Anaemia

Impaired Red Blood Cell (RBC) Production

  • In adequate supply of nutrients essential for erythropoiesis
  • Iron Deficiency
  • Vitamin B12 Deficiency
  • Folic Acid Deficiency
  • Protein
  • Depression of erythropoietic activity
  • Anaemia of Chronic Disorders
  • Anaemia of Renal Failure
  • Aplastic Anaemia
  • Anaemia Due to Replacement of Normal Bone Marrow (BM)

Leukaemia/Lymphoma
Myeloma
Carcinoma
Myeloproliferative Disorders

  • Inherited Disorders

Thalassaemia

Iron

  • Trace element found widely in the body
  • Compounds that serve as storage forms for iron
  • Compound that serve in metabolic or enzymatic functions
  • In an average 70kg man, the haemoglobin is distributed as follows: 65% in Hb; 4% in Mb (Methaemoglobin); 0.08% Cytochrome C; 0.15% Transferrin; 30% Ferritin (tissue)
  • The average total iron concentration in the body is 2-4g (1g iron per kg RBCs)
  • Total daily male requirement approx. 1mg/day (menstruation female approx 2mg/day; pregnant female approx. 3-4mg/day)
  • A woman stores approx. half as much iron as a man
  • Only small amounts of iron are used - once in Hb, it remains there until RBC degradation
  • A very small amount of iron is left in the epithelial cells, some is lost in the urine and some is stuck in the gut
  • Iron is recycled as macrophages take RBCs out of the system - principally in the:

Liver Spleen Bone Marrow

  • Porphyrins break down in the circulation to give bilirubin

New look for June 2003

Anaemia

The erythrocyte as seen by the routine haematologist is essentially at the final stage of maturation. The "cell" is anucleate, without mitochondria, lysosomes or endoplasmic reticulum (ER), and relies on the Embden-Meyerhof and Heptose Monophosphate pathways to maintain the cell's shape etc.

All cells (except for T cells) are produced in haematopoietic tissue called Bone Marrow. In a child, this is found in the tibia. In an adult, bone marrow is found in the anterior/posterior iliac crest, sternum, ribs, vertebrae and skull.

The bone marrow houses the most primitive haematological cells - called Stem cells. These cells are pluripotential, which means they are able to differentiate into a range of haematological cell types.

The bone marrow is influenced by Erythropoietin produced mostly in the kidneys, but also the liver.

Anaemia is functionally described as a decrease in the competence of blood to carry oxygen to tissues therefore causing tissue hypoxia.
The haematologist looks at haemoglobin, an indirect measure of anaemia:

M = P x S (L)

Where,
M = No./Mass of erythrocytes
P = Production of erythrocytes by the bone marrow
S (L) = Survival (loss) of erythrocytes

Anaemia can be due to a lack of the raw materials necessary for erythrocyte production (eg. iron, vitamin B12 or folate) or because of the presence of chemicals / drugs which affect the stem cell.
A decrease in "M" leads to anaemia.
An increase in "P" = increased erythropoiesis. The marrow can increase production 6-8x to compensate for an anaemic condition.
An increase in "L" can be due to an increase in haemolysis eg. haemolytic anaemia, or because of haemorrhage.

  • Acute Haemorrhage
  • 20% (1000mL) loss with no resting clinical anaemic signs
  • 30-40% (1500-2999mL) loss leads to circulatory collapse and shock
  • 50% loss - death
  • Chronic Haemorrhage
  • eg. Ulcer
  • The body adapts slowly using mechanisms that allow organ function with up to 50% haemoglobin loss. These mechanisms include:
  • Increased Oxygen Flow to Tissues
  • Increase the cardiac output, cardiac rate and circulation rate by decreasing viscosity and peripheral resistance together with preferential blood delivery.
  • Increased Oxygen Flow to Tissues
  • Increase 2,3 DPG producing a shift to the right in the oxygen dissociation curve

The Haematology Profile (HP)

To determine whether the patient is anaemic or not

Factors affecting the components of the HP include...
Haemoglobin (Hb)

Values are higher for a standing patient or if a tourniquet has been applied for too long resulting in haemoconcentration

Decreases after 65yrs, in 2nd and 3rd trimesters of pregnancy and in females
Red Cell Count (RCC)

