AnemiaLaboratory Diagnosis Presented byDr. Mohammed Abbas
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,
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
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)
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
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
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.
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
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 Drabkins 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.
:Normal hemoglobin values Men 14-17 gm% Women 13-15 gm% Infants 14-19gm% Children (1year) 11-13gm% Children (10-12 years0 12-14gm%
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
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
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
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.
)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
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)
)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)
)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
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.
Normal red cells (normochromic): have uniformly coloured haemoglobin in side the cell with a small clear paler region in the center
: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
Ecchinocytes: cells with 10-30 uniformly distributedspiculesCause: blood loss (acute), burns, DIC, carcinoma ofstomach
Elliptocytes: have a cigar shapeCause: hereditary elliptocytosis, leukemia, thalassaemia
Sickle cells:cells have a sickle with appoint at one endCause: sickle cell anaemia, haemoglobin S disease
Sphereocytes cells:are globe like rather than biconcave with an abnormalsmall dimpleCause: hereditary spheroytosis, autoimmune haemolyticanaemia, septicemia
Stomatocyte:cells are cup shaped with an abnormal area of centralpallor that may be oval, elongated, or slit likeCause: liver disease, alcoholism, hereditary spheroytosis
Target cells:cells have an increased ratio of surface to volume, due to ashape that looks like a cup, bellCause: iron deficiency, liver disease, haemoglopinopathies,post spleenectomy
Tear drop poikilocyte: cells have teardrop or pear shapeCause: myelofibrosis, extramedullary haemopoiesis,myeloid metaplasia
: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
Content of structure variationBasophilic stippling: appearance of fine blue dotsscattered in red cellsCause: haemoglopinopathies, lead poisoning, haemolyticanaemia, myelodysplasia
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
Heinz bodies: are denatured particles of haemoglobinattached to RBC membrane that appear when stained withcresyl blueCause: G6PD anaemia, drug induced, alpha thalassaemia
Howell jolly body:are nuclear fragment found in red cells, mostly single butsometimes multipleCause: post splenectomy, hyposplenism
Siderocytes granules (papenheimer bodies):are cells with mitochondrial concentration of ferritin (non-haemoglobin iron) depositthe cells are stained by Prussian blue reactionCause: disorder of iron metabolism as Sideroblasticanaemia. Postsplenectomy, burns, hemochromatosis
LEUCOCYTE AND PLATELET COUNTMeasurement 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 leukemias, the leucocyte counts are high and immature leucocytes appear in the blood.
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.
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
Indication of Bone marrow examination in case of anemia megaloblastic sideroblastic iron deficiency aplastic anemia
Special Investigations Biochemical Tests biochemical tests are aimed at identifying1-a depleted cofactor necessary for normal hematopoiesis (iron, ferritin, folate, B12),2-an abnormally functioning enzyme (glucose-6-phosphate dehydrogenase, pyruvate kinase), or3-abnormal function of the immune system (the direct antiglobulin [Coombs] test).
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
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
.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
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 Coombs antiglobulin test Electrophoresis for abnormal haemoglobin Estimation of HbA2
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
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