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1 Laboratoriumsmedizin, Krankenhaus Nordwest, Steinbacher Hohl 2-26, 60488 Frankfurt/Main, Germany.
aAuthor for correspondence. Fax 49-69787390; e-mail TH-books{at}t-online.de.
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Abstract |
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Methods: We studied 442 patients with various disease-specific anemias and 154 nonanemic patients. As indicators of red cell hemoglobinization, we measured the reticulocyte hemoglobin content (CHr) and the proportion of hypochromic red cells (HYPO), using an Advia 120 hematology analyzer. Ferritin, transferrin, transferrin saturation, and the concentration of the soluble transferrin receptor (sTfR) were determined by ELISA and immunoturbidimetric assay. The sTfR/log ferritin ratio (sTfR-F index) was used as an additional marker for biochemical identification of iron-deficient erythropoiesis.
Results: In a control group (n = 71), the 2.5 percentile values were 28 pg for CHr and 5% for HYPO. These values were used to indicate unimpaired red cell hemoglobinization and absence of functional ID. In patients with deficient red cell hemoglobinization but no acute-phase response (APR), iron-deficient erythropoiesis was indicated by serum ferritin and sTfR-F index values 
20.8 µg/L and >1.5, respectively. Corresponding values in patients with APR were 61.7 µg/L and >0.8, respectively. The positive likelihood ratios for the biochemical markers and the sTfR-F index for identifying iron-restricted erythropoiesis in patients with and without APR were 2.6–6.9 and 4.3–16.5, respectively.
Conclusion: In APR patients, biochemical markers demonstrate weaknesses in the diagnosis of functional ID as defined by hematologic indices. Use of diagnostic plots to illustrate the relationship between the sTfR-F index and CHr allows the progression of ID to be identified, regardless of whether an APR is present.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ACD is characterized by inadequate production of erythropoietin, inhibition of the proliferation of erythroid progenitor cells in the bone marrow, and disturbances in iron distribution (6). ACD results from activation of the immune and inflammatory systems. There is increased production of inflammatory cytokines, which in turn increases the concentration of C-reactive protein (CRP) (7). In ACD, like IDA, functional ID is the limiting factor of erythrocyte hemoglobinization. Functional ID is defined as an imbalance between the iron needs of the erythroid marrow and the iron supply, which is not maintained at a rate sufficient to allow normal hemoglobinization of the erythrocytes. This leads to reduced reticulocyte and erythrocyte cellular hemoglobin (Hb) content. In IDA, the iron supply depends on the amount of the iron stores, whereas in ACD, the supply depends on its rate of mobilization. In ACD, functional ID may occur even in the presence of large iron stores when iron release is impaired (8).
The hemoglobinization of erythrocytes is used to detect functional ID because the Hb content of reticulocytes and erythrocytes provides an evaluation of the bone marrow activity, reflecting the balance between iron and erythropoiesis (3). However, biochemical markers measure iron supply for the bone marrow and are only indirect indicators of the balance between iron and erythropoiesis (9).
Traditionally, the standard biochemical markers of iron metabolism have been serum or plasma iron, transferrin (Tf), transferrin saturation (TfS), ferritin, and more recently, measurement of serum soluble Tf receptor (sTfR). The diagnosis of IDA is based on the presence of anemia and erythrocyte morphology (hypochromia, microcytosis) in conjunction with low serum ferritin, decreased TfS, or increased sTfR.
Diagnosis of ID associated with serum ferritin concentrations within established reference intervals can be difficult in anemia that accompanies diseases in which there is an acute-phase response (APR). Ferritin is an acute-phase reactant and Tf a negative one (10)(11). Increased sTfR is also a useful indicator of ID. This marker may be increased in patients who display hyperproliferative erythropoiesis (12)(13). Finally, heterozygous ß-thalassemia (ß-thal trait) may mimic the usual hematologic indicators of ID by producing hypochromia and microcytosis. These diagnostic difficulties have led to efforts to develop clinical laboratory tests that are capable of measuring the functional iron availability at the site of Hb synthesis in the erythron, especially the erythrocyte and its precursors. Modern hematology analyzers, which measure individual erythrocytes rather than mean cell indices of the whole erythrocyte population, are able to provide acceptable identification of small subpopulations of iron-deficient erythrons (14)(15).
