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Study the serum erythropoietin, ferritin and transferrin concentration in patients with end stage renal disease undergoing hemodialysis

THE MINISTRY OF

THE MINISRY OF DEFENCE

EDUCATION AND TRAINING
MILITARY MEDICAL UNIVERSITY

PHAN THE CUONG

STUDY THE CONCENTRATION OF SERUM
ERYTHROPOIETIN, FERRITIN AND
TRANSFERRIN IN PATIENTS WITH END-STAGE
RENAL DISEASE UNDERGOING HEMODIALYSIS

Speciality : Nephro-Urology
Code : 62.72.01.46

MD-PhD. THESIS SUMMARY

HA NOI - 2016



The thesis was completed at:
MILITARY MEDICAL UNIVERSITY

Supervisors:

1. Prof. Dr. Nguyen Anh Tri
2. A. Prof. Dr. Hoang Trung Vinh

1st reviewer:

2nd reviewer

3rd reviewer:

The thesis will be upheld before the University Grade Thesis
Examination Board
At:

hour

on day month year 20161

The thesis can be found at:

1. National library
2. Military medical university’s library


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FOREWORDS
1. The necessity of the topic
Anemia is common in chronic kidney disease (CKD) patients
especially in the end-stage and increases mortality, cardiovascular
complications and reduces quality of life. Main cause of anemia in these
patients is the decrease in the excretion of erythropoietin (EPO) in the
kidneys. The invention of recombinant human erythropoietin (Rhu EPO) has opened a turning-point in the treatment of anemia for CKD
patients. However, the treatment of anemia for them especially dialysis
patients so far remains challenging for clinicians because of expensive
treatment costs and there are many factors that reduce the response to

Rhu-EPO treatment. Iron deficiency increases the severity of anemia
and is one of the main causes of reduced effectiveness of Rhu-EPO
treatment in CKD patients. In fact, the results of the Rhu-EPO are
different in various objects, including some cases even without using
Rhu-EPO or treated with low doses still achieved target Hb. This
phenomenon may be related to the preservation of EPO secretion
function and regulatory mechanisms in CKD patients. Furthermore, the
adequate intravenous iron supplement significantly affects the
efficiency of the treatment of anemia. Therefore, the study of the extent
and the factors related to the decreased excretion of EPO and iron
indices in CKD patients is very necessary, providing a scientific basis
for determining the time, the most suitable dose of Rhu-EPO and
intravenous iron to achieve high treatment efficiency and reduce the
costs of treatment. For this reason, we conducted this study with two
following objectives:
1. To investigate erythropoietin level, serum ferritin and transferrin
saturation (TSAT) in patients with end-stage renal disease (ESRD)
indicated chronic hemodialysis.
2 To assess the change of concentration of erythropoietin , ferritin and
TSAT in patients with end-stage renal disease after the first 3 months of
chronic hemodialysis combined with some other treatment .
2. New contributions of the topic
- Quantitatively determine the average serum erythropoietin levels in
patients with end-stage renal disease indicated chronic hemodialysis.


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and find out the difference of these values compared to normal people
and anemia patients without renal failure. Create equation calculating
estimated erythropoietin levels from a linear correlation between
erythropoietin and hemoglobin levels in anemia patients without renal
failure from which to assess the EPO response in anemia patients with
ESRD.
- Evaluate the results of iron deficiency treatment with bolus dose, iron
deficiency prevention with maintenance dose as well as variations in the
iron status of patients without iron supplementation through serum
ferritin and TSAT in patients after the first 3 months of chronic
hemodialysis.
3. Structure of the thesis: The thesis includes 121 pages. Forewords:
2 pages; Overview: 30 pages; Subjects and methods: 20 pages; Results:
32 pages; Discussion: 33 pages; Conclusions and recommendations: 3
pages. The thesis contains 41 tables, 18 charts, 4 figures. It reffered 146
references including 18 Vietnamese and 128 English.
Chapter 1. OVERVIEW
1.1. Erythropoietin levels in anemia patients with and without
chronic renal failure
1.1.1. The role and regulatory mechanism of erythropoietin
secretion
* The role of erythropoietin in the process of red blood cell
production in the bone marrow
Erythropoietin is a glycoprotein with a molecular weight 30,4kDa and
fold into a compact spherical structure consisting of four α helix bundle.
EPO stimulates red blood stem cells (Burst forrming units erythroid BFU – E, erythroid colony forming units - CFU - E) to produce,
develop morphological structure to return into proerythroblast,
erythroblast and reticulocyte .
* Regulatory mechanism of erythropoietin secretion
In adults, EPO is primarily produced in the justaglomerular cells
(90%), only a small amount (10 %) are produced in pericapillary cells
of the liver interstitial. The decrease of blood oxygen levels due to
anemia or arterial oxygen pressure (who resides in a high altitude ) will
stimulate renal EPO synthesis and secretion into the bloodstream.


