Hematology session 2

What are the characteristics of a red blood cell on a smear?

• 6-8 u

• 2/3-3/4 the side of a small lymphocyte

• salmon pink cytoplasm

• no nucleus

• center pale area 1/3 of diameter

What is anisocytosis?

variation in the size of a cell

What is being looked at in RBC microscopic evalutions?

size: normo-, macro-, micro- cytic

shape: normal vs abnormal

color: normochromic, hypochromic and polychromasia

distribution: agglutination, rouleaux

What is poiliocytosis?

Variation in shape

What are drop/dacrocytes?

What are target cells?

What are elliptocytes?

What are polychromatophils?

Immature RBCs, no nucleus

What are nRBCs?

nucleated RBCs

What appearance do to platelets have on a slide?

• 2-4 u

• no nucleus

• center filled with granules

What appearance to neutrophils have on a slide?

• 10-14 u

• clear cytoplasm with granules, pink to lavender

• dense nuclear chromatin

• segmented nucleus

What are band neutrophiles?

What are metamyelocytes?

What are myelocytes?

What are promyelocytes?

What are myeloidblasts?

What do eosinophils look like on a smear?

• 12-17 u

• cytoplasm filled with red-orange, spherical granules

• dense nuclear chromatin

• segmented nucleus

What do basophils look like on a smear?

• 10-14 u

• cytoplasm filled with large, blue-black granules

• granules may obscure nucleus, water soluble

• dense nuclear chromatin

• segmented nucleus

What do lymphocytes look like on a smear?

• size varies

• dense nuclear chromatin

• round-oval nucleus

• blue cytoplasm

• ± azurophilic granules

What do monocytes look like on a smear?

• 12-22 u

• blue gray cytoplasm

• irregular shaped nucleus

• less dense nuclear chromatin

• most likely to be vacuolated

What does a peripheral blood smear include?

• WBC estimate

• WBC differential

• RBC morphology evaluation

• platelet estimate

• WBC morphology evaluation

• calculation of WBC absolute values

What causes neutrophila?

• younger forms seen as severity increases

• bacterial infection

• inflammation leukemias

What cause neutropenia?

• drug induced

• viral illnesses

• overwhelming infection

What are some sources of error in the WBC differentials?

• non-random cell distribution

• individual variation in cell identification

• variation in method

• inherent error in evaluating 100 cells

What are some causes of lymphocytosis?

• viral

• infection

• leukemia

• childhood

What are some causes of lymphocyopenia?

• drug induced

• bacterial infection

What are some causes of moncytosis?

• fungal infections

• leukemia

• tb

• post chemotherapy

What causes eosinophila?

• allergies

• asthma

• parasitic infection

• leukemia

What causes basophila?

• allergies

• hypersensitivity reactions

• leukemia

Where is EP produced?

the kidneys

What is another name for a pronormoblast?

rubriblast

What is another name for a basophilic normoblast?

prorubricyte

What is another name for a polychromatophilic normoblast?

rubricyte

What is another name for an orthochromic normoblast?

metarubricyte

What is another name for a polychromatophilic erthrocyte?

reticulocyte

What is pronormoblast?

• 20-25 u

• high N:C ratio

• lacy chromatin

• nucleoli

• basophilic cytoplasm

• no granules

What is a basophilic normoblast?

• 16-18 u

• lower n:c ratio

• coarser chromatin

• nucleoli

• basophilic cytoplasm

• no granules

What is a polychromatic normoblast?

• 12-15

• lower N:C ratio

• coarser chromatin

• blue-gray cytoplasm

• no granules

WHat is the last RBC stage capable of mitosis?

polychromatic normoblast

What is an orthochromic normoblast?

• 10-15 u

• lower N:C ratio

• very coarse chromatin

• oink cytoplasm with blue tint

• no granules

What is polychromatophilic erythrocyte?

• 7-8 u

• no nucleus

• pink cytoplasm

• no granules

What is the function of a RBC?

• carry oxygen to tissues

• remove CO2

• 40% of blood volume

What is the RBC memembrane?

semi-permeable phospholipid layer

What is the major protein of the RBC cytoskeleton?

Spectrin, internal peripheral protein that helps the RBC maintain its shape and integrity

What is glycoprotein?

integral protein, RBC antigen and sialic acid attachment

What are the three layers of the RBC trilaminar membrane phospholipid bilayer?

• outer hydrophilic layer: phospholipid heads, glycolipid, glycoprotein, protein

• central hydrophilic layer: fatty acid tails, protein, cholesterol, phospholipid, lecithin/sphingomyelin

• inner hydrophilic layer: phospholipid heads, protein, attachment to cytoskeleton

What do cholesterol and phospholipids affect?

The surface area of the RBC

What does the LCAT do?

esterifies cholesterol so it cannot enter the membrane

What do increased levels of cholesterol and phospholipids do to the RBC?

Increase the membrane SA forming target cells

What do increased levels of cholesterol without an increase of phospholipids do the the RBC?

increase membrane rigidity and form acanthocytes

What is the function of the RBC membrane?

• maintain cell shape (deformability, oxygen exchange)

• attachment sites for enzymes, hormones and cell signals

• transport of anion and cations

• containment and transport of hemoglobin

What are the K and Na concentrations in the RBC compared to the plasma?

• Na: RBC 6 mmols, plasma 140 mmols

• K: RBC 100 mmols, plasma 4.5

Does the RBC maintain high Ca concentrations?

