Immune-Mediated Hemolytic Anemia (IMHA) - Part 1 & 2 - VETM 5291

Immune-mediated hemolytic anemia - part 1 VETM 5291 Cardio-Respiratory-Hemolymph System Learning objectives Students will be able to Recognize the clinical signs of anemia Use clinical signs and diagnostic algorithms to differentiate between 3 mechanisms of anemia – loss/hemo...

Immune-mediated hemolytic anemia - part 1 VETM 5291 Cardio-Respiratory-Hemolymph System Learning objectives Students will be able to Recognize the clinical signs of anemia Use clinical signs and diagnostic algorithms to differentiate between 3 mechanisms of anemia – loss/hemorrhage – destruction/hemolysis – decreased production Diagnose and treat immune-mediated hemolytic anemia Outline Algorithms – pallor, anemia Clinical signs CBC changes – regenerative anemia – hemorrhage – hemolysis IMHA  Pallor Pale mucous membranes Anemia Shock/hypoperfusion ↓ PCV, CRT < 2 secs Normal PCV, CRT > 2 sec Cardiogenic Regenerative Non-cardiogenic Hemolysis Hemorrhage Hypovolemia Dehydration, third spacing Non-regenerative Distributive Sepsis, SIRS, anaphylaxis Erythropoietin Bone marrow Obstructive depression pathology Pericardial effusion, PTE  Anemia ↓ PCV, CRT < 2 secs Regenerative Non-regenerative Reticulocytosis Normocytic, normochromic Polychromasia Macrocytic, hypochromic Erythropoietic depression Hemorrhage Hemolysis Bone marrow Iron transport ↓ PCV and TS ↓ PCV, normal TS affected pathology Other cell lines likely affected Clinical signs Degree of anemia Chronicity Changes in circulating volume Concurrent disorders Extent of physical activity Hypoxemia Lethargy Weakness and collapse Increased heart rate Mild-moderate heart murmur – grade I-III, hemic, altered blood viscosity Weak pulses Hypotension Regenerative anemia Red blood cell indices or parameters – size – mean cell volume – Hb content – mean cell Hb concentration, MCHC Macrocytosis or anisocytosis Polychromasia or hypochromasia Nucleated red blood cells Regenerative anemia – hemorrhage Initially normocytic, normochromic, non-regenerative Erythropoietin concentrations increase Regenerative response in 3-5 days, maximal 4-7 days ± Leukocytosis with left shift Anemia Panhypoproteinemia (albumin and globulin) Reticulocytosis Canine reticulocytosis – interpret in context of severity of anemia – mild 110-150K, moderate 150-300K, marked > 500K – (older references intervals were > 80K) External blood loss GI tract - melena if upper GI bleed – endoparasites, NSAIDs, Addison’s, IBD, neoplasia Urinary tract – trauma, neoplasia, idiopathic renal hematuria Respiratory tract – epistaxis can be swallowed, hemoptysis Skin – Ectoparasites Young animals often classified as anemic – adult reference interval; milk diet, growing, endoparasites Chronic external blood loss Microcytic, hypochromic – loss of iron decreases Hb concentration in RBC precursors – erythrocytes undergo extra division Eventually becomes non-regenerative Treat underlying cause Iron supplement – oral or IM injection, ouch! Internal blood loss Body cavities – thoracic, abdominal – pericardium, joints, CSF, subcutis Coagulopathies – congenital, hemophilia – acquired, rodenticide toxicosis – trauma – neoplasia Iron recycled and not depleted Extravascular hemolysis Normal destruction of senescent erythrocytes – spleen, bone marrow, liver Also triggered by – antibody attachment – blood parasite attachment – oxidative damage – intrinsic defect – membrane fragility