Anemia I-III+Notes+Anemia Case Discussion 1+IMHA discussion Notes🧪

•Blood transfusion -If clinical for anemia -Severe/Very severe •Red blood cell production (1° > 2°) -Vitamin B12 (injection or oral) -Iron (injection or oral) -Folic acid; Vit B9 (oral) -Erythropoietin supplementation (injection) •-Renal disease •-Erythroid hypoplasia/aplasia? •-Myelofibrosis?

Treatment General therapies for anemia: ________________________

Erythroid hypoplasia/aplasia (PIMA or PRCA) Myelophthisis Myelofibrosis

Treatment Primary (intrinsic) bone marrow •______________ -Immunosuppressive medications •____________ -Chemotherapy (neoplasia) -Antimicrobials/antifungals (infection) •___________________ -Immunosuppressive medications? -Intermittent blood transfusions

Anemia of Inflammatory disease (AID) Renal disease Endocrine disease

Treatment Secondary (extrinsic) bone marrow •_________________ -Treat underlying disease •__________________ -Erythropoietin supplementation •________________________ -Treat underlying disease •Iron deficiency anemia -Treat underlying disease: deworm, gastroprotectants -Iron supplementation (blood transfusion and/or injection)

False •Packed Cell Volume (PCV) and Hematocrit (HCT) used clinically If you Hct and PCV are not within ~4% agreement, you should consider performing another PCV from a new blood draw.

True or False? Packed Cell Volume and Hematocrit are not used clinically

•Decrease in red blood cell mass ANEMIA = ↓ RBC count, ↓ PCV/Hematocrit and ↓ Hemoglobin

What is anemia?

PCV/TP

_____________ in any patient suspected to be anemic!!

Positive

_____________ saline agglutination test

Anemia of inflammatory disease Cats

________________ is COMMON! Usually mild. ___________ are an exception, as they can have a more moderate anemia with AID alone

Mycoplasma

_________________ spp. (cat >>>dog)

Intravascular hemolysis

ghost cells 👻 hemoglobinemia (not as specific) hemoglobinuria (more characteristic) a. Intravascular hemolysis b. extravascular hemolysis c. either

Either

high bilirubin, either in blood or urine aka hyperbilirubinemia autoagglutination a. Intravascular hemolysis b. extravascular hemolysis c. either

ü **Check reticulocyte count** ü Check RBC indices (MCV and MCHC) ü Check blood smear-red cell morphology (polychromasia?) Picture: Absolute Reticulocyte Count (Not %age)

o DETERMINE if REGENERATIVE or NON-REGENERATIVE ANEMIA How?

PCV

percentage of whole blood composed of erythrocytes

Extravascular hemolysis

spherocytes (macrophage took nibble out of RBC) a. Intravascular hemolysis b. extravascular hemolysis c. either

EXTRAVASCULAR HEMOLYSIS

• Usually IgG-mediated or C3(complement)-mediated • IgG or C3 coated erythrocytes are removed by the MPS o These macrophages have Fc receptors that recognize the Fc portion of of IgG o Primarily occurs in the spleen +/- liver o Erythrophagocytosis by the MPS can either be partial (resulting in a spherocyte) or complete o Hyperbilirubinemia and bilirubinuria o Hemoglobin enters the bilirubin pathway before entering the circulation, so hemoglobinemia and hemoglobinuria do NOT occur o More common form; associated with better prognosis compared to intravascular

Neonatal Isoerythrolysis

•Cats ("fading kitten syndrome") -UNCOMMON!!! -Type B queen has naturally occurring anti-A antibodies! And vice versa. -Type B queen has the antibodies in colostrum/milk that when ingested by Type A kitten results in RBC lysis

IMHA -Loss of immunologic tolerance toward self antigen? -Alteration in the RBC surface antigen -> looks foreign? • In the normal dog and cat, autoantibodies directed against red blood cell antigens are identified by the mononuclear phagocyte system in the spleen and liver, resulting in destruction and removal of senescent ("old") erythrocytes. • Average erythrocyte life span is ~120 days in dogs, ~80-90 days in cats • IMHA is a pathologic condition resulting in PREMATURE destruction of erythrocytes due to an inappropriate immune response. PATHOPHYSIOLOGY • Type II hypersensitivity reaction o Anti-RBC antibodies (IgG, IgM) o Components of the complement system • Results in premature RBC destruction (hemolysis) o Many patients have a component of BOTH intravascular and extravascular RBC destruction (hemolysis) Extravascular more common than intravascular Extravascular -> mononuclear phagocyte system (spleen, liver) Intravascular -> complement mediated (often assoc with IgM) • Results in systemic inflammation that can result in any or all of the following: o Fever o Severe neutrophilia (can be as high as 70,000-100,000/uL) Often have many band neutrophils (1000-8000/uL) Cats with primary IMHA usually have a normal leukocyte count o Thrombocytopenia - 25-70% of cases Usually mild/moderate Can be severe with concurrent ITP ("Evan's syndrome") o Risk of thromboembolism Many reasons for a hypercoagulable state (more on pg 13) o Systemic inflammatory response syndrome (SIRS) Disseminated intravascular coagulation (DIC): 14-45% of dogs Multiple organ dysfunction syndrome (MODS) • Acute kidney injury (AKI) • Liver disease/dysfunction Non-cardiogenic pulmonary edema (ARDS/ALI) • Common in dogs. Rare in cats. • Can be primary or secondary to other diseases (eg; infection, neoplasia) or any immune stimulus!

•Senescent RBC normally removed via monocyte phagocytic system (MPS) •(answer 1) is antibody-mediated PREMATURE RBC destruction -"Type II hypersensitivity" -IgG or IgM -> extravascular hemolysis -IgM -> activation of complement -> membrane attack complex (MAC) -> intravascular hemolysis •Why does it happen?? _______________________

Non-Regenerative Anemia NON-REGENERATIVE ANEMIA Normocytic (normal MCV), normochromic (normal MCHC), normal reticulocyte count (~ Dog <60,000/uL absolute retic; Cat <45,000/uL absolute retic)

•Usually chronic •May be an incidental finding •MANY causes! -Pre-regenerative -Primary bone marrow ("instrinsic") -Secondary bone marrow ("extrinsic") •Species differences -Cat > Dogs •Bone marrow evaluation may be indicated

spherocytes

1.Extravascular hemolysis -> _______________

Hyperbilirubinemia

1.______________ does NOT help to differentiate between extravascular and intravascular hemolysis -AND you can have BOTH extra and intravascular hemolysis with IMHA

hemoglobinemia/uria

2.Intravascular hemolysis -> ___________

Spherocytosis (moderate or marked) and/or autoagglutination

2.__________________are diagnostic for IMHA (with few exceptions)

BOTH intravascular and extravascular

3.See hyperbilirubinemia/uria with __________________

Euthanasia and thromboembolism

4.___________________ are the 2 most common causes of death in canine IMHA with mortality rates varying depending on severity

The blood film has been reviewed and reported cell counts appear accurate. There is marked anemia with evidence of strong regeneration given marked polychromasia. NRBC's are proportional to the degree of polychromasia, and are attributed to RBC regeneration. Moderate numbers of spherocytes are present and are consistent with immune-mediated hemolysis. No hemoparasites are seen. The actual platelet count may be slightly higher than the reported automated count due to the presence of occasional small platelet clumps and macroplatelets observed on blood smear. However, subjectively platelets appear mildly to moderately decreased. The presence of macroplatelets is supportive of thrombopoiesis. -moderate to severe anemia in a dog -macrocytic, borderline hyperchromic , moderate to severe anemia of 17%, no reticulocyte account, but has polychromasia and there are a lot of enucleated red cells so regenerative. Acanthocytosis (cells of many sizes). Low thrombocytopenia. Moderate to marked neutrophilia, with high amounts of bands, monocytosis with normal lymphocyte count. Toxic changes to neutrophils. If total protein is normal but has a high bilirubin would be IMHA. The banded neutrophils can be normal with IMHA

