Molecular and cytogenetic analysis are essential techniques for diagnosing and managing hematological disorders. Key methods include DNA extraction, PCR, FISH, and next generation sequencing. Clinical applications involve investigating diseases like sickle cell anemia, thalassemias, leukemias, lymphomas, and coagulation disorders. Molecular analysis allows identification of genetic mutations and translocations that underlie these conditions and guides treatment decisions. While providing critical diagnostic information, these techniques also have limitations like risk of infection and interference from therapies.

1. MOLECULAR AND CYTOGENETIC
ANALYSIS OF HAEMATOLOGICAL
DISORDERS:
SUPERVISOR: Mr EMMANUEL
PRESENTERS:
 MARLIUS M SANGU
 SARAFINA SANGA
 MICHAEL M SHAGEMBE
 JULIUS D DONARD
 GHATI M CHACHA
 GODFREY P. JENGELA

2. OUTLINES
• Introduction
• Methodologies used in molecular and cytogenetic analysis.
• Clinical applications of molecular and cytogenetic analysis.
• Advantages and disadvantages of molecular and cytogenetic analysis.

3. Introduction
Cytogenetics is the study of chromosomes and the related disease
states caused by abnormal chromosome number and/or structure.
Chromosomes: complex structure located in the nucleus, composed of
DNA, histone and non histone proteins, RNA and polysaccharides
The accuracy of cytogenetic analysis has been significantly improved
over the last 30 years due to technical advances regarding culture
methodology and banding techniques.
Cytogenetic analysis is essential in the diagnosis and prognosis of
hematologic malignancies.

4. Introduction
Examples of haematological abnormalities
• Leukemia
• AML t(8;21), t(15;17),inv(16),t(9;11),inv(3),t(6;9),t(1;22),-
5/5q,-7/7q
• CML t(9;22)
• ALL t(4;11),t(1;19),t(v;11q23q),t(12;21)

5. Introduction
• Acquired chromosomal abnormalities, structural or numerical, are
detected in malignant bone marrow cells in more than 75% of
patients with heamatologic malignancies, with an increasing
incidence due to the application of complementary detection
methods provided by molecular cytogenetics.
• Cytogenetic analysis involves the study of both the number and
structure of the chromosomes.
• Tissues that are appropriate for chromosome study:
Tissues that can be stimulated to undergo cell division in-vitro, eg,
chorionic villi, amniotic fluid, peripheral blood( lymphocytes), skin(
fibroblast), bone marrow.

6. METHODOLOGIES
DNA extraction
DNA can be extracted from blood or tissue sample
The quantity and quality of DNA obtained will vary depending on the
size, time from collection and cell count of the sample
DNA is tightly associated with many protein as chromatin, it
important to remove these as well as other cellular proteins to extract
the DNA .
This is achieved through the use of organic solvent, salt precipitations
or DNA affinity columns.

7. DNA Extraction cont…
An aqueous solution of DNA is obtained from which further purified
by precipitation
Currently, there are number of commercially available DNA extraction
kit for general and specialist application
These kits ,they produce good quality DNA from various starting
materials, they are also well reliable and cost effective
In additional automation can achieve simultaneous extraction of
larger number of sample which save time and by pass the use of
organic solvent and provide good quality control of the reagents used.

8. 1.CONVECTIONAL CYTOGENETIC
ANALYSIS(CCA)/CHROMOSOMAL BANDING
ANALYSIS
examines the patient’s chromosomes in a sample of cells.
Counting the number of chromosomes and evaluating their structure
(banding patterns) results in the construction of a karyogram
(chromosome card) and karyotype (formula).
The technique requires a sufficient number of dividing cells or
mitoses of acceptable quality, i.e. at the metaphase stage of the cell
cycle.
malignant cells derived from peripheral blood, bone marrow, lymph
node, spleen, effusions or extramedullary tissues are cultured in
order to obtain chromosomes

9. STEPS FOR CCA…..(summarized)

10. Advantages and Disadvantages of
conventional cytogenetic technique
• • Advantages
1. Enable the entire genome to be viewed at one time.
2. Suitable when a specific anomaly is suspected ( e.g. Philadelphia in
CML ) and as a general diagnostic tool to detect additional
chromosome abnormalities commonly seen in disease progression
of CML.
• Disadvantages
1. Detect major structural abnormalities (one band = 6mb of DNA ~
150 genes ).
2. Labor intensive and highly dependent upon operator experience
and skills.

