International Journal of Laboratory HematologyVolume 38, Issue 4 p. e86-e88 Letter to the Editor Results of a prepilot study of potential test material for the external quality assessment of reticulocyte haemoglobin content R. F. Hinchliffe, R. F. Hinchliffe UK NEQAS Haematology General Scientific Advisory Group, Watford, UKSearch for more papers by this authorA. Mahon, A. Mahon UK NEQAS Haematology, Watford, UKSearch for more papers by this authorW. Thomas, W. Thomas UK NEQAS Haematology General Scientific Advisory Group, Watford, UKSearch for more papers by this authorC. J. Doré, C. J. Doré orcid.org/0000-0001-9796-4970 UK NEQAS Haematology, Watford, UKSearch for more papers by this authorC. Briggs, C. Briggs UK NEQAS Haematology General Scientific Advisory Group, Watford, UKCarol Briggs died on 25th February 2015.Search for more papers by this authorB. De la Salle, B. De la Salle barbara.delasalle@whht.nhs.uk UK NEQAS Haematology, Watford, UKSearch for more papers by this authorK. Hyde, K. Hyde UK NEQAS Haematology, Watford, UKSearch for more papers by this author R. F. Hinchliffe, R. F. Hinchliffe UK NEQAS Haematology General Scientific Advisory Group, Watford, UKSearch for more papers by this authorA. Mahon, A. Mahon UK NEQAS Haematology, Watford, UKSearch for more papers by this authorW. Thomas, W. Thomas UK NEQAS Haematology General Scientific Advisory Group, Watford, UKSearch for more papers by this authorC. J. Doré, C. J. Doré orcid.org/0000-0001-9796-4970 UK NEQAS Haematology, Watford, UKSearch for more papers by this authorC. Briggs, C. Briggs UK NEQAS Haematology General Scientific Advisory Group, Watford, UKCarol Briggs died on 25th February 2015.Search for more papers by this authorB. De la Salle, B. De la Salle barbara.delasalle@whht.nhs.uk UK NEQAS Haematology, Watford, UKSearch for more papers by this authorK. Hyde, K. Hyde UK NEQAS Haematology, Watford, UKSearch for more papers by this author First published: 16 June 2016 https://doi.org/10.1111/ijlh.12514Citations: 1 Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume38, Issue4August 2016Pages e86-e88 RelatedInformation

AIMS:To derive reference values for red cell variables and platelet counts from a cohort of infants sampled at precise ages during the first 13 months of life. METHODS:Blood counts, reticulocyte counts and zinc protoporphyrin concentrations were obtained from healthy term infants of North European ancestry at 2, 5 and 13 months of age. RESULTS:Mean cell volume (MCV) and mean cell haemoglobin (MCH) values did not differ significantly between 5 and 13 months and MCH concentration was unaffected by age. Values of all other variables at any one age differed significantly from those at the other two. Haemoglobin, mean cell haemoglobin, zinc protoporphyrin and platelet values (95% ranges) at 2 (n=119), 5 (n=97) and 13 months (n=42) were, respectively, 91-125, 101-129 and 105-133 g/L; 28.6-33.1, 24.5-28.7 and 24.3-28.7 pg; 36-116, 25-91 and 27-57 micromol/mol haem; and 216-658, 241-591 and 209-455×10(9)/L. At 2 and 5 months, respectively, 26.9% and 10.8% of subjects had platelet counts >500×10(9)/L. Reticulocyte counts at 2 months and MCV and MCH values at 5 months were significantly higher in girls. In boys, red cell distribution width values were significantly higher at 5 months, and zinc protoporphyrin values at both 2 and 5 months. CONCLUSIONS:These findings indicate the value of obtaining reference data at precise ages during infancy and confirm and extend earlier reports indicating a gender difference in laboratory measures used to assess iron status in early infancy.

