professionals with clear guidance on the management of FID, in particular with respect to patients with CKD but also to other disease states in which ESAs have been used. The guidance may not be appropriate to patients with inflammatory diseases and in all cases individual patient circumstances may dictate an alternative approach. This guideline is only applicable to adults, not children. The laboratory classification of iron status breaks down in the presence of inflammatory disease, as most of the variables used to define iron deficiency become abnormal despite the presence of adequate body iron reserves. ACD typically develops, a consistent feature of which is the retention of iron within body stores. As a result the supply of iron to the erythroid marrow becomes inadequate. This is the major form of FID; a second type often occurs when the erythroid marrow is stimulated by ESAs. Since the discovery of the iron regulatory peptide, hepcidin, a 25-amino acid peptide synthesized in the liver, our understanding of the biology of ACD has greatly improved (Goodnough et al, 2011). Hepcidin is upregulated in the setting of chronic inflammation and cancer, resulting in its increased synthesis in the liver stimulated by cytokines of which interleukin (IL) 6 is the most important. By degrading ferroportin, hepcidin decreases iron absorption from the gastrointestinal tract and decreases the accessibility of stored iron from macrophages. Where FID and inflammatory illness coexist it is likely that increased hepcidin synthesis will restrict the absorption of oral iron. Intravenous iron preparations might overcome this block. In patients treated with ESAs for the anaemia associated with CKD, the response rate improves when intravenous rather than oral iron supplements are given, and often allows a reduction in the ESA dose (Nemeth et al, 2004; van Wyck et al, 2004; Henry, 2010; Qunibi et al, 2010). Intravenous iron is routinely used in ESA-treated patients with CKD on dialysis and this practice is endorsed in national and international guidelines (NICE, 2011; National Kidney Foundation, 2006; Kidney Disease: Improving Global Outcomes (KDIGO) Anemia Work Group 2012). There are now a considerable number of red cell indices available for the assessment of iron status. Used in isolation as a diagnostic test, none is capable of differentiating between iron deficiency and FID. All are confounded by α°- and β-thalassaemia heterozygosity and in homozygotes and some heterozygotes for α+ thalassaemia. Patients with combined iron and vitamin B12/folate deficiencies or sideroblastic anaemia may also prove problematic. Once a diagnosis has been made, however, some of these variables may be used to monitor the response to ESA therapy and the requirement for iron. These indices can be divided into five groups; the traditional measures of MCV, MCH and mean cell haemoglobin concentration (MCHC): measures based on increased hypochromia: indices of reticulocyte volume and Hb content: red cell zinc protoporphyrin (ZPP) concentration: and recently introduced indices, such as red cell size factor (Rsf). The MCH is derived from the red blood cell (RBC) count and haemoglobin concentration (Hb), both of which are measured with considerable accuracy and precision by modern analysers. Values obtained from differing types of analyser are therefore largely interchangeable whereas MCV values, which are derived using differing analytical principles, are much less so. Unlike MCV, MCH is unaffected by several days of storage. As both MCV and MCH are derived from the entire circulating red cell mass, they are slow to change and have no value in detecting either the acute development of iron lack or the early response to iron therapy in patients treated with ESAs. The MCHC is of limited or no use in assessing changes in iron availability associated with ESA therapy. As originally identified using Siemens technology, hypochromic red cells are those with Hb <280 g/l. The clinical utility of this variable in detecting FID in patients with chronic renal failure treated with ESAs has long been recognized (Macdougall et al, 1992). A value of ≥6% was found to be superior to measurements of soluble transferrin receptor (sTfR), ZPP, ferritin and total iron-binding capacity (TIBC) in differentiating between iron-deficient and iron-sufficient patients with chronic renal failure receiving maintenance doses of ESAs (Tessitore et al, 2001). This value was incorporated into UK guidelines on anaemia management in this group (NICE, 2006). The measure is effective in the detection and monitoring of FID secondary to ESA therapy in anaemic subjects with advanced acquired immunodeficiency syndrome (Matzkies et al, 1999) and rheumatoid arthritis (Arndt et al, 2005). An increase in %HRC in subjects with low-risk myelodysplastic syndromes treated with ESAs may however reflect improved survival of a pre-existing population of abnormal hypochromic red cells, rather than ESA-induced FID (Ljung et al, 2004). Two other variables are now available for the assessment of hypochromia. One, %Hypo-He, is produced by some Sysmex blood counter analysers (XE 5000 and XN), and defines those red cells having MCH <17 pg. The measure has clinical utility in the differential diagnosis of anaemia (Urrechaga et al, 2009). The second, Low Haemoglobin Density (LHD%), is a variable based on a mathematical transformation of the MCHC value and is available on some Beckman Coulter instruments. Values correlate highly with those of %HRC (Urrechaga, 2010). The great increase in precision of the automated reticulocyte count and the provision of measures of immature reticulocyte fraction and the reticulocyte-specific indices of volume and haemoglobin content provide an opportunity to assess the effects of changing iron status on this transient population. The reticulocyte count itself cannot provide information about a patient's iron status. However a reticulocyte increase of ≥40 × 109/l from baseline by week four of ESA therapy in cancer patients has been shown to predict an adequate response, defined by a ≥ 6% rise in haematocrit above baseline, and it implies adequate iron stores (Henry et al, 