9 year old FS Australian Shepherd •9 day history of lethargy •Increased respiratory effort over the past 1-2 days. Worsening lethargy. •Owner noticed gums were "pale." •Not currently on any medications. •UTD on vaccinations. •No significant medical history Path Review ____________________________________

-Clopidogrel (Plavix®) - 1-4 mg/kg SID 🌟 •-Irreversible platelet inhibition •-Better than aspirin? --contraindicated if the patient has Evan's syndrome where not only are RBCs being destroyed but also all WBCs are being destroyed -Low dose aspirin (0.5-1 mg/kg SID - DOG) •-Irreversible platelet inhibition •-Questionable efficacy in dogs? And cats? •-Not routinely used at OSU •Heparin or rivaroxaban (Xarelto®)? -Clinician dependent -Some evidence improves survival -Not routinely used at OSU TREATMENT of IMHA Platelet inhibitors (one OR the other) • Low dose aspirin o Dogs: 0.5-1 mg/kg PO q 24 hours o Cats: 0.5-1 mg/kg PO q 48-72 hours • Clopidogrel (Plavix®) o Dogs: 1-2 mg/kg PO q 24 hours o Cats: 18.75 mg/cat PO q 24 hours Heparin • Better for venous thrombosis (in theory) • Unfractionated heparin o Variable efficacy between patients o 200-300 units/kg SC q 6-8 hours o Monitoring PTT: goal 1.5-2 x upper limit of normal Anti-10a concentration (better!) Monitor for bleeding! o Evidence that individual dosing based on Anti-10a concentration improves survival • Low-molecular weight heparin (dalteparin, enoxaparin) o More predictable efficacy o Dalteparin: 100-150 units/kg SC q 12 hours o Enoxaparin: 0.8 mg/kg SC q 6 hours o CRAZY expensive!!! Warfarin - don't use in dogs or cats • Narrow therapeutic index - requires frequent monitoring

Acute Management IMHA 2. Thromboprophylaxis •Platelet inhibition ______________________

-Vascular injury - cytokines, hypoxia -Blood stasis - hospitalized, IV catheter -Hypercoagulable - free hemoglobin, RBC transfusions, glucocorticoids, etc pulmonary thromboembolism TREATMENT of IMHA o Thromboprophylaxis therapy The most common cause of natural death in patients with IMHA is thromboembolism (especially PTE)! Patients with IMHA are at risk of thromboembolism via multiple mechanisms • Vascular injury from release of inflammatory cytokines and tissue hypoxia • Blood stasis exacerbated in hospitalized patients (cage confinement, IV catheters) • Hypercoagulability secondary to free hemoglobin (intravascular hemolysis), blood transfusions, and administration of glucocorticoids • And many more...

Acute Management IMHA 2. Thromboprophylaxis 🌟 •Risk of thromboemoblism _______________________ •Leading cause of natural death in IMHA! -Specifically ______________

1.Immunosuppression 🌟 2.Thromboprophylaxis 🌟 (they have too many reasons to make clots) 3.Supportive care (can be very expensive dogs) 4. +/- Adjunctive immunomodulatory therapy

Acute Management of IMHA

Saline agglutination test

Autoagglutination _______________: 1 drop saline + 1 drop EDTA blood

HCT Hematocrit is a calculated value, so significant alterations in MCV can result in a falsely increased or decreased Hct result. (MCV X RBC) = Hct 10 Example: MCV can be markedly increased in patients with autoagglutination, because the CBC machine assesses a "clump" of multiple RBCs as 1 large RBC and this results in an increased MCV. In that case, the Hct may be falsely increased.

Calculated value (MCV X RBC)÷10

1= •Hyperbilirubinemia? •Hemoglobinuria? 2= •IMHA •Spherocytosis? •Autoagglutination? 3=•Evidence of bleeding? 4=•Low total protein

Categories of Regenerative anemia

1= •Normocytic, normochromic •Microcytic, hypochromic (when iron deficient, but the most common cause of iron deficiency is blood loss) 2=•Other cytopenias? 3=•Chronic?

Categories of anemia

1=•Macrocytic, hypochromic 2= •Reticulocytosis •Polychromasia •nRBC 3= •Evidence of bleeding? •Low total protein?

Categories of anemia

1=•Hemolysis 2=•Blood Loss 3=•Pre-Regenerative 4=•2° Bone Marrow (Extrinsic) (bone marrow works find, it just doesn't have the components to make RBCs; animals who don't have enough erythropoietin, like in Chronic kidney disease, iron deficiency, B12 deficiency) 5=•1°Bone Marrow (Intrinsic) (actually something wrong with the bone marrow; neoplasia, histo, IMHA at the level of the BM)

Categories of anemia 🌟

Immune-mediated hemolytic anemia

Categories of hemolytic anemia 1. ______________________ We will come back to this one...

Immune-mediated hemolytic anemia (IMHA)

Categories of hemolytic anemia 1.______________________ •Intravascular vs. extravascular -Often a combination of both! •Primary vs. secondary

-Babesia spp. -Mycoplasma hemocanis (increased risk after splenectomy) -Mycoplasma hemofelis and M. haemominutum 🌟 •-Formally known as "hemobartonella" -Cytauxzoon felis (geographical) -Blood smear - organism identification -Blood PCR -Serology and PCR for Babesia

Categories of hemolytic anemia 2. Erythrocyte organisms •DOGS: __________________ •CATS: _______________________ •DIAGNOSIS: _________________________

heinz-bodies increased fragility intravascular

Categories of hemolytic anemia 3. Toxins (oxidative damage) •🌟 Oxidative injury -> _____________ form -> RBC less deformable ->_______________ -> hemolysis (typically more ____________) -Zinc (dog) •-Pennies after 1982 •-Zinc oxide -Onions/garlic -Acetominophen •Eccentrocytes and mild spherocytososis may also be present in some cases

🌟 Schistocytes or keratocytes -Disseminated intravascular coagulation (DIC) -Hemangiosarcoma -Vasculitis (uncommon, has to be severe!) -Heartworm caval syndrome (uncommon) thrombocytopenia -Due to consumption of platelets

Categories of hemolytic anemia 4. Microangiopathic or fragmentation anemia •Mechanical trauma to RBC secondary to fibrin strands or other (eg; heartworms) •____________________ •Causes: _______________________ •Doesn't usually cause severe anemia •Often have concurrent ________________

🌟 Hypophosphatemia (decreased phosphorus)

Categories of hemolytic anemia 5. Increased erythrocyte fragility •______________________ -Usually VERY low <1.5 (normal 2-5) -Aggressive insulin administration (DKA) -Re-feeding syndrome FYI •Congenital RBC disorders (rare) -Pyruvate kinase deficiency (PK) -Phosphofructakinase deficiency (PFK) -Hereditary osmotic fragility syndrome

•Form of malignant histocytosis/disseminated histiocytic sarcoma •Phagocytic neoplastic histiocytes from spleen and bone marrow -> RBC destruction •Breeds: Bernese Mt Dog, Rotties, Golden, Lab •Often misdiagnosed as IMHA •Difficult to diagnose! -Often looks like IMHA without classic dx features

Categories of hemolytic anemia FYI 6. Hemophagocytic syndrome

1.Immune-mediated hemolytic anemia -Intravascular vs. Extravascular -Primary vs. Secondary 🌟 2. Erythrocyte organisms 3. Toxins (oxidative damage-garlic and onions in dogs, acetaminophen in cats, zinc in dogs, etc.) 4. Microangiopathic or fragmentation anemia (patients that have shearing of their red cells like in DIC or hemangiosarcoma-usually mild anemias with a mild increase in bilirubin but shouldn't have agglutination) 5. Increased erythrocyte fragility 6. Hemophagocytic syndrome-looks like IMHA, not IMHA. Macrophages that are neoplastic that are running around eating red cells. So technically not IMHA but looks the same, doesn't usually have autoagglutination

