1 INTRODUCTION Chronic kidney disease (CKD) is increasing rapidly in the world as well as in Vietnam due to the rapid increase of kidney damage causes such as diabetes and hypertension Based on the changing of glomerular filtration rate, chronic kidney diseases are divided into five sequential and successive stages From stage to stage of chronic kidney disease (glomerular filtration rate < 60 ml/min) is considered as chronic renal failure When glomerular filtration rate < 15 ml / min, patients need kidney replacement therapy such as hemodialysis or kidney transplantation Anemia is common in patients with chronic kidney disease Anemia is often caused by a combination of three causes: dysfunction of hematopoietic organs, lack of hematopoietic materials and bleeding, in which lack of erythropoietin and iron deficiency are two important factors Iron deficiency oftens occurs in patients with renal failure (glomerular filtration rate < 60 ml/min) Patients with end stage chronic kidney disease often have high proportion of iron deficiency, but in hemodialysis patients, iron overload often occurs Both situations affects to the quality of anemia treatment in this group of patients As the recommendation of the National Kidney Foundation Disease Outcomes Quality Initiative (NKF-K/DOQI), to treat anemia effectively in patients with chronic kidney disease, besides providing erythropoietin, patients should be supplemented with amino acids, trace elements and iron in particular In patients with chronic kidney disease, it is necessary to determine the status of serum iron storage and its classification: iron deficiency, enough iron and iron overload This recommendation also mention that sufficient iron compensation should be based on ferritin and transferin saturation Transferrin saturation is calculated through serum iron and Total Iron Binding Capacity (TIBC) Thus, there are three most necessary quantities to assess iron deficiency status: iron, ferritin and TIBC In Vietnam, there have been many studies on the status of serum iron, ferritin and transferrin in patients with chronic renal failure, but there have not been many studies that mention to TIBC and the status of iron storage which is recommended by the K/DOQI in patients with chronic kidney disease who have not undergoing renal replacement therapy For the above reasons, we conducted the study "Study on the concentration of plasma iron, ferritin, and total iron binding capacity in chronic kidney disease patients without renal replacement" Objectives of the study The study was conducted in patients who were diagnosed with chronic kidney disease (glomerular filtration rate < 60 ml/min) and had not been treated with kidney replacement therapy in the Department of Nephrology and Hemodialysis, Military Hospital 103, with the following two objectives: - Describe the characteristics of anemia, the concentration of plasma iron, ferritin, TIBC and assess the status of iron storage according to the KDIGO in stage to chronic kidney disease patients without renal replacement - Determine the relationship between concentrations of plasma iron, ferritin, TIBC, the iron storage status with some characteristics of chronic kidney disease patients without renal replacement The urgency of the study Anemia is a common manifestation in CKD patients, especially in patients with GFR < 60 ml/min (renal failure) The mechanism of anemia in CKD patients involves Erythropoietin deficiency and lack of hematopoietic materials including iron Iron supplementation for anemia treatment in CKD patients requires a scientific basis and is a difficult problem for clinicians As the recommendation of the World and Vietnam Kidney Association, it is necessary to determine the status of iron storage before iron compensation Assessment of iron status should be based on serum ferritin concentration and transferin saturation, in which transferin saturation is calculated by serum iron and TIBC concentrations