Nonregenerative Anemias

Nonregenerative Anemias

• These anemias occur when the bone marrow doesn't respond appropriately to the anemia.
• There's no increase in polychromasia/reticulocytes, limiting their usefulness as indicators in equines.
• Absence of basophilic stippling occurs in ruminants.
• Erythroid hyperplasia is not present in the bone marrow, though ineffective erythroid hyperplasia can occur where the bone marrow is producing a lot of cells but they can't get out.

Causes of Nonregenerative Anemia

• Decreased RBC production usually requires several weeks to manifest clinical signs, but patients can compensate for gradual decreases in hematocrit.
• Other cell lines may be involved with general bone marrow disorders like pancytopenia.
• Insufficient time for regeneration can cause early/acute anemias from any cause (hemolysis, blood loss, etc.) to be nonregenerative.
• Nonregenerative anemia is sometimes called "preregenerative" anemia, and is difficult to prove clinically except by waiting to see if regeneration occurs, rechecking CBC after two or three days is advised to assess regeneration
• Iron deficiency and chronic hemorrhage, also known as iron-restricted erythropoiesis, can cause nonregenerative anemia.
• Anemia of inflammatory or chronic disease, chronic renal disease, chronic liver disease, bone marrow diseases, and endocrine diseases can all lead to nonregenerative anemia.

Iron Deficiency

• Iron deficiency can be caused by chronic blood loss due to parasites (GI or external) or GI hemorrhage, which can sometimes go undetected.
• Excessive blood donation can cause iron deficiency, consider as you evaluate
• It starts as a regenerative anemia, then gradually becomes nonregenerative making it difficult to interpret results
• Dietary deficiency is the least common cause of iron deficiency
• Dietary deficiency is rare in adult animals but, neonates have low Fe stores, and milk has little Fe; it is most common in piglets, where iron injections may be needed.

Diagnosis of Iron Deficiency

• Anemia is diagnosed using RBC Parameters/indices
• It can also be diagnosed using Iron/transferrin testing (‘iron panel")
• Bone marrow iron assessment is also used
• As a clinician, identifying the cause of iron deficiency is vital, whether it's parasites, GI lesions, or other sources of chronic blood loss.

Iron Deficiency Anemia

• Anemia happens with lack of sufficient iron for hemoglobin production.
• It classically exhibits microcytic (low MCV) and hypochromic (low MCHC) RBCs, but these may not always be present.
• Hypochromasia can occur without microcytosis if anemia is regenerative.
• Microcytic, hypochromic anemia is also common with portosystemic shunting/liver disease, which is a functional iron deficiency.
• Nonregenerative anemia is typical, but it may be regenerative, especially in early stages.
• Microcytosis occurs after depletion of iron stores.
• Extra RBC cell divisions occur because of low Hb concentration, which reduces RBC size with increased divisions.

Iron Deficiency: Reticulocyte Indices

• Hematology analyzers can evaluate hemoglobin in the reticulocyte population rather than in all RBCs.
• This may be a more sensitive way of evaluating Fe deficiency.
• Advia( Siemens analyer), CHr and CHCMr are types of tests
• Reticulocyte hemoglobin equivalent is found in iDEXX ProCyte and correlates with iron deficiency.

CHr and CHCMr

• Advia hematology analyzer can measure Hb content in reticulocytes (CHr) and reticulocyte Hb concentration (CHCMr) as the cells are counted.
• Low CHr or CHCMr in dogs may create a suspicion for Fe deficiency:
• CHr
  • Iron deficiency is ≤ 20.1 pg
  • Anemia of the inflammatory disease is ≥ 21.8 pg
  • Portosystemic shunt is ≥ 19.2 pg
  • Breed-associated microcytosis is ≥ 21 pg, these include breeds Akita, Shiba Inu, Shar Pei, Chow Chow)
• CHCMr
  • Iron deficiency is ≤ 26 g/dL
  • Anemia of the inflammatory disease is ≥ 26 g/dL
  • Portosystemic shunt is ≤ 24.1 g/dL
  • Breed-associated microcytosis is ≥ 27.7 g/dL

Iron Pools

• Functional iron includes Hemoglobin (approx. 2/3 of body Fe), Myoglobin(muscle), and Cytochromes etc
• Transport iron is transferrin
• Storage includes Ferritin and Hemosiderin

