Names: SERAHO MBULUYO

Student number: 220048061

Vaal university Of technology

Haematology Assignment
PRACTICAL 1 : LEUCOCYTE DIFFERENTIAL COUNT(MANUAL METHOD)
1)
a. No stain precipitate is present on the smear
b. Stain should not be too dark or too pale.
c. the slide is neither too thin nor too thick.
d. It should end about two-thirds to three-fourths of the way down the slide
2) wright stain, Group- Romanowsky stain
3) result of practical
a) 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) Sickle cells and target cells present

c) Metamyelocytes and Band neutrophils present

 d) Giant Platelets present

4) correct WBC = Total WBC/ (100 + NRBC) *100

= 7.2/ (100+12) *100
= 6.4x10^9/L
5)
a) Neutrophilia: Rheumatoid Arthritis and Pregnancy
b) Neutropoenia: HIV and Tuberculosis
c) Lymphocytosis: Hepatitis and Hepatitis
d) Lymphocytopenia: AIDS and COVID19
e) Monocytosis: Mononucleosis and Lupus
f) Eosinophilia: Parasitic and fungal disease
g) Basophilia: Myelofibrosis and Polycythaemia vera
6.REFERENCES
 Adewoyin AS, Nwogoh B. Peripheral blood film - a review. Ann Ib Postgrad Med.
2014 Dec;12(2):71-9. PMID: 25960697; PMCID: PMC4415389.
 A. V. Hoffbrand, Paul A. H. Moss 2016. Hoffbrand’s Essential Haematology, Seventh
Edition. Wiley Blackwell.

PRACTICAL 2 : ACUTE LEUKAEMIA

1)

2) The basic principle of flow cytometry is the single-file passage of cells in front of a laser
for detection, counting, and sorting. The laser excites fluorescently labelled cell
components, which then emit light with a range of wavelengths. The quantity and kind of
cells present in the specimen are then determined by measuring the fluorescence. Finally,
we examine the antigens that are located on the cell. On the surface of every one of our
cells are proteins. If our cell contains that antigen, we add an antibody that has been
labelled with a fluorescent marker, and the antibody will bind to the cell and release light.
The presence of the antigen on the cell is shown by the presence of light
3)
 CD 34- negative
 HLA-DR- negative
 CD 13- positive
 CD 33- positive
 CD 45- positive
 CD 117- positive
 MPO- weakly positive
4)
a) MYELOPEROXIDASE: Myeloperoxidase in the leukocyte granules oxidizes
substrates from an insoluble blue/brown derivative to a colorless form when
hydrogen peroxide is present. Substrates include benzidine, 3,3′-
diaminobenzidine, and p-phenylenediamine dihydrochloride.
b) NEUTROPHIL ALKALINE PHOSPHATASE: Neutrophilic-Leukocyte The diazotization-
coupling concept is the foundation of alkaline phosphatase stain. Alkaline
Phosphatase converts naphthol AS-BI Phosphoric Acid into -naphthol at pH 9.2–
9.8. In the cytoplasm, an insoluble colored precipitate will be formed by the
stable diazonium salt and naphthol.
c) SUDAN BLACK: Sudan Black is a somewhat basic dye that reacts with acidic
groups in complex lipids to stain them as well. a substitute for the Sudan Black B
stain. 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) Fluorescent in situ hybridization: The basic idea is to use a nucleic acid probe to hybridize
nuclear DNA from either interphase cells or metaphase chromosomes attached to a
microscopic slide. The probes are either directly labelled by incorporating a fluorophore or
indirectly by incorporating a hapten. Following denaturation, the labelled probe and the
target DNA are combined, allowing complementary DNA sequences to anneal.
6)
a) A brown cytoplasmic granulation that is darker than the background staining of
red blood cells is visible in the cell precursors.
b) Sudan Black: At the location of granule formation, mature myeloid precursors
exhibit intracellular staining that is black and dark blue.
 c) NAP: Mature neutrophils' secondary granules exhibit a blue granular response in
the smear.
7) Results of acute leukaemia morphology (only count the AML slide, but look at ALL slide to
compare):
a) Relative WCC = Number of cells counted/ 100* 100
Relative WCC = 3/ 100* 100
Relative WCC = 3%
b) haemoglobin level is low, causing anaemia, and red blood cells are very few and
poikilocytic
c) Eosinophilic big immature lymphocytes are the type of white cells seen on the ALL
slide. The white cells seen on the APL slide are myeloblasts, which lack granules and
have basophilic cytoplasm and a bilobed nucleus.

