Reticulocyte hemoglobin content in the diagnosis of iron deficiency in Chinese pre-menopausal women

Pre-menopausal women are at risk for iron deficiency due to menstrual blood losses. The prevalence rates (PR) of iron depletion, iron deficiency anemia (IDA), and iron deficiency were 34.4%, 15.1% and 49.5% in pre-menopausal non-pregnant women respectively in China.¹ Traditionally, the diagnosis of iron deficiency relies on the hematological markers (hemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), red cell distribution width (RDW)) and biochemical markers (serum ferritin (SF), serum iron (SI), transferrin saturation (TS), total iron-binding capacity (TIBC)). Recently, the reticulocyte hemoglobin content (CHr) is considered to be used as a marker of iron deficiency.² The aim of this study was to compare the diagnostic efficiency of CHr with the old markers in diagnosis of iron deficiency in Chinese pre-menopausal women.

Study subjects
All study subjects were gynaecological outpatients who were enrolled from September 2004 to November 2005 at Peking Union Medical College Hospital, and by review of their medical history (without any systemic diseases), physical examination (without ECG signs of angina pectoris, myocardial infarction, or other cardiovascular disease) and by routine laboratory studies (hematologic, liver, renal studies). The eligibility criteria was >18 years and≤45 years, pre-menopausal non-pregnant women only. In this study, iron deficiency included non-anemic iron deficiency and IDA. Non-anemic iron deficiency was defined as ferritin ≤14 µg/L and hemoglobin ≥110 g/L, and IDA as ferritin ≤14 µg/L and hemoglobin <110 g/L. The controls had ferritin >14 µg/L and hemoglobin ≥110 g/L. The subjects who had infectious, inflammatory disease or any other disease were excluded from this study. At last, 71 iron deficient subjects (41 non-anemic iron deficient women (mean (30.5±5.4) years), 30 IDA women (mean (30.9±6.1) years)) and 71 healthy controls (mean (30.0±7.2) years) were selected.

Laboratory assays
After overnight fasting, venous blood samples were collected separately in 3.6% EDTA tubes and serum separator tubes. Samples in tubes containing 3.6% EDTA were used to examine the haematology parameters, including erythrocyte (HGB, MCV, RDW) and reticulocyte indices (including CHr) on Advia 120 haematology analyzer (Bayer diagnostics, Dublin, Ireland) within 2 hours. Reticulocytes were measured by staining the reticulocyte RNA, with the dye oxazine, and measured the cell adsorption by flow cytometry. Samples in serum separator tubes were centrifuged and separated at once after collection, and serum were stored at –70˚C. SF was measured by immunochemiluminescence (IMX, Abbott, USA) while the SI and TIBC were measured by colorimetric method on chemical spectrophotometer.

Statistical analysis
All analyses were performed with SPSS v.11.0. The normality of the distribution of continuous variables was tested with the Kolmogorov-Smirnov test. Normal distributions of continuous variables were expressed as mean ± standard deviation (SD). Skewed distributions of variables were expressed as median. One way analysis of variance (ANOVA) was used to compare the levels of CHr, HGB, HCT, MCV, and TIBC from 3 groups. Kruskal-Wallis test (non-parametric statistics) was used to compare the levels of SF, SI, TS, and RDW from 3 groups. Receiver operator characteristic (ROC) curve was used to describe the reliability of CHr in diagnosis of iron deficiency and non-anemic iron deficiency. A two-tailed P value <0.05 was considered statistically significant.

Hematological and biochemical indicators in non-anemic iron deficient group, iron deficient anemia group and normal group
The results of hematological and biochemical indicators in 3 groups are shown in Table. The values of CHr in the non-anemic iron deficient group was significantly lower than the normal group (P<0.001) and higher than the IDA group (P<0.001). The HGB, HCT, MCV, SI, TS values in the non-anemic iron deficient group was significantly lower than the normal group (P<0.001, respectively) and higher than the IDA group (P<0.01, respectively). The SF values in the non-anemic iron deficient group was significantly lower than the normal group (P<0.001). No significant difference was observed in the SF between the non-anemic iron deficient group and the IDA group (P=0.234). The RDW values in the non-anemic iron deficient group was higher than the normal group (P<0.001) and lower than in the IDA group (P<0.001). TIBC showed no significant difference among 3 groups (P=0.448).

ROC curve analysis in diagnosing iron deficiency
ROC curve analysis was performed to evaluate the reliability of different indicators in diagnosing iron deficiency. Lower test results of CHr, TS, HGB, MCV, HCT and SI as well as higher test results of RDW indicated positive test result. ROC curve in our study was built on the condition of smaller test results indicating more positive tests, so RDW was at the bottom of Fig. 1. The area under the curve for CHr, RDW, TS, HGB, MCV, HCT, SI, TIBC was 0.928, 0.915, 0.915, 0.9, 0.886, 0.87, 0.868, 0.434, respectively (Fig. 1).

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Fig. 1. Receiver operating characteristic curves for CHr and the conventional indicators in the diagnosis of iron deficiency.

ROC curve analysis in diagnosing non-anemic iron deficiency
The area under the curve for CHr, TS, RDW, HGB, MCV, HCT, SI, TIBC was 0.892, 0.865, 0.856, 0.827, 0.817, 0.776, 0.798, 0.441, respectively (Fig. 2).

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Fig. 2. Receiver operating characteristic curves for CHr and the conventional indicators in the diagnosis of non-anaemic iron deficiency.

