**3. Results**

240 Chronic Kidney Disease

sTfR was measured with Access sTfR assay in the Access immunochemical analyzer

Statistical software package SPSS (SPSS; Chicago, IL, USA) version 17.0 for Windows was

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

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

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

Cohen's Kappa Index of Inter-rater Reliability (κ index) was calculated to determine the

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

MCH pg MCHC

31.3 (1.53)

22.5 (4.23)

21.5 (1.3)

31.9 (2.23)

31.1 (2.23)

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

g/L

343 (5.2)

320 (17.3)

327 (9.2)

343 (10)

325 (8)

Iron µmol/L

> 16.5 (0.62)

> 4.8 (2.15)

5.1 (3.5)

10.0 (6.8)

9.8 (4.47) Transf g/L

> 2.53 (0.2)

3.31 (0.53)

2.78 (0.28)

2.68 (0.66)

1.87 (0.43) Ferritin µg/L

> 75 (2.8)

> > 14 (9)

37 (25)

522 (704)

335 (204) Sat %

> 31 (1.9)

> 6 (3.6)

> 9 (5.6)

> > 15 (5)

21 (10)

(Beckman Coulter Inc., Miami Fl, USA).

**2.2 Statistical evaluation of analytical results** 

applied for statistical analysis of the results.

the Gaussian distribution of LHD% values.

considered to be statistically significant.

concordance between LHD% and sTrR.

Hb g/L

154 (6.4)

95 (14.2)

96 (12.1)

> 101 (11)

112 (8.5) MCV fL

91.1 (2.55)

70 (10.3)

75.8 (3.7)

93.2 (6.0)

95.6 (6.67)

specificity.

satisfactory.

RBC

Health 4.9

IDA 4.6

ACD 3.5

CKD 3.5

disease (ACD).

IDA APR 1012/L

(0.27)

(0.61)

4.4 (0.43)

(0.48)

(0.45)

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

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). Reference range 0 - 4.4 %.

Fig. 7. Low hemoglobin density (LHD %) values in a population of 120 healthy adult subjects. The values showed a non Gaussian distribution (Kolmogorov-Smirnoff test, p=0.034).

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 included in the study.

Assessing Iron Status in CKD Patients: New Laboratory Parameters 243

No statistic difference was found between IDA group and IDA patients with acute phase

Receiver operating characteristic (ROC) curve analysis for LHD% in the diagnosis of iron deficiency, defined by %Hypo > 5% AUC 0.954, cut off 6.0 %, sensitivity 96.6%, specificity

Discriminant efficiency of biochemical parameters and classical erythrocyte indices: mean cell hemoglobin (MCH), AUC 0.89; mean cell volume, (MCV), AUC 0.822; serum ferritin,

In the group including 85 ACD patients, significant differences were detected when iron replete ACD patients (LHD% 10.5 %) were compared to the group with both ACD and IDA

Table 3 exhibits sTfR values, mean and standard deviation (SD) and LHD % values, median

ROC analysis for LHD% in the detection of iron deficiency rendered area under curve (AUC) 0.903; at a threshold value 5.5 % sensitivity was 88.6 % and specificity 76.9 %. The

> LHD% MCH MCV Ferritin Iron

Using the cut off 5.5 % for LHD% the k index obtained in comparison to sTfR was 0.65.

0 20 40 60 80 100

100-Specificity

Fig. 9. Receiver operating characteristic (ROC) curve analysis for LHD% and Biochemical parameters and classical erythrocyte indices in the diagnosis of iron deficiency, defined by %Hypo >5%. LHD% Ares under curve (AUC) 0.954; mean cell hemoglobin (MCH), AUC 0.89; mean cell volume, (MCV), AUC 0.822; serum ferritin, AUC 0.722; serum iron, AUC

response (p=0.578).

83.3% (Figure 9).

(LHD% 24.1 %, p<0.0001).

100

80

60

40

Sensitivity

0.683.

20

0

AUC 0.722; serum iron, AUC 0.683 (Figure 9).

and 5th - 95th interquartiles in these patients.

ferropenic state was defined by a sTfR > 21 nmol/L.


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

IDA, iron deficiency anemia; IDA APR, iron deficiency anemia and acute phase response; ACD, anemia of chronic disease; CKD, chronic kidney disease.

Correlation between %Hypo and LHD% values, r = 0.869 (Spearman method) (p<0.001). y = 1.338 x + 4.40 (Figure 8).

Independent samples U test was performed in order to detect statistical deviations between the groups of patients.

Significant differences in LHD % values (p<0.001) were detected when groups with iron deficiency (IDA, median 29.6 % and IDA with APR, median 27.3 %) were compared with patients undergoing therapy (ACD, median 7.3 %; CKD, median 9.6 %) and the healthy subjects (median 2.1 %).

Fig. 8. Relationship between %Hypo and LHD% values (Spearman correlation) r = 0.869 y = 1.338 x + 4.4.

