Erythrocytes (Red Blood Cells): Shapes, Lifespan

• Erythrocytes are also know as red blood cells (RBCs) or red corpuscles
• Function is to transport oxygen to tissues

RBC Shapes

• In most mammals, RBCs are biconcave discs with central depression
• Cats, cattle, and horses have RBCs with less concavity than dogs, resulting in less central pallor on blood films
• Camelids have oval/elliptical RBCs
• Birds, reptiles, amphibians, and fish have oval nucleated RBCs
• Normal mammalian RBCs lack nuclei
• Llamas and alpacas have normal (anucleated) RBCs

Approximate RBC Lifespans

• Reptiles RBC lifespan is 600-800 days
• Cattle RBC lifespan is 160 days
• Goat RBC lifespan is 125 days
• Horse RBC lifespan is 150 days
• Dog RBC lifespan is 120 days (approximately 3 to 4 months)
• Cat RBC lifespan is 70 days
• Rat RBC lifespan is 65 days
• Mouse RBC lifespan is 45 days
• Avian RBC lifespan is 35-45 days
• Herbivores tend to have longer RBC lifespans than dogs and cats
• Rodents have somewhat shorter RBC lifespans
• Metabolic rate is more closely associated with RBC lifespan, rather than patient size

RBC Metabolism

• Mammalian RBCs lack mitochondria, and obtain most energy through anaerobic glycolysis
• Glycolysis produces lactate as a byproduct
• Glycolysis produces ATP used to maintain cell membrane
• Glycolysis produces ATP used to maintain Na/K ATPase (Na:K gradient)
• Glycolysis produces ATP to aid in maintenance of Fe in reduced (ferrous) state
• Glycolysis produces ATP for production of 2,3 DPG
• Mature/fully mature RBCs cannot produce more Hb

Na/K ATPase in RBCs

• Na/K ATPase in RBCs is a cell membrane transporter that maintains gradient between cytoplasm and ECF
• Most cells have high intracellular K+ and low intracellular Na+
• Most species have relatively high Na/K ATPase activity in RBCs, with low Na+ and high K+ intracellularly
• Dogs and cats generally have low Na/K ATPase activity in RBCs, with low intracellular K+
• Shiba Inu, Akita, and Jindo dog breeds may have high Na/K ATPase activity with high intra-RBC K+
• Hemolysis in species with high RBC K+ (humans, horses) can lead to artifactual hyperkalemia
• Cats and most dogs do not exhibit artifactual hyperkalemia from hemolysis, because their RBCs are low in potassium

Hemoglobin (Hb)

• Hemoglobin is vital to the basic function of RBCs aiding in oxygen transport
• Hemoglobin components are Heme, Iron, and Globin (protein)
• Heme is synthesized in mitochondria, while globin is synthesized in ribosomes
• Mature RBCs cannot produce more Hb
• Polychromatophilic cells/reticulocytes are still producing hemoglobin

Assessment of Erythrocytes

• Erythrocytes are assessed by number/mass, indices, and morphology
• Goal of RBC assessment is aiding diagnosis of disease

RBC Assessment: Hematocrit

• Hematocrit is also known as Packed Cell Volume (PCV) or "crit"
• Blood is centrifuged in small tubes to separate RBCs from plasma
• Micro-hematocrit tubes are used to measure spun hematocrit, which distinguishes it from the instrument "calculated" hematocrit
• Percentage of RBC volume vs. plasma is measured to determine hematocrit
• Hematocrit, or PCV, is reported as % (in USA) or L/L (40% PCV = 0.40 L/L)
• PCV, or Hct, is typically about 3x the Hb concentration in g/dl
• Spun hematocrit is considered the gold standard
• Packing time of spun hematocrit is the duration until it stops getting smaller

Calculated Hematocrit

• Hematocrit is calculated by hematology analyzers based on volume and number of RBCs
• Calculated hematocrit is sometimes referred to as "machine hematocrit"
• Can be erroneous if RBC count or MCV is incorrect
• Calculation: RBC count (M/ul) x Mean Cell Volume (MCV) in fL, then divide by 10 to get hematocrit in %
• Spun hematocrit is commonly performed to check calculated hematocrit

Abnormalities of RBC Number/Volume

• Anemia is indicated by decreased Hct/PCV, or hemoglobin
• Veterinary medicine typically uses decreases in hematocrit to diagnose anemia
• Human medicine typically uses decreases in hemoglobin to diagnose anemia
• Polycythemia or erythrocytosis is indicated by increased Hct/PCV
• Anemia is much more common than polycythemia
• Anemia can be a primary disease or can be secondary to other conditions

