**1.3.1 Siemens**

232 Chronic Kidney Disease

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

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

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

The Hemogram is one of the more required tests by the clinicians; the analysis nowadays is totally automated and the correct interpretation of the results requires to unite the knowledge about the characteristics of the equipment and the clinical meaning of the results. The suppliers contribute innovations, providing new parameters that can help the clinicians to

The professionals of the Clinical Laboratory must obtain the maximum yield of the new

Automated blood cell counters have changed substantially during the last 20 years. Technological progress has meant that in recent years modern analyzers, fully automated, have been available. These analyzers report new parameters that provide further information from the traditional count; this information must be evaluated to prove the

When a state of iron deficiency proceeds red blood cells are continuously produced in the bone marrow and as the iron stores progressively decrease, mean cell volume (MCV), mean cell hemoglobin (MCH) and red blood cell count (RBC) count tend to decline. In iron deficient erythropoiesis, synthesis of hemoglobin (Hb) molecules is severely impaired leading to the production of erythrocytes with low Hb concentration (hypochromic cells). Because of their long life span of approximately 3 months, several cohorts of normochromic and increasingly hypochromic red cells coexist in the peripheral blood leading to anisocytosis; red cell distribution width (RDW) reflects the variation of size of the red cells. Flow cytometry provides information about individual cell characteristics. This is in contrast to previous measurements of MCV, MCH, and MCHC which only calculate mean indices

MCV is the mean of the volumes of all erythrocytes; RDW refers to the variety of volumes present in the red cell population, so the whole picture is clear and the contribution of

marginal sized subpopulations to the calculated mean value can be assessed.

make a diagnosis in a fast, cheap and useful manner (Buttarello & Plebani, 2008).

technologies obtaining as much information as possible.

potential clinical utility in different clinical situations.

*et al*., 2002) and this reason would make sTfR a more reliable test than serum ferritin.

technology.

hypochromic red cells is open.

**1.3 Technology at a glance**

for the total red cell population.

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 & Thomas, 2002).

Reticulocyte hemoglobin content (CHr) and the percentage of hypochromic red blood cells (%Hypo) reflect iron availability and are reliable markers of functional iron deficiency (Cullen *et al*., 1999).

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

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

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

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.

Assessing Iron Status in CKD Patients: New Laboratory Parameters 235

Fig. 3. Volume/Hemoglobin Concentration (V/HC) cytogram (Mie Map) is a linear version of the RBC scatter cytogram. Hemoglobin concentration is plotted along the x axis and cell

volume is plotted along the y axis. Only red blood cells appear on this cytogram. Markers organize the cytogram into 9 distinct areas of red blood cell morphology. On the x axis, hemoglobin concentration markers are set at 280 g/L (3) and 410 g/L (4). Red blood cells with a hemoglobin concentration less than 280 g/L are hypochromic, while

cells with a hemoglobin concentration greater than 410 g/L are hyperchromic.

Red blood cells with a volume less than 60 fL are microcytic, while cells with a volume

CHr and %Hypo have been used as a diagnostic tool, together with biochemical markers, to distinguish IDA from ACD, and are incorporated to the guidelines for the monitoring of recombinant human erythropoietin rHuEpo therapy (Macdougall *et al*., 2000; Kotisaari 2002;

Sysmex XE analyzers (Sysmex Corporation, Kobe, Japan) employ flow cytometry technology. In the reticulocyte channel blood cells are stained by a polymethine dye, specific for RNA/DNA, and analysed by flow cytometry using a semiconductor laser. A bidimensional distribution of forward scattered light and fluorescence is presented as a

Forward scatter correlates with erythrocyte and reticulocyte hemoglobin content (Ret He,

On the y axis, RBC volume markers are set at 60 fL (1) and 120 fL (2).

scattergram, indicating mature red cells and reticulocytes (Figure 4).

greater than 120 fL are macrocytic.

Locateli *et al*., 2004).

