Practical Hematology Practical Hematology Anemia 101 1. Anemia 101 2. Blood Loss Anemia Wendy Blount, DVM 3. Hemolysis 4. Non-Regenerative Anemias 5. Transfusion Medicine 6. Polycythemia 7. Bone Marrow Disease 8. Coagulopathy 9. Central IV Lines 10.Leukophilia 11.Leukopenias 12.Splenic Disease

DDx Anemia RBC Indices Regenerative Non-Regenerative • MCV – mean corpuscular volume – RBC size • Blood Loss • Secondary Anemia • MCH – mean corpuscular Hb • External bleeding Anemia of inflammatory Dz • MCHC – mean corpuscular Hb concentration – • Internal bleeding Chronic renal disease RBC color intensity • Hemolysis Chronic hepatic disease • Immune mediated Endocrine disease • Microcytic – low MCV • Iron Deficiency • Cold hemagluttinin Dz • Normocytic – anemia with normal MCV • Bone Marrow Disease • Blood parasites Immune mediated • Macrocytic – high MCV Mycoplasma, Babesia, Pure red cell aplasia Cytauxzoon • Hyperchromic – high MCHC • Oxidation – Heinz, Myelodysplasia, Myelofibrosis MetHb Aplasia,Necrosis • Normochromic – anemia with normal MCHC • Heavy metals – Zn, Cu Myelophtisis, neoplasia • Hypochromic – low MCHC (pale RBC) Macrophage proliferation • Hypophosphatemia • Polychromic – more RNA (blue) and often less • Drug Induced Dyscrasia – • Hereditary estrogen, bute, sulfas Hb (orange-red) PK deficiency, PFK • Infection – FeLV, FIV, Ehrlichia, deficiency parvovirus

Diagnosis Diagnosis When is anemia significant? • “Anemia” is not a diagnosis • Cats – PCV persistently <20-25% • It’s a symptom • Dogs – PCV persistently <30-35% • Puppies PCV 28-30% and 3-4% reticulocytes • Treating anemia without knowing the diagnosis • St Bernard normal PCV 35-40% doesn’t often work out very well • Sight Hound normal PCV 52-60% Greyhound Borzoi What is the most common treatment for Italian Greyhound Afghan anemia? • Very few anemias require treatment with iron Whippet Basenji** • Iron supplementation will significantly help very Scottish Deerhound Pharoah Hound** few anemias Irish Wolfhound Ibizan** • Contraindicated for anemia of chronic Saluki Rhodesian Ridgeback** inflammatory disease Sloughi

1 Diagnosis Diagnosis When is anemia significant? Symptoms secondary to anemia

• Mild Anemia - Cats PCV 20-25%, Dogs 30-35% when to run a CBC • May or may not be a primary problem • Reduced oxygen carrying capacity • Often secondary to chronic inflammation, • Tachypnea, dyspnea, syncope, weakness, confusion malignancy, organ failure, or endocrine disease • hypoxia without cyanosis • Moderate Anemia – Cats PCV 14-19%, Dogs • Pallor PCV 20-29% • Reduced blood volume (blood loss anemia) • Weak peripheral pulses ==>> shock death • Severe Anemia – Cats PCV <13%, Dogs • Pallor, slow CRT (Capillary Refill Time) PCV<20% Related to decreased blood viscosity • Very Severe Anemia – Cats <10%, dogs <13% • Heart murmur Related to underlying disease – pica, Hburia

Diagnosis Diagnosis 2 parts of a CBC 2 parts of a CBC

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

Diagnosis Making & Reading the Blood Smear 2 parts of a CBC • Blood smear examination – EDTA • Use good slides with smooth edges • within 30 minutes is best – air dry • Wipe the glass dust off both slides first • Blood smear of any age can still yield valuable information • Let the slide air dry • on all CBCs with significant abnormalities • Avoid the very edge where RBC are • RBC and WBC morphology damaged and distorted • Hemoparasites • Avoid the smear where it becomes thick • capillary blood best yield (ear prick, foot pad) • Read RBC morphology in the monolayer • Inclusions – Dohle bodies, CDV inclusions • I have better luck with a smaller drop of • Differentiate WBC cell lines blood • Sometimes there are cells that the counter can not identify

2 Making & Reading the Blood Smear Making & Reading the Blood Smear

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

Making & Reading the Blood Smear Making & Reading the Blood Smear

• Use good slides with smooth edges • Use good slides with smooth edges • Wipe the glass dust off both slides first • Wipe the glass dust off both slides first • Let the slide air dry • Let the slide air dry • Avoid the very edge where RBC are • Avoid the very edge where RBC are damaged and distorted damaged and distorted • Avoid the smear where it becomes thick • Avoid the smear where it becomes thick • Read RBC morphology in the monolayer • Read RBC morphology in the monolayer

Feathered• I have Edge better - Don’t luck Read with Morphology a smaller Here drop Monolayer• I have – Read better Morphology luck with Here a smaller drop