Non-immunologic hemolytic anemia Microangiopathic HA Phospholipases Heartworm disease Snake venom, spider bites, bee stings Hemangiosarcoma Schistocytes Oxidative stress Onion or garlic Zinc RBC intrinsic defect/fragility ‒ pennies after 1983 RBC enzyme deficiency ‒ sunscreen ‒ phosphofructokinase Heinz bodies ‒ pyruvate kinase Osmotic fragility Hypophosphatemia ‒ refeeding syndrome or DKA Outline Algorithms – pallor, anemia Clinical signs CBC changes – regenerative anemia – hemorrhage – hemolysis IMHA Immune mediated hemolytic anemia Pathophysiology Diagnosis Differentiating primary vs secondary IMHA Treatment – acute stabilization – anti-thrombotic and immunosuppressive drugs – splenectomy/therapeutic plasma exchange Precursor IMA What must go wrong for autoimmune hemolytic anemia to occur? Recognition of erythrocyte self-antigen Loss of immunological tolerance Diagnostic approach to IMHA 1. Diagnose anemia 2. Confirm that regenerative and/or hemolytic [Exception = precursor immune-mediated anemia] [Exception = feline anemia of immune-mediated origin] 3. Rule out secondary or associative causes 4. Primary or non-associative IHMA is a diagnosis of exclusion  Summary - pathogenesis of IMHA Predisposing factors Neoplasia Infection, vaccine, drug e.g. genetics, age, environment B Immune T Immune response dysregulation T B Anti-drug or microbe Ab Primary / Secondary / Non-associative Associative Autoreactive Ab RBC Day M. in Schalm’s Veterinary Hematology (6th ed.) 2010: 216-225 Drugs Hapten effect Penicillins, e.g. Clavamox Cephalosporins, e.g. Cephalexin Trimethoprim sulfa Methimazole Vaccines?  J Vet Intern Med 1996;10:290-295 Temporal relationship between vaccination and IMHA Correlation ≠ causation Study failed to confirm association between vaccination and ITP However, underpowered study – sample size was small Recommend performing titers to see if vaccines should be given Legal obligation to recommend rabies vaccines Pathogenesis of IMHA Extravascular Intravascular Extravascular hemolysis + Opsonization of erythrocytes → spherocytes Extravascular hemolysis Heme metabolized and excreted in canine kidneys Kidneys can excrete conjugated bilirubin Small amounts of urinary bilirubin – normal in healthy dogs Bilirubinuria is not normal in cats IMHA - excess hemoglobin enters bilirubin pathway – pre-hepatic cause of hyperbilirubinemia – icterus or jaundice, bilirubin > 1.5-2.0 mg/dl Intravascular hemolysis Activation of complement Hemolyzed erythrocytes → hemoglobemia → hemoglobinuria Why this matters – typically worse prognosis IMHA Pathophysiology Diagnosis Differentiating primary vs secondary IMHA Treatment – acute stabilization – anti-thrombotic and immunosuppressive drugs – splenectomy/therapeutic plasma exchange Precursor IMA History Diet – onion or garlic-containing foods? Medications – including flea and tick preventatives Phospholipases can cause intravascular IHMA – bee stings, spider bites, snake venom Criteria for IMHA 1. PCV < 25% and 2. Spherocytes [dogs only] or 3. Autoagglutination or 4. Positive Coombs’ test Autoagglutination Macroagglutination – hemaggglutination visible on blood tube – saline agglutination test (1 drop saline: 1 drop blood) Microagglutination – visible when slide examined under microscope – tap slide to differentiate from rouleaux Direct/Serum antiglobulin test (DAT/SAT) or Coombs’ test Anti-canine immunoglobulin + YY antibody Patient RBCs + autoantibodies Agglutination