Clinical laboratory

1. ‫بسم ا الحمن الرحيم‬

2. Anemia
Laboratory Diagnosis
Presented by
Dr. Mohammed Abbas

3. 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,

4. 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

5. 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)

6. 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

7. 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

8. Normocytic Hypochromic

9. 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

10. Normocytic Normochromic

11. Macrocytic Normochromic
:Causes
)Megaloblastic anemia (B12 or folate deficiency
Alcoholism
Liver disease
Reticulocytosis
Chemotherapy
Myelodysplastic syndromes
Multiple myeloma
Hypothyroidism

12. Macrocytic Normochromic

13. 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.

14.  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

15. 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.

16. :Normal hemoglobin values
 Men 14-17 gm%
 Women 13-15 gm%
 Infants 14-19gm%
 Children (1year) 11-13gm%
 Children (10-12 years0 12-14gm%

17. 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

18. 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

20. 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

21. 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 < 80 fl) or
macrocytic (MCV > 95 fl).
 The term hypochromia refers to RBC populations with
MCH < 27 pg/RBC or MCHC < 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.

22. )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

23. 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)

24. )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)

25. )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

26. 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.

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

28. :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

29. Shape variation
Acanthocytes
with irregular, thorny speculated membrane surface projections
bulbous round ends
Cause: abetalipoproteinemia, renal failure, liver disease, haemolytic
anaemia

30. Ecchinocytes: cells with 10-30 uniformly distributed
spicules
Cause: blood loss (acute), burns, DIC, carcinoma of
stomach

31. Elliptocytes: have a cigar shape
Cause: hereditary elliptocytosis, leukemia, thalassaemia

32. Sickle cells:
cells have a sickle with appoint at one end
Cause: sickle cell anaemia, haemoglobin S disease

33. Sphereocytes cells:
are globe like rather than biconcave with an abnormal
small dimple
Cause: hereditary spheroytosis, autoimmune haemolytic
anaemia, septicemia

34. Stomatocyte:
cells are cup shaped with an abnormal area of central
pallor that may be oval, elongated, or slit like
Cause: liver disease, alcoholism, hereditary spheroytosis

35. 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

36. Tear drop poikilocyte: cells have teardrop or pear shape
Cause: myelofibrosis, extramedullary haemopoiesis,
myeloid metaplasia

37. :Size variation
 Normal: normal size (6-8u) is known as
normocytic
 Macrocyte: increase size of cells having
diameter > 8 u and MCV > 95u
 Cause: folic acid anaemia, following
haemorrhage, liver disease
 Microcyte: decrease size of cells having
diameter < 6 u and MCV < 80u
 Cause: haemoglopinopathies, iron deficiency,
thalassaemia

38. Content of structure variation
Basophilic stippling: appearance of fine blue dots
scattered in red cells
Cause: haemoglopinopathies, lead poisoning, haemolytic
anaemia, myelodysplasia

39.  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

40. Heinz bodies: are denatured particles of haemoglobin
attached to RBC membrane that appear when stained with
cresyl blue
Cause: G6PD anaemia, drug induced, alpha thalassaemia

41. Howell jolly body:
are nuclear fragment found in red cells, mostly single but
sometimes multiple
Cause: post splenectomy, hyposplenism

42. Siderocytes granules (papenheimer bodies):
are cells with mitochondrial concentration of ferritin (non-
haemoglobin iron) deposit
the cells are stained by Prussian blue reaction
Cause: disorder of iron metabolism as Sideroblastic
anaemia. Postsplenectomy, burns, hemochromatosis

43. 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.

44. 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.

45. 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

47. Indication of Bone marrow examination in case of
anemia
 megaloblastic
 sideroblastic
 iron deficiency
 aplastic anemia

48. 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).

49. 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

50. 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 ( α globulin binding protein) is
reduced or absent
 Plasma lactic acid dehydrogenase is raised
 Evidence of intravascular haemolysis in the form of
haemoglobinaemia, haemoglobinuria,
haemosiderinuria

51. .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

52. 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

53. 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

54. 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

55. Thank You