Practical HematologyAnemia 101Wendy Blount, DVM

Practical Hematology

1. Anemia 1012. Blood Loss Anemia3. Hemolysis4. Non-Regenerative Anemias5. Transfusion Medicine6. Polycythemia7. Bone Marrow Disease8. Coagulopathy9. Central IV Lines10.Leukophilia11.Leukopenias12.Splenic Disease

DDx Anemia

Regenerative• Blood Loss

• 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 DzChronic renal diseaseChronic hepatic diseaseEndocrine disease

• Iron Deficiency• Bone Marrow Disease

Immune mediatedPure red cell aplasiaMyelodysplasia, MyelofibrosisAplasia,NecrosisMyelophtisis, neoplasiaMacrophage 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 intensity

• Microcytic – low MCV• Normocytic – anemia with normal MCV• Macrocytic – high MCV

• Hyperchromic – high MCHC• Normochromic – anemia with normal MCHC• Hypochromic – low MCHC (pale RBC)• 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?• 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

• Often 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 anemia)

• 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 totally 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 of blood

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 dropFeathered 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 dropMonolayer – 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

1. Platelet Estimate – 8-30/HPF (100x)

• Platelet clumping at feathered edge

• Platelet morphology

2. RBC morphology

3. 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 that you count

RBC Morphology

K9 RBC (discocyte)

feline RBC polychromatophil reticulocyte(NMB stain)

spherocyte

schistocyteschizocyte

blister cellkeratocyte

helmet cellkeratocyte

crenationechinocyte

burr cell

acanthocytespurr cell

budding fragmentation

eccentrocyte leptocyte Target cell (codocyte)

Mycoplasma haemofelis

Heinz body (NMB stain)

Howell Jolly Body

dacryocyte

RBC Morphology

normal normal regenerative response

regenerative response

IV hemolysis

DICangiopathy

oxidation oxidation artifactmetabolic dz

liver disease

DIC, angiopathy,IDA, marrow dz

oxidationSplenic dzhepatic dz

regeneration

Mycoplasma haemofelis

oxidation Increased nRBC

Cases

Shelter Cat

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

Shelter Cat

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

dry lung alveolar-interstitial lung fluid

Shelter Cat

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 fluid25% caval bounce Enlarged hepatic vein

Shelter Cat

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

Attendee, DVMCity TX

15 month DLH cat – tachypnea, lethargy

Shelter Cat

5.1 + 4.0 = 9.1

Shelter Cat

15 month DLH cat – 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

15 month DLH cat – 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, apex shifted right

Shelter Cat

15 month DLH cat – tachypnea, lethargy

• Heart Failure??• no LHF, maybe impending RHF

• Diagnosis• TFAST3® showed dilation of LV & RV, but not LA• Flea Anemia (PCV 10%)

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

Severity of Symptoms due to anemia

• 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

Sequellae of Severe Anemia

Hypoxic Injury

• Liver compromise (worsens icterus)

• Myocardial hypoxia – Arrhythmia

• Pancreatic hypoxia – pancreatitis

• Brain injury

Toxic Injury (lactic acidosis, etc.)

• Liver compromise, pancreatitis

• Coagulopathy (DIC, direct toxicity)

• SIRS• Systemic Inflammatory Response Syndrome

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

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

Assessing the Regenerative Response

Corrected Percent Reticulocytes (CPR/CRP)

• If you don’t know the RBC and can not calculate absolute retics, you can still correct retic % for anemia

CPR/CRP = % retics x patient PCV

normal PCV

Cat normal PCV = 37%, Dog normal PCV = 45%

• Normal animals <0.4% corrected retic %

• >1% is a regenerative response

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

RBC INDICES HAVE LOW SENSITIVITY FOR REGENERATION

– 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

Degree of Regeneration - Dogs

Acute Blood Loss – non-regenerative, then moderately regenerative 3-7 days later

Chronic Blood Loss – marked regeneration, until IDA sets in

Hemolysis – moderate to marked regeneration

% ReticsCorrected

% ReticsK9 Absolute

Retics

Non-regenerative <1% <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 – ARC Cats vs. Dogs

K9 ReticsFeline

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

Regeneration300-500,000/ul 100-200,000/ul 1-1,500,000/ul

Marked Regeneration

>500,000/ul >200,000/ul >1,500,000/ul

Regenerative Anemia

Regenerative Anemia

Use ARC to monitor regenerative anemias

Aggregate Retics live 1-3 days

So you know your anemia is

Regenerative…

Now What???

Julie Henderson, DVMHallsville TX

Summary

PowerPoint - .pptx, .pdf 1 slide per page, .pdf 6 slides per page

Client Handout• Anemia in Cats• Anemia in Dogs

Vet Handouts• Ear Prick for Capillary Blood• Lip Prick for Capillary Blood• Willard – Diagnostic Approach to Anemia• Blount – Diagnostic Chart for Classifying

Anemia

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

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