**1.3.2 Sysmex** 

RBC He).

Hypochromic red cells percentages have been correlated with iron deficiency. %Hypo is reported by Siemens Advia 120 hematology analyzer based on the optical cell-by-cell hemoglobin measurement (Figures 2 and 3).

Fig. 2. RBC Scatter Cytogram.


The RBC Scatter cytogram is the graphical representation of two light-scatter measurements: the high-angle light scatter (5° to 15°) is plotted along the x axis, and the low-angle light scatter (2° to 3°) is plotted along the y axis. (Figure 2).

The RBC map shows the relationship between the light-scatter measurements and the cellby-cell characteristics of volume and hemoglobin concentration. The map grid encompasses RBC volumes between 30 fL and 180 fL and hemoglobin concentrations between 190 g/L and 490 g/L. (Figure 3).

The measurement of %Hypo (defined as the percentage of red blood cells with Hb concentration less than 280 g/L) is a sensitive method for quantifying the hemoglobinization of mature red cells. Because of the long circulating life span of mature erythrocytes %Hypo values are related to iron status in the last 2-3 months, and have been recognised as an indicator of iron deficiency (Macdougal 1998; Bovy *et al*., 2005; Bovy *et al*., 2007). %Hypo < 5% is considered normal. Two different criteria, more specifically, %Hypo >5% and >10% have been used. %Hypo >10% has been more commonly used for defining absolute iron deficiency and functional iron deficiency (Locatelli *et al*., 2004).

Fig. 3. Volume/Hemoglobin Concentration (V/HC) cytogram (Mie Map) is a linear version of the RBC scatter cytogram. Hemoglobin concentration is plotted along the x axis and cell volume is plotted along the y axis. Only red blood cells appear on this cytogram. Markers organize the cytogram into 9 distinct areas of red blood cell morphology. On the x axis, hemoglobin concentration markers are set at 280 g/L (3) and 410 g/L (4). Red blood cells with a hemoglobin concentration less than 280 g/L are hypochromic, while cells with a hemoglobin concentration greater than 410 g/L are hyperchromic. On the y axis, RBC volume markers are set at 60 fL (1) and 120 fL (2). Red blood cells with a volume less than 60 fL are microcytic, while cells with a volume greater than 120 fL are macrocytic.

CHr and %Hypo have been used as a diagnostic tool, together with biochemical markers, to distinguish IDA from ACD, and are incorporated to the guidelines for the monitoring of recombinant human erythropoietin rHuEpo therapy (Macdougall *et al*., 2000; Kotisaari 2002; Locateli *et al*., 2004).

#### **1.3.2 Sysmex**

234 Chronic Kidney Disease

Hypochromic red cells percentages have been correlated with iron deficiency. %Hypo is reported by Siemens Advia 120 hematology analyzer based on the optical cell-by-cell

The RBC Scatter cytogram is the graphical representation of two light-scatter measurements: the high-angle light scatter (5° to 15°) is plotted along the x axis, and the low-angle light

The RBC map shows the relationship between the light-scatter measurements and the cellby-cell characteristics of volume and hemoglobin concentration. The map grid encompasses RBC volumes between 30 fL and 180 fL and hemoglobin concentrations between 190 g/L

The measurement of %Hypo (defined as the percentage of red blood cells with Hb concentration less than 280 g/L) is a sensitive method for quantifying the hemoglobinization of mature red cells. Because of the long circulating life span of mature erythrocytes %Hypo values are related to iron status in the last 2-3 months, and have been recognised as an indicator of iron deficiency (Macdougal 1998; Bovy *et al*., 2005; Bovy *et al*., 2007). %Hypo < 5% is considered normal. Two different criteria, more specifically, %Hypo >5% and >10% have been used. %Hypo >10% has been more commonly used for defining absolute iron

hemoglobin measurement (Figures 2 and 3).