An hematologic index that has gained merit in assessing functional iron status is the measurement of the proportion of hypochromic red cells (HYPO). Because erythrocytes have a lifespan of 
120 days, the HYPO index is able to provide information over a several-month period and is a late indicator of iron-restricted erythropoiesis (16). A HYPO value <10% in association with low serum ferritin is assumed to indicate that the iron supply for erythropoiesis is maintained at a rate sufficient for normal red cell hemoglobinization, although the body’s iron stores may have been considerably depleted (17).
Reticulocyte Hb content (CHr) is an early marker of functional ID, as reticulocytes exist in the circulation for only 1–2 days. The usefulness of this index in monitoring erythropoietic function is indicated by studies of the evaluation of iron status in hemodialysis patients (18)(19), in the diagnosis of ID in children (3), and in the diagnosis and treatment of various hematologic disorders (20).
The intent of this study was to evaluate the clinical efficacy of the majority of standard biochemical markers for ID in anemic patients to determine the best marker for distinguishing ID from ACD and the combined state of functional ID/ACD. The diagnosis of functional ID for all patients was based on an examination of erythrocyte hemoglobinization using the CHr test and the HYPO test as the gold standard (3)(15)(16)(17)(21).
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Materials and Methods |
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. Diagnoses were unknown to the analysts until the laboratory findings were completed. Blood specimens were collected from the patients within 24 h of their hospitalization. Of the 602 patients selected, 596 (240 men and 356 women; mean ± SD age, 64 ± 15 years for the men and 54 ± 22 years for the women) were investigated for complete blood cell count, the standard biochemical markers of iron metabolism, and CRP. Six patients were excluded from the study because their specimens did not fulfill the preanalytical criteria of our clinical chemistry laboratory. Of the 596 patients, 442 (74%) were anemic and 154 (26%) were not anemic.
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It is important to apply inclusion criteria very carefully to individuals to ensure that reference intervals for the hematologic indices and biochemical markers of iron metabolism are correct. Reference intervals may not be appropriate for hospitalized patients if only entirely healthy individuals are enrolled. The controls for this study were selected by the following procedures:
140 g/L for men and 123 g/L for women (22); normocytic, normochromic red cells; a red cell distribution width (RDW) 15%; a corrected reticulocyte index (CRI) 2.6%; a leukocyte count 10 000/µL; and a CRP concentration 5 mg/L (23). We used ROC curve analysis to evaluate the ability of biochemical markers for ID to discriminate patients with functional ID from cases without ID. The patient group consisted of 227 patients with normal red cell hemoglobinization and 369 patients with reduced red cell hemoglobinization. The accuracy of the biochemical markers was determined according to gender and to whether the concentration of the acute-phase reactant CRP was within the reference interval or increased. The most informative biochemical markers and hematologic indices of ID were selected to divide the 442 anemic patients into diagnostic plot quadrants for classifying the advancing ID phases.
Because APR is an important influencing factor on iron metabolism and the proliferation and hemoglobinization of red cells, the anemic patients were primarily divided into groups with normal and increased CRP, and not as is usually done, into IDA and ACD. Most of the anemic patients with APR had either ACD or the ID/ACD combination.
EDTA-blood specimens were collected for the complete blood cell count and measurement of CHr and HYPO. The specimens were measured within 8 h of venipuncture. Plasma specimens were used to determine the standard biochemical markers of iron metabolism and CRP. Because sTfR is considered a sensitive analyte for detection of ID (24), the reliability of this marker was tested using three commercially available sTfR assays for comparison.