DISCUSSION

Iron deficiency, especially non-anemic iron deficiencies are easy to be neglected in women. In our study, ROC curves showed that CHr had a better predictive ability than TS, RDW, HGB, MCV, HCT, SI, TIBC in diagnosing iron deficiency and non-anemic iron deficiency in pre-menopausal women. Prussian Blue stain of bone marrow aspirate is the “gold standard” test for iron deficiency. Due to its invasive, painful, and high cost, Prussian Blue stain is limited. SF, SI, TS, and TIBC are always used to evaluate the iron status. SI exhibits diurnal variations, with higher concentrations at the end of the day, and may transiently reach reference values after ingestion of meat or oral iron supplements. Oral contraceptives are known to increase in serum transferrin and produce inappropriately low TS.³ SF was a reliable marker in diagnosing iron deficiency. However, SF is limited by its low sensitivity, because it is an acute phase reactant (APR). In APR patients, biochemical markers demonstrate weaknesses in the diagnosis of functional ID as defined by hematological indices.⁴ CHr is expected to provide a reliable direct marker of iron status, regardless the presence of APR.^4-6

Mast et al² demonstrated that CHr had the highest sensitivity and specificity among the peripheral blood tests (CHr, ferritin, transferrin saturation, and MCV) in predicting the absence of bone marrow iron stores. Some studies found that the use of CHr for iron deficiency anemia screening may increase the accuracy of diagnosis, enabling early detection in 9- to 12-month infants, children and adolescents.^7-9 CHr has been shown to be a sensitive，specific, stable marker of iron status in dialysis patients.¹⁰

It is known that the average survival of erythrocyte is about 120 days, which limits the sensitivity of red blood cell indicators to provide real-time information. However, reticulocytes are the earliest red blood cells to enter the circulation and exist for only 1 to 2 days. CHr expresses the content of hemoglobin in reticulocytes. CHr is calculated as the product of volume multiplies by the hemoglobin concentration of reticulocytes. CHr is more stable than hemoglobin concentration and the cell volume of the reticulocyte because the hemoglobin concentration increases as the cell volume decreases during reticulocyte maturation. It is likely that at early stage of iron deficiency, before the development of anemia, the impaired iron supply to the erythron produces transient bouts of iron-deficient erythropoiesis, which lead to the shift in the distribution of CHr to lower values and to the appearance of hypochromic cells. These changes are then followed by the development of frank microcytosis and hypochromia and are therefore helpful in identifying early stages of iron deficiency, especially in conditions where biochemical markers are not informative.¹¹ Therefore determination of the reticulocyte hemoglobin content (CHr) may allow a real-time evaluation of iron deficiency.

Automated analysis of reticulocytes by ADVIA 120 analyzer provides better precision, accuracy determination than the manual analysis before. The analyzer allows simultaneous determination of markers on both mature red blood cells and reticulocytes. CHr may be measured as a by-product of routine complete blood count at a little increment cost within the same EDTA-blood sample. This would result in a significant reduction in the expense and the amount of blood needed in the clinical diagnosis. Furthermore, all of the results of complete blood cells and reticulocyte counts (including CHr) can be gained in about an hour.

Our data showed that CHr was the best predictor of iron deficiency, especially non-anemic iron deficiency when compared to TS, RDW, HGB, MCV, HCT, SI, TIBC in Chinese pre-menopausal women. Diagnosing iron deficiency based on CHr is simple, inexpensive, rapid and practical to perform. We supposed that CHr is an ideal test to early predict iron deficiency, especially non-anemic iron deficiency. Further studies are needed to determine whether CHr should be the preferred screening tool in the early detection of iron deficiency in larger, unselected population of pre-menopausal women and children.

REFERENCES

1. Liao QK, Chinese Children, Pregnant Women & Pre-menopausal Women Iron Deficiency Epidemiological Survey Group. Prevalence of iron deficiency in pregnant and pre-menopausal women in China: a nationwide epidemiological survey. Chin J Hematol (Chin) 2004; 25: 653-657.

2. Mast AE, Blinder MA, Lu Q, Flax S, Dietzen DJ. Clinical utility of the reticulocyte hemoglobin content in the diagnosis of iron deficiency. Blood 2002; 99: 1489-1491.

3. Brugnara C. Iron deficiency and erythropoiesis: new diagnostic approaches. Clin Chem 2003; 49: 1573-1578.

4. Thomas C, Thomas L. Biochemical markers and hematological indices in the diagnosis of functional iron deficiency. Clin Chem 2002; 48: 1066-1076.

5. Markovic M, Majkic-Singh N, Subota V, Mijuskovic Z. Reticulocyte hemoglobin content in the diagnosis of iron deficiency anemia. Clin Lab 2004; 50: 431-436.

6. Mitsuiki K, Harada A, Miyata Y. Reticulocyte hemoglobin content in hemodialysis patients with acute infection. Clin Exp Nephrol 2004; 8: 257-262.

7. Ullrich C, Wu A, Armsby C, Rieber S, Wingerter S, Brugnara C, et al. Screening healthy infants for iron deficiency using reticulocyte hemoglobin content. JAMA 2005; 294: 924-930.

8. Brugnara C, Zurakowski D, DiCanzio J, Boyd T, Platt O. Reticulocyte hemoglobin content to diagnose iron deficiency in children. JAMA 1999; 281: 2225-2230.

9. Stoffman N, Brugnara C, Woods ER. An algorithm using reticulocyte hemoglobin content (CHr) measurement in screening adolescents for iron deficiency. J Adolesc Health 2005; 36: 529.

10. Tsuchiya K, Okano H, Teramura M, Iwamoto Y, Yamashita N, Suda A, et al. Content of reticulocyte hemoglobin is a reliable tool for determining iron deficiency in dialysis patients. Clin Nephrol 2003; 59: 115-123.

11. Brugnara C. A hematologic “gold standard” for iron-deficient states? Clin Chem 2002; 48: 981-982.