11. 2. Polymerase chain reaction
PCR refers to the molecular technique which involves the use of thermally
stable DNA polymerase enzymes extracted from thermus aquaticus to
amplify a specific DNA fragment.
 Purpose of PCR;
amplification of a specific DNA fragment such that it can be visualised
using intercalating SYBR Safe added to agarose gels.
 Ethidium bromide, a mutagenic product, is no longer in use for health and
safety reasons.

12. Requirements of PCR.
, Two oligo-primers( oligonucleotides)
 The DNA template- from which the DNA fragment will be amplified
The four deoxynucleotide triphosphates (dATP, dTTP, dCTP and dGTP)
building blocks of the newly synthesised DNA,
A salt buffer containing MgCl2 .
The thermostable DNA polymerase (Taq polymerase).

13. Procedures…..(summarized)

14. Modifications and development of PCR
• Multiplex. More than one fragment can be amplified in the same
tube simply by adding in further primer pairs.
• Nested PCR. two pairs of primers are used; the second pair, located
within the sequence amplified by the first, allows products to be
generated from as little as a single cell.
• Long-range amplification. Fragments upward of 10 kb can now be
generated by PCR using modified polymerases.
• Automation. Involves the use of robots and 96-well plate technology.
• Automated fragment analysis. Use of gel electrophoresis for the
detection of fluorescently labelled PCR products on DNA fragment
analysers

15. Interpretation
If the amplification has been successful, a discrete fragment of the
expected size is seen in a SYBR Safe-stained agarose gel in all samples,
except in the NTC lane.
 If a product is seen in the NTC, then one of the solutions has been
contaminated and the results cannot be relied on.

16. Problems in PCR
The absence of a fragment in all tracks, including the positive control;
This can occur for a number of reasons, including poor quality template or
omission of one of the essential reagents.
magnesium concentration is too low (standard concentration 1.5mM)
or if the annealing temperature is too high.

17. 3. GAP- PCR
 Used to detect Large deletions.
 Primers located 5′ and 3′ to the breakpoints of a deletion will be too
far apart on the normal chromosome to generate a fragment in a
standard PCR.
For example, detection of deletions in α0 thalassaemia.

18. GAP-PCR……….

19. 4. FLUORECENT IN SITU HYBRIDIZATION(FISH)
A process which distinctly paints and detects RNA as well as DNA
Structures, numbers and location in place in the cell or in situ.
• FISH may be used with:
• Morphologically preserved chromosome preparations (Metaphase).
• Fixed cells or tissue sections (Interphase)
 Aids in gene mapping, toxicological studies, analysis of chromosome
structural aberrations, and ploidy determination.
use non-dividing cells as targets (interphase FISH) , allowing for the
identification of both numerical and structural chromosome
abnormalities in a large number of nuclei.

20. FISH…………..
Tissue samples for FISH analysis;
• Peripheral blood
• Fibroblasts from skin biopsy
• Epithelial cells from buccal smear
• Bone marrow
• Solid tumor biopsies

21. PROCEDURES OF FISH
• Slides preparation from cultured (Metaphase) or uncultured
(Interphase) cells and fixed sing standard cytogenetic procedure.
• Fixed cells are exposed to a probe (60-200–kb fragment of DNA
attached covalently to a fluorescent molecule).
• Denature the chromosomes
• Denature the probe
• Hybridization: The probe will hybridize or bind to its complementary
sequences in the cellular DNA
• Fluorescence staining
• The bound probe can be visualized under a fluorescent microscope
in the nucleus of the cel

22. POCEDURES………..(Summarised)
• Summarized diagram for FISH

23. TYPES OF FISH PROBES

24. FISH……………
Telomeric probes
• DNA probes specific to the telomeres of all human chromosomes.
• Useful for the detection of chromosome structural abnormalities
such as cryptic translocations or small deletions that are not easily
visualized by standard karyotyping.