Iron panels

• Serum Iron- this is not representative of total body iron stores.
• It decreases with Fe deficiency or inflammation
• Total Iron Binding Capacity - Indirect measurement of transferrin, it Increases in Fe deficiency, and has less increase in dogs than in other species and it may be decreased with hepatic insufficiency
• Ferritin - Usually best serum indicator of iron stores

Transferrin

• Iron transport protein made in the liver (beta globulin), circulates in the plasma.
• Each Tf binds 2 Fe3+ (ferric) iron atoms.
• Tf enters cells by binding transferrin receptor, after which the Tf/Tf receptor complex undergoes endocytosis.
• Normally, around ~25-50% of transferrin Fe binding sites are saturated.
• Iron deficiency increases liver transferrin production, and decreased transferrin saturation.
• It is a negative acute phase protein, and decreases with inflammation.

Ferritin

• Protein that acts as an intracellular store for iron.
• Encloses Fe atoms in a protein "globe,"
• Fe rapidly mobilized from ferritin.
• It protects cells from formation of free radicals

Ferritin Levels

• Serum ferritin levels are correlated with total body iron stores and are measured by (species-specific) immunoassay.
• Decreased levels mean there is iron deficiency
• Increased levels may occur with inflammation or actue phase protein

Iron Within Macrophages

• Iron enters macrophages via transferrin/transferrin receptor
• Enters macrophages via from Hb in phagocytized erythrocytes
• Iron is stored as ferritin (or hemosiderin)
• Can be exported via ferroportin

Hemosiderin

• Large complexes of protein (including denatured ferritin) and iron.
• Fe is released more slowly than from ferritin as it is a relatively insoluble iron storage protein.
• Appears as golden-brown to blue-green granules with light microscopy, generally within macrophages.

Iron Deficiency

• Serum Fe may be decreased or WNL
• Transferrin/TIBC may increase
• Ferritin is decreased

Inflammation

• Serum Fe may be decreased or normal
• Transferrin/TIBC may decrease
• Ferritin may increase
• Mechanism may deprive infectious organisms of iron.

Progression of Iron Deficiency Anemia

• Anemia is initially regenerative (macrocytic, hypochromic, with increased polychromasia) when caused by blood loss.
• Over time, anemia tends to become nonregenerative (and microcytic) as iron stores are depleted.
• Patients with early Fe deficiency may have microcytic anemia, but still regenerative

Hemosiderosis/Hemochromatosis

• Increased iron stores.
• Large amounts of hemosiderin accumulate in tissues like the spleen, liver, and lymph nodes.
• It may be due to chronic hemolytic disease, large numbers of transfusions, or a genetic disorder with a lack of hepcidin.

Anemia of Inflammatory Disease

• Commonly referred to as anemia of chronic disease where its caused by almost any type of inflammation, including bacterial, fungal, viral, protozoal infections, autoimmune disease, neoplasia, or trauma.
• The onset can occur in as little as ~3 days, so the time frame may not necessarily look "chronic".
• It is a mild to moderate nonregenerative anemia.
• Usually Normocytic (rarely microcytic), and normochromic.
• Patients often exhibit inflammatory leukogram.
• Proteins tend to be increased (immunoglobulins +/- fibrinogen or other acute phase proteins).
• The Mechanisms of decreased iron availability inhibits erythropoiesis with the increase in ferritin, decreasing transferrin and serum Fe while also increasing in hepcidin production.
• There is Production of anti-erythropoietic inflammatory mediators like IL-1, TNFα, and interferon.
• Results in reduces RBC lifespan
• Reguarly considered a minor/secondary problem
• Diagnosis based on Diagnosis of underlying inflammatory disease
• Also Diagnosis based on Typical CBC findings (mild nonregenerative anemia) and Lack of evidence of other causes of anemia

Anemia of Renal Disease

• Patients with chronic renal disease are often anemic.
• it is Primarily the result of decreased erythropoietin (EPO) production.
• May also involve anemia of inflammation like inflammatory renal diseases.
• May also involve decreased RBC survival like fragmentation of RBCs in glomerulonephritis.
• Ranges from Mild to severe (depending on duration) nonregenerative anemia, usually with a gradual onset.
• Normocytic, normochromic (no increase in reticulocytes)
• Diagnosed due to the finding nonregenerative anemia with evidence of renal disease like Azotemia increased urea, creatinine) and the Loss of urine concentrating ability , having a low specific gravity but there is no Low EPO level test currently available

