الحمن الله الحمن بسم الله بسمالرحيمالرحيم

AnemiaAnemiaLaboratory DiagnosisLaboratory Diagnosis

Presented byPresented byDr. Mohammed AbbasDr. Mohammed Abbas

DefinitionDefinition

Anemia (a decrease in the number of RBCs, Hb Anemia (a decrease in the number of RBCs, Hb content, or Hematocrit) below the lower limit of content, or Hematocrit) below the lower limit of the normal range for the age and sex of the the normal range for the age and sex of the individual. individual.

In adults, the lower extreme of the normal In adults, the lower extreme of the normal haemoglobin is taken as 13.0 g/ dl for males and haemoglobin 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 level Newborn infants have higher haemoglobin level and, therefore, 15 g/dl is taken as the lower limit and, therefore, 15 g/dl is taken as the lower limit at birth, at birth,

Classification of AnemiaClassification of Anemia

Several types of classifications of anaemias Several types of classifications of anaemias have been proposed. Two of the widely have been proposed. Two of the widely accepted classifications are based on accepted classifications are based on

The pathophysiology and The pathophysiology and The morphologyThe morphology

The pathophysiological The pathophysiological classificationclassification

Depending upon the pathophysiologic Depending upon the pathophysiologic mechanism, anaemias are classified into 3 mechanism, anaemias are classified into 3 groups: groups:

I. Anaemia due to increased blood loss I. Anaemia due to increased blood loss II. Anaemias due to impaired red cell II. Anaemias due to impaired red cell

productproductionion III. Anaemias due to increased red cell III. Anaemias due to increased red cell

destruction (Haemolytic anaemias)destruction (Haemolytic anaemias)

The Morphological classificationThe Morphological classification

Based on red cell size, haemoglobin Based on red cell size, haemoglobin content and red cell indices anaemias are content and red cell indices anaemias are classified into 3 types:classified into 3 types:

I. Microcytic, hypochromic I. Microcytic, hypochromic II. Normocytic, normochromic II. Normocytic, normochromic III. Macrocytic, normochromicIII. Macrocytic, normochromic

Microcytic HypochromicMicrocytic Hypochromic

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

Normocytic HypochromicNormocytic Hypochromic

Normocytic NormochromicNormocytic 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

Normocytic NormochromicNormocytic Normochromic

Macrocytic NormochromicMacrocytic NormochromicCauses:Megaloblastic anemia (B12 or folate deficiency)AlcoholismLiver diseaseReticulocytosisChemotherapyMyelodysplastic syndromesMultiple myelomaHypothyroidism

Macrocytic NormochromicMacrocytic Normochromic

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.

In most laboratories the initial investigation and tentative In 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 or Screening is usually done with the CBC or ""complete blood complete blood

countcount". ". The exact procedures in a CBC depends upon the The exact procedures in a CBC depends upon the

instrumentation in the laboratoryinstrumentation in the laboratory. . Most laboratories now use automated, multiparameter Most laboratories now use automated, multiparameter

instruments which will provide results for the following instruments which will provide results for the following parametersparameters::

hemoglobin hemoglobin hematocrit hematocrit red cell count red cell count MCV , MCH ,MCHC MCV , MCH ,MCHC RDW RDW white cell and platelet count white cell and platelet count automated differential automated differential histograms histograms

HAE MOGLOBIN ESTIMATIONHAE MOGLOBIN ESTIMATION

The first and foremost investigation in any suspected The 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 and Several methods are available but most reliable and accurate is the cyanmethaemoglobin (HiCN) method accurate 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 the If the haemoglobin value is below the lower limit of the normal range for particular age and sex, the patient is normal range for particular age and sex, the patient is said to be anaemic. said to be anaemic.

In pregnancy, there is haemodilution and, therefore, the In 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.