1995). Automated blood counters capable of producing reticulocyte data generally group cells depending on RNA content. The immature reticulocyte fraction (IRF) is the component with highest RNA content. Immature reticulocytes are released during periods of intense erythropoietic stimulation, such as following haemorrhage or haemolysis, or in response to therapy with iron or ESAs. The IRF increases several days before the reticulocyte count (Davies, 1996) and is thus an early indicator of response to therapy. Nevertheless, the test is little used, probably because of a lack of standardization of methods and instrument- or method-specific reference ranges. CHr, the term used to describe the reticulocyte MCH as derived by Siemens analysers, was the first automated reticulocyte measure available for routine use. Among patients undergoing bone marrow examination for diagnostic purposes CHr had a better predictive value for iron depletion than MCV, serum ferritin or transferrin saturation values (Mast et al, 2002). CHr has been used as the standard against which other emerging variables have been assessed (Brugnara et al, 2006). Among subjects with presumed FID, CHr compared favourably with other measures of iron status in predicting a response to intravenous iron (Mittman et al, 1997; Chuang et al, 2003). The variable received US Federal Drug Administration approval in 1997 and was incorporated into the revised European Best Practice Guidelines for the management of patients with chronic renal failure (Locatelli et al, 2004). A target CHr of 29 pg was recommended (evidence level B). This value is indicative of the adequacy of iron incorporation into the developing erythron, although some patients with CHr values >29 pg responded to intravenous iron therapy, leading to a suggested cut-off value of 32 pg (Fishbane et al, 2001). In a study of sample stability, a small but statistically insignificant fall in CHr values over 24 h was demonstrated (Lippi et al, 2005). An alternative measure of reticulocyte haemoglobin content, Ret-He, is available on some analysers manufactured by the Sysmex Corporation. Although expressed in the standard unit of cellular haemoglobin content, (pg), Ret-He is a natural log transformation of Ret-Y, a measure of volume obtained from measurement of forward light scatter of reticulocytes and itself expressed in arbitrary units. A number of studies (Canals et al, 2005; Thomas et al, 2005; Brugnara et al, 2006; Garzia et al, 2007; Maconi et al, 2009; Miwa et al, 2010) have found excellent concordance between Ret-He and CHr in subjects with both iron deficiency and chronic renal failure. However Brugnara (2003) has reported that it is less clear that either low Ret-He or CHr values are predictive of response to intravenous iron or whether ESA usage can thus be reduced to the minimum required (Mast et al, 2008). Canals et al (2005) studied 504 patients with ACD or other iron-restricted states. Ret-He alone was able to distinguish iron-deficient and iron-sufficient subjects using a cut-off of 25 pg with reasonable sensitivity (0·76). However the groups that included ACD, mild iron deficiency anaemia and reduced iron stores showed significant overlap. The interquartile range for the ACD group in this study was below that of the reference range and the values of the ACD group were significantly different from those of the iron deficiency group. Although less sensitive (80% agreement with sTfR and sTfR/log ferritin values), a Ret-He cut-off of 25 pg may also help to distinguish iron deficiency (values <25 pg) from ACD (values >25 pg). A Ret-He cut-off value of pg is a better of response to intravenous iron than baseline serum ferritin or transferrin saturation values in CKD patients undergoing et al, 2010). This variable is derived from the of the of the of and It with CHr, with better sensitivity and for the detection of iron-restricted Patients with values were more likely to have ACD, those with values to have iron deficiency (Urrechaga, 2009). protoporphyrin (ZPP) is a of synthesis and that limits iron supply to the erythroid marrow or synthesis leads to an increased concentration of in circulating red cells. The incorporation of iron into protoporphyrin is the of and an increase in is an indicator of at this The measure is therefore and values in iron FID, and in many iron-sufficient subjects with α°- and β-thalassaemia et al, There are of the measurement of by the used method of to the of the with found in the presence of et al, and in chronic renal failure a and increase in values is seen as Hb below about and many subjects with or anaemia have values of iron status. These can be overcome by to or the Hb concentration to a standard but sample is to lack of not be used in isolation as a diagnostic test, but a diagnosis is it may be used to monitor response to therapy. The concentration the entire circulating red cell population and is thus less sensitive than %HRC or CHr to acute changes in iron availability (Fishbane of body iron stores is both as a diagnostic and to monitor the effects of therapy with iron ESAs. This may be by of the iron content in bone or by use of the serum ferritin studies using iron have been from as they are these days and can be An assessment of iron stores in the bone marrow can be by use of Although a test, assessment can be insufficient is or more be available for review et al, and few haematologists can they this number on was a major factor in a study that that over of of of stainable iron were et al, 2001). the presence of stainable iron not define much can be and incorporated into the developing bone marrow examination is and not such as and The serum ferritin has become the standard test for the assessment of iron stores in serum from from or and in there is a between the such that each of ferritin in serum is to of iron in stores variables may this and the level of stored iron in some the it is derived are of the of serum ferritin but the by which this variable from the true measure of the iron stores is less often the ferritin value cannot in CKD, is to when stores to supply the term implies the patient will not to iron have it may be to an of serum ferritin