Anemia

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AnemiaLaboratory Diagnosis
Dr. Mohammed Iqbal Musani
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Definition

• Anemia (a decrease in the number of RBCs, Hb
  content, or Hematocrit) below the lower limit of
  the normal range for the age and sex of the
  individual.
• In adults, the lower extreme of the normal
  haemoglobin is taken as 13.0 g/ dl for males and
  11.5 g/dl for females.
• Newborn infants have higher haemoglobin level
  and, therefore, 15 g/dl is taken as the lower
  limit at birth,

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Classification of Anemia

• Several types of classifications of anaemias
  have been proposed. Two of the widely accepted
  classifications are based on
• The pathophysiology and
• The morphology

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The pathophysiological classification

• Depending upon the pathophysiologic mechanism,
  anaemias are classified into 3 groups
• I. Anaemia due to increased blood loss
• II. Anaemias due to impaired red cell production
• III. Anaemias due to increased red cell
  destruction (Haemolytic anaemias)

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The Morphological classification

• Based on red cell size, haemoglobin content and
  red cell indices anaemias are classified into 3
  types
• I. Microcytic, hypochromic
• II. Normocytic, normochromic
• III. Macrocytic, normochromic

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Microcytic Hypochromic

• Causes
• Iron deficiency
• Thalassemia minor
• Anemia of chronic disease
• Lead poisoning
• Congenital sideroblastic anemia
• ß-Thalassemia intermedia and major
• Hemoglobin H or E disease

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Normocytic Hypochromic
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Normocytic Normochromic

• causes
• Anemia of chronic disease
• Early iron deficiency
• Renal failure
• Acquired immunodeficiency syndrome
• Aplastic anemia
• Pure red cell aplasia
• Bone marrow infiltration
• Leukemia
• Lymphoma
• Cancer
• Granulomatous diseases
• Myeloproliferative disorder

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Normocytic Normochromic
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Macrocytic Normochromic

• Causes
• Megaloblastic anemia (B12 or folate deficiency)
• Alcoholism
• Liver disease
• Reticulocytosis
• Chemotherapy
• Myelodysplastic syndromes
• Multiple myeloma
• Hypothyroidism

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Macrocytic Normochromic
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Laboratory Investigation

• Anemia is not a diagnosis, but a sign of
  underlying disease.
• The objective of the laboratory is to
• determine the type of anemia as an aid in
  discovering the cause.

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• In most laboratories the initial investigation
  and tentative diagnosis is made with a relatively
  small number of tests.
• The precise diagnosis is made with further
  special tests.
• Screening is usually done with the CBC or
  "complete blood count".
• The exact procedures in a CBC depends upon
  the instrumentation in the laboratory.
• Most laboratories now use automated,
  multiparameter instruments which will provide
  results for the following parameters
• hemoglobin
• hematocrit
• red cell count
• MCV , MCH ,MCHC
• RDW
• white cell and platelet count
• automated differential
• histograms

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HAE MOGLOBIN ESTIMATION

• The first and foremost investigation in any
  suspected case of anaemia is to carry out
  haemoglobin estimation.
• Several methods are available but most reliable
  and accurate is the cyanmethaemoglobin (HiCN)
  method employing Drabkin's solution and a
  spectrophotometer.
• If the haemoglobin value is below the lower limit
  of the normal range for particular age and sex,
  the patient is said to be anaemic.
• In pregnancy, there is haemodilution and,
  therefore, the lower limit in normal pregnant
  women is less (10.5 g/ dl) than in the
  non-pregnant state.

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Normal hemoglobin values

• Men
  14-17 gm
• Women
  13-15 gm
• Infants
  14-19gm
• Children (1year)
  11-13gm
• Children (10-12 years0 12-14gm

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Clinical significance of Hb measurement

• A decrease or increase in hemoglobin
  concentration must be reported ,as it is a sign
  of disease requiring investigations
• A decrease in Hb concentration is a sign of
  anemia
• While an increase can occur due to
• Haemochromatosis (loss of body fluid as in severe
  diarrhea)
• Reduced oxygen supply (congenital heart disease ,
  emphysema)
• Polycythemia

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Haematocrit or Packed Cell Volume

• It is the amount of packed red blood cell,
  following centrifugation, expressed as a total
  blood volume
• Normal value
• Male 42-52
• Female 36-49
• Roughly, the haematocrit value is 3 times the Hb
  concentration

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Clinical significance

• A decrease in the haematocrit value is a
  suitable measurement for detection of anaemia,
  also in case of hydremia (excessive fluid in
  blood as in pregnancy)
• An increase is an indication decrease oxygen
  supply (as in congenital heart disease,
  emphysema) or as in polycythemia and dehydration
• The value of haematocrit is used with haemoglobin
  and red cell count for the calculation of MCV,
  MCH and MCHC