Making & Reading the Blood Smear Making & Reading the Blood Smear

• Use good slides with smooth edges 1. Platelet Estimate – 8-30/HPF (100x) • Wipe the glass dust off both slides first • Platelet clumping at feathered edge

Thick Body• – Let Don’t the Read slide Morphology air dry Here • Platelet morphology • Avoid the very edge where RBC are 2. RBC morphology damaged and distorted 3. WBC estimate – 20-50/LPF (10x) dogs, • Avoid the smear where it becomes thick 10-40/LPF (10x) cats • Read RBC morphology in the monolayer • Manual WBC Diff if what you see does not • I have better luck with a smaller drop correlate with the automated count • Count nRBC, but don’t include them in the 100 WBC that you count

3 RBC Morphology RBC Morphology

polychromatophil reticulocyte spherocyte K9 RBC feline RBC normal normal regenerative regenerative IV hemolysis (NMB stain) (discocyte) response response

schistocyte blister cell helmet cell crenation acanthocyte liver disease schizocyte keratocyte keratocyte echinocyte spurr cell DIC oxidation oxidation artifact burr cell angiopathy metabolic dz dacryocyte

leptocyte Splenic dz budding eccentrocyte Mycoplasma Heinz body Howell Target cell DIC, angiopathy, oxidation hepatic dz Mycoplasma oxidation Increased fragmentation haemofelis (NMB stain) Jolly Body (codocyte) IDA, marrow dz regeneration haemofelis nRBC

Cases Shelter Cat

5 month DLH cat – tachypnea, lethargy • VetBLUE® ultrasound

Shelter Cat Shelter Cat

5 month DLH cat 5 month DLH cat – tachypnea, lethargy – tachypnea, lethargy • VetBLUE® ultrasound • VetBLUE (Bedside Lung US Exam) • Dry lungs • AFAST3 (Abdominal Focused ASessment for Trauma, Triage and Tracking) • Abdominal fluid score 0/4 • Gall bladder and urinary bladder normal • No25% retroperitoneal caval bounce fluid, no pleural Enlarged hepatic fluid, vein no dry lung alveolar-interstitial lung fluid pericardial fluid

4 Attendee, DVM Shelter Cat City TX

5 month DLH cat – tachypnea, lethargy • VetBLUE® (Bedside Lung US Exam) • Dry lungs • AFAST3® (Abdominal Focused ASessment for Trauma, Triage and Tracking) • Abdominal fluid score 0/4 • Gall bladder and urinary bladder normal • No retroperitoneal fluid, no pleural fluid, no pericardial fluid • Caudal vena cava 25% bounce, tree trunk • Chest x-rays

Shelter Cat Shelter Cat

15 month DLH cat 15 month DLH cat – tachypnea, lethargy5.1 + 4.0 = 9.1 – tachypnea, lethargy • Skeletal & cranial abdomen • Airways, Lung fields • No abnormalities noted • Great vessels • caudal vena cava somewhat enlarged • Smaller vessels • No abnormalities noted • Cardiac silhouette • Generalized cardiomegaly

Shelter Cat Shelter Cat

15 month DLH cat 15 month DLH cat – tachypnea, lethargy – tachypnea, lethargy • Skeletal & cranial abdomen • Airways, Lung fields • Heart Failure?? • No abnormalities noted • no LHF, maybe impending RHF • Great vessels • caudal vena cava somewhat enlarged • Diagnosis • Smaller vessels 3® • No abnormalities noted • TFAST showed dilation of LV & RV, but not LA • Cardiac silhouette • Flea Anemia (PCV 10%) • Generalized cardiomegaly, apex shifted right

5 Shelter Cat Lesson From Shelter Cat

• Chronic severe anemia can result in DCM like syndrome in the cat • Usually reversible when anemia treated • VetBLUE® usually much safer than x- rays for dyspneic cat – Allows treatment of pulmonary edema or pleural effusion prior to further diagnostics

Diagnosis Diagnosis Severity of Symptoms due to anemia Things that can mask anemia • Rapidity of onset • Dehydration • Severity of Anemia • Acute hemorrhage • Degree of physical activity (cats vs. dogs) • Shock, splenic contraction • Concurrent disease affecting respiratory • Cannot mask a severe anemia exchange • Look at plasma protein • Respiratory disease • Assuming there is no concurrent • Cardiovascular disease hypoprotenemia Pseudoanemia • Mild decrease in PCV due to plasma volume expansion, RBC normal • Congestive heart failure, pregnancy, glucocorticoid therapy, IV fluid therapy

Sequellae of Severe Anemia Diagnosis The First Question Hypoxic Injury • Is the anemia regenerative? • Liver compromise (worsens icterus) • i.e., is the body losing RBCs or not making • Myocardial hypoxia – Arrhythmia them or both? • Pancreatic hypoxia – pancreatitis • At maximum stimulation, the bone marrow can • Brain injury 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 Toxic Injury (lactic acidosis, etc.) • An acute regenerative anemia can look • Liver compromise, pancreatitis non-regenerative during the first week • Coagulopathy (DIC, direct toxicity) • Reticulocyte enumeration is the most • SIRS consistent way to evaluate regeneration • Systemic Inflammatory Response Syndrome • Run retics if PCV<30% in the dog or <20% in the cat