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1.1.2. Change in serum EPO levels of anemic patients without renal
failure
When patients with intact renal function suffer from anemia due to
any other cause, such as decreased production of red blood cells in the
bone marrow (Myelodysplasic syndromes or lack of material to produce
red blood cells), bleeding or hemolysis, the oxygen in tissues reduced,
stimulating EPO synthesis in the kidneys and secretion into the
bloodstream to increase red blood cell production in the bone marrow.
In this case, levels of EPO increase by up to tens or even hundreds of
times higher than normal. The studies showed that serum EPO levels
correlate closely and conversely with Hb levels but inversely and closely
correlated with the production of red blood stem cells (CFU - E).
1.1.3. Change in serum EPO levels of patients with ESRD
Initially, it is expected that serum EPO levels in anemic patients with
ESRD were lower than normal. In fact , serum EPO levels in most
patients with ESRD are equal to or higher than normal and this led
scientists to assume the presence of bone marrow inhibitors in patients
with ESRD. This also showed that the reductions of EPO response in
patients with ESRD can not be assessed through testing serum EPO
levels. When the glomerular filtration rate falls below 40ml / min, there
is no linear correlation between serum EPO and Hb levels or Hct. The
observations then showed that serum EPO levels in anemic patients
without kidney failure many times higher than those in ESRD patients
with the same degree of anemia. This is the basis for the scientists to
demonstrate the decrease of EPO secretion in patients with ESRD.
When glomerular filtration rate decreases, the EPO response will
decrease. However , there was no linear correlation between serum EPO
with glomerular filtration rate or serum creatinine. To assess the EPO
response in anemic patients with ESRD, their serum EPO levels are
compared with estimated serum EPO levels based on EPO response in
chronic anemic patients without renal failur. The EPO response in CKD
patients decreases with degree of renal failure, as CKD progressed to
stage 3 , 4 and 5 , the EPO response also decreased respectively: 15 %;
4 % and 3 %. The decrease in the EPO response in CKD patients is the
real cause of anemia when the glomerular filtration rate < 30ml/ min.


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1.2. The changes of serum ferritin and TSAT in patients with ESRD
1.2.1. Features of iron metabolism in patients with ESRD
In normal individuals, the daily cycle of iron metabolism of the body
almost formed "closed recycling". Transferrin obtain iron mainly from
reticuloendothelial system, where macrophages destruct "old" RBC and
release iron into the blood. Then, transferrin carries iron to the bone
marrow to produce new red blood cells. Only a small amount of iron
(about 1-2 mg) daily loss and is compensated by the absorption of iron
in the gastrointestinal tract. In hemodialysis patients, daily cycle of iron
metabolism has no longer feature "closed recycling". The characteristics
of the change in iron metabolism in hemodialysis patients include:
- Increased amount of supplemented intravenous iron.
- Increased the amount of iron transported to the bone marrow due to
Rhu-EPO treatment.
- Increased iron liberation from the destroyed red blood cells into
blood due to decreased erythrocyte life by about a third compared with
normal people.
- Increased iron loss due to blood loss (blood samples taken
periodically, blood loss during dialysis, bleeding from fistula needle
positions after the hemodialysis sessions, gastrointestinal bleeding ... ).
- Inflammation, malnutrition in hemodialysis patients increase the rate
of functional iron deficiency.
1.2.2. Evaluate iron status in patients with ESRD
* Diagnosis of iron deficiency
Iron deficiency includes 2 types: “absolute iron deficiency"when iron
stores are not enough to supply the bone marrow with iron to produce
red blood cells and “functional iron deficiency"when the iron stores in
the body are not lacking, but iron is not transported enough to the bone
marrow to produce red blood cells. International Society of Nephrology
recommends testing ferritin and TSAT levels for monitoring and
diagnosis of iron deficiency .
+ Absolute iron deficiency:
- Non-dialysed CKD patients: serum ferritin < 100ng/ml and or
TSAT < 20%.
- Hemodialysed patients: serum ferritin < 200ng/ml and or TSAT < 20%.
+ Functional iron deficiency: serum ferritin ≥ 200ng/ml and TSAT < 20%.


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* Diagnosis of iron overload in patients with ESRD
The applied diagnostic criteria:
- Blood tests : TSAT ≥ 50 % and or serum ferritin ≥ 500ng/ml
(caution is advised when treating intravenous iron
supplementation )
- Liver biopsy : Liver iron concentration - LIC > 7 mg/g dry
weight liver.
1.3. Studies of erythropoietin levels and iron status in CKD patients
1.3.1. Research world-wide
* Studies of EPO levels in CKD patients
Most of the research was to evaluate the role of reduced EPO
excretion leading to anemia in CKD patients or to assess the decreased
EPO secretion in various stages of CKD in relation to the degree of
anemia.
Early studies only showed that serum EPO to be equal or higher
than healthy people (Radtke H.W. et al. (1979), McGonigle R.J. et al.
(1984), Fukushima Y. Et al. (1986). From these results, the authors
concluded that in CKD patients and even hemodialysis patients EPO
excretory function of the kidneys still remained. Then, Seguchi C. et al
(1992) found that, in anemia patients with CKD serum EPO is much
lower than that in anemic patients without renal failure having the same
degree of anemia. Fehr T. et al (2004) showed that glomerular filtration
rate the more decreased the inverse correlation between the serum EPO
and Hb levels the more decreased and when the glomerular filtration
rate falls below 40 ml/min, this correlation did not exist as in anemic
patients without renal failure, this negative correlation is very strong.
Artunc F. et al (2007) conducted a study comparing serum EPO levels
in CKD patients with estimated serum EPO calculated from the linear
regression equation in anemic patients without renal failure and found
that the EPO response in patients with CKD stages 3,4 and 5 decreased
gradually 15%; 4% and 3% respectively.
Differences in the treatment of anemia, particularly in hemodialysis
patients also make researchers more interested in understanding and