Thus, the study on the concentration of iron, ferritin and TIBC, thereby determining the status of iron storage in CKD patients who have not been treated by kidney replacement therapy, is a necessary study for clinical practice New contributions of the thesis This is the first study in Vietnam that quantifies serum TIBC concentration, that recommended by the Kidney Associations to use along with serum iron and ferritin concentrations to assess the iron storage status of CKD patients who have not been treated by kidney replacement therapy The results of the study confirmed that mean value of serum TIBC concentration in CKD patients was lower than that of normal control group The results also showed high proportion of patients with lack of iron storage There are some factors related to TIBC and iron storage reduction Based on this results, clinicians will have a strategy to compensate for iron, improve the effectiveness of anemia treatment for patients with chronic kidney disease Thesis structure: The thesis includes 116 pages Introduction: pages, literature review: 34 pages, subjects and methods: 16 pages, results: 30 pages, discussions: 31 pages, conclusions and recommendations: pages In the thesis, there are 43 tables, 14 charts, diagram, pictures The thesis has 136 references, including 21 Vietnamese and 115 English references Chapter 1: LITERATURE REVIEW 1.1 Anemia in patients with chronic kidney disease According to the World Health Organization, anemia is a condition that reduces circulating hemoglobin in peripheral blood below normal levels of people with the same sex, age and in the same living environment According to International Society of Nephrology, in patients with chronic kidney disease, anemia was diagnosed when Hb < 130g/l for men, Hb < 120g/l for women and Hb < 110g/l for pregnant women 1.2 Assessing the status of iron storages in patients with chronic kidney disease Clinically, there are many indicators assessing iron function of healthy people, CKD and kidney failure patients KDOQI and KDIGO recommend some commonly used indicators such as serum iron, ferritin, transferin, TIBC concentration and transferrin saturation (TSAT) To assess the status of iron storage, International Society of Nephrology recommends that we mostly based on ferritin and TSAT Calculation of TSAT according to the following formula: Plasma iron (µmol/l) x 100 TSAT (%) = -Plasma TIBC (µmol/l) + Absolute iron deficiency: - CKD patients who have not been treated by kidney replacement therapy: Plasma ferritin concentration < 100ng/ml and/or serum transferrin saturation level < 20% - Patients with hemodialysis: Plasma ferritin concentration < 200 ng/ml and/or serum transferrin saturation level < 20% + Functional iron deficiency: plasma ferritin concentration ≥ 200ng/ml and/or serum transferrin saturation level < 20% + Iron overload in CKD patients who have not been treated by kidney replacement therapy: serum ferritin concentration ≥ 500ng/ml and/or TSAT ≥ 50% 1.3 Studies on concentrations of serum iron, ferritin and TIBC in CKD patients + In the world, there had been many studies on the use of serum TIBC, iron and ferritin concentrations to assess iron storage status in patients with and without dialysis Numerous studies had evaluated the results of iron compensation in anemia treatment in patients with chronic kidney disease + In Vietnam: there were some studies on serum iron and ferritin levels, however, there had been no studies on TIBC and iron storage status in chronic renal failure patients who have not been treated by kidney replacement therapy Chapter 2: SUBJECTS AND METHODS 2.1 Subjects The study was conducted on 190 subjects who was divided into groups: - Study group: 124 patients with stage to chronic kidney disease, who have not been treated by kidney replacement therapy (GFR < 60 ml/min), at the Department of Nephrology and Hemodialysis, Military Hospital 103 - Normal control group: 66 healthy people - Research