No, maintains low intercellular concentrations

What is the life span of hemoglobin out of the RBC and in the plasma?

200 minutes

What are the four metabolic pathways in the RBC?

• anaerobic glycolysis

• hexose monophosphate shunt

• rapoport luebering

• methemoglobin reductase

What is the purpose of the embden-myerhoff pathway (anaerobic glycolysis)?

• To provide the RBC with the energy to maintain the membrane and pumps

• 1 mole of glucose is turned to lactate which produces 2 ATP.

What is the purpose of the hexose monophosphate shunt?

• prevent oxidative denaturation of hemoglobin

• produces reduced NADPH and GSH

• 5% of glycolysis, oxidizes glucose-6-phosphate

What RBC inclusion occurs when there is a problem with the hexose monophosphate shunt?

Heinz bodies, hemoglobin precipitates out of cell and attaches to the cell membrane

What does the methemoglobin reductase pathway do?

maintains iron in the functional state as methemoglobin can not carry oxygen

What does the Rapoport/Luebering shunt do?

Produces 2,3 DPG which affect oxygen affinity

What is erythropoietin

A glycoprotein made in the kidneys

What does EPO regulate?

• differentiation of CFU-E to rubriblasts

• rate of release of cells into the blood

What stimulates the release of EPO?

• RBC lysis

• hypoxia

WHat inhibits the release of EPO?

hypertransfusion, high oxygen tension

What does the EPO response directly depend on?

• bone marrow function

• severity and duration of hypoxia/anemia

• adequacy of the EPO response

• amount of iron available

What else regulates the rate of erthropoiesis?

• steroids (testosterone and other androgenic steroids stimulate EPO)

• iron availability (ceiling on EPO response)

What are the five main ways erythropoiesis is regulated?

• oxygen

• iron

• epo

• steroids

• vitamins- folate and B12, help nucleus develop

What is the normal mechanism of destruction in RBCs?

Glycolytic enzymes

• alter membrane

• increase the permeability (net cation on influx with h20 causing osmotic lysis)

• hole in the membrane, surface area to volume increase, sphere shape forms and is removed by the spleen

What is the extravascular mechanism of hemoglobin catabolism?

• porphyrin ring is cleaved

• CO and biliverdin is released

• biliverdin is reduced to bilirubin and released

• bilirubin is bound to albumin in the plasma

• bilirubin is carried to the liver and conjugated

• excreted in the bile

• converted to urobilinogen in the intestine

• most is excreted in the feces but some is reabsorbed and excreted in the urine

In liver damage urobilinogen will?

Be seen in increased amounts in the blood and urine

In hemolysis urobilinogen will?

Be seen in increased amounts in the urine and feces

What happens to free hgb in intravascular catabolism?

What are two stains used for staining reticulocytes?

• supravital stain

• wrights stain

Why does the reticulocyte contain increased amounts of rna?

• to direct protein synthesis after extrusion of the nucleus

What is the purpose of a reticulocyte count?

to index bone marrow activity and RBC production

What is the principle of a reticulocyte counts?

• living cells are exposed to supra vital stain which stains rRNA

• RNA is precipitated and stained

• RNA appears as blue granules in the blue RBCs

• retics counted and reported as %

How many cells are usually counted in a reticulocyte count?

1000 cells

What is the dilution for supravital stain?

equal parts stain and blood

What two components are usually found in supravital stain?

• NMB (new methylene blue)

• BCB (brilliant cresyl blue)

How long after a sample is drawn do you have to preform a blood stain?

8 hours

How long most the retic samples be incubated with the stain?

2-3 minutes

What is the % retic count equations?

number of retics / 10 = % of reticulocyte count

In the miller’s disk method how many rbcs are counted?

109

In the millers disk where are blood cells counted? where are retics?

RBCs are counted in the center square with any retics in the square counted as a rbc. Retics are counted in the whole square.

What is the normal range for retics in the blood?

0.5-2.5%

What do you report if no retics are seen?

<0.1%

What is the corrected retic count for anemia?

% retics x (pt. HCT/45%) = corrected retic

Why must the retic count be corrected for anemia?

fewer blood cells with the same amount of retic = false increase in retic count

What is the reticulocyte production index (RPI)?

A correction performed for the early release of retics from the bone marrow.

What is the RPI equation?

RPI= corrected retic % / F

What is F in the RPI equation?

It is a value determined by the patient’s hct and corresponds to the number of days early retics are released

What does a RPI of 2.0 or more indiacte?

The bone marrow is responding to anemia

What is the equation for absolute retic count?

relative retic count % x RBC count

What are some technique-borne sources of error with the retic count?

• stain: must be supravital, filtered before use and used the the appropriate concentration

• artifacts: refractile inclusions

What are some additional retic parameters on automated instruments?

• CHr or Ret He

• MCVr

• IRF

What are some cell borne sources of error in the retic count?

• Howell-jolly bodies

• pappenheimer bodies

• Heinz bodies

• basophilic stippling

What are howell-jolly bodies?

nuclear remnants that appear when the spleen is absent or impaired

What is basophilic stippling?

Aggregates of ribosomes or ribosomal rna in the rbc cytoplasm

What are pappenheimer bodies?

Abnormal basophilic iron granules inside the rbc

What are Heinz bodies?

Denatured hemoglobin that has precipitated out of the cell and attached to the surface