Fig. 2. RBC Scatter Cytogram. 1. Low-angle light scatter (2° to 3°) 2. High-angle light scatter (5° to 15°)

3. Mie map containing RBCs 4. Platelets detected in RBC method

and 490 g/L. (Figure 3).

scatter (2° to 3°) is plotted along the y axis. (Figure 2).

deficiency and functional iron deficiency (Locatelli *et al*., 2004).

Sysmex XE analyzers (Sysmex Corporation, Kobe, Japan) employ flow cytometry technology. In the reticulocyte channel blood cells are stained by a polymethine dye, specific for RNA/DNA, and analysed by flow cytometry using a semiconductor laser. A bidimensional distribution of forward scattered light and fluorescence is presented as a scattergram, indicating mature red cells and reticulocytes (Figure 4).

Forward scatter correlates with erythrocyte and reticulocyte hemoglobin content (Ret He, RBC He).

Assessing Iron Status in CKD Patients: New Laboratory Parameters 237

cells. Derived from this technology four new RBC extended parameters or erythrocyte

%Hypo-He indicates the percentage of hypochromic red cells with an Hb content equivalent to less than 17pg. Recent studies confirm the clinical reliability of the hypochromic red cells, reported by the Sysmex XE 5000 counter, as markers of iron deficiency in hemodialysis patients; 2.7 % is the cut off value which defines iron deficiency (Buttarello *et al*., 2010).

Fig. 5. A bi-dimensional distribution of forward scattered light and fluorescence is presented as a scattergram, indicating mature red cells and reticulocytes. Forward scatter correlates with the hemoglobin content. A new algorithm divides the RBC He signal in three areas.

The percentages of red cells subsets can be calculated and the new parameters %Hypo-He

%Hypo-He indicates the percentage of hypochromic red cells with a Hb content < 17 pg. %Hyper-He indicates the percentage of hyperchromic red cells with a Hb content > 49 pg. %Micro R indicates the percentage of microcytic red cells with a volume less than 60 fL. %Macro R indicates the percentage of macrocytic red cells with a volume greater than 120 fL. The new Symex XE 5000 analyzer reports the percentages of hypochromic red cells; the reference range and the values in different types of anemia have been published (Urrechaga

subsets are now available in this analyzer.

Figure 5 shows s a scattergram of the reticulocyte channel.

*et al*., 2009).

and %Hyper-He obtained.

Ret He is the mean value of the forward light scatter histogram within the reticulocyte population obtained in a reticulocyte channel on the Sysmex XE-2100 hematology analyzer. Measurements of Ret He provides useful information in diagnosing anemia, iron restricted erythropoiesis and functional iron deficiency and response to iron therapy during r-HuEpo (Buttarello *et al*, 2004; Canals *et al*, 2005; Brugnara *et al*, 2006; Thomas *et al*, 2006; Garzia *et al*., 2007).

Ret He, generated by all Sysmex XE analysers (Sysmex Corporation, Kobe, Japan), has been recognised as a direct assessment of the incorporation of iron into erythrocyte hemoglobin and a direct estimate of the recent functional availability of iron into the erythron, thus provides the same information as CHr (Thomas *et al*., 2005; David *et al*., 2006). Twenty nine pg is the cut off value that defines deficient erythropoiesis Several studies have demonstrated that Ret He and CHr have the same clinical meaning (Mast *et al*, 2008; Maconi *et al*., 2009; Miwa *et al*., 2010).

Fig. 4. In the reticulocyte channel blood cells are stained by a polymethine dye, specific for RNA/DNA, and analysed by flow cytometry using a semiconductor laser. A bi-dimensional distribution of forward scattered light and fluorescence is presented as a scattergram, indicating mature red cells and reticulocytes. Forward scatter correlates with erythrocyte and reticulocyte hemoglobin content (RBC He, Ret He).