period: 01/2014 – 04/2018 + Inclusion criteria - Control group: healthy adults who not have kidney or urinary diseases, agree to participate in the study - Study group: Patients with stage to chronic kidney disease who have not been treated by kidney replacement therapy, age of 16 years old and above, agree to participate in the study + Exclusion criteria - Control group: Acute blood loss within the previous months or bleeding People who are pregnant or have just given birth within the last months - Study group: Blood transfusion during the previous months 5 Acute blood loss within the previous months or bleeding during the study period Patients have been taking iron or iron preparations Patients have an indication for emergency dialysis 2.2 Methods - A descriptive, cross-sectional, comparative case-control study - Sample size calculation: Percentage of patients with chronic renal disease with iron deficiency from 54.4% to 63.5% in previous studies Calculation of the sample size according to the following formula: (Z1-α/2)2 x p (1-p) n = D2 In which: Z = 1,96, with a reliability of 95% p = 0,544 (lowest value in previous studies) D = 0,1, the desired accuracy According to the calculation, the study must have at least 96 patients In the study, n = 124 patients were used 2.2.1 Study targets Patients admitted to hospital, were asked for medical conditions and were taken medical examination according to the research form The following criteria were collected: - Age, gender, history of disease - Measuring for blood pressure, height, weight and BMI calculation - Laboratory tests include: hematology, biochemistry (glucose, urea, creatinine, albumin, uric acid, four blood lipid indices, electrolytes, hsCRP ) - Quantitative determination of plasma iron, ferritin, and TIBC levels: venous blood was taken when patients were hungry in the early morning Plasma iron and ferritin concentration were quantified on Cobas 6000 system, which using the kit of Roche company, in Department of Biochemistry, Military Hospital 103 Serum TIBC concentration were quantified by ELISA method at Department of Pathophysiology, Vietnam Military Medical University for both study and control groups - Calculating glomerular filtration rate under the guidance of NKFK/DOQI using the MDRD formula which was based on creatinine, age, gender and race - Calculation of transferrin saturation by plasma iron and TIBC concentrations - The iron storage status was divided base on the recommendation of KDIGO and KDOQI throughout the ferritin and TSAT indices - The diagnostic, classification and evaluation standards were based on recommendations of national and international specialized associations - Evaluation of increasing or decreasing plasma iron, ferritin and TIBC levels were based on control group results 2.2.2 Data processing - The data was processed according to the medical statistical method using SPSS software program version16.0 - The algorithms were used include: multivariate logistic regression analysis, calculating mean values, standard deviations, percentages, comparing two average values and percentage by t-test, compare ratios by Chi-square test, comparing mean values by Anova test, calculating the correlation coefficient (r) 2.2.3 Ethics in research - The study did not violate ethics in medicine, serving for screening for chronic kidney disease patients - The study was approved by the Department of Heart - Kidney - Joints - Endocrinology, Vietnam Military Medical University before implementation - TIBC test fee was payed by myself Chapter 3: RESULTS 3.1 General characteristics of subjects - Age and gender characteristics: + Control group included 66 people with average age of 41.86 ± 5.68 years, men accounted for 69.7% and women accounted for 30.3% + The research group included 124 patients whose average age was 52.65 ± 17.95 years, men accounted for 72.6% and women accounted 27.4% - The cause of CKD: Chronic glomerulonephritis accounted for 