Making the diagnosis in Hematology Part 1

Think of…

• Too much• Too little• Poor function

RBC

• Low Hb- Anemia

• High Hb- Polycythaemia

Anemia

• Microcytic anemia• Normocytic anemia• Macrocytic anemia• Aplastic anemia• Hemolytic anemia

Causes of microcytic anemia

• Sideroblastic anemia• Anemia of chronic disease• Thalassemia• Iron deficiency

Main lab features of iron deficiency anemia

• Low serum iron.• Increased TIBC.• Increased Transferrin.• Low transferrin saturation.• Low Ferritin (confirms the diagnosis)• High Serum soluble transferrin receptors.

Main lab features of anemia of chronic disease

• Low serum iron and TIBC• Ferritin normal/raised• Serum soluble transferrin receptor normal.

Main features of sideroblastic anemia

• Presence of ring sideroblast.

Thalassaemias

Genetic defect:• β-thalassaemia -point mutations

• α-thalassaemia- gene deletions

** Serum ferritin and iron stores are NORMAL

Types of Hemoglobin

β-thalassaemia

Homozygous:• No normal B chain produced (β0), or • Very reduced (β+).• Excess alpha chains.• Result: High Hb A2(α2δ2) and Hb F(α2γ2) with

small amounts of Hb A(α2β2).

Heterozygous• Symptomless microcytosis with or without mild

anaemia

β-thalassaemia

β-thalassaemia

• Thalassaemia minor (trait)-asymptomatic.(Raised HbA2 and HbF)

• Thalassaemia intermedia- moderate anaemia.

• Thalassaemia major (Cooley’s anemia)- severe anaemia (Homozygous β-thalassaemia)

α -thalassaemia

• Gene deletion.• Deletion of one alpha chain (α+) or• Deletion of both alpha chain (α0)-most

common.

**Normal people have Four alpha globin chains.2 from mum, 2 from dad.

α -thalassaemia

• 1 gene deletion - Normal Hb. Normal blood picture.

• 2 gene deletion (trait) – Hb H bodies seen or Hb A present. mild anemia.

• 3gene deletion – Hb H disease.(Hb A2 normal or reduced.)

• 4 gene deletion – Hb Barts. Hydrops fetalis (incompatible with life)

Causes of normocytic anemia

• Anemia of chronic disease• Hypopituitarism• Hypothyroidism• Hypoadrenalism• Aplastic anemia• Some hemolytic anemia• Acute blood loss

Causes of macrocytic anemia

• megaloblastic marrow

• normoblastic marrow

Causes of macrocytosis (Raised MCV) with megaloblastic marrow

• B12 deficiency• Pernicious anemia• Folate deficiency• Congenital enzyme deficiencies in DNA

synthesis (e.g. orotic aciduria), • Drugs: hydroxyurea, azathioprine, zidovudine • Myelodysplasia due to dyserythropoiesis.

Causes of macrocytosis (Raised MCV) with normoblastic marrow

Physiological: Pregnancy.

Pathological causes:• alcohol excess• liver disease• Reticulocytosis• Hypothyroidism• aplastic anaemia• sideroblastic anaemia• pure red cell aplasia• Drugs (e.g. cytotoxics – azathioprine)• Spurious (agglutinated red cells measured on red cell

counters)• Cold agglutinins due to autoagglutination of red cells.

Megaloblast vs normoblast.

Megaloblast:• Peripheral blood film shows oval macrocytes with

hypersegmented polymorphs with six or more lobes in the nucleus.

• Bone marrow shows megaloblastic erythropoeisis.

Normoblast:• Normal serum B12 and folate.• Normoblastic bone marrow.

** Alcohol excess can be both.

Lab features in pernicious anemia

• Raised serum bilirubin and LDH.

• Raised Serum methylmalonic acid (MMA) AND homocysteine (HC) in B12 deficiency

(only HC raised in folate deficiency).

• Low serum vitamin B12

Principles of Schilling’s test.• Oral Radioactive B12 is given.

• Then i.m. injection of non-radioactive B12 to saturate B12 binding proteins and to flush out 58Co-B12.

• The urine is collected for 24 hours.

• Normal person will excrete > 10% of the oral dose.

• If <10, repeat test with addition of oral IF.

• If now normal, the diagnosis is pernicious anaemia or gastrectomy.

• If still abnormal, the lesion must be in the terminal ileum or bacterial overgrowth.

Other causes of B12 deficiency

Causes of aplastic anemia

Lab findings in aplastic anemia

• Pancytopenia• The virtual absence of reticulocytes• A hypocellular or aplastic bone marrow with

increased fat spaces

Causes for pancytopenia

Causes of Hemolytic anemia

Lab findings suggestive of hemolysis.