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 supplementation is not advised is not evidence-based (Dukkipati & Kalantar-Zadeh, 2007). Most guidelines use a value of >500 μg/l (NICE, 2006) or >800 μg/l (target range 200–500 μg/l; National Kidney Foundation, 2002) for CKD patients on ESA therapy, citing that above these values there is a greater risk of exacerbated iron overload with further therapy. However anaemic CKD patients (Hb <110 g/l) with ferritin concentrations of 500-1200 μg/l showed an increase in Hb when treated with intravenous iron (Coyne et al, 2007). These patients all had transferrin saturation (TSat) levels <25%, taken to indicate the presence of FID. The best indicator of underlying IRE in this group was the response to intravenous iron. The serum ferritin concentration was unhelpful in predicting response to ESA therapy in cancer-related anaemia (Littlewood et al, 2003). The transferrin receptor is highly expressed on erythroid precursors. Increased levels are found in disorders associated with an expanded erythroid marrow (Kohgo et al, 1987; Huebers et al, 1990) and also in iron deficiency. The clinical utility of sTfR measurement has been hampered by the lack of agreement concerning the source both of standards and of antigens used to raise antibodies. Use of the recently developed first World Health Organization Reference Reagent for sTfR should improve this situation (Thorpe et al, 2010). The major clinical role of the assay is in differentiating the anaemia of iron lack from that caused by inflammation, which has little or no effect on sTfR values, and in detecting the presence of iron lack when the two coexist (Ferguson et al, 1992; Punnonen et al (1997). Although in some studies estimation of sTfR did not prove superior to the serum ferritin assay in the detection of iron deficiency in patient groups typical of those seen in clinical practice (Mast et al, 1998; Means et al, 1999; Lee et al, 2002), a recent systematic review concluded that its use improves the diagnosis of iron deficiency, especially in the presence of chronic disease or gastrointestinal malignancy (Koulaouzidis et al, 2009). In patients with stable chronic renal failure and stable kidney disease not receiving iron or ESAs, the sTfR concentration alone proved inferior to that of serum ferritin in detecting those with coexisting iron deficiency (Fernandez-Rodriguez et al, 1999). However in a group of patients receiving maintenance doses of ESAs, and with stable erythropoiesis, the measure proved superior to ZPP, transferrin saturation and serum ferritin, but inferior to %HRC and CHr, in differentiating between iron-deficient and -sufficient subjects (Tessitore et al, 2001). The TfR index, a ratio of the ferritin concentration to that of sTfR (Punnonen et al, 1997), was found to be superior to ferritin alone in predicting response to intravenous iron in renal patients on long-term ESA therapy (Chen et al, 2006). This approach helps to overcome one drawback to the use of sTfR as a single test in monitoring iron status during ESA therapy, that of the therapy itself leading to increased concentrations via expansion of the erythroid marrow (Chiang et al, 2002). The TfR index has been used, along with a measure of iron availability to the erythroid marrow, such as %HRC or CHr, in the production of a so-called diagnostic plot (Thomas & Thomas, 2002). Sequential testing can be used to monitor the effects of, or need for, supplementation with iron or ESAs (Thomas et al, 2006). The %Sat value is derived from serum iron and TIBC values and is the most widely used of the three. The serum iron concentration falls markedly within hours of the onset of inflammatory illness and TIBC also falls but to a lesser degree. This results in reduced %Sat values that persist for the duration of the illness. Although a measure of iron in transport and not of iron in stores, %Sat values of <20 indicate the need for parenteral iron in the setting of anaemia treated with ESAs (Macdougall et al, 1990). Used in isolation, %Sat has poor sensitivity and specificity in detecting those who respond to intravenous iron (Low et al, 1997; Tessitore et al, 2001), although combination with another variable (e.g sTfR) produces improved accuracy and may be a useful alternative where RBC and reticulocyte variables are not available. Serum erythropoietin (Epo) levels are not routinely measured, particularly in the setting of CKD and are not recommended in patients with anaemia and CKD (National Kidney Foundation, 2006). What is clear is that for patients with the various stages of CKD with anaemia there is a relative lack of serum Epo. However low Epo levels have limited clinical value given that some cancers and arthritides are associated with suppression of Epo levels (Hochberg et al, 1988; Miller et al, 1990). In