diagnostic testing
Blood counts were measured by assaying one specimen from every patient for Hb, red blood cells, hematocrit, mean cellular volume (MCV), CH, RDW, and white blood cells on the Advia 120 (Bayer Diagnostics) automated hematology analyzer. Reticulocyte measurements included percentage of reticulocytes (% retic), mean Hb concentration of reticulocytes (CHCMr), and mean cell volume (MCVr). Calculated indices were absolute reticulocyte count ( retic = red blood cells x % retic), Hb content per reticulocyte (CHr = MCVr x CHCMr), and total reticulocyte Hb (RetHb = CHr x retic). Plasma Tf (reference interval, 2.0–3.6 g/L) (23) was assayed with the Image immunonephelometer (Beckman), using calibrators assigned against the IFCC plasma protein calibrator (BCR CRM 470). Serum iron (reference interval, 7.2–27.7 µmol/L) and CRP (reference values 5 mg/L) (23) were measured using the Vitros clinical chemistry analyzer (Ortho Diagnostics). Ferritin was determined on a Cobas Core analyzer (Roche Diagnostics); the reference intervals in our hospital are 15–150 µg/L for women and 30–350 µg/L for men. The sTfR concentration for each specimen was determined using three commercially available assays. Two of the assays (Dade Behring and Roche Diagnostics) use microagglutination of latex particles coated with an anti-sTfR monoclonal antibody. The third test (Nichols Institute) uses a noncompetitive sandwich-type assay with luminescence-labeled monoclonal antibodies to sTfR. The Dade Behring reagent was measured on a BN ProSpect immunoanalyzer, the Roche reagent on an Hitachi 917 analyzer, and the Nichols reagent on an Advantage analyzer. The reference intervals (2.5–97.5 percentiles) given by the manufacturers were 0.4–1.8 mg/L (Dade Behring), 0.7–4.2 mg/L (Nichols), and 1.9–4.4 mg/L for women and 2.2–5.0 mg/L for men (Roche).
The CVs for interday precision near the upper bounds of the reference intervals for HYPO, CHr, ferritin, and Tf were 4.8%, 5.6%, 4.2%, and 3.9%, respectively. The interday CV was 3.2% for sTfR (Dade), 3.6% for sTfR (Roche), and 4.1% for sTfR (Nichols) at means of 1.6, (Dade), 3.3 (Nichols), and 3.6 mg/L (Roche).
TfS was calculated based on the formula: TfS (%) = Fe (mg/L) x 70.9/Tf (g/L) (11), where Fe is the iron concentration. The CRI was calculated as the reticulocyte count (percentage of red cells) multiplied by the hematocrit and divided by 45.
It is normal for CHr to be greater than the mean, mature CH in individuals who do not have anemia. Patients with inverted values (CHr < CH) showed evidence consistent with recent development of ID (19). The development of an inverted CHr/CH ratio was used as an helpful indicator of recently developed functional ID. The ratio of sTfR to log ferritin (sTfR-F index) was also determined because this index has been reported to be an excellent marker for biochemical identification of ID (13)(25).
Diagnosis of ß-thal trait was facilitated by determination of the ratio of the percentage of microcytic cells to the percentage of hypochromic red cells. Patients with a ratio >0.9 and with >20% microcytic red cells were suspicious for ß-thal trait (26). Column chromatography with minicolumns was used for confirming the identification of HbA2.
statistical analysis
Data were evaluated using standard parametric tests, and calculations were performed with the MedCalc statistical software package for biomedical research (27).
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Results |
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). Median HYPO was 4.6% [mean (SD), 10.5% (14.2%)], and the distribution of values was positively skewed. The three methods used to determine sTfR were compared in 376 patients and showed a close correlation (Fig. 2
). The sTfR concentration increased in hyperproliferative erythropoiesis. However, there was no correlation between CRI and the sTfR concentration (Fig. 3
).
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hematologic indices in the control group
The control group consisted of 71 patients [30 men and 41 women; mean (SD) age, 55 (19) years]. Frequency distributions of RDW, CH, and CHr were normal, but those of Hb, HYPO, and CRI were not (Table 2
). The 2.5 percentile for CHr was 28 pg, whereas the 97.5 percentiles for HYPO and CRI were 5% and 2.6%, respectively. On the basis of cutoffs of 28 pg for CHr and 5% for HYPO, a CHr <28 pg and a HYPO >5% were used as indicators of functional ID.
