Erythrocytes aka RBC’s Laboratory Procedures
What is Blood? Whole Blood = fluid and cells Fluid Component = PLASMA Cel ular Component • Erythrocytes (RBC)• Leukocytes (WBC)• Thrombocytes (Platelets)
How Are These Cells Produced? • Hematopoesis : the production of blood cel s and platelets. • Erythropoesis : the production of erythrocytes (RBC) In juveniles, blood produced in liver, spleen, thymus and bone marrow. In adults, primary site of erythropoesis is in the bone marrow.During times of hematopoetic stress, the slpeen and liver may produce RBC
Leukopoiesis : the production of leukocytes or WBC Thrombopoiesis : the production of thrombocytes or platelets
Al of these cel s come from ONE CELL!! Proteins cal ed Cytokines are responsible in determining the fate of al stem cel s.
These cytokines determine in the cell will be either : Myeloid : Erythroblasts – Erythrocytes (Erythropoietin) Megakaryocytes –Platelets Myeloblasts –Leukocytes / Monocytes
Cel ular Maturation Diagram
Blood Composition • Separates into three components: • Red Blood Cel s (RBC’s)• White Blood Cel s and platelets (buffy coat) • Plasma • Bottom 1/3 to ½ of tube contains the heaviest of cel ular material (the RBC’s).
Hematocrit=PCV (Packed Cell Volume) • To determine hematocrit, whole blood is centrifuged to pel et the red blood cel s. • Plasma remains on the top of the red cel s.• The fraction of blood that is packed is the hematocrit and is read as a percentage.
Hemoglobin (Hgb) • Normal values are usual y 1/3 of the hematocrit.• Each hemoglobin molecule has 4 heme units attached to globulins. • Abnormal heme groups, cannot carry oxygen.• Carboxyhemaglobin- Hgb has a higher affinity for CO than O . 2 • Bright red blood • Methemoglobin- The Fe molecule is oxidized to Fe+3. • Blood becomes brown.• Tylenol toxicity in cats.
Red Blood Cells • Function: • Carry oxygen to the tissues• Oxygen must be carried at enough pressure to permit rapid diffusion of oxygen. • The RBC is a vehicle for hemoglobin which is the carrier molecule for oxygen. • The sigmoid shape of curve is a result of the cooperative binding from the 4 hemoglobin molecules.
Erythrocytes The morphological features of mature red blood cells of dogs, cats, horses, and ruminants are generally very similar in that they all lack nuclei, stain reddish to reddish-orange. Erythrocytes are biconcave discoid-shaped cells.
The major differences are in the size of the red blood cel s and the degree of central pal or. Listed from largest to smal est in size are : Dog Cat Horse Cow Sheep Goat
Erythrocytes • Maturation of a RBC.• Only occurs in the bone marrow of normal adult animals. • Occurs in the spleen and liver of the fetus. • Maturation time usual y takes 5 days.• Regulated by erythropoietin (EPO) which is increased in the presence of hypoxia. • In most species, the kidney is the sensor organ and major site of EPO.
Red Blood Cells (Erythrocytes) • No nucleus due to have to fold and squeeze through tight spaces. • Normocytes- cel s look normal
Erythrocyte Life Span • Dog- 110 days• Cat- 70 days• Cow- 160 days• Horse- 145 days• Man- 120 days• Mouse- 30 days
Erythrocyte Life Span • Stem Cel → Rubriblast → Prorubricyte → Metarubricyte → Rubricyte → Reticulocyte → RBC • Rubricyte- nucleated RBC releases in severe anemia. • No more mitotic division takes place after this stage. • .
Normal Erythrocytes • Morphologic features vary from species to species • Dogs: Biconcave disk shape with central pal or • Cats: General y round with little central pal or. • Birds: Contain a nucleated RBC
Categories of Erythrocyte Characteristics 1. Cel arrangement on the blood film. 2. Size3. Color4. Shape.5. Presence of structures on erythrocytes
Cell Arrangement on Blood Film • Rouleaux • Rouleaux formation is a group of erythrocytes in stacks. This can be a sign of increased fibrinogen or globulin concentration secondary to inflammation. Can be a norm al • It can also be an occurence!! artifact seen in blood that is held too long before preparing the blood slide or in blood that has been refrigerated. M ost C om m only Seen in H orses!!!
Cell Arrangement Continued • Agglutination • Agglutination, which appears as rouleaux, occurs in immune- mediated disorders. An antibody coats the cel causing bridging or clumping. • If you add a drop of saline to a drop of blood rouleaux formation wil disperse and agglutination wil not
Size • Terms: • Anisocytosis: • Variations in size• Can indicate anemia • Macrocytosis: • Larger than normal cel size• Liver disease or Vitamin B12 deficiency • Microcytosis: • Smal er than normal cel size• Iron deficiency
MCV • Mean Corpuscular Volume • Describes cel s as normocytic, microcytic, or macrocytic. Calculates the average volume of rbc’s. • MCV=(Hematocrit x 10)/RBC count in mil ions • Normal: 66-77
Color • Polychromasia (polychromatophilic): • Polychromatic erythrocytes exhibit a bluish tint. The tint is due to a small amount nucleus retained in the cytoplasm. These are young cells and may appear as a reticulocyte • Hypochromasia: • is a decrease in color, due to a decreased staining intensity caused by insufficient hemoglobin within the cell. • Iron deficiency is the most common cause. • Hyperchromasia (hyperchromatophilic): • refers to cell that appears darker than normal cells. This gives the appearance that the cell is over saturated with hemoglobin. The erythrocyte has a fixed maximum capacity for hemoglobin and over saturation can NOT occur.