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assessment of EPO secretion function and its regulatory mechanism in
circumstance of hypoxia due to anemia. A. Kato et al. have found that
when Hb levels decreased from 8,9 g /dl to 5,8 g/dl in 11 hemodialysis
patients, the serum EPO increased 52.2 times higher than normal. This
shows that the ability of EPO secretion was partly preserved in patients
with ESRD. Ifudu O. et al studied the changes of EPO lavels in
hemodialysis patients treated with Rhu-EPO. After a 6-week dialysis
dose increase, hematocrit levels increased and EPO levels decreased
from 9,1 mU/ ml to 6,1 mU/ ml. The author commented: negative
feedback phenomenon regulating EPO secretion still remained in
dialysis patients.
1.3.1.2. Studies of serum ferritin and TSAT in CKD patients
The research on iron status in patients can be divided into several
groups such as research evaluating iron status, studies assessing the
effectiveness of treatment of iron deficiency and iron maintenance therapy,
… The typical multinational studies evaluating iron status may be listed
such as studies NHANES (National Health and Nutrition Examination
Survey) of Hsu C. Y. et al conducted in the US (2002), ESAM 2003
(European Survey on Anemia Management 2003) carried out by Jacob C.
et al in Europe (2005) and GSAM in 2005 (The Gulf Survey on Anemia
Management) conducted by Alsuwaida A. et al in the Arab countries (2007)
... These studies have shown a panorama of anemia, iron deficiency and the
related factors in CKD patients.
The studies of effectiveness of treatment of iron deficiency in
hemodialysis patients showed that the majority of patients achieve
adequate iron with intravenous iron loading dose (Van Wyck D.B. et al
(1989), Sunder -Plassmann G. et al (1995), Park L. et al (1998), ...
Maintenance dose 100-200 mg/month can prevent iron deficiency and
improve the effectiveness of treatment of anemia in CKD patients
(MacDougall I.C. et al (1996), Kosch M. et al (2001), Harmankaya
O.va et al (2002), ...


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1.3.2. Studies in Vietnam
Until now, in Vietnam no research of serum EPO levels in CKD
patients published. Studies on serum iron levels, ferritin and TSAT in
non-hemodialysis and hemodialysis patients were conducted by a lot of
authors during about recent 10 years such as Hoang Thi Oanh (2004),
Tran Thi Thuan (2010), Ngo Quan Vu (2011), Nguyen Thi Huong
(2006) and Nguyen Van Hung et al. (2011). Most of the studies
assessed the relationship between iron indicies and clinical, laboratory
parameters. There were some follow-up studies, but they did not show
the change of the iron indicies in subjects treated with intravenous iron
with different treatment regimes as well as did not evaluate the
effectiveness of iron deficiency therapy or prevention of iron deficiency
iron deficiency when combined Rhu-EPO treatment in hemodialysis
patients who have high risk of iron loss.
Chapter 2. SUBJECTS AND METHODS
2.1. Subjects
We studied 222 subjects and divided into 3 groups:
- Study group: 105 patients with ESRD indicated hemodialysis.
Patients were studied at the beginning and after first 3 months of
hemodialysis treatment combined other treatment measures.
- Anemia control group: 51 anemic patients without renal failure
suffering from Myelodysplasic syndromes - Refractory anemia treated at
National Hematology and Blood Transfusion Institute from 10/2008 to
12/2011.
- Healthy control group: 66 staff members of Hemodialysis
Department of Bachmai Hospital and the Bank for Agriculture and Rural
Development.
2.1.1. Selection criteria
* Healthy control group
- 66 healthy people including both genders
- Age ≥ 18. The age and gender distribution is equivalent to the
study group.


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- The test of blood count, serum urea and creatinine are within
normal limits.
- Voluntarily participated in the research.
* Anemia control group
- 51 anemic patients without renal failure suffering from
Myelodysplasic syndromes - Refractory anemia. Age ≥ 18, the age and
gender distribution is equivalent to the study group.
- Serum urea and creatinine are within normal limits.
- Voluntarily participated in the research.
* Study group
- Patients with ESRD starting hemodialysis trearment.
- Age ≥ 18.
- The causes of renal failure: chronic glomerulonephritis, chronic
pyelonephritis, hypertention, diabetes melitus, lupus nephritis.
- Voluntarily participated in the research.
- Patients were followed-up for the first 3 months of hemodialysis
with the coordinated measures: dialysed under a unified regime, use of
Rhu-EPO, iron supplementation as recommended by K/DOQI, antihypertensive medications and symptomatic treatment.
2.1.2. Exclusion criteria
* Healthy control group and anemia control group
- Had blood transfusions, acute blood loss, surgical interventions
or invasive procedures in the previous 3 months.
- Who is pregnant or had given birth within the previous 6 months.
- Suffer from polycystic kidneys and liver, chronic hepatitis,
cirrhosis, chronic cardio-pulmonary disease, peptic ulcer, acute or
chronic colitis, malignant blood diseases or cancer, cognitive disorders
or mental disorders, HIV infection.
- Refuse to participate in research.
* Study group
- Had blood transfusions, iron intravenous supplementaion, acute
blood loss, surgical interventions or invasive procedures in the previous
3 months.


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- Suffer from polycystic kidneys and liver, chronic hepatitis,
cirrhosis, chronic cardio-pulmonary disease, peptic ulcer, acute or
chronic colitis, malignant blood diseases or cancer, cognitive disorders
or mental disorders, HIV infection.
- Who is pregnant berore and during the study or had given birth
within the previous 6 months.
- Suffer from acute diseases within 3 months before or during the
study, such as myocardial infarction, acute heart failure, brain stroke ...,
vascular access does not guarantee the blood flow ≥200 ml/min, medical
emergency conditions indicated acute dialysis during the study due to acute
pulmonary edema, hyperkalemia, ..., cognitive disorders or mental
disorders.
- Refuse to participate in research, death or transfer to another
centers during the study.
2.2. Methods
Study design: this is a cross-sectional descriptive study compared with
healthy controls, prospective, longitudinal combined intervention study
group.
2.2.1. Research content
2.2.1.1. Healthy control group
Subjects must pass health examination included the following
contents: ask about medical history; clinical examination; laboratory
tests (blood count, blood chemistry). After being selected and included
in the study, subjects were tested for serum EPO by method
chemiluminescent enzyme-labeled Immuno-metric assay on the
automated system of Immulie 2000 with chemicals of Siemens Corp. in
the Department of Biochemistry, National Institute of Hematology and
Blood Transfusion.
2.2.1.2. Anemia control group
- Patients were diagnosed with anemia due to Myelodysplasic
syndromes - Refractory anemia and treated at the National Institute of
Hematology and Blood Transfusion.