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biochemical markers in the absence of functional id
Ferritin, Tf, TfS, and sTfR, the standard biochemical markers of ID, as well as the sTfR-F index were determined in 211–227 patients with normal red cell hemoglobinization. Some of the markers could not be determined in all of the patients because there was insufficient specimen available. The 2.5 percentile for ferritin differed significantly between male and female patients (P = 0.0097), but not the 97.5 percentile for sTfR (P = 0.16–0.19, depending on the assay used; Table 3
). In male and female patients, serum ferritin concentrations indicating ID were <20.8 and <11.2 µg/L, respectively.
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sTfR concentrations in male and female patients, depending on the sTfR assay, were >1.8, >5.5, and >5.5 mg/L, respectively. Cutoffs for the sTfR-F index were not gender specific, and sTfR-F index indicated ID at values of >1.5, >3.5, and >3.8 in the Dade, Nichols, and Roche assays, respectively.
biochemical markers in functional id
After eliminating results for 10 patients with ß-thal trait, we performed ROC analyses for ferritin, sTfR, and the sTfR-F index to evaluate the accuracy of these tests at identifying functional ID (CHr <28 pg, HYPO >5%) in patients with and without APR. The group consisted of 154 nonanemic and 432 anemic patients. Of the anemic patients, 288 had APR, 263 had reduced red cell hemoglobinization, and 195 had both.
As can be seen from Fig. 4
and Table 4
, the biochemical markers displayed a greater sensitivity, specificity, positive likelihood ratio, and area under the ROC curve (AUCROC) in patients without APR. The sTfR-F index was the best marker for biochemical identification of functional ID. Optimal cutoffs (highest sum of sensitivity and specificity from ROC curves) for functional ID in anemic patients with reduced red cell hemoglobinization are shown in Table 4
for ferritin, sTfR, and the sTfR-F index [data for comparison of the markers with CHr 28 pg and HYPO >5% are available with the online version of this article at Clinical Chemistry Online (http://www.clinchem.org/content/vol48/issue7/]. The cutoffs were similar to the 2.5 percentile for ferritin and the 97.5 percentiles for sTfR and the sTfR-F index of the control group with normal red cell hemoglobinization (Table 3
). Women had lower ferritin cutoff values than men irrespective of APR. In these patients, based on a HYPO >5%, functional ID was indicated by a ferritin concentration <97.1 µg/L in men and <22.9 µg/L in women; based on a CHr of <28 pg, functional ID was indicated by ferritin concentrations <61.7 and <22.9 µg/L in male and female patients, respectively (Table 4
). The sTfR-F index criterion value decreased from 1.5 in patients who had no APR to 0.8 in patients with a CRP concentration >5 mg/L.
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ch inversion
The CHr/CH ratio was inverted in 22% of 586 patients who did not have ß-thal trait (Table 5
). Only 3.5% of patients with normal red cell hemoglobinization had inverted values vs 34% of those with reduced red cell hemoglobinization. In this group, 26% of patients without APR displayed CH inversion; this increased to 38% in patients with APR. These findings suggest that CH inversion is stimulated not only by functional ID, but also by APR.
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diagnostic plots
In anemic patients, the sTfR-F index was the most accurate marker for biochemical identification of functional ID. However, as shown from the AUCROC and the positive likelihood ratios in Table 4
, the accuracy of the sTfR-F index for identifying functional ID was greater in patients with ID alone than in those with combined ID and APR. To evaluate the performance of the sTfR-F index in combination with CHr and HYPO for identification of the different phases of advancing ID in anemic patients with and without APR, we matched these markers in diagnostic plots (Fig. 5
). The plots, which are distributed into four quadrants, were generated as follows: For the hematologic indices, cutoff values for functional ID were CHr <28 pg and HYPO >5%, as determined in patients with a normal, complete blood cell count (Table 2
). The sTfR-F index cutoffs for functional ID, selected from ROC analyses of patients with IDA and combined functional ID/ACD, were 1.5 and 0.8, respectively. The idea was that data points in quadrants 1 and 2 would be consistent with normal red cell hemoglobinization in repleted or recently depleted iron stores. Data points in quadrants 3 and 4 would include patients with reduced red cell hemoglobinization with either depleted (quadrant 3) or repleted iron stores (quadrant 4).