Categories of hemolytic anemia ___________________________

•Gradual taper of immunosuppressive drugs -Do NOT start taper until PCV is normal! •-Evaluate for continued destruction -Typically reduce by 20-25% every 2-4 weeks -Usually takes 4-6 months to taper completely OFF of medications •-Some patients may have to stay on low dose immunosuppression long-term -If on multiple drugs, taper steroids first •-Unless not tolerating the other drug(s) LONG-TERM TREATMENT • Tapering of medications: Prednisone alone o DO NOT begin reducing prednisone dose until PCV is normal for at least 1-2 weeks and there is no evidence of regeneration o Should recheck full CBC with reticulocyte count and blood smear review (polychromasia? Spherocytosis?) until normal and periodically throughout taper o Reduce dose by 20-25% every 2-4 weeks Make sure to check PCV/TP before each taper, cannot rely on clinical signs alone!! Some clinicians will evaluate PCV/TP 1 week after each taper (especially in beginning) to monitor for relapse • Tapering of medications: Prednisone + 2nd line immunosuppressive o Generally taper prednisone first because of outward side effects o Using second-line immunosuppressive as "steroid sparing" in most cases -> may allow you to taper prednisone sooner if know you have another agent on board o Goal would be to taper off of prednisone completely and maintain on low dose of the 2nd line immunosuppressive agent long-term • Monitoring of potential adverse effects of 2nd line medications o Generally evaluate for adverse effects at 2, 6, and 12 weeks and then ever 3-6 months depending on how patient is doing and dose being used Azathioprine - Hepatotoxicity, bone marrow suppression • CBC and liver/chemistry panel • Dose dependent toxicity Cyclosporine - opportunistic infections, hepatotoxicity? • Consider cyclosporine trough blood concentration o Draw blood before next dose 3-7 days after starting therapy o Recheck trough blood conc if alter dose or formulation • Consider monitoring liver and renal values o Hepatoxicity and nephrotoxicity reported in humans o Haven't been reported in dogs and cats o Suspicious for hepatotoxicity in a few patients Mycophenolate - hepatoxicity, bone marrow suppression • CBC and liver/chemistry panel • Dose dependent toxicity? Leflunomide - bone marrow suppression, hepatotoxicity? • CBC and liver/chemistry panel

Chronic Management of IMHA

•You can't!! •Difficult to control initially? •Already relapsed? -During taper of meds -After off of meds •Concern for relapse? Ideal to TRY to get a patient off of therapy completely! • How long should you continue anti-thrombotic therapy? o Clinician and patient dependent... o Typically continue platelet inhibitor medication as long as patient is on prednisone or at least until prednisone dose is <0.5 mg/kg/day o Various protocols for heparin Patient and clinician dependent Some clinicians only administer in hospital If document thromboembolism may continue for weeks/months Considering tapering over 1-2 weeks when discontinue

Chronic Management of IMHA When do you determine that a patient CAN'T be tapered off of immunosuppressive therapy completely?

•VERY important! •Not a "quick-fix" disease -Initial management can be expensive -Long-term medications and follow-up •Glucocorticoid side effects •Importance of gradual taper of medications •Importance of follow-up •Predisposition to immune-mediated diseases? CLIENT EDUCATION • Importance of client education cannot be emphasized enough! o Need and importance for follow-up appointments Including length of follow-up: generally 6-12 months! Importance of gradual taper of medications to reduce the risk of relapse o Warn them of side effects of glucocorticoids Increased thirst/urination Increased appetite Panting Muscle wasting Weight gain Joint laxity o Short-term and long-term cost can be HUGE! As much as $1500-5000 initially Medications and recheck appointments add up!

Client Education of IMHA _____________________

Abdominal radiographs Thoracic radiographs Abdominal ultrasound Second wave of diagnostics (this will be based on results of above testing) · Imaging o FAST scan US -> assess for free fluid in a cavity o Abdominal radiographs § Zinc ingestion § Peritoneal effusion § Abdominal mass o Thoracic radiographs § Neoplastic disease § Pulmonary hemorrhage -> interstitial to alveolar lung pattern. May be more "patchy" in appearance § Pleural effusion o Abdominal ultrasound § Peritoneal effusion § Mass -> not uncommon to find a mass on ultrasound that was not felt on abdominal palpation OR seen on radiographs § GI ulceration -> RARELY visualized on US

Diagnostics •___________________ -Zinc toxicity (penny ingestion) (causes hemolytic anemia) -Peritoneal effusion - blood?? -Abdominal mass •________________ -Pulmonary hemorrhage -Pleural effusion (hemothorax?) -Neoplasia •________________________________ -Mass -Gastrointestinal ulceration? (difficult to see on US) -Peritoneal effusion -> hemoabdomen? (could be caused by rodenticide toxicity)

Macroscopic hypochromic regenerative 🌟 Spherocytes DIAGNOSIS of IMHA • Usually not that challenging o Challenging aspect is determining primary vs. secondary • Complete blood count o Moderate to severe anemia (typically <25%) Usually macrocytic, hypochromic regenerative • Regenerative anemia o Polychromasia o Nucleated RBCs - not specific for regeneration o Dogs: >60,000/uL absolute reticulocyte count o Cats: >40,000/uL absolute reticulocyte count What if non-regenerative? • Likely due to acute hemolysis so "pre-regenerative" o Takes 3-5 days to start making new RBCs! • Could be associated with IMHA at the level of the bone marrow or "intra-marrow IMHA." o Moderate to marked neutrophilia +/- band neutrophils (dogs>cats) o Thrombocytopenia Usually mild to moderate • Consider associated vector-borne disease • Consumption from disseminated intravascular coagulation If moderate to severe consider "Evan's syndrome" - concurrent IMHA and ITP

Diagnostics for IMHA •Complete blood count (CBC) -(Macroscopic/Microscopic?), (hypochromic/hyperchromic?) anemia -(Regenerative/Non-regenerative?) •-Polychromasia •-Reticulocyte count -RBC morphology •-________________ -> extravascular hemolysis •-Ghost cells -> intravascular hemolysis •-RBC organisms? -Often thrombocytopenic (mild/moderate) -Often have leukocytosis (can be marked)

•IV fluids -Hydration, electrolyte balance •RBC transfusion(s) PRN -Packed RBC or whole blood -Some patients may need multiple •Others PRN -Gastroprotectants -Anti-emetics TREATMENT of IMHA o Supportive care Intravenous fluids • Maintain hydration • Prevent renal injury from pigmenturia, DIC, dehydration • Correct electrolyte imbalances RBC transfusion • Most cases need at least one RBC transfusion. Some patients may need multiple transfusions (2-5). • pRBC (if able) better than whole blood (WB) o WB increases risk of transfusion reaction and may provide hypercoagulable state o pRBC: ~10 mL/kg IV over 4 hours (faster if critical) o WB: ~20 mL/kg IV over 4 hours o Cats: 60 mL WB/cat over 4 hours (faster if critical) • Cross match or type?? o Dogs First transfusion: No cross match required If receiving 2nd transfusion >3-5 days after first transfusion MUST cross match to donor o Cats MUST type ALL cats!! Most will be "type A" If give type A blood to a type B cat will likely die quickly - all it takes is 1 mL!!! Anti-emetic therapy • Maropitant, ondansetron/dolasetron, metoclopramide Gastroprotectants • Acid suppressant - famotidine or omeprazole • Sucralfate if concern for GI bleeding/ulcer o Remember may alter absorption of other drugs! Antibiotics?? • Usually not indicated in cases or primary IMHA • If suspect vector-borne disease, mycoplasma or leptospirosis o Doxycycline 5 mg/kg q 12 hr or 10 mg/kg q 24 hr • If suspect other bacterial infection o Broad spectrum antibiotics Unasyn or Clavamox +/- Fluoroquinolone • Secondary IMHA o Treat underlying disease! o May still need immunosuppression and other therapies listed above Will depend on severity

Acute Management 3. Supportive care ___________________________

Human Intravenous Immunoglobulin (IVIg) TREATMENT of IMHA o Adjunctive immunomodulatory therapy Human Intravenous immunoglobulin (hIVIg) • Purified product of pooled human plasma from multiple healthy donors - 90% IgG o Many different formulations • Multiple proposed mechanism of action o Known mechanism in dogs:IgG blocks Fc receptor and therefore reduces ability of Fc receptor to recognized anti-RBC antibodies and destroy RBC • Dose: 0.5-1.5 gram/kg administered IV over 6-12 hours • EXPENSIVE!! Medium/Large breed dog = $2000-3000 • Evidence for use in canine IMHA? o One study compared prednisone + hIVIg vs. prednisone alone and found no difference is days to stabilization of PCV or duration of hospitalization • Tend to reserve for more severe cases • Potential adverse effects: o Hypersensitivity reaction o Thromboembolism o Proceed with caution in patients with moderate/severe cardiac disease (risk of volume overload) o Do NOT repeat dosing!