The Sysmex XE 5000 analyzer incorporates flow fluorescence cytometry technology, which enables independent measurement of the volume and hemoglobin content of individual red

Ret He is the mean value of the forward light scatter histogram within the reticulocyte population obtained in a reticulocyte channel on the Sysmex XE-2100 hematology analyzer. Measurements of Ret He provides useful information in diagnosing anemia, iron restricted erythropoiesis and functional iron deficiency and response to iron therapy during r-HuEpo (Buttarello *et al*, 2004; Canals *et al*, 2005; Brugnara *et al*, 2006; Thomas *et al*, 2006; Garzia *et al*.,

Ret He, generated by all Sysmex XE analysers (Sysmex Corporation, Kobe, Japan), has been recognised as a direct assessment of the incorporation of iron into erythrocyte hemoglobin and a direct estimate of the recent functional availability of iron into the erythron, thus provides the same information as CHr (Thomas *et al*., 2005; David *et al*., 2006). Twenty nine pg is the cut off value that defines deficient erythropoiesis Several studies have demonstrated that Ret He and CHr have the same clinical meaning (Mast *et al*, 2008; Maconi

Fig. 4. In the reticulocyte channel blood cells are stained by a polymethine dye, specific for RNA/DNA, and analysed by flow cytometry using a semiconductor laser. A bi-dimensional distribution of forward scattered light and fluorescence is presented as a scattergram, indicating mature red cells and reticulocytes. Forward scatter correlates with erythrocyte

The Sysmex XE 5000 analyzer incorporates flow fluorescence cytometry technology, which enables independent measurement of the volume and hemoglobin content of individual red

and reticulocyte hemoglobin content (RBC He, Ret He).

2007).

*et al*., 2009; Miwa *et al*., 2010).

cells. Derived from this technology four new RBC extended parameters or erythrocyte subsets are now available in this analyzer.

%Hypo-He indicates the percentage of hypochromic red cells with a Hb content < 17 pg. %Hyper-He indicates the percentage of hyperchromic red cells with a Hb content > 49 pg. %Micro R indicates the percentage of microcytic red cells with a volume less than 60 fL. %Macro R indicates the percentage of macrocytic red cells with a volume greater than 120 fL.

The new Symex XE 5000 analyzer reports the percentages of hypochromic red cells; the reference range and the values in different types of anemia have been published (Urrechaga *et al*., 2009).

%Hypo-He indicates the percentage of hypochromic red cells with an Hb content equivalent to less than 17pg. Recent studies confirm the clinical reliability of the hypochromic red cells, reported by the Sysmex XE 5000 counter, as markers of iron deficiency in hemodialysis patients; 2.7 % is the cut off value which defines iron deficiency (Buttarello *et al*., 2010).

Figure 5 shows s a scattergram of the reticulocyte channel.

Fig. 5. A bi-dimensional distribution of forward scattered light and fluorescence is presented as a scattergram, indicating mature red cells and reticulocytes. Forward scatter correlates with the hemoglobin content. A new algorithm divides the RBC He signal in three areas.

The percentages of red cells subsets can be calculated and the new parameters %Hypo-He and %Hyper-He obtained.

Assessing Iron Status in CKD Patients: New Laboratory Parameters 239

Fig. 6. the Beckman -Coulter cube in which the cells are classified according to the volume, conductivity and laser scatter signals. ERIT , erythrocytes; PLQ, platelets; RET, reticulocytes;

A group of 132 IDA patients fulfilled traditional diagnostic criteria for iron deficiency anemia diagnosis, serum iron < 7.5 μmol/L, transferrin saturation < 20 %, ferritin < 50 µg/L, and Hb < 110 g/L (Cook, 2005), were included before iron treatment. This group was divided into a non acute phase response group (n=72, CRP < 5 mg/L) and acute phase response group (n=60, CRP > 5 mg/L). Acute phase response included inflammation or

CKD patients were managed according to the recommendations of the NKF-K/DOQI guidelines (Locatelli *et al*., 2004). All patients were treated with a variety of erythropoietin doses, the majority of them were treated with a maintenance dose of intravenous iron