Caviezel et al. JVIM 2014;28:583-591 Flow cytometry Side scatter (cell granularity) Relative cell number Negative control Positive Anti-canine signal globulin Ab “Gating” Forward scatter (cell size) Anti-dogs Ab fluorescence Weiss D. JVIM 2007;21:440-4. Weiss D. Vet Clin Path 2008;31:72-80. Diagnostic testing – patient side MM color, CRT, pulse quality, and blood pressure PCV/TS – hemorrhage vs hemolysis – serum icteric or hemolyzed? Blood smear – polychromasia, spherocytosis (dogs), nRBCs, hemoparasites – evaluate WBCs and platelets too! Saline agglutination test Diagnostic tests CBC – RBC indices, reticulocytosis, WBC, platelets Chemistry – albumin/globulin – hemolysis vs hemorrhage – renal parameters – chronic renal disease – liver disease (hepatic and post-hepatic hyperbilirubinemia) – Na, K for Addison’s disease – P for refeeding syndrome Diagnostic tests Urinalysis – USG – renal failure? – bilirubinuria – pigmenturia hemogloburinia hematuria (spin down) myoglobinuria (rare in SA) – sediment exam neoplasia? IMHA – primary or secondary? Screen for neoplasia (and rule out zinc FB) ‒ thoracic and abdominal radiographs ± abdominal ultrasound or CT ± ECG and echocardiogram Rule out infectious disease ‒ antibodies/titers/serology (serum) ‒ PCR, antigen within cells/plasma Infectious disease testing Blood smear – organisms not always visible Serology Mycoplasma hemocanis, Babesia not typical rickettsiae on a 4Dx SNAP test Leptospira Snap tests – FeLV antigen/FIV antibody Mycoplasma hemofelis PCR assay – Mycoplasma hemofelis and canis, Babesia, Leptospira – fresh feline bone marrow for occult FeLV Poor prognostic factors ↑ BUN – pigment nephropathy, GI bleed Icterus – indicates severity of hemolysis Band neutrophils, petechiae – severity of systemic inflammatory response IMHA also seen with other immune-mediated diseases – Evan’s disease = IMHA and immune-mediated thrombocytopenia (ITP) – systemic lupus erythematous, SLE IMHA Pathophysiology Diagnosis Differentiating primary vs secondary IMHA Treatment – acute stabilization – anti-thrombotic and immunosuppressive drugs – splenectomy/therapeutic plasma exchange Precursor IMA Volume support Crystalloids – deficiency + ongoing losses + maintenance – canine maintenance ~60 ml/kg/day – feline maintenance ~45 ml/kg/day – rate of replacement reflects disease timeline – also helps kidneys with pigment nephropathy (bilirubin/Hb) Blood products Only if indicated Use clinical signs (HR, RR) not absolute PCV % – although I do use ~15% as a trigger Ideally type canine donor and recipient for 1st transfusion – do not have to do this in emergency Should crossmatch for next transfusion Always type feline donor and recipient for 1 st transfusion Canine transfusions Incompatible 1st transfusion Donor Patient Sensitization + _ = DEA 1.1+ Y Subsequent transfusion Hemolysis + _ = DEA 1.1+ Y Small animal blood typing Visible hemagglutination – patient RBC surface antigen – known monoclonal or polyclonal antisera Methods – card and ELISA Type A Type B If autoagglutination already present …? Sandwich or indirect ELISA Lateral flow test – similar to home pregnancy or COVID test http://alvedia.com/en Blood transfusions Screen donor for blood borne infectious diseases Use filter to remove microclots Give blood product over 4-6 hours Monitor for transfusion reactions – HR, RR, temperature, BP – fever, facial swelling, vomiting, tachypnea – hemolysis Reading IMHA review article Lecture