Varies according to altitude (usually increasing proportionally), age (usually decreasing with increasing age) or smoking (usually increasing)
Haematocrit (Hct)

An absolute increase in plasma volume or an absolute decrease in red blood cells (RBC) may give a low Hct

An absolute decrease in plasma volume with a normal RBC volume may give an elevated Hct

Acute haemorrhage (decreased RBC and plasma volume) may appear normal
Mean Cell Volume (MCV)

Reliable

Relating volume to diameter - spherocytes may have anormal volume but appear with a much smaller diameter
Mean Cell Haemoglobin (MCH)

Does not take into account the size of the cell

Best used with MCV
Mean Cell Haemoglobin Concentration (MCHC)

Concentration of haemoglobin relative to its size

Hb / Hct = Hb / (MCV+RCC) Therefore if increased MCV, MCHC decreases and if decreased MCV, MCHC increases

RBC clumps produce increased MCHC, MCH and MCV with a decreased RCC, and a normal Hb, ie a smaller cell will show a smaller central pallor (hyperchromic-the only cell described as this is the microspherocyte) whilst a larger cell will show a larger central pallor (hypochromic), with a constant Hb

Microspheocytes have decreased MCV and therefore an increased MCHC (constant Hb)
Red Cell Distribution Width (RDW)

Equivalent to anisocytosis in a blood film
White Cell Count (WCC)

A leukaemia can crowd out RBC production leading to anaemia

Validation and Description

Check the results with the request film and the blood film

X10 Power

Scan for evidence of errors in the blood film production
Estimate WCC (1field/4 or 5 fields/20)

X40 Power

Differential
If normal, may stop here
If immature cells are present:

  • Anisocytosis - compare to RDW - Normocytic? (7um), Macrocytic? (>9um), Microcytic? (6um)
  • Poikilocytosis - slight, moderate, marked
  • Haemoglobinisation - colour of RBCs in relation to central pallor (1/3 of area = normal)
  • Immature Forms - NRBC's (nucleated red blood cells); polychromasia -reticulocytes
  • Inclusion Bodies
  • Basophilic Stippling - aggregated ribosomes visualised as bluish-black granular inclusions throughout cellular volume. Can be proportional to chain precipitation in thalassaemia
  • Sideroblastic anaemia
  • Lead poisoning
  • Thalassaemia
  • Abnormal haemoglobin synthesis
  • Enzyme deficiencies
  • Heinz Bodies
    Precipitated or denatured Hb
    Visible fresh as refractile bodies
    Visible in methyl violet/brilliant cresyl blue/new methylene blue/crystal violet (supravital stains)
    Invisible with Romanowsky stain
  • Howell Jolly Bodies - dark purple/violet, spherical, nuclear remnants in RBC (usually 2 or less, resulting from mitotic problems)
  • Pappenheimer bodies - small, irregular, iron containing granules seen with Romanowsky staining, confirmed with Prussian blue staining
  • Malarial parasites

Megaloblastic Anaemia

Diagnosis

  • Many cases are seen that have a decreased serum B12 with a normal haematological profile and possibly normal clinical findings
  • Test for methylmalonic acid in the serum as an indicator of vitB12 deficiency
  • Test for homocysteine to check folate levels

Haematological Profile

  • Hb/RCC/Hct - Dec (due to Dec. DNA production)
  • MCV/MCH - Inc. (As precursors are megaloblastic)
  • MCHC - Normal
  • RDW - Inc. (Anisocytosis due to microcytes, normocytes and oval macrocytes)
  • Plt./WCC - Dec
  • Pancytopaenia with a normochromic, macrocytic anaemia
  • Intramedullary haemolysis due to ineffective erythropoiesis (reason for hypercellular BM)
  • Poikilocytosis - ++ (tear drops and schistocytes)
  • Anisocytosis - +++ (oval macrocytes)
  • Hypersegmented neutrophils ("Shift-to-the-Right" - seen in 91% of megaloblastic anaemias where >5% of Neutrophils/Eosinophils have 5/3 lobes - diagnostic with oval macrocytes)

A "Shift-to-the-Left" indicates an Inc. in band form WBCs
"Hypersegmented" refers to >5% nuclei with 6 (or more) lobes

