Understanding anemia causes, diagnosis and treatment

(non-regenerative anemia)

Asst. Prof. Phudit Maneesaay, DVM., MA., DTBVP

Department of Pathology, Faculty of Veterinary Medicine

Kasetsart University

Non-regenerative anemia

• Slow progressive disorder with secondary adaptive response

• Mild clinical signs and no hypoxia, pale pink to pink membrane

• Normocytosis, normochromasia (normal MCV, MCHC)

• PCV 20-25%, no increased RDW

Red cell production• Rubriblast: unipotential progenitor cell

sitmulate by CFU-E, five division produce 8-32 differential cells

• Prorubricyte: progenitor cell that have limit capacity to self- renewal and differentiation

• Rubricyte: no capacity to self-renewal but proliferate while differentiation into mature cell (3 division)

• Metarubricyte: large state of red cell that have nucleus

• Reticulocyte: remain in marrow 2-3 day before mature in peripheral blood

• Erythrocyte

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Reticulocyte

• Immature RBC that contained residual amounts of mRNA• Larger than mature RBC (8-10μm), anucleated cells with basophilic

reticulum, no nucleoli and have large amount of blue-pink staining hemoglobin cytoplasm• Reticulocyte count: an index of bone marrow response or effective

erythropoiesis

Reticulocyte???MCV, MCHC, RDW, (Retic)

Reticulocyte count by mixed equal part of blood or EDTA blood in new methylene blue or brill, incubate at room temperature for 15 minutes, smear and count (X100) for 500-1000 red cells = reticulocyte (%)

• Corrected reticulocyte percentage (CRP)

• The corrected reticulocyte percentage compensates for the degree of anemia, but it makes assumptions as to what the normal hematocrit of the patient is (45% for a dog and 35% for a cat). This is useful in practice, where a packed cell volume (PCV) can be readily obtained from an animal, in-house

• CRP = reticulocyte % x (patient’s HCT ÷ normal HCT)

• Reported reference intervals for CRP are < 1% in the dog and < 0.4% in the cat

- Absolute reticulocyte count (ARC)ARC = Ret % (a/100) X RBC count

DOG Aggregated retic (%) CAT

NONE < 93,000 < 1 < 62,000

Mild 110,00-150,000 1.0-2.5 80,000-100,000

MODERATE 150,000-300,000 2.5-5.0 100,000-200,000

MARKED >500,000 > 5.0 > 200,000

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Non-regenerative anemia: classification

• Pre-regenerative condition• acute erythrocytes loss with acute and marked clinical signs: tachypnea,

weakness and exercise intolerance relative to degree of anemia

• Primary marrow disorders• Aplastic anermia, leukemia, neoplasia, pure red cell aplasia, myelofibrosis,

myelodysplastic syndrome, FeLV infection in cat

• Secondary to inflammatory or metabolic diseases• Anemioa of inflammatory disease, anemia of chronic renal failure, anemia of

endocrine diseases

• Anemia due to nutritional deficiencies

Non-regenerative anemia caused by decrease marrow production• Insufficiency production or activity of erythropoietic cytokine:

chronic kidney diseases• Suppression of erythropoiesis: drugs, cytokines, tumors or immune

cells• Deficiency of minerals/vitamins/nutrients: iron, copper, folate,

vitamin B12, malnutrition• Defective hemoglobin synthesis: secondary to iron deficiency• Defective DNA synthesis or nuclear maturation: dietary deficiency,

drugs, myelodysplastic syndrome

• Destruction of marrow erythropoietic cells: immune-mediated, drugs, neoplasia, infectious, ischemia

• Replacement of hematopoiesis: neoplasia, myelophthisis• Drugs: Actinomycin D, amphoteracin B, azidothymidine, busulfan,

carboplastin, cephalosporin, chloramphenicol, chlorambucil, cyclophosphamide, cytarabine, doxorubicin, estrogen, fenbendazole, griseofulvin, meclofenamic acid, methothexate, nitrosureas, phenobarbital, phenothiazine, propylthiouracil, quinidine, rhEOP, thiacetarsamide• Toxins: Benzene, tricholoethylene toxicity• Heavy metals: Lead poisoning

