Laboratory haematology

Laboratory haematology


Automated FBC


Erythrocyte analysis

§        Hb concentration

§        Cells lysed – then Potassium ferocyanide (or alternative less toxic reagent) added, which converts all forms of haemoglobin to cyanmethaemoglobin and the absorbance is then measured at 540nm using photospectrometer. Confounded by SHb

§        RBC

§        Calculated by either aperture impedance (Coulter counter) or light scatter

§        Problems

§        Coincidence (i.e. 2 cells go through the counter at the same time) – can be corrected statistically or by ensuring the cells are counted in single file

§        Recirculation of cells can also be a problem so that they are counted twice

§        Agglutination – reduced RBC and increases MCV

§        Bubbles, lipid droplets, particles, microorganisms and white cells can be counted incorrectly (white cells not normally a problem unless count very high)

§        May have difficulties distinguishing platelets from rbs if the MCV is very low

§        MCV – measured directly by either impedance or light scatter

§        Can be artificially increased by hyperglycaemia, agglutination and sickling

§        May be underestimated both in impedance or light scattering in cells with a low Hb concentration

§        CHCM – cellular haemoglobin concentration mean – can be calculated directly by some analysers by light scatter

§        Derived parameters:

§        PCV (HCT) calculated by red cell number x red cell volume

§        MCH calculated by [Hb]/red cell count

§        MCHC calculated by [Hb]/PCV (may not fall in iron deficiency as expected due to ‘plasma trapping’ leading to elevations in measured PCV

§        RDW coefficient of variation of red cell size distribution


Leukoyte analysis

§        Variety of mechanisms involving light scatter at different angles, peroxidase staining, electrical conductivity

§        Counts performed on diluted whole blood in which the red cells are either lysed or rendered transparent

§        Multiple channel analysis (cells exposed to lytic reagents or a cytochemical reaction prior to analysis)

§        3 channel splits into granulocytes (large), lymphocytes (small) and monocytes (middle)

§        5 or 7 allows differentiation of neutrophils, eosinophils, basophils, lymphocyts and monocytes

§        Specimens flagged for morphological review – blasts, nucleated red cells etc.


Platelet analysis

§        Numbers

§        Often counted on the basis of MCV ie between 2-20 fL

§        Falsely increased by

§        Red cell fragments

§        Bacteria / fungi

§        Falsely low counts can occur:

§        Giant platelets may not be counted

§        Platelet clumping due to platelet-EDTA antibodies

§        Platelet satellitism

§   MPV (mean platelet volume)

§ Increased in

§ Hyperthyroidism

§ MPDs


§ Bernard-Soulier (uniformly increased)

§ Decreased

§ Megakaryocytic hypoplasia

§        Platelet distribution width (PDW)

§        May be helpful in distinguishing ET (increased) from reactive (normal)


Reticulocyte analysis

§        Methylene blue or fluorescent dyes that bind RNA are used


Blood morphology

§        Wright or May-Grunwald-Geimsa



Cytochemical stains

§        Myeloperoxidase

§        Primary granules of neutrophils and secondary granules of eosinophils

§        Monocytici lysosomal granules stain faintly

§        Sudan black

§        Stains intracellular phospholipids and other lipids

§        Similar pattern to myeloperoxidase

§        Lymphoblast azurophilic granules may be positive

§        Napthol AS-D cholroacetate esterase (specific esterase)

§        Granulocytes stain, monocytes do not

§        Can be used to stain granulocytes and mast cells

§        Alpha-napthylbutyrate / acetate (non-specific esterase)

§        Stain monocytes, macrophages and histiocytes but do not stain neutrophils

§        Megas stain with the acetate but not the butyrate

§        TdT (terminal deoxynucleotidyl transferase)

§        Intracellular enzyme that stains thypmocytes and lymphoblasts

§        Some myeloblasts also stain

§        TRAP (tartrate-resistent acid phosphatase

§        Stains an acid phosphatase isoenxyme

§        Positive in hairy cell leukaemia, Gaucher cells and activated T lymphocytes

§        Periodic acid-Schiff (PAS)

§        Detects intracellular glycogen and neutral mucosubstances

§        Positive staining in ALL, AML, erythroleukaemia and Gaucher cells

§        Toluidine blue

§        Detects acid mucopolysaccharides (positive in mast cells and basophils)

§        Tryptase

§        Positive in mast cells

§        Negative in basophils

§        Mast cells in systemic mast cell disease frequently have a spindled shape

