Report on Cellular Haematology: Diagnosing Beta-Thalassaemia

This report delves into the realm of cellular haematology, specifically focusing on the diagnosis of Beta-Thalassaemia. It begins with an introduction to cellular haematology and defines Beta-Thalassaemia as a haemolytic anaemia caused by reduced or absent beta-globin chain synthesis. The report then explores the diagnostic methods used, including High-Performance Liquid Chromatography (HPLC) and various molecular tools like PCR and NGS. HPLC is presented as a standard method for detecting and quantifying haemoglobin variants, while molecular tools are discussed for identifying gene mutations. The report highlights the advantages and limitations of each method, concluding that molecular tests are essential for diagnosis, especially when other less expensive tests like HPLC are not sufficient. The discussion emphasizes the importance of choosing the right diagnostic approach based on the clinical setting to aid in routine diagnosis, treatment, and prognosis.

CELLULAR HAEMATOLOGY

Table of Contents
INTRODUCTION...........................................................................................................................1
Βeta-Thalassaemia..................................................................................................................1
High Performance Liquid Chromatography (HPLC)............................................................1
Molecular Tools for Diagnosis...............................................................................................2
CONCLUSION................................................................................................................................3
REFERENCE...................................................................................................................................5

INTRODUCTION
The cellular haematology is defined as the branch of medicine which deals in the
different term and cause of prognosis, treatment and prevention of disease related to the blood. In
comprise treating disease affect the production of blood and its components. The elements of
blood are blood cells, haemoglobin, blood proteins, bone marrow, blood vessels, spleen and the
mechanism of coagulation. In the case of this, there are different term of the anaemias. They are
classified sickle cell anaemia or Thalassaemia or Acquired: warm and cold autoimmune or
alloimmune. In medical science there are many method and technique which is used in the
diagnosis of different type of anaemias. Which helps to identified the long term of diagnosis of
haemolytic anaemias. In this, we focus on the diagnosis of β-Thalassaemia within this essay
(Hussein and et. al., 2018).
Βeta-Thalassaemia
Beta-Thalassaemia is haemolytic anaemia which is caused by the decreased synthesis or
no synthesis of beta globin chain. As we go through the physiology, haemoglobin is made up of
beta globin of 2 units and 2 units of alpha globin chain and there is ferrous group also. Which
made to make up the tetrameric molecule that called haemoglobin. Βeta-Thalassaemia associated
with many genetic mutation either it may be enhancer, promoter or the gene complex. Beta-
Thalassaemia is divided in many sub group like Beta-Thalassaemia major , intermedia and one
of them is minor. According to Hoffbrand and Moss the clinical severity of this disease is
dependent on the type of Thalassaemia. Needs and Lynch elaborated that the physiology trait in
beta Thalassaemia is an enhanced synthesis of Hb F and HB A, in this case the anaemia is caused
by the no production of Hb A. They also explains about the causes ineffective Erythropoiesis and
due to this pathophysiology, severe anaemia can occur (Ravandi and et. al., 2016).
High Performance Liquid Chromatography (HPLC)
HPLC is a technique which is used in the separation, identification, and quantification of
present compounds, there are different kind of HPLC method used now day in the different
company. In which such as normal phase HPLC which helps to separate out the compound
which is analysed base on their polarity. They operate based in the on compound size, ion
charges and hydrophilic interaction. The mode of detection is through UV spectroscopy where
the organic analytes will absorb the UV lights of a particular wavelength. HPLC explained by
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Hoffbrand and Moss that it is a diagnostic tool used as a standard method of determining
haemoglobinopathies, since HPLC involves detection, measurement and separation of different
variants of haemoglobin. This method use to confirm that the separation test. In this the
limitation can be found in the blood count. And peripheral blood smear, as they are not very
specific in identifying the subtype of anaemia, but only indicative, making it difficult. While on
the basis of cell morphology it is difficult to differentiate the beta-Thalassaemia major, and
Thalasseaemia intermedia. Moreover the use of method of HPLC in this play a significant role
used to distinguish between haemoglobinopathies (Smith and et al., 2018). On this addition,
when HPLC is performed, a decrease in normal Hb A should be seen in patient suggestive of
Thalassaemia intermedia or minor. According to the smith, HPLC technique take 10 to 30
minute on the run time, being quicker than other chromatography technique. HPLC have high
accuracy and resolution and can be performed with minimum training in different setting of
diagnostic tools. Also give a data about how HPLC have beneficial tool to differentiate various
haemoglobin variants. Which also have to quantify the abnormal and normal haemoglobin
fractions, which is observed using the retention time window of this test. Moreover some of the
Hb variants have similar movement seen by the electrophoresis and HPLC such as Hb E and Hb
