RESEARCH PROPOSAL

PERFORMACE EVALUATION OF BIO-RAD VARIANT II BY USING HIGH

PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) METHOD ON
HEMOGLOBIN ANALYSIS IN HOSPITAL UNIVERSITY KEBANGSAAN MALAYSIA

PROPOSED BY: NUR AMIRAH NAZIFA BINTI LOKMAN

ID: 4212003411

IN PARTIAL FULFILLMENT FOR THE DEGREE

PROGRAM BACHELOR OF BIOMEDICAL SCIENCE (HONOURS)

 SUPERVISOR:

MOHD JAAMIA QAADIR BIN MOHD BADRIN

FACULTY OF HEALTH SCIENCES

UNIVERSITY SELANGOR (UNISEL)

40000, SELANGOR,

MALAYSIA

NO TEL: 03-5522 3400

CO-SUPERVISOR:

WAN AHMAD SHUKRI BIN WAN ABDUL AZIZ

MEDICAL LABORATORY TECHNOLOGIST

DEPARTMENT OF HEMATOLOGY

HUKM
Table of Contents Page

1.0 Introduction

1.1 Problem Statement 3-4

1.2 Objective 5

1.3 Hypothesis 5

1.4 Significance of Study 5

1.5 Background of Study 5-6

2.0 LITERATURE REVIEW 6

3.0 METHODOLOGY

3.1 Materials 7

3.2 Selection of Subject 7

4.0 Laboratory Investigation 7

4.1 Bio-Rad Variant-II Analyzer 7

4.1.1 Collection of Patient Sample

4.1.2 Specimen Preparation

4.1.3 Reagent Preparation 8

4.1.4 Procedures 8

4.1.5 Results 9

4.2 Sebia Capillary 2 Flex Piercing Analyzer 10

4.2.1 Collection of Patient Sample 10

4.2.2 Specimen Preparation 10

 4.2.3 Reagent Preparation 10

4.2.4 Procedures 11

4.2.5 Results 11

5.0 Data Collection and Analysis 12

6.0 Performance Statistics 13 - 15

7.0 Estimated Budget 16

8.0 Gantt Chart 17

References 18 - 19
1.0 INTRODUCTION

The various forms of hemoglobin in the bloodstream can be measured and identified using a
bloodtest called a hemoglobin electrophoresis test. The protein called hemoglobin found inside
red bloodcells is in charge of delivering oxygen to tissues and organs. The body may create
hemoglobin thatis wrongly produced as a result of genetic alterations. The tissues and organs
may not receive enough oxygen as a result of this aberrant hemoglobin. Hemoglobin comes in
hundreds of varieties. It is comprising F hemoglobin. Fetal hemoglobin is yet another name for
this. It is the kind that develops in developing fetuses and infants. Soon after birth, hemoglobin
A takes it is place. Adult hemoglobin is another name for hemoglobin A and this is the most
typical type of hemoglobin. Both healthy children and adults have it. Rare aberrant hemoglobin
types induced bygenetic abnormalities include hemoglobin C, D, E, M, and S. The amount of
hemoglobin in bloodcannot be determined using a hemoglobin electrophoresis test. In a full
blood count, it is carried out. The percentages of the various forms of hemoglobin that may be
present in blood are the amounts that a hemoglobin electrophoresis test refers to. Babies and
adults experience this differently. In fetuses, hemoglobin F makes up the majority of the blood
protein. The majority ofthe hemoglobin in infants is still hemoglobin F. By the time the baby
is a year old, it rapidly declines. Newborn has 60 to 80% Hemoglobin F percentage and for 1+
year has 1 to 2%. The normal levels of the types of hemoglobin in adults are hemoglobin A
95% to 98%. Hemoglobin A2 2% to 3%. Hemoglobin F 1% to 2%. Hemoglobin S and
hemoglobin C 0%.

