Lecture 7: Laboratory evaluation of erythrocytes- indices, boombs, methemoglobin, iron

How are hematocrit (Hct), hemoglobin concentration (Hb), and erythrocyte count (RBC) determined, and how are they interrelated?

- Hct: Measured by centrifugation (PCV) or calculated from RBC × MCV.

- Hb concentration: Measured by spectrophotometry after RBC lysis.

- RBC count: Direct enumeration by automated counters.

- Interrelation: All three reflect erythron mass—Hct is proportionate to RBC count × MCV; Hb correlates strongly with Hct. Discrepancies suggest analytic error or abnormal RBC morphology.

What does the presence of immunoglobulin on erythrocytes cause, and how is it detected?

- Effect: Leads to RBC agglutination.

- Autoagglutination: Occurs spontaneously, often in immune-mediated hemolytic anemia (IMHA).

- No visible autoagglutination: Detected by Direct Antiglobulin Test (Coombs test) or flow cytometry to identify surface-bound immunoglobulin or complement.

What is macrocytosis (high MCV), and what are its common causes?

Macrocytosis = increased average erythrocyte size. Causes include:

- Regenerative anemia, especially hemolytic (reticulocytosis).

- FeLV-positive cats (often nonregenerative macrocytosis).

- Myeloproliferative disorders incl. MDS (nonregenerative).

- Dogs with nonregenerative IMHA or myelofibrosis.

- Poodle dogs (non-anemic hereditary trait).

- Hereditary stomatocytosis in dogs.

- Folate deficiency (e.g., cats on strict plant-based diets).

- Abyssinian & Somali cats with ↑ erythrocyte osmotic fragility.

- Fetal/neonatal animals (physiologic).

- Congenital dyserythropoiesis in Hereford calves.

Why do FeLV-positive cats often exhibit macrocytosis, and is it usually regenerative?

FeLV infection alters erythroid precursor maturation, producing larger erythrocytes. Unlike hemolytic causes, this macrocytosis is typically nonregenerative, reflecting ineffective erythropoiesis.

Why do fetal and neonatal animals often exhibit macrocytosis

Their erythrocytes are physiologically larger due to immature hematopoiesis and higher fetal hemoglobin content—this is not pathologic.

What hereditary and breed-related conditions are associated with macrocytosis?

- Poodle dogs (benign, non-anemic macrocytosis).

- Hereditary stomatocytosis in dogs.

- Abyssinian and Somali cats (↑ RBC osmotic fragility).

- Congenital dyserythropoiesis in Hereford calves.

What are three major causes of erroneous macrocytosis (falsely high MCV) on hematology analyzers?

1. Autoagglutination of RBCs → analyzer sizes clumped RBCs as one large cell.

2. Prolonged blood storage → RBCs swell over time.

3. Persistent hypernatremia (dogs & cats) → higher plasma osmolality than analyzer diluent → RBCs swell when diluted for assay.

List causes of true microcytosis (low MCV) in veterinary patients.

- Chronic iron deficiency anemia.

- Portosystemic shunts (dogs; variable mild anemia).

- Anemia of inflammatory disease (minimal effect).

- Breed-related: Oriental dog breeds (Akita, Shiba Inu, Jindo, Tosa, Chow Chow, Shar-Pei); slightly lower in normal Abyssinian cats.

- Erythrocyte fragmentation.

- Cats with hepatic lipidosis.Heme synthesis inhibition: lead, chloramphenicol, hydroxyzine, vitamin B6 deficiency.

- Rare hereditary disorders: elliptocytosis in dogs; dyserythropoiesis in English Springer Spaniels & Labrador Retrievers.

What can cause spurious (false) microcytosis on hematology analyzers?

Platelets counted as RBCs in severely anemic patients (very low RBC count).

Persistent hyponatremia in dogs → blood osmolality lower than analyzer diluent → RBC shrinkage when diluted.

What are the common causes of low MCHC values in veterinary patients?

- Regenerative anemia (reticulocytes contain less Hb; usually also macrocytic).

- Chronic iron deficiency anemia (also microcytic).

- Hereditary stomatocytosis in dogs (swollen RBCs; also macrocytic).