  • Hypercellular bone marrow (M:E at 1:1 or reversed)
  • Erythron
  • Large cells with fine chromatin (Megaloblasts)
  • Nuclear(N)/Cytoplasmic(C) dissociation with N lagging C - normal cytoplasmic maturation
  • Multiple Howell-Jolly bodies (diagnostic if observed)
  • Leucon
  • Giant myelocytes and metamyelocytes
  • Thrombon
  • Often not obvious
  • The peripheral blood (PB) and bone marrow (BM) findings do not distinguish between a vitamin B12/cobalamin deficiency and a folic acid (folate) deficiency

Treatment

  • Shotgun therapy may be used where the causative agent is unclear
  • Folate, then vitB12 - attempts to make the patient well
  • Intramuscular hydroxy-cobalamin daily for a week (regime will depend on the cause of the anaemia)
  • Monitor the reticulocyte count (should return to normal in 2-3 weeks) and the Hb concentration (20-30g/L rise every week until normal)
  • If no response occurs, the diagnosis is incorrect
  • If a response occurs and then reaches a plateau, an iron deficiency is complicating RBC production

Causes of Vitamin B12 Deficiency

  • Reflected by:
  • Impaired DNA synthesis
  • Defective fatty acid degradation (excessive demyelination)
  • Administration of folate will correct the anaemia but will not reverse any neurological disease - approximately 5% show no other symptoms but neurological disorder
  • Symptoms include:
  • Anaemia
  • Diarrhoea/constipation
  • Glossitis (shiny tongue)
  • Sterility/Infertility
  • Numb, tingling fingers (paresthesia), wobbly gait and potential paralysis
  • "Megaloblastic madness" due to mental problems
  • Nutritional
  • Rare
  • Malabsorption
  • Gatsric Disease
  • Gastrectomy - usually preceeded by iron deficiency
  • Pernicious Anaemia (PA):
  • Juvenile or adult
  • Can be fatal
  • More common in females
  • Autoimmune disease whereby Ab to parietal cells leads to gastric atrophy (loss of epithelial cells and intrinsic factor (IF)) or an Ab that blocks the function of IF
  • Most frequent in Northern Europe, running in families

  • The vitB12 Journey.
    IF-Intrinsic Factor; Cbl-vitB12/ or Cobalamin; TC II-transcobalamin II)
  • Testing
  • Lack of gastric HCl is an indirect measure of lack of IF as it is due to atrophy of gastric cells
  • Look for IF Ab's (Blocking-prevents B12 binding to IF; Binding-prevents B12-IF complex binding to the ileum). 70% of PA sufferers have these Ab's
  • If negative (30%) do a Schilling Test.
  • Patient's vitB12 binding proteins are saturated by giving a large dose of unlabelled B12 intravenously
  • Absorption (via the ileum) of cobalamin is measured following ingestion of radioactively labelled B12.
  • In a normal system, more than 10% of the radioactive cobalamin will be excreted in the urine over 24hours.
  • If less than 10% is excreted, the patient is given cobalamin + IF. If the patient is suffering from PA, greater than 10% of the labelled cobalamin will appear in the urine within 24hours, otherwise the patient can be said to have an absorption problem.
  • Low results may occur in patient's with kidney problems
  • Terminal ileal disease
  • Ileal resections
  • Ulcerative colitis (may be autoimmune)
  • Tropical sprue (generalised malabsorption syndrome - seen in New Guinea)
  • Competitive parasites
  • Diphylobothrium latum
  • Pancreatic failure (enzymes from the pancreas are required to allow binding of IF to vitB12)
  • Drug induced
  • Bacterial overgrowth (competition for vitB12)
  • Increased Requirement for B12
  • Pregnancy
  • Infancy
  • Cancer
  • Haemolytic anaemia
  • Leukaemia
  • Exfoliative dermatitis
  • Multiple myeloma
  • Disorders of Transport and Storage

Folic Acid Deficiency

  • Folate is obtained from leafy vegetables
  • Deficiency results in anaemia in approx. 20 weeks
  • Require 50-100ug/day
  • Serum folate assay can be variable and should be carried out with a RBC folate assay (which indicates folate levels at the time of production of the RBCs)
  • Polyglutamate folate is the most common form of folate in food. The broken down, monoglutamate is transported across the intestinal mucosa, mostly at the jejunum and is primarily stored in the liver (capacity of 5-10mg)