§        Reticulin

§        Reticulin fibers stain black

Red cell abnormalities


Shape abnormalities

§        Acanthocytes

§        Due to altered membrane lipids or abnormalities in membrane protiens

§        Infantile pkynocytosis

§        Vitamin E deficiency

§        Abetalipoproteineamia

§        McLeod Phenotype (due to Kx deficiency and hence absent Kell expression)

§        MDS

§        Pyruvate kinase deficiency

§        Splenectomy

§        Anorexia

§        Ecchinocytes

§        Storage change

§        Renal +/- liver impairment

§        Pyruvate kinase deficiency

§        Nutritional phosphate deficiency

§        HUS

§        Burns

§        Post cardiac by-pass

§        Post-transfusion

§        Tear drops

§        Marrow fibrosis

§        Severe dyserythropoiesis

§        Haemolytic anaemia

§        Esp. megaloblastic anaemia and thal major

§        Bite cells

§        Smooth semicircle taken from one edge

§        Due to Heinz body pitting by the spleen

§        G6PD defeicincy

§        Drug induced oxidant haemolysis

§        Eliptocytosis

§        Not seen in reticulocytes (which are round) in the hereditary causes

§        Hereditary eliptocytosis

§        Hereditary pyropoikilocytosis (only one of the many types of the many poikilocytes present)

§        SE asian ovalocytosis (along with stomatocytes and macro-ovalocytes)

§        Irregularly contracted red cells

§        Drug / toxin induced haemolytic anaemia

§        G6PD (extreme contraction in favism e.g. hemighost cells)

§        HbC and E homozygotes

§        Small numbers in b-thal trait and Hb C / E trait

§        Unstable haemoglobins (Hb Koln, Hb St Mary’s)

§        Pyknocytes – similar to irregularly contracted cells – seen in premature neonates

§        Keratocytes

§        Pair of spicules that appear like horns – formed by removal of a Heinz body by the spleen or by mechanical damage (similar to schistocytes)

§        Leptocytosis

§        Unusually thin red cells

§        Ring of membrane with unstained central area

§        Poikilocytosis

§        Megaloblastic anaemia

§        Iron deficiency

§        Thalassaemia (alpha/beta)

§        Eliptocytosis

§        Ovalocytosis

§        CDA

§        Myelofibrosis

§        HbC disease (specifically the Hb C poikilocyte)

§        Seen in iron deficiency and thalassaemia

§        Schistocytes / helmet cells / fragments

§        Distorted, fragmented cells with 2-3 pointed ends

§        Microangiopathic haemolytic anaemia (Thrombotic thrombocytopenic purpura (TTP), DIC etc.)

§        Caused by mechanical distortion in the microvasculature by fibrin strands or damage by mechanical heart valves

§        Severe burns (due to direct injury – often round like microspherocytes and blebbing of the normal red cells may occur).

§        Thalassaemia

§        CDA (congenital dyserythropoietic anaemia)

§        Herediatary pyropoikilocytosis

§        Megaloblastic anaemia

§        Spherocytes

§        Spherical cells with dense appearance and absent central pallor due to decreased membrane reduncancy

§        AIHA (autoimmune haemolytic anaemia)

§        Hereditary spherocytosis 

§        Bacterial toxins (C. Perfringens and clostridium welchii)

§        Fresh water drowning

§        Microspherocytes due to complement / antibody mediated damage to the red cell membrane or fragmentation

§        Spheroechinocytes

§        Dense cells with crenation

§        Artefact

§        Splenectomised HS patients

§        Transfused blood film

§        Stomatocytes

§        Mouth like deformity due to membrane defects caused by abnormal cation permeability

§        May be artefactual

§        Hereditary stomatocytosis

§        SE asian ovalocytosis

§        AIHA

§        MDS

§        Liver disease

§        Alcohol

§        Target cells

§        Central round stained area and a peripheral rim of staining due to increased membrane redundance

§        Chronic liver disease (due to red cell membrane loading with cholesterol)

§        Hypobetalipoproteinaemia

§        Post splenectomy

§        Thalassaemia

§        HbC, Hb SC, Hb C beta thal

§        Hb E, Hb H

§        Sideroblastic anaemia

§        Hereditary xerocytosis

§        Analphalipoproteinaemia


Red cell inclusions

§        Basophilic stippling

§        Punctuate basophilic inclusions due to precipitated ribosomes – doesn’t stain with Pearls stain (in contrast to Pappenheimer bodies)