Non-regenerative anemia caused by decrease marrow production

Clues to identify the causes of non-regenerative anemia

• Clinical history• Presence of underlying diseases: hepatopathy, CKD, neoplasms,

endocrinopathies• Severity of anemia

• Mild to moderate: Anemia of inflammatory disease (ACD)• Severe: non-regenerative immune-mediated anemia (PIMA/PRCA)

• Red blood cell indices: normochromic, normocytic and/or hypochromic, microcytic anemia

• Other cytopenia: Neutropenia, thrombocytopenia in primary marrow diseases (immune-mediated, acute leukemia, infiltrative neoplasia, infection of E.canis)

• Abnormal cells: myelodysplatic syndromes, acute leukemia

Non-regenerative anemia: Extra-marrow diseases

• Chronic kidney disease: severe normocytic, normochromic non regenerative anemia with significant clinical signs • Decreased erythropoietin production, increased hepcidin, suppression of

erythropoiesis, decreased RBC lifespan, hemorrhage and malnutrition

• Endocrine diseases• Cortisol and thyroxin enhance effect of erythropoietin• Hypothyroidism: mild anemia • Hypoadrenocorticism: mild to moderate anemia but may be masked by

hemoconcentration• More severe in secondary GI bleeding

Non-regenerative anemia: Extra-marrow diseases

• Anemia from nutritional deficiencies: deficiency of protein, energy, vitamin B and minerals• Iron deficiency is the most common• Deficiency of folate and cobalamin: macrocytic normochromic anemia

• Cobalamin deficiency: dog: autosomal recessive disorders in Giant Schnauzers, Border Collies, Shar Peis and Australian Shepherds resulting in ileal absorption defects with mild to moderate non-regenerative anemia, increased RDW, some macrocyte and neutropenia

• Cobalamin deficiency: cat: exocrine pancreatic insufficiency and severe ileal disease

• Non-regenerative anemia associated with feline viral infections• Various mechanisms: leukemia, myelodysplasia, aplastic anemia,

secondary pure red cell anemia and IMHA, FeLV related immunosuppression leading to anemia of chronic disease

Non-regenerative anemia: Extra-marrow diseases Anemia of chronic disease and iron deficiency anemia

• Anemia of chronic inflammatory disease (AID):• Mild to moderate normocytic normochromic non-regenerative anemia• Anemia within 10 days of inflammatory disorders: chronic infection,

tissue trauma, tissue necrosis secondary to malignant neoplasms• Iron deficiency anemia (IDA): • Inadequate hemoglobinization of red cells and release of microcytic

hypochromic red cells in circulation• Low MCV, MCHC with increased RDW, persistent thrombosis and

hypoproteinemia• Leptocytes, target cells (codocytes), schistocytes

Anemia from chronic diseases/ chronic inflammatory diseases (ACD/ ACID)• Shortened red cell life span: moderate reduction• Increased oxidative damage or binding of IG to red cells• Activation of macrophages by TNF-α, resulting in

erythrophagocytosis• Inhibition of iron metabolism by hepcidin via production of IL-1, IL-6• Host immune response limit microbial access to iron• Inhibit gastrointestinal iron absorption• Retention of iron within macrophages

• Impairment of bone marrow function by IL-1 or TNF• Impaired proliferation and differentiation of erythroid precursors

by altered responsiveness to EPO• Cytokine-mediated induction of apoptosis• Down-regulation of EPO receptor expression on erythroid

precursor cells• Reduced expression of other pre-hematopoietic factors: stem-cell

factors, CFU-E

Iron-deficiency anemia

• Chronic external blood loss• GI bleeding ulcers, colonic ectasia, bloodsucking parasites• Heavy flea infestation in young animals• Chronic urinary tract hemorrhage