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The data point distribution of 432 anemic patients with and without APR is shown in Fig. 5
. Most data points are located in quadrants 1 and 3. Quadrant 1 patients had normocytic and normochromic red cells, but the percentage of patients with HYPO >5% was higher (13% vs 24%) in the population with APR. Most patients with normal hemoglobinization of red cells, CRP 5 mg/L, and data points in quadrant 2 had either ferritin concentrations <20 µg/L or a CRI >2.6%, resulting from hemolysis or recently started oral iron therapy. Quadrant 2 patients with a CRP >5 mg/L mainly suffered from cancer-related anemia (CRA) with reduced hemoglobinization of red cells (HYPO >5%). In these patients, erythropoiesis was monitored after chemotherapy, at a time when erythropoiesis recovered and changed from hypoproliferation to hyperproliferation.
Patients with data points in quadrant 3 had anemia with biochemically and hematologically identified ID. Among 134 quadrant 3 patients with increased CRP, 102 (76%) had a CHr <28 pg and a HYPO >5%, indicating that ID had been present for several months, and 57 (43%) had an inverted CHr/CH ratio, showing evidence of the recent development of ID. In quadrant 3 patients with CRP within the reference interval, 50 of 62 (81%) had a CHr <28 pg and a HYPO >5%, and 20 (32%) revealed CH inversion.
Fourteen of 144 patients (10%) with CRP within the reference interval had data points in quadrant 4. In six of these patients, the CHr was <28 pg because of CH inversion. Of the nine patients with a combination of CHr <28 pg and HYPO >5%, seven had the combined state of functional ID/ACD, and two were suffering from refractory IDA. Quadrant 4 contained 61 of 288 patients with APR (21%). Combinations of CHr <28 pg with HYPO >5% and CHr <28 pg with an inverted CHr/CH ratio were identified in 32 (57%) and 29 (48%) quadrant 4 patients, respectively. Both combinations were typical markers in quadrant 3 patients, with ID occurring as a result of depletion of storage and functional iron compounds. Therefore, patients with a CRP >5 mg/L and data points in quadrant 4 who met one of these criteria (CHr <28 pg and HYPO >5%; CHr <28 pg and CH inversion) were classified as functionally iron deficient; nevertheless, their storage compounds were repleted. These patients were typically those with combined functional ID/ACD. In contrast to patients with data points in quadrant 4, in whom the CH inversion rate was 48%, patients with normal red cell hemoglobinization and storage iron compound repletion (data points in quadrant 1) had an inversion rate of only 4.5%. This further demonstrates that an inverted CHr/CH ratio is also a marker of functional ID in patients with repleted iron compounds.
To investigate the selectivity of diagnostic plots for disease-specific anemias, the distributions of patients with IDA, CRA, ACD without CRA, and third-trimester pregnancy were recorded (Table 6
). The disease-specific anemias were selected according to clinical findings and laboratory criteria. Thus, there were differences in the sTfR-F index between patients with and without APR. The patient groups were separated into subgroups based CRP 5 mg/L or >5 mg/L. Data points for patients with IDA were localized in quadrant 3, which is indicative of storage depletion and functional iron compounds. In ACD without CRA and in CRA, the majority of the data points were localized in quadrant 1, indicating normal red cell hemoglobinization in the iron-repleted state. Increased CRP in these diseases was the cause of the data point distributions in quadrants 3 and 4, representing decreased red cell hemoglobinization in the iron-depleted or -repleted states, respectively.
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Approximately 65% of pregnant women displayed depletion of storage and functional iron compounds (quadrant 3). In 7 of 47 (15%) pregnancies, data points were localized in quadrant 2, indicating the increased sTfR concentration during pregnancy, which reflects increased erythropoietic activity in the last trimester (28). In the 20 pregnancies with increased CRP concentrations, there were no cases of ID in which iron stores had been repleted.
Of the 10 patients with ß-thal trait, 7 had data points in quadrant 4. Three pregnant women with ferritin concentrations <10 µg/L had data points in quadrant 3.