§        Thalassaemia trait esp. B and thal major

§        Lead poisoning

§        Haemolytic anaemia

§        Liver disease

§        Megaloblastic anaemia

§        Unstable Hb

§        Pyramidine 5 nucleotidase deficiency

§        Dyserythropoiesis

§        Including CDA, SA, erythroleukaemia and MF

§        Erythropoietic porphyria

§        Howell-Jolly bodies

§        Small, discrete, basophilic dense inclusions – due to nuclear remnants

§        Post splenectomy

§        Coeliac disease

§        Haemolytic anaemia

§        Megaloblastic anaemia

§        neonates

§        Cabot ring

§        Circular, blue, thread like inclusion with dots – due to nuclear remnant

§        Post splenectomy

§        haemolytic anaemia

§        megaloblastic anaemia

§        Heinz bodies

§        Not normally seen with normal stains (can be seen after splenectomy) – due to precipitation of unstable haemoglobin

§        G6PD

§        Unstable haemoglobins

§        Pappenheimer bodies

§        Small, peripherally dense basophilic granules – due to iron containing sideromes or mitochondrial remnants. Can be confirmed by Pearl’s stain.

§        Splenectomy

§        Lead poisoning

§        Sideroblastic anaemia

§        Iron overload

§        Dohle bodies

§        May-Hegglin

§        Sepsis



Size abnormalities

§        Macrocytosis

§        Megaloblastic anaemia

§        Neonatal cells

§        Aplastic anaemia

§        MDS (myelodysplasia)

§        CDA (particularly type III)

§        Drugs (hydroxyurea, alcohol)

§        Liver

§        Benign familial

§        Reticulocytosis

§        Microcytosis

§        Iron deficiency

§        Thalassaemia (alpha and beta)

§        Hb C and Hb E beta thal

§        Sideroblastic anaemia

§        Fragments

§        Zinc toxicity

§        Copper deficiency

§        Nucleated red cells

§        Immature red cells in the peripheral circulation

§        Severe anaemia (except aplastic anaemia)

§        HDN

§        Premature infants (normal)

§        Myelofibrosis

§        Carcinomatosis

§        Post splenectomy

§        Sickle cell anaemia

§        Sepsis



Blood film findings in different diseases


Iron deficiency

§   Initially normochromic normocytic, anisocytosis precedes anaemia

§   Hypochromic microcytosis

§   Poikilocytes, elliptocytes – often very narrow cells – pencil cells

§   Thrombocytosis

§   Target cells are uncommon unless the patient has HbC or S trait

§   Basophilic stippling is uncommon

§   Polychromasia is sometimes present


Differential diagnosis

§   Thalassaemia trait (basophilic stippling and target cells favour)

§   Anaemia of chronic disease (rouleaux, background staining and other signs of inflammation)

§   Congenital sideroblastic anaemia (hypochromia, microcytosis, occasionally target cells and basophilic stippling, pappenheimer bodies – diagnosed with a bone marrow + Pearl’s stain)

§   Lead poisoning (may show hypochromia and microcytosis, basophilic stippling is prominent, Pappenheimer bodies may also be present)  DD includes 5 pyramidine nucleosidase deficiency



§        B thal trait

§        Normal ranging to markedly abnormal with:

§        Microcytosis

§        Anisocytosis, poikilocytosis

§        Hypochromia

§        Basophilic stippling (more severe)

§        Target cells (more severe)

§        Irregularly contracted cells (more severe)

§        Elliptocytes move commonly found in iron deficiency

§        Differential

§        Dbthal trait


§        Bthal intermedia

§        Similar to Bthal trait but more severe:

§        Hypochromia

§        Microcytosis, anisocytosis, poikilocytosis

§        Basophilic stippling

§        Polychromasia

§        Possibly circulating erythroblasts


§        Bthal major

§        Hypochromia / hypochromic microcytes

§        Marked anisocytosis

§        Poikilocytosis including fragments, teardrops

§        Target cells

§        Basophilic stippling

§        Pappenheimer bodies

§        NRBCs with dyserythropoietic features

§        Increased white count  (but may have neutropenia and thrombocytopenia if massive splenomegaly)

§        Hyposplenic features if previous splenectomy (Howell-Jolly bodies, target cells, lymphocytosis, thrombocytosis, giant platelets)

