2 Professor, Department of Internal Medicine, Jos University Teaching Hospital, Jos, Nigeria
3 Professor, Department of Hematology and Blood Transfusion, College of Medicine, University of Lagos, Lagos, Nigeria
Methods: 215 participants were recruited from the wards and outpatient clinics, 125 cases (i.e. individuals with chronic diseases) and 90 controls consisting of individuals being seen for an annual or physical examination. Venous blood was collected for full blood counts, serum Iron (spectrophotometric), serum Ferritin (ELISA) and serum Erythropoietin (ELISA). Appropriateness of Erythropoietin response was determined by establishing an exponential relationship between erythropoietin levels and Hemoglobin (Hb) levels of controls (log EPO = 2.446 – 0.11 x Hb g/l). Inappropriate Erythropoietin secretion was indicated by an observed/ predicted log (serum EPO) ratio of less than 0.8 (O/P< 0.8).
Results: The result showed 72% of participants with chronic disease had anemia and 65.5% had anemia of chronic disease. Of the anemic participants, 33% had an Erythropoietin O/P ratio < 0.8, which corresponded with inappropriate Erythropoietin secretion. The mean circulating serum Erythropoietin level for control subjects was 13.1 ±10.1mu/l, significantly lower than that of subjects with chronic disease, 32.1±42.1mμ/l (P =0.00). Correlation studies showed a significant negative relationship between Hemoglobin and Packed Cell Volume.
Conclusion: The study showed that about two-thirds (65.5%) of the patients with anemia had Anemia of Chronic Disease, and a third showed evidence of diminished response endogenous Erythropoietin.
Keywords: Anemia; Inappropriate Erythropoietin Response; Erythropoietin; Anemia of Chronic Disease; Chronic Disease; Iron; Ferritin
The mechanism of inhibition of the EPO axis in ACD has been linked to inhibitory effects of proinflamatory cytokines, such as Tumor necrosis factor α (TNF α), Interferon γ (INF γ) and Interleukin 1 (IL1). More recently the protein Hepcidin, has been implicated as the major cytokine. Hepcidin interferes with EPO gene expression, but also has a major impact on Iron metabolism and determines the reticuloendothelial Iron supply for erythropoiesis. Hepcidin is a key regulator for iron metabolism and mediator of anaemia during inflammation. This small peptide produced in the liver, is induced by inflammatory cytokines and its over production results in macrophage iron retention and, consequently, less iron is available for erythropoiesis. This reticuloendothelial iron block cannot be overcome by oral iron administration, whereas intravenous iron may be effective for this purpose. [8, 9, 10, 11].
The patients were enrolled from all outpatient clinics and all Medical and Surgical Wards at the Jos University Teaching Hospital. 5mls of blood were collected from all participants in the morning (to account for the circadian rhythm of iron) to determine full blood count, serum iron, serum ferritin levels, and serum EPO levels (after an overnight fast). Haematological profile was determined with automated counters, while Serum iron was assayed using spectrophotometric method designed by TECO diagnostics, 1268 N. Lakeview Avenue. Anaheim, CA 92807. Ferritin was assayed by a direct sandwich ELISA method, using diagnostic kit of clinotec® and pharmaceuticals, Inc 2101-11871 Horseshoe way Richmond B.C. V7A 5H5 Canada.
Linear regression was performed, using the REGRESS command to test the relationship between variables. E.g. EPO levels and Hb levels. Chi square test was used to test significance of association of discrete variables. P values of < 0.05 was considered significant. All our procedures were in accordance with the ethical standards of the institutional committee on human experimentation and with the Helsinki Declaration of 1975, as revised in 1996.
Table 1 shows the demographic distribution of cases and controls by age (ranging from 1 – 80 yrs) and gender. There were 47 males (52.2%), 43 (41.8) females amoung cases, and 51 males (56.7%) and 39 females (43.3%) in controls.
Age group |
Male |
Female |
||
Case |
Control |
Case |
Control |
|
0 - 15 |
8 |
12 |
4 |
5 |
16 - 30 |
14 |
12 |
21 |
15 |
31 – 45 |
18 |
11 |
11 |
11 |
46 – 60 |
4 |
12 |
6 |
7 |
61 – 75 |
2 |
4 |
1 |
1 |
≥ 76 |
1 |
- |
- |
- |
Table 2: Diseases Diagnosed In the study group |
||
Diagnosis |
Frequency |
% |
TB |
25 |
27.8 |
Malignancies |
14 |
15.6 |
Osteoarthritis |
5 |
5.5 |
PID + UTI |
6 |
6.7 |
Rheumatoid arthritis |
6 |
6.7 |
AIDS |
10 |
11.1 |
AIDS + Cryptococcal meningitis |
5 |
5.5 |
TB/AIDS |
13 |
14.4 |
Hepatitis |
6 |
6.7 |
Parameter |
Control |
Case |
Reference value |
Test of statistical significance |
N=90 |
N=90 |
|||
Hb (g/dL) |
13.1± 0.91 |
9.16± 1.69 |
13.6 ± 4.9 |
P=0.000 |
PCV (L/L) |
0.42 ± 0.03 |
0.29 ± 0.05 |
0.42 ± 0.78 |
P=0.000 |
RBC count (x1012/L) |
5.0 ± 0.57 |
3.6 ± 0.8 |
4.5 ± 1.7 |
P=0.000 |
Retic index (%) |
1.16± 1.0 |
0.5±0.3 |
0.2 – 2.0 |
P=0.000 |
Platelets (x109/L) |
275.5±98.4 |
242.5±95.9 |
135.5±40.11 |
P=0.024 |
Iron (µmol/L) |
18.98±8.77 |
6.80±3.76 |
13-32 |
P=0.000 |
Ferritin (µg/L) |
157.9±165.6 |
436±242 |