By inheriting gene mutations on the genes that create hemoglobin, many aberrant forms of
hemoglobin can be produced. To find out if have a disease that results in the creation of
abnormalhemoglobin, perform a hemoglobin electrophoresis test. This is due to identify blood
diseases. Any aberrant hemoglobin types in the blood can be found with the use of the test.
These might besymptoms of illnesses such sickle cell anemia, thalassemia, and polycythemia
Vera. Besides, thistest also to check for genetic disorders. Before having children, those who
have a family history of hereditary anemias such thalassemia or sickle cell anemia may decide
to get screened for thesegenetic conditions. If there are any aberrant forms of hemoglobin
brought on by hereditary diseases, a hemoglobin electrophoresis will reveal this. These genetic
hemoglobin abnormalities are frequently checked for in newborns as well.
Iron-carrying hemoglobin, often known as Hgb, is a protein found in red blood cells.
Hemoglobinis a crucial part of blood due to the iron's capacity to store oxygen. Cells do not get
enough oxygenwhen the blood's hemoglobin content is insufficient. By examining a sample of
blood, ascertain the hemoglobin level. Age, gender, and medical history are only a few of the
1
variables that determine hemoglobin levels. Men have a slightly higher average hemoglobin
level than women have in adulthood. G/dL, or grams per deciliter, is the unit of measurement.
Whereas male has 13or higher, female has 12 or higher. Hemoglobin levels are also often lower
in older persons. This could be caused by a variety of things, such as chronic inflammation
lowering iron levels, inadequate diet, pharmaceutical side effects, and a high prevalence of
chronic conditions includingkidney disease. Hemoglobin levels in infants are typically higher
than in adults. This is due to thefact that they require more red blood cells to deliver the oxygen
due to the greater oxygen levels in the womb. But after several weeks, this amount starts to
decline.

When the production of healthy red blood cells in the blood is below average, anemia is a
disorder that results. The body does not receive enough oxygen-rich blood if have anemia.
Feeling weak or fatigued can result from a lack of oxygen. Additionally, it may cause
headaches, nausea, fainting, or an irregular heartbeat. Hemolytic anemia, iron-deficiency
anemia, vitamin B12-deficiency anemia, and other kinds of anemia exist. Anyone might
become afflicted with mild anemia, which is a common and manageable illness. It can be
brought on by food, medication use, or another medical condition, and it can happen quickly or
gradually. Chronic anemia is a type of anemia thatpersists for a very long time and might never
fully disappear. Some anemia forms are hereditary. Iron-deficiency anemia is the most
prevalent kind of anemia. Anemia is more common in some persons, particularly in pregnant
and menstruating women. Additionally, those who take particularmedications or treatments, do
not get enough iron, do not get certain vitamins, and are at higher risk. Anemia might
potentially indicate a more serious ailment, such as gastrointestinal bleeding, infection-related
inflammation, kidney illness, cancer, or autoimmune diseases. The kind and severity of anemia
determine the course of treatment. Some mild to severe anemia forms may require iron
supplements, vitamins, or drugs that increase red blood cell production from the body.

