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ZPP/H for iron deficiency anemia diagnosis
Discussion
In this study of healthy rural community-dwelling non- anemic Indian women and children and their counterparts with biochemically defined IDA, we 1) report the median values for the iron biomarkers erythrocyte ZPP/H and serum hepcidin, 2) analyze ROC curves for erythrocyte ZPP/H and serum hepcidin, and 3) define the ZPP/H ratio and serum hepcidin cut-off values for IDA diagnosis and estimate the post-test probability of IDA for these cut-off values. Overall, these findings demonstrate the utility of erythrocyte ZPP/H as a POC biomarker for IDA diagnosis, particularly in women and children from low-middle income settings.
We found similar median ZPP/H levels in non-anemic children to those reported previously (47.5 and 58 mmol/mol heme).15,16 Although evaluated systematical- ly in children and non-anemic pregnant women with iron deficiency,5,17 ZPP/H levels have not been studied either in women or preschool children using rigorous criteria for nutritional IDA. Only one large Indian study of tribal adults and children (<18 years) previously used ZPP/H to detect IDA in a subset (n=100) of anemic individuals (mean Hb 8.4) with normal Hb phenotype.18 The authors reported a higher mean ZPP/H value (214.9 ± 120.1) than in our study. This discrepancy may be explained by either the difference in the two study populations (sever- ity of anemia or undetected Hb disorders) or methodolog- ical differences (i.e., whole blood vs. washed erythro- cytes).19 The median ZPP/H ratios reported in our study probably reflect values encountered in healthy women and children residing in rural Indian communities.
Serum hepcidin values in non-anemic children in our study are concordant with reports in European chil- dren,20,21 but higher than values reported in Asian22 and African children.23,24 The inclusion of <12-month-old non-anemic children in the latter studies explain these differences, since hepcidin concentrations are decreased between three and six months of age.24 Children with IDA in our study had serum hepcidin levels comparable with those reported in anemic children from Asia25 and Africa.23 Non-anemic women in our study had variable levels compared with those previously reported in European studies,26,27 discrepancies that are possibly explained by socioeconomic and dietary differences between these populations. Some of the inter-study vari- ability is also possibly attributable to differences in the methodological assays used to estimate hepcidin.28 Women with IDA in our study had very low median hepcidin levels concordant with previously published studies.29
Using ROC analysis, we selected cut-off values for ZPP/H that detected IDA in both women and children with >90% specificity. Cut-off values selected in recent studies were lower (>40 mmole/mole30,31 heme and >70 mmol/mole heme6) and lacked specificity (56% and 60%, respectively), perhaps because they were selected to detect iron deficiency, not IDA. Another recent study uti- lized a ZPP/H cut-off value of 70 mmole/mole to diagnose IDA in a pediatric population, but this value had a low specificity (75%).32 The large Indian study referenced pre- viously used a cut-off value of >80 μmole/mole heme to define IDA in a mixed population of healthy individuals and those with sickle cell trait or sickle cell anemia.18 The scientific rationale for this cut-off value and the validity
of ZPP/H as a stand-alone diagnostic assay for IDA in individuals with sickle cell anemia, and possibly α-thalas- saemia,33 is uncertain. In contrast, our study used rigorous biochemical criteria to define IDA in a representative sample of healthy community-dwelling women and chil- dren. Consequently, this is the first study to demonstrate the utility of ZPP/H as a biomarker of IDA and define cut- off values with which to establish an IDA diagnosis in healthy rural women and children.
The hepcidin cut-off values selected to diagnose IDA in children in our study were higher than those selected pre- viously in Korean children (≤6.85ng/mL vs. ≤2.735ng/mL) and yielded higher AUCs (0.97 vs. 0.90).25 The higher AUC value indicates a better discriminative power of hepcidin in detecting IDA. However, these cut-off values were sim- ilar to values that detect IDA in six to 60-month-old Gambian and Tanzanian children (5 and 8 ng/ml, respec- tively).7
The proposed cut-off values, with their high sensitivity and specificity, increase the probability of a diagnosis of IDA. However, we also determined the predictive values of these tests by estimating their likelihood ratios. The likelihood ratio indicates how many times more likely a particular test result is, in a patient with that particular condition, with a likelihood (LR) ratio value >10 providing robust diagnostic evidence.34 Assuming a pre-test probabil- ity of IDA of 50% in any given population, the selected ZPP/H and hepcidin cut-off values had likelihood ratios of ~10, which according to the Fagan nomogram corre- sponded with a 90% post-test probability of having an IDA diagnosis.35 Thus, even in populations with lower pre-test probabilities of having IDA, these diagnostic cut- offs are valid.
Measuring erythrocyte ZPP/H is procedurally simple, technically feasible by field health workers possessing <12th-grade education, and could provide rapid results at the primary health center. Rapid diagnosis would facilitate therapeutic decision-making in a single visit and favor patient convenience, an important consideration in low- middle income settings. Although not a prior study objec- tive, informal assay cost estimates in our laboratory indi- cate that ZPP/H measurement is cheaper than hepcidin. Thus, our findings suggest that ZPP/H has greater utility as a POC diagnostic test to detect IDA in women and chil- dren. Although hepcidin is extremely useful in predicting iron absorption and incorporation into erythrocytes,8 potential limitations of its use in this setting include higher costs, lack of hepcidin standardization and the require- ment to convert the immunological assay into a POC assay.36
The strengths of this study are its inclusion of a large representative sample of healthy community dwelling women and children, a random selection of blood samples for biomarker measurements, and rigorous definition of IDA using multiple biomarkers.4,37 Using a combination of low Hb with ferritin as a gold standard for IDA instead of bone marrow aspiration with perls staining raises poten- tial concerns regarding diagnostic accuracy. Reassurance against this concern is provided by the sTfR/log ferritin index >2 and near normalization of Hb in response to iron therapy in children with IDA after six months treatment (baseline Hb for children in the IDA group = 9.6±0.8g/dL; six-month post-treatment Hb = 10.5±1.3g/dL). Finally, estimates of the diagnostic accuracy for the proposed cut- off values highlight the clinical applicability of these find-
haematologica | 2018; 103(12)
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