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- The patients were asked about medical history; clinical examination;
laboratory tests (blood count, blood chemistry). After being selected and
included in the study, subjects were tested for serum EPO.
2.2.2.3. Study group
Research content included following steps:
+ Step 1: Ask about history of some diseases such as kidney and
urinary diseases, blood diseases and chronic diseases (chronic hepatitis,
tuberculosis, chronic cardio-pulmonary disease...), history of alcoholism
and drug addiction; history of blood loss, transfusion and Rhu-EPO
injection, the treatment of anemia, iron supplementation.
+ Step 2: Physical examinations include genaral condition
assessment, cardiovascular, respiratory, digestive, musculoskeletal,
urology, hematology systems, vascular access, nutritional status, acute
infections, skin diseases, thyroid, peripheral lymph nodes, spleen, ...,
signs or symptoms of acute blood loss such as black stool, blood
vomiting, coughing up blood, nose bleeding, hematuria, ...
+ Step 3: Establish medical records according to unified form
+ Step 4: Laboratory tests
- The blood samples of each patient study were taken at the same
time on the same day to test blood count, blood chemistry. In case of
resampling regain blood at the same time of the day (8 am), but not
more than 3 days after the previous test samples.
- For EPO tests, serum ferritin, transferrin blood samples were
taken after injection of intravenous iron with dose of 100 mg and RhuEPO injection for at least 7 days.
+ Step 5: Treatment
- Hemodialysis treatment: 3,5 hour x3 times/ week; polysulfone
membrane, surface area 1,3 m2; Kuf: 13 ml/mmHg/hour; Dialysate
bicarbonate, Dialysate flow: 500 ml/min; Anticoagulation: heparrin
standard; Blood flow ≥ 200 ml/min; Ultrafiltration ≤ 5% dry weight.
- Intravenous iron therapy
o Bolus dose:


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Indication: serum ferritin < 200 ng/ml or 200ng/ml ≤ serum ferritin
< 500 ng/ml and TSAT < 20%.
Dose: 1000 mg iron sucrose (Venofer) divided into 10 injections in
10 consecutive dialysis sessions then maintained: 200 mg iron sucrose/
month, divided into 2 equally spaced injections.
o Maintenance dose:
Indication: 200 ng/ml ≤ serum ferritin < 500 ng/ml and TSAT ≥
20% or serum ferritin > 500ng/ml and TSAT < 20%.
Dose: 200 mg iron sucrose/month, divided into 2 equally spaced
injections.
o No treatment: Serum ferritin ≥ 500 ng/ml and TSAT ≥ 20%.
- rHu-EPO: Neo-recormon 2000UI x 3 syringes/week;
subcutaneously (intravenously if there are adverse events) after each
dialysis session. Stop injecting when anaphylaxis happened; Increased
dose by 25% every 4 weeks if Hb level did not increase by 10-20 g/l /
month or reduced dose by 25% while Hb level increased more than 1020 g/l / month or achieved target Hb (100-120 g/l).
+ Step 6: Evaluation after 3 months of treatment
- RBC, hemoglobin, hematocrit.
- Ure, creatinin, protein, albumin, hs-CRP, serum iron, serum
ferritin, transferrin, EPO level.
2.2.2. The diagnostic criteria and classifications, formulas used in
the study
- Criteria for diagnosis of stages of CKD according to the NKF /
KDOQI 2003.
- Evaluate iron status according to American Society of Nephrology
Table 2.2. Criteria for assessing the serum ferritin and TSAT
Level
Serum ferritin (ng/ml)
TSAT (%)
Low
<200
<20
Normal
200 – 499,9
20 – 49,9
High
≥500
≥50
o Absolute iron deficiency: serum ferritin < 200 ng/ml


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o Functional iron deficiency: serum ferritin ≥ 200 ng/ml and TSAT < 20%.
o Enough iron: serum ferritin 200-499,9 ng/ml and TSAT 20%-49,9%.
o Iron overload: serum ferritin ≥ 500 ng/ml and (or) TSAT ≥ 50%.
- Diagnosis and division level of anemia according to WHO
- Grading of malnutrition in dialysis patients (Dialysis malnutrition
Score-DMS) according to Kalantar-Zadeh K.
- Diagnose myelodysplasic syndromes - refractory anemia
according to WHO.
- Evaluate the abnormalities of serum EPO levels based on the
healthy control gpoup: the EPO levels within the limits X ± 2SD were
considered normal. Patients with EPO levels > X + 2SD or < X - 2SD
were considered raising or falling.
- Formula to calculate glomerular filtration rate based on the Levey
formula adjusted from Cockcroft and Gault formula (Modification of
Diet in Renal Disease - MDRD)
- Calculate TSAT according to Thomas L. formula
- Evaluate EPO response according to Artunc formula:
Serum EPO level
EPO response (%) =
x100 (%)
Estimated EPO level
Serum EPO level: serum EPO carried from the test result (mU/ml);
Estimated EPO level: EPO level calculated from the linear regression
equation between serum EPO levels and Hb levels in the anemia control
group (mU/ml). In this study, the linear regression equation to calculate
the estimated EPO levels concentrations was following:
Estimated EPO level = - 13,062 x Hb level + 1519,470