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

IDA, iron deficiency anemia; IDA APR, iron deficiency anemia and acute phase response;

Correlation between %Hypo and LHD% values, r = 0.869 (Spearman method) (p<0.001). y =

Independent samples U test was performed in order to detect statistical deviations between

Significant differences in LHD % values (p<0.001) were detected when groups with iron deficiency (IDA, median 29.6 % and IDA with APR, median 27.3 %) were compared with patients undergoing therapy (ACD, median 7.3 %; CKD, median 9.6 %) and the healthy

Fig. 8. Relationship between %Hypo and LHD% values (Spearman correlation) r = 0.869

 LHD % Median (IQ)

% Hypo Mean (SD)

ACD, anemia of chronic disease; CKD, chronic kidney disease.

1.338 x + 4.40 (Figure 8).

the groups of patients.

subjects (median 2.1 %).

y = 1.338 x + 4.4.

Healthy 0.13 (0.15) 2.1 (0.9-4.1) IDA 17.2 (17.4) 29.6 (7.5-76) IDA APR 16.8 (15.5) 27.3 (8.3-71.2) ACD 4.1 (4.4) 7.3 (5.1-30) CKD 5.1 (6.7) 9.6 (5.6-27)

No statistic difference was found between IDA group and IDA patients with acute phase response (p=0.578).

Receiver operating characteristic (ROC) curve analysis for LHD% in the diagnosis of iron deficiency, defined by %Hypo > 5% AUC 0.954, cut off 6.0 %, sensitivity 96.6%, specificity 83.3% (Figure 9).

Discriminant efficiency of biochemical parameters and classical erythrocyte indices: mean cell hemoglobin (MCH), AUC 0.89; mean cell volume, (MCV), AUC 0.822; serum ferritin, AUC 0.722; serum iron, AUC 0.683 (Figure 9).

In the group including 85 ACD patients, significant differences were detected when iron replete ACD patients (LHD% 10.5 %) were compared to the group with both ACD and IDA (LHD% 24.1 %, p<0.0001).

Table 3 exhibits sTfR values, mean and standard deviation (SD) and LHD % values, median and 5th - 95th interquartiles in these patients.

ROC analysis for LHD% in the detection of iron deficiency rendered area under curve (AUC) 0.903; at a threshold value 5.5 % sensitivity was 88.6 % and specificity 76.9 %. The ferropenic state was defined by a sTfR > 21 nmol/L.

Using the cut off 5.5 % for LHD% the k index obtained in comparison to sTfR was 0.65.

Fig. 9. Receiver operating characteristic (ROC) curve analysis for LHD% and Biochemical parameters and classical erythrocyte indices in the diagnosis of iron deficiency, defined by %Hypo >5%. LHD% Ares under curve (AUC) 0.954; mean cell hemoglobin (MCH), AUC 0.89; mean cell volume, (MCV), AUC 0.822; serum ferritin, AUC 0.722; serum iron, AUC 0.683.

Assessing Iron Status in CKD Patients: New Laboratory Parameters 245

The treatment of renal anemia with rHuEpo has improved the quality of life and outcome of hemodialysis patients. The efficacy of this therapy depends on the identification and correction of resistance factors, such as vitamin deficiency, inflammation, hyperparathyroidism. The major cause of resistance to rHuEpo is iron deficiency. The assessment of functional iron deficiency remains a daily challenge for nephrologists and their need to be careful of an appropriate use of the resources and the need to optimize

A better understanding of iron homeostasis enhance treatments for anemia. Subsequently, evidence-based diagnostic strategies must be developed, using both conventional and innovative laboratory tests, to differentiate between the various causes of distortions of iron

Efforts have been made to evaluate some readily available and relatively inexpensive laboratory parameters as indirect markers of iron restricted erythropoiesis and iron availability in a clinical context influenced by inflammation and acute phase reaction.

The assessment of iron requirements and monitoring of therapy require accurate markers. It is desirable to seek alternative markers for iron status widely available. LHD% is related to iron availability for erythropoiesis in the previous weeks, derived from MCHC, it could be

The data exposed show the reliability of LHD% in distinguishing iron deficient patients with and without inflammation. This parameter could help to the correct classification of patients with iron deficiency when the traditional markers become unreliable: it is particularly challenging the accurate assessment of iron status in chronically ill patients such

LHD % correlates with the percentage of hypochromic erythrocytes as reported by Siemens analyzers (%Hypo) and comparing the results obtained for LHD% with those of sTfR the reliability of LHD% in distinguishing iron deficient patients with and without inflammation

In conclusion, these results show that the new LHD% parameter is useful for diagnosing iron deficiency and a reliable parameter recognizing subsets of patients and therefore improving the diagnosis and management of anemia.The analysis of LHD% can be performed simultaneously in the course of routine blood counts, with no incremental costs and no additional needs of more blood sampling. In conjunction with standard blood cell counts and iron parameters could enable the diagnosis to be made rapid and

More prospective and longitudinal studies are needed in order to verify the results obtained, to determine their reliability for clinical purposes or whether the additional information provided could be used in managing the iron requirements of patients in

Iron metabolism is a dynamic process which cannot be defined by one laboratory test only. The analysis of these new parameters can be performed simultaneously in the course of

patient treatment.

calculated in different hematological counters.

metabolism.

as CKD.

has been stated.

accurately.

different clinical situations.

**5. Conclusion** 


Table 3. sTfR values, mean and standard deviation (SD) and LHD % values, median and 5th - 95th interquartiles in a group of 120 healthy subjects, 85 anemia of chronic disease patients (ACD), 61 of them iron replete and 24 iron deficient (ACD/IDA).