• Dietary deficiency of iron• uncommon cause of iron deficiency in animals• secondary to acidosis, excess vitamin C or zinc

• Copper deficiency: required for absorption of iron from the GI tract and for release of iron from stores in macrophages in the body• Chronic lead poisoning: secondary by incorporation of iron into

porphyrin ring of heme

Can Vet J (2012)53:250-256 Journal of Small Animal Practice (2016) 57, 348-353

Pathogenesis of iron deficiency anemia

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Laboratory testing for ACD and IDA

• Serum iron: total amount of iron bound to both transferrin and ferritin• Ferritin: concentration in serum correlate with body iron store • Total iron binding capacity (TIBC): indirect measurement of transferrin

• Serum sample id flooded with excess iron binds to all the available iron sites on transferrin

• Unbound iron is removed by chemical method and the iron concentration in remaining sample is evaluated

• The amount of iron measured is proportional to the amount of transferrin present

• Percentage transferrin saturation: calculated by dividing the serum concentration by the TIBC• Marrow hemosiderin store: staining a bone marrow aspiration with

Prussian blue (blue aggregates within macrophages) = most accurate method of assessing body iron store in dog

PCV Iron store Type of anemia

PCV >20%: mild to moderate anemia

Low serum ironLow total iron binding capacityHigh bone marrow ironHigh percentage transferrin saturation

Anemia ofinflammatory disease

PCV >20%: mild to moderate anemia

Low serum ironNormal to high total iron binding capacityLow bone marrow ironLow percentage transferrin saturation

Iron deficiency anemia

PCV <20% : severe anemia

Iron deficiency anemia

Non-regenerative anemia: Primary bone marrow disorders

• Aplastic anemia: aplastic pancytopenia• Damage of stem cells or marrow microenvironment: marrow failure

and fibrosis• Causes by infections, drugs, toxins, immune-mediated• Marrow aspirate: mixed fat cells, macrophages, endothelial cells,

plasma cells, lymphocytes and mast cells• Acute form: destruction of progenitor and deviding cells with

leukopenia (within 5 days), thrombocytopenia (8-10 days), gradually anemia, recover and repopulate within 3 weeks

• Chronic form: progressive stem cell destruction and replacement of fat, neutropenia, thrombocytopenia, moderate to severe anemia

Non-regenerative anemia: Primary bone marrow disorders

Drugs Infections Idiopathics

Estrogen (dogs)Phenylbutazone (dogs)Trimethroprim-sulphadiazine (dogs), Chloramphenicol (dogs), sulfonamides (cat)Albendazole (dogs and cats), Fenbendazole (dogs)Griseofulvin (cats) Azathioprine (dogs)Meclofenamic acid (dogs)Methimazole (cat)Quinidine (dogs)

ParvovirusEhrlichiosisSepsisFeLV

Possible immune-mediated destruction

Causes of aplastic anemia

Villiers E. and J. Ristic BSAVA Manual of Canine and Feline Clinical Pathology 3 rd edition

Non-regenerative anemia: Primary bone marrow disorders

• Non-regenerative IMHA (NRIMHA) or precursor directed immune-mediated anemia (PIMA)

• Immune-mediated destruction of red cell precursors in bone marrow• No clinical signs of hypoxia and evidence of regeneration in blood and normal

RBC morphology • Severe non-regenerative (normochromic normocytic) anemia (HCT 15-20%),

may be neutropenia (cat) or thrombocytopenia • Usually Coombs’ negative, some spherocytes in dog, response to

immunosuppressive therapy but delayed • Bone marrow aspiration:

• 1) Left shift maturation in erythroid progenitors (more immature form: metarubricytes) = maturation arrest

