3. DefinitionDefinition
Anemia (a decrease in the number of RBCs, HbAnemia (a decrease in the number of RBCs, Hb
content, or Hematocrit) below the lower limit ofcontent, or Hematocrit) below the lower limit of
the normal range for the age and sex of thethe normal range for the age and sex of the
individual.individual.
In adults, the lower extreme of the normalIn adults, the lower extreme of the normal
haemoglobin is taken as 13.0 g/ dl for males andhaemoglobin is taken as 13.0 g/ dl for males and
11.5 g/dl for females.11.5 g/dl for females.
Newborn infants have higher haemoglobin levelNewborn infants have higher haemoglobin level
and, therefore, 15 g/dl is taken as the lower limitand, therefore, 15 g/dl is taken as the lower limit
at birth,at birth,
4. Classification of AnemiaClassification of Anemia
Several types of classifications of anaemiasSeveral types of classifications of anaemias
have been proposed. Two of the widelyhave been proposed. Two of the widely
accepted classifications are based onaccepted classifications are based on
The pathophysiology andThe pathophysiology and
The morphologyThe morphology
5. The pathophysiologicalThe pathophysiological
classificationclassification
Depending upon the pathophysiologicDepending upon the pathophysiologic
mechanism, anaemias are classified into 3mechanism, anaemias are classified into 3
groups:groups:
I. Anaemia due to increased blood lossI. Anaemia due to increased blood loss
II. Anaemias due to impaired red cellII. Anaemias due to impaired red cell
productproductionion
III. Anaemias due to increased red cellIII. Anaemias due to increased red cell
destruction (Haemolytic anaemias)destruction (Haemolytic anaemias)
6. The Morphological classificationThe Morphological classification
Based on red cell size, haemoglobinBased on red cell size, haemoglobin
content and red cell indices anaemias arecontent and red cell indices anaemias are
classified into 3 types:classified into 3 types:
I. Microcytic, hypochromicI. Microcytic, hypochromic
II. Normocytic, normochromicII. Normocytic, normochromic
III. Macrocytic, normochromicIII. Macrocytic, normochromic
13. Laboratory InvestigationLaboratory 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 tentativeIn most laboratories the initial investigation and tentative
diagnosis is made with a relatively small number of testsdiagnosis is made with a relatively small number of tests..
The precise diagnosis is made with further special testsThe precise diagnosis is made with further special tests..
Screening is usually done with the CBC orScreening is usually done with the CBC or ""complete bloodcomplete blood
countcount".".
The exact procedures in a CBC depends upon theThe exact procedures in a CBC depends upon the
instrumentation in the laboratoryinstrumentation in the laboratory..
Most laboratories now use automated, multiparameterMost laboratories now use automated, multiparameter
instruments which will provide results for the followinginstruments which will provide results for the following
parametersparameters::
hemoglobinhemoglobin
hematocrithematocrit
red cell countred cell count
MCV , MCH ,MCHCMCV , MCH ,MCHC
RDWRDW
white cell and platelet countwhite cell and platelet count
automated differentialautomated differential
histogramshistograms
15. HAE MOGLOBIN ESTIMATIONHAE MOGLOBIN ESTIMATION
The first and foremost investigation in any suspectedThe first and foremost investigation in any suspected
case of anaemia is to carry out haemoglobin estimation.case of anaemia is to carry out haemoglobin estimation.
Several methods are available but most reliable andSeveral methods are available but most reliable and
accurate is the cyanmethaemoglobin (HiCN) methodaccurate is the cyanmethaemoglobin (HiCN) method
employing Drabkin's solution and a spectrophotometer.employing Drabkin's solution and a spectrophotometer.
If the haemoglobin value is below the lower limit of theIf the haemoglobin value is below the lower limit of the
normal range for particular age and sex, the patient isnormal range for particular age and sex, the patient is
said to be anaemic.said to be anaemic.
