Evaluating Biomarkers Of Iron Deficiency Anemia In Anemia Of Inflammation

Low middle income countries like India have a high prevalence of childhood anemia (74% in children <5years of age) (NFHS-3 survey 2006) and the predominant cause is iron deficiency anemia (IDA) (Pasricha et al, Pediatrics 2010). Additionally, in this setting there is a large burden of infectious diseases in children. Thus, several community dwelling Indian children have anemia and infection, where the use of standard markers such as ferritin to diagnose IDA is challenging. Using HIV infected children as a model for anemia of inflammation (AI), we explored the utility of zinc protoporphyrin/heme (ZPP/H) as a biomarker for the diagnosis of IDA superimposed on AI.

We prospectively enrolled 241 HIV-infected children in a multicentric study to assess anemia in HIV infected children. Of these, 221 children (aged 2-12 years, mean 7.86 years) had evaluable blood samples that were used to measure hematological and biochemical parameters. Stored samples of washed erythrocytes were used to measure Zinc protoporphyrin/Heme ratio (ZPP/H) with a hematofluorometer (AVIV Biomedical). Using standard biochemical assays, we measured serum ferritin, high sensitivity C-reactive protein and serum transferrin receptor levels. Anemia was defined using WHO age specific hemoglobin-based criteria (2011). The soluble transferrin receptor log ferritin index (sTfR-F index ≥1.5) was used to identify those having pure IDA (index ≥ 1.5) and non-iron deficient AI (sTfR-F index < 1.5). Statistical analysis was performed using SPSS software (version 21.0 for Mac). The performance of ZPP/H ratio was compared to sTfR-F index as a gold standard using Receiver Operator Characteristics (ROC).

Among 221 evaluable patients, the mean age was 7.9 ± 2.5 years with a range of 2-12 years. Based on an age-stratified hemoglobin criteria (WHO, 2011), 65 children (29.4%) were classified as anemic, of whom 56% were males. Comparisons between anemic and non-anemic children are presented in tables 1 and 2. Using previously published biochemical criteria (sTfR index > 1.5; Punnonen et al, Blood 1997); we found that the sTfR index detected pure IDA in 43% cases. Using an alternative algorithm based on the serum ferritin value (Goodnough et al, NEJM 2005), the patients were reclassified into pure IDA (46.2%, <30mg/l), pure AI (20.0%, >100mg/l) and mixed IDA and AI (33.8%, 30-100mg/l). High sensitivity CRP (>1.0mg/dl indicating inflammation) biochemically defined AI, in 35.4% patients. An ROC for ZPP/H levels was plotted using the sTfR index as a gold standard (n=65), with an area under the curve (AUC) of 0.80 and a standard error of 0.05. The ZPP/H cutoff value of 59.5μmol/mol heme, on the efficiency curve, identified IDA with 96.4% sensitivity and 78.4% specificity.

Our studies suggest that a ZPP/H cutoff value of 59.5 μmol/mol heme can diagnose IDA in anemic HIV infected children with acceptable sensitivity and specificity. In low middle-income countries, ZPP/H is a relatively cost efficient point of care test that can guide the use of iron supplements. However, further studies in a healthy pediatric population are needed to assess the validity of these cut off values.

No relevant conflicts of interest to declare.