ACD group included patients with a variety of diseases: chronic infections (tuberculosis); neoplasic disorders (Hodgkin's disease, breast carcinoma); non infectious inflammatory diseases (rheumatoid arthritis, systemic lupus erythematosus). ACD patients received treatment to maintain normal erythropoiesis and presented the traditional diagnostic criteria for 'Functional iron-deficiency' diagnosis Transferrin saturation < 20%, Hb < 110 g/L and serum ferritin values normal or over the reference range (Weiss & Goodnough,

In a second phase of the study ACD group was extended to 85 patients. This group was further subdivided based on sTfR levels. ACD patients with sTfR higher than 21 nmol/L were considered to have storage iron depletion (iron deficiency associated, n=24) and

patients with normal sTfR were considered to have functional iron deficiency (n=61).

weekly, in order to maintain Hb at the recommended level 110 - 120 g/L.

**PLQ**

infectious conditions, in addition to ferropenic status.

**ERIT**

LEU, leucocytes.

2005).

**LEU**

**RET**

%Hypo-He indicates the percentage of hypochromic red cells with a Hb content < 17 pg. %Hyper-He indicates the percentage of hyperchromic red cells with a Hb content > 49 pg.

#### **1.3.3 Beckman-Coulter**

The percentage of hypochromic red cells are only available on Siemens analyzers (Siemens Medical Solutions Diagnostics, Tarrytown N.Y., USA) and on the new Sysmex analyzer XE 5000 (Sysmex Corporation, Kobe, Japan); this fact limits its generalized use. Beckman Coulter (Beckman Coulter Inc. Miami, Fl, USA) applies the Volume Conductivity Scatter technology to this field and new parameters are now available on the LH series analyzers.

Low hemoglobin density (LHD %) derives from the traditional mean cell hemoglobin concentration (MCHC), using the mathematical sigmoid transformation

$$\text{LHD} \,\%= 100 \times \sqrt{1 - \left[1/\left(1 + \text{e} \,\text{1.8}\left(30 - \text{MCHC}\right)\right)\right]}$$

MCHC is an all inclusive measure of both the availability of iron over the preceding 90–120 days, and of the proper introduction of iron into intracellular hemoglobin. In the same way LHD% is related to iron availability and the hemoglobin concentration of the mature red cells. In this equation defining LHD %, in addition to the standard sigmoid function, a square root is applied to further enhance numerical resolution in the region corresponding to the lower end, to improve the differentiation between the normal and the abnormal among the blood samples having relatively low values of LHD %.

The reference range for LHD % and the values in normal population and different types of anemia have been established (Urrechaga, 2010). Then a study was conducted to investigate its clinical usefulness in the assessment of iron status in terms of correlation with %Hypo (Urrechaga *et al*., 2010) and sTfR (Urrechaga *et al*., 2011).

Cells are identified and classified by simultaneous three-dimensional analysis using Volume, Conductivity, and Light Scatter (Figure 6). Volume, as measured by direct current, is used to identify the size of the cell. Conductivity, or radio frequency measurements, provides information about the internal characteristics of the cell. Light scatter measurements, obtained as cells pass through the helium-neon laser beam, provide information about cell surface characteristics and cell granularity.

#### **2. Materials and methods**

#### **2.1 Criteria for selecting the groups of patients**

Samples from 120 healthy individuals, 72 iron deficiency anemia (IDA), 60 IDA with acute phase response (IDA APR), 71 chronic kidney disease (CKD) and 58 anemia of chronic disease (ACD) were randomly extracted from the routine workload and run sequentially on both LH 750 (Beckman Coulter Inc. Miami, Fl, USA) and Advia 2120 (Siemens Medical Solutions Diagnostics, Tarrytown N.Y., USA) analyzers within 6 hours of collection.

Healthy group: 54 male and 66 female adult subjects, with no clinical symptoms of disease and with results of the complete blood count and biochemical iron metabolism markers within reference ranges.