A. this technique contain some limitations, such as not being sensitive for alpha Thalassaemia. If
the disease is present in the mild form, the Hb A two does not increase to higher level until 6
month of age. The iron deficiency in patient may lead to lower level of Hb A two production,
they didn't cause nay problem in diagnosing a patient which is suffered from the beta-
Thalassaemia traits. The diagnosis of beta-Thalassaemia is confirm by the khera et. al. (2014)
between the abnormal haemoglobin variants by separate independent tests.
Molecular Tools for Diagnosis
In another term, they have different molecular assay that generally used as a confirmatory
test for the haemoglobin. To identify the type gene mutation in the patient in question which is
arises by the khera. These are DNA blot analysis, polymerase chain reaction (PCR), sanger
sequencing and next generation sequencing (NGS). As discussed above, the most simple and
common mutation that lead to beta-Thalassaemia are hit and point mutation which is observed in
this case. This create advantage for those who are suffering from beta-Thalassaemia carriers of
the defective gene, to have for better identification. Where any one suffering from beta-
Thalassaemia so its have full surety that it will happens to offspring also and born are facing
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severe Thalassaemia syndrome (El‐Khazragy and et., al., 2019). The Thalassaemia can be
confirm by using the non molecular conventional method, molecular tools for diagnosis are
being practised for genetic counselling and prenatal diagnosis. PCR (polymerase chain reaction)
involves in DNA amplification, that can be sampled from various tissue types, which is in very
small quantity of DNA are needed for the amplification. The test is very sensitive. Every PCR
require require a primers and a DNA template with DNA polymerase enzymes, reaction buffer
and nucleotide. The DNA needs to be heated up to 95 degree Celsius. And they denature to
separate the double helix into 2 single strand of line (Vollack and et. al., 2017). Te next step is
called hybridisation and it involves annealing of primers and complementary nucleotides to the
single strand of DNA. If the mutation in question is present for this to occur the section made up
of primer + single strands need to contain a free hydroxyl group (-OH) on the 3' side for Taq
DNA polymerase to catalyse annealing of free nucleotide fourthly the result implies in 2 copies
of the original DNA strand. Thus qPCR follow the real time pace, so it is no only about to detect
the presence of the PCR product as qualitative means of analysis through gel electrophoresis, but
also help in the quantification of PCR product through the means of fluorescence analysis.
Moreover, it is a fast method of DNA amplification cross contamination is decreased due to no
manipulation of sample after amplification, multiplexing is also an advantage which helps to
implies the amplification of millions to millions copies of DNA. PCR does have their limitation
because of the result which is appear due form of contamination this is comprise due to the high
sensitivity of this assay. Thus, this assay can used for basic quantification of DNA of known
beta-Thalassasemia mutation. In other word, the limitation is that the primers can anneal to non
specific sequences that similar, but no identical. So cannot detect the point mutation , moreover,
more than 90% of the mutation that cause Thalassaemia have been identified, so it would still
beneficial to use the PCR for known mutation (Slatter and et al., 2016).
CONCLUSION
As per the above discussion, PCR is not much useful for the point mutation, which prefer
to advantage in clinical and molecular diagnosis because most of the beta-Thalassaemia subtype
are linked with the point mutation. The alternative molecular tests can be sought such as NGS
which can analyse the actual mutation sequencing. Moreover, the molecular tests are necessary
for beta-Thalassaemia diagnosis, especially when other less expensive tests can be performed
( such as HPLC) because this not be so useful in areas with limited healthcare resources. The
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total matter is all dependent on the clinical settings requiring diagnosis of the beta-Thalassaemia.
In the case of this the diagnosis id important but for the most molecular testing would be
advantageous , to aid the routine diagnosis, treatment and prognosis, in which HPLC be more
diagnostically suitable
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REFERENCE
Books and journals
Hussein and et. al., 2018. Effects of Lipopolysaccharides of Pseudomonas Aeruginosa and
Aqueous Extract of Ginkgo Biloba, Ginkgoaceae, on Cellular Immune Response in
Mice Balb/c. Gazi Medical Journal, 29(3).
Ravandi and et. al., 2016. Minimal residual disease assessed by multi‐parameter flow cytometry
is highly prognostic in adult patients with acute lymphoblastic leukaemia. British
journal of haematology, 172(3), pp.392-400.
Smith and et al., 2018. Anti-inflammatory cellular targets on neutrophils elucidated using a novel
cell migration model and confocal microscopy: a clinical supplementation study.
Journal of Inflammation, 15(1), pp.1-10.
El‐Khazragy and et., al., 2019. Interaction between 12p chromosomal abnormalities and Lnc‐
HOTAIR mediated pathway in acute myeloid leukemia. Journal of cellular
biochemistry, 120(9), pp.15288-15296.
Vollack and et. al., 2017. Anti‐Fcγ RIIB (CD 32) Antibodies Differentially Modulate Murine
FVIII‐Specific Recall Response in vitro. Scandinavian journal of immunology, 86(2),
pp.91-99.
Slatter and et al., 2016. Haploidentical CD3 TCRαβ and CD19-depleted second stem cell
transplant for steroid-resistant acute skin graft versus host disease. Journal of Allergy
and Clinical Immunology, 138(2), pp.603-605.
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