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%

Clinical significance of Hb Clinical significance of Hb measurementmeasurement::

A A decreasedecrease or or increaseincrease in hemoglobin in hemoglobin concentration must be reported ,as it is a sign of concentration must be reported ,as it is a sign of disease requiring investigationsdisease requiring investigations

A A decreasedecrease in Hb concentration is a sign of in Hb concentration is a sign of anemiaanemia

While an While an increaseincrease can occur due to; can occur due to; Haemochromatosis (loss of body fluid as in Haemochromatosis (loss of body fluid as in

severe diarrhea)severe diarrhea) Reduced oxygen supply (congenital heart Reduced oxygen supply (congenital heart

disease , emphysema)disease , emphysema) PolycythemiaPolycythemia

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 a following 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 times Roughly, the haematocrit value is 3 times

the Hb concentration the Hb concentration

Clinical significanceClinical significance

A decrease in the haematocrit value is a suitable A decrease in the haematocrit value is a suitable measurement for detection of anaemia, also in measurement for detection of anaemia, also in case of hydremia (excessive fluid in blood as in case of hydremia (excessive fluid in blood as in pregnancy)pregnancy)

An increase is an indication decrease oxygen An increase is an indication decrease oxygen supply (as in congenital heart disease, supply (as in congenital heart disease, emphysema) or as in polycythemia and emphysema) or as in polycythemia and dehydrationdehydration

The value of haematocrit is used with The value of haematocrit is used with haemoglobin and red cell count for the haemoglobin and red cell count for the calculation of MCV, MCH and MCHCcalculation of MCV, MCH and MCHC

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), and mean corpuscular Hb (MCH), and mean corpuscular Hb concentration (MCHC). mean corpuscular Hb concentration (MCHC). RBC populations are termed RBC populations are termed microcyticmicrocytic (MCV < 80 fl) or (MCV < 80 fl) or

macrocyticmacrocytic (MCV > 95 fl). (MCV > 95 fl). The term The term hypochromiahypochromia refers to RBC populations with refers to RBC populations with

MCH < 27 pg/RBC or MCHC < 30%. MCH < 27 pg/RBC or MCHC < 30%. These quantitative relationships can usually be These quantitative relationships can usually be

recognized on a peripheral blood smear and, together recognized on a peripheral blood smear and, together with the indices, permit a classification of anemias that with the indices, permit a classification of anemias that correlates with etiologic classification and greatly aids correlates with etiologic classification and greatly aids diagnosis.diagnosis.

Mean Cell Volume(MCV)Mean Cell Volume(MCV)

It is calculated from PCV and red cell It is calculated from PCV and red cell count as follows:count as follows:

MCV = PCV/RBC fl MCV = 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 fl 80-95 fl It decrease in iron deficiency anaemia and It decrease in iron deficiency anaemia and

haemoglopinopathieshaemoglopinopathies It is increase in megaloblastic anaemia It is increase in megaloblastic anaemia

and chronic haemolytic anaemia and chronic haemolytic anaemia

Mean Cell Haemoglobin Concentration Mean Cell Haemoglobin Concentration (MCHC)(MCHC)

It is calculated from the haemoglobin and It 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/dl 32-35.5 g/dl It is usually decrease in iron deficiency It is usually decrease in iron deficiency

anaemia (microcytic hypochromic anaemia (microcytic hypochromic anaemia)anaemia)

Mean Cell Haemoglobin (MCH)Mean Cell Haemoglobin (MCH) It is calculated from the haemoglobin and It is calculated from the haemoglobin and

erythrocyte count as follows:erythrocyte count as follows: MCH = Hbx10/RBC pg MCH = Hbx10/RBC pg A pictogram (pg) is 10-12 of a gram A pictogram (pg) is 10-12 of a gram Normal value:Normal value: 27-32 pg 27-32 pg It is decrease in iron deficiency anaemia and It 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 cell It is recognized by the pale colour of the red cell

in the peripheral blood film in the peripheral blood film It is increase in microcytic anaemia (vitamin B 12 It is increase in microcytic anaemia (vitamin B 12

and folic acid)and folic acid)

Red Cell Distribution width (RDW)Red Cell Distribution width (RDW)

RDW reflects the variation of RBCs RDW reflects the variation of RBCs volumevolume

it is usually performed by modern it is usually performed by modern analysersanalysers

Normal RDW varies between 12 to 17Normal RDW varies between 12 to 17 Severe iron deficiency anemia is Severe iron deficiency anemia is

associated with increased RDWassociated with increased RDW Thalassemia and anemia of chronic Thalassemia and anemia of chronic

disease are associated with normal RDWdisease are associated with normal RDW

PERIPHERAL BLOOD FILM EXAMINATIONPERIPHERAL BLOOD FILM EXAMINATION

Normal RBC :Normal RBC :

The normal human erythrocytes are biconcave The normal human erythrocytes are biconcave disc, 7.2 um in diameter, and the thickness of disc, 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 quite The biconcave shape render the red cell quite flexible so that they can pass through capillaries flexible so that they can pass through capillaries whose minimum diameter is 3.5 um whose minimum diameter is 3.5 um

more than 90% of the weight of the red cell more than 90% of the weight of the red cell consist of haemoglobin. consist of haemoglobin.

