Lecture 7 Flashcards
Problems in any of these components can cause changes to RBC shape, size, HGB content or cell lifespan
- Nucleus (immature)
- Membrane
- Cytoplasm
- External forces that can alter RBCs
- Central pallor occupies
approximately 1/3 of the cell’s
diameter - wellfilled’ or Normochromic
RBC Nucleus mutations
- Genetic mutations result in abnormal protein production
- Nutritional deficiencies
can affect DNA synthesis - Early release of immature
RBCs - Abnormal cell division can
cause increased or decreased RBC counts - Can also affect the size:
‒Less divisions = larger cells
‒More divisions = smaller cells
Shape of RBC and its characteristic
Biconcave
greater surface area - crucial to RBC gas exchange function
Lipid-Bilayer
Composed of Phospholipids and Cholesterol and its characteristic
-Semi-permeable
-Elasticity & Tensile strength
-Passive transportation of gases, water & glucose
-Active transportation of ions
-Phospholipids can rapidly reseal if there is membrane damage more phospholipids content can change membrane shape
Cholesterol provides tensile strength - but more tensile strength means less elasticity with shorter lifespan
Transmembrane Proteins and its characteristic
-Transport sites, adhesion sites, and signaling receptors found in or on membrane
-vertical membrane stability - membrane integrity
-Adhesion sites – changes permit RBCs to adhere to each other and vessel wall- promoting cell fragmentation, reducing flexibility, and shorten cell lifespan
-loss of transport sites can increase viscosity
Cytoskeletal Proteins and its characteristic
-Skeleton or anchorage structure on cytoplasmic side of membrane
-Overall horizontal membrane stability-supports membrane elasticity
RBC Cytoplasm and its characteristic
- Hemoglobin abnormalities
‒ Globin chain production E.g., Thalassemias
‒ Heme production E.g, Iron deficiency
‒ Assembly of the Hgb molecule ie Enzyme or protoporphyrin
abnormalities - Enzyme mutations
‒ Energy pathway(s) affected E.g., G6PD or PK deficiency
any problem in the cytoplasm has the potential to change the HCB content and shorten the lifespan
Sickle Cell Anemia
-a mutation of the Hgb molecule which causes the Hgb to polymerize, harden and form long protein chains which
elongate within the RBC
membrane
Guidelines for Grading Abnormal Findings:
- 0 – 5 % abnormalities -normal
- 5 – 10% non specific not report
*10 – 25% abnormality moderate - > 25% abnormalities marked
Abnormal RBC ‘Arrangement
Rouleaux
- RBCs in a stacked formation. *Resemble roll of coins
- Increased plasma protein levels causes RBC to stack up and stick together
*four or more RBCs stacked in tail, monolayer, and head of the PBF
* Length of ‘stack’ exceeds the width of RBC
* Central pallor is obscured due to overlapping cells
* blue background stain‒ Excess plasma protein in the smear picks up the stain
- Caused by increased amounts of plasma proteins
‒ Changes surface membrane charge
‒ Reduces zeta potential
‒ Allows cells to become loosely
joined together - Biconcave shape allows RBCs to ‘stack’ in rows rather than ‘clump
- Report only if seen in monolayer
*Seen in ‘head’ or thick end of smear as artifact
‒Multiple Myeloma/Plasma cell Myeloma
‒Chronic Liver Disease
‒Lymphomas
‒Acute and chronic inflammatory disease
Abnormal RBC ‘Arrangement Agglutination
‒RBCs clumped together with irregular mass– haphazard grouping of cells
* RBC antibodies attach to RBC antigens and cause them to randomly agglutinate
- Seen in the tail, monolayer, and head of the PBF
- Caused by IgM antibodies called Cold agglutinins
‒ IgM binds to antigens on the RBC membrane
‒ Antibodies against Ii Blood Group System - Seen in samples at room temperature
‒ Antibody thermal range is 20° to 30° C
‒ No agglutination in patient’s blood at 37° C - IgM antibodies are large and can span the gaps (or zeta potential) between RBCs
- They form a lattice joining RBCs into irregular clumps
‒ Cold Autoimmune Hemolytic Anemia
‒ IgM associated Lymphomas
‒ Multiple Myeloma
‒ Paroxysmal Cold Hemoglobinuria (rare)
Looking for Rouleaux or Agglutination
- Look under 10x
*Confirm under 40x - Look for Background staining with Rouleaux
*Mark as Moderate-obvious with many cells or Marked - most cells
‒Report Background staining
Problems with Arrangement how youd miss it
Rouleaux
Agglutination
- Rouleaux is seen as artifact in the thick area of the
PBF – make sure that you are looking at the right end
of the smear . Look macroscopically and find the tail - If Marked Agglutination is present, WBC & PLT can be
caught up in the clumps or obscured:
‒Do not perform Estimates or Differential - ‘Unable to examine WBC or platelets due to RBC agglutination’ - We must report the abnormal Arrangement
‒Increased protein in plasma - R
‒Cold autoantibodies – A - performing counter-measures (or reflex testing)
