Practical Clin Path Anemia 101 Wendy Blount, DVM

DDx Anemia Regenerative Non-Regenerative Blood Loss Secondary Anemia External bleeding Internal bleeding Hemolysis Immune mediated Cold hemagluttinin Dz Blood parasites Mycoplasma, Babesia, Cytauxzoon Oxidation – Heinz, MetHb Heavy metals – Zn, Cu Hypophosphatemia Hereditary PK deficiency, PFK deficiency Non-Regenerative Secondary Anemia Anemia of inflammatory Dz Chronic renal disease Chronic hepatic disease Endocrine disease Iron Deficiency Bone Marrow Disease Immune mediated Pure red cell aplasia Myelodysplasia, Myelofibrosis Aplasia,Necrosis Myelophtisis, neoplasia Macrophage proliferation Drug Induced Dyscrasia – estrogen, bute, sulfas Infection – FeLV, FIV, Ehrlichia, parvovirus

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)

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

Diagnosis When is anemia significant? Greyhound Italian Greyhound Cats – PCV persistently <20-25% Dogs – PCV persistently <30-35% Puppies PCV 28-30% and 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**

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% Severe Anemia – Cats PCV <13%, Dogs PCV<20% Very Severe Anemia – Cats <10%, dogs <13%

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

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

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

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

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

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

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

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 

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 

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 

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

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

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

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

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 hypoprotenemia

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

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

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

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

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Assessing the Regenerative Response If you can’t calculate an ARC, then corrected retic % (CRP/CPR) is second best Reticulocyte Production Index (RPI) No longer used very much early retics live longer than those made later Increased RDW (red cell distribution width) Objective measure of anisocytosis If increased, you have one of the following: Normal + large RBC – regenerative Small + normal RBC – developing IDA All 3 cell sizes – chronic blood loss

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**

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

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

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

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

Moderate Regeneration 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 % Retics Corrected K9 Absolute Retics Non-regenerative <1% <60,000/ul Mild Regeneration 1-4% 1-2.5% 60-100,000/ul Moderate Regeneration 5-20% 2.5-5% 100-300,000/ul Marked Regeneration 21-50% >5% >300,000/ul

Regenerative Anemia Degree of Regeneration – Absolute Reticulocytes K9 Retics Feline Aggregate Retics Feline Punctate Retics Non-regenerative <60,000/ul <15,000/ul <200,000/ul Mild Regeneration 60-100,000/ul 15-50,000/ul 200-500,000/ul Moderate Regeneration 100-300,000/ul 50-100,000/ul 500-1,000,000/ul Moderate to Marked Regeneration 300-500,000/ul 100-200,000/ul 1-1,500,000/ul Marked Regeneration >500,000/ul >200,000/ul >1,500,000/ul

Regenerative Anemia The most common error in interpretation of reticulocytes is to conclude a regenerative response based on slight increases in one of the ways of assessing retics

Use ARC to monitor regenerative anemias So you know your anemia is Aggregate Retics live 1-3 days So you know your anemia is Regenerative… Now What???

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 Sequellae of anemia Corrected Percent Reticulocyte Count Client Handouts Anemia in Dogs Anemia in Cats

Summary Vet Handouts Diagnostic Chart – Classifying Anemia Diagnostic Algorithm – Diagnosing Anemia Capillary Blood Collection – Ear Prick Capillary Blood Collection – Lip Prick

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

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