25. FISH………..
Whole xsome painting probes(paints)
oComplex mixture of sequences from the entire length of specific
xsome.
oUsed to clarify complex translocations.
oCan’t detect intrachromosomal structural anomalies or alterations
involving centromeric and telomeric regions.

26. FISH…………

27. FISH…………
• Advantages of Interphase FISH
• Interphase cells for FISH do not require culturing of the cells and
stimulating division to get metaphase spreads
• interphase FISH is faster than methods using metaphase cells
• valuable for analysis of cells that do not divide well in culture, including
fixed cells.
• 200–500 cells can be analyzed microscopically using FISH
• the sensitivity of detection is higher than that of metaphase
procedures, which commonly examine 20 spreads.
• Monitor recurrent or residual disease in BMT pt.

28. Spectral karyotyping (SKY) and multiple
fluorescent hybridization (M-FISH)
• By mixing combinations of five fluorophores and using special
imaging software, can distinguish all 23 chromosomes by
chromosome specific colors.
• This type of analysis can be used to detect abnormalities that
affect multiple chromosomes as is sometimes found in cancer
cells or immortalized cell lines.

29. SKY………….
Advantages:
• Mapping of chromosomal breakpoints.
• Detection of subtle translocations.
• Identification of marker chromosomes, homogeneously staining regions, and
double minute chromosomes.
• Characterization of complex rearrangements.
 Disadvantages:
• Very expensive equipment.
• The technique is labor intensive.
• Dose not detect structural rearrangements within a single chromosome. • Low
resolution (up to 15 mb ).
• Specific, not a screening method.

30. Limitations of FISH
• The inability to identify chromosomal changes other than those at the
specific binding region of the probe.
• Preparation of the sample is critical in interphase FISH analysis
• To permeabilize the cells for optimal probe target interaction
• To maintain cell morphology.
• Cannot detect small mutations.
• Miss Inversions.
• Probes are not yet commercially available for all chromosomal regions
• Relatively expensive.

31. 5.FUSION GENE ANALYSIS
Detects genes that break and translocate to another chromosome
hence the formation of new fusion proteins with oncogenic effects.
 used in an analysis of minimal residual disease(MRD) in chronic
myeloid leukaemia (CML) and other leukaemias.
The presence of the fusion gene BCR-ABL( eg, in CML) can be
measured by detecting BCR-ABL RNA in the blood.

32. 6.Next Generation Sequencing
Sanger Sequencing could sequence only a single DNA fragment at a time by
capillary electrophoresis.
In NGS millions of DNA fragments can be sequenced simultaneously. Thus, it is
highly throughput parallel sequencing technique and allows thousands of
genomes to be studied in a short time .
Through NGS one can study not only the genome but also the transcriptome
(from RNA) and epigenome ( DNA methylation sites).
The common used NGS includes
 Genomics –whole genome sequencing, exome sequencing , targeted sequencing
 Transcriptomics-mRNA sequencing
 Epigenomics-CHIP sequencing ( chromatin immunoprecipitation) to study DNA-protein interactions
NGS is unraveling several novel mutations in genes involved in hematological
malignancies. The list of such mutated genes like IDH1, IDH2, DNMT3A and
SF3B1 is commonly increased

33. NGS :STEPS
Figure : Next-Generation Sequencing
NGS includes four steps:
(A) library preparation,
(B) cluster generation,
(C) sequencing, and
(D) alignment and data analysis.