*report both Pre- treatment and Posttreatment results
Troubleshooting
Rouleaux
- Cause - increased plasma proteins
- This indicates a specific
disorder - We must reduce the samples plasma protein content to ‘disperse’ the Rouleaux
Saline Replacement
* Replace High-protein plasma with saline
‒ Test small aliquot of WholeBlood
‒ Analyze and make a new PBF
* No excess protein = No Rouleaux
* Now Estimates, Diff & Morph
assessment can be completed
Troubleshooting
Agglutination
- Cause - cold-reacting Autoantibodies
- This indicates a specific disorder
- We must stop the antibody from reacting with RBC to prevent Agglutination
Warm or Pre-Warm Technique
* Pre-warm EDTA sample to 37C
‒ IgM reacts in cold temps
* Warm, mix well & re-analyze
* Make a ‘pre-warmed’ PBF
* IgM can’t react so Agglutination no longer seen
* Now Estimates, Diff & Morph assessment can be completed
Polychromasia
-Large and lumpy compared to the mature RBC
-Lack characteristic central pallor and have a blue hue to their cytoplasm
Polychromatic RBCs or Reticulocytes
* Last stage of RBC maturation
* R.I. Adults 0.5 – 2.5%
Can be increased when:
‒ RBCs lost, damaged or destroyed prematurely
– Simply reported as: Increased Polychromasia
‒Normal or Decreased not reported
Reflex test
* Reticulocyte count -% Retic
‒More accurate than assessing PBF
- Manual- Supravital staining and manual counting
- Automated methods- Immature Reticulocyte Fraction (IRF) – use of fluorescent/ supravital dyes that stain nucleic acid in Retics before counted using fluorescence or absorbance and light scatter
Hypochromasia (Hypochromic)
- > 1/3 central pallor
- decrease or abnormal HGB
synthesis = less HGB/cell - Central pallor is exaggerated. Gradual clearing of central pallor
- Decreased MCH & MCHC on CBC
- Iron Deficiency anemia
- Thalassemia
- Sideroblastic anemia
- Lead poisoning
- Some cases of Anemia of Chronic Inflammation
When reporting consider the degree of hypochromasia and how many RBC are showing the abnormality
* Moderate ½ to 2/3 central pallor
Marked ¾+ central pallor
-to report it should be seen consistently throughout all fields
Hyperchromasia
- Does NOT really exist
- you cant add more HBG into RBCs but the change in shape or membrane loss can concentrate the amount of hgb
- No central pallor – darker colour
Anisocytosis
- Term that refers to variation in RBC volume or RBC diameter on PBF
‒Microcytes, Macrocytes - When two distinct populations of RBCs are seen: dual population or a dimorphic population
‒Typically, a normal population along with an abnormal
population
*E.g., Normochromic /Normocytic and hypochromic/microcytic cell
populations are seen in a treated Iron Deficiency anemia patient
Report, ‘Dual Population’ in RBC Morph box
MICROCYTES
*Caused by More divisions = smaller cells
‒ Due to HGB errors - cells continue to divide trying to reach critical HGB levels
-Decreased MCV (< 80fL)
- Smaller, biconcave RBCs ‒NOT spheroid
- increased central pallor due to low cellular HGB concentration
- Smaller but similar sized
throughout -Thalassemia - normal RBC distribution - Smaller & of varying sizes ‒E.g., IDA with increased RDW
- Iron Deficiency anemia
- Thalassemia (minor)
- Some cases of Anemia of
Chronic Inflammation - Lead poisoning
- Some hemoglobinopathies
- Sideroblastic anemia
MACROCYTES
I. Less divisions = larger cells
‒ Precursor cells are larger
‒ Cells normally decrease in size with divisions & maturation- here they do not. Increases in cell membrane cholesterol = increased size and surface area Increased MCV (> 100fL)
- not as obvious as microcytes
‒RBC can only get so big - Two types- round/oval
‒Round caused by:
* Increased membrane lipids
‒ Increased size and surface area
* Retain shape but more membrane = larger cell E.g., Liver disease
* Reported with SIZE
* MCV moderately increased
‒Oval caused by:
* Less cell divisions
‒DNA replication problem
‒Cytoplasm increases but nuclear division stalled E.g., Megaloblastic Anemia
* Reported as a variation in SHAPE (not size)
* MCV greatly increased
- Liver disease
- Vitamin B12 deficiency
- Folate deficiency
- Neonates (normal newborns)
- Reticulocytosis
Polychromatic Erythrocytes
- naturally larger than mature RBCs ‒Can look oval as well
- not considered ‘macrocytes’ but will affect the MCV and RDW in
increased numbers - We report this as ‘Increased
Polychromasia,’ not as
Macrocytes
Size & Colour Related
-RBCs appear normal ‘Normochromic / Normocytic’
or N/N
● Anemic patients can have N/N RBCs, but the anemia (low HGB) is a result of less numbers of RBCs
-RBCs appear small and with exaggerated central pallor on PBF. Size and color are reported seperately but the RBC are reported as :
●‘Hypochromic / Microcytic’ or ‘Hypo / Micro
● Anemia is result of abnormal HGB production/metabolism
‒ Increased cell divisions with less HGB in cells