A reduction in the production of the alpha or beta chains of hemoglobin (Hb) results in a
heterogeneous grouping of hereditary illnesses known as thalassemia. The oxygen-carrying
protein in red blood cells is called hemoglobin. Two proteins, an alpha and a beta make up
this
substance. The formation of red blood cells is compromised if the body does not produce
enoughof either of these two proteins, which results in anemia that manifests in infancy and
persists throughout life. Since thalassemia is an inherited condition, at least one parent must
carry the disease's gene. Either a genetic mutation or the loss of specific important gene
segments is the root cause. Alpha-globin gene deletion, which leads in diminished or
2
nonexistent production of alpha-globin chains, is the cause of alpha thalassemia. There are four
variants of the alpha globin gene, and the severity of the condition varies from mild to severe
depending on how many deletions there are. The most severe variant, four allele deletion,
results in no alpha globin production and the formation of tetramers from extra gamma chains
that were present throughout fetal development. It causes hydrops fetalis and is incompatible
with life. The smallest and most clinically silent variant is one allele deletion. The beta-globin
gene experiences point mutations that cause β-thalassemia. In accordance with the zygosity of
the beta-gene mutation, it is categorized into three categories. Beta-plus thalassemia, a
heterozygous mutation that causes underproduction of beta chains, is known as β-thalassemia
minor. It normally has no symptoms and is minor. A homozygous mutation (beta-zero
thalassemia) in the beta-globin gene, which results in the complete absence of beta chains, is
the cause of β-thalassemia major. Jaundice, growth retardation, hepatosplenomegaly,
endocrine problems, and severe anemia necessitating constant blood transfusions are some of
its clinical symptoms. β-thalassemia intermedia, with mild to moderate clinical signs, is the
name used to describe the illness that falls between these two categories.

3
1.1 Statement of problem

The most useful test for identifying β-thalassemia carriers is quantitative HbA2 measurement.
Several techniques have been established, but just a few are currently advised due to their
accuracy.It should be made clear that utilizing densitometry scanning after cellulose acetate
electrophoresisto determine HbA2 is imprecise and inaccurate, and that its usage should be
avoided. Although laborious and time-consuming, isoelectric focusing (IEF) has exceptional
resolution that allows for accurate measurement. For the diagnosis of hemoglobinopathies,
capillary electrophoresis (CE) is being employed more and more often in clinical diagnostic
settings. For both qualitative and quantitative hemoglobin analysis, studies have shown a strong
association between capillary electrophoresis and high-performance cation-exchange
chromatography (HPLC). The most used
methods for measuring HbA2 are HPLC and CE. These procedures are quick, easy, and
extremelyaccurate. Additionally, CE and HPLC detect and quantify a wide variety of variant
hemoglobin, including the frequently occurring variations HbS, HbC, HbE, and Hb D-Punjab.

Although not all hospitals in Malaysia offer this advanced test, several of the larger hospitals
do. Understanding the results of a Hb study is crucial for detecting anemia, a condition in which
the body produces fewer red blood cells than usual. One of Malaysia's five university hospitals
is Universiti Kebangsaan Malaysia Medical Centre (UKMMC), also known as Hospital
Canselor Tuanku Muhriz UKM (HCTM) and formerly known as Hospital Universiti
Kebangsaan Malaysia. It is managed by Universiti Kebangsaan Malaysia and is situated in
Bandar Tun Razak, Kuala Lumpur. The Hematology Unit, Department of Diagnostic
Laboratory Services UKM Medical Center offers Hb analysis Screening tests. The project
sample will be taken randomly from the blood sample and will involve different races and
genders.

4
1.2 Objectives

General Objectives:

1. To evaluate the performance of Bio-Rad Variant-II analyzer.

2. To compare the results Bio-Rad Variant-II with Sebia Capillary 2 Flex Piercing
analyzeras reference.

Specific Objectives:

1. To evaluate the performance of Bio-Rad Variant-II analyzer with determination of

sensitivity, specificity, accuracy and precision by using ion-exchange high performance
liquid chromatography (HPLC) method.
2. Detecting and determining the level of hemoglobin type using the Sebia Capillary 2
Flex Piercing analyzer with Capillary Electrophoresis (CE) method.

1.3 Hypothesis

The Bio-Rad Variant-II analyzer's performance using the ion-exchange high performance liquid
chromatography (HPLC) method is reliable and accurate enough to be used in medical reports.

1.4 Significance of Study

This study will help laboratory staff in hospitals and health centers to identify easier and
accurate diagnostic methods for testing hemoglobin analysis and explains the method of
analyzing normaland abnormal hemoglobin protein contained in red blood cells such as A, A2,
F, H, S, Bart and others.