a study by Rose et al (1995), patients with myelodysplasia and baseline Epo levels <100 μ/ml were most likely to respond to ESA therapy. It remains to be seen whether such patients' refractoriness to ESA therapy relates to FID in cancer patients, but supplementation with iron appears to improve response rates (Henry, 2010). Recently, hepcidin has emerged as the master regulator of iron availability to the bone marrow (Ganz, 2007). Assays for its measurement in serum or plasma have improved considerably and this has generated optimism that hepcidin quantitation might be a superior alternative to traditional markers of iron status. Broadly speaking, the assays that have been developed include radioimmunoassay, enzyme-linked immunosorbent assays and mass spectrometry techniques (Macdougall et al, 2010). The main advantage of immunoassays is that they are technically easier to perform, readily accessible (several are commercially available) and cheaper to implement. Their main disadvantage is that the antibodies used cross-react with both the biologically inactive hepcidin-22 and -20 fragments, thus overestimating the true bioactive hepcidin-25 level (Macdougall et al, 2010). This is acceptable when the same assay is used to measure changes over time, with or without intervention, but is less satisfactory if absolute hepcidin values are required. Mass spectrometry techniques are more accurate, detecting only hepcidin-25, but they are costly, labour-intensive and time-consuming. Serum levels of hepcidin are elevated in acute and chronic inflammatory states, such as infection, rheumatoid arthritis, inflammatory bowel disease and CKD, and are usually undetectable in conditions causing iron overload, such as hereditary haemochromatosis (Ganz, 2007). Other factors, however, may affect hepcidin levels, and it is not yet clear whether this novel biomarker has any advantages in determining iron status or in ascertaining the need for supplemental iron. Indeed, in a study of haemodialysis patients, hepcidin measurement was inferior to %HRC in predicting the response to intravenous iron (Tessitore et al, 2010). The hepcidin level in CKD patients may not be of greater diagnostic value than the ferritin level, but further studies are needed (Coyne, 2011; Peters et al, 2012). At present there are no UK EQA schemes for hepcidin assays. There is also a lack of harmonization of reference values across the different assay platforms. The failure of adequate iron incorporation into the developing erythron is just one component of ACD and cancer-related anaemia. With these disorders the actions of γ-interferon, transforming growth factor-β and tumour necrosis factor produce a down-regulation of the erythron, early erythroid precursor cell death and reduced Epo sensitivity. Increased levels of both hepcidin and IL6 have the additional effect of reducing iron transfer to developing erythroblasts. It is therefore difficult to predict the response to intravenous iron therapy in these patients. Despite this however there is evidence to suggest that ACD patients with biochemical markers of FID do benefit from iron supplementation (Thomas et al, 2005). The development of FID in anaemic cancer patients blunts the response to ESA therapy unless supplementary iron is provided (Cazzola et al, 1992). Oral iron therapy has been the mainstay of treatment for patients with iron deficiency. Intravenous iron is safe and effective and in patients with ACD is superior to oral iron when used in conjunction with ESAs. Physicians treating patients with chronic inflammatory diseases or cancer with ESAs should be aware of the potential development of FID. Using the variables discussed above to help guide therapy seems entirely appropriate even though evidence is less clear than for CKD. Fig 1 provides an example of an algorithm for use in CKD patients. Internal quality control (IQC) and EQA for all reported variables should be available. This is the case for traditional variables such as Hb, MCV, MCH and reticulocyte count but, for newer variables used to detect or monitor FID and IRE, EQA is not available and in some instances neither is IQC. While the advice and information in these guidelines is believed to be true and accurate at the time of going to press, neither the authors, the British Society for Haematology nor the publishers accept legal responsibility for the content of these guidelines. Strong (grade 1): Strong recommendations (grade 1) are made when there is confidence do or do not outweigh harm and burden. Grade 1 recommendations can be applied uniformly to most patients. Regard as ‘recommend.’ Weak (grade 2): Where the magnitude of benefit or not is less certain a weaker grade 2 recommendation is made. Grade 2 recommendations require judicious application to individual patients. Regard as ‘suggest.’