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RED CELL INDICES

• The type of anemia may be indicated by the RBC
  indices
• mean corpuscular volume (MCV),
• mean corpuscular Hb (MCH), and
• mean corpuscular Hb concentration (MCHC).
• RBC populations are termed microcytic (MCV lt 80
  fl) or macrocytic (MCV gt 95 fl).
• The term hypochromia refers to RBC populations
  with MCH lt 27 pg/RBC or MCHC lt 30.
• These quantitative relationships can usually be
  recognized on a peripheral blood smear and,
  together with the indices, permit a
  classification of anemias that correlates with
  etiologic classification and greatly aids
  diagnosis.

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Mean Cell Volume(MCV)

• It is calculated from PCV and red cell count as
  follows
• MCV PCV/RBC fl
• A femtoliter (fl) is 10 15 of a liter
• Normal value 80-95 fl
• It decrease in iron deficiency anaemia and
  haemoglopinopathies
• It is increase in megaloblastic anaemia and
  chronic haemolytic anaemia

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Mean Cell Haemoglobin Concentration (MCHC)

• It is calculated from the haemoglobin and PCV as
  follows
• MCHC Hb/PCV g/dl
• Normal value 32-35.5
  g/dl
• It is usually decrease in iron deficiency anaemia
  (microcytic hypochromic anaemia)

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Mean Cell Haemoglobin (MCH)

• It is calculated from the haemoglobin and
  erythrocyte count as follows
• MCH Hbx10/RBC pg
  
• A pictogram (pg) is 10-12 of a gram
• Normal value 27-32 pg
• It is decrease in iron deficiency anaemia and
  thalassaemia (microcytic hypochromic anaemia)
• It is recognized by the pale colour of the red
  cell in the peripheral blood film
• It is increase in microcytic anaemia (vitamin B
  12 and folic acid)

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Red Cell Distribution width (RDW)

• RDW reflects the variation of RBCs volume
• it is usually performed by modern analysers
• Normal RDW varies between 12 to 17
• Severe iron deficiency anemia is associated with
  increased RDW
• Thalassemia and anemia of chronic disease are
  associated with normal RDW

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PERIPHERAL BLOOD FILM EXAMINATION

• Normal RBC
• The normal human erythrocytes are biconcave
  disc, 7.2 um in diameter, and the thickness of
  2.4 um at the periphery and 1 um in the center.
  The biconcave shape render the red cell quite
  flexible so that they can pass through
  capillaries whose minimum diameter is 3.5 um
• more than 90 of the weight of the red cell
  consist of haemoglobin.

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• Normal red cells (normochromic) have uniformly
  coloured haemoglobin in side the cell with a
  small clear paler region in the center

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Colour variation

• Anisochromasia is a variable staining
  intensities indicating unequal haemoglobin
  content
• Cause iron deficiency anaemia treated by
  transfused blood
• Hyperchromasia presence of cells having a
  smaller than normal area of central pallor,
  demonstrate higher than normal pigmentation
• Cause dehydration, chronic inflammation,
  spheroytosis
• Hypochromasia presence of cells having a larger
  than normal area of central pallor, demonstrate
  less than normal pigmentation
• Cause iron deficiency anaemia, decreased
  haemoglobin concentration
• Polychromasia the red cells are grey coloured
  and may be slightly larger than normal
• Cause reticulocytosis

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Shape variation Acanthocytes with
irregular, thorny speculated membrane surface
projections bulbous round endsCause
abetalipoproteinemia, renal failure, liver
disease, haemolytic anaemia
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Ecchinocytes cells with 10-30 uniformly
distributed spiculesCause blood loss (acute),
burns, DIC, carcinoma of stomach
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Elliptocytes have a cigar shapeCause
hereditary elliptocytosis, leukemia, thalassaemia
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Sickle cells cells have a sickle with appoint
at one end Cause sickle cell anaemia,
haemoglobin S disease
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Sphereocytes cells are globe like rather
than biconcave with an abnormal small
dimpleCause hereditary spheroytosis, autoimmune
haemolytic anaemia, septicemia
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Stomatocyte cells are cup shaped with an
abnormal area of central pallor that may be oval,
elongated, or slit likeCause liver disease,
alcoholism, hereditary spheroytosis
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Target cells cells have an increased ratio
of surface to volume, due to a shape that looks
like a cup, bell Cause iron deficiency, liver
disease, haemoglopinopathies, post spleenectomy
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Tear drop poikilocyte cells have teardrop or
pear shape Cause myelofibrosis, extramedullary
haemopoiesis, myeloid metaplasia
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Size variation