Assessment of Anemias

• Anemias can be regenerative or nonregenerative
• Regenerative anemia indicates bone marrow that is responding to anemia by increasing RBC production
• Regenerative anemia usually indicates increased destruction or loss of RBCs
• Nonregenerative anemia results from decreased hematopoiesis/erythropoiesis
• Preregenerative anemia is anemia that will become regenerative but bone marrow hasn't had time to respond yet

RBC Indices

• RBC Indices are measurements of RBC size and hemoglobin content
• They provide clinical utility and may assist in diagnosing the causes of RBC abnormalities like anemia
• RBC volume is measured by Mean Cell Volume or MCV, in femtoliters (fl)
• RBC hemoglobin content is measured by Mean Cell Hemoglobin or MCH, in picograms (pg)
• RBC hemoglobin concentration is measured by Mean Cell Hemoglobin Concentration or MCHC in pg/fl
• Red cell Distribution Width or RDW (%) measures variation in RBC size (anisocytosis)

Mean Cell Volume: MCV

• Mean Cell Volume (MCV) is the average volume of individual erythrocytes
• MCV is typically directly measured by hematology analyzers
• MCV may be calculated from RBC count and PCV using the formula MCV = (PCV/RBC) X 10

MCV Variations

• Dog: 70 fl
• Pig: 63 fl
• Cow: 52 fl
• Cat: 45 fl
• Horse: 45 fl
• Llama: 25 fl
• Goat: 19 fl
• Amphibians and reptiles can have very large RBCs
• Whales tend to have large red blood cells

MCV Assessment

• Normal MCV indicates normocytic RBCs
• Increased MCV indicates macrocytic/macrocytosis
• Decreased MCV indicates microcytic/microcytosis

Macrocytic/Microcytic Erythrocytes

• Enlarged RBCs (macrocytes) or small RBCs (microcytes) may be detected visually during blood film examination
• Subtle size differences may be missed on blood film examination
• Macrocytes or microcytes may not correlate with abnormal MCV because MCV reflects an average cell volume
• MCV is generally considered more sensitive to overall size changes than visual assessment

Macrocytosis: Causes

• Macrocytosis is most commonly caused by regenerative anemia and increased release of larger, immature RBCs
• FeLV-infected cats may exhibit macrocytosis resulting in nonregenerative macrocytic anemia
• Breed differences like Toy/miniature poodles and miniature horses may have higher MCVs
• Uncommon causes are folate or cobalamin deficiency
• Drugs that interfere with DNA synthesis, like hydroxyurea, may cause macrocytosis
• Myelodysplasia or dyserythropoiesis may cause macrocytosis
• Artifacts like hyperosmolality and hypernatremia may cause macrocytosis
• If hyperosmolality develops slowly, cell volume is maintained by increasing intracellular osmoles to balance ICF/ECF
• If RBCs adapted to high osmolality are suspended in isotonic fluid, they will swell as water enters, increasing the measured MCV

Microcytosis

• Smaller RBCs may be found in breeds like Akita, Shiba Inu, Sharpei, and Siberian Husky dogs, or Abyssinian cats
• Causes of microcytic anemia include iron deficiency/chronic blood loss (most common)
• Other causes of microcytic anemia include portosystemic shunting, copper deficiency, and lead poisoning

Red Cell Distribution Width (RDW)

• RDW is a measurement of the variation in size of RBCs
• RDW (%) is calculated by (Standard deviation ÷ mean) cell volume x 100
• Increased RDW indicates increased anisocytosis
• Anisocytosis is also evaluated visually, which can be somewhat subjective

Mean Cell Hemoglobin: MCH

• MCH is the average amount of Hb in each RBC calculated by (Hb/RBC count) x 10
• MCH doesn't account for cell size, but larger cells tend to have more Hb
• MCHC is generally preferred for RBC assessments
• MCH is not very useful in clinical applications

Mean Cell Hemoglobin Concentration: MCHC

• MCHC is the average concentration of Hb in the RBCs
• MCHC = (Hb/PCV) x 100
• Normal MCHC is classified as normochromic
• Increased MCHC is classified as hyperchromic
• Hyperchromic RBCs do not really exist
• Decreased MCHC is classified as hypochromic
• CHCM, or Cell Hemoglobin Concentration Mean, is the direct measurement of Hb concentration inside RBCs

Hypochromasia: Causes

• Hypochromasia can be caused by regenerative anemias where immature RBCs have less hemoglobin
• Hypochromasia is caused by iron deficiency
• Hypochromic RBCs may be visible on blood film showing larger central pallor area
• MCHC is a more sensitive indicator than morphology

Hyperchromasia

• Increased MCHC is caused by artifacts like hemolysis, Heinz bodies, lipemia, and RBC agglutination
• Large clumps of RBCs aren't counted as erythrocytes by analyzer, but their Hb is measured.