In pregnancy, there is haemodilution and, therefore, theIn pregnancy, there is haemodilution and, therefore, the
lower limit in normal pregnant women is less (10.5 g/ dl)lower limit in normal pregnant women is less (10.5 g/ dl)
than in the non-pregnant state.than in the non-pregnant state.
16. Normal hemoglobin valuesNormal hemoglobin values::
Men 14-17 gm%Men 14-17 gm%
Women 13-15 gm%Women 13-15 gm%
Infants 14-19gm%Infants 14-19gm%
Children (1year) 11-13gm%Children (1year) 11-13gm%
Children (10-12 years0 12-14gm%Children (10-12 years0 12-14gm%
17. Clinical significance of HbClinical significance of Hb
measurementmeasurement::
AA decreasedecrease oror increaseincrease in hemoglobinin hemoglobin
concentration must be reported ,as it is a sign ofconcentration must be reported ,as it is a sign of
disease requiring investigationsdisease requiring investigations
AA decreasedecrease in Hb concentration is a sign ofin Hb concentration is a sign of
anemiaanemia
While anWhile an increaseincrease can occur due to;can occur due to;
Haemochromatosis (loss of body fluid as inHaemochromatosis (loss of body fluid as in
severe diarrhea)severe diarrhea)
Reduced oxygen supply (congenital heartReduced oxygen supply (congenital heart
disease , emphysema)disease , emphysema)
PolycythemiaPolycythemia
18. Haematocrit or Packed Cell VolumeHaematocrit or Packed Cell Volume
It is the amount of packed red blood cell,It is the amount of packed red blood cell,
following centrifugation, expressed as afollowing centrifugation, expressed as a
total blood volumetotal blood volume
Normal valueNormal value
Male: 42-52 %Male: 42-52 %
Female: 36-49%Female: 36-49%
Roughly, the haematocrit value is 3 timesRoughly, the haematocrit value is 3 times
the Hb concentrationthe Hb concentration
19.
20. Clinical significanceClinical significance
A decrease in the haematocrit value is a suitableA decrease in the haematocrit value is a suitable
measurement for detection of anaemia, also inmeasurement for detection of anaemia, also in
case of hydremia (excessive fluid in blood as incase of hydremia (excessive fluid in blood as in
pregnancy)pregnancy)
An increase is an indication decrease oxygenAn increase is an indication decrease oxygen
supply (as in congenital heart disease,supply (as in congenital heart disease,
emphysema) or as in polycythemia andemphysema) or as in polycythemia and
dehydrationdehydration
The value of haematocrit is used withThe value of haematocrit is used with
haemoglobin and red cell count for thehaemoglobin and red cell count for the
calculation of MCV, MCH and MCHCcalculation of MCV, MCH and MCHC
21. RED CELL INDICESRED CELL INDICES
The type of anemia may be indicated by the RBC indices:The type of anemia may be indicated by the RBC indices:
mean corpuscular volume (MCV),mean corpuscular volume (MCV),
mean corpuscular Hb (MCH), andmean corpuscular Hb (MCH), and
mean corpuscular Hb concentration (MCHC).mean corpuscular Hb concentration (MCHC).
RBC populations are termedRBC populations are termed microcyticmicrocytic (MCV < 80 fl) or(MCV < 80 fl) or
macrocyticmacrocytic (MCV > 95 fl).(MCV > 95 fl).
The termThe term hypochromiahypochromia refers to RBC populations withrefers to RBC populations with
MCH < 27 pg/RBC or MCHC < 30%.MCH < 27 pg/RBC or MCHC < 30%.
These quantitative relationships can usually beThese quantitative relationships can usually be
recognized on a peripheral blood smear and, togetherrecognized on a peripheral blood smear and, together
with the indices, permit a classification of anemias thatwith the indices, permit a classification of anemias that
correlates with etiologic classification and greatly aidscorrelates with etiologic classification and greatly aids
diagnosis.diagnosis.