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Discussion |
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The markers HYPO and CHr have been investigated primarily by monitoring the erythropoietic function in hemodialysis patients. A HYPO >10% is consistent with functional ID, which is an important limiting factor in the effectiveness of recombinant human erythropoietin (rHuEPO) therapy (17)(30)(31)(33). Further studies will, however, be necessary to evaluate the role of this test (34). CHr has been shown to be a sensitive and specific indicator of functional ID in healthy individuals (29) and in patients with end-stage renal failure treated with rHuEPO (18)(19)(30)(31)(35). In healthy individuals administered rHuEPO, the decrease in CHr was inversely correlated to the baseline serum ferritin log value, which shows that CHr is a useful early indicator of iron-deficient erythropoiesis (29). In hemodialysis patients on rHuEPO therapy, CHr was a much more stable indicator of functional ID than either serum ferritin or TfS (30)(31). Less use of intravenous iron was required in iron management using CHr than in management with serum ferritin and TfS (31). In one study, the usefulness of CHr in hemodialysis patients was, however, found to be somewhat limited (35). Studies on healthy individuals and hemodialysis patients on rHuEPO therapy have shown that HYPO and especially CHr are direct measures of iron sufficiency (16)(29)(31). On the basis of these data, we used these indices as reference tests for functional ID in anemic patients with a broad spectrum of diseases. Although tests for these indices are available only on analyzers from a single manufacturer, various cutoff values for functional ID are reported in the literature, ranging from 2% to 10% for HYPO (16)(35) and from 23 to 29 pg for CHr (3)(15)(18)(19)(31)(35). The corresponding values in this study, using the 97.5 and 2.5 percentiles for the control group, were 5% for HYPO and 28 pg for CHr. In most studies, the ability of biochemical markers to identify functional ID and evaluate the phases of ID from storage to functional ID was compared with indirect methods. This was the case in studies in which iron staining of the bone marrow was used or iron therapy was used as the gold standard for iron depletion (13)(25)(33)(36).
In this study, we used normal red cell hemoglobinization (CHr 28 pg, HYPO 5%), a direct marker of functional ID, as the standard. In patients with normal red cell hemoglobinization, cutoff values for biochemical markers indicating ID (Table 3
) were in close agreement with the data published for ferritin (13)(36)(37) and sTfR (38). Significant gender-specific differences could be seen for ferritin, but not for sTfR or the sTfR-F index. This was also observed in studies in which iron staining of the bone marrow or iron therapy was used as the standard (13)(25)(36). The cutoff values for biochemical markers of ID in patients with normal red cell hemoglobinization (shown in Table 3
) were confirmed by the criterion values for functional ID in the ROC curves of patients with reduced red cell hemoglobinization. However, in patients with anemia and APR (CRP >5 mg/L), cutoff values for ferritin that indicated ID were skewed to higher concentrations from approximately <20 µg/L to <100 µg/L in men and from approximately <10 µg/L to <20 µg/L in women (Table 4
). The cutoff value for sTfR did not change significantly in patients with APR, whereas the sTfR-F index decreased from 1.5 to 0.8. These findings do not agree with data in the literature in which only one sTfR-F index value was used to distinguish IDA from ACD and the combined state of functional ID/ACD (13)(25). However, the use of different sTfR-F indices in the diagnosis of ID in patients with and without APR is plausible. The increase of serum ferritin in APR decreases the sTfR-F index because sTfR is unaffected.
Biochemical markers have high diagnostic accuracy for ID when iron staining of the bone marrow is used as the criterion (13)(24). In our study, in which the diagnostic accuracy of the biochemical markers was based on red cell hemoglobinization, the results were different (Table 4
). The reason might be that investigations of stainable bone marrow iron mainly depict disturbances of the iron metabolism in which iron stores and iron turnover are involved. In such a situation, biochemical markers of ID could be expected to show a good response. However, for reduced bone marrow activity attributable to APRinduced disturbances of iron distribution and disturbances of iron utilization of the erythroid precursors, markers such as ferritin, Tf, and sTfR do not reflect the balance between iron and erythropoiesis.