1.5 Background of Study

Hemoglobin electrophoresis has been replaced by Bio-Rad Variant-II, which uses High-
performance liquid chromatography (HPLC) as a screening method for hemoglobinopathies
and thalassemia. The HPLC's great resolution and consistency, which lead to precise diagnosis,
are its advantages. However, this method occasionally yields patterns that are rather
complicated and challenging to decipher. The hemoglobin fractions are divided by Sebia
Capillary 2 Flex Piercing.Employing Capillary Electrophoresis (CE) and an alkaline buffer
based on their electrophoretic mobility. It offers high throughput analysis with high resolution
separation of different hemoglobintypes. Clinical investigations contrasting HPLC and CE have
discovered that these two techniquesare complimentary and can be used together for accurate
and exact detection of hemoglobin variants.

5
The Bio-Rad Variant-II uses the β-thalassemia Short Programme for HPLC analysis to separate
hemoglobin variations using cation exchange chromatography with a salt gradient. Daily
adjustments were made to guarantee proper retention times and precise measurement using
HbA2and HbF single point calibrators. Before patient samples were analysed, Bio-Rad level 1
(HbA, F,and A2) and level 2 (HbA, F, A2, and S) QC materials were also run daily.

The Sebia Capillary 2 Flex Piercing was used to conduct capillary electrophoresis (CE) using
theCapillary Hemoglobin kit. The device is capable of re-suspending, lysing, and separating
hemoglobin variations for use in EDTA whole blood analysis. The lysed red cells are
electrophoresed in an alkaline buffer (pH 9.4), allowing pH and endosmosis to control the
direction of separation. Calibration is not necessary every day for CE. Every day, before
running new QC or patient samples, each capillary is examined through to verify correct charge
and operation of the capillaries.

2.0 LITERATURE REVIEW

Primary hemoglobin A (HbA), a little amount of hemoglobin A2 (HbA2), and traces of fetal
hemoglobin (HbF) are all present in adult blood. Hemoglobin A2 and F levels in those with β-
thalassemia can exceed 3.5% and 2% of total hemoglobin, respectively. The most efficient way
to identify carriers of the β-thalassemia gene is to look for concurrently high levels of
hemoglobin A2 and F. Anion-exchange column chromatography and electrophoresis are two
methods for quantifying hemoglobin A2. Techniques for measuring hemoglobin F Include
radial immunodiffusion (RID), alkali denaturation, and electrophoresis. The determination of
several hemoglobin, including hemoglobin A2 and F, has been done using high-performance
liquid chromatography (HPLC), which can be a relatively quick and reliable procedure. The
Bio-Rad Variant-II is a fully automated HPLC system that may be used to qualitatively identify
aberrant hemoglobin and separate and calculate area percentages for hemoglobin A2 and E.
Hemoglobin D, S, C, and E are the four most prevalent hemoglobin subtypes. Retention time
windows, such asa "D Window," "S Window," and "C Window," are used to presumptively
identify these hemoglobin variations. Using the Bio-Rad Variant-II β-thalassemia Short
Programmed,hemoglobin E, the second most common hemoglobin variant, elutes within the
hemoglobin A2 retention time window. With careful study of the area percent computation on
the sample report, hemoglobin A2 can be distinguished from it. Hemoglobin E is commonly
present in a range of 30to 35% in heterozygous individuals (phenotype AE).

6
3.0 METHODOLOGY

3.1 Materials

Samples will be randomly selected. The target is to obtain ten abnormal blood samples and ten
blood samples are normal. The aimed at assessing the performance between the two analyzers.
Meanwhile, to identify the normal range of hemoglobin analysis assay, a total of twenty
samples from screening test will be used.

