Guideline for the laboratory diagnosis of functional iron deficiency
Year: 2013
Type: article
Abstract: 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 30·6 pg is a better predictor of response to intravenous iron than baseline serum ferritin or transferrin saturation values in CKD patients undergoing thrice-weekly haemodialysis (Buttarello et al, 2010). This variable is derived from the square root of the product of the MCVs of mature RBC and reticulocytes. It shows good correlation with CHr, with slightly better sensitivity and identical specificity for the detection of iron-restricted erythropoiesis (IRE). Patients with values >87·7 fl were more likely to have ACD, those with lower values to have iron deficiency (Urrechaga, 2009). Zinc protoporphyrin (ZPP) is a trace by-product of haem synthesis and any condition that limits iron supply to the erythroid marrow or stimulates porphyrin synthesis leads to an increased concentration of ZPP in circulating red cells. The incorporation of iron into protoporphyrin IX is the final stage of haem synthesis, and an increase in ZPP is an indicator of defect(s) at any point along this pathway. The measure is therefore non-specific and raised values occur in iron deficiency, FID, lead poisoning and in many iron-sufficient subjects with α°- and β-thalassaemia traits (Graham et al, 1996). There are two important limitations of the measurement of ZPP by the commonly used method of haematofluorometry. First, plasma constituents contribute to the magnitude of the ZPP value, with potentially misleading elevations being found in the presence of hyperbilirubinaemia (Buhrmann et al, 1978) and in chronic renal failure (Garrett & Worwood, 1994). Second, a spurious and progressive increase in ZPP values is seen as Hb falls below about 100 g/l and many subjects with moderate or severe anaemia have raised ZPP values irrespective of iron status. These shortcomings can be overcome by washing RBC prior to testing or adjusting the Hb concentration to a standard value, but sample manipulation is time-consuming. Due to lack of specificity, ZPP should not be used in isolation as a diagnostic test, but once a diagnosis is made it may be used to monitor response to therapy. The ZPP concentration reflects the entire circulating red cell population and is thus less sensitive than %HRC or CHr to acute changes in iron availability (Fishbane & Maessaka, 1997). Assessment of body iron stores is essential both as a diagnostic tool and to monitor the effects of therapy with iron and/or ESAs. This may be achieved by cytological evaluation of the iron content in aspirated bone marrow, or by use of the serum ferritin assay. Ferrokinetic studies using radiolabelled iron have been excluded from discussion as they are rarely employed these days and can be cumbersome. An assessment of iron stores in the bone marrow can be made by use of Perls' Prussian blue reaction. Although considered a 'gold standard' test, assessment can be misleading if insufficient material is available: seven or more particles should be available for review (Hughes et al, 2004), and few haematologists can honestly say they invariably manage this number on their aspirate films. Inadequate material was a major factor in a study that concluded that over 30% of reports of absence of stainable iron were inaccurate (Barron et al, 2001). Also, the presence of stainable iron does not define how much can be re-solubilized and incorporated into the developing erythron. Furthermore, bone marrow examination is uncomfortable and not without complications, such as post-biopsy pain and bleeding. The serum ferritin assay has become the standard test for the assessment of iron stores (Cavill, 1999). Ferritin in serum results from leakage from tissue or intracellular fluids and in health there is a relationship between the two, such that each μg/l of ferritin in serum is equivalent to ~8–10 mg of iron in stores (Cook & Skikne, 1982; Worwood, 1997). Confounding variables may alter this leakage and mask the level of stored iron or, in some cases, the organ(s) it is derived from. Most clinicians are aware of the 'acute phase' nature of serum ferritin but the degree by which this variable deflects from the true measure of the iron stores is less often considered. What the ferritin value cannot do, particularly in CKD, is to indicate when sufficient stores exist to supply erythropoiesis. Here the term 'sufficient' implies the patient will not respond to additional iron supplementation, usually intravenously. Some authors have argued it may be counterproductive to set an upper limit of serum ferritin concentration to provide a 'sufficiency level' (Kalantar-Zadeh et al, 2005), such that it may still be safe to give intravenous iron, and some CKD patients may respond to this therapy despite raised ferritin values. There is also evidence to suggest that CKD patients with low serum ferritin concentrations have a poorer outcome compared to patients with values in the 200–1200 μg/l range (Kalantar-Zadeh et al, 2005). Additionally, some patients with CKD may have excess iron in the liver and spleen, yet paucity within the bone marrow available for erythropoiesis (Ali et al, 1980, 1982). Therefore the setting of an upper limit of serum ferritin concentration above which intravenous iron is not is not & Most guidelines use a value of μg/l (NICE, or μg/l 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 concentrations of μg/l showed an increase in Hb when treated with intravenous iron et al, These patients all had transferrin saturation levels to indicate 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 should this et al, 2010). The major clinical of the assay 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 two coexist et al, et al Although in some studies of sTfR not prove superior to the serum ferritin assay in the detection of iron deficiency in patient groups of those seen in clinical practice (Mast et al, et al, et al, a review concluded that its use improves the diagnosis of iron deficiency, 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, 1999). 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 concentrations of the erythroid marrow et al, 2002). The has been used, along with a measure of iron availability to the erythroid marrow, such as %HRC or CHr, in the production of a diagnostic & 2002). testing 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 falls within of the of inflammatory illness and also falls but to a This results in reduced values that for the of the Although a measure of iron in transport and not of iron in stores, values of indicate the for iron in the setting of anaemia treated with ESAs (Macdougall et al, Used in has sensitivity and specificity in detecting those respond to intravenous iron et al, 1997; et al, although with variable improved accuracy and may be a alternative where RBC and reticulocyte variables are not available. levels are not routinely particularly in the setting of CKD and are not recommended in patients with anaemia and CKD Kidney Foundation, 2006). What 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 respond 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 plasma 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 assay is used to measure changes over with or without but is less if hepcidin values are are more detecting only but they are and time-consuming. levels of hepcidin are in acute and chronic inflammatory such as rheumatoid inflammatory disease and CKD, and are usually in iron such as however, may hepcidin and it is not yet clear whether this has any in iron status or in the for iron. in a study of haemodialysis 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 assay 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 additional of iron to developing It is therefore difficult to predict the response to intravenous iron therapy in these this however there is evidence to suggest 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 safe 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 should be aware of the development of FID. the variables above to help therapy appropriate evidence is less clear than for an of an for use in CKD quality control and for all reported variables should 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 made when there is or not and can be to most as Where the magnitude of or not is less a is to individual as
Source: British Journal of Haematology
DOI: 10.1111/bjh.12311
Language: English
Subfield: Hematology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2007839859