Erythrocytes Flashcards
RBC anomaly characterized by an increased number of cells with variation in size
Anisocytosis
Normal RBC diameter
7-8um (seen when MCV is 80-100 fL)*
RBC diameter smaller than the nucleus of the small lymphocytes
Microcytes
Average volume of individual RBCs
MCV (Mean cell volume)
Reference range for MCV
80-100fL
Formula employed for the computation of MCV
(Hct/RBC ct. ) x 100
Normal RDW-CV for normocytes
11.5-14.5%
Parameters computed using the RBC histogram
MCV and RDW
Basis of anisocytosis
RDW (Red cell distribution width)
Microcytosis:
Shift to the left/right ?
Left
MCV: 62.3 fL
RDW: 12.4%
Anisocytosis / No anisocytosis?
No anisocytosis * because RDW is normal range (11.5-14.5%)
Refers to the degree of anisocytosis
RDW
Four ways of detecting anisocytosis
- PBS
- MCV
- RDW
- RBC Histogram
RBC Histogram: Shift to the right
Macrocytosis
Tests that can determine the size of the red blood cell
MCV, RBC Histogram
Variation in the normal coloration of RBCs
Anisochromia
Normal color of RBCs
Salmon Pink
Pale area at the center of RBCs
Central pallor * Occupies 1/3 of the diameter of the RBC
Central pallor of >1/3 diameter that is usually microcytic
Hypochromic
Grading of hypochromia which indicates an area of central pallor equal to 3/4 of diameter
3+
central pallor area = 2/3 of the diameter is equal to what grading?
2+
RBCs that lack central pallor
Hyperchromic cells
Increased number of red blood cells with variation in shape
Poikilocytosis
Almost spherical in shape, lacks the central pallor
Spherocyte
True / False:
Natural RBC death can result also to a spherocytic RBC
True
Increased MCHC poikilocytosis
Hereditary spherocytosis
Hereditary spherocytosis characteristics
Hyperchromic, microcytic
MCHC value in hereditary spherocytosis
35-38 g/dL
Elongated RBCs with a slit like central pallor
Stomatocyte aka Mouth cells
Most common form of stomatocytosis
Dehydrated stomatocytosis
Other term of dehydrated stomatocytosis
Hereditary xerocytosis
Difference of normal stomatocyte and hereditary xerocytosis
Hereditary xerocytosis have a puddled end * HX is also a stomatocyte
Red blood cells with irregularly spiculated surface
Acanthocytes (aka thorn / spur cell)
Absence of LDL, VLDL, and chylomicrons in the plasma
Abetalipoproteinemia
Condition that exhibits acanthocytosis
- Abetalipoproteinemia , McLeod Syndrome, Pyruvate kinase deficiency
Bassen-Kornzweig Syndrome
Hereditary acanthocytosis or abetalipoproteinemia
RBCs with regularly spiculated surface
Burr cells / Echinocytes
Pyruvate kinase deficiency may exhibit _ & _ (poikilocytosis)
Burr and Thorn cells
RBC metabolic pathway Pyruvate kinase is related to
Embden-meyerhof pathway
Egg / Oval–shaped RBCs
Ovalocytes
Type of cell exhibited in southeast asian ovalocytosis
Ovalocyte
Cigar-shaped RBCs
Elliptocytes
RBC abnormalities found in Hereditary leptocytosis
- Elliptocyte
- Leptocyte
- Dacrocytes
- Target cells
- Thalassemia is also known as mediterranean anemia / hereditary leptocytosis
Pear/ teardrop shaped RBCs
Dacrocyte / Teardrop cells
Associated condition with dacrocytes
- Primary Myelofibrosis *
2. Megaloblastic anemia
Fragmented RBCs
Schistocytes / Schizocytes
Type of schistocyte present in MAHAs (Microangiopathic hemolytic anemias)
Helmet cells
Generalized over-activation of the coagulation and fibrinolytic systems
Disseminated intravascular coagulation
Kind of leukemia that may produce DIC later on
Acute promyelocytic leukemia
Sickle / crescent-shaped RBCs
Drepanocytes / Sickle cells
Two forms of drepanocytes
- Irreversible sickle cells
2. Oat-shaped cells
Form of drepanocyte that has crescent shape with long projections
Irreversible sickle cells
Form of drepanocyte that can revert to an original biconcave disk when reoxygenated
Oat -shaped cells