22. Mean Cell Volume(MCVMean Cell Volume(MCV))
It is calculated from PCV and red cellIt is calculated from PCV and red cell
count as follows:count as follows:
MCV = PCV/RBC flMCV = PCV/RBC fl
A femtoliter (fl) is 10 15 of a literA femtoliter (fl) is 10 15 of a liter
Normal value:Normal value: 80-95 fl80-95 fl
It decrease in iron deficiency anaemia andIt decrease in iron deficiency anaemia and
haemoglopinopathieshaemoglopinopathies
It is increase in megaloblastic anaemiaIt is increase in megaloblastic anaemia
and chronic haemolytic anaemiaand chronic haemolytic anaemia
23. Mean Cell Haemoglobin ConcentrationMean Cell Haemoglobin Concentration
(MCHC(MCHC))
It is calculated from the haemoglobin andIt is calculated from the haemoglobin and
PCV as follows:PCV as follows:
MCHC = Hb/PCV g/dlMCHC = Hb/PCV g/dl
Normal value:Normal value: 32-35.5 g/dl32-35.5 g/dl
It is usually decrease in iron deficiencyIt is usually decrease in iron deficiency
anaemia (microcytic hypochromicanaemia (microcytic hypochromic
anaemia)anaemia)
24. Mean Cell Haemoglobin (MCHMean Cell Haemoglobin (MCH))
It is calculated from the haemoglobin andIt is calculated from the haemoglobin and
erythrocyte count as follows:erythrocyte count as follows:
MCH = Hbx10/RBC pgMCH = Hbx10/RBC pg
A pictogram (pg) is 10-12 of a gramA pictogram (pg) is 10-12 of a gram
Normal value:Normal value: 27-32 pg27-32 pg
It is decrease in iron deficiency anaemia andIt is decrease in iron deficiency anaemia and
thalassaemia (microcytic hypochromic anaemia)thalassaemia (microcytic hypochromic anaemia)
It is recognized by the pale colour of the red cellIt is recognized by the pale colour of the red cell
in the peripheral blood filmin the peripheral blood film
It is increase in microcytic anaemia (vitamin BIt is increase in microcytic anaemia (vitamin B
12 and folic acid)12 and folic acid)
25. Red Cell Distribution width (RDWRed Cell Distribution width (RDW))
RDW reflects the variation of RBCsRDW reflects the variation of RBCs
volumevolume
it is usually performed by modernit is usually performed by modern
analysersanalysers
Normal RDW varies between 12 to 17Normal RDW varies between 12 to 17
Severe iron deficiency anemia isSevere iron deficiency anemia is
associated with increased RDWassociated with increased RDW
Thalassemia and anemia of chronicThalassemia and anemia of chronic
disease are associated with normal RDWdisease are associated with normal RDW
26. PERIPHERAL BLOOD FILM EXAMINATIONPERIPHERAL BLOOD FILM EXAMINATION
Normal RBC :Normal RBC :
The normal human erythrocytes are biconcaveThe normal human erythrocytes are biconcave
disc, 7.2 um in diameter, and the thickness ofdisc, 7.2 um in diameter, and the thickness of
2.4 um at the periphery and 1 um in the center.2.4 um at the periphery and 1 um in the center.
The biconcave shape render the red cell quiteThe biconcave shape render the red cell quite
flexible so that they can pass through capillariesflexible so that they can pass through capillaries
whose minimum diameter is 3.5 umwhose minimum diameter is 3.5 um
more than 90% of the weight of the red cellmore than 90% of the weight of the red cell
consist of haemoglobin.consist of haemoglobin.