The weak correlation between CHr and the biochemical markers sTfR and sTfR-F index suggest that the classic iron markers are relatively insensitive in diagnosing functional ID. Of the quadrant 4 patients with increased CRP and an iron-repleted state (Fig. 5
), only 15% had increased sTfR, although 57% displayed reduced red cell hemoglobinization and 48% revealed an inverted CHr/CH ratio, the indicators of functional ID. The reason for the high percentage of sTfR concentrations within the reference interval is the hypoproliferation of erythroid marrow in APR. Functional ID in the presence of hypoproliferative erythropoiesis does not cause an adequate increase in sTfR compared with the extent of functional ID. This might also be the reason for the inadequately low sTfR-F index in ACD that is accompanied by APR.
Differentiation of functional ID into IDA, ACD, and combined ID/ACD is possible only by measuring CHr, HYPO, and CH inversion. The use of these indices in combination with diagnostic plots identifies functional ID. The sTfR-F index reflects the iron store status and allows differentiation of functional ID in the iron-depleted and -repleted states. On the basis of the present data, we recommend the use of diagnostic plots to classify ID, especially in patients with APR. Plots of the data points enable iron status to be differentiated into the four categories, as shown in Fig. 6
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Data points in quadrant 1 suggest normal red cell hemoglobinization, a condition seen mainly in patients with ACD and CRA, and in hemodialysis patients in the absence of functional ID. Data points in quadrant 2 indicate three possible conditions, according to our results:
28 pg, HYPO 5%). Data points in quadrant 3 suggest a reduced iron supply for erythropoiesis as being the cause of functional ID attributable to depleted iron stores (the typical situation in IDA). Patients with data points in quadrant 4 are iron-repleted but have functional ID (seen mainly in anemia accompanying infection or chronic inflammation, and in APR that accompanies CRA).
Patients with ß-thal trait and those with the combined state of ID/ACD are microcytic and hypochromic, and have data points in quadrant 4. Therefore, as a screening test to avoid mismatching, patients with data points in this quadrant must be investigated for ß-thal trait by determining the ratio of percentage of microcytic to percentage of hypochromic red cells.
The diagnostic plot used in this study has several advantages over other diagnostic diagrams used to detect advancing ID and the division of anemic patients into those with and without ACD (13)(25). ACD can be separated from the combined state of functional ID/ACD. A combination of hematologic indices and biochemical markers of iron metabolism in the diagnostic plot can indicate an early and sensitive erythropoietic response to changes in the iron supply. In pregnant women with increased sTfR, hyperproliferative erythropoiesis can be distinguished from ID.
According to preliminary studies, the practical therapeutic implications of the quadrants depicted in Figs. 5
and 6
are as follows:
(a) Patients with data points in quadrants 2 and 3 should be administered oral iron supplements. The exceptions to this are pregnant women and patients with reticulocytosis who have data points in quadrant 2. The response to treatment of IDA is indicated by the data point shifting from quadrant 3 to quadrant 2 within 10 days, and from quadrant 2 to quadrant 1 (iron-repleted state) within 4–6 weeks.
(b) Anemic patients with data points in quadrants 1 and 4 can be effectively treated with rHuEPO. In patients with data points in quadrant 4, the response-limiting factor is functional ID. Therefore, intravenous iron supplements should preferably be given concurrently with rHuEPO. In patients with data points in quadrant 1, rHuEPO administration is generally started without iron supplementation as long as the data points remain in that quadrant. Where there is an inadequate response, the data point shifts to quadrant 3 or 4, and iron supplements must be administered.
In conclusion, biochemical markers of ID are of limited value in diagnosing functional ID, especially in diseases with APR. The combination of the hematologic indices CHr and HYPO with the sTfR-F index in a diagnostic plot provides an attractive tool for diagnosis and therapeutic monitoring of functional ID.
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Footnotes |
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References |
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, HYPO <5%; •, HYPO >5%; 
, inverted CHr/CH ratio; 
, combination of HYPO >5% and inverted CHr/CH ratio.