49.2%, chronic renal pyelonephritis accounted for 16.9%, hypertension accounted for 15.3%, diabetes accounted for 12.9 %, the lowest due to polycystic kidneys and Gout which accounted for only 3.2% and 2.4% respectively - The proportion of stage CKD patients accounted for 79.8%, stage and accounted for 20.2% The average glomerular filtration rate was 8.3 ml/min 7 - Up to 89.5% of patients had hypertension in the study, only 10.5% of patients did not have hypertension - The group of patients with normal BMI accounted for the highest rate (70.2%), the proportion of overweight and obesity accounted for only 15.3% and underweight accounted for 14.5% The average value of BMI was 20.05 3.2 Characteristics of anemia, concentrations of plasma iron, ferritin, TIBC and iron storage status following to KDIGO in patients with chronic kidney disease 3.2.1 Characteristic of anemia in study group Table 3.1 The percentage of patients on the severity of anemia (n = 124) Severity of anemia Number Percentage No anemia 05 4.0 Anemia 119 96.0 Mild 34 27.4 Moderate 43 34.7 Severe 42 33.9 Average Hemoglobin (g/l) 91.13 ± 22.01 - The proportion of anemia was 96.0%, the average Hb concentration was 91.13 g/l - Mild, moderate and severe anemia accounted for 27.4%, 34.7% and 33.9% respectively Table 3.2 Percentage of patients according to the size of red blood cells (n=119) Erythrocyte size Number Percentage Microcytic 12 10.1 Normocytic 107 89.9 Macrocytic 0 - In group of anemia patients, there were 10.1% of patients with microcytic anemia and 89.9% of patients with normocytic anemia Table 3.3 Percentage of patients according to the amount of hemoglobin in each cell (n=119) Anemia classification Number Percentage Hypochromic anemia 42 35.3 Normochromic anemia 74 62.2 Hyperchromic anemia 2.5 - Among patients with anemia, hypochromic anemia accounted for 35.3%, this proportion of hyperchromic anemia was 2.5% 8 - The highest proportion was normochromic anemia with 62.2% 3.2.2 The concentrations of plasma iron, ferritin and TIBC in the study subjects Table 3.4 Comparison of concentrations of plasma iron, ferritin and TIBC between study group and control group Control group Study group Indices p (n=66) (n=124) Iron* Median 15.81 10.7 < 0.001 (µmol/l) (IQR) (11.56 – 19.26) (6.62 – 15.25) Min 7.3 2.1 Max 31.4 41.6 Ferritin* 198.45 403.73 Median (ng/ml) (68.05 – (211.36 < 0.001 (IQR) 255.22) 548.42) Min 16.5 31 Max 383.4 1070.1 TIBC* Mean 67.58 ± 11.58 50.64 ± 19.79 < 0.001 (µmol/l) Min 46.12 17.5 Max 90.63 98.21 * Approximate normal range by control group: Iron (Percentiles 2.5% 97.5%): 7.31 - 29.8 µmol/l; Ferritin (Percentiles 2.5% - 97.5%): 16.77 - 375.16 ng/ml; TIBC: Mean ± 1.96xSD: 44.89 - 90.27 µmol/l - Plasma iron and TIBC concentrations in study group were significant lower than in control group, p < 0.001 - In contrast, plasma ferritin concentration in study group was sighnificant higher than in control group, p < 0.001 Table 3.5 The proportion of patients with increasing or decreasing concentrations of plasma iron, ferritin and TIBC compared to control group (n=124) Indices Number Percentage Decrease 36 29 Iron (µmol/l) Normal 84 67.7 Increase 3.2 Decrease 0 Ferritin (ng/mL) Normal 56 45.2 Increase 68 54.8 TIBC (µmol/L) Decrease 59 47.6 Normal 63 50.8 Increase 1.6 - The proportion of patients with normal concentration of plasma iron was 67.7% Up to 29% of patients had decreasing and 3.2% of patients increasing plasma iron concentration compared to control group - Meanwhile, 54.8% of patients increased plasma ferritin concentration compared to control group - The proportion of patients with decreasing TIBC concentration was 47.6% There was only 1.6% of patients who had increasing TIBC concentration 3.2.3 Assessment of serum iron storage status according to KDIGO guideline in the study group Table 3.6 Characteristics of transferin saturation in study group (n=124) Both gender Female Male (n=90) (n=124) (n=34) Characteristics P n % n % n % Low 38 30.6 26 28.9 12 35.3 Normal 76 61.3 55 61.1 21 61.8 > 0.05 High 10 8.1 10 2.9 22.31 22.3 22.02 Median (IQR) (17.77 – (18.5 – (13.29 – > 0.05 33.5) 34.47) 26.68) - The proportion of patients with low TSAT (< 20%) was 30.6% There was 8.1% of patients with high TSAT (> 50%), while normal TSAT accounted for a major proportion (61.3%) - There was no difference of TSAT characteristics between men and women - The median value of TSAT was 22.31% Table 3.7 Characteristics of iron storage status in study group according to KDIGO guideline (n=124) Iron storage status * Number Percentage Iron deficiency 46 37.1 Enough iron 30 24.2 Iron overload 48 38.7 * Iron deficiency: Ferritin < 100 and/or TSAT < 20%; Iron overload: Ferritin > 500 and/or TSAT > 50% - Based on the recommendation of KDIGO for patients with nondialysis chronic kidney disease, the proportion of patients with iron 10 deficiency in the study was 37.1%, however, there were also 38.7% of patients with iron overload 3.3 The relationship between concentrations of plasma iron, ferritin, TIBC, iron storage status and some characteristics of patients with chronic kidney disease 3.3.1 The relation to the stage of chronic kidney disease Bảng 3.8 Comparison of mean values of plasma iron, ferritin and TIBC concentrations between stages of chronic kidney disease (n=124) TIBC Ferritin Stages of Iron (µmol/L) (µmol/L) (ng/mL) CKD Median Median X ± SD 10.16 (7 – 252.6 (177.89 3+4 (n = 25) 68.44 ± 16.36 14.58) – 437) 10.7 (6.6 – 435.5 (250.49 (n = 99) 46.15 ± 18.02 15.6) – 557.68) p > 0.05 < 0.001 < 0.05 - Plasma iron concentration was not related to the stages of chronic kidney disease with p > 0.05 - However, plasma ferritin concentration in patients with stage chronic kidney disease was significant higher than that of patients in stage and (p < 0.05) - In contrast, the concentration of TIBC in patients with stage chronic kidney disease was lower than that of patients in stage and (p < 0.001) Table 3.9 The proportion of patients with increasing or decreasing concentration of plasma iron, ferritin and TIBC between stages of chronic kidney disease (n=124) Stage + Stage Indices p (n = 25) (n = 99) Decrease (28) 29 (29.3) (n, %) Iron > 0.05 (mmol/l) Normal 18 (72) 66 (66.7) Increase (0) (4) Decrease (0) (0) Ferritin Normal 17 (68) 39 (39.4) < 0.05 (ng/mL) Increase (32) 60 (60.6) Decrease (12) 56 (56.6) < 0.001 11 Normal 22 (88) 42 (41.4) TIBC (µmol/L) Increase (0) (2) - There was no difference in the proportion of patients with with increasing or decreasing concentration of plasma iron concentration between stages of chronic kidney disease - In contrast, the proportion of increasing plasma ferritin concentration in stage chronic kidney disease patients was significant higher than that of patients in stage and 4, p < 0.05 - The proportion of decreasing TIBC concentration in patients with stage chronic renal disease was significant higher than that of patients in stage and 4, p < 0.001 Table 3.10 The relationship between iron storage status and stages of chronic kidney disease in study group (n=124) Stages of Iron deficiency Enough iron Iron overload CKD (n, %) (n, %) (n, %) +4 (n=25) 15 (60) (20) (20) (n=99) 31 (31.3) 25 (25.3) 43 (43.4) p < 0.05 - The proportion of iron deficiency in stage and CKD patients was sighnificant higher than that of stage CKD patients In contrast, the proportion of iron overload in stage CKD patients was significant higher than that of stage and (p 0.05 Ferritin (ng/ml) -0.134 > 0.05 TIBC= 0.168*Hemoglobin + TIBC (µmol/L) 0.208 < 0.05 32.474 - Plasma iron and TIBC concentration had a weak positive correlation while plasma ferritin concentration had a weak negative correlation with hemoglobin concentration (p 0.05 - Plasma iron concentration in patients with decreasing albumin concentration was significantly lower than that of patients with normal albumin concentration (p 0.05 Table 3.13 The relationship between concentrations of plasma