Cell Hemoglobin Concentration Mean (CHCM)

• CHCM is obtained by direct measurement of Hb concentration inside RBCs
• CHCM is similar to MCHC and uses laser backscatter to measure Hb in intact RBCs
• CHCM is less susceptible to hemolysis artifact

RBC Morphology

• Evaluation of a blood film should include RBC morphology assessment
• Morphology is evaluated for size, color, central pallor, abnormal shapes called poikilocytes, and presence of inclusions or infectious organisms

Reticulocytes and Polychromasia

• Most animals accelerate the release of immature RBCs from bone marrow in anemia when marrow response is normal
• Horses don't typically release significant numbers of reticulocytes
• Increased polychromasia or reticulocytosis can be seen in the blood
• Polychromatophilic RBCs are reticulocytes that are named based on the stain used to assess cells

Polychromasia

• Polychromasia are basophilic (bluish-staining) RBCs seen in blood film when using normal hematology stain
• Increased polychromasia indicates increased release of immature RBCs from bone marrow during regenerative anemia
• Polychromatophilic cells contain lower Hb and higher RNA content correlating to decreased MCHC
• Polychromatophilic cells are larger and have less hemoglobin than mature RBCs, which tends to cause ↑MCV, ↓MCHC, and ↑RDW

Basophilic Stippling

• Basophilic stippling is basophilic, dot-like inclusions in RBCs
• Basophilic stippling is caused by aggregated ribosomes
• Basophilic stippling may occur with erythrocyte regeneration in ruminants, or lead poisoning

Poikilocytes

• Abnormally-shaped RBCs are called poikilocytes, or poikilocytosis
• The type of poikilocytes may provide a clue about the causes of anemia/disease
• Echinocytes or crenated RBCs are the most common type of poikilocytosis
• Echinocytes are characterized by uniformly-sized and spaced bumps or “spikes.”
• Echinocytes are most commonly present as a drying artifact without clinical significance
• Echinocytes may occur in clinical conditions like uremia, snake envenomation, glomerulonephritis, RBC dehydration, Electrolyte losses in horses due to sweating and neoplasia
• They are commonly found in normal pigs
• Acanthocytes are similar to echinocytes, but with irregularly-sized or spaced projections
• The theoretical mechanism and cause is increased cholesterol content in RBC membrane
• Acanthocytes may occur with hypercholesterolemia, liver disease (especially in cats- lipidosis), erythrocyte fragmentation, turbulence injury with schistocytes, and young goats, deer or cattle with Hb crystallization

Schistocytes

• Schistocytes, or schizocytes, are small, irregularly-shaped RBC fragments
• They are generally formed by fragmentation of RBCs via increased fragility interacting with fibrin strands, abnormal vascular channels, and turbulent blood flow.
• Schistocyte causes Hemangiosarcoma, Disseminated intravascular coagulation, Glomerulonephritis, Fe deficiency, Heartworm disease/caval syndrome, Liver disease in cats, Vasculitis, and Burns in horses.
• Keratocytes are Acanthocyte-like cells with two “horns” or spikes
• Keratocytes may appear with horns connected as a prekeratocyte or "blister cell"
• Their theoretical mechanisms come from causes are similar to schistocytes
• Eccentrocytes occur when RBC membrane oxidizes and fuses together, excluding cytoplasm from that area.
• Eccentrocytes are Found in oxidative cell injury
• They May form a spherocyte if membrane “flap” is removed