Anemia of Bone Marrow Disorders

• Disorders of erythrocyte production, or hematopoiesis in general, typically cause a nonregenerative anemia.
• Many disorders affect multiple cell lines.
• Bicytopenia or pancytopenia
• RBCs usually last cell line to decrease

Myelophthisic Disease

• Myelophthisis is the active replacement of normal hematopoietic tissue with abnormal tissue.
• Hematopoietic tissue is “crowded out".
• Myelophthisis is NOT the replacement of active marrow with adipose tissue.
• Causes include hematopoietic neoplasia or leukemias, metastatic neoplasia or lymphoma, and Myelofibrosis fibroblasts, collagen, reticulin.
• It is diagnosed via Bone marrow aspirate/biopsy
• Biopsy required for diagnosis of myelofibrosis

Myelofibrosis

• Causes include chronic bone marrow inflammation and PIMA or ehrlichiosis
• It can be caused by chronic overstimulation of RBC production ("burnout”) from (e.g., polycythemia vera, pyruvate kinase deficiency, or EPO excess) and also Bone marrow necrosis Neoplasia with (lymphoma or leukemia) or myelodysplasia
• Drug treatment (phenobarbital, phenytoin, phenylbutazone, colchicine, EPO) or maybe Idiopathic
• Clinical course is often treated for immune-mediated disease unless know inciting cause

Neoplastic Myelophthisis

• Commonly due to round cell neoplasms like Acute leukemias, which include myeloid or lymphoid
• Can be due to Lymphoma stage V or Histiocytic sarcoma or and Plasma cell myeloma
• It is Uncommon in other types of neoplasia e.g., metastatic carcinoma

Aplastic Anemia

• Aplasia of all three cell lines- erythroid, myeloid, megakaryocytes
• Results in pancytopenia where Neutropenia happens berfore thrombocytopenia and anemia
• Bone marrow is severely hypoplastic, meaning there is adipose tissue rather than hematopoietic cells.

Cause of Aplastic anemia

• Immune-mediated (pluripotent stem cell destruction) and/or drug/toxin-induced.
• Estrogen
• Ehrlichia canis infection
• Parvovirus infection (transient)
• FeLV
• Radiation
• Idiopathic

Drug/Toxin-Induced Marro Aplasia

Including Chemotherapy agents Azathioprine Trimethoprim-sulfa, Phenylbutazone toxicity, Bracken fern, Albendazole, methimazole(cats), Griseofulvin in cats

• Captopril
• Cephalosporins
• Mycotoxins
• Estrogens

Estrogen-Induced Marrow Hypoplasia/Aplasia

• Estrogen causes myelosuppression by unknown mechanism.
• Causes include estrogen administration, like with diethylstibestrol, and estrogen-secreting tumors.
• Examples include Sertoli cell tumors in males, granulosa cell tumor and cystic ovaries in females, and prolonged estrus in ferrets.

Pure Red Cell Aplasia/PRCA

• Aplasia of the Erythrocyte line only
• Leukocytes and platelets are normal or increased
• Seen in dogs and cats.
• Causes include immune-mediated response against RBC precursors, FeLV subgroup C infection in cats, rhEPO treatment (where anti-EPO antibodies may develop), and it may also be idiopathic and immune-mediated.

Nonregenerative IMA/PIMA

• Immune-mediated anemias are usually strongly regenerative.
• Mature RBCs are destroyed, but marrow is not affected
• Iron is recycled
• Destruction of developing erythrocytes can cause nonregenerative anemia or precursor-targeted IMA PIMA
• Bone marrow findings depend on which RBC stage is targeted by the immune system.
• If against intermediate stages, its "Maturation arrest'
• If against stem cells or early precursors isred cell aplasia

Nonregenerative IMA/PRCA/PIMA

• It is Moderate to severe nonregenerative anemia
• Coombs' test may be positive or negative
• Spherocytes may or may not be present
• Patients may also develop myelofibrosis in chronic cases

PIMA vs PRCA

• Terminology is usually based on marrow findings
• Erythroid aplasia is based on pure red cell aplasia
• Ineffective erythropoiesis or erythrooid hypoplasia or maturation arrest is PIMA
• Diffcult to definitively diagnose if Coombs negative
• Includes Diagnosis of exclusion, reponse to treatment and may take weeks to months to respond