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Chapter 3. RESULTS
3.1. General characteristics of the subjects
- The average age of patients was 44.9±15.9; the percentage of male
was 52.4% and women 47.6%. There was no significant difference in the
age and gender between the groups of this study.
- Chronic glomerulonephritis are the most common causes of chronic
kidney disease (59%), the next ones were diabetes and pyelonephritis
accounted for 14% of cases.
- Proportion of patients with high CRP-hs level accounted for 35.2%;
and with low serum albumin (<38g/l) accounted for 47.6%.
- The Hb concentration in patients with ESRD was 85.4±15.7 g/l.
Proportion of patients with mild, moderate and severe anemia were 23.8;
42.9 and 33.3% respectively and was no diference in compare with the
anemia controled group (p>0.05).
3.2. Erythropoietin, ferritin levels and TSAT in the subjects of study
3.2.1. The features of serum EPO in the subjects of study
Table 3.5. The comparision of the average of EPO levels in the subjects of
study
The subjects of study
n
p
X ± SD
Healthy controls (1)
66
10.2±4.7
p1-2<0.01
The study group (2)
105
15.8±10.2
p2-3<0.001
Anemia controls (3)
51
366.2±248.2
p1-3<0.001
EPO concentrations in patients with end-stage renal disease was significant
higher than that in the healthy control group, but lower than in the anemia
control group.
Table 3.7. Correlation between serun erythropoietin and red blood
cells, hemoglobin levels and hematocrit
The study
Anemia
Healthy
subjects
group (n=105)
Index
RBC (T/l)
Hb (g/l)
Hct (%)

r

p

-0.09
-0.05
-0.07

>0.05
>0.05
>0.05

controls
(n=51)
r
p
-0.66
-0.75
-0.74

<0.001
<0.001
<0.001

controls (n=66)
R

p

0.21
-0.05
0.06

>0.05
>0.05
>0.05


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There were no a correlation between erythropoietin and RBC, Hb and Hct
in the study group and the healthy controls but there was correlation in the
anemia controls.
Rate of patients %

< 5%

< 10%

< 15%

EPO respose
quantiles

Biểu đồ 3.8. Distribution of the rate of patients with ESRD beyond EPO
respose quantiles
EPO respose quantiles were below the 20th percentile in 99% of the
patients with ESRD.
- The EPO response was no significant difference in patients with different
causes of chronic kidney disease and ranging from 2.9±1.6% to 5.3±3.9%.
- In patients with ESRD, serum erythropoietin wasn᾿t correlated with
glomerular filtration rate but moderately inversely correlated with serum
ferritin (r= -0.31; p< 0.001).
3.2.2. Serum ferritin level and TSAT in patients with ESRD
- The mean level of ferritin and TSAT in male (545.0±394.9 ng/ml and
23.7±11.1%) was significant higher in female (379.8±313.9ng/ml and
19.6±7.8%) with p<0.05
- The rate of patients with high ferritin level and low TSAT were the highest
propotions (38.1% và 52.4%). The rate of patients with low ferritin level
and high TSAT were 31.4% và 1.9%.
- Proportion of female patients with low ferritin levels and TSAT was
significant higher than of male patients but proportion of female with high
ferritin level was significant lower than of male (p <0.05).
- Proportion of patients with ferritin levels ≥ 500ng/ml and TSAT <20% in
group of CRP-hs≥0.5mg/dl was significant higher than in group of CRP-hs
<0.5mg/dl (p<0.05).
- Serum ferritin moderately correlated with serum CRP-hs in patient with
serum ferritin ≥200ng/ml (r= 0.33; p < 0.001).
- TSAT moderately correlated with serum ferritin huyết thanh (r= 0.30; p <
0.01).


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3.3. Changes of serum erythropoietin, ferritin and TSAT after 3
months of treatment
3.3.1. Outcome of first three-month hemodialysis combined with other
treatment methods
- RBC, Hb and Hct after treatment (3.47±0.59T/l; 104.6±17.8g/l; 31.7±5.5%
respectively) were increased significantly compared to befor treatment
(2.98±0.59T/l; 85.4±15.7g/l; 26.1±4.8% respectively) with p<0.001.
- Comparison of proportions of patients with different degree of anemia
before and after treatment showed significant changes in the rate of patients
with mild anemia and no anemia after treatment (65.7%) compared to
before treatment (23.8%) (p <0.001). Proportions of moderate and severe
anemia after treatment (42.9% and 33.3% respectively) reduced significantly
compared to before treatment (25.7 and 8.6% respectively) (p <0.05)
- Proportion of patients with high CRP-hs level before treatment was
35.2% decreased significantly to 16.2% after treatment with p <0.01.
3.3.2. Changes in level of serum EPO after treatment in patients with
end-stage renal disease
EPO (mUI/ml)

Chart 3.15. Comparing the average value of serum EPO concentration
before and after
p<0,01treatment (n = 105)
After treatment, EPO level was significantly decreased compared to befor
treatment with p<0.05.
Delt
a
EPO
(mU
I/ml)

r = -0,36; p < 0,001
y = - 0,253X + 1,247

Delta hemoglobin (g/l)