d) Platelets-smear: Giant platelets, extremely low platelet count, the slides

demonstrate thrombocytopenia
e) Because ALL slides have thrombocytopenia due to the low platelet count (75 x
109/L), Because WCC is high (35x 109/L), ALL slides have leucocytosis. There are not
many red cells on the slide because Hb is low (8g/dL).
7) cellular feature used to differentiate between AML and ALL: Auer 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

PRACTICAL 3: CHRONIC LEUKAEMIA

1)
a) Relative WCC = Number of cells counted/ 100* 100
Relative WCC = 50/100*100
Relative WCC = 50%
b) Nucleated RBCS, Rouleaux production in red cells under anisopoikilocytosis
conditions.
c) White cells are neutrophils with hypersegmented nuclei, some with band cells, and
others with vacuoles and myelocytes. The cells are immature and granulopoiesis
d) Platelets are giant, come in a variety of sizes, shapes, and sizes.
 e) CML stage 1
- the slide shows enormous proliferation of granulocytic cells (neutrophil, Eosinophil,
and basophil) and mature neutrophils and myelocytes.

2)
a) PCR: BCR-ABL mRNA
b) Cytogenetics: Philadelphia chromosome t(9:22)
c) FISH: pH chromosome positive

3)
a) Relative WCC = number of cells counted/ 100*100
Relative WCC = 100/100*100
Relative WCC = 100%

b) - spherocytes showing polychromasia

- Normocytic Normochromic cells

c) White cells are mostly lymphocytes and smudge cells, which are shattered
lymphocytes during slide preparation.
d) Thrombocytopenia

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

5.REFERENCES
 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

Under the influence of an electrical current, serum electrophoresis can separate the various
components of blood proteins into bands or zones based on their electrical charge. Protein
zones are segregated from neighbouring zones. The stains then highlight these zones.
2)

3)
 It has a round eccentric nucleus
 It has an abundant basophilic cytoplasm
 It has a perinuclear halo
 clockface chromatin is also present

4)
a) Relative WCC = Number of cells counted/ 100* 100
Relative WCC= 21/100* 100
Relative WCC = 21%
b) Low haemoglobin levels indicating anaemia, The red cells have a rouleaux formation
and a changing size centre pallor. Some red cells are macrocytic (bigger than regular
red cells), whilst others are microcytic. Poikilocytosis is minor among the red cells.
c) Leukopenia, Atypical Plasma Cells , The white cells contain neutrophils with
multilobed nuclei, as well as a few monocytes with eccentric nuclei and lymphocytes
with a high nucleus to cytoplasmic ratio.
d) Abnormal platelet in size and shape, thrombocytopaenia

5.)
a) Red cells have rouleaux development, Poikilocytosis-spherocytes, teardrop and
elliptocytes cells, Hypochromia
b) White cell-plasma cells dominate the smear, with an eccentric nucleus and basophilic
cytoplasm. There are also lymphocytes with a high nucleus-to-cytoplasm ratio. Atypical
lymphocytes, Leukocytosis
c) Platelet- There are a few big platelets scattered around the slide.
d) Plasma cell leukaemia is defined by abnormally high numbers of plasma cells in the blood,
whereas multiple myeloma is defined by plasma cell accumulation in the bone marrow

5.REFERENCES

 Burtis,C.A. & Bruns,D.E (2008). Tietz Fundamentals of CLINICAL CHEMISTRY AND

MOLECULAR DIAGNOSTICS.7th edition. Elsevier. Missouri

 Hoffbrand, A.V. and Moss, P.A.H( 2016). Essential Haematology. 7th edition.
Blackwell Science. West Sussex

PRACTICAL 5: MYELOPROLIFERATIVE DISORDERS

1)
a) some are teardrop-shaped, while others appear undeveloped or juvenile. Other red cells
are target cells because they have a round shape with a circular core pallor. A nucleated red
cell is also present.
b) White cells feature a non-segmented nucleus with granules (eosinophils), a banded and
segmented nucleus, and a high nucleus to cytoplasmic ratio (lymphocytes).
c) Platelets- there are big and unusual platelets on the smear.
d) Bone marrow is typically unavailable due to a dry tap.
e) Three probable myelofibrosis genetic mutations
(1) CALR
(2) JAK2V617F
(3) MPL

2)
a) Red cells: the majority of them have typical forms, with only a few having irregular
morphology.
b) White cells: There are fewer neutrophils with non-segmented nuclei. They contain fewer
segments than mature neutrophils.
c) Platelets - There are numerous normal, tiny, and unusual platelets on the slide. Giant
platelets, thrombocytosis
d) Increased large hyper lobulated megakaryotes will be found in bone marrow

3)
 Have neutrophil leucocytosis, nucleated red blood cells,
 Have an abundance of normal, big, and unusual platelets
 Normal white cell morphology
 The smear shows circulating erythroid progenitor cells
 Have hypercellular bone marrow

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