§        Post splenectomy may contain alpha chain precipitates (can be seen pre splenectomy but less frequent)

§        Leptocytes seen post splenectomy – very flat cells with striking hypochromia

§        Transfused cells

§        Bone marrow

§        Severe dyserythropoiesis with nuclear lobulatoin and fragmentation, basophilic stippling

§        Phagocytic macrophages

§        Pseudo-Gaucher cells

§        Increased iron stores


Alpha thal trait

§   Alpha + thal trait likely to be normal

§   Alpha 0 heterozygosity and alpha + thal homozygosity - similar features to beta thal trait but basophilic stippling often less prominent


Haemoglobin H disease

§   Moderate anaemia (Hb 6-10)

§   Marked hypochromia, microcytosis and poikilocytosis

§   Target cells, tear drop cells and fragments

§   Basophilic stippling and polychromasia (reticulocytosis)


§   Beta thal (intermedia)

§   CDA (but they have normocytic or macrocytic cells and no reticulocytes) or HPP (but microspherocytes, elliptocyete and red cells with bud like projections) may show similar degrees of poikilocytosis


Sickle cell

§   Anisocytosis

§   Sickle cells

§   Boat shaped cells

§   Target cells

§   Polychromasia, NRBC

§   Basophilic stippling

§   Occasional irregularly contracted cells and spherocyte

§   Hyposplenic features (past infancy) – Howell-Jolley bodies, Pappenheimer bodies and numerous target cells.  (Acanthocytes are unusual in sickle cf other causes of hyposplenism)


§   HbS alpha thal (difficult to distinguish)

§   HbS beta thal (probably more microcytosis and hypochromia, with more prominent Pappenheimer bodies)

§   HbSC


HbSC disease

§   Hb higher but overlaps with HbSS

§   Few sickle cells and less NRBC and polychromasia

§   Less evidence of hyposplenism

§   Target cells prominent

§   Boat shaped cells prominent

§   SC poikilocytes – usually infrequent but may be present in large numbers

§   Haemoglobin C crystals


HbCC disease

§   Mild to moderate anaemia

§   Large numbers of target cells and irregularly contacted cells

§   Polychromasia and nucleated red cells may be present

§   Haemoglobin C crystals may be present but are uncommon


Hb C trait

§   Hb normal

§   Target cells

§   Hypochromic microcytic red cells


Hb E disease

§   Mild anaemia

§   Hypochromia and microcytosis with variable target cells (similar to beta thal trait)


HbE Beta thal

§   Hb 7-9

§   Marked hypochromia and microcytosis

§   Basophilic stippling, anisocytosis and poikilocytosis (including keratocytes and teardrop cells)



Megaloblastic anaemia

§        Peripheral blood

§        Oval macrocytes with anisocytosis and poikilocytosis and fragmentation

§        Hypersegmented neutrophils (>5)

§        Leucopenia and thrombocytopenia

§        Howell-Jolley bodies

§        Polycythaemia usually absent despite marked anaemia

§        Bone marrow

§        Hypercellular with accumulation of primitive cells

§        Nuclear; cytoplasmic asynchrony in erythroblasts

§        Nucleus has an open, fine lacy appearance

§        Nuclear fragments

§        Giant metamyelocytes and hyperpolypoid megakaryocytes

§        Can be confused with erythroleukaemia


Differential diagnosis

§   B12 / Folate

§   Drugs (MTX)

§   Alcohol (macrocytes round rather than oval, no hypersegmented neutrophils, thrombocytopenia, rouleaux, target cells, stomatocytes, spur cell haemolytic anaemia – advanced liver disease)

§   MDS (dysplastic neutrophils, population of hypochromic microcytes due to sideroblastic anaemia)


Causes of red cell fragmentation


§   TTP (thrombotic thrombocytopenic purpura - not to be missed - 90% mortality if untreated)

§   DIC (sepsis, malignancy)

§   HUS Haemolytic uraemic syndrome (diarrhoea +: E. Coli 0517, shigella, campylobacter / diarrhoea -ve: pneumococcus)

§   Pregnancy associated (HELLP, Pre eclampsia, eclampsia)

§   Drugs (mitomycin C, bleomycin, quinolone….. CSA, tacro…)

§   Pancreatitis

§   Renal small vessel disease

§   Malignant hypertension

§   Acute glomerulonephritis (PAN, SLE, Wegeners granulomatosis)