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Chart 3.17. The correlation between delta EPO with delta hemoglobin
after-before treatment
Delta EPO correlated inversely with delta Hb after- befor treatment
3.3.3. Changes in levels of iron, ferritin and TSAT after treatment
- Compareration of levels of iron, ferritin and TSAT befor and after
treatment we found that the averages of iron, ferritin and TSAT after
treatment (13.0±3.5 µmol/l; 570.1±239.5 ng/ml and 29.2±8.9%
respectively) were increased compared to befor treatment (10.2±4.2
µmol/l; 466.3±366.4 ng/ml and 21.8±9.9% respectively) with p<0.001.
- The proportion of patients with low ferritin level and low TAST after IV
iron therapy (1% and 6.7% respectively) were decreased compared to
before treatment (31.4 and 52.4% respectively) with p <0.001. The
proportion of patients with high TSAT and high ferritn level before and
after treatment difference was not changed significantly (p> 0.05)
Table 3.25. Comparison of ferritin and TSAT levels before and after
treatment beyond on iron supplementation
IV iron
supplements

Ferritin (ng/ml)
Befor
After
therapy
therapy

p

Befor
therapy

TSAT (%)
After
therapy

p

Bonus (n=45) 176.8±118.8 537.8±210.6 <0.001 16.3±4.9 28.0±6.9 <0.001
Maintenance
(n=31)
No (n=29)

444.4±146.
520.0±206.6 <0.05 23.3±6.4 29.9±11.5 <0.01
0
939.1±294.
674.0±286.4 <0.001 28.6±13.5 30.2±7.2 >0.05
8

The mean levels of ferritin and TSAT after treatment were significantly
increased compared to before treatment in both bolus and maintenance
group. In group without iron supplementation, ferritin level was
significantly reduced after three-month but TSAT was not changed.
Table 3.26. Comparing the rates of patients with high, normal and low
ferritin level before and after treatment
Ferritin (ng/ml)
<200
200-499.9
≥500
IV iron supplements
n(%)
n(%)
n(%)
Bonus
Befor therapy
33 (73.3)
12 (26.7)
0
(n=45)
After therapy
0
30 (66.7)
15 (33.3)
P
<0.001**
<0.001*
<0.001**


19
Maintenanc Befor therapy
e
After therapy
(n=31)
P
No (n=29) Befor therapy
After3 month
P

0
1(3.2)

20 (64.5)
14 (45.2)

11 (35.5)
16 (51.6)

0
0
-

>0.05*
0
10 (34.5)
<0.001**

>0.05*
29 (100)
19 (65.5)
<0.001**

(*) Chi square test (**)Fisher,s exact test
In the bonus subgroup, the proportion of patients with ferritin level <200
ng / ml after treatment was significantly reduced compared to before
treatment (p <0.001); In the subgroup without iron supplement, the
proportion of patients with ferritin ≥ 500 ng / ml after 3 months (65.5%)
decreased significantly compared to pre-treatment (100%) (p <0.001).
Table 3.27. Comparing the rates of patients with high, normal and low
TSAT level before and after treatment
TSAT (%)
<20 n(%) 20-49.9 n(%) ≥50 n(%)
IV iron supplements
37 (82.2)
8 (17.8)
0
Bonus
Befor therapy
4 (8.9)
41 (91.1)
0
After therapy
(n=45)
P
<0.001**
<0.001*
Maintenanc Befor therapy
10 (32.3)
21 (67.7)
0
2 (6.5)
28 (90.3)
1 (3.2)
e
After therapy
(n=31)
P
<0.05**
<0.05**
>0.05**
No
8 (27.6)
19 (65.5)
2(6.9)
Befor therapy
1
(3.4)
28
(96.6)
0
(n=29)
After3 month
P
<0.05**
<0.01**
>0.05
(*) Chi square test (**)Fisher,s exact test
In bonus and maintenance subgroup, the proportion of patients with low
TSAT after treatment was significantly reduced compared to before
treatment (p <0.001).

Chapter 4. DISCURSION
4.1. General chracteristics of the subjects
We studied 222 objects divided into 3 groups which were similar in
age. The mean age of patients with ESRD in study group was 44,9 ± 15,9.


20
Most patients starting hemodialysis treatment in our study were at working
age. Proportions of men and women between the research groups also did
not differ significantly (p> 0,05). The causes of renal failure of the patients
in study group were various excluded only polycystic kidney disease. The
most common cause of kidney failure is chronic glomerulonephritis
accounting for 59%, followed by diabetes, chronic pyelonephritis and
hypertension with the corresponding ratios: 14,3%; 14,3% and 9,5%.
4.2. Serum erythropoietin, ferritin and TSAT of the subjects
4.2.1. Serum erythropoietin and its relation with some clinical and
laboratory parameters
* Serum erythropoietin of the subjects
The study results showed that, with quantitative method
“chemiluminescent enzyme-labeled Immuno-metric assay"mean serum
EPO levels in anemia patients with ESRD were 15,8 ± 10,2 mU/ml,
significantly higher compared with healthy controls, p < 0,05 (table 3.5).
EPO secretion in the kidneys is regulated by the oxygen level in the
blood. The blood oxygen reduction will stimulate the secretion of EPO and
serum EPO increases. This is obvious that although patients with end- stage
renal disease had reduced endocrine function, but in the case of anemia the
kidneys were still stimulated to secrete EPO leading to increase serum EPO
higher than healthy people.
When compare serum EPO in patients with ESRD with that in anemic
patients without renal failure we saw a huge difference (15,8 compared to
366,2 mU/ml) (p <0,001) (Table 3.5) while the RBC, hemoglobin and
hematocrit of the two groups did not differ significantly (p> 0,05). This
showed that, in patients with ESRD EPO secretion did not correspond to
the degree of anemia.
* The correlation between serum EPO with hematological indices
and the EPO secretion in patients with end-stage renal disease
When renal function is intact, blood oxygen reduction will stimulate
the kidneys to increase secretion of EPO, hence serum EPO increases. Thus
serum EPO inversely correlated with haematological indices such as RBC,
hemoglobin and hematocrit. In our study, serum EPO in patients with
ESRD has no relation to the RBC, hemoglobin and hematocrit with / r / <
0,3; p > 0,05 (Table 3.7). In contrast, in the anemia control group, there is