The percentage of hypochromic red cells are only available on Siemens analyzers (Siemens Medical Solutions Diagnostics, Tarrytown N.Y., USA) and on the new Sysmex analyzer XE 5000 (Sysmex Corporation, Kobe, Japan); this fact limits its generalized use. Beckman Coulter (Beckman Coulter Inc. Miami, Fl, USA) applies the Volume Conductivity Scatter technology to this field and new parameters are now available on the LH series analyzers. Low hemoglobin density (LHD %) derives from the traditional mean cell hemoglobin

LHD % 100 1 1 1 e 1.8 30 MCHC

MCHC is an all inclusive measure of both the availability of iron over the preceding 90–120 days, and of the proper introduction of iron into intracellular hemoglobin. In the same way LHD% is related to iron availability and the hemoglobin concentration of the mature red cells. In this equation defining LHD %, in addition to the standard sigmoid function, a square root is applied to further enhance numerical resolution in the region corresponding to the lower end, to improve the differentiation between the normal and the abnormal

The reference range for LHD % and the values in normal population and different types of anemia have been established (Urrechaga, 2010). Then a study was conducted to investigate its clinical usefulness in the assessment of iron status in terms of correlation with %Hypo

Cells are identified and classified by simultaneous three-dimensional analysis using Volume, Conductivity, and Light Scatter (Figure 6). Volume, as measured by direct current, is used to identify the size of the cell. Conductivity, or radio frequency measurements, provides information about the internal characteristics of the cell. Light scatter measurements, obtained as cells pass through the helium-neon laser beam, provide

Samples from 120 healthy individuals, 72 iron deficiency anemia (IDA), 60 IDA with acute phase response (IDA APR), 71 chronic kidney disease (CKD) and 58 anemia of chronic disease (ACD) were randomly extracted from the routine workload and run sequentially on both LH 750 (Beckman Coulter Inc. Miami, Fl, USA) and Advia 2120 (Siemens Medical

Healthy group: 54 male and 66 female adult subjects, with no clinical symptoms of disease and with results of the complete blood count and biochemical iron metabolism markers

Solutions Diagnostics, Tarrytown N.Y., USA) analyzers within 6 hours of collection.

concentration (MCHC), using the mathematical sigmoid transformation

among the blood samples having relatively low values of LHD %.

information about cell surface characteristics and cell granularity.

(Urrechaga *et al*., 2010) and sTfR (Urrechaga *et al*., 2011).

**2.1 Criteria for selecting the groups of patients**

**2. Materials and methods** 

within reference ranges.

%Hypo-He indicates the percentage of hypochromic red cells with a Hb content < 17 pg. %Hyper-He indicates the percentage of hyperchromic red cells with a Hb content > 49 pg.

**1.3.3 Beckman-Coulter** 

Fig. 6. the Beckman -Coulter cube in which the cells are classified according to the volume, conductivity and laser scatter signals. ERIT , erythrocytes; PLQ, platelets; RET, reticulocytes; LEU, leucocytes.

A group of 132 IDA patients fulfilled traditional diagnostic criteria for iron deficiency anemia diagnosis, serum iron < 7.5 μmol/L, transferrin saturation < 20 %, ferritin < 50 µg/L, and Hb < 110 g/L (Cook, 2005), were included before iron treatment. This group was divided into a non acute phase response group (n=72, CRP < 5 mg/L) and acute phase response group (n=60, CRP > 5 mg/L). Acute phase response included inflammation or infectious conditions, in addition to ferropenic status.

CKD patients were managed according to the recommendations of the NKF-K/DOQI guidelines (Locatelli *et al*., 2004). All patients were treated with a variety of erythropoietin doses, the majority of them were treated with a maintenance dose of intravenous iron weekly, in order to maintain Hb at the recommended level 110 - 120 g/L.