Intravascular hemolysis:• Raised plasma Hb.• Raised hemosiderinuria.• Low haptaglobin• Presence of methaemalbumin (positive

Schumm’s test)

Conditions with intravascular hemolysis

• Mismatched blood transfusion• PNH• Septicemia• MAHA• March hemoglobinuria• G6PD deficiency.

Result of increased red cell breakdown

• Anemia• Raised serum bilirubin• Raised LDH• Raised urobilinogen• Positive urinary hemosiderin• Reduced plasma haptaglobin.

Result of increased red cell production

• Reticulocytosis/Polychromasia• Erythroid hyperplasia of bone marrow.

Causes of inherited hemolytic anemia

• Hereditary spherocytosis• Thalassemias• Sickle syndromes• G6PD deficiency• Pyruvate kinase deficiency

Causes of acquired hemolytic anemiaImmune:• Autoimmune hemolytic anemia- warm and cold.• Drug induced immune hemolytic anemia.• Alloimmune hemolytic anemia

Non-immune:• Paroxysmal noctural hemoglobinuria (PNH).

• Mechanical hemolytic anemia: mechanical heart valves MAHA March hemoglobinuria.

How to find the cause of hemolysis?

• Look at the blood film• Direct antiglobulin test.• +/- osmotic fragility test, Hb electrophoresis.

Specific features of each condition.

Hereditary spherocytosis.• Spherocytes on blood film• Positive osmotic fragility test.• Negative Coomb’s test.

Hereditary elliptocytosis.• Elliptical red cells.

Sickle cell disease.• Blood film show hyposplenism.• Positive sickle solubility test.• Hb electrophoresis (confirms diagnosis)

shows Hb SS (80-95%), No HbA, Hb F (2-20%).

G6PD deficiency• Blood count normal between attacks.• During attack, blood film show bite cells, blister

cells, Heinz bodies and reticulocytosis.

Pyruvate kinase deficiency.• Blood film shows prickle cells and reticulocytosis.• Low pyruvate kinase activity.

Warm AIHA.• Spherocytosis.• IgG antibodies predominate.• Positive DAT (IgG +/- complement)• Autoantibodies- specific for Rh blood grp system.• Autoimmune thrombocytopenia.

Cold AIHA• Red cells agglutinate in the cold temperature.• DAT is positive with complements (C3d) only.• Monoclonal IgM antibodies.

Drug induced hemolytic anemia.• Antibodies to drug only: quinidine, rifampicin.• Antibodies to the cell membrane only: methyldopa.• Antibodies to both: penicillin. hemolysis typically occurs over 7-10 days.

Diagnosis: Must have:1) Temporal association between administration of a drug and haemolytic anaemia,

and recovery after withdrawal of the drug.

2) Positive DAT.

3) Drug dependent red cell antibodies detectable in the first and third mechanisms described above.

In the second mechanism, the antibodies are not drug-dependent and are indistinguishable from autoantibodies.

Paroxysmal nocturnal hemoglobinuria:• Mutation in the X-linked gene PIG-A• Evidence of intravascular haemolysis.• Flow cytometric analysis of red cells with anti-CD55 and anti-CD59 has

replaced the Ham’s test.• Hypoplastic bone marrow

Major clinical signs:• Intravascular hemolysis, venous thrombosis, hemoglobinuria.

• Characteristically only the urine voided at night and in the morning on waking is dark in colour.

• Rx with eculizumab.

Causes of MAHA

• When fragmentation of red cells occurs in an abnormal microcirculation.

• Malignant hypertension• Eclampsia• HUS• TTP• Vasculitis • DIC

The Peripheral Blood filmAnisocytosis• is variation in RBC size, eg megaloblastic anaemia, thalassaemia, IDA.

Acanthocytes:• RBCs show many spicules due to an unstable red cell membrane lipid

structure (eg in abetalipoproteinaemia).

Basophilic RBC stippling:• Denatured RNA found in RBCs, indicating accelerated erythropoiesis or

defective Hb synthesis. Seen in lead poisoning, megaloblastic anaemia, myelodysplasia, liver disease, haemoglobinopathy eg thalassaemia.

Blasts:• Nucleated precursor cells. They are not normally in peripheral blood, but

are seen in myelofibrosis, leukaemia or malignant infiltration by carcinoma.

Burr cells:• Irregularly shaped cells occurring in uraemia.