21
strong negative correlation between serum EPO and RBC, hemoglobin and
hematocrit / r /> 0,7; p <0,001 (Table 3.7).
From the results of previous research in the world, we calculate the
estimated EPO concentrations from the linear regression equation between
the relationship between Hb level and serum EPO in patients of the anemia
control group and compared with serum EPO measured in patients with
ESRD. The results showed that mean EPO response in these objects is
from 2,9±1,6% to 5,3±3,9%, depending on the cause of renal failure,
therein 99,0% of patients have EPO response <15% (chart 3.8).
* Relationship between serum EPO with some clinical and laboratory
parameters
We found that there is no correlation between serum EPO and
glomerular filtration rate in patients with ESRD (r = -0,05; p> 0.05). Thus,
the serum EPO seems to be unrelated to the glomerular filtration rate of
these objects.
Evaluate the relationship between the causes of renal failure with
serum EPO levels in patients with ESRD, we found that mean serum EPO
levels in the groups with different causes were not different with p > 0,05.
Evaluate the relationship between serum EPO with iron status we
found that serum EPO was inversely and moderately correlated with serum
ferritin (r = -0,312; p < 0,01). The results of this study showed that there
was a relationship between the body's iron stores and the serum EPO in
patients with ESRD.
4.2.2. Serum iron, ferritin and TSAT in patients with ESRD and their
relations with some clinical and laboratory parameters

* Serum iron, ferritin and TSAT in patients with ESRD
In our study, the mean serum iron, ferritin and TSAT 10,2 ± 4,6 μmol/l;
466,3 ± 366,4 ng/ml and 22,8 ± 9,9 % respectively. In hemodialysis
patients, iron balance tends to be negtive. It means the iron loss due to blood
loss more than the amount of iron absorbed from the gastrointestinal tract
including the case of oral iron supplementation.
Iron stores are typically higher when the patients are treated with
intravenous iron therapy or blood transfusions or they suffer from
pathological disorders of iron metabolism. In case of infection, the
majority of patients have high serum ferritin combined with low TSAT.


22
Our results showed that the proportions of patients having low
serum ferritin (< 200 ng/ml) were high for both men and women (21,8%
and 42% respectively). Those with low TSAT (< 20%) were also met
with a high rate (male: 41,8% female: 64%). Comparing the proportions
of patients with TSAT <20% and ferritin < 200 ng/ml between men and
women, the percentage of women is higher significantly than men (p <
0,05). Serum ferritin and TSAT were also significanttly lower in women
compared to men (p < 0,05).
* The relationship between serum iron, ferritin and TSAT with a
number of clinical and laboratory parameters in patients with ESRD
Iron status in patients with ESRD is reflected in the amount of iron
stores (serum ferritin) and iron readily transported to the bone marrow for
erythropoiesis (TSAT). Both these indicators are afected by many factors
such as inflammation, malnutrition,…
The study results showed that the proportion of patients with TSAT <
20% in hs-CRP ≥ 0,5mg/dl group significantly higher than that in hs-CRP
< 0,5 mg/dl group (p < 0,01) and the proportion of patients with ferritin ≥
500 ng/ml in hs-CRP ≥ 0,5 mg/dl group were also significantly higher than
that in hs-CRP < 0,5 mg/dl group (p <0,05).
The results are consistent with the scientific evidence which has
proved that when there is inflammation inflammatory substances dominate
the iron binding sites on transferrin molecules associated with increased
hepcidin synthesis (hepcidin prevents the liberation of iron from
reticuloendothelial system into the bloodstream and reduces iron absorption
from the gastrointestinal tract into the bloodstream) in liver leading to
reduced TSAT and increased serum ferritin.
Evaluating the relationship between hs-CRP levels and serum ferritin
in 72 patiets with serum ferritin ≥ 200 ng/ml, we found that there is a
positive moderate correlation between the two indices (r = 0,33 ; p < 0,05).
Evaluating the relationship between TSAT and serum ferritin in
patients with serum ferritin ≥ 200 ng/ml, we also found that there is a positive
moderate correlation between the two indices (r=0,30; p<0,01). Thus, when
there is not absolute iron deficiency, the TSAT positively correlated with serum
ferritin.
4.3. Changes of serum EPO, ferritin and TSAT after 3-month
hemodialysis treatment


23
4.3.1. Outcomes of first 3-month hemodialysis combined with other
treatment methods
Most patients with ESRD indicated hemodialysis had anemia and are
going to have blood loss related to dialysis treatment, so anemia therapy is
very important. Effectiveness of anemia treatment is usually assessed
through RBC, hemoglobin and hematocrit. The study results showed that at
the end of the study all 3 indices are significantly higher than those before
treatment (p < 0,001). This showed that, the combination treatment
intravenous iron and Rhu-EPO has improved effectiveness of anemia
treatment.
4.3.2. The changes in serum erythropoietin of hemodialysis patients
The study results showed that when Hb level increased from 85,4 g/l
to 104,6 g/l, the serum EPO significantly decreased from 15,8 mU/ml to
12,2 mU/ml (p < 0,01) (chart 3.15). We found that there is a negative
moderate correlation between delta serum EPO and delta Hb after-before
treatment (r= -0,36; p< 0,001) (chart 3.17).
This showed that the changes of Hb levels have affected the changes
of serum EPO in opposite direction, or in other words, the regulatory feedback mechanisms of EPO secretion in hemodialysis patients still remained,
the stimulation of EPO secretion in case of hypoxia and inhibition of that in
case of enough oxygen existed.
Two possibilities can occur simultaneously in this case: firstly, the
presence of EPO secretion inhibitors in patients with ESRD, which has
partly eliminated by dialysis treatment leading to partial recovery of
regulatory EPO secretion; secondly, patients had adapted to the anemia, so
the changes in Hb levels also led to the changes of serum EPO in the
opposite direction. If it is true, it is clear that adequate dialysis and
maintaince stable target Hb levels in patients with ESRD is very necessary
because the patients will adapt to the target Hb levels and regulatory EPO
secretion mechanisms will play certain role in maintaining Hb levels in
case of blood loss.
4.3.3. The changes in serum ferritin and TSAT of hemodialysis patients
In this study, we rely on the recommendations to apply 3 methods of
treatments for subjects with different iron status and it seems to be very
effective in reducing the proportion of patients with low iron stores and low


