Regional Clinical Center of Miners Health Protection
 18


Search
Surzhikova G.S., Klochkova-Abelyants S.A.

ALGORITHM FOR LABORATORY DIAGNOSTICS OF HYPOCHROMIC ANEMIA


Novokuznetsk Institute of Postgraduate Training,

Novokuznetsk, Russia

According to WHO recommendations, anemia is defined as decrease in hemoglobin level lower 130 g/L in men and 120 g/L in women. The reason for this definition is values of middle level of hemoglobin in healthy individuals.

As for the prevalence, hypochromic anemia takes one of the leading parts among types of anemia. Hypochromic anemia is presented in view of iron deficiency anemia (IDA) and anemia of chronic disease (ACD). A great variety of the factors forming the basis of anemia results in importance of differential diagnostics of hypochromic anemia. Anemia of chronic disease is the leading part of so called stress-syndrome. It develops in most patients with chronic infectious diseases and inflammatory processes of non-infectious origin [1, 2, 4]. In clinical practice ACD is often associated with IDA, because it has such IDA laboratory signs as decreasing hemoglobin level in red blood cells, microcytosis, decrease in levels of serum iron and transferrin saturation ratio [3]. Diagnostic significance of some iron metabolism decreases sharply in most inflammatory states and does not allow identifying an actual cause of iron metabolism disorders [4].

Objective – to study the parameters of iron metabolism for development of diagnostic algorithm of IDA and functional iron deficiency.

MATERIALS AND METHODS

The examination included 296 women (age of 16-90) with anemic syndrome. IDA was identified in 103 women, ACD in 193. 121 women had anemia, which developed at the background of rheumatoid arthritis (RA), 72 ACD at the background of bacterial infections (chronic tonsillitis, bacterial endocarditis, chronic pyelonephritis). The control group included 79 almost healthy women.

Prelaboratory clinical examination was performed with the questionary including sections about patients’ complaints, history of life and diseases, presence of concurrent diseases and the data of objective study.

The scientific research was performed in concordance with the constitutional provisions of Russian Federation, the clause 32 Foundations of Russia Legislation about Protection of Population Health, Declaration of Helsinki and World Medical Association “Recommendations for Physicians Dealing with Biomedical Research with Human Participation”. The study protocol was approved by the ethic committee of Novokuznetsk Institute of Postgraduate Training. Before the study all participants gave their written informed consent, which was confirmed by the ethic committee of Novokuznetsk Institute of Postgraduate Training.

The exclusion criteria were the age < 16 or > 60, other types of anemia, presence of malignant diseases and refusal from participation.

The examination of hematologic values in peripheral blood was conducted with Becman Culter blood analyzer (USA), with evaluation of the following parameters: hemoglobin contents (HGB), amount of red blood cells (RBC), hematocrit (HCT), mean cell volume (MCV), mean cell hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood count (WBC), mean platelet volume (MPV), red cell distribution width (RDW). Morphological research was performed with optic microscopy technique with peripheral blood preparation staining with azure-eosin according to Kryukov-Pappenheim. Reticulocyte amount was calculated in the optical microscope with supravital staining. Blood serum iron and total iron binding capacity (TIBC) were estimated with ferrozin technique using Teco diagnostic sets (USA) in KONELAB-60 automatic biochemical analyzer, with following calculation of latent iron-binding capacity (IBC) and transferrin saturation ratio (TSR). Iron reserve was evaluated with serum ferritin level using immunoenzyme method and Orgentec diagnostika test systems (Germany). Soluble transferrin receptors (STR) were assessed with immunoenzyme method and Vector-Best test systems.

The mathematic preparation of results was performed with Microsoft Excel 2000 and Biostat 4.03. The mean (M) and the error of mean (m) were calculated. The results were prepared with Students test. P value was defined. P < 0.05 was considered as significant. Kolmogorov-Smirnov test was used for testing hypothesis about normalcy of distribution in the groups.

RESULTS

79 apparently healthy women were examined for development of referential meanings of examined values. In this group the hemogramm values were within the normal range, the levels of reticulocytes were within the range of 6.74 ± 0.4 ‰, the absolute number of reticulocytes 27.05 x 109/L 1.67. The morphophysiological values of erythrocytes (MCV, MCH, MCHC, RDW) were within the normal range (Table 1). Iron metabolism parameters were within the normal range in healthy women, serum ferritin was 33.55 2.59 ng/ml, ranging from 26.31 to 51.25 ng/ml. The levels of soluble transferrin receptors varied from 0.9 to 1.75 mcg/ml, with mean value of 1.14 0.12 ng/ml (table 2).

Table 1
Hemogram values in patients with IDA and ACD
1.jpg
Note: * reliability of differences in values compared to the control group's values, with p < 0.05; • reliability of differences in values in persons with ACD compared to values in persons with IDA, with p < 0.05.

