Practical Clin Path Anemia 101

1. Practical Clin PathAnemia 101 Wendy Blount, DVM

2. RBC Indices • MCV – mean corpuscular volume – RBC size • MCH – mean corpuscular Hb • MCHC – mean corpuscular Hb concentration – RBC color • Microcytic– low MCV • Normocytic– anemia with normal MCV • Macrocytic– high MCV • Hyperchromic– high MCHC • Normochromic– anemia with normal MCHC • Hypochromic– low MCHC • Polychromic– more RNA (blue) and often less Hb (orange-red)

3. Diagnosis • “Anemia” is not a diagnosis • It’s a symptom • Treating anemia without knowing the diagnosis doesn’t often work out very well What is the most common treatment for anemia? • Very few anemias require treatment with iron • Iron supplementation will significantly help very few anemias • Contraindicated for anemia of chronic inflammatory disease

4. Diagnosis When is anemia significant? • Cats – PCV persistently <20-25% • Dogs – PCV persistently <30-35% • Puppy Normals - PCV 28-30%, 3-4% reticulocytes • St Bernard -normal PCV 35-40% • Sight Hound - normal PCV 52-60% Greyhound Italian Greyhound Whippet Scottish Deerhound Irish Wolfhound Saluki Sloughi Borzoi Afghan Basenji** Pharoah Hound** Ibizan** Rhodesian Ridgeback**

5. Diagnosis When is anemia significant? • Mild Anemia - Cats PCV 20-25%, Dogs 30-35% • May or may not be a primary problem • Secondary to chronic inflammation, malignancy, organ failure, or endocrine disease • Moderate Anemia – Cats PCV 14-19%, Dogs PCV 20-29% - needs to be worked up • Severe Anemia – Cats PCV <13%, Dogs PCV<20% • Potentially life threatening • Very Severe Anemia – Cats <10%, dogs <13% • needs transfusion to allow time to diagnose and treat

6. Diagnosis Symptoms secondary to anemia when to run a CBC • Reduced oxygen carrying capacity • Tachypnea, dyspnea, syncope, weakness, confusion • hypoxia without cyanosis • Pallor • Reduced blood volume (blood loss) • Weak peripheral pulses ==>> shock death • Pallor, slow CRT (Capillary Refill Time) Related to decreased blood viscosity • Heart murmur Related to underlying disease – pica, Hburia

7. Diagnosis 2 parts of a CBC • Automated count - EDTA or citrate • Should be run within 3 hrs - refrigerate after • not reliable >24 hrs • RBC swelling at 6-24 hrs • inc. PCV & dec. MCHC • Do not run samples with clots in them • Inaccurate automated counts • Clog the machine • If your HCT does not match your patient, spin a HCT tube (11-15K rpm x 5 min) • Blood smear examination - EDTA

8. Diagnosis 2 parts of a CBC • Automated count - EDTA or citrate • Should be run within 3 hrs - refrigerate after • not reliable >24 hrs • RBC swelling at 6-24 hrs • inc. PCV & dec. MCHC • Do not run samples with clots in them • Inaccurate automated counts • Clog the machine • If your HCT does not match your patient, spin a HCT tube • Blood smear examination - EDTA

9. Diagnosis 2 parts of a CBC • Blood smear examination – EDTA • within 30 minutes is best – air dry • Blood smear of any age can still yield valuable information • on all CBCs with significant abnormalities • RBC and WBC morphology • Hemoparasites • capillary blood best yield (ear prick, foot pad) • Inclusions – Dohle bodies, CDV inclusions • Differentiate WBC cell lines • Sometimes there are cells that the counter can not identify

10. Making & Reading the Blood Smear • Use good slides with smooth edges • Wipe the glass dust off both slides first • Let the slide air dry • Avoid the very edge where RBC are damaged and distorted • Avoid the smear where it becomes thick • Read RBC morphology in the monolayer • I have better luck with a smaller drop

11. Making & Reading the Blood Smear • Use good slides with smooth edges • Wipe the glass dust off both slides first • Let the slide air dry • Avoid the very edge where RBC are damaged and distorted • Avoid the smear where it becomes thick • Read RBC morphology in the monolayer • I have better luck with a smaller drop Autoagglutination