§   Familial HUS – complement H deficiency

§   Antiphospholipid syndrome

§   Dysfibrinogenaemia

§   Disseminated carcinoma (particularly mucin secreting tumours – esp gastric Ca)

§   Bone marrow transplantation

Malformations of large vessels

§   Haemangiomas (eg hepatic)

§   Prosthetic heart valves (rarely AS or severe mitral valve disease)

§   Mechanical fragmentation



§        Tissue hypoxia

§        Primary

§        Inherited

§        Increased sensitivity to EPO

§        Acquired

§        PRV

§        Essential / idiopathic erythrocytosis

§        Secondary

§        Inherited

§        Congenital methaemoglobinaemia

·        Haemoglobin M

·        NAD / NADH linked methaemoglobin reductase

§        Impaired release of oxygen from haemoglobin

§        Acquired

§        Hypoxia (high altitude, cyanotic heart disease, chronic hypoxic lung disease, sleep apnoea, hepatic cirrhosis

§        Carbon monoxide poisoning

§        Methaemoglobinaemia

§        Increased EPO production

§        Inherited

§        Familial inappropriate EPO secretion

§        Acquired

§        Renal (hypernephroma, Wilms tumour, renal adenoma, renal haemangioma, renal cystis including ADPKD

§        Cerebellar haemangiomas

§        Hepatic lesions (hepatoma)

§        Uterine fibroids

§        Adrenal tumours

§        Phaeochromocytoma

§        Atrial myxoma

§        Cushings

§        Unknown

§        Mid-Volga familial polycythaemia

§        Monge’s disease


In babies

§        Intra-uterine twin-twin transfusion

§        Intra-uterine maternal to fetal transfusion

§        Placental insufficiency and intrauterine hypoxia

§        Small for dates babies

§        Post mature babies

§        Maternal pregnancy associated hypertension

§        Maternal smoking

§        Maternal diabetes

§        Chromosomal abnormalities

§        Downs

§        Trisomy 13, 18

§        Neonatal thyrotoxicosis

§        Congenital adrenal hyperplasia



White cell abnormalities



§        Hyperlobated neutrophils

§        5 or more nuclear segments

§        Ureamia

§        Iron deficiency

§        Cytotoxic treatment (esp methotrexate occasionally hydroxycarbamide)

§        Hypolobated

§        Pelger-Huet anomaly (benign inherited condition)

§        MDS

§        AML with dysplasia

§        Accelerated phase CML

§   Granules

§        Toxic granulation

§        Bacterial infection

§        GCSF

§        Aplastic anaemia / myelofibrosis – coarse granules

§        Alder-Reilly anomaly

§        Large discrete, deep red granules which obscure the nucleus – function normally

§        Cheidiak Higashi

§        Giant, scanty azurophilic granules

§        Hypogranular

§        MDS

§        Pyknotic neutrophils

§        Dead or dying cells (may be mistake for nucleated red cells)

§        Artefact

§        Sepsis

§        Vacuolation

§        Severe sepsis

§        Artefact due to prolonged storage



§        85% T cells or NK cells

§        Activated lymphocytes (Turk cells)

§        Large cell with round nucleus and basophilic cytoplasm often scalloping around adjacent red cells

§        Infection (EBV)

§        Smear cells

§        CLL – small lymphocytes

§        Lobulated (Flower)

§        Storage artefact

§        ATLL / HTLV-1 infection

§        Cerebriform

§        Sezary syndrome



§        Anisocytosis

§        MPD

§        Enlarged

§        Increased platelet production (eg ITP)

§        Hyposplenism

§        Bernard Soulier syndrome

§        May-Hegglin syndrome

§        Hypogranular

§        Grey platelet syndrome





§        Herediatery neutrophilia

§        Inherided deficiency of CR3 complement receptors

§        Deficinet surface expression of leucocyte adhesion molecules CD11b or CD15



§        Infections (bacterial, viral, fungal, parasitic)

§        Tissue damage / acute inflammation (trauma, surgery, pancreatitis, myocardial infarction, inflammatory disorders – chrons, UC, RA, Gout)

§        Haemorrhage

§        Malignancy

§        Myeloproliferative disorders

o        CML

o        CMML

o        Neutrophilic leukaemia

§        Metabolic disorders (DKA, ARF, cushings, thyrotoxicosis)

§        GCSF and other cytokine administration

§        Adrenaline, corticosteroids, lithium, desmopressin

§        Cigarette smoking

§        Exercise