27. Normal red cellsNormal red cells (normochromic): have(normochromic): have
uniformly coloured haemoglobin in sideuniformly coloured haemoglobin in side
the cell with a small clear paler region inthe cell with a small clear paler region in
the centerthe center
28. Colour variationColour variation::
Anisochromasia:Anisochromasia: is a variable staining intensities indicatingis a variable staining intensities indicating
unequal haemoglobin contentunequal haemoglobin content
Cause: iron deficiency anaemia treated by transfused bloodCause: iron deficiency anaemia treated by transfused blood
HyperchromasiaHyperchromasia: presence of cells having a smaller than normal: presence of cells having a smaller than normal
area of central pallor, demonstrate higher than normal pigmentationarea of central pallor, demonstrate higher than normal pigmentation
Cause: dehydration, chronic inflammation, spheroytosisCause: dehydration, chronic inflammation, spheroytosis
Hypochromasia:Hypochromasia: presence of cells having a larger than normalpresence of cells having a larger than normal
area of central pallor, demonstrate less than normal pigmentationarea of central pallor, demonstrate less than normal pigmentation
Cause: iron deficiency anaemia, decreased haemoglobinCause: iron deficiency anaemia, decreased haemoglobin
concentrationconcentration
Polychromasia:Polychromasia: the red cells are grey coloured and may be slightlythe red cells are grey coloured and may be slightly
larger than normallarger than normal
Cause: reticulocytosisCause: reticulocytosis
30. Ecchinocytes:Ecchinocytes: cells with 10-30 uniformly distributedcells with 10-30 uniformly distributed
spiculesspicules
Cause: blood loss (acute), burns, DIC, carcinoma ofCause: blood loss (acute), burns, DIC, carcinoma of
stomachstomach
31. ElliptocytesElliptocytes: have a cigar shape: have a cigar shape
Cause: hereditary elliptocytosis, leukemia, thalassaemiaCause: hereditary elliptocytosis, leukemia, thalassaemia
32. Sickle cells:Sickle cells:
cells have a sickle with appoint at one endcells have a sickle with appoint at one end
Cause: sickle cell anaemia, haemoglobin S diseaseCause: sickle cell anaemia, haemoglobin S disease
33. Sphereocytes cells:Sphereocytes cells:
are globe likeare globe like rather than biconcave with an abnormalrather than biconcave with an abnormal
small dimplesmall dimple
Cause: hereditary spheroytosis, autoimmune haemolyticCause: hereditary spheroytosis, autoimmune haemolytic
anaemia, septicemiaanaemia, septicemia
34. Stomatocyte:Stomatocyte:
cells are cup shaped with an abnormal area of centralcells are cup shaped with an abnormal area of central
pallor that may be oval, elongated, or slit likepallor that may be oval, elongated, or slit like
Cause: liver disease, alcoholism, hereditary spheroytosisCause: liver disease, alcoholism, hereditary spheroytosis
35. Target cells:Target cells:
cells have an increased ratio of surface to volume, due to acells have an increased ratio of surface to volume, due to a
shape that looks like a cup, bellshape that looks like a cup, bell
Cause: iron deficiency, liver disease, haemoglopinopathies,Cause: iron deficiency, liver disease, haemoglopinopathies,
post spleenectomypost spleenectomy
36. Tear drop poikilocyte:Tear drop poikilocyte: cells have teardrop or pear shapecells have teardrop or pear shape
Cause: myelofibrosis, extramedullary haemopoiesis,Cause: myelofibrosis, extramedullary haemopoiesis,
myeloid metaplasiamyeloid metaplasia
37. Size variationSize variation::
Normal:Normal: normal size (6-8u) is known asnormal size (6-8u) is known as
normocyticnormocytic
Macrocyte:Macrocyte: increase size of cells havingincrease size of cells having
diameter > 8 u and MCV > 95udiameter > 8 u and MCV > 95u
Cause: folic acid anaemia, followingCause: folic acid anaemia, following
haemorrhage, liver diseasehaemorrhage, liver disease
