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TESTS IN HAEMATOLOGY

TESTS IN HAEMATOLOGY. Lab 3. OVERVIEW. 1. Blood smear evaluation 2. Packed cell volume (PCV)/haematocrit (Hct) and total proteins 3. Erythrocyte (RBC) count, haemoglobin (Hb) and indices 4. Reticulocyte count 5. Clinical approach to anaemias.

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TESTS IN HAEMATOLOGY

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  1. TESTS IN HAEMATOLOGY Lab 3

  2. OVERVIEW 1. Blood smear evaluation 2. Packed cell volume (PCV)/haematocrit (Hct) and total proteins 3. Erythrocyte (RBC) count, haemoglobin (Hb) and indices 4. Reticulocyte count 5. Clinical approach to anaemias These tests are included in the HAEMOGRAM which is defined as a selection of tests on blood cells (erythrocytes, leukocytes and platelets) for diagnostic purposes. This selection can vary with the laboratory. CBC (complete blood count) is used by some authors as a synonym of haemogram.

  3. HOW TO MAKE A BLOOD SMEAR

  4. MAIN PARTS OF A BLOOD SMEAR Feathered edge Base or head Monolayer

  5. ABLOOD SMEAR SHOULD PROVIDE: • An area where cell morphology can be observedwith optimal detail because cells: • Are in a monolayer (about 50% are in contact with each other) • Do not overlap each other (base/head) • -Are not damaged or distorted (feathered edge)

  6. STEPS TO EVALUATE A BLOOD SMEAR -Selection of monolayer area -Detection of platelet agreggation, RBCs rouleaux/agglutination At 100x magnification -WBC number estimation/differential count At 400x magnification -Detailed evaluation of RBCs, WBCs and platelets At 1000x magnification

  7. Slide Agglutination Test Mix one drop of EDTA blood with 2 drops of saline Examine grossly & by microscopy

  8. 2.PCV/HctAND TOTAL PROTEINS

  9. PACKED CELL VOLUME (PCV) PCV is the ratio of erythrocyte volume to whole blood volume - PCV is measured by whole blood centrifugation - Haematocrit is calculated from RBC count and mean cell volume by electronic cell counters In clinical terms PCV = Haematocrit They are used to detect polycythaemia and anaemia

  10. *PCV MEASUREMENT PROCEDURE (Microhaematocrit Method) -Fill the capillar tube up to ¾ of its length -Seal with plastiline or in a bunsen flame -Centrifuge for 5 minutes at >10,000 r.p.m. (10 min. in ruminants) -Calculate PCV= length of packed erythrocytes/total length of blood column Some sources of error: -Delay in reading of PCV - Centrifuge does not reach adequate speed -Excessive or inadequate filling of capillary tube * Haematocrit (Hct) Measurement By Electronic Cell Counters: - Hct may be slightly lower (0.01-0.03 L/L) than obtained by the microhaematocrit method since there is no trapped plasma between erythrocytes. -Counters designed for use with human blood must be validated for animal species (RBC are smaller in animals, except in the dog). Platelets may be counted as erythrocytes and yield falsely increased results especially in cats.

  11. ADVANTAGES OF PCV MEASUREMENT • -It is a very simple, precise and accurate method • It provides more than just a PCV (see next slide) Plasma area Erythrocytic area Buffy coat Buffy coat includes leukocytes and platelets

  12. PRACTICAL USES OF DIFFERENT SECTIONS OF CAPILLARY TUBE Packed Cell Volume determination ERYTHROCYTIC SECTION -Increase suggests leukocytosis or thrombocytosis -May be used to make concentrated WBCs smears and to aid detection of Microfilaria BUFFY COAT -Analysis of total plasma proteins/ fibrinogen -Its colour can provide clinical information: •icterus, carotene (cows) •haemolysis •lipaemia PLASMA SECTION

  13. CAPILLARY TUBE SECTIONS AND PLASMA COLOURS Leukocytosis or thrombocytosis icterus dehydration haemolysis lipaemia plasma buffycoat erythrocytes

  14. TOTAL PLASMA PROTEINS (TPP) Can be determined from the plasma at the top of a centrifuged capillary tube using a refractometer high concentration of glucose/urea lipaemia or haemolysis values <25 g/L or >95 g/L Sources of error

  15. INTERPRETATION OF RESULTS 1. Increase in PCV.............. POLYCYTHAEMIA + TPPdehydration 2. Decrease in PCV.............. ANAEMIA + TPPblood loss Example: PCV and TPP used to monitor hydration status in horses with colic Values Situation ================== ====================== -PCV<0.40, TPP>75 Fluid treatment not required -PCV=0.40-0.55, TPP=75-95 Fluid treatment required -PCV>0.55, TPP>95 Intensive fluid treatment -PCV>0.60, TPP>10 Very poor prognosis Units: PCV (L/L), TPP (g/L)

  16. Blood Transfusion Volume Required = Bl Vol (ml) x (Req PCV - Recip PCV) PCV of donated blood Volume Required by 2.5 kg cat = 165 x (18 - 6) = 66ml 30

  17. 3. ERYTHROCYTE (RBC) COUNT, HAEMOGLOBIN (Hb) AND INDICES

  18. RBC COUNT AND Hb CONCENTRATION • -Can be determined: • - by manual methods • - by haematological analysers: • • laser cell counters • • quantitative buffy coat analysers • Give similar clinical information as the PCV • Are used to calculate RBC indices

  19. RBC INDICES MCV (MEAN CELL VOLUME) MCV (fl) = PCV(L/L) x 1000 / RBC COUNT (1012/L) Indicates the average size of RBCs: • indicates MACROCYTOSIS • values within reference range indicates NORMOCYTOSIS • indicates MICROCYTOSIS MCV is directly measured by electronic cell counters.

