Myeloperoxidase (MPO): is present in the first and second granules of granulocytes.

With acute
leukaemia demonstrating the presence of myeloid component. Staining granules and Auer rods
brown. Used to differentiated between AML (+ve) and ALL (-ve)

Sudan black: It’s a lipophilic dye, which stains unidentified component in myeloid granules, some
monocytes and eosinophils. Provides the same information or results as MPO (+ve for myeloblastic
leukaemia, granulocytes and monocyte and –ve for ALL). Stains granules and Auer rods black

Reticulocytes stain: It’s a supravital stain which exposed unfixed cells to new methylene blue. The
reticulation (RNA) stains blue-purple, while the RBC stains greenish blue. Done by mixing equal
volume of blood and stain (methylene blue). Followed by incubation in water bath (15-20 mins)
Make a smear, allow to dry and observe under the microscope.

Perl’s Prussian blue: Used to stain haemosiderin (iron). Containing ferrocyanide, staining iron blue.
Normally, there is no stainable iron in mature peripheral RBC. The developing erythroblast in the BM
contains 1.5 granules of iron. Sideroblasts are nucleated RBC which contains iron. Siderocyte are
mature RBC containing iron
Periodic acid schift: PAS stains the carbohydrates ( more especially glycogen). Providing positive
staining seen in lymphocytes, abnormal erythroblast, megakaryocytes (platelets) and neutrophils.
Reaction product colouration is red, pink or purple. Used in the diagnosis of ALL (+ve), but the
immunophenotyping results is more appropriates for diagnosis. L1 and L2 (+ve) and L3 (-ve),
myeloblast (-ve).

Acid Phosphatase: This enzyme is presence in all haemopoietic cells and is used to diagnose T-ALL
and hairy cells leukaemia (TRAP/ tartrate resistant). B-cells leukaemia (-ve) or showing weak
reaction. Reaction product red.

Romanowsky stains: Commonly used in routine staining and this stain depends on 2 component;
azure B and eosin Y. 1-A basic or cationic dye (blue or violet colour), methylene blue or azure B.
Binds to the nucleic acids (DNA/RNA), granules of granulocytes (neutrophils and basophils). 2-An
acidic or anionic dye (red or orange colour), eosin y. Binds to granules of eosinophils and
heamoglobin

Heinz bodies staining

•Prepared by dissolving 0.5g of methyl violet dye in 100ml of 9 g/l of NaCL and filter.
•Followed by the addition of 1 volume of blood to 4 parts or volume of methyl violet solution.
•Let the solution stand for 10 minutes at RT and made a smear with a drop of the mixed solution
and air dry.
•Heinz bodies (denatured Hb) will appear purple.
•Heinz can stain with supravital, methyl violet, new methylene blue, brilliant cresyl blue and
rhodanile blue.
•Mostly (Heinz bodies) found in splenectonized individuals or patients.
•Also commonly seen due to exposure of oxidant drug (G6PD deficiency).
•Heinz bodies are larger than pappenheimer bodies

Haemoglobin H inclusion staining

•Prepared by mixing 2 parts of blood and 1 part of stain solution.
•Followed by incubation at RT (370C) for 4 hours.
•Make a smear, allow to dry and examine for reticulocyte count.
•Inclusion bodies will appear as multiple greenish-blue dots appearing like golf ball (can be
distinguished or differentiated from retics as they exhibit an uneven reticular infrequent fine dots.
•HBH are abnormal Hb tetramer of beta chains (alpha chains absent).
•Mostly observed (HbH diseases) in alpha thalassaemia, and thalassaemia trait (few cases).

Leishman stain
•Prepared by weighing out 0.2g of the powered dye
•Transferring into 200-250 ml conical flask.
•Addition of 100ml of methanol
•Heat the mixture at 500C for 15 minutes.
•Allow to cool and filter.
•Make a blood smear and air dry it.
•Stain the smear for 2 minutes.
•After 2 minutes, pour a equal (double) volume of both stain and water for 5-7 minutes.
•Wash or rinse in a stream water buffered solution still a pinkish tinge coloration is observed for 2
minutes.
•Blot dry the smear in upright position and observe under the microscope

Giemsa stain
•Prepared by weighing 1g of powdered dye
•Transferring into 200-250ml conical flask and
•Adding 100ml of methanol
•This mixture ( powered dye and methanol) are heated at 500C for 15 minutes.
•Allow to cool and filter.
-staining procedure
1. prepare blood film, air dry and place in a staining rack
2. fix with methanolfor 3 minutes
3. dilute the stain 1 in 10 with distilled water or buffer solution of pH 6.8
4. cover the blood film with diluted Giemsa stain for 10 min
5. wash with distilled water, dry and examine under microscope

T-cell prolymphocytic leukaemia: Similar to B-cell PLL, having elevated WCC. Lymphadenopathy is
more marked, skin lesions and serous effusions are common. CD4+
B-cell prolymphocytic leukaemia: Similar to CLL, but has the appearance of prolymphocytes in
blood. These prolymphocytes is twice the size of CLL lymphocytes with central nucleolus. B-cell PLL is
greater than T=cell PLL. PLL is characterized by massive splenomegaly, without lymphadenopathy.