3.2 Selection of Subject

Selection of blood samples will be by convenience sampling of EDTA blood samples with
minimum 1ml in Hematology unit, UKM Medical Centre. Using Bio-Rad Variant-II analyzer
as screening tests and Sebia Capillary 2 Flex Piercing analyzer as reference. All management,
sampling methods and subject samples that will be received and processed in the laboratory
will

comply with the standard operating procedure (SOP) criteria, references are from Laboratory
manual guideline version 2017 (Department of Diagnostic Laboratory Services UKM Medical
Center) and Quality Documents (Blood Collection Division) MS ISO15189.

4.0 LABORATORY INVESTIGATION

4.1 Hemoglobin Analysis Screening Test (Bio-Rad Variant-II analyzer.)

4.1.1 Collection of Patient Sample

The laboratory will receive a blood sample in a K2EDTA anticoagulant tube, volume 1ml, with
acomplete test application form. Sample acceptance criteria will be observed before proceeding
with the test.

4.1.2 Specimen Preparation

It is not necessary to prepare the sample. Selection of blood samples will be by convenience
sampling of EDTA blood samples with minimum 1ml.

7
4.1.3 Reagent Preparation

• Elution buffers and wash solution

§ Elution buffer 1 is to transport the Hb
§ Elution 2 to separates Hb and displacer
§ Before starting the assay, let the wash/diluent solution and elution buffers get to
roomtemperature (15–30 °C). Before installation, gently invert each bottle to mix
it.
§ When kept unopened at 15–30 °C, the Elution Buffers and Wash/Diluent Solution
willremain stable until the expiration date.
§ Install one bottle of each reagent in a new reorder pack.
§ Different reorder pack amounts of the Wash/Diluent Solution can be
usedinterchangeably.
• Whole Blood Primer
§ To prepare the cartridge for analysis at the start of each run, use the Whole
BloodPrimer
§ When kept unopened at 2-8 °C, the Whole Blood Primer will remain stable until
theexpiration date.

§ To prepare the Whole Blood Primer, fill each vial with 1.0 ml of deionized water.
§ Permit to stand at 15 to 30 °C for 10 minutes.
§ To dissolve and ensure thorough mixing, gently swirl the mixture.
§ Indicate the label with the reconstitution date. When kept between 2 and 8 °C,
thereconstituted Whole Blood Primer is stable for 21 days.

4.1.4 Procedures

1. Eight normal sample tubes and eight abnormal sample tube are positioned on the
conveyorbelt of the Bio-Rad Variant-II analyzer Sampling Station and loaded into the
VARIANT IIsample racks. Use special adapters and rack inserts for pediatric tubes
measuring 10 mm, 13 mm, and 14 mm.

8
4.1.5 Result Analysis

1. Each analysis's total area should be between 1.0 million- and 3.0-million-volt seconds.
Results that fall outside of this range should not be reported.
2. The average adult HbA2 range is 1.75–3.25% of total hemoglobin. HbA2 values in
heterozygous β-thalassemia patients range from 4 to 9%. HbF in adults normally ranges
between <1% of total hemoglobin. HbF levels of 1-5% and 80-100% are produced in
β- thalassemia heterozygous and homozygous situations, respectively.
3. Despite the wide linear range shown in the linearity experiments, the acceptable range
forHbA2 is 1–13% and for HbF is 1-40%. The selection of these ranges was made to
be congruent with a commercially available reference technique.
4. HbA and HbF readings below the range should be reported as 1.0% in order to be
consistentwith the specified reportable ranges. HbA and HbF readings below the range
should be reported as 1.0% in order to be consistent with the specified reportable
ranges., respectively.

Interpret the normal and abnormal hemoglobin found in patient samples, analyte identification
"windows" are used. The "windows" are predetermined time periods during which the Bio-Rad
Variant-II analyzer β-thalassemia Short Programmed has recorded the elution of common
variations. The window's centre is the retention time. From the moment the sample is injected
to each peak's maximum point, retention time is calculated. The band is the window's half-
width.