MCHC • Mean Corpuscular Hemoglobin Concentration • describes cel s as normochromatic or hypochromatic. • MCHC= (Hgb)/(Hct) x 100 • Normal is 31-36% • (we wil come back to this calculation again)
Hypochromasia Usually associated with iron deficiency. Especially in Llamas
Hypochromasia continued • Hypochromatic should be differentiated from cel s with the center “punched out”. A punched out appearance can be an artifact due to improper smear technique
Shape • Poikilocytosis • Poikilocytosis is a major deviation in the normal shape of the erythrocyte. The term poikilocytosis is an umbrel a term that is used for any and al abnormal y shaped erythrocytes and does not suggest a specific diagnosis
Schistocytes (Fragmented Cells) • Also known as poikilocytes.• RBC’s with abnormal shape.• Formed as a result of shearing of the cel by fibrin strands. This occurs when red blood cel s rapidly pass through microvasculature that is lined or meshed with strands. • They are observed in fragmentation hemolysis caused by DIC, vascular neoplasia, endocarditis, and possibly iron deficiency anemia.
Acanthocytes (Spur Cells) • The term acanthocyte is derived from the Greek word “acanthi” meaning “thorn” Acanthocytes are cel s with five to ten irregular, blunt, finger-like projections. • The projections with vary in width, length and surface distribution. These cells are seen in animals with altered lipid metabolism such as cats with hepatic lipidosis or dogs with liver disease.
Echinoctyes (Burr Cell) • Echinocytes have multiple, smal , delicate regular shaped spines evenly distributed around the cel and are indistinguishable from artificial y crenated cel s.
Echinoctyes Continued • Echinocyte formation can be artificial, often seen with slow drying blood films or if the EDTA tube was underfilled. This artifact is then termed crenation. • Echinocytes have been associated with renal disease, lymphosarcoma and rattlesnake bites in dogs. • They can be seen after exercise in horses.
Crenation • Identified as the presence of many irregular membrane projections involving most RBC’s. • It is usual y an artifact due to slow drying of the blood film. • Commonly observed in pig blood but can be seen in any species.
Drepanocytes (Sickle cell) • These cel s are crescent shaped with pointed ends. • Drepanocytes are often seen in normal blood of deer and goats. It is thought to be a result of low oxygen tension.
Keratocyte (Helmet/Blister Cells) • Also cal ed blister cel s or bite cel s. Keratocytes are associated with trauma especial y cel ular damage from contact with fibrin strands.
Prekeratocytes • Cel s with pseudovacuoles are cal ed blister cel s or pre-keratocytes.
Spherocytes • Cel s have a spheroid shape instead of the usual biconcave disk shape. • Have reduced cel membrane and are hypochromatic. • Seen most frequently in autoimmune hemolytic anemia (AIHA). When WBC partial y remove antibody-coated membranes. • Usual y seen in dogs.
Stomatocytes • The appearance of stomatocytes with their oval or rectangular central pal or has been compared to a smiling face, a fish mouth, and a coin slot. • Stomatocytes are associated with an hereditary condition but are also seen in liver disease, acute alcoholism (humans), and electrolyte imbalances.
Anulocytes • These are bowl shaped erythrocytes that form as a loss of membrane flexibility that does not al ow the cel to return to a normal shape after passing through a capil ary. They can occur due to lowed hemoglobin concentration or as an artifact.
Dacryocytes (tear drop cells) • These tear drop shaped cel s are seen in myeloproliferative diseases. These cel s can be produced as an artifact but can be identified by the direction of their tail
• Dacryocytes produced as an artifact have their tails pointing in the same direction.
Target Cells and Folded Cellsaka Codocytes • Two types of cel s observed mainly in dogs. • Represent cel s with an outfolding of the rbc membrane. • The cel membrane is thin and flimsy.• Can be associated with liver dz and reticulocytosis.
Target Cells (Mexican Hat Cells) • Also cal ed codocytes• Thin, bel -shaped cel s• Central y stained area• May be seen as artifacts when smearsmade in high humidityor if blown dry. Can indicate liver disease or hemoglobinopathies.
Howell-Jolly Bodies • Nuclear remnants observed in young erythrocytes.• Often observed in cats and horses.• Can be seen in regenerative anemic animals.• Also may be seen with splenic disease or in an animal with the spleen removed.
Nucleated Red Blood Cells (NRBC’s) • Nucleated red blood cel s (NRBC) usual y represent early release of immature red blood cel s during anemia. • These are nuclear remnants seen in young erythrocytes during a response to anemia.
Nucleated Erythrocytes • Metarubricytes are sometimes found in the peripheral blood. • Usual y observed with regenerative anemias. • May be found in non-regenerative states such as lead poisoning and hypoxia.
Basophilic Stippling • Observed in RBC’s that contain abnormal aggregation of RNA. • Can be observed in cases of heavy metal poisoning with non-regenerative anemias or intense erythrogenesis in dogs, cats, and ruminants.
Heinz Bodies • Particles of denature hemoglobin protien.• They stain with new methylene blue and appear as colorless bumps with quick stain. • May be caused by oxidant drugs and chemicals.• Normal cat blood may have 2-3%.• Spleen recognizes as abnormal and starts to lyse the cel s.
Reticulocytes • These are immature red blood cel s that contain organel es (ribosomes) that are lost in the mature cel . Cats have two forms of reticulocytes. • The aggregate form contains large clumps of reticulum. • The punctate form, unique to cats, contains two to eight singular granules
Reticulocytes in the Peripheral Blood • Non-nucleated cel containing RNA which can be easily seen when stained with methylene blue. • Hal mark of erythrocyte regenerative response.