The Investigative Approach to Anaemia.docx

**[The Investigative Approach to Anaemia: The Preparation and examination of Bloods Smears:]** 1. **[Learning Objectives:]** Welcome to Week Two, Lesson One of Clinical Pathology and Laboratory Medicine. Throughout Week Two we are going to focus on anaemia. During this lesson, we will explore th...

**[The Investigative Approach to Anaemia: The Preparation and examination of Bloods Smears:]** 1. **[Learning Objectives:]** Welcome to Week Two, Lesson One of Clinical Pathology and Laboratory Medicine. Throughout Week Two we are going to focus on anaemia. During this lesson, we will explore the machine generated values pertinent to our understanding of the type of anaemia present and spend some time evaluating and understanding the importance of blood smear examination. Nick Bexfield BVetMed PhD DSAM DipECVIM-CA PGDipMEdSci PGCHE FHEA MRCVS **By the end of this lesson, you should be able to:** 1. Describe the investigative approach to anaemia; 2. Explain the correct process for preparing, staining and examining blood smears for optimal results. 2. **[Introduction:]** In the majority of cases anaemia is suspected on the basis of the patient\'s history and clinical findings. Nick Bexfield Clues about the possible cause are often present and our investigations will be based on our clinical assessment. Anaemia can be acute or chronic. Patients with acute anaemia can present with pallor, collapse, weakness, tachycardia, poor pulses, hypovolaemic shock (acute haemorrhage), icterus (acute haemolysis) and/or haemic murmur. These patients have little time to compensate. In comparison patients with chronic anaemia present with pallor, bounding peripheral pulses, pica, haemic murmur and lethargy and have had longer to compensate so can appear remarkably normal. 3. **[Definition and Classification of Anaemia:]** Anaemia is a reduction in the number of erythrocytes and is indicated by a decreased red blood cell count (RBC), haemoglobin concentration and/or haematocrit (HCT). Explore these parameters in more detail on the tabs below. Nick Bexfield 1. **[RBC Count: ]** It\'s important to be aware that haematology machines count RBCs as they pass through a gap. Feline red blood cells and platelets have a considerable size overlap and a variable, but considerable, proportion of platelets may therefore be included in the RBC numbers which can lead to a false thrombocytopenia. 2. **[Haemoglobin concentration:]** Haemoglobin is the most accurate way of measuring the ability to carry oxygen. Values are influenced by lipaemia and intravascular haemolysis. 3. **[Haematocrit/ PCV:]** Haematocrit is a machine generated figure which takes into account the number of RBCs and their average size. It is similar to PCV, but because it is a calculated value rather than measured there can be some differences between the two parameters. 4. **[Classification:]** Anaemia may be classified morphologically on the basis of red cell indices, such as Mean Cell Volume (MCV), Mean Corpuscular Haemoglobin (MCH) and Mean Corpuscular Haemoglobin Concentration (MCHC). Alternatively, it can be classified on a pathophysiological and aetiological basis taking into account the degree of bone marrow responsiveness (regenerative or non-regenerative). We will spend lessons two and three looking at regenerative and non-regenerative anaemias in detail. In this lesson we are going to review the important factors which ensure we have confidence in our blood results and the preparation, staining and interpretation of the blood smear.  Normal canine blood smear Do you remember the factors we discussed in Week One? Nick Bexfield Which of the following is most likely to significantly affect the results of our data when investigating anaemia? - **A clot in the sample** - Age of patient - Lipaemic sample - Size of patient  There is very little in the literature on the effects of large clots on laboratory parameters. Experience shows that the presence of clots can affect platelets, red cell parameters and leucocytes. There is also a relationship with maintenance personnel in the laboratory.  **ALWAYS CHECK BEFORE YOU RUN SAMPLES!**  Explore the blood sample with a capillary tube if any doubt that there may be a clot present. 