Analyte name Retention time Band (minutes) Window (minutes)

(minutes)
F 1.10 0.12 0.98-1.22
P2 1.39 0.11 1.28-1.50
P3 1.70 0.20 1.50-1.90
A0 2.50 0.60 1.90-3.10
A2 3.60 0.30 3.30-3.90
D-WINDOW 4.10 0.20 3.90-4.30
S-WINDOW 4.50 0.20 4.30-4.70
C-WINDOW 5.10 0.20 4.90-5.30
Table 1: Analyte identification Window p2 and p3 are minor peak associated with HbA.

9
 Patient State HbA2 Level HbF Level
Heterozygous β-thalassemia 4 – 9% 1 – 5%
Homozygous β-thalassemia Normal to Increased 80 – 100%
Heterozygous HPFH <1.5% 10 – 20%
Homozygous HPFH Absent 100%
Table 2: Expected value Ranges.

4.2 Hemoglobin Analysis Screening Test (Sebia Capillary 2 Flex Piercing).

4.2.1 Collection of Patient Sample

It is recommended to draw fresh, anticoagulated whole blood samples for analysis in K2EDTA
test tubes. Anticoagulants containing iodoacetate should not be used. The same procedures that
have been developed for clinical laboratory testing must be followed when drawing blood. For
upto 7 days, samples can be stored between 2 and 8 °C.

4.2.2 Specimen Preparation

Use directly whole blood samples. Check that all the tubes contain 1mL minimum of blood
and are perfectly closed.

4.2.3 Reagents Preparation

• Deionized or distilled water
§ Sebia Capillary 2 Flex Piercing, automated equipment for capillary
electrophoresis rinses capillaries. It is advised to use a 0.45 m filter to filter
distilled or deionized water before using.
§ Change the water every day to stop the growth of microbes.
• Capiclean
§ The vial of CAPICLEAN concentrated solution contains, proteolytic enzymes,
surfactants and additives nonhazardous at concentrations used, necessary for
optimum performances.
§ For sample probe cleaning in automated instrument Sebia Capillary 2 Flex
Piercingfor CE, during the CAPICLEAN cleaning sequence.
• Sodium Hypochlorite Solution
§ Prepare a sodium hypochlorite solution (2 % to 3 % chloride) by diluting 250 mL
9.6 % chloride concentrated solution to 1 liter with cold distilled or deionized water.
§ For the sample probe cleaning in the Sebia Capillary 2 Flex Piercing instrument
(weekly maintenance in order to eliminate adsorbed proteins from the probe)

10
 • Capillarys / Minicap Wash Solution
§ Each vial of the stock Wash Solution should be diluted up to 750 mL with
distilled or deionized water. After dilution, the wash solution contains an
alkaline solution pH12.
§ For washing the capillaries of Sebia Capillary 2 Flex Piercing. This additional
reagent is needed when the number of tests in series is below 40.

4.2.4 Procedure

Hemoglobin on parallel capillaries is examined using the multiparameter Sebia Capillary 2

Flex Piercing apparatus. The samples are passed through eight capillaries during the
hemoglobin testing. The sequence of automated steps is as follows:

• Bar code scanning of sample racks and tubes (up to 8 tubes).

• Blood samples being mixed before analysis.
• Collect dilution and hemolysis samples from primary tubes.
• Washing of capillaries.
• Injecting hemolyzed sample material.
• The separation of hemoglobin and the immediate detection of the separated
hemoglobinon capillaries.