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

Colour variationColour variation:: Anisochromasia:Anisochromasia: is a variable staining intensities indicating is a variable staining intensities indicating

unequal haemoglobin content unequal 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 normal presence 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 haemoglobin Cause: iron deficiency anaemia, decreased haemoglobin concentrationconcentration

Polychromasia:Polychromasia: the red cells are grey coloured and may be slightly the red cells are grey coloured and may be slightly larger than normallarger than normal

Cause: reticulocytosisCause: reticulocytosis

Shape variationShape variation

Acanthocytes Acanthocytes with irregular, thorny speculated membrane surface projections with irregular, thorny speculated membrane surface projections bulbous round endsbulbous round endsCause: abetalipoproteinemia, renal failure, liver disease, haemolytic Cause: abetalipoproteinemia, renal failure, liver disease, haemolytic anaemiaanaemia

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

ElliptocytesElliptocytes: have a cigar shape: have a cigar shapeCause: hereditary elliptocytosis, leukemia, thalassaemiaCause: hereditary elliptocytosis, leukemia, thalassaemia

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

Sphereocytes cells:Sphereocytes cells:

are globe likeare globe like rather than biconcave with an abnormal rather than biconcave with an abnormal small dimplesmall dimpleCause: hereditary spheroytosis, autoimmune haemolytic Cause: hereditary spheroytosis, autoimmune haemolytic anaemia, septicemiaanaemia, septicemia

Stomatocyte:Stomatocyte: cells are cup shaped with an abnormal area of central cells 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 likeCause: liver disease, alcoholism, hereditary spheroytosisCause: liver disease, alcoholism, hereditary spheroytosis

Target cells:Target cells: cells have an increased ratio of surface to volume, due to a cells have an increased ratio of surface to volume, due to a shape that looks like a cup, bell shape that looks like a cup, bell Cause: iron deficiency, liver disease, haemoglopinopathies, Cause: iron deficiency, liver disease, haemoglopinopathies, post spleenectomypost spleenectomy

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

Size variationSize variation::

Normal:Normal: normal size (6-8u) is known as normal size (6-8u) is known as normocyticnormocytic

Macrocyte:Macrocyte: increase size of cells having increase size of cells having diameter > 8 u and MCV > 95udiameter > 8 u and MCV > 95u

Cause: folic acid anaemia, following Cause: folic acid anaemia, following haemorrhage, liver diseasehaemorrhage, liver disease

Microcyte:Microcyte: decrease size of cells having decrease size of cells having diameter < 6 u and MCV < 80u diameter < 6 u and MCV < 80u

Cause: haemoglopinopathies, iron deficiency, Cause: haemoglopinopathies, iron deficiency, thalassaemiathalassaemia

Content of structure variationContent of structure variation

Basophilic stippling:Basophilic stippling: appearance appearance of fine blue dots of fine blue dots scattered in red cellsscattered in red cellsCause: haemoglopinopathies, lead poisoning, haemolytic Cause: haemoglopinopathies, lead poisoning, haemolytic anaemia, myelodysplasiaanaemia, myelodysplasia

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 or thread 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

Heinz bodies:Heinz bodies: are denatured are denatured particles of haemoglobin particles of haemoglobin attached to RBC membrane that appear when stained with attached to RBC membrane that appear when stained with cresyl bluecresyl blueCause: G6PD anaemia, drug induced, alpha thalassaemiaCause: G6PD anaemia, drug induced, alpha thalassaemia

Howell jolly body:Howell jolly body: are nuclear fragment found in red cells, mostly single but are nuclear fragment found in red cells, mostly single but sometimes multiplesometimes multipleCause: post splenectomy, hyposplenismCause: post splenectomy, hyposplenism

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) deposit haemoglobin iron) deposit the cells are stained by Prussian blue reactionthe cells are stained by Prussian blue reactionCause: disorder of iron metabolism as Sideroblastic Cause: disorder of iron metabolism as Sideroblastic anaemia. Postsplenectomy, burns, hemochromatosis anaemia. Postsplenectomy, burns, hemochromatosis

LEUCOCYTE AND PLATELET COUNTLEUCOCYTE AND PLATELET COUNT

Measurement of leukocyte and platelet count helps to distinguish pure Measurement of leukocyte and platelet count helps to distinguish pure

anaemia from pancytopenia in which red cells, granulocytes and anaemia 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 count In 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 in the leucocyte counts are high and immature leucocytes appear in

the blood. the blood.

