Erythropoietin, Iron and Red Blood Cell Production: Laborato
Carlo Brugnara, MD
Children's Hospital & Harvard Medical School
Boston, Massachusetts, USA
Several clinical settings have furthered our understanding of the relationship between erythropoietin, iron, and the erythropoietic response to anemia. These settings include hereditary hemolytic anemia, hemochromatosis, autologous blood donation, and therapy with recombinant human erythropoietin (r-HuEPO).
The knowledge acquired in these settings has allowed us to identify a ≥relative iron deficiency≈ that occurs when increased erythron iron requirements exceed the available supply of iron, even in the presence of storage iron. The identification of this relative (or functional) iron deficiency is important for the proper assessment of r-HuEPO use.
Known indicators of iron metabolism and r-HuEPO can be broken down into two main categories: biochemical indicators, which include serum iron, transferrin, transferrin saturation (Tsat), ferritin, and circulating transferrin receptor (TfR), and hematological indicators, which include erythrocyte zinc protoporphyrin (ZPP), percent hypochromic cells (%hypo), and reticulocyte hemoglobin content (CHr).
Of the biochemical indicators, one of the more recent developments has been in the area of circulating transferrin receptor (TfR), which is a truncated form of the transferrin receptor that circulates in plasma, usually attached to transferrin. Several studies have shown that the levels of TfR are elevated in iron deficiency anemia, in some people living at high altitude, and in disorders with expanded erythropoiesis. TfR levels have also been shown to be decreased slightly in the setting of iron overload and in disorders with reduced erythropoiesis.
Clinical research on the clinical values of TfR in the setting of r-HuEPO therapy has found that TfR is predictive of responses to the initiation of r-HuEPO therapy,1 and that r-HuEPO therapy brings about an increase in TfR. This increase is of lower magnitude in patients with delayed or absent response to r-HuEPO therapy.2
The advantages of using measurements of TfR to assess r-HuEPO use are that it is a good, dynamic test that reflects changes in real time and is a good index of erythropoietic mass at a given location. The disadvantages of this method include the facts that the assays used for TfR are better suited to research, rather than clinical use, and that although there is a good correlation between different methods of testing, these methods all use different units. Additionally, the accuracy of using TfR to identify iron deficiency in the setting of r-HuEPO-driven erythropoiesis has yet to be determined.
Erythrocyte zinc protoporphyrin (ZPP), a red cell parameter long used to detect lead poisoning in children, has been the subject of numerous studies in the context of EPO use. It has been shown that patients taking EPO in a clinical setting showed modest but significant increases in ZPP.
The use of ZPP to follow patients on EPO is, however, limited. A paper in Clinical Chemistry in 1992 showed that a large number of substances interfere with the measurement of ZPP and that very different measurements are obtained if red cells are washed and resuspended in saline. For these reasons, measurement of ZPP is not suited to following patients on EPO or to identify iron deficiency.
Another parameter that has been studied is the percentage of hypochromic red cells (%hypo). This is the percentage of cells that have a hemoglobin concentration below an arbitrary cut-off point of 28 g/dL.
One of the first papers to examine the concept of functional iron deficiency showed that patients on dialysis treated with EPO tended to develop functional iron deficiency, as identified by the increased appearance of hypochromic red cells.3 These cells had abnormally low hemoglobin concentrations, very similar to those seen in cases of iron deficiency.
A similar concept was shown in a study that used flow cytometric assessment of volume and volume concentration. In this study, it was found that the newly formed cells associated with EPO administration had increased ZPP, decreased volume concentration, and decreased red cell volume.3 This suggests that, in normal subjects, use of EPO can result in the production of red cells that are indistinguishable from those seen in patients with iron deficiency anemia.
Several studies have suggested that %hypo measurement can be useful in studying iron deficiency and EPO use.4,5 A number of other studies, however, have shown no value for this parameter in the setting of r-HuEPO.6,7
%Hypo has the advantages of already being used in Europe and of being part of the regular CBC. This parameter does, however, have apparent disadvantages, such as incorporating both hypochromic red cells and normal reticulocytes and increasing with storage at room temperature. Also, %hypo can only be measured using Bayer instruments.