Acute Management 4. Adjunctive immunomodulatory therapy •_________________ -Primarily IgG -Many mechanisms of action •-Blocks Fc receptors in splenic macrophages •-Prevents extravascular hemolysis -Minimal evidence for use in canine IMHA -Expensive! -Do not repeat infusion -> risk of hypersensitivity reaction

No strong evidence to support WHICH agent is chosen for a particular patient.

Acute Management IMHA 1.Immunosuppression Which immunosuppressive agent is best??

-Cost? -How long to take effect? -Formulation? •-Tablet/capsule size •-Liquid? -Adverse effects?

Acute Management IMHA 1.Immunosuppression •Considerations _________________________

-Prednisone -Dexamethasone (often used in hospital) •-Remember 7-10 x more potent than prednisone!! -Azathioprine -Cyclosporine -Mycophenolate -Leflunomide TREATMENT of IMHA • Primary/Idiopathic IMHA o Immunosuppressive therapy Prednisone 2 mg/kg/day (q 24 hr or divided q 12 hr) • Prednisolone in cats • Dexamethasone IV or SQ in hospital (0.2-0.3 mg/kg/day) +/- 2nd line therapy • No strong evidence to support WHICH agent is chosen for a particular patient. • Factors to consider: o Cost? o Potential adverse effects - consider your patient! o Ease of dosing - tablet/capsule size, liquid avail? o How quickly will it start working? • Azathioprine o Starting dose: 2 mg/kg/day PO q 24 hr x 10-14 days o Then reduce to 1 mg/kg/day OR 2 mg/kg every other day NO MATTER what!! o Takes 1-3 weeks to start working o Do NOT use in cats • Cyclosporine o 3-5 mg/kg PO q 12 hours (higher dose compared to skin and GI diseases) o Variable bioavailability - consider checking a cyclosporine trough blood concentration to determine efficacy or toxicity • Mycophenolate o 6-10 mg/kg q 12 hr • Leflunomide o 1-4 mg/kg PO q 24 hrs o Wide dose range: typically start low and increase if needed • Cyclophosphamide - no longer used for this purpose

Acute Management IMHA 1.Immunosuppression •Glucocorticoid 🌟 _________________________ •+/- 2nd immunosuppressant agent __________________________

-Improved immunosuppression (severe cases) -Steroid sparing •-Adverse effects ---Especially important in medium/large dogs •-Concurrent diseases ---Diabetes mellitus ---Severe cardiac disease

Acute Management IMHA 1.Immunosuppression •Why add a 2nd line drug?

Process of elimination Consider your differential diagnoses for non-regenerative anemia and consider the following: • Clinical signs o A dog with hypothyroidism resulting in anemia is going to have clinical signs consistent with hypothyroidism (lethargy, weight gain, heat seeking, alopecia, etc). • Physical exam findings o Many patients with GI bleeding severe enough to result in iron deficiency anemia are going to have melena o Most dogs with lymphoma have enlarged and firm peripheral lymph nodes • CBC o Evaluate RBC indices o Other cytopenias? o Inflammatory leukogram? o Any infectious organisms seen? • Chemistry panel o Renal disease can result in anemia o Addison's disease often results in electrolyte abnormalities (increased K, decreased Na) o Hypothyroidism often results in hypercholesterolemia and hypertriglyceridemia Look at the entire CBC! If patient has other cytopenias (either 2 or 3 lines), then the likelihood of a primary bone marrow disease is increased. • There are exceptions to this rule though! For example, a septic patient may have pancytopenia, but in reality their bone marrow is functioning normally. Their neutrophils are being consumed faster than they can make them and they may have mild thrombocytopenia from various mechanisms (usually consumption in the case of sepsis) and a mild anemia from AID. • Use your reticulocyte count and markers of bone marrow regeneration for your other cell lines to help you determine this. o Band neutrophils often indicate neutrophil regeneration (some exceptions) o Large or giant platelets often indicate regeneration (some breed exceptions) Evaluate the severity of the anemia Secondary bone marrow diseases/disorders tend to be a "mild" anemia (occasionally moderate) • Exceptions: o Cats with anemia of inflammatory disease can be moderate o Iron deficiency anemia can be mild, moderate OR severe A moderate anemia almost always rules-out endocrinopathy-associated anemia, but doesn't help you to sort out other causes. • Timing is everything! Keep in mind that everything is determinant on when you determine the patient is anemic in the course of their disease. Severe anemia tends to be due to primary bone marrow disease and generally are more chronic in their course. • Exceptions: o Iron deficiency anemia can be mild, moderate, or severe. Typically once they are truly iron-deficient (microcytic, hypochromic, non-regenerative), these patients tend to have a moderate or severe anemia o Concurrent causes of anemia can be more severe. For example, a cat with anemia of inflammatory disease secondary to histoplasmosis that also likely has bone marrow infiltration of histoplasmosis

How do you sort out whether the patient is anemic from primary vs. secondary bone marrow disease?

Primary IMHA

IMHA •_______________ -Idiopathic •-No identifiable underlying cause/trigger •-COMMON IN DOGS! 60-75% of canine IMHA cases •-Predisposed: ---Cockers, Miniature Schnauzers, Poodles, other small breeds ---Young/middle aged: 2-7 years ---Females > Males? •-Rare in cats! Diagnosis of exclusion!! -As a component of SLE ("lupus") •-Multisystemic immune-mediated disease •-ITP, IMPA, GN, Myositis, Meningitis, IM skin disease

Secondary IMHA

IMHA •___________________ -ANY disease process that alters the immune system -Neoplasia - LSA, Leukemia, histiocytic sarcoma -Infectious diseases •-Vector-borne diseases ---Including RBC organisms •-Other bacterial infections? -Drugs - antibiotics (B-lactams, sulfas), ANY -Vaccines - weak association

•Signs associated with anemia and systemic inflammation •Icterus (total bilirubin >2 mg/dL) •Pigmenturia -"Port wine" = hemoglobinuria -"Darker/orange" = bilirubinuria •May have GI signs -Anorexia, vomiting, diarrhea •Hepatomegaly, splenomegaly •+/- Lymphadenopathy - mild (if present at all) •+/- Fever CLINICAL PRESENTATION • Increased occurrence of disease in the warmer months o 57% in April-September • Clinical signs and PE typically reflective of the anemia and systemic inflammation o Lethargy, depression, weakness, anorexia o Tachycardia, tachypnea, +/- systolic murmur o Pigmenturia (bilirubin vs. hemoglobin - "port wine" appearance) o Mucous membrane pallor, icterus o Bilirubin greater than ~2 mg/dl will manifest as clinical icterus o Hepatosplenomegaly, lymphadenopathy • Cats - lethargy, anorexia, pica

IMHA - Clinical Presentation

Extravascular

IMHA: ________________ hemolysis - Antibody bound to RBC 🌟 Spherocytosis Hyperbilirubinemia Bilirubinuria