RBCs with centrally stained area and a thin outer rim of hemoglobin
Leptocyte / Target cell / Bull’s eye cell / Mexican hat / Codocyte
Stained central hemoglobin
Target cell
Another term for bite cells
Degmacytes
RBC with a semicircular defect in edge
Bite cells
Particular pathway G6PD is associated to
Hexose monophosphate shutn
Folded RBCs
Biscuit cell
* Found in Hb SC disease
Content of basophilic stipplings
Aggregated RNA
Irregular dark blue to purple granules evenly distributed within an RBC
Basophilic stippling / Punctate Basophilia
Stains used for basophilic staining
- Wright
2. Supravital
Clinical conditions associated with basophilic stippling
Lead poisoning *
also found in megaloblastic anemias, thalassemia, alcoholism, arsenic poisoning
Two forms of basophilic stippling
- Fine stippling
2. Coarse stippling
Multiple dark blue irregular granules in Prussian blue iron staining
Siderotic granules
Color of siderotic granules when stained with Wright’s stain
Pale blue
Difference pf Pappenheimer bodies and Siderotic granules
Pappenheimer bodies: Uses New Methylene Blue / Wright’s stain ; Siderotic granules : Uses Iron Stains
Fragments of pyrenocyte left inside the RBC
Howell-Jolly Bodies
Stains used for Howell-Jolly Bodies
- Wright stain
- New Methylene Blue (NMB)
- (+) Feulgen Reaction
Reaction of Howell-Jolly bodies in the feulgen reaction
+
Mitotic spindle remnants
Cabot Rings
Difference of Heinz and Howell-Jolly Bodies
Heinz bodies are situated at the periphery of the RBC, while Howell-Jolly Bodies are situated near the center of the RBC
Pitted Golf ball appearance
Hb H inclusion bodies
Not demonstrated by Wright’s stain
- Heinz
- Hemoglobin H
H= hindi nadedemonstrate nf Wright Stain (HH)
A complex of globin, protoporphyrin, and iron
Hemoglobin
How many O2 molecules can one hemoglobin carry?
4
Component of hemoglobin that is excreted as bilirubin
Protoporphyrin ring
RBC energy production
Anaerobic glycolysis
Shortened RBC survival
Hereditary nonspherocytic hemolytic anemia
RBC transmembrane protein for glucose
Glut-1
Net ATP of the Embden-Meyerhof pathway
2
- 4 produced, 2 consumed
Give the metabolic pathway:
Glucose is converted to pyruvate (pyruvic acid)
EMP
Three diversion pathways
- Hexose monophosphate pathway (HMP) / Pentose phosphate shunt
- Methemoglobin reductase pathway
- Rapoport-Luebering pathway
Give the metabolic pathway:
Diverts glucose-6-phosphate (G6P) to g-phosphogluconate (6-PG) by the action of G6PD
HMP
Give the metabolic pathway:
NADP is converted to NADPH (reduced form)
HMP
Most common inherited RBC enzyme deficiency
G6PD deficiency
Anemia that results from G6PD deficiency
Hereditary nonspherocytic anemia
- G6PD –> NADPH –> GSH
GSH = reduced glutathione from GSSG oxidized glutathione. Aid of G6PD removes oxidation
Oxidizes heme iron from the ferrous (2+) to the ferric state
Peroxide
Ferric state of hemoglobin
Methemoglobin
Another name for methemoglobin reductase
Cytochrome b5 reductase
Reduces methemoglobin
NADPH with the aid of Methemoglobin reductase
A shunt that generates 2,3-BPG or 2,3-DPG
Rapoport-Luebering Pathway
- BPG=bisphosphoglycerate
- DPG=diphosphoglycerate
Enzyme that diverts 1,3-BPG to 2,3-BPG
Bisphosphoglycerate mutase
Diameter of normal RBCs
7-8 um
The average surface area of RBCs
140 um
Normal MCHC (mean cell hemoglobin concentration)
32-36%
MCHC signifies an increase in the internal _
Viscosity
^ Hb = ^ viscous
True/ False
MCHCs greater than 36% shorten the RBC lifespan because viscous cells become damaged as they stretch to pass through narrow capillaries or splenic pores
True
- Seen in cases of hereditary spherocytosis
Confers to the tensile strength of the RBC lipid bilayer
Cholesterol
- ^ cholesterol = ^ strength BUT loses elesticitiy