SummaryAccurate platelet counts are essential for the safe management of severe thrombocytopenia (platelet counts ≤20 × 109/l). The effect of carry over on platelet counting in severe thrombocytopenia was investigated by performing counts before and after saline rinses on three Bayer Advia 120 automated blood counters. Counts were performed in both primary and manual closed tube system modes on two instruments and in manual open tube mode on a third. A total of 194 samples with platelet counts ≤20 × 109/l were studied. First counts were significantly higher in all groups. The magnitude of the difference varied both by analyser and counting mode. Carry over was minimal with one analyser in primary mode and second counts were on average only 5.5% lower; on a second analyser in manual closed tube system mode second counts were on average 37.7% lower. A first count of ≥10 × 109/l fell to <10 × 109/l on the second count in 35 of 145 samples (24.1%). In five such samples, all tested on one analyser, the second count was <50% of the value of the first count. Two of 49 (4.1%) first counts of <10 × 109/l increased to ≥10 × 109/l on repeat. These results show a variable and often potentially clinically important carry‐over effect on severely thrombocytopenic samples using the Advia 120.

British Journal of HaematologyVolume 135, Issue 4 p. 421-421 A subject with populations of both myeloperoxidase-positive and -negative neutrophils Roderick Hinchliffe, Roderick Hinchliffe Paediatric Haematology Department, Sheffield Children's Hospital, Sheffield, UK E-mail: [email protected]Search for more papers by this authorAjay Vora, Ajay Vora Paediatric Haematology Department, Sheffield Children's Hospital, Sheffield, UK E-mail: [email protected]Search for more papers by this author Roderick Hinchliffe, Roderick Hinchliffe Paediatric Haematology Department, Sheffield Children's Hospital, Sheffield, UK E-mail: [email protected]Search for more papers by this authorAjay Vora, Ajay Vora Paediatric Haematology Department, Sheffield Children's Hospital, Sheffield, UK E-mail: [email protected]Search for more papers by this author First published: 16 October 2006 https://doi.org/10.1111/j.1365-2141.2006.06241.xCitations: 2Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume135, Issue4November 2006Pages 421-421 RelatedInformation

Clinical & Laboratory HaematologyVolume 27, Issue 5 p. 353-354 Automated counting of cells in cerebrospinal fluid G. J. Bellamy, G. J. Bellamy Department of Paediatric Haematology, Sheffield Children's NHS Trust, Western Bank, Sheffield S10 2TH, UKSearch for more papers by this authorS. J. Clark, S. J. Clark Department of Haematology, Christie NHS Trust, Wilmslow Road, Withington, Manchester M20 4BX, UKSearch for more papers by this authorP. S. Simpkin, P. S. Simpkin Department of Paediatric Haematology, Sheffield Children's NHS Trust, Western Bank, Sheffield S10 2TH, UKSearch for more papers by this authorP. Batstone, P. Batstone Department of Haematology, Christie NHS Trust, Wilmslow Road, Withington, Manchester M20 4BX, UKSearch for more papers by this authorR. F. Hinchliffe, R. F. Hinchliffe Department of Paediatric Haematology, Sheffield Children's NHS Trust, Western Bank, Sheffield S10 2TH, UKSearch for more papers by this author G. J. Bellamy, G. J. Bellamy Department of Paediatric Haematology, Sheffield Children's NHS Trust, Western Bank, Sheffield S10 2TH, UKSearch for more papers by this authorS. J. Clark, S. J. Clark Department of Haematology, Christie NHS Trust, Wilmslow Road, Withington, Manchester M20 4BX, UKSearch for more papers by this authorP. S. Simpkin, P. S. Simpkin Department of Paediatric Haematology, Sheffield Children's NHS Trust, Western Bank, Sheffield S10 2TH, UKSearch for more papers by this authorP. Batstone, P. Batstone Department of Haematology, Christie NHS Trust, Wilmslow Road, Withington, Manchester M20 4BX, UKSearch for more papers by this authorR. F. Hinchliffe, R. F. Hinchliffe Department of Paediatric Haematology, Sheffield Children's NHS Trust, Western Bank, Sheffield S10 2TH, UKSearch for more papers by this author First published: 21 September 2005 https://doi.org/10.1111/j.1365-2257.2005.00700.xCitations: 3Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume27, Issue5October 2005Pages 353-354 RelatedInformation