4.2.5 Result Analysis

Individual hemoglobin fractions are automatically relative quantified at the end of the
procedure,and profiles can be examined. The hemoglobin fractions Hb A, Hb F, and Hb A2
are recognised automatically. In the midst of the review window, the Hb A fraction is modified.
Visual pattern irregularities are checked on the generated electrophoregrams. On the
instrument's screen and on the result ticket are indications of the prospective places of the
various hemoglobin variations, which are classified into zones denoted Z1 to Z15. The table in
the section under "Interpretation"lists known variants that could exist in each corresponding
zone. The name of this zone is generated when the software locates a hemoglobin fraction in a
predetermined zone. In order to make patterns easier to comprehend, they are automatically
modified in relation to Hb A and Hb A2 fractions:

• The adjustment is made using the position of the Hb A fraction on the two prior
patterns obtained with the same capillary when Hb A and Hb A2 fractions are
not identified on an electrophoretic pattern, resulting in the appearance of a
yellow warning signal. The fraction is then unknown, with the exception of
cases where Hb C is found. Hb A2 and Hb C fractions are recognised in this
11
 instance.
• The yellow warning signal does not show when Hb F is found on an
electrophoreticpattern without any Hb A detection. The correction is then made
using the positionof the Hb F fraction, and Hb F, Hb A, and Hb A2 fractions are
identified.
• Hb F and Hb A2 fractions are not identified when the adjustment is not possible,
resulting in the appearance of a red warning signal.
• For the purpose of determining the position of the Hb A fraction, a warning
message is displayed when the optical density (OD) on a migration control
electrophoretic pattern (obtained with the normal Hb A2 control, identified with
its bar code labelon the sample rack No. 0) is insufficient. The message asks the
user whether to consider or disregard this analysis. The Hb A and Hb A2
fractions are then undetected, and a purple danger signal shows on the review
pane.

• In every instance, neither the instrument's screen nor the ticket result provide
any indication of the various migration zones (Z1 to Z15).

On the electrophoretic pattern, the curves of Hb A2 and Hb C fractions, are calculated and
redrawn by fitting with adjustment (or fitted) and are overlaid with the native curve. This
display allows the Hb A2 fraction quantification if Hb C is present in the sample.

5.0 DATA COLLECTION AND ANALYSIS

The selected sample will undergo two required types of tests which is Bio-Rad Variant-II
analyzerby using High Performance Liquid Chromatography (HPLC) method and second tests
will be using Sebia Capillary 2 Flex Piercing by Capillary Electrophoresis (CE) method as
reference. TheBio-Rad Variant-II analyzer result will be compare with Sebia Capillary 2 Flex
Piercing. This comparison to know the performance of Bio-Rad Variant-II analyzer and to
identify the specific hemoglobin.

12
6.0 PERFORMANCE CHARACTERISTICS

• Precision

The precision of the Bio-Rad Variant-II analyzer β-thalassemia Short Program was evaluated
in astudy based upon the NCCLS EPS-T2 guideline "Evaluation of Precision Performance of
ClinicalChemistry Devices". Forty runs were performed over a ten-day period using to Bio-
Rad Variant- II analyzer. Within each run, replicates of three blood samples were analyzed
randomly. The results of the precision study are summarized in the tables below.

Low Patient Mid Patient High Patient

mean(%HbA2) 2.8 3.2 4.6
Within-Run % CV 1.64 1.88 0.88
Between-Run % CV 0.00 1.61 0.00
Between-Run % CV 1.38 1.06 1.98
total precision % CV 2.02 2.69 2.13
Table 3: Precision Results for HbA2

Low Patient Mid Patient High Patient

Mean(%HbF) 1.6 3.1 8.2
Within-Run % CV 2.05 1.20 0.59
Between-Run % CV 2.03 1.15 0.18
Between-Run % CV 2.55 1.45 1.24
total precision % CV 3.85 2.20 1.38
Tables 4: Precision Results for HbF

13
Analyte N Total No Mean RT SD RT % CV Observed Observed
of Analyte Minimum RT Maximum RT
F 3 138 1.12 0.02 1.57 1.09 1.19
P2 1 185 1.34 0.01 0.77 1.31 1.36
P3 1 185 1.73 0.02 0.91 1.66 1.78
A0 3 185 2.33 0.03 1.46 2.27 2.44
A2 3 185 3.65 0.01 0.33 3.63 3.69
D 5 4 4.15 0.01 0.17 4.14 4.15
S 41 25 4.54 0.05 1.12 4.49 4.64
C 30 10 5.13 0.05 0.72 5.09 5.18