iron, ferritin, TIBC and CRP concentration in study group (n=112) TIBC Ferritin CRP Iron (µmol/L) (µmol/L) (ng/mL) concentration (Median) (Median) ( X ± SD) 431.25 Increase > mg/l 6.87 (231.5 – 46.52 ± 19.73 (n=48) (4.35 – 12.34) 567.73) 385.2 No increase 11.9 (193.1 – 54.89 ± 19.45 (n=64) (8.8 – 18.6) 544.61) p < 0.001 > 0.05 < 0.05 - In the group of patients with increasing hs-CRP concentration, plasma iron and TIBC concentrations were significant lower than that of nonincreasing group (p < 0.05) - In contrast, plasma ferritin concentration in patients with increasing hs-CRP were not significant difference with non-increasing hs-CRP group (p > 0.05) 3.3.4 The plasma TIBC in evaluating iron storage in study group Albumin concentration Iron (µmol/L) (Median) 13 - Area under the curve AUC = 0.755; p < 0.001 Cut-off value= 48.03 Sensitivity Sp = 73.9% Specificity Se = 69.2% Chart 3.1 The ROC curve for iron storage deficiency diagnosis of plasma TIBC concentration Comment: At the cut-off value of 48.03µmol/L, plasma TIBC concentration had a diagnostic value for iron deficiency The higher TIBC concentration, the more valuable of diagnostic value (p 0.05 Female 0.595 0.217 – 1.630 > 0.05 Albumin< 35 g/l 1.411 0.522 – 3.814 > 0.05 CRP > 1.718 0.65 – 4.539 > 0.05 BMI < 18.5 0.439 0.116 – 1.654 > 0.05 14 First diagnosis GFR 0.05 < 0.001 1,287 0,284 – 5,826 > 0,05 Multivariate logistic regression model: Log ( ) = -2.625 + 0.327* Age of 60 and above - 0.519* Female + 0.344* Decreasing Albumin + 0.541* Increasing CRP - 0.823* BMI lower than 18.5 - 0.185* First diagnosis + 2.963* GFR below 15ml/min + 0.253* Ferritin lower than 100ng/ml - GFR 0.05 Albumin< 35 g/l 0.489 0.155 – 1.54 > 0.05 CRP > 2.579 0.805 – 8.267 > 0.05 BMI < 18.5 0.578 0.126 – 2.655 > 0.05 First diagnosis 12.177 3.407 – 43.516 < 0.001 GFR 0.05 TIBC > 48.03 9.612 2.974 – 31.073 < 0.001 µmol/L Multivariate logistic regression model: Log ( ) = -2.568 - 0.04* Age of 60 and above + 0.711* Female - 0.715* Decreasing Albumin + 0.948* Increasing CRP - 0.547* BMI lower than 18.5 + 2.5* First diagnosis - 1.041* GFR lower than 15ml/min + 2.263* TIBC higher than 48.03 - First diagnosis and increasing plasma TIBC concentrations were independent risk factors of iron deficiency (p 48.03 µmol/L were independent risk factors for iron deficiency (p < 0.01) Anemia in CKD patients is usually normochromic anemia, but the iron deficiency status was increased gradually along with the reduction of GFR, despite of increasing ferritin concentration Thus, first diagnosed CKD patients had a very high possibility of iron deficiency This results had a clinical significance in the diagnosis of iron storage deficiency and iron supplementation should be given to 22 patients when the patient is diagnosed with low-GFR CKD from the first time regardless of serum iron quantification LIMITATION OF THE STUDY Although the study had achieved the given objectives, however, the proportion of stage and CKD patients was quite small, so the change of plasma iron, ferritin, TIBC concentration and iron storage status in this group of patients has not been clear CONCLUSION Study on plasma iron, ferritin and TIBC concentrations in 124 nondialysis CKD patients and compare with 66 healthy people, we draw some conclusions: Characteristics of anemia, concentrations of plasma iron, ferritin, TIBC and iron storage status in study group * Characteristics of anemia + The proportion of anemia in study group accounted for 96%, including 33.9% of severe anemia, 34.7% of moderate anemia and 27.4% of mild anemia The mean value of hemoglobin concentration was 91.13 g/l + Characteristics of anemia: 10.1% of microcytic anemia and 35.3% of hypochromic anemia * Concentrations of plasma iron, ferritin and TIBC in patients with stage to chronic kidney disease + In study group, the mean value of plasma iron and TIBC concentration was lower, while the mean value of ferritin concentration was higher than that of control group (p