Aims: To search for laboratory evidence of hereditary spherocytosis (HS) among apparently healthy children with the chance finding of an isolated increase in hyperchromic red cells (cells with intracellular haemoglobin concentration > 410 g/litre). Methods: Blood and reticulocyte counts and Pink tests were performed on successive children found on routine counts to have > 4% hyperchromic red cells, and compared with age and mean cell haemoglobin concentration (MCHC) matched controls and children known to have HS. Results: Thirty four patients with > 4% hyperchromic red cells had significantly higher absolute numbers of such cells (p < 0.0001) and higher reticulocyte counts (p < 0.01) than age matched controls, together with higher MCHC (p < 0.0001) and haemoglobin distribution width (p < 0.0001) values and lower mean cell volume (p < 0.02) values. Significant differences were also found among hyperchromic red blood cell, reticulocyte, and haemoglobin distribution width values when subjects were compared with MCHC matched controls. Pink test values were higher in children with increased hyperchromic red blood cells, but not significantly so. In patients with HS, most variables measured were significantly different both from those of children with > 4% hyperchromic cells and controls. Despite the differences found, few MCHC, HDW, reticulocyte, or Pink test values were outside of the normal limits, and only one child with increased hyperchromic cells had both a mild reticulocytosis and a slightly raised Pink test value. Conclusions: Subjects with an isolated increase in hyperchromic red blood cells have a profile of red blood cell changes similar to that of patients with HS, but to a lesser degree. They may carry a recessive form of the disease but lack the laboratory features of clinically manifest HS.

Reference ranges for the total and differential leucocyte counts were determined from venous blood collected at 2, 5 and 13 months of age from a cohort of 112 healthy children of north European ancestry. At 2, 5 and 13 months, the ranges for neutrophils were found to be 0.7-4.7, 1.1-5.6 and 1.0-7.6 x 109/l, and for lymphocytes 3.3-10.5, 3.4-11.3 and 3.5-10.4 x 109/l, respectively. The upper limits for monocytes at each age were 1.2, 1.2 and 0.91 x 109/l, and for eosinophils 0.84, 1.0 and 0.88 x 109/l, respectively. Mean counts for all cell types, except monocytes, increased between 2 and 5 months of age. There was little change in mean counts between 5 and 13 months. Statistically significant correlations existed between the numbers of each cell type at 2 months of age, and were still present at 13 months between monocytes and each of the granulocyte series and between basophils and all other cell types. By comparison with older data these findings indicate a lower reference limit for neutrophils at 2 months of age, and a narrower range for this cell type at both 2 and 5 months of age. Reference ranges for lymphocytes and eosinophils are wider than indicated by some previous studies.

The study aimed to investigate local concerns about clinically important discrepancies between repeat HemoCue haemoglobin measurements from single drops of blood. Two biomedical scientists and two health visitors each obtained a series of paired haemoglobin values by fingerprick sampling from healthy volunteers. Seven of 20 paired values obtained by health visitors and three of 20 obtained by scientists from the first drop of blood forming at the puncture site differed by > or = 10 g/l; 11 of 20 paired values obtained by health visitors and one of 20 by the scientists from the fourth drop of blood differed by > or = 10 g/l. After collecting and mixing a number of drops in EDTA tubes before analysis, seven of 40 paired values differed by > 5 g/l, and none by > 10 g/l. Pooling drops of blood before analysis improves precision of HemoCue haemoglobin measurement and allows users to achieve results comparable to those obtained by experienced laboratory staff. Measurement of haemoglobin from single drops of skin puncture blood should be discontinued.

The Bayer H1 automated blood counter was used to assess the MCHC values of 40 non-anaemic patients with HbC trait, 21 with HbD trait, 23 with HbE trait and 69 with HbS trait. These were compared with values from controls with a normal Hb phenotype. Values were significantly higher in those with HbC, D and S traits and approached significance in those with HbE trait. In 45% of subjects with HbC trait the MCHC value was greater than or equal to 35 g/dl. Such values may prove a useful marker for this abnormality. In a further 12 patients with HbC, D, E or S traits and coexisting iron deficiency anaemia, MCHC values were usually higher and the percentage of hypochromic cells (red cells with CHC <28 g/dl, directly measured by the H1) usually lower than values derived from controls with a normal Hb phenotype and iron deficiency anaemia of similar degree. In individuals with HbC, D, E or S traits, the MCHC and proportion of hypochromic cells are less sensitive indicators of iron lack than in subjects with a normal Hb phenotype.

