Week 3 - RBC eval/Anemia

Erythrocyte Evaluation includes

• Packed Cell Volume (PCV)

• Hematocrit (Hct)

• Hemoglobin (Hgb)

• Red Blood cell count

PCV -definition

Percentage of whole blood composed of erythrocytes

• Used interchangeably with hematocrit

Difference between PCV and HCT

PCV is measures whereas, Hct is Calculated

Potential sources of errors for PCV

• Not mixing sample well; invert tube 5-10x

• Incorrectly reading results

• Centrifuge issues

• Inadequate sample to anti-coagulant ratio = inc. amount of anit-coagulant shrinks RBCs causes a false reading

PCV

• Accurate estimate of RBC mass

• Low cost and rapidly performed in 1-3 minutes

• Can be estimated by Hgb x 3

• Units = %

For a PCV get used to

• Estimating the PCV

• Evaluating the buffy coat

• Looking at plasma color

• EVERY TIME

What is this?

Hematocrit tube

Buffy Coat

Composed of leukocytes, platelets, metarubricytes and reticulocytes

Leukocytosis - buffy coat

Increased/thickened

Leukopenia - Buffy coat

Decreased/small

PCV procedure

1. Line where the clay sealant and RBCs meet at he 0% on the card reader

2. Line the top of the plasma at 100% on the card reader

3. Read PCV where RBCs and buffy coat meet

Buffy coat evaluation

Chromic Lymphocytic Leukemia

WBC - 350,000

Normal Plasma Color

Colorless to pale yellow

Equine plasma color

Typically yellow in color

• due to diet from carotene pigments

Icteric Plasma

Dark, more intense yellow color

Cause of Icteric plasma

Increase bilirubin concentration in the plasma

Bilirubin increase due to:

• Hemolysis

• Liver disease

• Bile duct obstruction

Hemolyzed Plasma

Pink or red colored plasma

What is this showing?

Icteric plasma

Causes for Hemolyzed Plasma

Erythrocytes being lysed/rupturing/destroyed/popping

Due to:

• improper blood collection

• Improper processing techniques

• Accelerated intravascular red blood cell destruction

What is this showing?

Hemolyzed plasma

Intravascular hemolysis

RBCs lysing inside the vessel → typically premature lysing

Extravascular Hemolysis

RBCs are lysed and phagocytized by macrophages outside the vessel → can be normal or premature

Cause of Lipemic Plasma

Excess lipid/fat in the plasma

Commonly found in canines and felines

Due to:

• Animal recently eating a meal

What is this showing?

Lipemic plasma

Lipemic plasma

Milky white in color

Hematocrit

A value that has been calculated by automated instruments

Hemoglobin

• Protein molecule composed of 4 globin chains each bound to a heme group

• Oxygen carrying capacity of the erythrocyte

  • This is the primary job of an erythrocyte

• Can be estimated by Hct/3

• Units = g/dL

Total Protein

TP or TS (Total solid)

TP - Functions of Plasma

• Transporting cells

• Transporting Nutrients

• Excretion of by products and waste

• Maintain homeostasis

• Stabilize body temperature and PH

Lipemia and/or hemolysis

Can falsely elevate TP

Materials for a TP

• Refractometer

• Hematocrit tube

• Kim wipes

How to read a Total Protein

1. Break hematocrit tube where buffy coat and plasma meet

2. Gently tap side of hand on counter to push plasma out of crti tube and onto the prism then close cover plate

OR

2. leave cover plate closed and gently tape the crit tube at the top of the refractometer and push plasma out

3. Read TP where blue and white intersect

4. Clean prism off with Kim wipe

5. UNITS = g/dL

What do we NOT do when we go to read a TP?

Tap the prism directly with the crit tube!!!