Tables 5: analyte Retention Time

Precision RT refers to the retention time

of the analyte

P2 and P3 are minor peaks associated with hemoglobin A

• Accurancy

HbA2 results obtained from the thalassemia Short Program using the Bio-Rad Variant-II
analyzer were compared to values obtained on VARIANT. The comparison study was
performed on 47 blood samples. The values for relative percent HbA2 in this study ranged
between 1.3% and 6.6%using the -thalassemia Short Program.

N x-coefficient y-intercept r
47 1.048 -0.027 0.987
Tables 6: HbA2 Linear Regression
• Linearity

Linearity studies were performed to evaluate the response of the Bio-Rad Variant-II analyzer
β- thalassemia Short Program to increasing concentrations of hemoglobin A2 and hemoglobin
F. In these studies, purified solutions of HbA2 A and F were serially diluted with hemolysate
that contained only hemoglobin A0 and analyzed. Observed results from the Bio-Rad Variant-
II analyzer were compared to theoretical values determined from the number of serial dilutions
and are as follows.

14
Briefly, for HbA2 the data shows that the VARIANT I &-thalassemia Short Program is linear
from1.6% through 18.7% HbA2.

For HbF, the data shows that the VARIANT II $-thalassemia Short Program is linear from
1.3% through 44.3% HbF.

THEORETICAL OBSERVED
1.7 1.6
1.8 1.8 Linearity of HbA
2.1 2.1
20
2.6 2.6
Observed % A2

15
3.7 3.7
5.7 5.7 10

9.9 10.0
18.7 18.7
SLOPE 0.999 10 15 20
Theoretical % A
Y-INTERCEPT 0.001
R2 1.000
Figure 1: HbA2 Linearity
THEORETICAL OBSERVED
1.3 1.3
2.3 2.2
4.2 4.0 Linearity of HbA
8.3 8.1 20
12.4 11.9
15
16.6 15.7
Observed % A2

20.9 20.8 10
25.3 24.9
27.6 27.2
29.9 29.3
32.2 31.5
10 15 20
42.7 44.3 Theoretical % A
SLOPE 1.01
Y-INTERCEPT -0.48
R2 0.998

Figure 2: HbF Linearity

15
7.0 ESTIMATED BUDGET

Budget Type Sub-Budget Description Total (RM)

Traveling & Traveling & Conference and 100.00

Transportation Transportation Workshop

Research Materials & Research Materials & Elution buffer 1 1200.00

Supplies Supplies (Bio-Rad
Variant-II analyzer) Elution buffer 2 1200.00
Variant II test kit 1120.00
Wash/diluent solution
and Whole blood
primer (16 tests)

Research Materials & Buffer Analyzer 2894.00

Supplies (Sebia
Capillary 2 Flex Hemolysing solution 1000.00
Piercing) capiclean 564.00

Supplies & EDTA test tube (16 240.00

Consumables test tube)

16
8.0 GANTT CHART

April 2023 August 2023 December 2023 January 2024

May June July Sept Oct Nov

Week 1
Week 2

Week 3
Week 4

Week 1

Week 2
Week 3
Week 4

Week 1
Week 2
Week 3

Week 4

Week 1
Week 2

Week 3

Week 4
Tasks
2023 2023 2023 2023 2023 2023

Title
Review of literature and
writing the preliminary report
Discussion with supervisor
and filed supervisor
Proposal writing
Materials checking
Proposal checking bysupervisor
Proposal submission
Writing report
Laboratory tests
Collecting and analyzedata
Submission of report (draft)
Writing and the report forwarded
to receiving feedback and
guidance from supervisor
Early presentation
Complete the project report
Submission of report on the
potal for assessment and viva
Final presentation
Final report correction
Submission of project thesis(final)

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