24
available iron, significantly limiting increase the rate of patients at risk for
iron overload.
In the subgroup treated with bolus dose aimed to supplement deficient
iron, expected iron to be mobilized to erythropoiesis and iron loss during
the first 3-month dialysis treatment, results showed that the average serum
ferritin after treatment (536,7 ± 212,6 ng/ml) increased with significantly
compared with that before treatment (176,8 ± 118,8 ng/ml) with p < 0,001
and the average TSAT after treatment (28,0 ± 7,7%) increased significantly
compared with that before treatment (16,3 ± 4,9 %) with p < 0,001 (table
3.26). The proportion of patients with ferritin levels < 200 ng/mL and
TSAT < 20% after treatment were 0% and 8,9%, decreased significantly
compared with that before treatment (73,3% and 82,2% respectively) (p <
0,001) (table 3.25 and 3.26).
In the subgroup treated with maintenance dose of 200 mg iron sucrose/
month, average serum ferritin and TSAT after treatment (520,9 ± 206,6 ng/
ml and 29,9 ± 11,5 respectively) also rose than those before treatment
(444,4 ± 146,0 ng/ml and 23,3 ± 6,4% respectively) with p < 0,01 (table
3.26). In this subgroup, proportions of patients with serum ferritin ≥ 500
ng/mL and TSAT ≥ 50% after treatment were increased more than those
before treatment, but not significantly (p > 0,05) (table 3.25 and 3.26).
This showed that maintainance intravenous iron dose can ensure
adequate iron stores but may cause iron overload, so patients should be
periodically tested for iron status at least once per 3 months to prevent the
risk of iron overload.
In the subgroup without intravenous iron supplement, serum ferritin
decreased significantly from 939,1 ± 294,8 ng/ml to 674,0±286,4 ng/ml)
after 3 months (p<0,001), but TSAT increased from 28,6 ± 13,5% to
30,2±7,2% (table 3.26). The phenomenon of decreased iron stores in our
study may be explained by the iron was transported to the bone marrow
producing new RBC or was lost due to blood loss. However, increased
TSAT is difficult to explain because normaly, high reserves iron or
intravenous iron therapy will increase the ability binding transferrin of iron
molecule and lead to TSAT increasing such as intravenous iron treatment
group. The only reason we can suppose in this case, the rate of inflamation
was decreased at the end of the study and iron binding sites on transferrin
molecules are released from cytokines leading to increase ability binding of
iron with transferrin molecules in this group


25
The study results also showed that after 3 months without iron
supplementation, the proportion of patients with serum ferritin ≥500ng / ml
(65.5%) was significantly decreased compared to before treatment (100%)
with p <0.001, and the proportion of patients with TSAT <20% after
treatment (3.4%) was significantly decreased compared to before treatment
(27.6%) with p <0.05 (table 3.27). Thus, the periodic monitoring of iron
status in this group to timely iron supplement to prevent iron deficiency
and ineffective rHu-EPO treatment.

CONCLUSION

1.1.

1. Erythropoietin, ferritin levels and TSAT in patients with end-stage
renal disease
Serum EPO in patients with end-stage renal disease
-The mean serum EPO concentration in patients with ESRD was 15.8 ± 10.2
mU / ml which was much lower than this in the anemia patients without renal
failure ((366.2 ± 248.2 mU/ml) mU / ml) with p <0.001 but was higher than
this in the normal people (10.2 ± 4.7 mU / ml) with p <0.05.
- In patients with ESRD, serum EPO levels wasn ᾿t correlated with
hemoglobin concentrations (r=-0.03; p>0.05) and glomerular filtration rate
(r=-0.05 và p>0.05) but moderately inversely correlated with serum ferritin
(r= -0.31; p< 0.001).
- The EPO response was no significant difference in patients with different
etiology of chronic kidney disease and ranging from 2.9±1.6% to 5.3±3.9%.
and EPO respose quantiles was below 15 th percentile in 99.0% of the
patients with ESRD.
1.2. Serum ferritin level and TSAT in patients with ESRD
- The rate of patients with low ferritin level and low TSAT were 31.4% and
52.4%. In the female, these rates were significantly higher than in the male.
- Proportions of patients with ferritin levels ≥ 500ng/ml and TSAT <20% in
group of CRP-hs≥0.5mg/dl (56.8% và 67.6% respectively) were
significantly higher than in group of CRP-hs <0.5mg/dl (27.9% và 44.1%)
with p<0.05.
- Serum ferritin moderately correlated with serum CRP-hs in patient with
serum ferritin ≥200ng/ml (r= 0.33; p < 0.001).
- TSAT moderately correlated with serum ferritin huyết thanh (r= 0.30; p < 0.01).
2. Changes of serum erythropoietin, ferritin and TSAT after first 3
months of hemodialysis treatment


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