Primary Proteins

1. Albumin

2. Globulin

Albumin

• Created in the liver

• Keeps fluid inside the vessels

• Binds and transports ions and hormones

• Provides amino acids to tissues

Globulin

• Includes alpha, beta, and gamma globulins

• Created in the liver

• Gamma globulins (immunoglobulins/IgG) play an important role in the body’s immune defense system

Fibrinogen

• Clotting factor

• Specific beta globulin

• Created in the liver

• Marker for increased inflammation

• Heat precipitation → used in LA

• Clottable (Clauss method) → used in SA

Most common blood tubes used

Red top

Purple Top

Green Top

Blue top

Green top tube

Contains heparin

• used for chemistry

Blue top tube

Contains sodium citrate

Purple top tube

Contains EDTA

• most commonly used for CBC

RBC indices include

MCV, MCH, MCHC

MCV

Volume (size) of average RBC

• Normocytic: normal sized RBCs

• Microcytic: RBCs smaller than normal

• Macrocytic: RBCs larger than normal

MCV equation

Hematocrit (%) x 10/ RBC count

MCV units

femtoliters (fL)

MCH

Amount/weight of hemoglobin in average RBC

• Normochromic: normal amount of Hgb in average RBC

• Hypochromic: decreased amount of Hgb in an average RBC

MCV and MCHC

What we focus on when we describe erythrocytes

MCHC

Concentration of hemoglobin in average RBC; how saturated RBCs are with Hgb

• Most accurate of the indices becuase RBC count is not used in the calculation

• Normochromic: normal conc. of Hgb present in the average RBC

• Hypochromic: decreased conc. of Hgb present in the average RBC

MCH equation

Hemogoblin (g/dL) x 10 / RBC count

MCH units

Picograms (pg)

MCHC equation

Hemoglobin(g/dL) x 100 / Hematocrit (%)

MCHC units

grams per deciliter (g/dL)

Hyperchromasia?

DOES NOT EXIST? the limit does not exist

• false elevation due to:

  • lipemia

  • Hemolysis

  • incorrect calculation

MCH or MCHC truly above RI?

Use Normochromic to describe

What is the MCV?

73.2fL

What is the MCH?

25.4pg

What is the MCHC?

34.6g/dL

Describe the blood cell picture

Macrocytic and hypochromic

Describe the blood cell picture

Macrocytic and Normochromic

Anemia

Decrease number of erythrocytes in circulation

Causes of anemia

• Decrease Production

• Increased destruction (hemolysis)

• Blood Loss (hemorrhage)

Hgb, Hct, RBC count

Values we look at for aniema

• At least 2 have to be decreased to be anemic at the time

• If only 1 is decreased, it may be a sign that the patient may be becoming anemic in a couple of days

Regenerative Anemia

Bone marrow is responding to peripheral demand

• BM increases RBC production

• Release of immature RBCs and into curculation

• Look at RBC indices AND RBC morphology

Non-Regenerative Anemia

Inadequate/no bone marrow response to peripheral demand

“The BIG 6” Morphologic Feature of Regeneration

1. Polychromasia

2. Anisocytosis; not regeneration specific

3. Target cells

4. Howell Jolly bodies

5. Basophilic stippling

6. Nucleated Red blood cells/NRBCs

What does this show?

Polychromasia

What does this show?

Howell-Jolly bodies

What does this show?

Target cells

What does this show?

Anisocytosis

What does this show?

NRBC

What does this show?

Basophilic stippling

What patient (A or B) would be anemic and why?

Patient B

• You can clearly see that there are significantly fewer red blood cells present in patient B’s blood smear than Patient A’s even though they are both being viewed in the monolayer.

Information used to determine the type of anemia

• Microscopic evaluation of the peripheral blood film

• Erythrocyte indices

• Bone marrow response

• Pathophysiologic mechanism

  • functional changes associated with or resulting from disease or injury

Polychromasia

• Macrocytic cell

• Immature RBC

• Basophilic in color

• Large amounts of RNA; given it the blue/purple hue

What is true are polychromatophils and reticulocytes?