• 2) Erythroid hyperplasia with complete maturation of erythroid series and destruction of reticulocytes, increased marrow iron (dog), mild reactive lymphocytosis (cats) and plasmocytosis (dogs) = ineffective erythropoiesis

Non-regenerative anemia: Primary bone marrow disorders

• Pure red cell anemia (PRCA)• Dogs, cats, horses and ferrets and unknown mechanism• More severe form of PIMA and selective destruction of early

erythroid precursors• Absence of erythroid precursors or small numbers of rubriblasts or

prorubricytes in bone marrow (<5% of marrow cells)• No hemostasis abnormalities and/or hypercoagulability• Aggressive immunotherapy, recurrence, refractory• Secondary PRCA: recombinant human erythropoietin (rhEPO),

parvovirus, FeLV sub C

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Non-regenerative anemia: Primary bone marrow disorders

• Bone marrow neoplasia• Leukemia, lymphoma,disseminated histiocytic sarcoma, metastatic

neoplasm (mammary carcinomas, mast cell tumor)• Combination cytopenia• Leukemias: neoplastic transformation of hematopoietic precursor cell of

one cell line in BM • Clonal expansion and releasing of large number of neoplastic cells into the

circulation• Marrow were crowded out by neoplastic cells and suppressed normal

hematopoiesis• Neoplastic cells complete nutrients, release inhibiting substances = anemia,

neutropenia, thrombocytopenia together with circulating atypical (leukemic) cells

Non-regenerative anemia: Primary bone marrow disorders

• Myelofibrosis: proliferation of fibroblasts, deposition of reticulin and collagen fibers in hematipoietic space• Non-regenerative anemia, often without leukopenia and

thrombocytopenia • Fibrosis secondary to IMHA, PRCA, neoplasia, toxic marrow damage

from phenobarbital and inherited red cell defect PK deficiency• Idiopathic myelofibrosis: rare and involve megakaryocytes and

granulocytes• Bone marrow aspiration

• Dry tap: blood with individual hematopoietic precursors• Core biopsy: fibrous tissue extending throughout hematopoietic spaces

Non-regenerative anemia: Primary bone marrow disorders

• Myelodysplastic syndrome (MDS): mutation of hematopoietic stem cells lead to ineffective hematopoiesis, dysplasia and premature cell death in one or more cell lines• Cytopenia often bi-or pancytopenia• Dysplastic features: asynchonous nuclear/cytoplamic development with

immature nuclei and mature cytoplasm, altered granularity, giant nuclei, ring-shaped nuclei, fragmented nuclei, multinucleation, blasts (<20%)

• Marrow: hypercellular, mostly immature precursor, abnormal morphology

• MDS-refractory anemia: anemia only, good response to EPO therapy • MDS-excessive blasts: 5-20% in marrow, bi-or pancytopenia, poor

prognosis

Bone marrow exanimation

• In cases of non-regenerative anemia• Non-regenerative/pre-regenerative anemia• Underlying causes of secondary anemia: CKD or AID

• History and physical finding• Fever of unknow origin, suspicious neoplasia or drug toxicity

• Abnormal serum finding• Hypercalcemia, hyperglobulinemia, decreased serum iron

• Abnormal CBC finding• Unexplained cytopenia, unexplained cytosis, abnormal cell

morphology, atypical cellular reaction

Bone marrow exanimation

• Bone marrow aspirate and core biopsy• Aspiration: single cell, hypo/hyperplasia, leukemia, MDS• Core: relation of cellular population to each other and to stromal

cells• General anesthesia or sedation and local anesthesia• Anesthetic risks, bleeding disorders, thrombocytopenia, infection

• Iliac crest, proximal humerus, femur• 10-12 slides and quick air dried• Unsuccessful sampling: • Poor technic• Myelofibrosis/myelophthisis

Management of non-regenerative anemia

• Treatment of underlying causes• Blood transfusion in severe anemia and hypoxia• EPO therapy (Darbepoetin): 44-132 U/kg 3 times/week• Start with 88 U/kg, monitor PCV weekly or reticulocyte count