Hairy cell leukaemia

•Uncommon B-cell lymphoproliferative disorder of male dominance (40-60 years).
•Anaemia, infection and splenomegaly and lymphadenopathy is very uncommon.
•Present of pancytopenia and monocytopenia
•Variation number of lymphocytes present in blood film with villous cytoplasmic projections.
•Flow cytometry reveals CD19+, CD5+, CD22, FMC7 and CD 103+.
•Stain +ve for TRAP
•BM shows mild fibrosis and diffuse cellular infiltrate.

Large granular lymphocytic leukaemia (LGL-L)

•Characterized by the present of circulating lymphocytes, having abundant cytoplasm and large
azurophilic granules.
•Expression of CD16, CD56 and CD57.
•Cytopenia (neutropenia) is the main clinical problem.
•Anaemia, splenomegaly and arthropathy is common
Adult T-cell Leukaemia/lymphoma (ATLL): Associated with human retrovirus, human T-cell
leukaemia/lymphoma virus type 1. Lymphocytes of ATLL is bizzare in morphology with convoluted
clover leaf nucleus and consistent CD4+ phenotype. Hypercalcaemia, skin lessions,
hepatosplenomegaly and lymphadenopathy.

Multiple myeloma
•Neoplastic disorder characterized by plasma cell accumulation in BM.
•Myeloma cell; post-germinal centre plasma cell undergoing immunoglobulin class switching,
somatic hypermutation and paraprotein secretion in serum.
•With the presence of monoclonal protein in serum and urine.
•Common at the age of 40 years
-clinical feature:
•Bone pain, infection anaemia, hypercalcaemia, polyuria, mental disturbance, thrombocytopenia.
•Abnormal bleeding tendency, amyloidosis, purpura, visual failure, heart failure
Lymphomas
•These are group of diseases caused by malignant lymphocytes which accumulate in the lymph
nodes.
•Lymphadenopathy is the main clinical characteristic.
•Infiltrates in organ outside the lymphoid tissue.
•Subdivided into Hodgkin and non-Hodgkin lymphoma.
•Reed-Sternberg cells is the main histological findings or features.

Hodgkin Lymphoma (HL)

•Characterized by reed Sternberg cells, having large mirror image nuclei) and non malignant reactive
cells (lymphocytes, eosinophils, neutrophils, fibroblast, histocytes, collagen and plasma cells.
•It localizes from the beginning and later spreads to other lymphoid tissues.
•HL arises in the lymph nodes and commonly seen in adults.
•The tumour cells appear as ringed with T-lymphocytes
-Clinical features of HL
•Enlarged lymph nodes and splenomegaly
•Fever, pruritus, weight loss, profuse sweating, weakness, anorexia, fatigue, and cachexia
Classical types consist of 4 subtypes.
1. Nodular sclerosis are frequent mostly.
2. Mixed cellularity having pleomorphic infiltrate eosinophils, histocytes plasma cell, fibroblast, and
lymphocytes
3. Lymphocyte rich histology with good prognosis having reed Sternberg cells (RS)
4. Nodular lymphocyte which are depleted with no RS cells, with many features of non-Hodgkin
lymphoma (poor prognosis).

Chronic myeloid leukaemias

•Clonal disorder of pluripotent stem cell, accounting for 15% of leukaemias and occur at any age.
•Rare below 20 years and median with age onset of 40-50 years.
•Resulting in BCR-ABL at translocation between 9 and 22 of Philadelphia chromosome
t(9;22)(q34;q11).
- Clinical features: Anaemia with pallor, dyspnoea and tachycardia, Bruising, epistaxis, menorrhagia
or haemorrhage, Splenomegaly associated with pain and discomforts. Hypermetabolism such as
weight loos, anorexia or night sweats. Gout or renal impairment due to hyperuricaemia, (due to
excessive purine breakdown)