RBC membrane component which bear the blood group antigens such as the BH and the Lewis blood group systems
Glycolipids (sugar-bearing lipids)
Effect of the disruption in the transport protein function
Osmotic tension of the cytoplasm (results to high viscosity and loss of deformability)
Functions of transmembrane proteins
- Transport
- Adhesion
- Signal
Principal cytoskeletal protein of the RBC
beta and alpha spectrin
- forms an antiparallel heterodimer (tetramer)
Stability of RBC Membrane
Horizonal / lateral membrane stability
Other name for cytoskeletal proteins
Peripheral proteins ( bec. they do NOT penetrate the bilayer)
RBC Impermeable cations
Na+, K+, and Ca+
RBC permeable ions
water, bicarbonate (HCO3-) , and Chloride (Cl-)
Associated disease linked with the decrease in aquaporin 1
Hereditary spherocytosis
95% of the cytoplasmic content of RBCs
Hemoglobin
Normal concentration of hemoglobin with the RBCs
34 g/dL
- MW= 64,000 Daltons
Component of the heme that reversibly combine with one oxygen molecule
Ferrous iron (Fe2+)
- Once ferrous is oxidized to ferric, it can no longer bind to oxygen
Heme ring of carbon, hydrogen and nitrogen
Protoporphyrin IX
Oxidized hemoglobin
Methemoglobin
Predominant Adult hemoglobin
HbA1
Globin composition of HbA
2Alpha, 2Beta
Most characterized of the glycated hemoglobins
HbA1c
Site of heme synthesis
Mitochondria
*through condensation of glycine & succinyl coA
Final step of heme production
Ferrous iron combines with protoporphyrin IX in the presence of ferrochelatase (heme synthase) to make a heme
Plasma protein that carries iron in the ferric state
Transferrin
In the human genome, there is one copy of each globin gene per chromatid EXCEPT
Alpha and gamma globin genes
Highest affinity for beta chain
Alpha chain
- Alpha = pos. charge ; beta= positive neg.
Two alpha and two delta chains
HbA2
Major hemoglobin present from _ months of age until adulthood
HbA1; 6 months
* (<3.5% of total hb)
Two alpha and two gamma chains
HbF (present in 1-2% of total hb)
Volume of oxygen bound by each gram of hemoglobin
1.34 mL
Reference interval for arterial oxygen saturation
96%
State when the hemoglobin tetramer is fully oxygenated
R state (relaxed)
Lower pH reduces/increases the affinity of hemoglobin to oxygen
Reduces
* Hemoglobin readily releases oxygen more
A shift in the curve because of a change in pH / hydrogen ion concentration
Bohr effect
Which has a higher affinity to oxygen?
a. myoglobin
b. hemoglobin
Myoglobin
RBC enzyme which facilitates the diffusion of CO2 into the RBC and combine with water to form carbonic acid
Carbonic anhydrase
Secreted by vascular endothelial cells and causes relaxation of the vascular wall smooth muscle and vasolidation
Nitric oxide
Three types of dyshemoglobins
- Methemoglobin
- Carboxyhemoglobin
- Sulfhemoglobin
Dysfunctional hemoglobins that are unable to transport oxygen
Dyshemoglobins
Type of hemoglobin formed by the reversible oxidation of heme iron to the ferric state
Methemoglobin
Methemoglobin:
Reversible / Irreversible
Reversible
Give the predominant methemoglobin reduction system
NADH-cytochrome b5 reductase 3 / NADH-methemoglobin reductase
<25% methemoglobin level
Asymptomatic
> 30% methemoglobin level
Cyanosis & symptoms of hypoxia
> 50% methemoglobin level
coma / death
Increase in methemoglobin
Methemoglobinemia
Other name for acquired methemoglobinemia
Toxic methemoglobinemia (individuals exposed to an exogenous oxidant)
Administered to patients with toxic methemoglobinemia
Intravenous methylene blue
- Met.blue reduces ferric to ferrous
Common enzyme affected in hereditary methemoglobinemia
NADHY-CYB5R3 (cytochrome b5 reductase 3)
*this enzyme reduces ferric to ferrous / reverts methem to normal
Hb M
Homozygous cytochrome b5 reductase deficiency
Absorption peak of methemoglobin
630 nm.