slides ECLINPATH – anemia Immune-mediated hemolytic anemia - part 2 VETM 5291 Cardio-Respiratory-Hemolymph System IMHA Pathophysiology Diagnosis Differentiating primary vs secondary IMHA Treatment – acute stabilization – anti-thrombotic and immunosuppressive drugs – splenectomy/therapeutic plasma exchange Precursor IMA Anti-thrombotic drugs Dog with IMHA die of thromboembolic complications Typically, in first 1-2 weeks of disease Mortality rates of 50+% Anti-thrombotic drugs – clopidogrel (anti-platelet), 1-2 mg/kg PO q 24h – aspirin (anti-platelet), low dose PO since giving with steroids, mixed efficacy – rivaroxaban (anti-Xa factor), parenteral and PO, $$$ but generic available soon – heparin, parenteral When to stop??? After 2 weeks … or when d/c steroids Piek et al, JVIM 2008; 22:366 Corticosteroids e.g. prednisone Genomic and non-genomic mechanism of action Affects multiple components of immunity – lymphocytes, macrophages, complement, neutrophils, NK cells … Very efficacious, work quickly Cheap Rhen & Cidlowski NEJM 2005;353:1711-1723 Prednisolone Prednisone converted to prednisolone in the liver Prednisolone (theoretically) preferable in – cats – 30% cannot metabolize prednisone – patients with hepatic disease/dysfunction Little more expensive that standard prednisone Prednisone or prednisolone dosing per day Dose = mg/kg Don’t exceed 60 mg total/dog 2-4 0.5-1 Immunosuppressive 0.2-0.4 Anti-inflammatory Physiologic Treating immune-mediated Short course for disease Maintenance flea allergy for Addisonian dermatitis patient Corticosteroids - dosing Alternate doing is 40 mg/m2 if > 25-30 kg Weight 2 mg/kg Max of 60 mg/d 40 mg/m2 10 kg 20 mg - ~ 20 mg 30 kg 60 mg 60 mg ~ 40 mg 50 kg 100 mg 60 mg ~ 55 mg Aim for “remission” or PCV > 30% for 2 weeks then – taper 25-50% every 2 to 4 weeks – discontinue once dose 0.25 – 0.5 mg/kg q 48h – complete withdrawal should take months McCullough. Vet Clin North Am 2003: 33: 1295-1315 Ettinger (Garden, O.) 2010; www.expertconsult.com Taper 25-50% every 2- 4 weeks 10 kg dog starts at 2 mg/kg/day = 20 mg Taper 50 % q 2 weeks Total prednisone mg/day Taper 25-33 % q 4 weeks Total prednisone mg/day Weeks 1-3 20 (2mg/kg/d) Weeks 1-4 20 (2mg/kg/d) Weeks 4-5 10 (1 mg/kg/d) Weeks 5-8 15 Weeks 6-7 5 (0.5 mg/kg/d) Weeks 9-12 10 Weeks 8-9 2.5 (0.25 mg/kg/d) Weeks 13-16 7.5 Week 10 2.5 every other day Weeks 17-20 5 Stop at 2.5 months Weeks 21-24 2.5 Weeks 25-26 2.5 every other day Weeks 27-28 2.5 every third day Stop at 6.5+ months Taper 25-50% every 2- 4 weeks 10 kg dog starts at 2 mg/kg/day = 20 mg Taper 50 % q 2 weeks Total prednisone mg/day Taper 25-33 % q 4 weeks Total prednisone mg/day Weeks 1-3 20 (2mg/kg/d) Weeks 1-4 20 (2mg/kg/d) Weeks 4-5 10 (1 mg/kg/d) Weeks 5-8 15 Weeks 6-7 5 (0.5 mg/kg/d) Weeks 9-12 10 Weeks 8-9 2.5 (0.25 mg/kg/d) Weeks 13-16 7.5 Week 10 2.5 every other day Weeks 17-20 5 Stop at 2.5 months Weeks 21-24 2.5 Weeks 25-26 2.5 every other day Weeks 27-28 2.5 every third day Stop at 6.5+ months Dexamethasone Injectable formulation, if can’t take oral medications x7-10 times as potent as prednisone Must decrease dose – 10 kg dog – 2 mg/kg prednisone > 20 mg prednisone – 0.2 mg/kg dexamethasone > 2 mg dexamethasone Feldman & Nelson. Canine and Feline Endocrinology & Reproduction ,3rd ed. 2004:482. Corticosteroids – adverse effects Iatrogenic hypercortisolemia / Cushing’s disease “Acceptable” – polyuria/polydipsia – polyphagia – alopecia, muscle wasting – hepatomegaly, panting Aim for once daily dosing – ↑owner compliance, ↓ nocturia 1. Torres et al. JAVMA 2005; 227: 239-43  Corticosteroids – adverse effects “Unacceptable” – GI bleeding - NEVER combine steroids with NSAIDs in dogs and cats – utility of gastroprotectants, e.g. proton pump inhibitor = omeprazole? – give steroids with meal Torres et al. JAVMA 2005; 227: 239-43 CBC changes with IMHA and corticosteroids IMHA + high dose steroids – often cause a leukemoid response – extreme leukocytosis, e.g. dog > 60 x 103/μl Prednisone medication = hypercortisolemia Seg neutrophils – expect a stress leukogram Monocytes Increase Important to monitor CBC Lymphocytes Eosinophils – expect left shift to ↓ with time Decrease – do not expect to see toxic change infection in immunosuppressed dog Chemistry changes with corticosteroids ALKP increases in dogs – steroid isoenzyme and cholestasis due to hepatocyte glycogen accumulation – do not expect an increased ALKP in cats treated with corticosteroids – cats do not have this isoenzyme and ALKP half life is very short Hyperlipemia, hypercholesterolemia Hyperglycemia (glucocortocoids) Urinalysis changes with corticosteroids Decreased urine specific gravity Pollakuria - increased frequency If urinary tract infection ± bacteria – very dilute urine ± pyuria (WBCs), hematuria (RBCs) – anti-inflammatory action  Corticosteroids – “unacceptable” adverse effects Short term – infections, e.g. skin, urinary tract – soft tissue catabolism - decreased wound healing, ligament rupture – aggression – Na retention – cardiac dz; care in cats! – insulin resistance – diabetics; care in cats > dogs! Long term – uroliths, e.g. calcium oxalate – gall bladder mucoceles – calcinois cutis Torres et al. JAVMA 2005; 227: 239-43 Using immunosuppressive drugs Start with prednisone – 1 mg/kg PO q 12h (dogs), or 2 mg/kg q 24h – 2mg/kg PO q 24h (cats) When to use an adjunctive therapy – to spare prednisone effects in (large) dogs – IMHA with autoagglutination – intravascular IMHA – to spare prednisolone in cats (risk of diabetes mellitus) Cyclosporin 5mg/kg PO q 12-24h – mainly affects T lymphocytes function – binds calcineurin and stops clonal expansion – days to effect, $$$ although generic available – microemulsified for predictable absorption – liquid and oral capsules forms available – adverse effect: GI, vomiting and diarrhea – put in freezer, give with food or anti-emetics to control GI signs – dogs: gingival hyperplasia and hirsuitism – cats: Toxoplasma reactivation Mycophenolate mofetil 10 mg/kg IV or PO q 12h – inhibits purine synthesis in B + T lymphocytes – parenteral (injectable) and oral forms available – days to effect – $$$ – adverse effects mainly GI, vomiting and diarrhea – decrease with time, probiotics, ± metronidazole if very severe Azathioprine 2mg/kg PO q 24h for 14 days then q 48h – purine antagonist – delayed clinical efficacy (3+ weeks) – $ – but hepatotoxic and myelosuppressive – should monitor CBC and liver enzymes q 2-4 weeks – 50mg tablets may need compounding – tell owner to wear gloves when administering, teratogenic – DO NOT USE IN CATS X  Adjunctive immunosuppressive drugs Drug Dog and/or cat? Mechanism of action Dose, route Adverse effects Contraindications #1 #2 #3 #4 Which adjunctive immunosuppressive? Adjunctive drug – consider comorbid disease(s) and metabolism routes – more is not necessarily better! – often dictated by $, patient size, adverse effects Formulation? – parenteral (injectable) helpful when oral meds can’t be used Efficacy – generics cheaper – compounding can help dosing BUT beware efficacy Tapering immunosuppressive drugs Reassess PCV/Hct before taper One drug at a time, unless fulminant infection – typically prednisone first due to side effects – if $ more of an issue, adjunctive drug first – 25-50% every 2- 4 weeks Can decrease steroid to physiologic dose if an emergency – e.g. 0.25 mg/kg day Ettinger. 