- Abyssinian & Somali cats with osmotic fragility (also macrocytic).

- Spurious in dogs & cats with persistent hypernatremia → RBCs swell in analyzer diluent, diluting Hb concentration.

Why is MCHC often low in regenerative anemia?

Because reticulocytes are larger (macrocytic) but contain proportionally less hemoglobin → falsely lowers the average Hb concentration per RBC.

What are the main artifactual causes of high MCHC values?

- Intravascular hemolysis (plasma Hb overestimates Hb concentration).

- In vitro hemolysis (sample handling artifact).

- Heinz bodies (interfere with Hb measurement).

- Lipemia (spectrophotometric interference).

- Erythrocyte agglutination in electronic counters (low HCT + falsely high Hb).

How do erroneous analyzer calculations produce artifactually high MCHC values?

Formula: MCHC = (Hb / HCT) × 100.

If Hb is overestimated (e.g., hemolysis, lipemia) or HCT is underestimated (e.g., agglutination), MCHC will be artifactually increased.

What is the purpose of erythrocyte indices (MCV, MCHC, RDW) in veterinary hematology?

They provide quantitative information on RBC size and hemoglobin concentration. They are primarily used to classify anemias (microcytic, normocytic, macrocytic; hypochromic, normochromic) and guide differential diagnosis.

Define MCV (Mean Cell Volume), how it is measured, and its units.

Definition: Average volume of a single erythrocyte.

Measured: Directly by hematology analyzers in most mammals.

Manual calculation needed in camelids and non-mammals:

- Formula = (HCT / RBC count) × 10.

- Units: femtoliters (fL).

Example calculation of MCV in a mammal:

HCT = 45%

RBC count = 6.9 × 10⁶/µL

MCV = (HCT ÷ RBC count) × 10= (45 ÷ 6.9) × 10= 65 fL

How is MCV used in anemia classification?

Microcytic anemia: Iron deficiency, portosystemic shunt, breed-related.

Normocytic anemia: Most nonregenerative anemias.

Macrocytic anemia: Regeneration, FeLV, dyserythropoiesis, folate deficiency, some breeds.

Define MCH (Mean Cell Hemoglobin) and describe its utility.

- Definition: Average amount of hemoglobin (pg) per RBC.

- Calculated: (Hb / RBC count) × 10.

- Limited utility because it reflects both RBC size and Hb concentration → largely redundant with MCV & MCHC.

- Lowest values seen in iron deficiency (low MCV + low MCHC).

Example calculation of MCH in a mammal:

Hb = 15 g/dL

RBC count = 6.9 × 10⁶/µL

MCH = (Hb ÷ RBC count) × 10= (15 ÷ 6.9) × 10= 22 pg

Define MCHC (Mean Cell Hemoglobin Concentration) and explain how it differs from MCH.

- Definition: Average Hb concentration per unit RBC volume.

- Independent of cell size, unlike MCH.

- Clinical use: More reliable than MCH for detecting hypochromia.

Low MCHC: Regenerative anemia, iron deficiency, stomatocytosis.

High MCHC: Almost always artifactual (hemolysis, lipemia, Heinz bodies, agglutination).

What is RDW (Red Cell Distribution Width), and how is it used in anemia diagnosis?

- Definition: A measure of variation in RBC size (anisocytosis).

- High RDW = increased size variability → often seen with regenerative anemia (reticulocytosis) or mixed microcytic + macrocytic populations.

- Low/normal RDW = more uniform RBC population.

How do erythrocyte indices guide the differential diagnosis of anemia?

- Microcytic, hypochromic anemia: Iron deficiency, portosystemic shunt.

- Normocytic, normochromic anemia: Anemia of chronic disease, endocrine disorders, renal disease.

- Macrocytic, hypochromic anemia: Regenerative anemia (reticulocytosis).

- Macrocytic, normochromic anemia: FeLV, myelodysplasia, congenital dyserythropoiesis, folate deficiency.

Define MCHC (Mean Cell Hemoglobin Concentration) and explain how it is calculated.

- Definition: Average Hb concentration per unit volume of packed erythrocytes.

- Reported: g/dL of packed RBCs (not whole blood).