RETICULOCYTE COUNTRETICULOCYTE COUNT

Reticulocyte count (normal 0.5-2.5%) is Reticulocyte count (normal 0.5-2.5%) is done in each case of anaemia to assess done 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 of the reticulocyte response is indicative of impaired marrow function.impaired marrow function.

BONE MARROW EXAMINATIONBONE MARROW EXAMINATION

Bone marrow aspiration is done in cases Bone marrow aspiration is done in cases where the cause for anaemia is not where the cause for anaemia is not obvious. obvious.

The procedures involved marrow The procedures involved marrow aspiration and aspiration and

trephine biopsytrephine biopsy

Indication of Bone marrow examination in case of Indication of Bone marrow examination in case of anemiaanemia

megaloblastic sideroblastic iron deficiency aplastic anemia

Special InvestigationsSpecial Investigations

Biochemical TestsBiochemical Tests biochemical tests are aimed at identifying biochemical tests are aimed at identifying 1-a depleted cofactor necessary for normal 1-a depleted cofactor necessary for normal

hematopoiesis (iron, ferritin, folate, B12), hematopoiesis (iron, ferritin, folate, B12), 2-an abnormally functioning enzyme 2-an abnormally functioning enzyme

(glucose-6-phosphate dehydrogenase, (glucose-6-phosphate dehydrogenase, pyruvate kinase), or pyruvate kinase), or

3-abnormal function of the immune system 3-abnormal function of the immune system (the direct antiglobulin [Coombs'] test). (the direct antiglobulin [Coombs'] test).

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

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 no is raised but there is no biliruninuriabiliruninuria

Faecal StercobilinogenFaecal Stercobilinogen is raised is 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 raised is raised Evidence of intravascular haemolysisEvidence of intravascular haemolysis in the form of in the form of

haemoglobinaemia, haemoglobinuria, haemoglobinaemia, haemoglobinuria, haemosiderinuriahaemosiderinuria

Tests of increased red cell productionTests of increased red cell production..

Reticulocyte countReticulocyte count reveals reticulocytosis reveals reticulocytosis which indicate marrow erythroid hyperplasiawhich indicate marrow erythroid hyperplasia

Routine blood filmRoutine blood film shows macrocytosis, shows macrocytosis, polychromasia, normoblasts polychromasia, normoblasts

Bone marrowBone marrow show erythroid hyperplasia with show erythroid hyperplasia with raised iron storesraised iron stores

X ray of bonesX ray of bones shows evidence of expansion shows evidence of expansion of marrow spaces especially in tubular bones of marrow spaces especially in tubular bones and skulland skull

Tests of damage to red cellsTests of damage to red cells

Routine blood filmRoutine blood film shows a variety of shows a variety of abnormal morphological appearances of red abnormal morphological appearances of red cellscells

Osmotic fragilityOsmotic fragility is increased is increased AutohaemolysisAutohaemolysis test test Coomb'sCoomb's antiglobulin test antiglobulin test ElectrophoresisElectrophoresis for abnormal haemoglobin for abnormal haemoglobin Estimation of HbAEstimation of HbA22

Tests for shortened red cell life spanTests for shortened red cell life span

Tested by 51Cr labeling method normal Tested by 51Cr labeling method normal RBC life span of 120 days is shortened to RBC life span of 120 days is shortened to 20-40 days in moderate haemolysis and 5-20-40 days in moderate haemolysis and 5-20 days in severe haemolysis20 days in severe haemolysis

LABORATORY ERRORSLABORATORY ERRORS

1 1 .Errors in reporting or recording of .Errors in reporting or recording of results results

2 2 .Inadequate study of the blood film.Inadequate study of the blood film 3 3 .Failure to assess indices.Failure to assess indices 4 4 .Failure to do retic count.Failure to do retic count 5 5 .Failure to note rouleux.Failure to note rouleux

Thank YouThank You