Dimorphic picture:• Two populations of red cells. Seen after treatment of

Fe, B12 or folate deficiency, post-transfusion, or with primary sideroblastic anaemia, where a clone of abnormal erythroblasts produce abnormal red cells, alongside normal red cell production.

Howell-Jolly bodies:• DNA nuclear remnants in RBCs, which are normally removed by the

spleen. Seen post-splenectomy and in hyposplenism (eg sickle cell disease, coeliac disease, congenital, UC/Crohn's, myeloproliferative disease, amyloid). Also in dyserythopoietic states: myelodysplasia, megaloblastic anaemia.

Hypochromia:• Less dense staining of RBCs due to reduced Hb synthesis, seen in

IDA, thalassaemia, and sideroblastic anaemia (iron stores unusable).

Left shift:• Immature neutrophils are sent out of the marrow, eg in infection.

Leucoerythroblastic anaemia:• Immature cells (myelocytes, promyelocytes,

metamyelocytes, normoblasts) seen in film. Due to marrow infiltration (eg malignancy) when these cells are displaced; also seen in anorexia, sepsis, severe haemolysis.

Leukaemoid reaction:• A marked leucocytosis (WCC>50). Seen in severe illness eg

with infection or burns, and also in leukaemia.

Pappenheimer bodies:• Granules of siderocytes containing iron. Seen in lead

poisoning, carcinomatosis, and post-splenectomy.

Poikilocytosis:• is variation in RBC shape, eg in IDA, myelofibrosis, thalassaemia.

Polychromasia:• RBCs of different ages stain unevenly (young are bluer). This is a

response to bleeding, haematinic replacement (ferrous sulfate, B12, folate), haemolysis, or marrow infiltration. Reticulocyte count is raised.

Reticulocytes:• (normal range: 0.8-2%). Young, larger RBCs (contain RNA) signifying

active erythropoiesis. Increased in haemolysis, haemorrhage, and if B12, iron or folate is given to marrow that lack these.

Right shift:• Hypermature white cells: hypersegmented polymorphs

(>5 lobes to nucleus) seen in megaloblastic anaemia, uraemia, and liver disease.

Rouleaux formation:• Red cells stack on each other (it causes a raised ESR).

Seen with chronic inflammation, paraproteinaemia and myeloma.

Spherocytes:• Spherical cells found in hereditary spherocytosis and

autoimmune haemolytic anaemia.

Schistocytes:• Fragmented RBCs sliced by fibrin bands, in intravascular

haemolysis. Look for microangiopathic anaemia, eg DIC , haemolytic uraemic syndrome, thrombotic thrombocytopenic purpura or pre-eclampsia.

Target cells:• (also known as Mexican hat cells). These are RBCs with

central staining, a ring of pallor, and an outer rim of staining seen in liver disease, hyposplenism, thalassaemia and, in small numbers, in iron-deficiency anaemia.

A normal blood film, with a neutrophil, normal red cells, and platelets (arrows).

Microcytic hypochromic cells in iron deficiency anaemia

Poikilocytosis and anisocytosis seen in iron deficiency anaemia.

Pathological ring sideroblasts in the bone marrow, with a perinuclear ring of iron granules, found in

sideroblastic anaemia

Acanthocytosis.

Basophilic stippling.

Burr cells

Left-shift: presence of immature neutrophils in the blood

Pappenheimer bodies

Reticulocytes. RNA in RBCs; supravital staining (azure B; cresyl blue) is needed

Rouleaux formation

Film in hyposplenism: target cell (short arrow), acanthocyte (long arrow) and a Howell-Jolly body

(arrow head).

A Cabot ring; these red/purple-staining filamentous figure-of-8 rings are often seen in RBCs with basophilic stippling.

They occur in severe or megaloblastic anaemia, leukaemia, and lead poisoning.

Megaloblastic anaemia: peripheral blood film showing many macrocytes and one hypersegmented neutrophil (normally there

should be <5 segments).

A bite cell in G6PD, following removal of Heinz bodies by the spleen. Heinz bodies are formed from oxidized, denatured Hb

during oxidative crises

Blister cells (arrows) in G6PD, following removal of Heinz bodies. Also contracted red cells (arrowheads).

Microangiopathic anaemia eg from DIC: numerous cell fragments (schistocytes) are present

Fibrin strands, deposited in HUS & TTP, slice up passing red cells (microangiopathic anaemia).

Hereditary spherocytosis.

Hereditary elliptocytosis

Blood film in sickle-cell anaemia: there are sickle cells,

target cells, and a nucleated red cell.

Leg ulcers in sickle cell disease.