Transfusion and Apheresis Science 58 (2019) 375–385

Contents lists available at ScienceDirect

Transfusion and Apheresis Science

journal homepage: www.elsevier.com/locate/transci

Review

Diagnosis of anemia—A synoptic overview and practical approach T

Verena Jansen
LADR Laborzentrum an den Immanuel Kliniken, MVZ Laborverbund GmbH, Neuendorfstraße 16A, D-16761 Hennigsdorf bei Berlin, Germany

A R T I C LE I N FO A B S T R A C T

Keywords: Anemia is common in everyday clinical practice. In the following, the characteristics of apparently proven as
Anemia well as new biomarkers are presented – for diagnosis and therapy control, considering their diagnostic value. In
Biomarker spite of new diagnostic tools, the importance of microscopy in hematological manifestations is illustrated. Based
Diagnostic value on a classification of anemia, a strategy is proposed for an economic diagnosis of different types of anemia and
Classification
their predisposition.

1. Introduction
A WHO report on the global prevalence of anemias in 2011, pub-
lished in 2015, identified 1.5 billion people with iron deficiency anemia
and between 2.0 and 4 billion with manifest iron deficiency. About 50%
of the anemia cases are due to iron deficiency, followed by anemia due
to vitamin deficiencies, acute and chronic infections, congenital or ac-
quired disorders of hemoglobin synthesis or erythropoiesis disorders. In
developing countries, iron deficiency anemia promotes higher maternal
and neonatal mortality, which in turn accounts for 3 million deaths in
201. [1].
In Germany, iron deficiency anemia (IDA), anemia of chronic dis-
ease (ACD) and mixed-type anemia (IDA + ACD) are the most common
types of anemia. Every year, up to 30 million iron assessments are
performed in German laboratories, and 3.37 million RBC concentrates
were transfused in 2017 in German hospitals and medical practices [2].
Worldwide, about 75 million RBC concentrates are transfused each
year.
During the past twelve months, 84,000 anemia biomarkers have
been requested in our laboratory sites. Almost two-thirds of outpatient
care account for the parameters ferritin, transferrin, iron and transferrin
saturation (Fig. 1). In inpatient care, these parameters account for 44%
of the requests, with an additional 48% for vitamin B12 and folic acid.
The soluble transferrin receptor (sTfR) was rarely requested (Fig. 2),
almost exclusively in inpatient nephrology and combined with re-
ticulocyte hemoglobin (Ret-He/CHr) for the iron plot (Thomas plot)
[3,4].
The spectrum of requested anemia biomarkers and the frequency of
selected parameters only partially reflect current recommendations,
which focus on new markers to assess iron status, iron supply of ery-
throcytes and reticulocytes, to predict and evaluate the responsiveness
to therapeutic iron, rHuEPO or vitamin B12 supplementation.
2. Predisposition and risk population
The selection of appropriate diagnostic parameters to clarify man-
ifest anemia should be consistent with clinical findings and patient
history. Thus, the anemia diagnostics in otherwise healthy girls or
women who introduce themselves as outpatients and complain of hy-
permenorrhoea, will first be focused on the iron metabolism. In elderly
patients, especially those who are hospitalized, another diagnostic ac-
cess could be necessary. With increasing age, chronic diseases

Abbreviations: ACD, anemia in chronic disease; AIDS, acquired immunodeficiency syndrome; AIHA, autoimmune hemolytic anemia; AML, acute myeloid leukemia;
B12, vitamin B12; CE, Communauté européenne, product label according to standards in the European Union; CHr, content of hemoglobin in reticulocytes; CML,
chronic myeloid leukemia; CRP, C-reactive protein; DAT, direct antiglobulin test; DCT, direct Coombs test; ELISA, enzyme-linked immunosorbent assay; G6PD,
glucose-6-phosphate dehydrogenase; GC–MS, gas chromatography-mass spectrometry; HD, H(a)emodialysis; HDFN, hemolytic disease of the fetus and newborn;
Holo-TC, holo-transcobalamin; IBD, inflammatory bowel disease; IDA, iron deficiency anemia; IgG, immunoglobulin G; IVD, in vitro diagnostics; LC–MS, liquid
chromatography-mass spectrometry; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume;
MDS, myelodysplastic syndrome; MHN, morbus haemolyticus neonatorum; MMA, methylmalonic acid; PEI, Paul-Ehrlich-Institut; PKM, pyruvatkinase isozymes M1/
M2; RBC, red blood cells; RDW, red cell distribution width; Ret-Hb, reticulocyte hemoglobin; Ret-He, reticulocyte hemoglobin equivalent; rHuEPO, recombinant
human erythropoietin; ROC, receiver operating characteristic; RPI, reticulocyte production index; SELDI-TOF-MS, surface-enhanced laser desorption/ionization time-
of-flight mass spectrometry; SF, serum ferritin; sTfR, soluble transferrin receptor; sTfR-F, sTfR/log ferritin index; TC, transcobalamin; TIBC, total iron-binding
capacity; TS, transferrin saturation; WHO, World Health Organization; ZnPP, zinc protoporphyrin
E-mail address: v.jansen@ladr.de.

https://doi.org/10.1016/j.transci.2019.06.012

1473-0502/ © 2019 Elsevier Ltd. All rights reserved.

V. Jansen Transfusion and Apheresis Science 58 (2019) 375–385

• Proton pump inhibitors

• H receptor antagonists
2

• After nitrous oxide inhalation

3. Diagnostic challenge and selection of parameters

The selection of laboratory parameters and the diagnostic procedure

depend on the question whether manifest anemia or a predisposition for
anemia is the focus. In case of manifest anemia, the choice of suitable
biomarkers is important for therapeutic decisions, follow-up and
prognosis.
Reliably and rapidly differentiating manifest anemia, if necessary
gradually, depends on several factors that may differ between out-
patient and inpatient care:

• Existing restoring samples in the laboratory for supplementary tests

• Analyte stable for additional laboratory requests
• Reduction of diagnostic blood sampling
Fig. 1. Anemia biomarkers requested in outpatient care.

