2023

SEKGOBELA TIISETSO
221003584
HAEMATOLOGY ASSIGNMENT
PRACTICAL 1 : LEUCOCYTE DIFFERENTIAL COUNT(MANUAL METHOD

1.characteristics of a good smear

➢ A good blood smear must be thick at the drop end and thin at the opposite
end.
➢ The blood smear should occupy the central portion of the slide and should not touch
the edge nor extend into the last 15 mm of the slide.
➢ Does not contain any lines or holes
➢ A good smear is tongue shaped
➢
2. STAIN USED TO PREPARE THE SMEAR
➢ Eosin Stain – Cytoplasmic stain
➢ Methylene blue – Cationic stain
3. a) Relative white cell count and Absolute white cell count

Relative WCC = Number of cells counted/ 100* 100

Relative WCC = 100/ 100* 100
Relative WCC = 100 %
Absolute WCC = Total WCC x Relative WCC
Absolute WCC = 7.2 x 100%
Absolute WCC = 720 x10^9/L
b) red cells and abnormalities observed- There is rouleaux formation

c) white cells and abnormalities observed- Some neutrophils are hypersegmented while
others have a band nucleus

d) Platelets and abnormalities observed - The picture shows large platelets

4 CORRECTED WHITE BLOOD CELL COUNT
𝑇𝑜𝑡𝑎𝑙 𝑊𝐶𝐶 𝑋 100
Corrected white cell count = (100+𝑁𝑅𝐵𝐶)

= 7.2/ (100+12) *100

= 6.4x10^9/L

5. POSSIBLE CAUSES
a) Neutrophilia
➢ Bacterial infections
➢ Pregnancy
b) Neutropaenia
➢ Bone marrow failure
➢ Chemotherapy
c) Lymphocytosis
➢ Acute or chronic infections
➢ Lymphoid leukaemias
d) Lymphocytopaenia
➢ Severe bone marrow failure
➢ Corticosteroids and other immunosuppressive therapy
e) Monocytosis
➢ Chronic bacterial infections: tuberculosis,
➢ Connective tissue diseases: SLE, rheumatoid arthritis

f) Eosinophilia
➢ Allergic diseases
➢ Parasitic diseases
g) Basophilia
➢ Myxoedema, during chickenpox infection
➢ Ulcerative colitis.

6.REFERENCES
➢ Lewis, S.M., et.al (2017) Dacie and Lewis Practical Haematology. 12th
Edition. Churchill.Elsevier.China
➢ Hoffbrand, A.V. and Moss, P.A.H( 2016). Essential Haematology. 7 th Edition.
Blackwell Science. West Sussex
➢ Drew Provan, et al.(2004). Oxford Handbook of Clinical Haematology. 2nd
edition.Oxford University Press.New York
PRACTICAL 2 : ACUTE LEUKAEMIA
1.FBC SCATTERGRAM

2. The principle of flow cytometry is a technique used to analyze and quantify the physical
and biochemical properties of individual cells or particles suspended in a fluid. It involves the
use of fluid dynamics, optics, and fluorescence detection to examine and measure
characteristics such as cell size, granularity, and specific markers.
Flow cytometry works by passing cells or particles through a narrow, focused stream of fluid.
As they pass through a flow cell, they are illuminated by laser beams, which generate
scattered light and fluorescence emission. The scattered light provides information about cell
size and granularity, while fluorescence emission allows the detection and quantification of
specific markers or molecules within the cells.