Intravascular hemolysis

IMHA: ___________________ -- Antibody (IgM) bound to RBC -- Activates complement -- Membrane attack complex (MAC) punches holes in RBC Hyperbilirubinemia 🌟 Hemoglobinemia 🌟 Hemoglobinuria Ghost cells Worse prognosis

IMHA is antibody-mediated! Look for spherocytes and autoagglutination -autoagglutination doesn't tell you whether intra or extravascular HEMOLYTIC ANEMIA · IMHA is only ONE cause of hemolytic anemia! · Other conditions to consider that result in hemolysis, but are NOT immune-mediated in etiology: o Microangiopathic or "fragmentation" anemia § Consider diseases that "damage" or cause "shearing" of the RBC · Large thrombus or DIC · Hemangiosarcoma · Vasculitis · Heartworm caval syndrome § Typical RBC morphology - schistocytes +/- keratocytes § Usually results in consumptive thrombocytopenia o RBC organisms § These typically result in secondary IMHA § Babesia spp (dogs), Mycoplasma spp (cats>dogs), Cytauxzoon felis (cats; Missouri/Oklahoma/Arkansas) o Toxins § Oxidative injury -> heinz bodies form à erythrocytes less deformable à increased fragility -> hemolysis § Dogs: Onions, garlic, zinc (penny after 1982, zinc oxide ingestion), acetaminophen, benzocaine § Cats: acetaminophen, benzocaine § May also see eccentrocytes and occasional spherocytes § NOTE: Other diseases may result in Heinz body formation (eg; hyperthyroidism), but don't usually result in Heinz body anemia o Increased RBC fragility § Hypophosphatemia (typically has to be <1.5) · Aggressive insulin treatment (DKA) · "Refeeding syndrome" § Congenital RBC disorders - RARE! · Pyruvate kinase deficiency (PK) o English Springer Spaniel, Basenji, Beagle o Usually diagnosed at 2-4 years of age o Prolonged, chronic hemolytic anemia o Most do not show clinical signs until have myelofibrosis from "bone marrow burnout" from chronic hemolysis o PCR testing available · Phosphofructokinase deficiency (PFK) o English Springer Spaniel, American Cocker Spaniel o Exacerbated by anything that causes a "respiratory alkalosis" (eg: exercise, hyperventilation, and high ambient temperature) o PCR testing available · Hereditary osmotic fragility syndrome o Abyssinian cats o Hemophagocytic anemia/syndrome § May or may not have evidence of spherocytosis § Generally associated with malignant histiocytosis/disseminated histiocytic sarcoma § Invasive of phagocytic, neoplastic histiocytes arising from spleen and bone marrow § Predisposed breeds: Bernese Mountain Dog, Rottweilers, Golden, Labrador, Flat-coated Retrievers § DIFFICULT to definitively diagnosis -> need biopsy

Intravascular IMHA NOTE: Many causes of hemolytic anemia result in intravascular hemolysis and can mimic intravascular IMHA So, what is the difference?? ____________________________________

-RBC organisms -Morphology changes (schistocytes, spherocytes, heinz bodies)

LOOK at the RBCs....they can help you! How?

entire

Look at the __________ CBC

REGENERATIVE ANEMIA

Macrocytic (↑ MCV), hypochromic (↓ MCHC), polychromasia, increased absolute reticulocytes

Feline IMHA WHAT IS DIFFERENT ABOUT FELINE IMHA? • Most are secondary IMHA o Should always search for an underlying disease! • Typically young (<6 yrs, many 1-2 yrs) and male • May have other cytopenias o Thrombocytopenia o Neutropenia • Few have leukocytosis • Spherocytes difficult to detect o Normal feline RBC does not always have distinct central pallor • Treatment: Prednisolone +/- cyclosporine o MANY need cyclosporine! • Type B cats have STRONG anti-A antibodies o Neonatal isoerythrolysis Type-A kittens born to Type-B queen o Risk of blood transfusion reaction (even if first transfusion!) Especially Type-A blood to a Type-B cat!!

Most are secondary! -Infections -> Mycoplasma spp., FeLV, Cytauxzoon felis If primary: •Typically young (<6 yr; often 1-2 yrs) and male •May be non-regenerative initially •May have other cytopenias? -Thrombocytopenia -Neutropenia •Few have leukocytosis

•Good history •Re-evaluation of reticulocyte count •How sick are they? • PRE-REGENERATIVE • Takes 2-5 days to see regenerative response Species differences -> Cats take longer than dogs Blood loss takes longer than hemolysis • WHY? Losing all components of RBC when lose whole blood vs. hemolysis where can recycle RBC components • Peak reticulocyte response 4-8 days after onset of anemia

Non-Regenerative Anemia Categories 1. Pre-regeneration •Acute blood loss •Acute hemolysis Takes 2-5 days to start making RBCs! How do you determine this?? ______________________

•Precursor immune mediated anemia (PIMA) or Pure Red Cell Aplasia (PRCA) or other erythyroid hypoplasia 🌟 •Myelofibrosis •Myelophthisis (marrow is being crowded out by something, usually neoplasia) 🌟 •Myelodysplasia- • Primary (intra-marrow) Disease Moderate to severe anemia May have concurrent leukopenia and/or thrombocytopenia Primary processes include: • Immune mediated o This may affect multiple cell lines OR just the red cell line. Occasionally see "intramarrow IMHA" where immune-system attacks RBC precursors instead of peripheral RBCs. • Neoplasia o Lymphoma, leukemia, multiple myeloma, and histiocytic sarcoma most common • Infectious o Cats -> FeLV, FIV, histoplasmosis o Dogs -> Ehrlichia canis (chronic form), Leishmania (uncommon in USA), histoplasmosis (uncommon form in dogs but does occur) • Estrogen toxicity o Endogenous -> sertoli cell tumor o Exogenous -> ask owner about estrogen creams, patches, etc. • Drugs/Toxins (MANY) • Myelodysplastic syndromes • Myelofibrosis o Often secondary to another bone marrow disease Check bone marrow aspirate +/- core biopsy Aspirate • One of the most important diagnostic tests in patients with non-regenerative anemias presumed to have primary bone marrow disease • Provides cellular information o Maturation of cell lines o Appropriate response from the bone marrow? MUST do a CBC within 24 hours of a bone marrow! Must know what is going on peripherally so that you can adequately evaluate the bone marrows response o Evaluate for infectious organisms o Evaluate for neoplastic cells Biopsy • Provides information regarding structure of the bone marrow (eg; fibrosis, necrosis, etc) • Often obtain at the same time as bone marrow aspirate, even if don't end up submitting! • After collection, typically do a "roll prep" on a slide and then place core biopsy in formalin o Remember that formalin and aspirate slides should not be stored or shipped close to one another, as the formalin can result in damage/alterations to your cytology sample

Non-Regenerative Anemia Categories 2. Primary (intrinsic) bone marrow __________________________________________________________________ •Thought to be due to Immune mediated destruction of RBC precursors in most cases •Toxins/drugs •Infections (FeLV, FIV) Anemia only! ________________________ -"Idiopathic" -Often secondary to other diseases (eg; toxin, neoplasia) __________________________ -Neoplasia (lymphoma, leukemia, multiple myeloma) -Infection (Ehrlichia canis, Histoplasmosis) ______________ Other cytopenias (usually)

•Anemia of Inflammatory disease (AID) 🌟 •Renal disease •Endocrine disease Iron deficiency anemia • Secondary Bone Marrow Disease Mild to moderate anemia (often MILD) Secondary to other disease processes • Anemia of inflammatory disease (AID) o "Anemia of chronic disease" o Common cause of anemia in dogs and cats o Can result in more moderate anemia in cats • Chronic kidney disease o The more advanced the kidney disease, the more severe the anemia • Endocrinopathies (hypothyroidism, hypoadrenocorticism) usually VERY mild • Iron deficiency anemia o Can remain regenerative for a long time before it becomes truly "non-regenerative." • Vitamin B12 deficiency (due to decreased intestinal absorption of cobalamin or vitamin B12) o RARELY results in clinical anemia in dogs and cats (not uncommon in humans - known as "pernicious anemia") o Specific congenital disorders that result in vitamin B12 deficiency secondary to intestinal cobalamin malabsorption (eg; giant schnauzers) that can result in clinical anemia. Check for underlying disease Many listed above can be determined with a good history, physical examination, and a minimum database (CBC, chemistry panel, and urinalysis) May need to evaluate for specific infectious diseases in some patients • Examples: o ANY anemic cats should have a FeLV/FIV test! o Any cat living in Oklahoma with weight loss, lethargy, and anemia should have a histoplasmosis urine antigen test.