• Turn-around time of further tests

• Resubmission date for outpatients
• Days/weeks of hospital stay
• Severity of anemia
• Urgency of therapy indication
• Availability of further investigations (on site or in a cooperating
medical laboratory)
• Organizational, logistical and economic effort of further investiga-
tions
• Planning of elective surgical interventions
• Patient Blood Management and restrictive transfusion regime
As diverse as the spectrum of available biomarkers, so numerous are
the limitations which range from sophisticated pre-analytics, low sen-
sitivity, poor standardizability, elaborate methodology, ambiguous
Fig. 2. Anemia biomarkers requested in inpatient care. predictability to organizational overhead (Table 1 with references
[6–44]).
Sufficiently sensitive biomarkers are available for reliable diagnosis
(autoimmune diseases, kidney disease, inflammation, infections), solid
of uncomplicated iron deficiency. The identification of mixed-type an-
tumors, hematological neoplasms and multimorbidity become more
emia, the detection of iron deficiency in ACD represents a diagnostic
likely. Accordingly, the risk of anemia in chronic disease (ACD) or renal
challenge because ferritin is a positive and transferrin a negative acute
anemia or combined forms increases. In elderly patients, deficiencies
phase reactant and therefore not sufficient to diagnose iron deficiency.
such as iron deficiency, vitamin B12 and/or folic acid deficiency can
Especially in dialysis patients, indication and therapy control of re-
occur as a result of malnutrition or – in case of vitamin B12 deficiency –
combinant human erythropoietin (rHuEPO), combined with in-
more commonly, in combination with ACD or renal anemia.
travenous iron, are complex. Available biomarkers show different di-
Nephrological patients, especially dialysis patients, may develop renal
agnostic sensitivity, specificity and predictive values with respect to
anemia and represent a risk population for functional vitamin B12 de-
diagnostic aims (Table 2 with references [45–50]). In nephrological
ficiency [5]. Functional vitamin B12 deficiency has to be considered in
patients an additional functional vitamin B12 deficiency occurs fre-
the following risk populations:
quently and has to be diagnosed early [5].
The transferrin saturation is a ratio calculated by serum iron and
• Elderly patients transferrin. The reliability of this index is limited by intraindividual and
• Dementia day-to-day variability of iron levels [7]. In case of manifest iron defi-
• Unexplained anemia ciency higher or increased levels of transferrin may be absent in con-
• Macrocytic anemia comitant (chronic) inflammations, infections or malignancies leading to
• Unexplained neuropsychiatric symptoms decreasing transferrin concentrations. While transferrin is a negative
• Nephrological patients acute phase reactant, ferritin concentration is upregulated during acute
• Ovolacto-vegetarians phase. Ferritin as a sensitive mirror for the storage iron can therefore be
• Vegan less or not demonstrably reduced in mixed-type anemia (IDA + ACD).
• Alcoholics Successful treatment of dialysis patients with recombinant human
• Endogenous depression erythropoietin (rHuEPO) requires intact bone marrow iron supply. The
• After gastric resection determination of the percentage of hypochromic erythrocytes and re-
• Crohn's disease ticulocyte hemoglobin (Ret-He/CHr) has been proposed to be sensitive
• Helicobacter pylori infection parameters for the assessment of iron-dependent erythropoiesis. H*3
• Chronic atrophic gastritis analyzers provide these results at the time of a routine blood count
• Diarrhea [40].
• Anorexia
• Other gastrointestinal manifestations
376
 Table 1
Biomarkers of anemia – indication and limitation.
V. Jansen

Hematology and immunohematology

Biomarker Indication Indicator, predictor Limitation Expenditure

Mean corpuscular volume Initial classification of anemia Decreased in iron deficiency anemia and beta- Differential diagnosis of iron deficiency anemia – Integral part of blood count
(MCV) thalassemia, rarely in myelodysplastic syndrome thalassemia, morphological evidence is later detectable
than deviating biomarkers.
Mean corpuscular Initial classification of anemia Decreased in iron deficiency anemia No early marker Integral part of blood count
hemoglobin (MCH)
Microscopy of peripheral Characterization of erythrocyte Characteristic diagnosis pointing results Subjective report by visual reading, morphological Skilled staff, personnel-intensive method, consensus
blood smear morphology, identification of evidence is later detectable than deviating biomarkers, training
leucocyte pathology as in beginning clinical manifestation.
Reticulocytes Chronic Anemia, chronic hemolytic Elevated in severe acute bleeding, elevated in chronic Causes of absent reticulocyte reaction: bone marrow Routine laboratory
Anemia hemolysis, missing elevation in anemic patients indicates hypoplasia or carcinosis, lymphoma, myeloma, acute
impaired bone marrow function or deficiency inspite of leucemia, tuberculosis, iron, vitamin B12, folate or
erythropoietin stimulus. erythropoietin deficiency, myxedema, protein
[6] deficiency
thalassaemia maior, myeloblast anemia, MDS,
myelofibrosis, congenital anemia, chronic
inflammation or malignancies
Direct antiglobulin test Detection or exclusion of auto- or Positive detection of sensitization of erythrocytes by Numerous causes of weakly positive DCT results Further differentiation recommended (IgG, C3d
(DAT), alloimmune hemolytic anemia, allo- or autoantibodies or complement factors, complement, IgG1 and IgG3, DCT dilution, elution
Direct Coombs test suspicion of HDFN (or exclusion) differentiation of isotypes (IgG, IgA, IgM) and IgG and identification), medical specialist’s
(DCT) subclasses possible (IgG1 and IgG3) interpretation necessary