20-300 |
P=0.000 |
EPO (mU/L) |
13.1±10.1 |
32.1±42.1 |
4.2±27.8 |
P=0.00 |
Based on the calculated O/P ratio (see appendix A), 30 (33.3%) of the 90 cases with anemia showed evidence of inappropriate or diminished response to EPO. TB was the commonest disease amongst patients with ACD followed by malignancy (table 2). This was also observed when considering table 5, with 28% (7) of the patients with TB exhibiting an inappropriate EPO response. Participants with a variety of malignancies had the second lowest EPO response (6), while those with TB and AIDS co-infection (5) had the third lowest response to EPO (See Table 4). [15, 16, 17, 18, 19, 20]
Disease |
O/P ratio<0.8 (inappropriate/diminished EPO response) |
O/P ratio>0.8 |
TB |
7 |
18 |
Malignancy |
6 |
8 |
TB+AIDS |
5 |
8 |
AIDS |
4 |
6 |
AIDS +Meningitis |
2 |
3 |
PID+UTI |
3 |
3 |
Osteoarthritis |
1 |
4 |
Rheumatoid arthritis |
1 |
5 |
Hepatitis |
1 |
5 |
Of all hematological parameters estimated, only serum ferritin appeared to be discriminatory between subjects with ACD, who had appropriate EPO response to anemia and those who had inappropriate EPO response (i.e. a diminished response) to their anemia. Mean serum ferritin levels for the appropriate responders, 395 ± 220μg/L was significantly lower than that for the inappropriate responders (516 ± 268.01 μg/L) P=0.025 (Table 5). On observation (although not of significant levels P=0.11) the inappropriate responders demonstrated lower Hb concentration 8.76 ± 1.77, than anemic but appropriate responders with a higher mean Hb concentration of 9.37 ± 1.63. (Table 5)
Parameters |
Inapp. Response N=30 |
App. Response N=60 |
P value |
||||||
O/P ratio <0.8 |
O/P ratio ≥0.8 |
|
|||||||
Mean |
SD |
Min. |
Max. |
Mean |
SD |
Min. |
Max |
||
Hb |
8.76 |
1.77 |
5.49 |
11 |
9.37 |
1.63 |
5.59 |
13.5 |
=0.111 |
PCV |
28.4 |
5.73 |
19 |
38 |
29.92 |
5.45 |
15 |
39 |
=0.225 |
MCV |
82.67 |
10.24 |
56 |
112 |
81.43 |
9.71 |
55 |
100 |
=0.579 |
MCH |
25.5 |
3.65 |
20 |
36 |
25.47 |
2.73 |
18 |
34 |
=0.961 |
Platelet |
220.1 |
80.32 |
98 |
375 |
253.73 |
101.63 |
112 |
545 |
=0.117 |
Serum Ferritin |
516.5 |
268.01 |
15 |
870 |
395.83 |
220.20 |
10 |
820 |
=0.025 |
WBC |
56.06 |
38.88 |
20 |
187 |
77.5 |
92.8 |
9 |
550 |
=0.229 |
The result of our study confirms the prevalence of anemia in the 125 participants with chronic disease as 72%, the prevalence of ACD at 65.5%, based on the criteria serum iron (<13mmol/L), ferritin (>20μg/L) and erythropoietin(>4.2U/L), and establishing a 33.3% prevalence of inappropriate EPO response to anaemia in these individuals. When compared to the different studies on the prevalence of ACD, ours’ was closeset to figures quoted by Opasich et al 57%73 and 52.32% by Cazzola. [9, 19, 20] Other figures were 6% by the American Family Physician Journal, 25% by Cash and Sears and 20% by Guralink et al. [1, 8, 18, 19, 20, 21, 22]
This study confirms the inverse relationship between Hb and serum EPO levels in healthy individuals and some of the cases. Serum ferritin levels were reported to be significantly high in our study population, with that of cases being twice as high as in the controls (Table 3), while the mean serum ferritin levels for the inappropriate responders was shown to be about 1.5 times higher than that for appropriate responders (Table 5). However serum ferritin levels are not a very reliable indicator for the diagnosis of ACD, because of the role proinflamatory cytokines (such as TNF α, INF γ, IL-1 and Hepcidin) play in Iron homeostasis, but in Sub- Saharan Africa, it may serve to increase the index of suspicion of ACD cases.
The study also demonstrates 33.3% (O/P ratio < 0.8) of cases as inappropriate responders to EPO. Serum EPO and SF levels were significantly higher in cases than controls and when comparisim was made between appropriate and Inappropriate responders, EPO levels although higher in the Inapproriate group did not show a corresponding and appropriate increase in thier Hb levels, therfore giving raise to the expression diminished or blunted response to EPO. This observations is of clinical importance, as a combination of intravenous iron and subcutaneous administration of recombinant human EPO, in selected cases has been proven to be of benefit as a rational theraputic approach to managing the anaemia in these patients. [9, 22, 23, 24, 25, 26, 27]
The predominant red blood cell morphological type observed was normocytic normochronic. 60% of our cases exhibited the expected normocytic normochronic picture, while the remaining 40% represented a spectrum of the other morphological subtypes. This further enforces the point that in as much as one subtype is termed predominant (i.e. normocytic normochromic); this does not invalidate the fact that other morphological types may be a form of presentation in ACD. The morphological picture of patients with ACD is also dependent on the length of the illness, severity and the presence of other co-morbid states. [3, 4, 7, 11, 13, 18, 20, 21, 22, 24, 25, 27] Figure 2 shows the Red cell morphology distribution of study group.
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