**1.2.2 New parameters for the diagnosis of anemia**

The question regarding anemia therapy in those patients is which are the best parameters to assess the iron available for erythropoiesis. New laboratory parameters are reported by different manufacturers as potential tools for anemia and iron restricted erythropoiesis diagnosis. These tests include reticulocyte hemoglobin content, percentage of hypochromic red cells and soluble transferrin receptor (Wish, 2006; Goodnough *et al*., 2010).

Serum transferrin receptor (sTfR) is a useful test for this purpose because it is not affected by inflammation so is a reliable marker of iron deficiency in mixed situations (Punnonen *et al*., 1997; Beguin, 2003; Skikne, 2008).

The sTfR test is based on the fact that erythroblasts in the bone marrow will increase the presentation of membrane transferrin receptor in the setting of iron deficiency. If a patient is not receiving sufficient iron and erythropoiesis is being stimulated by an ESA, then increased transferrin receptors will become expressed on the erythroblasts, some of which come off and will be detectable in the circulation. The sTfR correlates with this membrane expression of the

Assessing Iron Status in CKD Patients: New Laboratory Parameters 233

This is not the case for MHC. MCH is calculated from red blood cell count and hemoglobin and represents the average; the percentage subsets of erythrocytes can give complementary information of the contribution of cell with extreme values (hypochromic and hyperchromic cells) to the mean values, reflecting the fluctuations of iron availability to the erythron in the

Modern counters provide information about the reticulocyte counts but also about the characteristics of these cells (size or hemoglobin content) related to the quality of the

Nevertheless, each Company applies the technology in a different way in the analyzers, with different algorithms to translate the electronic signals to graphs and numerical values. For this reason these new parameters are exclusive of each manufacturer and they are patented.

On last decades, several new red blood cell and reticulocyte parameters have been reported having utilities in detection of iron deficiency and functional iron deficiency. Two of these parameters are hypochromic red cells (referred to as %Hypo) and CHr (reticulocyte hemoglobin content) reported by the Siemens ADVIA 120 hematology analyzer (Thomas &

Reticulocyte hemoglobin content (CHr) and the percentage of hypochromic red blood cells (%Hypo) reflect iron availability and are reliable markers of functional iron deficiency

CHr is defined by the formula (CHr = MCVr X CHCMr), wherein MCVr is the mean reticulocyte cell volume and CHCMr is the mean hemoglobin concentration of reticulocytes,

Reticulocytes are immature red blood cells with a life span of only 1 to 2 days. When these are first released from the bone marrow, measurement of their hemoglobin content can provide the amount of iron immediately available for erythropoiesis. A less than normal hemoglobin content in these reticulocytes is an indication of inadequate iron supply relative to demand. The amount of hemoglobin in these reticulocytes also corresponds to the amount of hemoglobin in mature red blood cells. CHr has been evaluated recently in numerous studies as a test for iron deficiency and functional iron deficiency and has been found to be highly sensitive and specific. However, exact threshold values have not been established, as the

The measurement of CHr is a direct assessment of the incorporation of iron into erythrocyte hemoglobin and thus an estimate of the recent functional availability of iron into the erythron; due to the life span of the reticulocytes CHr is a sensitive indicator of iron

Epoetin is effective in stimulating production, of red blood cells, but without an adequate iron supply to bind to heme, the red blood cells will be hypochromic, i.e., low in hemoglobin content. Thus, in states of iron deficiency, a significant percentage of red blood cells leaving the bone marrow will have a low hemoglobin content. By measuring the percentage of red blood cells with hemoglobin concentration <280 g/L, iron deficiency can be detected.

threshold values vary (28-30 pg), depending on the laboratory and instrument used.

deficient erythropoiesis (Fishbane *et al*., 1997; Mast *et al*., 2002; Brugnara 2003).

which is obtained by an optical cell-by-cell hemoglobin measurement.

previous weeks.

erythropoiesis.

**1.3.1 Siemens**

Thomas, 2002).

(Cullen *et al*., 1999).

transferrin receptor and also tends to be elevated in the presence of increased erythroid activity. It does seem to be a reasonable index of erythropoietic activity (Chiang *et al*., 2002; Tarng & Huang, 2002) and reflects the effect of stimulating bone marrow red cells production, before an increase in reticulocytes is noted and well before the Hb rises; therefore an increase in the sTfR may be the first detectable measure. It is not affected by inflammation (Beerenhout *et al*., 2002) and this reason would make sTfR a more reliable test than serum ferritin.

Direct consequence of an imbalance between the erythroid marrow iron requirements and the actual supply is a reduction of red cell hemoglobin content, which causes hypochromic mature red cells and reticulocytes. Interest has been generated in the use of erythrocyte and reticulocyte parameters, available on the modern analysers based on flow cytometry technology.

The modern hematological parameters contribute to the advanced study of the anemia and depend on the technology employed; the debate about other parameters with the same clinical meaning and potential utility as reticulocyte hemoglobin content and percentage of hypochromic red cells is open.