Biomarkers of screening and additional examinations

377
Biomarker Indication Indicator, predictor Limitation Expenditure

Iron Recommended only in combination with Isolated request obsolete Decreased in iron deficiency as well as in inflammation, Routine laboratory
transferrin [7] infections, neoplasm, diurnal variations and day-to-day
variations of iron concentration [7]
Transferrin Suspicion of iron deficiency Elevated in manifest iron deficiency Limited value in differention of mixed-type forms of iron Routine laboratory
[7–9] deficiency anemia + ACD: negative acute phase reactant
[7]
Soluble transferrin receptor Differentiation of mixed-type anemia Elevated in iron-deficient erythropoesis, Increased also in hemolysis, thalassemia, polycythemia Pre-analytics: specimens without
(sTfR) increasing during iron supplementation, rubra vera, sTfR in iron deficiency often normal lipemia, bilirubin or hemolysis ar
expressing stimulated erythropoiesis, [7] preferred.
parameter for differential diagnosis of iron-
deficient erythropoiesis.
[6–8,10,11]
Ferritin Mirror of iron storage Decreased in manifest iron deficiency Limited value in mixed-type forms of iron Routine laboratory
[6–8,11–16] deficiency + inflammation, infection, neoplasm: positive
acute phase reactant,
higher concentrations during pregnancy
Haptoglobin Intravascular hemolysis Most sensitive parameter for the diagnosis of Not in newborn (reference interval), limited value in Routine laboratory
intravascular hemolysis mixed-type forms of iron deficiency + inflammation,
infection, neoplasm: positive acute phase reactant
Vitamin B12 Differential diagnosis of macrocytic anemia Decreased in symptomatic, manifest vitamin B12 Holo-transcobalamin and methyl malonic acid more Standardization of pre-analytics
deficiency sensitive and more specific, vitamin B12 no early marker
[5]
Folate Differential diagnosis of macrocytic anemia Decreased in folate deficiency Falsely higher or falsely normal results already in Standardization of pre-analytics: avoid
[17,18] presence of discrete in vitro hemolysis, testing in hemolysis, protection of light
erythrocytes superior to testing in serum
(continued on next page)
Transfusion and Apheresis Science 58 (2019) 375–385
 Table 1 (continued)
Biomarkers of screening and additional examinations
Biomarker
Hemopexin
Holo-transcobalamin
Methylmalonic acid in serum
Methylmalonic acid in urine
Homocysteine in plasma
Erythropoetin in serum
378
Free hemoglobin in serum or
plasma
Free hemoglobin in urine
Zinc protoporphyrin (ZnPP) in
erythrocytes
ZnPP fluorescence
spectroscopic examination
in lips
Hepcidin-25, bioactive
Indication
Severity of intravascular hemolysis
Early diagnosis of vitamin B12 deficiency
Early diagnosis of vitamin B12 deficiency, therapy
control of vitamin B12 supplementation, preferably
in nephrological patients
In newborns with poor feeding, impaired
development, vomit, convulsions, suspicion of
methyl malonic aciduria
Therapy control of vitamin B12 supplementation,
preferably in patients with renal diseases
Identification of rHuEPO-responsive anemia,
rHuEPO therapy setting and therapy control
Manifest intravascular hemolysis, for example in
alloimmune hemolytic transfusion reaction or
AIHA
Quantification of hemoglobinuria
Suspicion of manifest iron deficiency, suspicion of
functional iron deficiency (impaired iron
distribution), suspicion of impaired iron
metabolism
Suspicion of functional iron deficiency, use in
pediatrics
Hereditary hemochromatosis, (secondary)
hemosiderosis, chronic hepatitis C, differential
diagnosis of iron deficiency anemia and ACD with
impaired iron distribution, chronic infections,
chronic renal diseases, competitive sportsmen
Indicator, predictor
Decreased during intense hemolytic processes
Decreased in functional vitamin B12 deficiency
[5]
Very sensitive biomarker of functional vitamin
B12 deficiency, elevated in functional vitamin B12
deficiency, indicator of the effectiveness of
vitamin B12-dependent reactions
[5,18,49]
Very sensitive biomarker of functional vitamin
B12 deficiency, elevated in functional vitamin B12
deficiency
[20,21]
Indicator of the effectiveness of vitamin B-
dependent intracellular methionine cycle,
elevated in functional vitamin B12 deficiency
[5,17–20,22]
Decreased in renal anemia, AIDS, cancer-related
anemia, chemotherapy, elevated in non-renal
anemia, elevated in paraneoplastic syndromes
Supplementary parameter
Risk marker of acute renal failure
Best indicator of iron-deficient erythropoesis,
normal ZnPP levels in non-renal patients exclude
iron-deficient erythropoesis, ZnPP elevated in
iron-deficient erythropoesis, correlation with
percentage of hypochromic erythrocytes
[7,8,11,23]
Non-invasive method, without blood sampling
[24]
Decreased levels in serum and urine in pure iron
deficiency anemia, additional information,
decreased in hereditary hemochromatosis, low in
(secondary) hemosiderosis, elevated in iron-
refractory iron deficiency anemia
[11,25–35]
V. Jansen
Limitation Expenditure
Further causes of decreased concentrations: No integral part of the analysis
chronic hepatic diseases, malabsorption syndrome, spectrum of every medical specialist
porphyria cutanea tarda, elevated in rapidly proliferating laboratory
malignen melanoma
Limited value therapy control in nephrological patients
because of impaired transcobalamin filtration with
consecutive accumulation of holo-TC and as a result to
falsely normal or falsely higher or increased
concentrations
Further causes of elevated concentrations: cobalamin No integral part of the analysis
genetic defects, classical methylmalonic acidemia spectrum of every medical specialist
laboratory, GC-MS, LC-MS
No integral part of the analysis
spectrum of every medical specialist
laboratory,
GC-MS, LC-MS
Critical pre-analytics,
parameter of limited stability, special tubes recommended
Avoid artificial in vitro hemolysis
Low sensitivity Standardization of pre-analytics: fresh
specimen (< 1 h)
ZnPP also strongly elevated in ACD with impaired iron No integral part of the analysis
distribution. After intravenous iron supplementation spectrum of every medical specialist
ZnPP in hemodialysis patients normalizes slowlier than laboratory, secure pre-analytics during
the percentage of hypochromic erythrocytes. sample shipment
In nephrological patients in the stage of uremia ZnPP
measurement may be impaired by unknown interference
factors with consecutive falsely higher or falsely increased
ZnPP results, increased in iron deficiency, ACD, MDS and
in lead poisoning, only useful for screening, not suitable
for differential diagnosis of anemia [7]
Strongly focussing on iron deficiency, new ly developed Expensive
method, up to now no routine procedure.
First CE-marked assays for in vitro diagnostics (IVD) Method-dependent: SELDI-TOF-MS and
available. LC-MS expensive, but (competitive RIA
Method-dependent age- and sex-specific reference ranges and) automated ELISA on random
necessary. Circadian rhythm and day-to-day variation, access analyzer available
increased in anemia of chronic kidney disease, in
neoplastic diseases, inflammation and sepsis
(continued on next page)
Transfusion and Apheresis Science 58 (2019) 375–385
 Table 1 (continued)
V. Jansen

Biomarkers of screening and additional examinations

Biomarker Indication Indicator, predictor Limitation Expenditure

Hemoglobin electrophoresis
Glucose-6-phosphate
dehydrogenase in
erythrocytes (G6PD)
and other enzymes
Genetic diagnostics
Suspicion of hemoglobinopathy, microcytic No definite results in newborns and infants during the
anemia first six months, heterozygous alpha-thalassemia is not
+ Mentzer index < 13 detectable by hemoglobin electrophoresis.
Blood transfusions may obscure or dilute abnormal
hemoglobin for 3-4 months.
Suspicion of enzyme defect as a cause of hemolytic Genetic advice, informed consent necessary, because the No integral part of the analysis
disease in Asian, Mediterraneans, African and testing aims at a genetic defect. spectrum of every medical specialist
Afro-american Further enzyme defects not detected laboratory, submit whole blood
Confirmation of enzyme defects and Genetic advice, informed consent Institute of medical and human
hemoglobinopathies genetics, expensive

Hematological indices, ratios and formulas

Biomarker Indication Indicator, predictor Limitation Expenditure

Red cell distribution width (RDW)
Reticulocyte production index (RPI)
Low practical importance
Classification of anemia into hypo-, normo- and
hyperregenerative forms, evaluaton of erythropoesis after
stem cell transplantation, evaluation of bone marrow
activity in normocytic anemia, for example in suspicion of
intravascular hemolysis, therapy control in iron
Degree of anisocytosis, Increased values are ambiguous. Integral part of blood count (flow
elevated in iron deficiency, hemolytic anemia, pernicious cytometry)
anemia, spherocytosis, osteomyelofibrosis, decreased
values without clinical relevance
In anemia an RPI higher than 3 indicates an adequate Calculated index based on
erythropoesis, an RPI lower than 2 an inadequate reticulocyte count, considering
erythropoiesis. hematocrit and maturation time of
The RPI better reflects the activity of erythropoesis than the eythrocytes
reticulocyte count.