3. 3.Pattern results
CD34 (-)
HLA-DR(-)
MPO(weakly positive)
CD13(-)
CD33(+)
CD45(weak positive)
,CD117(+)
4. a)Myeloperoxidase
Peroxidase is found primary and secondary granules of myeloid cells and azurophilic
granules of monocytes.Smears are incubated in buffered hydrogen peroxide with an
appropriate substrate.Presence of peroxidase oxidises the substrate, resulting in the
precipitation of a coloured product at the site of enzymatic activity. The reaction is
distinguished by a brown color and granular appearance .The stain is used to differentiate
between AML (>3% blasts are positive) and ALL (<3% blasts are positive)
b)Neutrophil Alkaline Phosphatase(NAP)
Alkaline phosphatase can be demonstrated in the tertiary granules of mature neutrophils and
bands from heparinised samples.The NAP level activity increases with maturity which is
when the enzyme hydrolyses the substrate naphthol-AS BI phosphate to phosphate and an
aryl naphtholamide which then binds to a diazonium salt (fast red-violet salt LB) and
precipitates out at the site of enzyme activity.The staining intensity of 100 neutrophils and
bands are then graded and is proportional to the amount of enzyme present.
c)Sudan black
This is a fat soluble dye that irreversibly stains lipids such as phospholipids, sterols and
fats,these are present in both primary and secondary granules of granulocytes and
lysosomal granules of monocytes.The reaction is seen as black and granular pigments , the
reaction gives a comparable staining pattern to myeloperoxidase .It is used to differentiate
between AML (>3% blasts are positive) and ALL (<3% blasts are positive)
5.FISH PRINCIPLE
The elements involved in FISH are a DNA probe and a target sequence ,prior to
hybridization of the DNA probe ,it is labelled indirectly with a hapten (left panel) or directly
using the incorporation of a fluorophore (right panel), the labelled probe and the target DNA
are then denatured. The combination of the denatured probe and the target allows the
annealing of the complementary DNA sequence .In the event that the probe is indirectly
labelled ,an extra step is employed to visualize the on-fluorescent hapten which uses an
enzymatic or immunological detection system .It is then that the signals are evaluated by a
fluorescent microscope.
6.REACTION SEEN
a) Myeloperoxidase -The reaction is brown and granular
b) Sudan black -The reaction is brown and granular pigments
c) NAP - The reaction is blue and granular
7.RESULTS OF ACUTE LEUKAEMIA MORPHOLOGY
a) Relative white cell count for promyelocytes
Relative WCC = Number of cells counted/ 100* 100
Relative WCC = 3/ 100* 100
Relative WCC = 3%
b) Red blood cells
c) White cells and abnormality sketch
d) Platelets -Smear : Giant platelets FBC: Thrombocytopaenia
e) Three cell lines on ALL slide-
Red blood cells- FBC:
White blood cell- FBC: leucocytosis
Platelets- FBC: Thrombocytopaenia
7. CELLULAR FEATURE USED TO DIFFERENCIATE BETWEEN AML AND ALL
-aure rods

8.REFERENCES
➢ Lewis, S.M., et.al (2017) Dacie and Lewis Practical Haematology. 12th
Edition. Churchill.Elsevier.China
➢ Hoffbrand, A.V. and Moss, P.A.H( 2016). Essential Haematology. 7 th Edition.
Blackwell Science. West Sussex
➢ Drew Provan, et al.(2004). Oxford Handbook of Clinical Haematology. 2nd
edition.Oxford University Press.New York

PRACTICAL 3: CHRONIC LEUKAEMIA

1.CML SLIDE RESULTS
a) Relative white cell count
Relative WCC = Number of cells counted/ 100* 100
Relative WCC = 50/100*100
Relative WCC = 50%
b) Red cells
There are nucleated red blood cells and there is rouleaux
c) White cells
The cells are Immature and granulopoiesis
d) Platelets
The platelets are giant
e) CML stage
Stage 4 because there are large platelets which indicates thrombocytopenia
2 EXPECTED RESULT FOR CML DIAGNOSIS
a) PCR- fusion gene BCL-ABL
b) Cytogenetics-philadelphia chromosome t (9;22)
c) FISH-ph chromosome positive

3 RESULTS ON CLL SLIDE

a) Relative white cell count
b) Red cells
- spherocytes showing polychromasia
- Normocytic Normochromic cells
c) White cells
- Lymphocytosis
-there are blast and smudged cells
d) Platelets
-thrombocytopenia

4 HCL SLIDE-
- Has cytoplasmic projection
-Has a high nucleus to cytoplasm ratio
-The nucleus is eccentrically located
- Chromatin is fine