Non-Regenerative Anemia Categories 3. Secondary (extrinsic) bone marrow ________________________ -"Anemia of chronic disease" -COMMON!! -shouldn't cause a bad anemia in dogs but will in cats __________________(decreased EPO production) _________________ -Hypothyroidism (dogs) - low thyroid hormone -Hypoadrenocorticism ("Addison's;" dogs) - low cortisol --need thyroid hormone and cortisol to create RBCs __________________ -Microcytic, hypochromic -Young - parasites (internal or external) -Older - chronic bleeding (eg; GI tract) -Fun fact: Usually have thrombocytosis! -Can be regenerative! Anemia only!

-Slide agglutination 🌟 -Direct Coomb's test-test to detect antibodies on the surface of RBCs -RBC surface antibody test (flow cytometry) -FeLV/FIV (cats!) 🌟 -Vector-borne disease testing -Fecal floatation (blood loss anemia - hookworms!) Non-regenerative Second wave of diagnostics (this will be based on results of above testing) · Infectious disease testing o Dogs § Vector-borne disease testing § Histoplasmosis (if clinically appropriate) § Fecal floatation to evaluate for intestinal parasites o Cats § **FeLV/FIV** -> every anemic cat deserves this! § Histoplasmosis (if clinically appropriate) · Bone marrow aspirate +/- core biopsy o Only in patients suspected to have bone marrow disease (typically) or potentially as staging for neoplasia · Immune-mediated hemolytic anemia o Direct Coomb's test o RBC surface antibody testing (flow cytometry)

Other diagnostics •Immune-mediated hemolytic anemia? ____________________________________________________________________ •Infectious disease? _____________________________________________________________________ •Bone marrow - (Regenerative/Non-regenerative?) anemias (typically) And so many more...............!!

•Pale (pallor) or pale pink (remember doesn't =anemia, it's just reduced perfusion like in a GDV) •Icterus -Usually with hemolytic anemia •Tachycardia •Tachypnea •Bounding pulses •Heart murmur (physiologic) -Occurs with more severe anemia (<25%) -Usually left basilar (systolic) -Usually < grade 2-3/6 •Weakness May be normal! Depends on severity • Physical exam findings in patients with anemia are variable depending on the severity and cause of the anemia. o Weakness -> some of these patients may be so weak that they are unable to walk on their own. o Tachycardia o Tachypnea o Bounding pulses o Pale mucous membranes Remember that pale mucous membranes can also be caused by decreased perfusion (generally hypotension) o Icterus (usually as a component of hemolysis) o Heart murmur -> physiologic due to turbulent blood flow that occurs when blood becomes "thin." Usually left basilar (location), systolic, and < grade 2-3/6. Should resolve when anemia resolves. Anemia should be moderate or severe to result in a heart murmur (typically <25%). Anemia may also increase the grade of a pre-existing heart murmur in a patient. For example, a patient with a grade 3/6 systolic heart murmur secondary to mitral valve degeneration may have an increase in the grade of this heart murmur to a 4/6 or 5/6 with concurrent anemia. o Cats with anemia often have systemic hypotension

Physical Exam

•Guarded to fair -Published mortality rate 20-70% •-Most say 30-50% -Leading causes of death •-Euthanasia ($$, poor prognosis) •-Thromboembolism -Variations in case severity -Intravascular hemolysis, autoagglutination, hyperbilirubinemia (severity), thrombocytopenia (severity), increased ALP PROGNOSIS • Variable - guarded to fair in most patients o Overall mortality rate for primary canine IMHA: 26-70% o Depends on the case, but I usually tell owners ~30% mortality • Factors that may be associated with a worse prognosis: o Autoagglutination o Thrombocytopenia o Leukocytosis with a left shift o Hyperbilirubinemia - higher the value = worse prognosis o Hypoalbuminemia o Increased ALP • Leading causes of death in patients with IMHA o #1: Euthanasia Cost is usually an important factor o #2: Thromboembolism (specifically PTE)

Prognosis of IMHA ____________________________ •Factors associated with worse prognosis ____________________________

Hyperbilirubinemia Intravascular INTRAVASCULAR HEMOLYSIS • Usually IgM-mediated o IgM is better at activating complement o Activation of complement leads to the "membrane attack complex" (MAC) that essentially punches holes into the cell membrane causing osmotic lysis of the RBC • Results in hemoglobinemia and hemoglobinuria (icterus can occur as well) o Keep in mind that hemoglobinemia can result from a hemolyzed sample (eg; unclean blood draw) and is NOT diagnostic for intravascular hemolysis! o Hemoglobinuria is diagnostic for intravascular hemolysis Many causes of intravascular hemolysis that are NOT immune-mediated in etiology. Must be differentiated from myoglobinuria rely on clinical signs and evaluate creatinine kinase (CK) on blood panel (should be moderately and markedly increased in cases of severe myopathies that result in myoglobuinemia and myoglobinuria). o More severe form - associated with worse prognosis o Less common form - 10-20% of IMHA patients DIAGNOSIS of IMHA • Chemistry panel o Hyperbilirubinemia Mild, moderate or severe • Will depend how fast RBCs are being destroyed and how fast bilirubin is able to be cleared by liver • Higher the bilirubin worse prognosis? May see a variety of other abnormalities • Increased ALT and/or ALP - likely associated with hypoxia from anemia • Dec albumin/Inc globulin - inflammatory • Electrolyte abnormalities o Secondary to anorexia, vomiting, diarrhea • Urinalysis o Bilirubinuria +/- hemoglobinuria

Diagnostics for IMHA •Chemistry panel -____________________ •-Can be VERY high •-BOTH extravascular and intravascular -+/- Increased ALT and/or ALP -+/- Electrolyte imbalances •-Anorexia, diarrhea, vomiting •Urinalysis -Bilirubinuria? •-BOTH extravascular and intravascular -Hemoglobinuria? •-(Intravascular/Extravascular?) ONLY

•PCV/TP oSerum icteric (usually) oTP normal •Saline agglutination oMacroscopic evaluation oMicroscopic evaluation §"Cluster of grapes" §Don't confuse with rouleaux ("stacking of coins") •Blood smear?? oRBC morphology