379
Reticulocyte hemoglobin equivalent
(Ret-He) = Content hemoglobin
of reticulocytes (CHr)
Percentage of hypochromic
erythrocytes
Transferrin saturation
sTfR/log ferritin index
Mentzer index
deficiency anemia
Anemia of nephrological patients and in chronic diseases,
monitoring of rHuEPO therapy and/or intravenous iron
substitution, indicator of reduced iron availability in
chronic kidney diseases during rHuEPO therapy
Suspicion of renal anemia
Suspicion of latent or manifest iron deficiency
Differentiation of mixed-type anemia
Differentiation of iron deficiency anemia and beta-
thalassemia
Indicator of functional iron deficiency, decreased in iron Flow cytometry:
deficiency and impaired iron distribution (early marker), measurement in reticulocyte channel
increasing during successful rHuEPO or iron therapy
[36–40]
Predictor of erythropoietic and iron response after Flow cytometry:
intravenous iron supplementation in maintenance measurement in reticulocyte channel
haemodialysis patients. Most sensitive parameter for
predicting hyporesponsiveness in CRP-positive HD
patients.
(8)
Indicator of iron turnover, Formula contains concentration of Calculated index of iron and
decreased in latent or manifest iron deficiency serum iron, which shows diurnal transferrin
[8,11,41,42] variation and day-to-day variation.
Indicator of iron supply of erythropoiesis Increased in diseases with stimulated Calculated index
[3,4,11,13,43] erythropoiesis [7]
MCV lower than 13: Simple ratio of MCV [fl] and red cell
suspicion of beta-thalassemia count [Tpt/l], inexpensive
[44]
Transfusion and Apheresis Science 58 (2019) 375–385
 Table 2
Biomarkers of anemia – diagnostic value.
V. Jansen

Biomarker Diagnostic criteria, Diagnostic Diagnostic Diagnostic value Positive predictive Negative Source
cut off sensitivity specifity value predictive value

Hemoglobin Iron deficiency in pre-school 41.7 % 89.1 % 35.7 % Yu 2011

Ferritin aged children, 41.7 % 96.4 % 62.5 % [45]
Zinc protoporphyrin (ZnPP) low hemoglobin levels, 91.7 % 60.2 % 33.3 %
ferritin < 15 μg/l,
ZPP/H ratio of
> 70 ZPP
μmol/mol heme
Iron Iron deficiency 63.5 % 38.6 % Serum iron and TIBC give no additional information in the diagnosis of Asif 2016
Total iron binding capacity 64.5 % 42.8 % iron deficiency anemia and these tests are redundant for the diagnosis of [46]
(TIBC) iron deficiency state, if serum ferritin is available.
Ferritin Ferritin at concentration of 80 % 99 %
30 ng/ml

Biomarker Diagnostic criteria, Diagnostic Diagnostic Diagnostic value Positive predictive Negative Source
cut off sensitivity specifity value predictive value
Transferrin Ferritin < 20 μg/l (males) and < 15 μg/l 36 % 97 % 79 % 83 % Withold 1994
(females) without anemia or with (hypochromic [9]
microcytic) anemia
Transferrin saturation (TS) Cut off ≤ 20 % 60.5 % Poor in all Kis 1998 [41]
studies
Transferrin saturation (TS) Iron status after intravenous iron dextran in 74 % 36 % The predictive value increased to 65 % when either 48 % for positive Low 1997
hemodialysis patients SF < 100 ng/ml or TS < 20 % were used. responses [42]
Ferritin (SF) TS < 20 % 60 % 30 % Both TS and SF are non-specific and insensitive 45 % for positive
ferritin indicators for accurate diagnosis of iron deficiency in responses

380
Transferrin saturation (TS) < 100 ng/ml 33 % 67 % hemodialysis patients in EPO. 47 % for positive
and ferritin (SF) TS < 20 % and ferritin responses
< 100 ng/ml

Biomarker Diagnostic criteria, Diagnostic Diagnostic Diagnostic value Positive predictive value Negative Source
cut off sensitivity specifity predictive value
Ferritin Uncomplicated iron deficiency, 82 % 95 % Recommended as a screening test Unsatisfactory at low prevalence Zanella 1989 [16]
confirmed by the response to iron
supplementation
Ferritin Ferritin cut off 86 % 90 % Better than transferrin, transferrin Baumann 1995 [8]
30 μg/l saturation, sTfR, percentage of
Stainable bone marrow iron hypochromic erythrocytes
Soluble transferrin Evaluation of anemia in 81 % 80 % These results suggests that the sTfR-F index Oustamanolakis 2011
receptor (sTfR) inflammatory bowel disease, seems to be very efficient in the detection and [47]
sTfR levels > 1.8 mg/l diagnosis of IDA, among patients with IBD.
sTfR/log ferritin index Evaluation of anemia in 91 % 92 % Oustamanolakis 2011
inflammatory bowel disease, [47]
sTfR/log ferritin index > 1.4

Biomarker Diagnostic criteria, Diagnostic Diagnostic Diagnostic value Positive Negative Source
cut off sensitivity specifity predictive value predictive value
sTfR/log ferritin index Cut off 1.70 […] using […] the transferrin/log (ferritin) ratio, the IDA and non-IDA Castel 2012 [43]
populations can be completely separated. The transferrin/log (ferritin)
ratio is a practical new tool that improves diagnosis of iron deficiency
when ferritin levels are inconclusive.
Reticulocyte Management of regular 81.5 % 61.6 % Useful marker and early predictor of response to intravenous iron Dalimunthe 2016
hemoglobin hemodialysis patients with iron supplementation in regular hemodialysis patients [48]
(Ret-He, CHr) deficiency anemia,
Ret-He cut off 31.65 pg
Transfusion and Apheresis Science 58 (2019) 375–385

(continued on next page)

 Table 2 (continued)
V. Jansen

Biomarker Diagnostic criteria, Diagnostic sensitivity Diagnostic specifity Diagnostic value Positive predictive Negative predictive Source
cut off value value
Percentage of hypochromic Identification of iron deficiency in patients with anemia of 72.7 % 70.4 % Torino 2015 [49]
erythrocytes chronic disease,
cut off 1.8 %
Percentage of hypochromic Prediction of iron deficiency in chronic inflammatory 77 % 90 % Baumann 1995 [8]
erythrocytes rheumatic disease anemia,
cut off 11 %

Biomarker Diagnostic criteria, Diagnostic Diagnostic Diagnostic value Positive Negative Source
cut off sensitivity specifity predictive value predictive value
Hepcidin-25, Differentiation IDA from ACD and ACD/IDA The combination of hepcidin-25 with CHr in the hepcidin-25 Thomas 2011
bioactive plot was useful for the differentiation of the states above: IDA, [34]
ACD, ACD/IDA.
Hepcidin-25, Competitive hepcidin ELISA, differential diagnosis of 100 % 67 % ROC curve analysis revealed that ELISA analysis of hepcidin is Karlsson 2017
bioactive iron deficiency anaemia with concurrent inflammation not superior to ferritin in the diagnosis of iron deficiency in [30]
Ferritin and anaemia of inflammation in elderly patients 91 % 83 % elderly anaemic patients with concurrent inflammation.
hepcidin cut off 21 μg/l
ferritin cut off
87 μg/l