4. **[Case Study: Max:]** Let\'s start with our first case study. Nick Bexfield 5. **[Meet Max: ]** - Max is a 5 year old male neutured Dalmatian. - He presented with a four day history of depression, anorexia and reluctance to exercise. He is weak and has poor peripheral pulses. - Max has not travelled abroad. - Max was admitted for further investigations. Match the missing words to the correct sentence to complete Max\'s history and the results of his clinical examination:  Icterus is a major problem in this case. In the activity below, sort the example causes into pre-hepatic, hepatic and post hepatic by dragging the cards to the correct tiles. **Pre-hepatic:** AIHA, Haemolysis, Onion toxicity, Babesiosis **Hepatic:** Primary liver disease **Post hepatic:** Pancreatitis, Cholelithiasis, Gall bladder rupture, bile duct obstruction, Pancreatic neoplasia Packed cell volume was measured and was markedly reduced at 15% (reference interval 37-55%). How will you investigate this dog's anaemia? What tests would you perform first? Think about or make a note of your answer before proceeding. Nick Bexfield 1. Take blood sample in EDTA. 2. Perform an automated analysis in house or at an external laboratory. 3. Evaluate machine generated values such as Mean Cell Volume (MCV) and Mean Corpuscular Haemoglobin Concentration (MCHC). 4. Examine blood smear. 6. **[Red Cell Indices: ]** MCV = Mean Cell Volume. The average size of red cells. Normal size = normocytic; Enlarged = macrocytic; Small = microcytic MCH = Mean Corpuscular Haemoglobin. The amount of haemoglobin per average red cell. MCHC = Mean Corpuscular Haemoglobin Concentration. The average haemoglobin concentration in the red blood cells. Normal colour = normochromic; Reduced colour = hypochromic; Increased colour = hyperchromic A decrease in MCHC causes hypochromasia though this is not usually observed in cats. An increase in MCHC is usually artefactual and due to lipaemia, haemolysis or the presence of Heinz bodies. Anaemia can be classified morphologically on the basis of these values as: - Macrocytic and hypochromic - Normocytic and normochromic - Macrocytic and normochromic - Microcytic and hypochromic 5. **[The Importance of Blood Smear Examination:]** The technology within automated haematology analysers is staggering and they often have outstanding performance. However, they are not infallible and can make mistakes. Nick Bexfield  Why is examining a blood smear so important? Click on the ticks to learn more. Nick Bexfield 1. Morphological changes are not detected by automated analysers (of ANY type). 2. Examination accelerates the clinical decision making process. 3. Examination enables identification of the discrepancies and/or errors that will happen with any analyser (Quality Assurance). 6. **[Making a Diagnostic Blood Smear:]** Watch the step by step presentation demonstrating how to make a diagnostic blood smear. Be sure to check for clots in your sample before you start. Nick Bexfield In this guide we\'ll explore how to make a blood smear. Click Start to begin.   Examine the slides below and indicate which are diagnostic. Nick Bexfield  Number 1 and 3 are diagnostic The criteria for a diagnostic blood smear include: - Adequate patient and sample identification; - Candle-flame shaped smear with even smearing; - The entirety of the smear must be within the stainable and viewable area of the slide (this is generally the middle two thirds of the slide). Parts of the smear that are not stained and cannot be viewed cannot be interpreted. 7. **[Blood Smear Hints and Tips:]** To show your understanding, tick off the following top tips for making a blood smear. Nick Bexfield - Use a capillary tube rather than a pipette to allow better control of the amount of blood taken up and dispensed onto the slide. - Place one to three drops of blood at the far end of the slide. - Draw the spreader slide back onto the drops and allow time for drops to run along edge. - With the spreader slide at a 30 -45º angle, push in a smooth move.  