Spherocytes

• On blood smear, spherocytes appear as small, dense RBCs lacking central pallor
• Spherocytes are typically only reported in dogs due to typical presence of central pallor in normal RBCs from other species
• Normal dog RBCs sometimes lack pallor area depending on blood thickness on slide
• Echinospherocytes also have “spikes”
• Spherocytes most commonly result from removal of part of erythrocyte cell membrane without loss of cytoplasm
• RBC has a higher ratio of cytoplasm to membrane, which causes it to form a sphere instead of biconcave disc
• MCV is not reduced, but cell appears smaller because it does not flatten on slide
• Immune-mediated anemia (IMA, IMHA) causing removal of section of RBC cell membrane by macrophages
• Underlying causes of IMA can be Autoimmune, Infectious organisms, or Hapten/drug-associated (penicillin, etc.)
• Spherocytes can be caused by fragmentation injury, oxidation from zinc toxicity, and bee stings or snake envenomation from phospholipases or IMHA
• RBC Ghosts are empty membranes from lysed RBCs
• RBC ghosts can be seen with intravenous or in vitro hemolysis
• RBCs lysed during smear preparation appear as smudged cells, not ghosts
• Leptocytes are abnormally thin (usually large) RBCs
• Leptocytes feature increased membrane surface area vs. volume, and they may appear thin and pale (hypochromic)
• Leptocytes may have excess membrane folds appearing as knizocytes or codocytes
• Codocytes, or target cells, feature excess membrane forms a “bump” in middle of cell
• Codocytes can be considered a type of leptocyte
• Codocyte causes include Immature RBCs, Increased membrane cholesterol, Splenectomy, and Iron deficiency
• Howell-Jolly Bodies are small, dark-staining, usually round inclusions
• Howell-Jolly Bodies are remnants of extruded nucleus
• Howell-Jolly have little clinical significance
• Howell-Jolly Bodies may be increased in regenerative anemias, or following splenectomy
• Heinz Bodies are RBC inclusions formed when hemoglobin is oxidized and precipitates
• They result from crosslinking of sulfhydryl groups in globin protein
• Heinz Bodies feature very indistinct staining with normal stain similar to normal cytoplasm, but lighter
• Heinz Bodies are more prominent on slides stained with New Methylene Blue (NMB)
• Heinz Bodies may appear as bumps on RBC edge, or "clown nose"

Causes of Heinz Bodies: Oxidants

• Onions and Garlic
• Acetaminophen toxicity, esp. in cats
• Zinc or copper toxicity
• Red maple leaf ingestion in horses
• Skunk musk
• Propofol in cats
• Propylene glycol in cats
• Napthalene
• Benzocaine
• Heinz bodies and Heinz body anemia are particularly common in cats due to more sulfhydryl groups in hemoglobin and less pitting function in the spleen
• Normal cats may have low numbers of Heinz bodies in circulation at around 1-5% of RBCs
• Increased Heinz bodies are seen in a variety of conditions in cats like oxidant ingestion, propofol anesthesia, and "metabolic” diseases like diabetes mellitus, lymphoma, and hyperthyroidism
• This is not necessarily associated with anemia
• Nucleated erythrocytes (NRBCs) may be found in circulation
• Most NRBCs are typically metarubricytes, however, earlier stages may be seen
• NRBCs are counted during the leukocyte differential and evaluated as NRBCs per 100 WBC
• NRBCs can appear inflated with very low WBC counts
• NRBCs may be found in normal animals in small numbers at about 0-2 per 100 WBC or 0-3 per 100 WBC
• Some dog breeds including miniature schnauzer, toy poodle, dachshund may have slightly higher numbers of NRBCs at about 0-7 per 100 WBC
• Increased NRBC numbers called metarubricytosis are often seen in conjunction with polychromasia as part of the regenerative response indicating appropriate metarubricytosis
• Increased NRBC number is isn't a sensitive or specific indicator of regeneration
• Other RBC forms like rubicytes may be seen along with metarubricytes
• Increased NRBCs without polychromasia is termed “inappropriate metarubricytosis"
• May result from damage to bone marrow or endothelium
• Presence of early RBC precursors like rubriblasts, and prorubricytes in blood is more suggestive of neoplasia

Inappropriate Metarubricytosis: Causes

• Splenic disease like hemangiosarcoma
• Heat stroke or severe pyrexia
• Hypoxia related to seizures
• Bone fractures if affected area has active marrow
• Endotoxemia
• Chemotherapy
• Lead poisoning
• Bone marrow neoplasia
• Hematopoietic neoplasia: Erythroleukemia, erythremic myelosis, or myelodysplasia of immature RBCs

Anemia

• Anemia is indicated by a decreased RBC mass, or hemoglobin
• Clinical signs include Pale mucous membranes, Weakness, exercise intolerance, Increased heart/respiration rate, and Pica
• Clinical signs are more severe in acute anemia

Compensation for Chronic Anemia

• Patients with chronic anemia typically display milder clinical signs than a patient with acute anemia due to compensation/adaptation
• Responses to chronic anemia include Increased levels of erythrocyte 2,3-Diphosphoglycerate (2,3 DPG), increased cardiac output, redistribution of blood flow, and increased oxygen extraction in tissues like the brain and muscle.
• Acute blood loss anemia also causes clinical signs due to hypovolemia
• Increased RBC 2,3-DPG levels reduce O2 affinity of hemoglobin
• This shifts the saturation curve to the right
• Results in increased release of O2 in tissues