FeLV and Anemia

• Feline leukemia virus infection is the most common cause of nonregenerative anemia in cats, but may also be regenerative.
• There are many causes, like Anemia of chronic/inflammatory disease and Immunosuppression or and secondary infection
• Also it can be caused by Aplastic anemia or PRCA, Induction of IMHA, leukemia, lymphoma and myelophthisis
• When seeing non-immune cats be sure to test for FeLV

FeLV and Macrocytic Anemia

• Cats with FeLV-induced anemia have macrocytic or nonregenerative anemia with Increased MCV +/- increased anisocytosis.
• Suspect FeLV infection in Anemia with an MCV> ~60 fl without regeneration

Anemia of Liver Disease

• Liver disease, particularly portosystemic shunting, may cause anemia.
• Usually mild, nonregenerative
• Microcytic (sometimes normocytic), normochromic

Anemia of Liver Diease Mechanism

• Altered Iron Metabolism
• Alteration in RBC membrane lipids
• EPO precursor is made in liver
• Also need to use Diagnosis: Evidence of liver disease where there is increaed bile acid or ammonia or and increased Liver Enzymes

Anemia of Endocrine Diseases

• Severe anemia or pancytopenia with hyperestrogenism.
• Mild anemia may occur with endocrine disorders like Hypothyroidism, where there is decreased EPO secretion with loss of thyroid hormone stimulation of erythropoiesis.
• Also it can be Hypoadrenocorticis or Addision's disease
• Or also Hypopituitarism

Copper Deficiency

• Rare cause of anemia (found in dogs and pigs)
• Ceruloplasmin and hephaestin contain copper
• Cu deficiency can interfere with Fe transport into and out of enterocytes

Lead Poisoning and Anemia

• Chronic lead poisoning may cause anemia.
• Hemolysis
• Suppression of heme synthesis
• Lead inhibits d-aminolevulinic acid dehydratase and ferrochelatase in the heme pathway.
• Resulting in Increased amount of erythrocyte protoporphyrin in RBCs
• Acute lead poisoning is usually not anemic.
• Results in increased NRBCs and basophilic stippling (without polychromasia) in blood

Polycythemia/Erythrocytosis

• PCV/Hct above reference interval
• Relative: Normal/mass of RBCs (increased) due to hemoconcentration.
• Absolute: Increased RBC #/production

Erythrocytosis

• Some dog breeds have higher Hct reference intervals than avereage in dogs
• Like Greyhounds, Slukia, or Afghan hound
• "Hot Blood Breeds" have higher Hct than "cold blood"

Relative Polycythemia/Erythrocytosis

• Dehydration where water loss decreases plasma volume and RBC volume unchanged leading to an increased Hct
• Albumin and plasma ptoteins should also increase
• Splenic Contraction by Sympathetic stimulation (Exercise ) where Splenic blood has a higher PCV than Venous Blood

Dehydration and Hct

• Dehydration/hemoconcentration should increase Hct (& albumin/total protein)
• These parameters may still be WNL in dehydrated patients, particularly if they were decreased or in lower or middle part of the RI before dehydration

Absolute Erythrocytosis: Causes

• Primary: Increased RBC production without EPO stimulation (polycythemia vera)
• Secondary: Caused by increased erythropoietin level and usually adaptive to low oxygenation/perfustion
• Adaption to altitude
• Cardiac Disease
• Chronic restrictive airway disease or pleura Effusion
• Persistent Methemoblobinemia
• EPO-secreting tumor (renal cysts, carcinoma)

Polycythemia Vera/Primary Erythrocytosis

• Rare, neoplastic-like proliferation of RBCs
• Reported in dogs and cats with a increased Hct/platelets and WBC WNLs/Neutrophilia that can occur
• Low/unmeasurable EPO levels

Primary Erythrocytosis

• High Hct/PCV often at 70-90%
• Increased blood viscosity/hyperviscosity
• Hyperemic Mucous Mems
• Neuro Signs
• Engorged retinal Vessels

DX for Primary Erythrocytosis

• Persistently elevated Hct
• Rule out other possible causes of polycythemia like No dehydration, Normal PaO2, Normal Cardiac Exam
• Treatment by therapeutic phlebotomy or hydroxyurea or a combination of both