concentration to provide a et al, such that it may be to intravenous and some CKD patients may to this therapy despite ferritin There is also to that CKD patients with low serum ferritin have a compared to patients with values in the range et al, 2005). some patients with CKD may have iron in the liver and within the bone marrow available for et al, the setting of an of serum ferritin concentration above which intravenous iron is not is not guidelines use a value of (NICE, or range National Kidney Foundation, for CKD patients on ESA therapy, that above these values there is a of iron with further therapy. However anaemic CKD patients with ferritin of showed an increase in Hb when treated with intravenous iron et al, These patients all had transferrin saturation levels to the presence of FID. The indicator of in this group was the response to intravenous iron. The serum ferritin concentration was in predicting response to ESA therapy in anaemia et al, 2003). The transferrin receptor is highly expressed on erythroid levels are found in associated with an erythroid marrow et al, et al, and also in iron The clinical utility of sTfR measurement has been by the lack of agreement the both of and of used to of the recently first Health for sTfR this et al, 2010). The major clinical of the is in differentiating the anaemia of iron lack from that by which has little or no on sTfR values, and in detecting the presence of iron lack when the coexist et al, et al Although in some studies of sTfR not prove superior to the serum ferritin in the detection of iron deficiency in patient groups of those seen in clinical practice (Mast et al, et al, et al, a review that its use improves the diagnosis of iron especially in the presence of chronic disease or gastrointestinal et al, 2009). In patients with chronic renal failure and kidney disease not receiving iron or the sTfR concentration alone to that of serum ferritin in detecting those with iron deficiency et al, However in a group of patients receiving maintenance doses of and with the measure superior to ZPP, transferrin saturation and serum but to %HRC and CHr, in differentiating between iron-deficient and subjects (Tessitore et al, 2001). The a of the ferritin concentration to that of sTfR et al, was found to be superior to ferritin alone in predicting response to intravenous iron in renal patients on ESA therapy et al, 2006). This to overcome to the use of sTfR as a test in monitoring iron status during ESA therapy, that of the therapy itself leading to increased of the erythroid marrow et al, 2002). The has been used, with a measure of iron availability to the erythroid such as %HRC or CHr, in the production of a diagnostic 2002). can be used to monitor the effects or with iron or ESAs et al, 2006). The value is derived from serum iron and values and is the most widely used of the The serum iron concentration within of the of inflammatory illness and also but to a This in reduced values that for the of the Although a measure of iron in transport and not of iron in stores, values of the for iron in the setting of anaemia treated with ESAs (Macdougall et al, Used in has sensitivity and in detecting those to intravenous iron et al, 1997; et al, although with variable improved accuracy and may be a alternative where and reticulocyte variables are not available. levels are not routinely in the setting of CKD and are not recommended in patients with anaemia and CKD Kidney Foundation, 2006). is clear is that for patients with the of CKD with anaemia there is a lack of serum However low levels have limited clinical value that some and are associated with of levels et al, et al, In a study by et al patients with and baseline levels were most likely to to ESA therapy. It to be seen whether such to ESA therapy to FID in cancer but with iron to response 2010). hepcidin has as the of iron availability to the bone marrow for its measurement in serum or have improved and this has that hepcidin might be a superior alternative to traditional of iron status. the that have been and (Macdougall et al, 2010). The of is that they are to are and to is that the used with both the inactive and thus the true level (Macdougall et al, 2010). This is when the is used to measure changes over with or but is less hepcidin values are are more detecting only but they are and levels of hepcidin are in acute and chronic inflammatory such as rheumatoid inflammatory disease and CKD, and are in iron such as however, may hepcidin and it is not clear whether this has in iron status or in the for iron. in a study of hepcidin measurement was to %HRC in predicting the response to intravenous iron (Tessitore et al, 2010). The hepcidin level in CKD patients may not be of diagnostic value than the ferritin but further studies are 2011; et al, 2012). there are no UK for hepcidin There is also a lack of of reference values the different The failure of adequate iron incorporation into the developing is component of ACD and With these the of and factor a of the erythron, early erythroid cell and reduced levels of both hepcidin and have the of iron to developing It is therefore difficult to predict the response to intravenous iron therapy in these this however there is to that ACD patients with of FID from iron et al, 2005). The development of FID in anaemic cancer patients the response to ESA therapy iron is et al, 1992). iron therapy has been the of for patients with iron Intravenous iron is and effective and in patients with ACD is superior to oral iron when used in with ESAs. patients with chronic inflammatory diseases or cancer with ESAs be of the development of FID. the variables above to help therapy appropriate is less clear than for an of an for use in CKD quality control and for all reported variables be available. This is the for traditional variables such as MCV, MCH and reticulocyte count for newer variables used to detect or monitor FID and is not available and in some is the and information in these guidelines is to be true and at the time of to the the British Society for Haematology the for the content of these are when there is or not and can be to most as Where the of or not is less a is to individual as