Microcyte:Microcyte: decrease size of cells havingdecrease size of cells having
diameter < 6 u and MCV < 80udiameter < 6 u and MCV < 80u
Cause: haemoglopinopathies, iron deficiency,Cause: haemoglopinopathies, iron deficiency,
thalassaemiathalassaemia
38. Content of structure variationContent of structure variation
Basophilic stippling:Basophilic stippling: appearanceappearance of fine blue dotsof fine blue dots
scattered in red cellsscattered in red cells
Cause: haemoglopinopathies, lead poisoning, haemolyticCause: haemoglopinopathies, lead poisoning, haemolytic
anaemia, myelodysplasiaanaemia, myelodysplasia
39. Cabot ringCabot ring: cells containing mitotic spindle remnants appearing as fine,: cells containing mitotic spindle remnants appearing as fine,
thread like filaments of bluish purple colour in the shape of a single ring orthread like filaments of bluish purple colour in the shape of a single ring or
double ring (figure of eight)double ring (figure of eight)
Cause: megaloblastic anaemia, haemolytic anaemiaCause: megaloblastic anaemia, haemolytic anaemia
40. Heinz bodies:Heinz bodies: are denaturedare denatured particles of haemoglobinparticles of haemoglobin
attached to RBC membrane that appear when stained withattached to RBC membrane that appear when stained with
cresyl bluecresyl blue
Cause: G6PD anaemia, drug induced, alpha thalassaemiaCause: G6PD anaemia, drug induced, alpha thalassaemia
41. Howell jolly body:Howell jolly body:
are nuclear fragment found in red cells, mostly single butare nuclear fragment found in red cells, mostly single but
sometimes multiplesometimes multiple
Cause: post splenectomy, hyposplenismCause: post splenectomy, hyposplenism
42. Siderocytes granules (papenheimer bodies):Siderocytes granules (papenheimer bodies):
are cells with mitochondrial concentration of ferritin (non-are cells with mitochondrial concentration of ferritin (non-
haemoglobin iron) deposithaemoglobin iron) deposit
the cells are stained by Prussian blue reactionthe cells are stained by Prussian blue reaction
Cause: disorder of iron metabolism as SideroblasticCause: disorder of iron metabolism as Sideroblastic
anaemia. Postsplenectomy, burns, hemochromatosisanaemia. Postsplenectomy, burns, hemochromatosis
43. LEUCOCYTE AND PLATELET COUNTLEUCOCYTE AND PLATELET COUNT
Measurement of leukocyte and platelet count helps to distinguish pureMeasurement of leukocyte and platelet count helps to distinguish pure
anaemia from pancytopenia in which red cells, granulocytes andanaemia from pancytopenia in which red cells, granulocytes and
platelets are all reduced.platelets are all reduced.
In anaemias due to haemolysis or haemorrhage, the neutrophil countIn anaemias due to haemolysis or haemorrhage, the neutrophil count
and platelet counts are often elevated. In infections and leukemia's,and platelet counts are often elevated. In infections and leukemia's,
the leucocyte counts are high and immature leucocytes appear inthe leucocyte counts are high and immature leucocytes appear in
the blood.the blood.
44. RETICULOCYTE COUNTRETICULOCYTE COUNT
Reticulocyte count (normal 0.5-2.5%) isReticulocyte count (normal 0.5-2.5%) is
done in each case of anaemia to assessdone in each case of anaemia to assess
the marrow erythropoietic activity.the marrow erythropoietic activity.
In acute haemorrhage and in haemolysis,In acute haemorrhage and in haemolysis,
the reticulocyte response is indicative ofthe reticulocyte response is indicative of
impaired marrow function.impaired marrow function.
45. BONE MARROW EXAMINATIONBONE MARROW EXAMINATION
Bone marrow aspiration is done in casesBone marrow aspiration is done in cases
where the cause for anaemia is notwhere the cause for anaemia is not
obvious.obvious.
The procedures involved marrowThe procedures involved marrow
aspiration andaspiration and
trephine biopsytrephine biopsy
46.