5.REFERENCES
Lewis, S.M., et.al (2017) Dacie and Lewis Practical Haematology. 12th Edition.
Churchill.Elsevier.China
Hoffbrand, A.V. and Moss, P.A.H( 2016). Essential Haematology. 7 th Edition. Blackwell
Science. West Sussex
Drew Provan, et al.(2004). Oxford Handbook of Clinical Haematology. 2nd edition.Oxford
University Press.New York

PRACTICAL 4: PLASMA CELL DISORDERS

1. PROTEIN ELECTROPHORESIS PRINCIPLE
Electrophoresis is the separation of analytes on a medium using an electrical current. It uses
the migration of charged solutes or particles in an electrical field to separate the different
proteins. Charged particles migrate as zones on a porous medium such as agarose gel after
the sample is mixed with a buffer. The process generates an electropherogram or display of
protein zones that are usually sharply separated from neighbouring zones on the supporting
medium. The zones are stained with a protein stain specific for the protein class of interest
2.PROTEIN ELECTROPHORESIS GRAPH
Graph showing normal serum protein electrophoresis ans that of multiple myeloma with an
indication of M spike
3.PLASMA CELL

Eccentric nucleus
Prominent nucleoli
Abundant basophilic cytoplasm
Nucleus is round or oval with clumped chromatin

4. MULTIPLE MYELOMA

a) Relative white cell count.

𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑
Relative white cell count % = × 100
100

N=49 % = 49
L =34%=34
M= 14%=14
E= 1% =1
B= 0%=0
Relative white cell count = 98%
Number of cells = 98 cells and 2 plasma cells

b) Red cell morphology

- Low haemoglobin levels indicating anaemia

- Rouleaux Formation
c) White blood cell morphology
- Leukopenia
-Atypical Plasma Cells
d) Platelet morphology
- Thrombocytopaenia
- Abnormal Platelet Size and Shape
5.PLASMA CELL LEUKAEMIA
a) Red cell morphology
- Rouleaux Formation
- Poikilocytosis-spherocytes,teardrop and elliptocytes cells
-Hypochromia
b) White cell morphology
- Atypical lymphocytes
-Leukocytosis
c) Platelet
-large morphology
d) Difference between plasma cell leukaemia and multiple myeloma
-Plasma cell leukaemia is a rare form multiple myeloma and is more aggressive

5.REFERENCES

➢ Hoffbrand, A.V. and Moss, P.A.H( 2016). Essential Haematology. 7th edition.
Blackwell Science. West Sussex
➢ Drew Provan, et al.(2004). Oxford Handbook of Clinical Haematology. 2nd
edition.Oxford University Press.New York
➢ Burtis,C.A. & Bruns,D.E (2008). Tietz Fundamentals of CLINICAL
CHEMISTRY AND MOLECULAR DIAGNOSTICS.7th edition. Elsevier.
Missouri

PRACTICAL 5: MYELOPROLIFERATIVE DISORDERS

1.
a) Red cell morphology
- There are tear drop shaped ,nucleated and immature
b) White cell morphology-
c) Platelet

- giant and bizarre platelets are present on the smear

d) Bone marrow findings-
e) Possible genetic mutations that attributed to the condition

-Mutations on JAK2, MPL, CALR, and TET2 genes, MPL

2. ESSENTIAL THROMBOCYTOPAENIA
a) Red cell morphology
- Most of them have normal shapes, and only a few have irregular morphology
b) White cell morphology
-giant cells
c) Platelet
- giant platelet
- Thrombocytosis
d) Bone marrow findings

- increased large hyper lobulated megakaryocytes

3. POLYCYTHAEMIA RUBRA VERA

a) Peripheral blood smear
-Has neutrophil leucocytosis

-nucleated red blood cells

- The smear shows circulating erythroid progenitor cells

- Normocytic and normochromic red cells
- Normal white cell morphology
Bone marrow findings
-The bone marrow shows hypercellularity and trilineage hyperplasia with abnormal
megakaryocytes (clustering with giant forms and increased ploidy),Increased fibrosis may be
present

4.REFERENCING

➢ Hoffbrand, A.V. and Moss, P.A.H( 2016). Essential Haematology. 7 th Edition.

Blackwell Science. West Sussex
➢ Drew Provan, et al.(2004). Oxford Handbook of Clinical Haematology. 2nd
edition.Oxford University Press.New York