If you have difficulties making blood smears you may find the following troubleshooting table helpful. Nick Bexfield ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **Problem** **Cause\ **Solution\ ** ** ----------------------------------------------------- ----------------------------------------------------------- ---------------------------------------------------------------------------- Smear too long\ Spreader speed to slow\ Move spreader slide faster\ \ \ \ Smear running beyond slide or out of stainable area Low PCV blood\ Hold spreader slide at greater angle (40-45º)\ \ \ Too big blood droplet to start Apply less blood Smear too short\ Spreader slide too fast\ Move spreader slide slower\ \ \ \ Smear too thick \ High PCV\ Hold spreader slide at a lower angle (20º) \ \ Very narrow or no monolayer Insufficient blood droplet applied Irregular feathered edge Uneven contact of spreader with slide\ Apply even and consistent downward pressure with spreader slide\ \ \ Spreader slide has dirty or rough edges Use fresh spreader slide Smear has holes Grease on slide\ Clean slide/avoid touching face of slide with fingers \ Lipaemic sample Smear thick at feathered edge Blood in front of spreading edge\ Proper technique\ \ \ Stopped moving spreader slide prior to smearing all blood Smooth and continuous motion of spreader slide until all blood is smeared. ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- To show your understanding, tick off the following top tips for staining a blood smear. Nick Bexfield - General use rapid stains (e.g. Diff -- Quik, Rapi - Diff) are all fairly similar. They should be used according to the manufacturer\'s instructions and replaced as frequently as indicated. - The stain should be rinsed well to reduce the likelihood of stain precipitate on the smear. - Use red cell staining to check that the eosin is correct and use a leucocyte nucleus to check the basic (dark) stain. - Keep two sets of stain (clean and dirty). - Using a slide holder uses up less stain. Afbeelding met violet, Kleurrijkheid, Magenta, Lila Automatisch gegenereerde beschrijving 8. **[Examining a Blood Smear:]** Blood smear examination allows the identification of morphological abnormalities, estimation of platelet count and a differential white blood cell count to be performed. It\'s very important to systematically examine the blood smear following the steps below. Nick Bexfield  1. Start small -- with low magnification and at the feathered edge of the smear scanning for platelet clumps, abnormal cells and organisms. 2. Go deeper - go two to three fields back to the body of the smear into the monolayer, where red cells are touching but not overlapping. Confirm WBC count from the machine. 3. Go bigger -- increase to oil (x1000) and evaluate platelet count (if no clumps) and RBC morphology. 4. Finish to the side -- go to the lateral edge and count 100 leucocytes into types, looking for abnormal forms as you go. 7. **[Geography of a Blood Smear: ]** In the activity below, click on the area that you think is the monolayer. Nick Bexfield Afbeelding met schermopname, tekst Automatisch gegenereerde beschrijving The monolayer is usually a distance of one 10x field behind the feathered edge and is the optimal area for examination of cells since this area dries quickly and the cells are well-spread (not overlapping) and not disrupted. Red blood cells in this area are separated or barely touching, with little overlap. 8. **[Main Parts of a Blood Smear: ]** Click on the icons to reveal the body (or base/head), monolayer and feathered edge of a blood smear. Nick Bexfield  1. Base or head: 2. Monolayer:  3. Feathered Edge: Afbeelding met schermopname, kaart Automatisch gegenereerde beschrijving 1. **[Body: ]** In this area the cells are densely arranged and are often overlying each other, preventing all cells from being viewed. The cells have dried slowly, resulting in shrinkage and poor preservation of morphology.  2. **[Feathered Edge: ]** The area where the smear ends. On blood smears, larger objects are often dragged the furthest, and so it is here that platelet clumps, clumping leucocytes, microfilaria, and large cells will be often found. The morphology of the cells in this area is often poorly preserved. Afbeelding met schermopname, handschrift Automatisch gegenereerde beschrijving 3. **[Monolayer:]** This is the region in between the body and the feathered edge. The erythrocytes are evenly spread with no overlying cells and approximately 50% of cells are touching. Cell preservation is optimal in this area, and this is where the morphology of the red cells, white cells and platelets is assessed, and manual platelet count estimates and white cell differential counts are performed.  