Biomarker Diagnostic criteria, Diagnostic sensitivity Diagnostic specifity Diagnostic value Positive predictive value Negative predictive value Source
cut off
Hepcidin-25, urinary Iron deficiency in children 88 % 88 % Non-invasive method Sanad 2011 [33]
stage 1 96 % 92 %
cut off ≤ 0.94 nmol/mmol Cr 96 % 100 %
stage 2
cut off ≤ 0.42 nmol/mmol Cr
stage 3

381
cut off ≤ 0.08 nmol/mmol Cr
Vitamin B12 Indicator of vitamin B12 deficiency in cancer 86 % 80 % 83 % Vashi 2016 [19]
Holo-transcobalamin (Holo-TC) Vit. B12 cut off 71 % 95 % 85 %
Methylmalonic acid (MMA), plasma 385 pg/ml 86 % 99 % 98 %
Holo-TC cut off
15.5 μmol/l
MMA cut off
413.5 nmol/l

Biomarker Diagnostic criteria, Diagnostic Diagnostic Diagnostic value Positive predictive Negative predictive Source
cut off sensitivity specifity value value
Holo-transcobalamin (Holo-TC) Young healthy graduate vegetarian Indians, none 77.78 % 71.05 % 19.6 % Naik 2018
Homocysteine was folate deficient, none has clinical signs of B12 82.72 % 89.7 % The combination of B12, holo-TC and 21.7 % [50]
deficiency homocysteine improves the diagnostic accuracy at
Vit. B12 cut off these cut offs.
100 pmol/l

Biomarker Diagnostic criteria, Diagnostic Diagnostic Diagnostic value Positive Negative Source
cut off sensitivity specifity predictive value predictive value
Methylmalonic acid Cobalamin deficiency in the newborn, high prevalence of 96.4 % Plasma MMA and urinary MMA B12 are the most robust 96.2% Kalay 2016
(MMA), plasma cobalamin deficiency in developing countries, plasma markers of cobalamin deficiency. As a non-invasive method, [21]
Methylmalonic acid cobalamin and serum folic acid in pregnant women over the 95.6 % urinary MMA is a sensitive method in demonstrating 96.9 %
(MMA), urinary last four prenatal weeks, plasma cobalamin, folic acid, cobalamin deficiency in the newborn.
Homocysteine homocysteine, MMA from umbilical cord blood and urinary 88.2 % 86 %
MMA in newborns were studied, plasma cobalamin low in
66 % of the mothers.
Transfusion and Apheresis Science 58 (2019) 375–385
V. Jansen Transfusion and Apheresis Science 58 (2019) 375–385

4. Soluble transferrin receptor (sTfR)

The transferrin receptor is a cellular glycoprotein located 80% on

erythrocyte precursors. In the absence of iron for erythropoiesis, the
receptor density on the membrane is upregulated. The receptors are
constantly released from the membrane into the blood stream, thus the
concentration of soluble transferrin receptor reflects the iron supply of
erythropoiesis. The parameter is not a sensitive indicator for the early
or intermediate stages of iron deficiency, but can be useful in the di-
agnosis of advanced IDA [10]. The sTfR is a suitable parameter for a
differential diagnosis of iron-deficient erythropoesis [7]. A calculated
ratio based on sTfR and ferritin, the sTfR/log ferritin index, is a useful
indicator of iron supply of erythropoiesis [13], but is increased in all
diseases with stimulated erythropoiesis [7]. This index is not mentioned
in the S1 Guideline Iron Deficiency Anemia 2016 [6].

5. Percentage of hypochromic erythrocytes

This parameter reflects the iron supply of erythropoiesis and – in

addition to ferritin as a mirror of the storage iron – provides additional
information for the characterization of complex iron metabolism dis-
orders [11]. The proportion of hypochromic erythrocytes is determined
by flow cytometry and can be analyzed simultaneously with the blood
count - with appropriate equipment of the medical laboratory.
The percentage of hypochromic erythrocytes is considered a pre-
dictor of erythropoietic and iron response after intravenous iron sup-
plementation in maintenance haemodialysis (HD) patients and the most
sensitive parameter for predicting hyporesponsiveness in CRP-positive
HD patients [7,51].

6. Reticulocyte hemoglobin equivalent (Ret-He, CHr)

This parameter is an advanced reticulocyte index that can be co-

determined and reported by modern hematology analyzers without
additional sample. Depending on the analytical system used, corre-
sponding names are used for reticulocyte hemoglobin: Ret-He for re- Fig. 3. (a) Iron plot (Thomas plot). (b) Iron plot (Thomas plot) during rHuEPO
ticulocyte hemoglobin equivalent or CHr for content of hemoglobin in and/or iron therapy.
reticulocytes. The parameter is used to diagnose iron-deficient ery-
thropoiesis [6,7,36–40]. 8. Bioactive hepcidin

Hepcidin is a peptide hormone that is produced in the liver and

7. The iron plot
With the iron plot (Thomas plot), a quadrant model of reticulocyte
hemoglobin (Ret-He/CHr) and sTfR/log-ferritin index, latent, manifest
and functional iron deficiency and different degrees of severity of an-
emia can be differentiated in chronic diseases (Fig. 3).
Depending on which quadrant the data point is originally located in,
a differential therapy can be recommended. The changing position of
the data point during or after therapy allows conclusions about the
response (responder, non-responder) and the therapeutic success (3, 4)
Oral iron therapy is recommended for patients with a data point in
quadrants 2 and 3 (Fig. 3a), and rHuEPO therapy for patients with a
data point in quadrants 1 and 4 (Fig. 3a). Patients with a data point in
quadrant 4 have functional iron deficiency and need combined therapy
with rHuEPO and intravenous iron supplementation. Under oral iron
therapy (Fig. 3b), the arrows 1 indicate the increase in hemoglobin
formation of the reticulocytes and the iron supply of erythropoiesis.
Under rHuEPO therapy (Fig. 3b), the direction of arrow 2 indicates a
non-responsiveness to the rHuEPO dosage (non-responder), arrow 3
indicates good responsiveness to rHuEPO (responder). The direction of
arrow 4 indicates a beginning functional iron deficiency after a good
response to rHuEPO. In patients with elevated CRP > 5 mg/L, the cut
off of sTfR / log ferritin has to be positioned on 2.0 (3, 4, Figs. 3a, b).
regulates iron metabolism by absorption, transport and distribution
[11]. It is subject to a classic feedback mechanism of hormonal control
circuits. Iron overload from hereditary hemochromatosis or secondary
hemosiderosis and excessive iron absorption from beta-thalassemia are
due to hepcidin deficiency or hepcidin resistance. In pure iron defi-
ciency anemia, hepcidin is decreased, but increased in iron refractory
anemia, ACD and renal anemia [25–29,32,35].
First CE-marked ELISAs for assessment of bioactive hepcidin-25
allow its integration into the diagnostic process although sex- and age-
specific reference intervals still have to be defined. Serum hepcidin-25
may replace sTfR/log ferritin index in the iron plot (hepcidin-25 plot)
and is proposed to be useful for the differentiation of IDA, ACD and
ACD/IDA [34].
9. Zinc protoporphyrin (ZnPP)
In heme synthesis, chelation of iron to protoporphyrin is catalyzed
by the enzyme ferrochelatase. In the absence of iron or insufficient iron
distribution, the same enzyme catalyzes the binding of zinc to proto-
porphyrin. The result is zinc protoporphyrin (ZnPP) [23].
This parameter is considered to be the best indicator of iron defi-
cient erythropoiesis, both because of actual iron deficiency and iron
distribution disorder in ACD. In nephrological patients, iron-deficient