- Formula: (Hb ÷ HCT) × 100.

Example: Hb = 15 g/dL; HCT = 45% → (15 ÷ 45) × 100 = 33 g/dL.

Why is MCHC considered more clinically useful than MCH?

MCHC is independent of cell size, so it reliably reflects RBC hemoglobinization.

- Low MCHC: true hypochromia (iron deficiency, regenerative anemia).

- High MCHC: usually artifactual (hemolysis, lipemia, Heinz bodies, agglutination).

Define RDW (Red Cell Distribution Width) and its clinical significance.

- Definition: Electronic measure of anisocytosis (coefficient of variation of RBC volumes).

- Clinical use: Detects mixed RBC populations and subtle size variability, supporting differential diagnosis of anemia.

What conditions cause an increased RDW in animals?

- Large reticulocytes (regenerative anemia).

- Microcytes (iron deficiency).

- Erythrocyte fragmentation.

- Post-transfusion with variable RBC sizes.

- Dyserythropoiesis (abnormal precursor maturation).

- Hereditary stomatocytosis.

What causes spurious increases in RDW on hematology analyzers?

Erythrocyte agglutination → analyzer counts clumps as large RBCs.

Platelets miscounted as RBCs in severely anemic patients.

What does the Coombs test (direct antiglobulin test) detect, and when is it used?

- Detects immunoglobulin or complement bound to erythrocytes in vivo.

- Used to confirm immune-mediated hemolytic anemia (IMHA) when autoagglutination is not obvious.

- A positive test = antibody/complement coating RBCs; false negatives possible if low antibody load.

What is a major cross-match in blood transfusion, and why is it the most important?

- Method: Donor RBCs tested against recipient serum.

- Detects recipient antibodies against donor RBC antigens.

- Incompatibility = risk of serious transfusion reactions → hemolysis, agglutination, shock.

- Essential before erythrocyte transfusions.

What is a minor cross-match, and in what clinical situations is it most critical?

- Method: Donor serum tested against recipient RBCs.

- Detects donor antibodies against recipient RBC antigens.

- Less likely to cause transfusion reactions (donor plasma is usually diluted).

Most important for:

- Neonatal isoerythrolysis testing (colostrum antibodies).

- Plasma transfusions, where donor antibodies are transfused in high amounts.

How is iron absorption regulated in the body under normal and altered states?

Normal: Only small amounts absorbed.

Increased absorption: Low total body iron, increased erythropoiesis.

Decreased absorption: High iron stores, presence of inflammation.

What is the role of hepcidin in systemic iron metabolism?

- Hepcidin = peptide hormone secreted by hepatocytes.

- Central regulator of iron availability.

- High hepcidin: inhibits iron release from enterocytes, macrophages, hepatocytes → ↓ plasma iron.

- Low hepcidin: increases iron release and absorption → supports erythropoiesis.

- Production modulated by total body iron and the magnitude of erythropoiesis.

Why might serum iron be low despite adequate body iron stores?

Inflammation → hepcidin production increases → traps iron in storage sites.

This results in anemia of chronic disease (functional iron deficiency).

What is the diagnostic utility of serum ferritin?

Reflects total body iron stores.

Low ferritin = strong evidence of true iron deficiency.

High ferritin = iron overload or inflammation (ferritin is an acute-phase protein).

How do plasma hepcidin concentrations change with body iron status and inflammation?

Decreased hepcidin → when iron is needed (low body iron, increased erythropoiesis).

Increased hepcidin → when iron is not needed (iron overload, inflammation).

Inflammation-driven hepcidin rise serves to limit plasma iron availability to bacteria.

What is the major source of plasma iron under normal physiologic conditions?

Nearly all plasma iron comes from macrophage release (RBC recycling).

- Only 0.2-4.5% comes from duodenal enterocyte absorption.

What proportion of plasma iron is used for hemoglobin synthesis, and what is its turnover rate?

~75% of plasma iron is utilized for hemoglobin synthesis.

Plasma iron pool turns over rapidly — within ≤3 hours.

What conditions are associated with increased serum iron concentrations in animals?