• Epoetin alpha: recombinant human (glycoprotein) erythropoietin• 100 U/kg SC 3 times/week for 4 month, following by 75-100 U/kg

SC 2-3 times/ week

Management of non-regenerative anemia

• Anabolic steroids• Oxymetholone: dog and cat at 1-5 mg/kg PO every 18-24 hr• Nandrolone decanoate• Dog: 1-1.5 mg/kg weekly IM• Cat: 1 mg/kg weekly IM

• ABOs: in cases of leukopenia/neutropenia, doxycycline• Immunosuppressive drugs: prednisolone or combination• Chemotherapeutic agents: cytarabine, cyclosporin, vincristine,

daunorubicin• Recombinant G-CSF, recombinant GM-CSF

• Hematonics- Iron in ferrous salt (sulfate, gluconate, fumarate) or chelated iron

- Dog: 15 mg iron salt/kg (5 mg elemental iron/kg) PO BID (or TID or 50-300 mg total dose every 24 hr)

- Cat: 50-100 mg/day- Iron dextran:

- Dog: 100 mg single IM or 10 mg elemental iron/kg weekly (or every 3-4 weeks)

- Cat: 50 mg/cat IM once every 3-4 weeks- Cobalamin (B12) for 3 months

- Dog: 250 µg for small dog to 10 Kg, 500 µg for 10-20 kg, 1000 µg for more tan 20 kg /day PO

- Cat: 250 µg/day PO - Folic acid (B9) (yeast, liver, kidney, green vegetables)

- Dog: 5 mg/day PO- Cat: 2.5 mg/day PO

• Iron supplement should be given several months

Treatment of leukemiaCOAP protocol for Acute lymphoid leukemia Cyclophosphamide Dogs: 50 mg/m² PO 4 days a week or every 48 hr for 8 weeks

Vincristine 0.5 mg/m² IV once a week for 8 weeks

Cytosine arabinoside 100 mg/m² SC or IV divided BID for 4 days

Prednisolone 40-50 mg/m² PO SID for 1 week, followed by 20-25 mg/m² PO every 48 hr for 7 weeks

COP protocol for Acute lymphoid leukemia Cyclophosphamide 50 mg/m² PO 4 days a week or every 48 hr or 300 mg/m² PO every 3 weeks

Vincristine 0.5 mg/m² IV once a week for 8 weeks

Prednisolone 40-50 mg/m² PO SID for 1 week, followed by 20-25 mg/m² PO every 48 hr for 7 weeks

Chronic lymphoid leukemiaChlorambucil 20 mg/m² PO every 2 week (with or without pred; 20 mg/m² PO every 48 hr)

Chronic lymphoid leukemia Cyclophosphamide 50 mg/m² PO 4 days a week or every 48 hr or 300 mg/m² PO every 3 weeks

Prednisolone 20 mg/m² PO every 48 hr

Acute myeloid leukemia Cytosine arabinoside 100 mg/m² SC every 12-24 hr or by IV drip over 8-12 hr

6-thioguanine 40-50 mg/m² PO every 24-48 hr)

Acute myeloid leukemia Cytosine arabinoside 100 mg/m² SC every 12-24 hr or by IV drip over 8-12 hr

6-thioguanine 40-50 mg/m² PO every 24-48 hr)

Doxorubicin 10 mg/m² IV on days2 and 4 of the cycle

Acute myeloid leukemia Cytosine arabinoside 100 mg/m² SC every 12-24 hr or by IV drip over 8-12 hr

Mitoxantrone 4-6 mg/m² by IV drip (both drugs are combined in the same saline bag) every 3 weeks. The mitoxanthrone is only used for 1 day, even if the Ara-C is used for 2 days

Chronic myeloid leukemia Hydroxyurea 50mg/kg PO divided in two daily doses every 24-48 hr