6 Assessing the Regenerative Response Assessing the Regenerative Response

Percent Reticulocytes Corrected Percent Reticulocytes (CPR/CRP) • Non-anemic animals <0.5% retics • If you don’t know the RBC and can not • >1% usually a regenerative response calculate absolute retics, you can still correct retic % for anemia • This method is not as reliable as…

Absolute Reticulocyte Count (ARC) CPR/CRP = % retics x patient PCV • RBC/ul x % retics = ARC normal PCV • Non-anemic animals <15-50,000/ul Cat normal PCV = 37%, Dog normal PCV = 45% • >200,000/ul highly regenerative • Automated counts are not always reliable • Normal animals <0.4% corrected retic % • This is the preferred single index for assessing regenerative response • >1% is a regenerative response

Assessing the Regenerative Response Assessing the Regenerative Response Increased MCV (mean corpuscular volume) = If you can’t calculate an ARC, then corrected macrocytosis retic % (CRP/CPR) is second best • Retics the most common macrocyte Reticulocyte Production Index (RPI) • Can also be increased due to: • No longer used very much • Prolonged storage (EDTA blood > 1 day) • early retics live longer than those made later • FeLV – RBC maturation arrest • marrow dysplasia – blasts, leukemia Increased RDW (red cell distribution width) • folate deficiency • Objective measure of anisocytosis • Phenobarbital therapy • If increased, you have one of the following: • Stomatocytes – liver disease • Normal + large RBC – regenerative • RBC leukemia – very, very rare • Small + normal RBC – developing IDA • **Atypical cells** • All 3 cell sizes – chronic blood loss

Assessing the Regenerative Response 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

RBC morphology – signs of regeneration • Anisocytosis – variation in RBC size • Polychromasia – blue-gray big RBCs • Polychromatophils = aggregate retics Howell-Jolly Bodies (HJB) and basophilic • >1/HPF (oil) indicates inc retics stippling are end stage nRBC

7 Regenerative Anemia Regenerative Anemia RBC morphology RBC morphology – semiquantitative scale

An abnormality should be present in nearly every field to be considered significant • 0 – not present • 1+ - mild – may not be clinically significant (5- Senescent cells can display any morphologic 10/HPF) abnormality • 2+ - mild to moderate (11-50/HPF) Poikilocytosis = increase in abnormally shaped • 3+ - moderate to marked (51-150/HPF) RBC cells • 4+ - marked (>150/HPF) RBC INDICES HAVE LOW SENSITIVITY FOR REGENERATION 2+ to 4+ are likely clinically significant – ONLY 8% of blood samples with regenerative anemia show increased MCV and decreased MCHC

Regenerative Anemia Regenerative Anemia

Degree of Regeneration - Dogs Degree of Regeneration – ARC Cats vs. Dogs

Corrected K9 Absolute % Retics Feline Feline % Retics Retics K9 Retics Aggregate Punctate Non-regenerative <1% <1% <60,000/ul Retics Retics Mild Regeneration 1-4% 1-2.5% 60-100,000/ul Non-regenerative <60,000/ul <15,000/ul <200,000/ul Moderate Mild Regeneration 60-100,000/ul 15-50,000/ul 200-500,000/ul 5-20% 2.5-5% 100-300,000/ul Regeneration Moderate 100-300,000/ul 50-100,000/ul 500-1,000,000/ul Regeneration Marked 21-50% >5% >300,000/ul Regeneration Moderate to Marked 300-500,000/ul 100-200,000/ul 1-1,500,000/ul Regeneration Acute Blood Loss – non-regenerative, then Marked >500,000/ul >200,000/ul >1,500,000/ul moderately regenerative 3-7 days later Regeneration Chronic Blood Loss – marked regeneration, until IDA sets in Hemolysis – moderate to marked regeneration

Regenerative Anemia Julie Henderson, DVM Hallsville TX Use ARC to monitor regenerative anemias Aggregate Retics live 1-3 days

So you know your anemia is Regenerative…

Now What???

8 Summary Acknowledgements

PowerPoint - .pptx, .pdf 1 slide per page, .pdf 6 Chapter 2: The Complete Blood Count, Bone slides per page Marrow Examination, and Blood Banking • Douglass Weiss and Harold Tvedten Client Handout • Small Animal Clinical Diagnosis by Laboratory • Anemia in Cats Methods, eds Michael D Willard and Harold th • Anemia in Dogs Tvedten, 5 Ed 2012

Vet Handouts Chapter 3: Erythrocyte Disorders • Ear Prick for Capillary Blood • Douglass Weiss and Harold Tvedten • Lip Prick for Capillary Blood • Small Animal Clinical Diagnosis by Laboratory Methods, eds Michael D Willard and Harold • Willard – Diagnostic Approach to Anemia Tvedten, 5th Ed 2012 • Blount – Diagnostic Chart for Classifying Anemia

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

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