Diagnostics of IMHA ______________________________

•Spherocytosis •Autoagglutination -Usually one or both of these is present •Detection of anti-erythrocyte antibodies -NOT indicated if see autoagglutination! -Coomb's test (Direct antiglobulin test - DAT) •-Many false positives and negatives -Flow cytometry detection of anti-RBC antibody •-Improved sensitivity •-Must be performed within 24 hrs of sample collection • How do you confirm diagnosis of IMHA? o Compatible CBC/Chemistry findings: Macrocytic, hypochromic, regenerative anemia Hyperbilirubinemia o Presence of spherocytes - extravascular hemolysis Spherocytes: small (<2/3 size of a normal erythrocyte), round, densely-stained erythrocyte with lack of central pallor due to phagocytosis of a piece of the cell membrane Extremely difficult to detect in cats because a normal feline erythrocyte does not always have distinct central pallor o Autoagglutination Occurs when an antibodies bound to two or more RBCs cause them to "stick" to one another and are NOT cleared by saline • Macroscopic agglutination o May occur in EDTA tube Rare cases of EDTA associated agglutination that is in vitro change o Saline agglutination tests: 1 drop saline + 1 drop EDTA normal dog should not "clump" • Microscopic agglutination o Evaluate saline agglutination slide under the microscope by using a cover slip o "cluster of grapes" appearance • Must differentiate between microscopic agglutination and rouleaux (artifact; "stacking of coins" appearance) Specific for antibodies bound to RBC surface, which is diagnostic for IMHA o Detection of Anti-erythrocyte antibodies Interpret with caution when the patient has: • Received a blood transfusion - false positive • Received immunosuppressive medications - false negative Do not rely on as the sole diagnostic tests Interpret in combination with clinical signs and other diagnostic test results compatible with IMHA Direct Coomb's Test (Direct antiglobulin test or DAT) • Detects species-specific IgG, IgM, and complement (C3) bound to RBC surface • Agglutination is the endpoint • No value in performing this test if patient has autoagglutination! • Results are reported as positive or negative or as a titer • Sensitivity - ~60-70% o Many false positives and false negatives Flow cytometry detection of Anti-RBC antibodies (K State) o Improved sensitivity compared to Coomb's test False positives and false negatives do occur o Disadvantages Must be performed within 24 hours of sample collection -> send overnight Expense ~$80-100

Diagnostics 🌟 How do you confirm diagnosis of IMHA?

RETICULOCYTE COUNT

Don't forget the _____________!!! Very important in differentiating between causes of anemia

-Autoagglutination -Spherocytes difficult/impossible to detect in cats -Rule-out secondary IMHA before dx primary IMHA -Primary: Prednisolone +/- cyclosporine -Secondary: Treat infection (doxycycline) +/- prednisolone Neonatal isoerythrolysis Type-A blood to a Type-B cat! -More on this later! : )

Feline IMHA •Diagnosis: ____________________ •Treatment: __________________ •________________ -Type-A kittens born to Type-B queen •Blood transfusion reaction -Especially ____________________

T Dogs Cats acute -Macrocytic, hypochromic regenerative anemia •-Most moderate/severe: PCV <25% -Hyperbilirubinemia (extravascular or intravascular) -+/- Hemoglobinemia/hemoglobinuria •-INTRAVASCULAR hemolysis only! HEMOLYSIS (marked regeneration, icteric or hemolyzed plasma, bilirubinemia, normal TP, bilirubinuria and/or hemoglobinuria) ü Check for autoagglutination § Saline agglutination test to assess for IMHA ü Check RBC morphology to try to identify underlying cause § Spherocytes à extravascular hemolysis (IMHA) § Ghost cells à intrascular hemolysis (IMHA) § Schistocytes à microangiopathia (DIC, hemangiosarcoma) § Heinz bodies and eccentrocytes à toxins resulting in oxidative damage § RBC organisms? ü Check Total Bilirubin ü Check thoracic radiographs, abdominal radiographs/ultrasound § Evaluate for secondary causes of IMHA § Evaluate for neoplastic disease that could result in microangiopathic (fragmentation) anemia § Important to evaluate for penny ingestion if concerned about zinc toxicity

Hemolytic anemia T/F? •Many causes of hemolytic anemia that are NOT immune-mediated! •(Dogs/Cats?) > (Dogs/Cats?) •Most cause (acute/chronic?) anemia •Typical CBC changes: ________________________________

-Iron deficiency = microcytic, hypochromic -AID: Inflammatory leukogram? -Other cytopenias? -Renal values (BUN, creatinine) -Hypothyroid: Cholesterol/triglycerides increased? -Addison's: Electrolytes? Albumin?

How do you narrow it down? 1. Process of elimination... •CBC ___________________ •Chemistry panel _____________________

•Anemia alone: 1° or 2° Bone Marrow •Multiple cytopenias: 1° Bone Marrow (usually)

How do you narrow it down? 2. Look at the ENTIRE CBC... ____________________

1=30-37% 2=20-29% 3=13-19% 4= < 13% 5=20-26% 6=14-19% 7=10-13% 8= <10%

How do you narrow it down? 3. Severity of anemia...

Primary (intrinsic) bone marrow Secondary (extrinsic) bone marrow

How do you narrow it down? Severe anemia (generally!) _________________ •Erythroid hypoplasia/aplasia •Myelophthisis •Myelofibrosis •Myelodysplasia _________________ •Iron deficiency anemia -OFTEN regenerative and mild/moderate in beginning!

•Anemia of Inflammatory disease (AID) •Renal disease (decreased EPO production) •Endocrine disease •Iron deficiency anemia -Cats with AID can sometimes have moderate anemia -Severe renal disease stress = high neuts, low lymphs epinephrine/excitement = high neuts and lymphs inflammatory = high neuts (at least 2-3x ref. interval) OR presence of bands

How do you narrow it down? Mild anemia (generally!) Secondary (extrinsic) bone marrow _______________________ Exceptions: _______________________

ALL THINGS GO!

How do you narrow it down?Moderate anemia (generally!)

Bone marrow aspirate 🌟 Bone marrow biopsy

Diagnostics •__________________ -Cellularity •-Maturation of cell lines •-Erythroid/myeloid ratio •-Neoplastic cells? •-Infectious organisms? ---Histoplasmosis •_________________ -Structure -> fibrosis, necrosis, etc Additional notes on procedure on moodle! (FYI)

•Depends on severity! -also depends on how fast they get there •Lethargy •Exercise intolerance •Weakness •Collapse •Pica (mainly cats) •Icterus •Bleeding May be normal! CLINICAL SIGNS OF ANEMIA The anemic patient often presents with non-specific signs of lethargy and/or weakness due to reduced oxygen delivery to tissues. There are other signs that may be specific to categories/types of anemia. Chronic, non-regenerative anemia may result in pica. This is usually due to the need or craving for iron. Dogs or cats may begin to eat dirt or clay-based cat litter. Usually these patients have a more a severe anemia that is chronic. Hemolysis (regardless of whether it is IMHA or another cause of hemolysis) may result in obvious hemoglobinuria and/or icterus that the owner notices. Remember that hemoglobinuria is often misinterpreted by owners as "hematuria" and as a clinician you MUST differentiate between these, as the diagnostic workup for these two conditions is different. Blood loss anemia patient may present for obvious bleeding observed by the owner (eg; epistaxis, urinary blood loss, GI). It is also possible they may present for abdominal or respiratory signs associated with bleeding into a cavity. Remember that the GI tract is a good place to lose a lot of blood without anyone noticing! It is also possible that a patient with a mild anemia or chronic moderate anemia may NOT have clinical signs and may be "normal" at home according to the owner. The anemia in these patients may be found incidentally when performing bloodwork as a component of a geriatric evaluation, pre-anesthetic evaluation, and/or evaluation of other, non-related symptoms. Remember that dogs and cats cannot talk to us, so it is possible that a PCV of 26% is causing a dog to be lethargic, but not enough so that the owner notices a change in their daily activities.

Clinical manifestation of anemia

•Chronic vs. acute? •Progressive? •Feces and urine character? •Concurrent condition(s)? •Medications? •Travel history? •Environment? •Flea/tick/parasite preventative? •Recent vaccination? •Toxin exposure? A complete history should be obtained from the primary caretaker for the patient. Although they should be part of every history, a thorough medication (remember over-the-counter medications and supplements), travel and environment history are extremely important when evaluating an anemic patient.

Complete History

non-regenerative

Consider the severity of the anemia...and how clinical the patient is for the degree of anemia. Remember that ____________ anemia tends to develop slowly because of the long life of RBCs

1=Normal-low 2=Normal-high 3=Common 4=Rare 5=No 6=Common 7=No 8=Common 9=No 10=Occasional

Criteria for Differentiating Blood Loss from Hemolytic Anemias

•PCV/TP •Serum icteric?