382
V. Jansen Transfusion and Apheresis Science 58 (2019) 375–385

Fig. 4. Classification of anemia.

Fig. 5. Diagnostic strategy.

Table 3
Recommendations of laboratory tests in frequent clinical settings.
Clinical setting Recommended parameters

Outpatient, girls and women, age app. 16-49 years, with microcytic hypochromic anemia, without clinical Ferritin
signs of inflammation and infection, with normal CRP
Elderly patients with chronic inflammation (rheumatic diseases, autoimmune diseases) and anemia sTfR/log ferritin index, hepcidin-25, RPI, Ret-He/CHr
Patients with solid tumors, leukemia or lymphoproliferative disorders and anemia sTfR/log ferritin index, hepcidin-25, RPI, Ret-He/CHr,
microscopic differential blood count
Nephrological or dialysis patients sTfR/log ferritin index, hepcidin-25, RPI, Ret-He/CHr, MMA,
with or without rHuEPO (and iron) supplementation homocysteine
Malnutrition sTfR/log ferritin index, holo-TC, folate
Marked microcytic anemia Mentzer index [44].
(MCV < 70 fl) and anamnestic cause (ethnicity, migration) If Mentzer index < 13:
→ hemoglobin electrophoresis, HbA2
Marked microcytic anemia (MCV < 70 fl) Microscopy of the erythrocyte morphology, Mentzer index.
If Mentzer index > 13:
→ ferritin, transferrin saturation
Macrocytic anemia Microscopic differential blood count, vitamin B12, folate.
If vitamin B12 is normal:
→ Holo-TC, MMA.
In case of pathologies in leukocytes or erythrocytes:
→ further hematological diagnostics
Patient of risk population of Holo-TC and MMA
functional vitamin B12 deficiency

erythropoiesis can be excluded at normal ZnPP concentrations. hypochromic erythrocytes. In uremic patients, ZnPP determination may
Increased ZnPP levels can be found in iron-deficient erythropoiesis be affected by unknown interfering factors that may lead to false higher
[11]. The ZnPP concentrations correlate with the proportion of or false increased ZnPP concentrations. ZnPP is a useful screening