12. Making & Reading the Blood Smear • Use good slides with smooth edges • Wipe the glass dust off both slides first • Let the slide air dry • Avoid the very edge where RBC are damaged and distorted • Avoid the smear where it becomes thick • Read RBC morphology in the monolayer • I have better luck with a smaller drop

13. Making & Reading the Blood Smear • Use good slides with smooth edges • Wipe the glass dust off both slides first • Let the slide air dry • Avoid the very edge where RBC are damaged and distorted • Avoid the smear where it becomes thick • Read RBC morphology in the monolayer • I have better luck with a smaller drop Feathered Edge - Don’t Read Morphology Here 

14. Making & Reading the Blood Smear • Use good slides with smooth edges • Wipe the glass dust off both slides first • Let the slide air dry • Avoid the very edge where RBC are damaged and distorted • Avoid the smear where it becomes thick • Read RBC morphology in the monolayer • I have better luck with a smaller drop Monolayer – Read Morphology Here 

15. Making & Reading the Blood Smear • Use good slides with smooth edges • Wipe the glass dust off both slides first • Let the slide air dry • Avoid the very edge where RBC are damaged and distorted • Avoid the smear where it becomes thick • Read RBC morphology in the monolayer • I have better luck with a smaller drop Thick Body – Don’t Read Morphology Here 

16. Making & Reading the Blood Smear • Platelet Estimate – 8-30/HPF (100x) • Platelet clumping at feathered edge • Platelet morphology • RBC morphology • WBC estimate – 20-50/LPF (10x) dogs, 10-40/LPF (10x) cats • Manual WBC Diff if what you see does not correlate with the automated count • Count nRBC, but don’t include them in the 100 WBC

17. RBC Morphology polychromatophil reticulocyte (NMB stain) spherocyte feline RBC K9 RBC (discocyte) acanthocyte spurr cell blister cell keratocyte crenation echinocyte burr cell helmet cell keratocyte schistocyte schizocyte dacryocyte leptocyte target cell (codocyte) budding fragmentation eccentrocyte Mycoplasma haemofelis Heinz body (NMB stain) Howell Jolly Body

18. RBC Morphology IV hemolysis normal regenerative response normal regenerative response liver disease DIC angiopathy oxidation artifact metabolic dz oxidation splenic, hepatic dz regeneration hypothyroidism oxidation Increased nRBC DIC, angiopathy, IDA, marrow dz oxidation Mycoplasma haemofelis

19. Diagnosis Severity of Symptoms • Rapidity of onset • Severity of Anemia • Degree of physical activity (cats vs. dogs) • Concurrent disease affecting respiratory exchange • Respiratory disease • Cardiovascular disease Pseudoanemia • Mild decrease in PCV due to plasma volume expansion, RBC normal • Congestive heart failure, pregnancy, glucocorticoid therapy, IV fluid therapy

20. Kirk ReeseWhitehouse TX

21. Diagnosis Things that can mask anemia • Dehydration • Acute hemorrhage • Shock, splenic contraction • Cannot mask a severe anemia • Look at plasma protein • Assuming there is no concurrent hypoproteinemia • Plasma proteins will be increased with hemoconcentration

22. Diagnosis The First Question • Is the anemia regenerative? • i.e., is the body losing RBCs or not making them or both? • At maximum stimulation, the bone marrow can make RBCs at 50x the usual rate • It takes at least a few days and up to a week for this to fully kick in • An acute regenerative anemia can look non-regenerative during the first week • Reticulocyte enumeration is the most consistent way to evaluate regeneration • Run retics if PCV<30% in the dog or <20% in the cat

23. Assessing the Regenerative Response Reticulocytes • RNA to make Hb retained for 1-3 days after the nRBC extrudes its nucleus • Macrocytic polychromic (blue) on DiffQuick • Mix EDTA blood with stain 1:1 (1:3 for birds) • New methylene blue (NMB) • Brilliant cresyl blue (BCB) • Incubate 10-15 min. for NMB, 15-30 for BCB • Air dry blood smears and stain • Count 500-1000 RBC • Report % retics of RBC counted