Treatment of CML: Tyrosine kinase inhibitors, Monitoring of response to imatinib, BCR-ABL mutation
screening, Second generation tyrokinase kinase inhibitors, Overall response to imatinib therapy,
Chemotherapy, Stem cell transplantation
Chronic neutrophilic Leukaemia
•Mild splenomegaly
•No inflammatory or other causes of neutrophilia
•No evidence of any myeloproliferative disorder
•Cytogenetic normal with variable prognostic

Chronic eosinophilic Leukaemia

•Clonal disorder of persistent eosinophilia
•With interstitial lesion in chromosome 4 resulting in the fusion gene FIP1L1-PDGFRA
•Less frequent cytogenetic or molecular defects
•More than 5% but less than 20% of blasts in BM.
•Various organs (skin, lung, GI,CNS) damage due to cell infiltration.
•Blast less than 5% is diagnosed as hypereosinophilic syndrome

Polycythaemia vera
•It is a clonal stem cell disorder where by there is an increased in red cell volume.
•Resulting from somatic mutation of single haemopoietic stem cell giving rise to progeny
proliferation.
•JAK2 mutation present in 95% in haemopoietic stem cell.
•With the over production of red cells, granulocytes and platelets.
Clinical features of PV
•Headaches, dyspnoeas, blurred vision, and night sweats
•Ruddy cyanosis, conjunctival suffusion, and retinal venous engorgement
•Splenomegaly, haemorrhage, hypertension, and gout.

Essential thrombocythaemia
•Clonal disorder of multipotent stem cell characterized by;
•Increased of platelet count due to megakaryocyte proliferation and over production of platelets.
•Ph chromosome or BCR-ABL1 rearrangement are absent.
•No collagen fibrosis in BM.
•JAK2 mutation is positive in half of the patients
(asymptomatic) and diagnosed accidentally during routine FBC.
Clinical features of ET
•Splenomegaly or splenic atrophy, burning sensation felt in the hand or feet.
•Haemorrhage, thrombosis, Budd-Chiari syndrome
Signs and symptom: itching, fatigue, blood clot, frequent headaches, enlarged spleen

Myelofibrosis (MF)
•Clonal stem cell disease characterized by the proliferation of megakaryocytes (predominantly) and
granulocytes in BM.
•The fibrosis of the BM is secondary to hyperplasia of abnormal megakaryocytes.
•Thus, fibroblast are stimulated by platelet derived growth factor and other cytokins which are
secreted by megakaryocytes and platelets.
•JAK2 mutation occurs in 50% of patients
Clinical Features MF: Anaemia common with older people, massive splenomegaly, weight loss, fever,
anorexia, and night sweats, Bleeding problems, gout and bone deformities, Lymphadenopathy

Non-Hodgkin lymphoma (NHL)

•Are large groups of clonal lymphoid tumours (85% of B cell and 15% of T or NK cells).
•Characterized by irregular pattern spread, with a significant proportion of individuals developing
extranodal disease.
•The cause of NHL is unknown, but infectious agents (Epstein Barr virus, HIV, hepatitis C, Malaria,)
are causes of some particular types.
•NHL can be divided as B or T cell in origin, and classified as low, intermediate and high grade.
•This disorder arises in lymph node (comprising mainly of lymphocytes) and histocytes (rarely)

Low- and High-grade non-Hodgkin lymphomas

•Diverse group of diseases, with highly proliferative and rapidly fatal diseases.
•Low grade are indolent (little or no pain) and response well to chemotherapy.
•High grade are aggressive and difficult to cure but often more curable.
•NB Lymphomas involves spleen, lymph nodes or other solid organs meanwhile leukaemias involves
bone marrow and circulating tumour cells.
•Clinical features of NHL; Lymphadenopathy, fever, night sweats, weigh loss, anaemia, infection
(neutropenia or purpura and thrombocytopenia).
•Splenomegaly, hepatomegaly, Sezary syndrome, mycosis fungoides.
Myelodysplasia
•Are group of clonal disorders characterized by haemopoietic (blood and BM) abnormalities.
•Which changes or evolves into acute leukaemia which was previously known as pre-leukaemia.
•Predominance in male slightly of age 70 and 25% of age 50 years.
•Pathogenesis is unclear, but assumed to begin with genetic damage to multipotent haemopoietic
progenitor cell.
Caused by:
•Primary; with unknown causes
•Secondary; due to chemotherapy, radiotherapy, exposure to benzene
Clinical features of Myelodysplasia: Anaemia with sign and symptoms of fatigue, pallor, Infection,
Bruising or bleeding, Impaired neutrophils, monocytes and platelets, Dysplastic features of BM