Condition / Serum B12 Assay / RBC Folate Assay
Pernicious Anaemia / Low / Decreased
Alcoholism / Normal / Low
Acute Folate Deficiency / Normal / Normal
  • Folate is taken into cells as N5-methyl-tetrahydrofolate(THF). This must be demethylated (which is mediated by cobalamin) before conjugation can occur. If N5-THF is not conjugated, it will leak out of the cell again.
  • If cobalamin is in short supply, demethylation is impaired
  • Normally, dUMP is entirely converted to dTMP. With decreased levels of folate, this conversion is limited and a build-up of dUMP occurs instead. As a consequence, dUTP concentration rises and begins to replace dTTP in DNA synthesis.
  • Error correction attempts to replace the U's but fails due to the lack of T's and DNA synthesis is interrupted
  • Note: Goat's milk is lower in folate than cow's milk

Causes

  • Nutritional
  • An incomplete diet is the most common cause
  • Alcoholics
  • Elderly - overcooking destroys folate
  • Malabsorption
  • Tropical (general bowel disruption) and non-tropical (densitive to gluten - coeliac disease) sprue
  • Alcohol - interferes with absorption
  • Drug related - convulsive drugs, folate antagonists (eg. methotrexate-used for treatment of some neoplastic diseases), oral contraceptives
  • Anatomic and functional intestinal abnormalities
  • Increased Folate Requirement
  • Pregnancy
  • Infancy
  • Haemolytic anaemia
  • Acute leukaemia
  • Exfoliative dermatitis
  • Multiple myeloma
  • Metastatic cancer
  • Haemodialysis
  • Defective Utilization of Folate
  • Enzyme Deficiency

Non Megaloblastic Macrocytic Anaemia

  • Macrocytic anaemias are characterised by large (in volume or size) RBCs
  • greater than 100fL
  • greater than 9m
  • Classified depending on precursors into:
  • Megaloblastic Anaemia and
  • Non-Megaloblastic Anaemia
  • Non-megaloblastic anaemia is a macrocytosis without an accompanying megablastosis

Physiological Causes

Newborn:

  • Usually not an actual anaemia
  • A newborn's blood always contains a large number of macrocytes

Pregnancy:

  • May be unrelated to a vitB12 or folate deficiency

Pathological Causes

Reticulocytosis:

  • Polychromasia seen on a blood film due to increased erythropoietin following haemorrhage or haemolysis

Liver Disease:

  • Round macrocytes and target cells
  • May get a normochromic microcytic anaemia

Alcohol:

  • Inc. RBC production may cover the causes of this anaemia such as hamolysis, impaired BM response (due to a direct toxic/suppressing effect), folate deficiency and blood loss
  • Significant Dec. in the length of RBC survival
  • May cause sideroblastic anaemia

Stem Cell Disorders:

  • Dyserythropoiesis (abnormality in morphology and function of RBCs)
  • Leukaemia
  • Myelodysplastic syndromes

Miscellaneous:

  • Hypoxia
  • Post splenectomy
  • Thyroid disorders
  • Note: - pseudomacrocytosis is associated with increased WCC, cold agglutinins, hyperglycaemia and increased protein levels

Hypoproliferative Anaemias

  • A pathological depression of all cellular elements of peripheral blood

Causes

Bone Marrow (BM) Production Decrease

  • Classical cause of pancytopaenia

eg. Aplastic Anaemia

Infiltration or Replacement of normal cells

eg. Acute leukaemia
Malignancy - lymphomas (may metastasize to the bone marrow
Myelofibrosis - fibrosis of the BM
Hairy cell leukaemia

Hypersplenism

  • Large, overactive spleen that allows stagnation of pooled of blood

Immune Destruction

  • Systemic Lupus Erythematosus (SLE) - a classical autoimmune disease

Ineffective Erythropoiesis

  • Megaloblastic anaemia (defective DNA synthesis)
  • Dyserythropoiesis - myelodysplastic syndrome (qualitative abnormalities of one or more cell lines in the peripheral blood)