9. **[Examining the monolayer under oil immersion lens (x1000): ]** - Verify previous findings. - Examine RBCs: size, shape, colour and any inclusions. - Examine WBCs, perform (or verify) the differential cell count and look for morphology changes and inclusions. - Look for parasites/inclusions if suspected. - Estimate platelet numbers and examine their morphology. 10. **[Platelet Counts:]** Checking platelet numbers in the anaemic patient is important. Platelets are generally small, round, pale basophilic structures that contain low numbers of fine pink granules. Platelets in cats often vary in size which may explain the difficulty automated analysers have in counting them. Very small platelets are potentially not recognised by analysers, and large (similar in size to red cell) and giant (larger than a red cell) platelets may be misidentified as red cells. A manual platelet estimate count should be performed to validate the analyser result.  How many platelets are there on average per high power field on this image?  - 5-10 - **10-12** - 15-18 - 20 The answer is 10-12. This means there are approximately 150 x 10^9^/l platelets in this patient, which is an adequate number. Afbeelding met tekst, Lettertype, schermopname, lijn Automatisch gegenereerde beschrijving 9. **[Red Blood Cell Morphology:]** Red cells, also known as erythrocytes, are bi-concave discs that contain haemoglobin, and should be evaluated in regard to their variation in size, colour and shape. Nick Bexfield  11. **[Anisocytosis:]** Anisocytosis is defined as a variation in cell size. It's important to determine if anisocytosis is accompanied by hypochromasia (decreased cell staining) polychromasia (variation in cell staining) and either macrocytosis or microcytosis. Afbeelding met schermopname Automatisch gegenereerde beschrijving 12. **[Macrocytosis:]** Macrocytosis is defined as larger than normal cell size. It is reflected by the MCV ( Mean Cell Volume).  Macrocytosis is indicative of a regenerative response especially when accompanied by other signs of regeneration e.g. polychromasia. In the absence of other regenerative signs, macrocytosis may be due to myeloproliferative disease e.g. FeLV, myelodysplasia. Familial macrocytosis is recognised in poodles. 13. **[Microcytosis:]** Microcytosis is defined as smaller than normal cell size which again is reflected by the MCV. Microcytosis is often associated with iron deficiency anaemia due to chronic blood loss and congenital portosystemic shunts. It can also be present alongside chronic inflammation ( anaemia of chronic disease). It is often accompanied by hypochromasia. Some Akitas and other oriental breeds have a low MCV but do not necessarily appear very microcytic on blood smear examination. Excess EDTA in the sample tube may artefactually decrease cell size. Afbeelding met kleding, stof, Lila Automatisch gegenereerde beschrijving 14. **[Polychromasia:]** Polychromasia is observed when immature red blood cells e.g. reticulocytes are released from the bone marrow. The immature red blood cells have a purple -grey hue to their cytoplasm and are referred to as polychromatophils.\   Polychromasia is sometimes seen in regenerative anaemias. In the absence of anaemia, excitement and acute stress can cause polychromasia due to adrenaline release. Hypochromasia is a reduction in cell staining often with a central pallor. Hypochromasia is seen in iron deficiency anaemia due to chronic external blood loss and portosystemic shunts as well as in regenerative anaemias.  Examining a blood smear enables changes such as anisocytosis , polychromasia and hypochromasia to be identified and is invaluable in detection of changes affecting small numbers of cells or mild changes affecting large numbers of cells. Machine generated values should always be verified by smear evaluation especially if the values are abnormal. Relying on machine counts alone can be misleading and lead to a delay in diagnosis. Results will depend on a good quality sample without the presence of clots.   Afbeelding met tekst, schermopname, Lettertype, Elektrisch blauw Automatisch gegenereerde beschrijving 10. **[Completion of Online Learning:]** You have now completed Week Two, Lesson One of Clinical Pathology and Laboratory Medicine. Nick Bexfield **To recap the learning objectives, you should now be able to:** 1. Describe the investigative approach to anaemia; 2. Explain the correct process for preparing, staining and examining blood smears for optimal results.