Table 2
Iron metabolism values in patients with IDA and ACD
2.jpg
Note: * reliability of differences in values compared to the control group's values, with p < 0.05; reliability of differences in values in persons with ACD compared to values in persons with IDA, with p < 0.05.



Anemia had hypochromic and microcytic characteristics in the women with identified iron deficiency anemia (table 1). Serum iron decreased from 8.44 0.32 mcM/L compared to 20.4 1.02 mcM/L in healthy persons. Increasing levels of TIBC and LIBC were up to 80.96 1.25 and 71.41 1.45 mcM/L compared to 65.65 1.83 mcM/L and 44.53 1.87 mcM/L in healthy women respectively. TIBC and LIBC increased in all women with iron deficiency anemia as opposed to other hypochromic anemias, which were conditioned by not iron deficiency, but associated with disorders of its inclusion into hemoglobin molecule or with iron redistribution or iron retention in macrophage system cells. Increase in TIBC and LIBC levels is associated with increasing transferrin synthesis as compensatory response of the body to iron deficiency in tissues.

Serum ferritin levels decreased sharply within the range of 4.91 0.66 ng/ml in the women with IDA. As long as iron depletion is the stage of IDA development, then decrease in ferritin level is one of the significant signs of iron deficiency characteristics of hypochromic anemias. At the same time, presence of concurrent inflammatory infectious or non-infectious process can disguise hypoferritinemia in patients with IDA. Examination of the levels of soluble transferrin receptors in IDA showed their increased levels up to 3.39 0.08 mcg/ml compared to 1.14 0.12 mcg/L in healthy individuals. It reflects compensatory increase in expression of transferrin receptors on the cell membranes of erythroid cells and decreasing intracellular iron. The calculated index combining soluble transferrin receptor and level of serum ferritin (sTFR/log SF) was significantly increased in the patients with IDA 4.91 0.09 compared to 0.75 0.08 in healthy individuals. Increase in this index is the significant diagnostic criterion of IDA (table 2).

In the women with ACD and infectious inflammatory processes anemia had hypochromic or, less frequently, normochromic characteristics and the following hemogramm values: count of red blood cells 3.29 0.28 x 1012/L, hemoglobin level 91.00 7.55 g/L, mean volume of erythrocytes 76.00 3.88 fL, MCH 26.80 1.97 pg, MCHC 34.10 0.89 g/dl, RDW 16.53 0.92 %. The number of reticulocytes was within the normal values (table 1). Examination of iron metabolism biochemical markers showed decreased levels of serum iron, TIBC and TSR. However, serum ferritin level increased and varied from 130.21 ng/ml to 182.12 ng/ml, with middle ferritin level of 155.81 24.56 ng/ml. The levels of soluble transferrin receptors did not differ significantly from healthy women and it was significantly lower than in case of IDA (table 2).

In patients with rheumatoid arthritis anemia had characteristics of ACD, and it was hypochromic and microcytic. The number of reticulocytes was 8.03 0.21 . The ferrokinetics indices (serum iron, TIBC and TSR) decreased significantly at the background of high levels of serum ferritin. The mean levels of ferritin were 210.64 30.1 ng/ml. It testifies decreasing level of serum iron (available for erythropoiesis) and significant increase in deposited iron. The level of soluble transferrin receptors was within the normal range and significantly lower compared to IDA (table 2).

Thus, iron functional deficit and changing synthesis of transferrin receptors on the erythroid cell are associated with disorders in iron metabolism in ACD at the background of infectious processes and rheumatoid arthritis. During infection and immunologic changes (PA) the anti-inflammatory cytokines (IL-6, IL-1, TNF) increase levels of acute phase proteins, including α-1- antitrypsin, which is able to reduce intensity of erythropoiesis by means of disorder of binding the complex transferrin-Fe++ to TFR, with subsequent formation (internalization) of TFR-transferrin complex [5].

CONCLUSION

Iron deficiency anemia and anemia of chronic diseases are the most common types of anemia hypochromic and microcytic (ACD is normochromic anemia less frequently). The differentiation depends on the values of iron metabolism. In differential diagnostics it is important to differentiate iron deficiency anemia from anemia of chronic diseases, with following adequate treatment. It is necessary to consider the influence of chronic diseases on results of examination of serum ferritin in case of iron-deficient erythropoiesis. Estimation of levels of soluble transferrin receptors significantly increases the accuracy of laboratory diagnostics of IDA and ACD. The index of sTFR/log ferritin > 1.5 is an adequate value of iron deficiency in the body. It should be used in the algorithm of differential diagnostics of hypochromic anemia (Fig.).

Figure

The algorithm for differential diagnostics of anemic syndrome (hypo-, normochromic anemia)

22.png