Haematological Reference Ranges

Red Cell Count (RCC): / Male: 5.5 +/- 1.0 x 1012/L / (4.5-6.5 x 1012/L)
Female: 4.8 +/- 1.0 x 1012/L / (3.8-5.8 x 1012/L)
Infant
(Full term, iron sufficient, cord blood): 5.0 +/- 1.0 x 1012/L / (4.0-6.0 x 1012/L)
Haemoglobin (Hb): / Male: 155 +/- 25 g/L / (130-180g/L)
Female: 140 +/- 25g/L / (115-165g/L)
Infant:
0-2 months: -
2-4 months: 100g/L (lower in premature babies 1.0kg-80g/L; 1.5kg-2.0-90g/L)
4-6 months: 105g/L
6 months-2 years: 110g/L
2-5 years: 110g/L
5-9 years: 115g/L
9-12 years: 120 g/L
Packed Cell Volume (Hct): / Male: 0.47 +/- 0.07 L/L / (0.40-0.54 L/L)
Female: 0.42 +/- 0.05L/L / (0.37-0.47 L/L)
Mean Cell Volume (MCV): / Adult: 86 +/- 10 fL / (76-96 fL)
Infant:
0-2 months: Macrocytes
2-4 months: 80 fL
4-6 months: 72 fL
6 months-2 years: 70 fL
2-5 years: 73 fL
5-9 years: 75 fL
9-12 years: 76 fL
Mean Cell Hamoglobin (MCH): / Adult: 29.5 +/- 2.5fL / (27-32 pg)
Mean Cell Haemoglobin Concentration (MCHC): / Adult/Children: 325 +/- 25 g/L / (300-350 g/L)
Red Cell Distribution Width (RDW): / 11.5-14.5%
Leucocyte/White Cell Count: / Adult: 7.5 +/- 3.5 x 109/L / (4-11x109/L)
Differential Leucocyte Count ("Diff"): / Adult:
  • Neutrophils: 2.0-7.5x109/L (40-75%)
  • Lymphocytes: 1.5-4.0x109/L (20-45%)
  • Monocytes: 0.2-0.8x109/L (2-10%)
  • Eosinophils:0.04-0.4x109/L (1-6%)
  • Basophils: less than 0.01-0.1x109/L (less than 1%)

Infant:
Lymphocytes
0-6 months: less than 15.0x109/L
0.5-1 years: less than 11.0x109/L
1-4 years: less than 9.0x109/L
4-10 years: less than 6.5x109/L
10-14 years: less than 6.0x109/L
Neutrophils:
Up to 20x109/L on the first day only
Lower lmits of normal:
less than 3 months: greater than 2.5.0x109/L
3 months-3 years: greater than 2.0x109/L
3-5 years: greater than 1.5x109/L
Eosinophils:
less than 5 years: less than 0.7x109/L
greater than 5 years: less than 0.5x109/L
Platelet Count (Plt): / Adult: 150-400 x 109/L
Reticulocyte Count: / Adult: 25-85 x 109/L / (0.2-2.0%)
Erythrocyte Sedimentation Rate (ESR): / Male: 2-10 mm/hr
Female: 2-15 mm/hr
Bone Marrow Cellularity: / 25-75% Cells
Fibrinogen: / 1-4g/L
Bleeding Time (BT): / Modified Ivy Technique: 1.5-4.5 min
Standardized Template Method: 2.5-9.5 min
Activated Partial Thromboplastin Time (aPTT): / Automated aPTT Reagent: 25-43 sec
Plasma Volume: / Adult: 50 +/- 10mL/kg bodyweight
Red Cell Mass/Volume: / Male: 30 +/- 5mL/kg bodyweight
Female: 25 +/- 5mL/kg bodyweight
Acute Leukaemia Distribution: / AML:
  • 20% of young children (2-10) with leukaemia
  • 40% of adults (especially >50yr) with leukaemia

ALL:
  • 80% of young children with leukaemia
  • 10-20% of adults with leukaemia

Chronic Leukaemia Distribution: / CML:
  • Rare in young children with leukaemia
  • 20-25% of adults with leukaemia

CLL:
  • Virtually unheard of in young children
  • 25-30% of adults with leukaemia

Shift To The Left: / N(seg) : N(nonseg) = 1 : 0.1-0.3
Immature cells include:
Blasts, promyelocytes, myelocytes,
metamyelocytes, pronormoblasts, basophilic normoblasts,
polychromatic normoblasts and
orthochromatic normoblasts
Shift To The Right/Hypersegmented: / Neutrophils:
  • More than 5% have 5 lobes / More than 5% have 6 or more lobes

Eosinophils:
  • More than 5% have 3 lobes / More than 5% have 4 or more lobes

New look for June 2003