Objective Spontaneous coronary artery dissection (SCAD) is an under-recognised but important cause of myocardial infarction and sudden cardiac death. We sought to determine the role of medical and molecular genetic screening for connective tissue disorders in patients with SCAD. Methods We performed a single-centre retrospective descriptive analysis of patients with spontaneous coronary artery disease who had undergone medical genetics evaluation 1984–2014 (n=116). The presence or absence of traits suggestive of heritable connective tissue disease was extracted. Genetic testing for connective tissue disorders and/or aortopathies, if performed, is also reported. Results Of the 116 patients (mean age 44.2 years, 94.8% women and 41.4% with non-coronary fibromuscular dysplasia (FMD)), 59 patients underwent genetic testing, of whom 3 (5.1%) received a diagnosis of connective tissue disorder: a 50-year-old man with Marfan syndrome; a 43-year-old woman with vascular Ehlers–Danlos syndrome and FMD; and a 45-year-old woman with vascular Ehlers–Danlos syndrome. An additional 12 patients (20.3%) had variants of unknown significance, none of which was thought to be a definite disease-causing mutation based on in silico analyses. Conclusions Only a minority of patients with SCAD who undergo genetic evaluation have a likely pathogenic mutation identified on gene panel testing. Even fewer exhibit clinical features of connective tissue disorder. These findings underscore the need for further studies to elucidate the molecular mechanisms of SCAD.

The clinical usefulness of the measurement of red cell zinc protoporphyrin (ZPP), an indicator of iron-deficient erythropoiesis, was assessed in a group of UK children undergoing investigation for red cell microcytosis. Of 213 children studied, 136 had increased ZPP values. Of these, 86 also had reduced iron stores as indicated by serum ferritin concentration. The 50 children with increased ZPP and normal ferritin values could be divided into two main groups. One group comprised 28 children who had evidence of coexistent infection or inflammatory disease. The other included 21 children who had beta-thalassemia trait (n = 19) or disease (n = 2). Among the 77 children with normal ZPP values, 22 had reduced serum ferritin concentrations and 45 did not, nor did they have evidence of beta-thalassemia. Microcytosis in some of these children could have been due to alpha-thalassemia trait. Measurement of ZPP is a simple, quick, and relatively cheap method of confirming the presence of iron-deficient erythropoiesis even when inflammation makes serum ferritin measurements unreliable. It is not as sensitive as the ferritin assay to the early stages of iron deficiency, and its specificity is reduced by the occurrence of raised values in most children with beta-thalassemia trait. Where there is microcytosis, normal values, together with normal hemoglobin A2 and serum ferritin concentrations, are likely to indicate alpha-thalassemia trait.

A study was carried out to determine the frequency of combined iron deficiency and beta-thalassaemia trait in a cohort of British Asian children to see whether the trait protects iron status. Of 470 consecutive children with red cell microcytosis, 77 had beta-thalassaemia trait and 26 (34%) of these also had evidence of iron deficiency. It was most common and profound in children under five years of age where the prevalence was 16 in 33 (48.5%). This suggests that iron deficiency is no less common in Asian children with beta-thalassaemia trait than in those without. It should not be presumed that the trait protects iron status or that the two are in any way mutually exclusive, at least in the early years.

Values for MCV, MCH, MCHC and red cell distribution width (RDW) derived from subjects with HbC and HbE traits using a Technicon H*1 automated blood count analyser were compared. Significantly higher MCH, MCHC and RDW values were found in those with HbC trait. By use of a simple discriminant function (MCHC2 x RDW/1000), 19/20 subjects with HbC trait gave a value > or = 16.3, and 20/21 subjects with HbE trait gave a value < or = 16.2. The function described may be of value as an adjunct to electrophoresis in intralaboratory quality control.

To determine whether skin puncture blood can be used reliably for CD4 lymphocyte counts, the numbers of the major subsets of lymphocytes were assessed in paired venous and skin puncture blood samples from 22 children and 10 adults. Paired values were highly correlated, with skin puncture values being about 7% higher than venous values for each cell type. Differences were of borderline statistical significance for total lymphocytes and for each subset except CD3+ CD8+ T lymphocytes. Nevertheless, the magnitude of the differences was small and unlikely to be of clinical importance, and it seems that skin puncture samples may be preferable for CD4 counts in children or adults with difficult venous access.