• Normal normal size (6-8u) is known as normocytic
• Macrocyte increase size of cells having diameter
  gt 8 u and MCV gt 95u
• Cause folic acid anaemia, following haemorrhage,
  liver disease
• Microcyte decrease size of cells having diameter
  lt 6 u and MCV lt 80u
• Cause haemoglopinopathies, iron deficiency,
  thalassaemia

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Content of structure variation Basophilic
stippling appearance of fine blue dots scattered
in red cellsCause haemoglopinopathies, lead
poisoning, haemolytic anaemia, myelodysplasia
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• Cabot ring cells containing mitotic spindle
  remnants appearing as fine, thread like filaments
  of bluish purple colour in the shape of a single
  ring or double ring (figure of eight)
• Cause megaloblastic anaemia, haemolytic
  anaemia

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Heinz bodies are denatured particles of
haemoglobin attached to RBC membrane that appear
when stained with cresyl blueCause G6PD
anaemia, drug induced, alpha thalassaemia
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Howell jolly body are nuclear fragment found
in red cells, mostly single but sometimes
multipleCause post splenectomy, hyposplenism
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Siderocytes granules (papenheimer bodies)
are cells with mitochondrial concentration of
ferritin (non-haemoglobin iron) deposit the
cells are stained by Prussian blue
reactionCause disorder of iron metabolism as
Sideroblastic anaemia. Postsplenectomy, burns,
hemochromatosis
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LEUCOCYTE AND PLATELET COUNT

• Measurement of leukocyte and platelet count helps
  to distinguish pure anaemia from pancytopenia in
  which red cells, granulocytes and platelets are
  all reduced.
• In anaemias due to haemolysis or haemorrhage, the
  neutrophil count and platelet counts are often
  elevated. In infections and leukemia's, the
  leucocyte counts are high and immature leucocytes
  appear in the blood.

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RETICULOCYTE COUNT

• Reticulocyte count (normal 0.5-2.5) is done in
  each case of anaemia to assess the marrow
  erythropoietic activity.
• In acute haemorrhage and in haemolysis, the
  reticulocyte response is indicative of impaired
  marrow function.

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BONE MARROW EXAMINATION

• Bone marrow aspiration is done in cases where the
  cause for anaemia is not obvious.
• The procedures involved marrow aspiration and
• trephine biopsy

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Indication of Bone marrow examination in case of
anemia

• megaloblastic
• sideroblastic
• iron deficiency
• aplastic anemia

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Special Investigations

• Biochemical Tests
• biochemical tests are aimed at identifying
• 1-a depleted cofactor necessary for normal
  hematopoiesis (iron, ferritin, folate, B12),
• 2-an abnormally functioning enzyme
  (glucose-6-phosphate dehydrogenase, pyruvate
  kinase), or
• 3-abnormal function of the immune system (the
  direct antiglobulin Coombs' test).

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Laboratory Investigation of Hemolytic anemia

• These are dividing into 4 groups
• I-Tests of increased red cell breakdown.
• II- Tests of increased red cell production.
• III- Tests of damage to red cells
• IV- Tests for shortened red cell life span

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Tests of increased red cell breakdown. these
include

• Serum bilirubin-unconjugated(indirect)bilirubin
  is raised
• Urine Urobilinogen is raised but there is no
  biliruninuria
• Faecal Stercobilinogen is raised
• Serum haptoglobin ( a globulin binding protein)
  is reduced or absent
• Plasma lactic acid dehydrogenase is raised
• Evidence of intravascular haemolysis in the form
  of haemoglobinaemia, haemoglobinuria,
  haemosiderinuria

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Tests of increased red cell production.

• Reticulocyte count reveals reticulocytosis which
  indicate marrow erythroid hyperplasia
• Routine blood film shows macrocytosis,
  polychromasia, normoblasts
• Bone marrow show erythroid hyperplasia with
  raised iron stores
• X ray of bones shows evidence of expansion of
  marrow spaces especially in tubular bones and
  skull

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Tests of damage to red cells

• Routine blood film shows a variety of abnormal
  morphological appearances of red cells
• Osmotic fragility is increased
• Autohaemolysis test
• Coomb's antiglobulin test
• Electrophoresis for abnormal haemoglobin
• Estimation of HbA2

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Tests for shortened red cell life span

• Tested by 51Cr labeling method normal RBC life
  span of 120 days is shortened to 20-40 days in
  moderate haemolysis and 5-20 days in severe
  haemolysis

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LABORATORY ERRORS

• 1 .Errors in reporting or recording of
  
  
  
  
  
  results
• 2 .Inadequate study of the blood film
• 3 .Failure to assess indices
• 4 .Failure to do retic count
• 5 .Failure to note rouleux

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• Thank You