2010 www.expertconsult.com Tapering immunosuppressive drugs Once prednisone ~0.5 mg/kg/d, or stopped Then taper adjunctive drug, 25-50% q 2- 4 weeks, or as formulation allows For 20 kg dog Week Prednisone mg/day Mycophenolate (AM) Mycophenolate (PM) 0-3 40 (2 mg/kg/d) 100 mg 100 mg 4-5 30 mg 100 mg 100 mg 6-7 20 mg 100 mg 100 mg 8-9 15 mg (0.5 mg/kg/d) 100 mg 100 mg 10-11 10 mg (0.5 mg/kg/d) 100 mg 100 mg 12-13 Maintain 100 mg 50 mg Ettinger. 2010 www.expertconsult.com Tapering immunosuppressive drugs If disease relapses – add in second immunosuppressive drug if only on steroid monotherapy – go back up one step – taper more slowly - decrease mg taper increments and/or increase interval Week Prednisone mg/day Mycophenolate (AM) Mycophenolate (PM) 0-3 40 (2 mg/kg/d) 100 mg 100 mg 4-5 30 mg 100 mg 100 mg 6-7 20 mg 100 mg 100 mg 8-9 15 mg (0.5 mg/kg/d) 100 mg 100 mg 10-11 10 mg (0.5 mg/kg/d) 100 mg 100 mg 12-13 Maintain 100 mg 50 mg Ettinger. 2010 www.expertconsult.com Using immunosuppressive drugs If use a second drug to spare effects of prednisone – then taper pred more rapidly than if monotherapy! Remember concurrent disease – might influence drug handling (hepatic or renal) – diabetes mellitus, cardiac disease, osteoarthritis and NSAIDS These are NOT benign medications – monitor for hematological + biochemical effects etc. – the secondary infection(s) may kill your patient, not the IM dz – ask the owner to wear gloves, teratogenic Immune mediated hemolytic anemia Pathophysiology Diagnosis Differentiating primary vs secondary IMHA Treatment – acute stabilization – anti-thrombotic and immunosuppressive drugs – splenectomy/therapeutic plasma exchange Precursor IMA Alternate therapies for IMHA Splenectomy – salvage option for unresponsive cases – I don’t triple immunosuppress patients – screen for tick-borne disease, especially Babesia since risk of reactivation! – decreased wound healing – modify anti-platelet drug doses in peri-operative period Therapeutic plasma exchange – removes antibodies – specialized equipment, $$$$$ Precursor-targeted immune-mediated anemia Confusing exception to the rule Immune response is aimed at RBC precursors ⎼ so anemia is non-regenerative Anemia ↓ PCV, CRT < 2 secs ⎼ spherocytes and agglutination much less common ⎼ may need a bone aspirate/biopsy to diagnose Regenerative Hemorrhage Hemolysis Anemia is chronic ⎼ patients can be stable with severe anemias, < 10%! Non-regenerative Erythropoietin Bone marrow depression pathology Assenmacher et al. 2019 JAVMA; 255: 366 Precursor-targeted immune-mediated anemia Treatment similar to “classic” IMHA 1. Blood products and supportive care 2. Anti-platelet drugs 3. Immunosuppression May take months to respond, transfusion dependent – patients often euthanized because of $ or drug adverse effects – splenectomy may be an effective alternate treatment beware impact of steroids on wound healing must stop anti-platelet drug ~ 3-7 days before surgery Assenmacher et al. 2019 JAVMA; 255: 366