34. STEPS FOR NEXT GENERATION SEQUENCING
1.Library preparation
The first step of the next generation sequencing allow the DNA or cDNA to
adhereto to the
sequencing flowing cell and allow the sample to be identified
2.Cluster generation
During the sequencing step of the NGS workflow, libraries are loaded onto a flow
cell and placed on the sequencer. The clusters of DNA fragments are amplified in a
process called cluster generation, resulting in millions of copies of single-stranded
DNA.

35. STEPS FOR NEXT GENERATION SEQUENCING
3.Sequencing
The process of determining the order of the nucleotide bases along a DNA
strand
4.Data allignment and analysis
is the process of subjecting a DNA, RNA or peptide sequence to any of a wide
range of analytical methods to understand its features, function, structure, or
evolution.

36. CLINICAL APPLICATIONS
1. Investigation of haemoglobinopathies;
a) Sickle cell diseases=Hbs genes detected by HPLC and sickling test.
=mutations –detected in PCR mutation rxn.
=no mutations-detected in wild-type PCR rxn.
b) β thalassaemia= commonly point mutations; detected by detected by
direct DNA sequence analysis.
c) α thalassaemia=mutations are large deletions.
 Although PCR amplification around the α globin locus has proved to be
rather difficult, the common deletions can now be identified by a
reasonably robust Gap-PCR. Now days multiplex PCR is used.

37. CLINICAL APPLICATIONS
2. Coagulopathies;
a) Thrombophilia screening; genetic risk factors found in patients with
venous thromboembolism (VTE). Mutations causing protein C,
protein S and anti-thrombin deficiency. An increased factor VIII level
is also a risk factor for VTE, but the genetic determinants of this are
unclear .
 Methodology-hydrolysis probe (TaqMan) based assay
b) Clotting disorders; Diverse mutations underlie haemophilia A and
haemophilia B .
 Methodologies; single-strand conformation polymorphism analysis
(SSCP), denaturing HPLC or direct DNA sequence analysis.

38. CLINICAL APPLICATIONS
3. Leukaemia and lymphoma;
a) Chronic myeloid leukaemia (CML)
The Philadelphia (Ph) chromosome resulting from the t(9;22)
translocation is detectable in 95% of cases of CML by routine
cytogenetic studies but the abnormality is sub-microscopic in the
remaining 5%.
presence of the BCR-ABL1 fusion gene can be confirmed by FISH, or
by detection of its transcript by RT-PCR.
Patients suspected of having CML should be tested for BCR-ABL1 for
definitive diagnosis.

39. CLINICAL APPLICATIONS
b) Follicular and mantle cell lymphomas.
- detection of a translocation involving BCL1 is indicative of mantle cell
lymphoma with t(11;14) (q13;q32), whereas identification of BCL2
involvement implies a follicular lymphoma with t(14;18)(q32;q21).
- Methodology: A fusion gene can be more readily demonstrated by
RT-PCR because exon-to-exon junctions are often highly consistent
and thus amenable to amplification using a common pair of PCR
primers.

40. CLINICAL APPLICATIONS
4. The lymphoproliferative disorders;
- DNA analysis may also help in determining whether a lymphocytosis
is monoclonal, oligoclonal or polyclonal.
- PCR has been used to detect rearrangement of the immunoglobulin
and TCR genes.

41. ADVANTAGES AND DISADVANTAGES OF
MOLECULAR AND CYTOGENETIC ANALYSIS
ADVANTAGES;
1. Help to diagnose diseases
2. Help to plan treatments
3. Help to find out how well treatments are working.
DISADVANTAGES.
1. Risk of infections during sample collection.
2. Interference of results by chemotherapies

42. REFERENCES
• Dace and Lewis practical haematology.
• Kearney L: The impact of the new fish technologies on the
cytogenetics of haematological malignancies. Br J Haematol 4: 648-
658, 1999.
• Chen Z and Sanderbrg AA: Molecular cytogenetic aspects of
hematologic malignancies: clinical implications. Am J Med Genet 115:
130-141, 2002.
• Shaffer LG, Slovak ML, Campell LJ, editors. ISCN 2009: an international
system for human cytogenetic nomenclature. Basel: Karger; 2009.