ACD group included patients with a variety of diseases: chronic infections (tuberculosis); neoplasic disorders (Hodgkin's disease, breast carcinoma); non infectious inflammatory diseases (rheumatoid arthritis, systemic lupus erythematosus). ACD patients received treatment to maintain normal erythropoiesis and presented the traditional diagnostic criteria for 'Functional iron-deficiency' diagnosis Transferrin saturation < 20%, Hb < 110 g/L and serum ferritin values normal or over the reference range (Weiss & Goodnough, 2005).

In a second phase of the study ACD group was extended to 85 patients. This group was further subdivided based on sTfR levels. ACD patients with sTfR higher than 21 nmol/L were considered to have storage iron depletion (iron deficiency associated, n=24) and patients with normal sTfR were considered to have functional iron deficiency (n=61).

Assessing Iron Status in CKD Patients: New Laboratory Parameters 241

RBC, red blood cells; Hb, hemoglobin; MCV, mean cell volume; MCH, mean cell hemoglobin; MCHC, mean cell hemoglobin concentration; Transf, transferrin; Sat, %

Table 1 shows the hematological and biochemical data, mean and (standard deviation). The parameters presented are of general use for every Laboratory in the evaluation of anemia. The patients included in the study sufferered common clinical situations in our daily practice: anemia of chronic disease (ACD), chronic kidney disease (CKD), iron deficiency

The healthy group was recruited to assess the reference range for the new parameter LHD

LHD % values in a population of 120 healthy adult subjects were not normally distributed and showed a non Gaussian distribution (Kolmogorov-Smirnoff test, p=0.034; figure 7).

Fig. 7. Low hemoglobin density (LHD %) values in a population of 120 healthy adult subjects.

Table 2 exhibits %Hypo values, mean and standard deviation (SD) and LHD % values, median and 5th - 95th interquartiles (IQ), in the variety of anemias and healthy subjects

The values showed a non Gaussian distribution (Kolmogorov-Smirnoff test, p=0.034).

anemia (IDA) iron deficiency anemia and acute phase response (IDA APR)

transferrin saturation.

Reference range 0 - 4.4 %.

included in the study.

**3. Results** 

%.

sTfR was measured with Access sTfR assay in the Access immunochemical analyzer (Beckman Coulter Inc., Miami Fl, USA).

#### **2.2 Statistical evaluation of analytical results**

Statistical software package SPSS (SPSS; Chicago, IL, USA) version 17.0 for Windows was applied for statistical analysis of the results.

Reference ranges were calculated from the results obtained in the group of healthy subjects (95 central percentiles of the distribution). Kolmogorov – Smirnoff test was applied to verify the Gaussian distribution of LHD% values.

When the parameters under study presented a non Gaussian distribution non parametric tests were applied. Correlation coefficients were calculated by Spearman method; independent samples Mann-Whitney U test was performed; p values less than 0.05 were considered to be statistically significant.

Receiver operating characteristic (ROC) curve analysis was utilized to illustrate the diagnostic performance of LHD% and other Laboratory tests in the detection of iron deficiency status; two analysis were performed; first iron deficiency was defined by %Hypo > 5 %, and second , including 85 ACD patients, the gold standard was sTfR > 21 nmol/L.

Cut off values were established based on the optimal combination of sensitivity and specificity.

Cohen's Kappa Index of Inter-rater Reliability (κ index) was calculated to determine the concordance between LHD% and sTrR.

κ has a range from 0-1.0, the larger values indicate better reliability; κ > 0.7 is considered satisfactory.


Table 1. shows the hematological and biochemical data, mean and (standard deviation), of the different groups. 120 healthy individuals, 72 iron deficiency anemia (IDA), 60 IDA with acute phase response (IDA APR), 71 chronic kidney disease (CKD) and 58 anemia of chronic disease (ACD).

RBC, red blood cells; Hb, hemoglobin; MCV, mean cell volume; MCH, mean cell hemoglobin; MCHC, mean cell hemoglobin concentration; Transf, transferrin; Sat, % transferrin saturation.