- Hemolytic anemia (macrophage recycling, erythroferrone ↑).

- Dyserythropoiesis (ineffective erythropoiesis → erythroferrone ↑).

- Hypoplastic or aplastic anemia (decreased utilization for erythropoiesis).

- Iron overload.

- Glucocorticoid administration (dogs, horses).

What conditions are associated with decreased serum iron concentrations?

- Iron deficiency.

- Anemia of inflammatory disease (↑ hepcidin sequesters iron).

- Portosystemic shunts (slight decrease in some dogs).

- Marked erythropoiesis with inadequate iron mobilization (e.g., EPO therapy).

- Glucocorticoid administration (cattle, goats).

- Intense exercise (dogs, horses).

What does serum ferritin concentration reflect, and how is it interpreted?

- Reflects total body iron stores.

- Low ferritin = diagnostic for iron deficiency.

- High ferritin = iron overload, hemolytic anemia, inflammation, malignant histiocytosis, or transiently post-exercise (horses, dogs).

Why can serum iron be misleading as a sole diagnostic test for iron status?

- Rapid turnover (within hours) makes it highly dynamic.

- Influenced by inflammation, erythropoiesis, hormones, and stress/exercise.

- Must be interpreted with ferritin, hepcidin, and clinical context.

What determines serum erythropoietin (EPO) concentration in animals?

Rate of production by the kidney (peritubular interstitial cells).

Rate of utilization by erythroid precursors in bone marrow.

In what situations are serum EPO concentrations increased?

Most anemias (hypoxia stimulates renal EPO production).

Secondary erythrocytosis (due to hypoxia-driven EPO, though levels may vary).

In what situations are serum EPO concentrations decreased?

Chronic renal disease (loss of functional renal tissue reduces EPO production → nonregenerative anemia).

Primary erythrocytosis (polycythemia vera; neoplastic erythroid proliferation not dependent on EPO, so feedback suppresses EPO).

Why is interpretation of serum EPO concentrations clinically limited?

- Assays are not standardized in veterinary medicine.

- Concentrations overlap among diseases.

- Results must always be interpreted with PCV/Hct, reticulocyte response, and renal function.

What are the two main causes of cyanotic-appearing skin in animals, and how are they differentiated?

Hypoxemia → low pO₂ in arterial blood (confirmed on blood gas).

Methemoglobinemia → normal pO₂ but hemoglobin unable to bind oxygen (detected via spot test or co-oximetry).

What are the major toxic causes of methemoglobinemia in veterinary species?

Dogs/cats: Acetaminophen, benzocaine, phenazopyridine, hydroxyurea; fermented bok choy (dogs); skunk musk (dogs).

- Sheep/goats: Copper toxicity.

- Horses: Red maple toxicity.

- Ruminants: Nitrite toxicity from nitrate ingestion.

What hereditary cause of methemoglobinemia is seen across species, and how is it inherited?

- Cytochrome b₅ reductase (Cb5R) deficiency → prevents reduction of methemoglobin back to hemoglobin.

- Reported in dogs, cats, horses, humans.

- Autosomal recessive inheritance.

What are the clinical signs of methemoglobinemia at different percentages of methemoglobin?

<50%: Cyanosis, decreased exercise tolerance.

50%: Lethargy, ataxia, tachycardia, tachypnea.

80%: Coma-like state, potentially fatal.

What additional clinical signs may be seen in toxic methemoglobinemia?

Vomiting, anorexia, diarrhea.

Cats with acetaminophen toxicity → subcutaneous facial edema.

What are the hallmarks of methemoglobin reductase (Cb5R) deficiency in animals?

- Recognized in many dog breeds and DSH cats.

- Clinical signs: Cyanotic skin, mucous membranes, brownish blood during surgery; often otherwise asymptomatic.

- Methemoglobin levels: 13-51% in dogs, 30-52% in cats.

- Cats may show mild erythrocytosis.

- Treatment: Usually not required; methylene blue may be given before anesthesia.

How can a cytochrome b₅ (Cb5) defect mimic Cb5R deficiency?

Rare cases reported in dogs → defective cytochrome b₅ itself rather than the reductase enzyme, producing similar methemoglobinemia.