Numerous studies have shown various effects of EPO on reticulocytes. These effects include a significant release of immature reticulocytes within 1.5 days of, and peaking 3.5 days after, EPO administration. EPO has also been shown to bring about an increase in the overall number of reticulocytes, peaking at 5 days after EPO administration, as well as a decrease in serum ferritin, with the nadir at 4.0 days after EPO administration.7
Several recent publications have addressed the use of reticulocyte indices and reticulocyte hemoglobin content (CHr) in dialysis patients. In the initial publications, measurements of CHr were obtained with the H*3 Bayer hematology analyzer. CHr has been shown to be a reliable indicator of iron status in patients undergoing chronic hemodialysis.7 Patients with CHr values lower than red cell MCH had lower transferrin saturation and lower hematocrit, and they all had a recent increase in r-HuEPO dose. With iron deficiency being defined as a state that shows an increase in reticulocyte counts following intravenous (IV) iron, CHr showed 100% sensitivity and 80% specificity. CHr was a much more accurate predictor of iron deficiency than serum ferritin, transferrin saturation, or percentage of hypochromic erythrocytes.7
In another study, a baseline CHr < 28 pg had 78% sensitivity and 71% specificity to diagnose functional iron deficiency, compared with 50% and 39% for traditional biochemical measures of iron deficiency.8
In patients treated with subcutaneous (SC) r-HuEPO and IV iron, CHr was shown to increase during IV iron therapy, indicating response to treatment and potential value as an early indicator of functional iron deficiency.9
More recent studies have used the ADVIA 120 Bayer hematology analyzer, which provides CHr data that are on the average 3 pg greater than the H*3 analyzer. This difference is important when cut-off values are used as decision points for anemia management in chronic dialysis patients. In a study by Fishbane et al., which was presented at the ASN 2000 meeting, management of iron therapy in dialysis patients based exclusively on CHr (< 29 pg) resulted in similar Hct and EPO dose and greatly reduced iron exposure compared with management based on biochemical parameters (ferritin < 100 ng/ml or transferring saturation < 20%).
In another report, a combination of %hypo > 6% and CHr < 29 pg provided the greatest diagnostic efficiency (90.4%), with 86.3% sensitivity and 93.2% specificity to identify hemodialysis patients who will respond to IV iron therapy.10
The advantages of the reticulocyte hemoglobin content parameter are that it provides a real-time estimate of iron availability and that it is provided as part of an automated reticulocyte count. Additionally, it is insensitive to storage-induced changes. It does, however, have disadvantages, namely that the measurement is available on only one class of instruments, it is not widely used in the United States or Europe, and it is not informative in patients with thalassemias or macrocytosis.
Looking at numerous studies, we can see that there are several indicators of r-HuEPO-driven erythropoiesis. These indicators, which were used in the indirect EPO-use detection method for the 2000 Olympic games, include: hemoglobin, transferrin receptors, percentage of hypochromic red cells, reticulocyte hematocrit (absolute retic count x MCVr), reticulocyte hemoglobin (absolute retic count x CHr), and serum EPO.
Examining all of the hematologic parameters discussed, one can ask the question of how they can be used together to differentiate EPO-driven erythropoiesis and EPO-associated iron deficiency. The bottom line is that several parameters are available, some of which can be used clinically in patients, and some of which may be useful in the detection of EPO use for blood doping.
Table 1 shows the effects of both EPO-driven erythropoiesis and EPO-associated iron deficiency on various hematological parameters.
In conclusion, we can see that hematological parameters can be reliable indicators of iron status and that these parameters may play an important role in the management of r-HuEPO therapy and the diagnosis of iron deficient states. In particular, %hypo and CHr may provide both long-term and real-time assessment of the balance between available iron and erythropoiesis.
Taken together, these parameters may play an important role in the management of r-HuEPO therapy and diagnosis of iron deficient states, although more studies are needed to validate their clinical use.
References:
1. Ahluwalia, et al. Am J Kidney Dis. 30:532-541, 1997.
2. Beguin et al. Br J Haematol. 89:17-23, 1995.
3. Macdougall IC, et al. Br. Med. J. 304:6821, 1992.
4. Nephrol. Dial. Transplant. 12:1173, 1997.
5. Bhandari S, et al. Am. J. Kidney Dis. 30:814, 1997.
6. Fishbane S, et al. Kidney Intern. 52:217, 1997.
7. Major A, et al. Br. J. Haematol. 87:605, 1997.
8. Mittman N, Sreedhara R, Mushnick R, et al. Am J Kidney Dis. 30:912-22, 1997.
9. Bhandari S, Turney JH, Brownjohn AM, et al. J Nephrol. 11:78-82, 1998.
10. Tessitore N, et al. Nephrology Dialysis Transplantation. 16:1416-1423, 2001.
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