47. Indication of Bone marrow examination in case ofIndication of Bone marrow examination in case of
anemiaanemia
megaloblastic
sideroblastic
iron deficiency
aplastic anemia
48. Special InvestigationsSpecial Investigations
Biochemical TestsBiochemical Tests
biochemical tests are aimed at identifyingbiochemical tests are aimed at identifying
1-a depleted cofactor necessary for normal1-a depleted cofactor necessary for normal
hematopoiesis (iron, ferritin, folate, B12),hematopoiesis (iron, ferritin, folate, B12),
2-an abnormally functioning enzyme2-an abnormally functioning enzyme
(glucose-6-phosphate dehydrogenase,(glucose-6-phosphate dehydrogenase,
pyruvate kinase), orpyruvate kinase), or
3-abnormal function of the immune system3-abnormal function of the immune system
(the direct antiglobulin [Coombs'] test).(the direct antiglobulin [Coombs'] test).
49. Laboratory Investigation of Hemolytic anemiaLaboratory Investigation of Hemolytic anemia
These are dividing into 4 groups:These are dividing into 4 groups:
I-Tests of increased red cell breakdownI-Tests of increased red cell breakdown..
II-II- Tests of increased red cell productionTests of increased red cell production..
III-III- Tests of damage to red cellsTests of damage to red cells
IV- Tests for shortened red cell life spanIV- Tests for shortened red cell life span
50. Tests of increased red cell breakdownTests of increased red cell breakdown..
these includethese include::
Serum bilirubinSerum bilirubin-unconjugated(indirect)bilirubin is-unconjugated(indirect)bilirubin is
raisedraised
Urine UrobilinogenUrine Urobilinogen is raised but there is nois raised but there is no
biliruninuriabiliruninuria
Faecal StercobilinogenFaecal Stercobilinogen is raisedis raised
Serum haptoglobinSerum haptoglobin ( α globulin binding protein) is( α globulin binding protein) is
reduced or absentreduced or absent
Plasma lactic acid dehydrogenasePlasma lactic acid dehydrogenase is raisedis raised
Evidence of intravascular haemolysisEvidence of intravascular haemolysis in the form ofin the form of
haemoglobinaemia, haemoglobinuria,haemoglobinaemia, haemoglobinuria,
haemosiderinuriahaemosiderinuria
51. Tests of increased red cell productionTests of increased red cell production..
Reticulocyte countReticulocyte count reveals reticulocytosisreveals reticulocytosis
which indicate marrow erythroid hyperplasiawhich indicate marrow erythroid hyperplasia
Routine blood filmRoutine blood film shows macrocytosis,shows macrocytosis,
polychromasia, normoblastspolychromasia, normoblasts
Bone marrowBone marrow show erythroid hyperplasia withshow erythroid hyperplasia with
raised iron storesraised iron stores
X ray of bonesX ray of bones shows evidence of expansionshows evidence of expansion
of marrow spaces especially in tubular bonesof marrow spaces especially in tubular bones
and skulland skull
52. Tests of damage to red cellsTests of damage to red cells
Routine blood filmRoutine blood film shows a variety ofshows a variety of
abnormal morphological appearances of redabnormal morphological appearances of red
cellscells
Osmotic fragilityOsmotic fragility is increasedis increased
AutohaemolysisAutohaemolysis testtest
Coomb'sCoomb's antiglobulin testantiglobulin test
ElectrophoresisElectrophoresis for abnormal haemoglobinfor abnormal haemoglobin
Estimation of HbAEstimation of HbA22
53. Tests for shortened red cell life spanTests for shortened red cell life span
Tested by 51Cr labeling method normalTested by 51Cr labeling method normal
RBC life span of 120 days is shortened toRBC life span of 120 days is shortened to
20-40 days in moderate haemolysis and20-40 days in moderate haemolysis and
5-20 days in severe haemolysis5-20 days in severe haemolysis
54. LABORATORY ERRORSLABORATORY ERRORS
11 .Errors in reporting or recording of.Errors in reporting or recording of
resultsresults
22 .Inadequate study of the blood film.Inadequate study of the blood film
33 .Failure to assess indices.Failure to assess indices
44 .Failure to do retic count.Failure to do retic count