383
V. Jansen
parameter, but not suitable for differential diagnosis of anemia, because
elevated ZnPP results are determined in IDA, ACD, MDS and lead poi-
soning [7].
10. Holo-transcobalamin (Holo-TC)
The ingested vitamin B12 is released during absorption into the
enterocytes and transferred to transcobalamin II (TC). The resulting
B12-TC complex is called Holo-TC and is available in the blood stream
for cellular uptake. This complex represents the metabolically active
vitamin B12. Only 30% of vitamin B12 is bound in this form. Lowered
holo-TC levels indicate an intracellular, metabolically manifest (func-
tional) vitamin B12 deficiency, which is also associated with MMA
elevations. Holo-TC is the earliest biomarker of a metabolically man-
ifest vitamin B12 deficiency. A decreased holo-TC associated with in-
creased MMA and elevated homocysteine indicates functional vitamin
B12 deficiency [5,20]. In nephrological patients, its diagnostic value is
limited, as disturbed transcobalamin filtration results in accumulation
of Holo-transcobalamin, resulting in false normal or false higher or
elevated concentrations. As a result, a functional vitamin B12 deficiency
is not recognized. Patients with kidney disease represent a risk group
for a vitamin B12 deficiency. In nephrological patients methylmalonic
acid in serum is the preferred parameter to detect functional vitamin
B12 deficiency [5,18,20].
11. Methylmalonic acid (MMA)
Vitamin B12 catalyzes the conversion of methylmalonic acid (MMA)
to succinyl-CoA and, together with folic acid, the conversion of
homocysteine to methionine [17,22]. In the presence of an intracellular
vitamin B12 deficiency, these enzymatic reactions are impaired, the
respective substrates, MMA and homocysteine, are metabolized to a
lesser extent, their levels rise in the blood (plasma). Functional vitamin
B12 deficiency is defined as the constellation of elevated levels of MMA
and homocysteine and normal (or even elevated) vitamin B12 levels in
the plasma. In patients with solid tumors or leukemia, elevated levels of
vitamin B12 are detected frequently, although there is a functional vi-
tamin B12 deficiency, actually [5,18,19].
12. Microscopic differential blood counts
Microscopic description of erythrocyte morphology represent a
subjective assessment method that can be standardized only to a limited
extent. In Germany, Gremienbeschlüsse des Sektorkomitees Medizinische
Laboratorien stipulate that in laboratory diagnostic examination pro-
cedures with subjective evaluation the comparability of the results has
to be ensured by consensus trainings at least twice-yearly [52]. The
determination of reticulocyte hemoglobin equivalent (Ret-He) or he-
moglobin content of reticulocytes (CHr) by flow cytometry during
routine blood count analysis (using the reticulocyte channel) and of the
percentage of hypochromic erythrocytes simultaneously is the metho-
dological answer to this challenge. Because of this further development
of instrumental analysis, standardization could be achieved and the
sensitivity significantly increased, beyond microscopic evaluation.
13. Therapy control
13.1. Therapy monitoring in iron deficiency anemia
One week after initiation of oral iron replacement therapy, the
blood count should show a typical dynamic: increase in hemoglobin
concentration, reticulocyte crisis. This provides rapid evidence for the
diagnosis of alimentary iron deficiency. If this dynamic does not persist
despite the patient's compliance, further diagnostics is necessary (S1
Guideline Iron Deficiency Anemia 2016). The S1 Guideline Iron Deficiency
Anemia 2016 [6] discusses the limited diagnostic value of ferritin in
384
Transfusion and Apheresis Science 58 (2019) 375–385
concomitant acute phase situations and recommends the parameters
sTfR and Ret-He/CHr. The guideline recommends urinary hepcidin as a
suitable biomarker in sideroblastic anemia and hereditary disorders of
the iron metabolism.
In order to determine the success of an oral iron therapy, the iron
plot is useful [3,4].
Additionally, patients with iron deficiency anemia show a normal-
ization of their ZnPP levels after successful supplementation [23].
13.2. Therapy monitoring for anemia due to chronic diseases
In non-renal patients with anemia due to chronic disease, successful
treatment of the underlying disease is also evident in ZnPP normal-
ization. In nephrological patients in the uraemic stage, the ZnPP de-
termination may be disturbed by unknown interfering factors leading to
false higher (false elevated) ZnPP results [7,23].
13.3. Monitoring rHuEPO therapy in dialysis patients
Based on the quadrant model of the Thomas plot rHuEPO re-
sponders can be distinguished from non-responders. In addition, a be-
ginning functional iron deficiency can be identified in the plot after a
good response to rHuEPO [3,4].
13.4. Therapy monitoring in vitamin B12 deficiency
The choice of therapy depends on the cause of the vitamin B12 de-
ficiency. In case of blocked or reduced oral bioavailability in the con-
text of pernicious anemia, intramuscular vitamin B12 injections are
required, otherwise oral substitution is useful. If a vitamin B12 defi-
ciency already exists, dietary measures alone are not sufficient and with
dietary habits of conviction (vegans, vegetarians) difficult to achieve.
Successful oral substitution is evident in increasing holo-TC and vitamin
B12 and decreasing MMA levels. This demonstrates the metabolic effi-
cacy of oral supplementation. In patients with kidney disease, suc-
cessful substitution therapy can be realized in declining MMA and
homocysteine levels. The holo-TC level is not suitable for therapy
monitoring in patients with kidney disease because holo-TC may ac-
cumulate due to impaired transcobalamin filtration and then not cor-
rectly reflect the vitamin B12 status [5].
13.5. Diagnostic strategy
The current request behavior of anemia biomarkers only partially
reflects the diagnostic value of new parameters. From a practical and
economic point of view, few parameters are sufficient to differentiate
the most common types of anemia and mixed-type anemia and to
identify risk groups for functional deficiencies reliably. Initial classifi-
cation of anemia with differentiation of microcytic, normocytic or
macrocytic anemia (Fig. 4) taking into account clinical and anamnestic
information may describe a clinical setting and promising biomarkers
(Fig. 5, Table 3). This algorithm may serve as a practical approach in
the diagnosis of anemia.
In addition to previous studies, further investigations will be needed
in order to understand the diagnostic potential of new biomarkers.
Declaration of Competing Interest
The author declares that there are no conflicts of interest.
References
[1] WHO. The global prevalence of anaemia in 2011. Geneva: World Health
Organization; 2015.
[2] Bundesamt Statistisches, Bonn Zweigstelle. Gesundheitsberichterstattung des
Bundes – gemeinsam getragen von RKI und Destatis. Verbrauch von Blutprodukten
V. Jansen
durch Transfusionen (Anzahl der eingesetzten Transfusionsbeutel), hier: Fremdblut
und Komponenten daraus. Jahr: 2017; Region: Deutschland; Quelle: Bericht zur
Meldung nach § 21 Transfusionsgesetz 16.04 Paul-Ehrlich-Institut. Stand; 2018.
[3] Thomas C, Thomas L. Biochemical markers and hematologic indices in the diagnosis
of functional iron deficiency. Clin Chem 2002;48(7):1066–76.
[4] Thomas L, Thomas C, Heimpel H. Neue Parameter zur Diagnostik von
Eisenmangelzuständen. Dtsch Ärztebl 2005;102:580–6. A.
[5] Herrmann W, Obeid R. Ursachen und frühzeitige Diagnostik von Vitamin-B12-
Mangel. Dtsch Ärztebl 2008;40:680–5.
[6] Behnisch W, Muckenthaler M, Kulozik A. S1-Leitlinie 025-021: Eisenmangelanämie.
Stand 01/2016. AWMF online S. 2016. p. 1–13.
[7] Hastka J, Heimpel H, Metzgeroth G. Eisenmangel und Eisenmangelanämie –
Leitlinie. Empfehlungen der Fachgesellschaft zur Diagnostik und Therapie