Diagnostics

•Neoplasia -Good physical exam! -Thoracic radiographs -Abdominal ultrasound •Drug and vaccine history •Infectious diseases -FeLV/FIV (cats) -Blood smear evaluation -Vector-borne diseases -Urine or blood cultures? -See your notes for specifics! OTHER DIAGNOSTICS - RULE OUT SECONDARY CAUSES of IMHA • Most important in atypical cases o Dogs <2 years >7 years o ANY cat! o Other abnormalities on PE or bloodwork that is atypical for IMHA • Complete history! o Recent drugs, vaccination, supplements, bee sting • Neoplasia o Thoracic radiographs (3-view!) o Abdominal ultrasound o +/- LN aspirate (if enlarged or suspicious of lymphoma) o +/- Bone marrow - usually NOT indicated for IMHA Situations when you might consider performing: • Non-regenerative anemia (AFTER rule-out pre-regenerative) • Bicytopenia or pancytopenia (without evidence of regeneration) • Abnormal cells in circulation (eg; lymphoblasts) • Refractory to standard therapy • Infectious o Consider diseases likely in your geographical region and travel history o Vector-borne diseases 4DX Plus Snap Test - Heartworm antigen, Ehrlichia spp antibody, Anaplasma spp antibody, Lyme (Borrelia burgdorferi) antibody Serology ("tick titers") +/- PCR o Oklahoma DOG diseases to consider Rocky Mountain Spotted Fever • Acute disease - dead or better in 2-3 weeks! • Must do convalescent serology • Blood PCR not useful - organisms in endothelium Ehrlichia ewingii and E. canis • Blood smear evaluation - RARELY find morulae in neutrophils (ewingii) or monocytes (canis) • 4DX, quantitative serology, blood PCR o Exposure can result is VERY high titers! Babesia spp. • Babesia gibsoni (Pit-bull babesia) more commonly causes IMHA • Thorough blood smear evaluation (piroplasms in RBCs) o Improved detection from small distal veins (eg; ear vein) • Serology, Blood PCR Bartonella spp? • Difficult to diagnosis and treat! • Consider a combination of serology, PCR, and specialized BAPGM culture • Associated with MANY diseases, but debate among clinicians whether it is primary or secondary pathogen Leptospirosis • Primarily associated with renal and liver disease • Potential to induce ITP and/or IMHA - UNCOMMON • Urine or blood PCR and/or serology (MAT) Mycoplasma hemocanis (dogs) • Uncommon; splenectomy is risk factor • Blood smear evaluation (RBC assoc organism) o Oklahoma CAT diseases to consider • Mycoplasma hemofelis and M. haemominutum o Thorough blood smear evaluation! o Blood PCR o Response to treatment • Cytauxzoon felis o Seasonal: April -> September o Fever, often pancytopenia, systemic disease that often results in death o Blood smear evaluation Piroplasms within RBC Schizonts in mononuclear cells o Blood PCR o IMHA alone can occur in "carrier" cats that have survived schizont phase of disease • Feline Leukemia (FeLV) o Should be performed in EVERY anemic cat! o FeLV antigen (FeLV/FIV combo test) o FeLV IFA on bone marrow sample • Feline Immunodeficiency Virus (FIV) o FIV antibody (FeLV/FIV combo test) Does not differentiate between vaccination and infection o Thorough evaluation for standard bacterial infections Complete history and PE! • Joint or back pain? • New heart murmur? Urine culture +/- Blood cultures o What about histoplasmosis??? Histo does lots of things, but should not cause IMHA. Can cause non-regenerative anemia if invade bone marrow and blood loss anemia if causes GI bleeding from GI infiltration

Diagnostics Evaluate for secondary causes of IMHA

•FeLV IFA or PCR on bone marrow sample •Direct Coomb's test or RBC surface antibody

Diagnostics ____________________ -"Focal" or "latent" FeLV infection -PCR on blood too! _____________________________ -Peripheral blood -Sometimes perform this test in dogs that are suspected to have immune mediated destruction of RBC at the level of the bone marrow...

Complete blood count (CBC) MCV (macrocytic=reticulocytes high) MCHC (macro chromic=lots of hemoglobin=cell color) Reticulocyte count 🌟 DIAGNOSTIC APPROACH TO ANEMIA The diagnostic approach in an anemic patient should ALWAYS include full CBC including RBC indices, blood smear evaluation (ideally by clinical pathologist or at minimum a trained technician) and reticulocyte count. The importance of a reticulocyte count cannot be emphasized enough, as this may not be included in a CBC (lab dependent). o Initial diagnostic evaluation: · PCV and Total protein o Provides lots of valuable information in 5 minutes! § Evaluate severity of anemia § Whether or not total protein is also decreased, which would potentially point you more in the direction of whole blood loss § Is serum icteric? Might point you more toward hemolysis · Blood smear o This is particularly important if you are sending out a CBC and may not receive a result for 24-48 hours. o Evaluation of RBC morphology to help determine if there is evidence of regeneration (polychromasia, nRBC), immune-mediated destruction (spherocytes), RBC organisms (eg; babesia spp., mycoplasma spp.), etc. · Saline agglutination if concerned about IMHA o Assess for both macroscopic and microscopic autoagglutination First wave of diagnostics: · CBC o Making sure to characterize the anemia § Macrocytic, normocytic, or microcytic § Normochromic, hypochromic · Remember that an increased MCHC ("hyperchromic") is NEVER an actual change, but may occur as a false increase from hemolysis § Regenerative or non-regenerative o Platelet count § If thrombocytopenic, consider whether this may be related to primary bone marrow disease, infectious disease, and/or if severely decreased, perhaps the cause of the anemia (ie; cause of bleeding) § Thrombocytosis is common in patients with iron deficiency anemia o Leukogram § Assess for inflammatory leukogram § Patients with IMHA and infectious disease may have a moderate or even markedly increased WBC count. § Lymphocytosis is often caused by lymphoid neoplasia (leukemia/lymphoma) in dogs. Less commonly, we occasionally see dogs with vector-borne disease that have a mild lymphocytosis. Cats often have a lymphocytosis with stress. § If leukopenic, consider primary bone marrow disease or toxin/drug reaction.

Diagnostics ____________________ -PCV and/or hematocrit -RBC indices -•_____________ - how big are the RBCs? -•_____________ - how much hemoglobin is in the RBCs? -RBC morphology -______________________ -•May have to request separately -Other cytopenias?

Chemistry panel Urinalysis First wave of diagnostics: · Chemistry panel o Assess total protein (typically low normal or decreased in blood loss anemia) o IMHA often results in hyperbilirubinemia o Assess for kidney disease and endocrinopathies o Other disease processes (eg; liver disease) · Urinalysis o Pigmenturia? Bilirubin and/or hemoglobin o USG -> may help to confirm renal azotemia, but otherwise may not be that helpful

Diagnostics •_______________ -Total protein (should always do with a PCV!) -Bilirubin -Kidney values -Electrolytes •__________________ -Bilirubinuria? -Hemoglobinuria? -USG?

ü Check total protein § Often decreased or low-normal in patients with blood loss anemia, because also losing plasma. Exception would be chronic blood loss, as the body is able to compensate and increase plasma protein production. ü Check thoracic radiographs, abdominal ultrasound, coagulation panel, platelet counts as indicated § Identify where patient is bleeding § Identify WHY patient is bleeding! · Importance of platelet count and coagulation panel (PT/PTT) ü Iron deficiency anemia § Microcytic (low MCV), hypochromic (low MCHC) § May or may not be regenerative

REGENERATIVE ANEMIA BLOOD LOSS (Moderate regeneration)

DOGS 🐶

Spherocytes ((DOGS/CATS?)!)

T Clinically, we rarely think in terms of RBC count or hemoglobin concentration. As a general rule, hemoglobin conc x 3 usually equals the Hct or PCV.

T/F? Rarely think in terms of RBC count or hemoglobin concentration

T

T/F? There are many causes of hemolytic anemia that are NOT immune-mediated.