ALL polychromatophils are reticulocytes, but not all reticulocytes are polychromatophils

Anisocytosis

• General, non-specific term

• Change in size of the cell

• Can be macrocytic or microcytic

Howell-Jolly bodies

Blue/purple, singular, perfectly round intracellular inclusion

• retained piece of nucleus

Nucleated Red blood cell

AKA Metarubricyte

• RBC that still has its nucleus

Target cells

“Bullseye target”

Can also be seen in liver disease and increased cholesterol

Basophilic Stippling

• Multiple, intracellular, blue/purple dots

• Contains dots of RNA

Microscopic evaluation of the peripheral blood film - anemia signs

Increased number of immature cells on film

Describe the blood cell picture and if this patient is anemic.

• Macrocytic and Hypochromic

• Patient is anemic - RBC count, Hgb, and Hct are all below the RI

Anemia - MCV

• Useful in horses (inc. MCV)

  • don’t release polychromatophils/retics

• Macrocytic anemia

  • usually indicated release of immature cells

• Microcytic anemia

  • Almost always result from iron deficiency

  • MCHC would be decreased

• Normocytic anemia

  • Usually non-regenerative anemia, OR…

  • Acute anemia (too soon for marrow response)

Anemia - MCH

• Tends to inc. and dec. with size(MCV)

• Not as helpful or accurate as MCHC

Anemia - MCHC

Hypochromasia

• increase in immature red cells

• Iron deficiency anemia

Hyperchromasia?

• “the limit does not exist!”

• inc. MCHC = Hgb artifact

  • lipemia, hemolysis, incorrect calculation

Iron Deficiency

• Iron is essential for hemoglobin production

• Inadequate amount of iron results in hypochromatic erythrocytes

• Increased level of central pallor

Causes of Iron deficiency

• Inadequate absorption from GI tract

• Chronic external blood loss

• Iron storages are depleted

• Not enough to create new Hgb

  • external parasites: ticks or fleas

  • GI bleeds

Questions to ask when determining if regenerative anemia

What should the MCV and MCHC be?

• MCV: increased

• MCHC: decreased

What should the blood film look like microscopically?

• macrocytic

• increased number of immature RBCs

3-5 days

The number of days it takes to see an increase in RBCs; the bone marrow needs time to respond to the depletion

7 Days

Peak erythrocyte production

Questions to ask when determining if non-regenerative anemia

Will we see any of “The Big 6”

What should the MCV and MCHC be?

• MCV: normocytic

• MCHC: Normocromic

What should the blood film look like microscopically

Regenerative Anemia Mechanisms

Blood Loss (Hemorrhage)

RBC Destruction (Hemolysis)

Blood Loss (Hemorrhage)

Ex. HBC

Extravascular loss

Chronic or acute: hemorrhoids or ulcers

Internal: into body cavity or tissues

External: skin, GI, urinary, respiratory tract

RBC Destruction (Hemolysis)

Premature RBC destruction

Immune medicated (body attacking itself)

RBC parasite (microfilaria)

Drugs/chemicals

• penicillin, antibiotics, thyroid meds

Often “oxidation” injury

Non-Regenerative Anemia Mechanisms

Defective Erythropoiesis (Internal Marrow Damage)

Immune mediated

Crowding

Decreased Erythropoiesis (external marrow effect)

Defective Erythropoiesis (Internal Marrow Damage)

BM not stimulated to produce new RBCs

Intramedullary; issue with BM

Extramedullary; disease suppressing BM response

Immune Mediated

Intramedullary

Body is attacking its own BM

Crowding

Extramedullary

Neoplastic cells; push out normal RBCs

Think of them as bullies!

Decreased Erythropoiesis (external marrow effect)

Chronic kidney disease (dec erythropoietin)

Inflammatory disease

• Basically, chronic inflammtion suppresses erythropoiesis, dec iron availability, dec. erythrocyte lifespan causing anemia

• Most common cause of non-reg anemia