  20. MCHC (MEAN CELL HAEMOGLOBIN CONCENTRATION) MCH (MEAN CELL HAEMOGLOBIN) MCHC (g/L) = Hb(g/L) / PCV(L/L) MCH (pg) = Hb(g/L) / RBC(1012/L) Indicates the average concentration of Hb in RBCs: • indicates HYPOCHROMASIA • values within reference range indicates NORMOCHROMASIA Apparent hyperchromasia (high MCHC) is usually due to an artifactual increase in the haemoglobin result, due to haemolysis, lipaemia, or large numbers of Heinz bodies. MCH is less commonly used in clinical practice

  21. RED CELL DISTRIBUTION WIDTH (RDW) A numeric representation of the variability in RBC size (RBC anisocytosis). Increased RDW Regenerative anaemia and MCV usually indicates similar RBC size different RBC size, increase RDW RDW is calculated from Standard Deviation of MCV/Mean of MCV

  22. 4. RETICULOCYTE COUNT AND EVALUATION

  23. Reticulocytes are young (immature) erythrocytes prematurely released to blood from the bone marrow. CLINICAL APPLICATIONS: Evaluation of erythropoiesis in bone narrow. Differentiation of regenerative and non-regenerative anaemia. • TECHNIQUES OF DETECTION: • Romanowsky stain • - New methylene blue

  24. ROMANOWSKY STAIN In dogs, an average of >10 polychromatic red cells per OIF suggests a markedregenerative response OIF = oil immersion field Romanowsky stain interact with RNA-protein complexes of reticulocytes and produce polychromasia

  25. NEW METHYLENE BLUE (NMB) STAIN Reticulocytes are non-nucleated erythrocytes in which NMB stain precipitates in RNA-protein complexes

  26. MANUAL ABSOLUTE RETICULOCYTE COUNT 1. Count number of reticulocytes per 500-1000 erythrocytes (blood smear) -e.g. 10 reticulocytes / 500 erythrocytes 2. Calculate % of reticulocytes -e.g. Reticulocyte % = 10 x 100 / 500 = 2 3. Calculate absolute reticulocyte count based on erythrocyte count from the haematology analyser -e.g. Absolute reticulocyte count (109/L)= Reticulocyte % x Erythrocyte count / 100

  27. INTERPRETATION If absolute reticulocyte count: Dogs >60x 109/L A sign of regeneration Cats>50x 109/L

  28. RETICULOCYTE PRODUCTION INDEX: CALCULATIONS - Corrected reticulocyte percentage (CRP) CRP= %reticulocytes x PCV ofsample/normal PCV - Reticulocyte Production Index (RPI) RPI= CRP/Maturation Index(MI) PCV MI (days) 0.45 1 (values for dogs) 0.35 1.5 0.25 2 0.15 2.5

  29. CRP. In patients with anaemia the RBC number and PCV are low. Therefore the percentage of reticulocytes can be misleading and the PCV of the patient must be taken into account RPI. Reticulocytes have a longer life span in peripheral blood as anaemia becomes more severe. Therefore the time that a reticulocyte will take to mature (Maturation Index) must be taken into account In dogs the maturation index (MI) depends on the PCV.

  30. RETICULOCYTE PRODUCTION INDEX: PRACTICAL EXAMPLE DogwithPCV=0.15L/Land reticulocytes =15% - Corrected reticulocytes percentage (CRP) CRP= 0.15 x 0.15/0.45 = 5% - Reticulocyte Production Index (RPI) RPI= 5/2.5 =2 RPI > 3 Very good regeneration RPI = 1-3 Good regeneration RPI < 1 Inadequate regeneration

  31. SPECIES VARIATIONS: IN CLINICALLY HEALTHY ANIMALS -Dogs. Low number of reticulocytes (<1%), aggregate only - Cats. Two types of reticulocytes: • aggregate: blue stained coarse clumping(0.5% of erythrocytes) • punctate: small, blue stained dots (1-10%). - Ruminants andhorses. Virtually no reticulocytes in blood.

  32. SPECIES VARIATION: in anaemic animals - Canine. Strong reticulocyte response in regenerative anaemias. Aggregated reticulocytes (indicate recent response) - Feline. Punctated reticulocytes (indicate response to anaemia occurring 3-4 weeks previously) - Ruminants andhorses. Reticulocytes may not appear even in very severe anaemias

  33. 5. Anaemia: A Clinician’s Approach • Initial Tests: • PCV • Total Protein • Slide agglutination test • Blood smear • Reticulocyte count

  34. Severity of Anaemia • Normal 24 - 45% 37 - 55% • Mild 20 - 24% 25 - 34% • Moderate 14 - 19% 19 - 24% • Severe 10 - 13% 15 - 18% • Very Severe <10% <15%

  35. Anaemia Classification • Non-Regenerative – few or no retics TP normal • Regenerative • Acute Haem few retics, TP low or N PCV not v low • Chronic Haem lots of retics TP N or low Fe def • Haemolytic lots of retics, TP N Plasma discoloured

  36. Investigating Non-regenerative Anaemia: • Look for systemic causes • Is it anaemia of chronic disease? • Test for FeLV • Blood smear – Pb poisoning, Fe def

  37. Investigating Blood Loss Anaemia • History and clinical examination • Rule out ext parasites • Investigate internal bleeding Faecal occult blood Endoparasites

  38. Investigating Haemolysis • Rule out toxins • Rule out parasites • Check smear for Heinz bodies /schistocytes • Rule out FeLV • Coomb’s test

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