24. Assessing the Regenerative Response Reticulocytes • Count only aggregates, not punctates in cats • Feline punctates have up to 10-15 blue dots that do not coalesce • Canine punctates have 1-2 blue dots that do not coalesce

25. Assessing the Regenerative Response Percent Reticulocytes • Non-anemic animals <0.5% retics • >1% usually a regenerative response • This method is not as reliable as… Absolute Reticulocyte Count (ARC) • RBC/ul x % retics = ARC • Non-anemic animals <15-50,000/ul • >200,000/ul highly regenerative • Automated counts are not always reliable • This is the preferred single index for assessing regenerative response

26. Assessing the Regenerative Response Increased MCV (mean corpuscular volume) = macrocytosis • Retics the most common macrocyte • Can also be increased due to: • Prolonged storage (EDTA blood > 1 day) • FeLV – RBC maturation arrest • marrow dysplasia – blasts, leukemia • folate deficiency • Phenobarbital therapy • Stomatocytes – liver disease • RBC leukemia – very, very rare • **Atypical cells**

27. Assessing the Regenerative Response nRBCs – aka - normoblasts, metarubricytes • Increased with: • Regenerative anemia • Splenic disease, Bone marrow disease, EMH • Iron deficiency anemia, lead poisoning • Heat Stroke, Sepsis, hyperadrenocorticism Howell-Jolly Bodies (HJB) and basophilic stippling are end stage nRBC

28. Assessing the Regenerative Response RBC morphology – signs of regeneration • Anisocytosis – variation in RBC size • Polychromasia – blue-gray big RBCs • Polychromatophils = aggregate retics • >1/HPF (oil) indicates inc retics

29. Regenerative Anemia RBC morphology An abnormality should be present in nearly every field to be considered significant Senescent cells can display any morphologic abnormality Poikilocytosis = increase in abnormally shaped RBC cells LOW SENSITIVITY – ONLY 8% of blood samples with regenerative anemia show increased MCV and decreased MCHC

30. Regenerative Anemia RBC morphology – semiquantitative scale • 0 – not present • 1+ - mild – may not be clinically significant (5-10/HPF) • 2+ - mild to moderate (11-50/HPF) • 3+ - moderate to marked (51-150/HPF) • 4+ - marked (>150/HPF) 2+ to 4+ are likely clinically significant

31. Regenerative Anemia Degree of Regeneration Acute Blood Loss – non-regenerative, then moderately regenerative 3-7 days later Chronic Blood Loss – marked regeneration Hemolysis – moderate to marked regeneration

32. Regenerative Anemia Degree of Regeneration – Absolute Reticulocytes

33. Summary PowerPoints • Anemia 101 - .pptx;.pdfs 1 & 6 slides per page • RBC Cases – feline DCM due to anemia • .pptx; .pdfs – 1 & 6 slides per page Hidden Slides • Complete list of causes of anemia • Sequellae of severe anemia • Corrected Percent Reticulocytes • Reticulocyte Production Index (RPI) • Pitfalls in assessing the regenerative response • Reticulocyte lifespan

34. Summary Client Handouts • Anemia in Dogs • Anemia in Cats Vet Handouts • Diagnostic Chart – Classifying Anemia • Diagnostic Algorithm – Diagnosing Anemia • Capillary Blood Collection – Ear Prick • Capillary Blood Collection – Lip Prick

35. Acknowledgements Chapter 2: The Complete Blood Count, Bone Marrow Examination, and Blood Banking • Douglass Weiss and Harold Tvedten • Small Animal Clinical Diagnosis by Laboratory Methods, eds Michael D Willard and Harold Tvedten, 5th Ed 2012 Chapter 3: Erythrocyte Disorders • Douglass Weiss and Harold Tvedten • Small Animal Clinical Diagnosis by Laboratory Methods, eds Michael D Willard and Harold Tvedten, 5th Ed 2012

36. Acknowledgements Chapter 59: Pallor • Wallace B Morrison • Textbook of Veterinary Internal Medicine, eds Stephen J Ettinger and Edward C Feldman, 6th Ed 2003 Challenging Anemia Cases • Crystal Hoh, ACVIM • Heart of Texas Veterinary Specialty Center • CAVMA CE