hämatologischer und onkologischer Erkrankungen. DGHO Deutsche Gesellschaft für
Hämatologie und Medizinische Onkologie e. V.; 2011. p. 1–21.
[8] Baumann Kurer S, Seifert B, Michel B, Ruegg R, Fehr J. Prediction of iron deficiency
in chronic inflammatory rheumatic disease anaemia. Br J Haematol
1995;91(4):620–6.
[9] Withold W, Neumayer C, Beyrau R, Heins M, Schausell S, Rick W. Efficacy of
transferrin determination in human sera in the diagnosis of iron deficiency. Eur J
Clin Chem Clin Biochem 1994;32(1):19–25.
[10] Choi JW. Sensitivity, specificity, and predictive value of serum soluble transferrin
receptor at different stages of iron deficiency. Ann Clin Lab Sci 2005;35(4):435–9.
[11] Graf L, Herklotz R, Huber AR, Korte W. Alte und neue Eisenparameter – Bedeutung
im Eisenstoffwechsel und in der Diagnostik. Therapeutische Umschau
2008;65:519–28.
[12] Babaei M, Shafiei S, Bijani A, Heidari B, Hosseyni SR, Sadeghi MV. Ability of serum
ferritin to diagnose iron deficiency anemia in an elderly cohort. Rev Bras Hematol
Hemoter 2017;39(3):223–8.
[13] Lee EJ, Oh EJ, Park YJ, Lee HK, Kim BK. Soluble transferrin receptor (sTfR), ferritin
and sTfR/log ferritin index in anemic patients with nonhematologic malignancy
and chronic inflammation. Clin Chem 2002;48(7):1118–21.
[14] Porter DR, Sturrock RD, Capell HA. The use of serum ferritin estimation in the
investigation of anaemia in patients with rheumatoid arthritis. Clin Exp Rheumatol
1994;12(2):179–82.
[15] Schaefer L, Schaefer RM. Diagnose der Rheuma-Anämie. Akt Rheumatol
2009;34(2):104–8.
[16] Zanella A, Gridelli L, Berzuini A, Colotti MT, Mozzi F, Milani S, et al. Sensitivity and
predictive value of serum ferritin and free erythrocyte protoporphyrin for iron
deficiency. J Lab Clin Med 1989;113(1):73–8.
[17] Herbert V, Das KC. The role of vitamin B12 and folic acid in hemato- and other cell-
poiesis. Vitam Horm 1976;34:1–30.
[18] Klee GG. Cobalamin and folate evaluation: measurement of methylmalonic acid and
homocysteine vs vitamin B12 and folate. Clin Chem 2000;46(8(B)):1277–83.
[19] Vashi P, Edwin P, Popiel B, Lammersfeld C, Gupta D. Methylmalonic acid and
homocysteine as indicators of vitamin B-12 deficiency in cancer. PLoS One
2016;11(1):e0147843.
[20] Hannibal L, Lynse V, Bjørke-Monsen A-L, Behringer S, Grünert SC, Spiekerkoetter U,
et al. Biomarkers and algorithms for the diagnosis of vitamin B12 deficiency. Front
Mol Biosci 2016;3(27):1–16.
[21] Kalay Z, Islek A, Parlak M, Kirecci A, Guney O, Koklu E, et al. Reliable and powerful
laboratory markers of cobalamin deficiency in the newborn: plasma and urinary
methylmalonic acid. J Matern Fetal Neonatal Med 2016;29(1):60–3.
[22] Allen RH, Stabler SP, Lindenbaum J. Metabolic abnormalities in cobalamin (vitamin
B12) and folate deficiency. FASEB J 1993;7(14):1344–53.
[23] Braun J. Erythrocyte zinc protoporphyrin. Kidney Int 1999;55(69):57–60.
[24] Hennig G, Homann C, Teksan I, et al. Non-invasive detection of iron deficiency by
fluorescence measurement of erythrocyte zinc protoporphyrin in the lip. Nat
Commun 2016;7:1–8.
[25] D’Angelo G. Role of hepcidin in the pathophysiology an diagnosis of anemia. Blood
Res 2013;48(1):10–5.
[26] Ganz T, Nemeth E. The hepcidin-ferroportin system as a therapeutic target in an-
emias and iron overload disorders. Hematol Am Soc Hematol Educ Program
2011;2011:538–42.
[27] Ganz T. Hepcidin. Rinsko Ketsueki 2016;57(10):1913–7.
[28] Girelli D, Nemeth E, Swinkels DW. Hepcidin in the diagnosis of iron disorders.
385
Transfusion and Apheresis Science 58 (2019) 375–385
Blood 2016;127(23):2809–13.
[29] Kali A, Charles MVP, Seetharam RSK. Hepcidin – a novel biomarker with changing
trends. Pharmacogn Rev 2015;9(17):35–40.
[30] Karlsson T. Evaluation of a competitive hepcidin ELISA assay in the differential
diagnosis of iron deficiency anaemia with concurrent inflammation and anaemia of
inflammation in elderly patients. J Inflamm (Lond) 2017;14:21.
[31] Papanikolaou G, Tzilianos M, Christakis J, et al. Hepcidin in iron overload dis-
orders. Blood 2005;105(10):4103–5.
[32] Robson KJ. Hepcidin and its role in iron absorption. Gut 2004;53(5):617–9.
[33] Sanad M, Gharib AF. Urinary hepcidin level as an early predictor of iron deficiency
in children: a case control study. Ital J Pediatr 2011;37:37.
[34] Thomas C, Kobold U, Balan S, Roeddinger R, Thomas L. Serum hepcidin-25 may
replace the ferritin index in the Thomas plot in assessing iron status in anemic
patients. Int J Lab Hematol 2011;33(2):187–93.
[35] Useda N, Takasawa K. Impact of inflammation on ferritin, hepcidin and the man-
agement of iron deficiency anemia in chronic kidney disease. Nutrients
2018;27(9):1–33. 10.
[36] Brugnara C, Schiller B, Moran J. Reticulocyte hemoglobin equivalent (Ret He) and
assessment of iron-deficient states. Clin Lab Haematol 2006;28(5):303–8.
[37] Brugnara C. Use of reticulocyte cellular indices in the diagnosis and treatment of
hematological disorders. Int J Clin Lab Res 1998;28(1):1–11.
[38] Garzia M, Di Mario A, Ferraro E, Tazza L, Rossi E, Luciani G, et al. Reticulocyte
hemoglobin equivalent: an indicator of reduced iron availability in chronic kidney
diseases during erythropoietin therapy. Lab Hematol 2007;13(1):6–11.
[39] Peerschke EIB, Pessin MS, Maslak P. Using the hemoglobin content of reticulocytes
(RET-He) to evaluate anemia in patients with cancer. Am J Clin Pathol
2014;142:506–12.
[40] Schaefer RM, Schaefer L. Hypochromic red blood cells and reticulocytes. Kidney Int
Suppl. 1999;69:44–8.
[41] Kis AM, Carnes M. Detecting iron deficiency in anemic patients with concomitant
medical problems. J Gen Intern Med 1998;13(7):455–61.
[42] Low CL, Bailie GR, Eisele G. Sensitivity and specificity of transferrin saturation and
serum ferritin as markers of iron status after intravenous iron dextran in hemo-
dialysis patients. 2019.
[43] Castel R, Tax MG, Droogendijk J, Leers MP, Beukers R, Levin MD, et al. The
transferrin/log(ferritin) ratio: a new tool for the diagnosis of iron deficiency an-
emia. Clin Chem Lab Med 2012;50(6):1343–9.
[44] Mentzer WC. Differentiation of iron deficiency from thalassaemia trait. Lancet
1973;7808:882–4.
[45] Yu KH. Effectiveness of zinc protoporphyrin/heme ratio for screening iron defi-
ciency in preschool-aged children. Nutr Res Pract 2011;5(1):40–5.
[46] Asif N, Iliaz A, Rafi T, Haroon ZH, Bashir S, Ayyub M. Diagnostic accuracy of serum
iron an total iron binding capacity (TIBC) in iron deficiency state. J Coll Physicians
Surg Pak 2016;26(12):958–61.
[47] Oustamanolakis P, Koutroubakis IE, Messaritakis I, Niniraki M, Kouroumalis EA.
Soluble transferrin receptor-ferritin index in the evaluation of anemia in in-
flammatory bowel disease: a case-control study. Ann Gastroenterol
2011;24(2):108–14.
[48] Dalimunthe NN, Lubis AR. Usefulness of reticulocyte hemoglobin equivalent in
management of regular hemodialysis patients with iron deficiency anemia. Rom J
Intern Med 2016;54(1):31–6.
[49] Torino ABB, de Fatima Pererira Gilberti M, da Costa E, Freire de Lima GA, Zerlotte
H, Grotto W. Evaluation of erythrocyte an reticulocyte parameters as indicative of
iron deficiency in patients with anemia of chronic disease. Rev Bras Hematol
Hemoter 2015;37(2):77–81.
[50] Naik S, Mahalie N, Bhide V. Identification of vitamin B12 deficiency in vegetarian
Indians. Br J Nutr 2018;119(6):629–35.
[51] Braun J, Lindner K, Schreiber M, Heidler RA, Hörl WH. Percentage of hypochromic
red blood cells as predictor of erythropoetic and iron response after i. V. Iron
supplementation in maintenance haemodialysis patients. Nephrol Dial Transplant
1997;12(6):1173–81.
[52] DAkkS – Beschlüsse des Sektorkomitees Medizinische Laboratorien zu
Anforderungen der DIN EN ISO 15189:2014 an die Qualität und Kompetenz von
Medizinischen Laboratorien 71 SD 3 025 | Revision: 1.4 | 17. Sicherstellung der
Vergleichbarkeit von Ergebnissen bei Verfahren mit subjektiver Bewertung; 2017.
November 2017 5.6-03.