Chronic lymphoid leukaemias

•Common in elderlies of age 60 and 80 years, with an unknown aetiology.
•CLL or also known as B cell disease is characterized by the infiltration of small lymphocytes in the
BM.
•Tumour cell appears to mature B cell with weak surface expression of IgM or IgD immunoglobulin.
•Cells accumulates in liver, spleen, blood, BM, and lymph nodes, which increases production and
prolongs lifespan with impaired apoptosis.
-Solitary plasmacytoma: isolated plasma tumours (soft tissue and bone)
-Plasma cell leukaemia: characterized by high number of circulating malignant plasma cells.
-Amyloidosis: characterized by extracellular deposition of protein in an abnormal fibrillar form..
-Systemic amyloid light chain amyloidosis: caused by deposition of monoclonal light chains, which is
produced from clonal plasma cell proliferation.
•Hyperviscosity syndrome: caused by polycythaemia having symptoms of confusion, muscle
weakness, visual disturbance, lethargy, congestive heart failure.

Mastocytosis
•Clonal neoplastic proliferation of mast cells, which accumulate in one or more organ.
•Mast cells are derived from haemopoietic stem cells.
•Systemic mastocytosis is the clonal myeloproliferative disorder of the BM, heart, spleen, lymph
nodes and skin.
•KIT mutation Asp816Val is detected in majority of individuals.
•Symptoms include; pruritus, flushing, abdominal pain and bronchospasm.
•Serum trypase is increased

Thick smear preparation

•Prepared by collecting blood (venipuncture) in a clean test tube.
•Collect blood with a capillary tube and add 2 large drops at the center of the slide.
•By holding the slide with your thumb and index finger, shake the slide gently to spread the blood
about 10mm in diameter.
•Allow the slide to air dry for 20-30minutes and stain with Romanowsky.
•Thick smear is more sensitive in detecting parasite as compared to thin smear (advantages).
•The morphologic features are not well seen and the malaria species are not well identified
(disadvantages)

Thin smear preparation

•Prepared by placing a small drop of blood on a clean glass slide.
•With the use of another clean glass slide (spreader) at an angle of 30-450, spread the drop along
the contact line of the slides.
•And quickly push the spread drop in a forward direction with the spreader to the lower side of the
glass slide.
•Allow the smear to dry and stain with Romanowsky.
•Thin smear is fast, simple and uses small volumes of blood (advantages) as compared to thick
smear.
•With the rapid air drying of smear to preserve the morphologies of the cells (advantages).
•The frequent application of this method produces or yield useful blood smears (advantages).
•The presence of grease spots formation (dirty slide) affect the morphologies and integrity of cells
(disadvantages).

Specific esterase/Chloroacetate esterase

•Prepared by making a smear, fixing in cold buffered formal acetone for 30 seconds and air dry.
•Followed by rinsing under running tap water and air dry.
•The smear is placed into a coplin jar containing the working substrate solution for 5 to 10 minutes.
•Rinse under running tap water (coplin jar) still it become clear and air dry.
•Later counterstain by using acqueous haematoxylin for 1 minute.
•Then blue in running tap water (1 minute) and allow it to air dry.
•The end or reaction product is bright red and provide the same information as myeloperoxidase
stain, which is positive for myeloid cells.

Non specific esterase/alpha-napthyl acetate esterase(ANAE)

•Prepared by making a smear, fixing in cold buffered formal acetone for 30 seconds and air dry.
•Followed by rinsing under running tap water and air dry.
•The smear is placed into a coplin jar containing the incubation medium for 30 to 60 minutes.
•Rinse under running tap water (coplin jar) after incubation still it become clear and air dry.
•Later counterstain by using acqueous haematoxylin for 2 to 5 minutes and observe under the
microscopes.
•The end or reaction product is red (pink/purple) and brown appearance.
•There will be a strong reaction stain with normal and leukaemic monocytes.
•But negative reaction with normal granulocytes.
•AML or MDS will stain positive with varying intensity.
•Platelets or megakaryocytes will stain strongly positive and leukaemic megakaryoblasts will stain
diffuse positive.
•T lymphocytes (most) and T lymphoblast (some) will present with focal dot-like positivity

Wright stain
•Make a blood film and add 1.0ml of wright stain solution on the blood film and allow the stain for 1
to 3 minutes.
•Followed by the addition of distilled water and wright stain phosphate buffer (Ph 6.8), and allow
the slide to stand for 6 minutes.
•Rinse the slide with running tap water, and allow to blot dry the slide with paper towel.
•Observe under the microscope.