Classification of Anemias

• Anemias are classified by RBC parameters like size andhemoglobin concentration
• Anemias can be regenerative or nonregenerative
• Regenerative anemias feature increased RBC production in marrow to correct anemia
• In "Pre-regenerative” anemias the time scale is too acute for a response, but regeneration should eventually occur
• Nonregenerative anemia indicates lack of an appropriate bone marrow response
• Anemias are Classified based on cause, if known

Regeneration

• Bone marrow shows increased erythroid production
• Increased numbers of immature RBCs, or reticulocytes, are released into circulation with regenerative anemia in most species
• Reticulocytes and Polychromasia can be detected visually or by measurements
• Polychromasia is not usually found in horses
• Reticulocytes can not be directly observed in horses
• Horses with regenerative anemia may have measurable macrocytosis like increased MCV and RDW
• Erythroid hyperplasia in bone marrow can indicate regeneration, but it's difficult to obtain samples

Assessment of Regeneration

• Increased reticulocyte count
• Blood film assessment of Polychromasia, Basophilic stippling particularly in ruminants, Anisocytosis, or increased NRBCs
• RBC indices showing increased MCV or macrocytosis, Decreased MCHC, and increased RDW

Reticulocytes vs. Polychromatophilic Cells

• Reticulocytes and polychromatophils are both terms for immature anucleate RBCs, named based on the stain used
• Routine hematology stain causes Polychromasia
• Reticulocyte stain uses new methylene blue to identify Reticulocytes
• Manual count of reticulocytes involves Mixing NMB stain and blood at a 1:1 ratio
• NMB precipitates and stains ribosomes for viewing
• Cells with visible precipitates are counted as reticulocytes
• 1000 RBCs are counted and the percentage of RBCs that are reticulocytes is calculated
• Advanced hematology analyzers can directly count reticulocytes detected by RNA staining in RBCs oxazine
• Using analyzers is More sensitive than manual count
• Reticulocytes may appear as aggregate reticulocytes, or large clumps of ribosomes, or punctate reticulocytes with small dots
• Feline reticulocytes stain as punctate reticulocytes longer than other species at about 7-10 days, even when the cell appears otherwise mature
• Feline punctate and aggregate reticulocytes are counted separately, and only the aggregate count is used to assess regeneration
• Reticulocyte percentage alone may overestimate the erythropoietic response to anemia
• Even where erythropoiesis is not increased like those with no regeneration, loss of RBCs will lead to increased reticulocyte %
• This issue can be avoided by using absolute or corrected reticulocyte counts
• Absolute reticulocyte count is the number of reticulocytes per microliter of blood
• Absolute reticulocyte count is calculated as RBC count x reticulocyte %
• This provides a more accurate assessment of regeneration than the reticulocyte percentage
• Regenerative response to anemia typically takes 3-5 days to begin
• 7-10 days is needed for full response; however, individual variation can complicate assessment

Regeneration Responses in Large Animals

• Horses do not release significant numbers of reticulocytes; only measurable macrocytosis like increased MCV and RDW may be present.

Regenerative Anemia: Basic Causes

• Hemorrhage is a basic cause of regenerative anemia
• Note that chronic blood loss leads to Fe deficiency & eventually nonregenerative anemia
• Another basic cause is increased RBC destruction via intravascular or extravascular Hemolysis

Acute Blood Loss

• Animals can tolerate loss of 25-30% blood volume
• Loss of 30-40% blood volume leads to hypovolemic shock
• Loss of >50% blood volume leads to death
• Loss of blood won't immediately affect CBC parameters
• Equal loss of RBCs and plasma causes normal PCV
• Normal RBC indices and lack of Polychromasia
• Spleen can contract in dogs, horses, and cats to increase hematocrit triggered by epinephrine release
• Fluid enters vascular space to restore lost circulatory volume via Shift of ECF to vascular space, Water consumption, and Fluid therapy
• The extra fluid dilutes the remaining blood with decreased PCV and decreased plasma proteins
• Anemia may take 12-24 hours to appear

Patterns in Acute Blood Loss

• Features low PCV and low total protein unless it is a very acute condition
• May have low protein but normal PCV in event of splenic contraction
• Internal blood loss such as hemoabdomen may cause low PCV with normal protein
• The absolute count is the number of reticulocytes per microliter of blood
• Calculation is the reticulocyte percentage times the red blood cell count
• Regenerative response typically takes 3-5 days to begin
• A full response typically needs about 7-10 days; however, variations among individuals can complicate evaluation
• Lack of macrocytosis does not rule out regeneration
• Bone marrow aspirate is used to evaluate regeneration. It also helps find causes of decreased production