High levels of methemoglobin color
chocolate brown
- does not revert back to red color after oxygen exposure
Irreversible oxidation of hemoglobin by drugs or exposure of sulfur chemicals in industrial/ environmental settings
Sulfhemoglobin
Results from the combination of CO with heme iron
Carboxyhemoglobin
Affinity of CO for hemoglobin
240x greater than oxygen
Carboxyhemoglobin level of smokers
15% (approx)
* As a result, smokers may have a higher hematocrit and polycythemia to compensate for hypoxia.
*Polycythemia - increased no. of RBCs
Spectral absorption of carboxyhemoglobin
540 nm
Color of the blood in carboxyhemoglobin
cherry red
Reference method for hemoglobin assay
Cyanmethemoglobin method
Normal distribution of hemoglobins in health adults
> 95% HbA, <3.5% Hb A2, 1-2% HbF
Three compartments of iron distribution
- Functional compartment
- Storage compartment
- Transport compartment
Reaction wherein iron reacts with peroxide
Fenton reaction
*peroxide=heme oxidizer
Iron storage of protein that mainly functions within the cell
Ferritin
Accumulates in red blood when iron is not incorporated in the heme and zinc binds to protoporphyrin IX instead
Zinc protoporphyrin
Anatomic sites of the functional compartment of iron
Hemoglobin in the blood ; Myoglobin in the plasma
Form and anatomic site of the storage compartment of iron
Ferritin and hemosiderin in macrophages and hepatocytes
Form of transfer compartment of iron
Transferrin
All cells can store iron except
Erythrocytes
Cage-like protein where ferric iron is stored
Apoferritin
Partially degraded ferritin
Hemosiderin
*Less metabolically available than ferritin
A dietary hormone that reduces ionic iron in the ferric form to ferrous before it can enter the enterocyte
Duodenal cytochrome b (dcytb)
A protein able to bind to ferroportin, leading to the inactivation of ferroportin
Hepcidin
- There will be an increased hepcidin production when the hepatocytes sense that there are adequate stores of iron
Protein that exports ferrous iron into the blood (true absorption)
Ferroportin
Only known protein that exports iron across in cell membranes
Ferroportin
A protein that is able to oxidize iron as it exits the enterocyte
Hephaestin
Once iron is oxidized by hephaestin, it is ready for plasma transport carried by _
Apotransferrin
*Once iron binds, the molecule is known as transferrin
A gene that is EPO responsive, and reduces hepcidin production and increases iron absorption in the small intestine
Erythroferrone
A cell receptor that has the highest affinity for differic transferrin
Tfr1 (transferrin receptor 1)
Plasma proteins that salvage plasma hemoglobin
Haptoglobin and Hemopexin
Haptoglobin receptor=macrophages; hemopexin receptor=hepatocytes
Luminal membrane carrier of ferrous iron in the enterocyte
DMT1 (divalent metal transporter 1)
RBC transmembrane / Integral protein(s) that are related to blood antigens
- Duffy
- Kell
- Kidd
- Rh
- RhAG
Most sensitive and specific test to diagnose hereditary spherocytosis
EMA binding test (eosin-5’-maleimide)
A measure of effective erythropoiesis
Reticulocyte count
Increased reticulocytes
Polychromasia
The first sign of accelerated erythropoiesis and hemolytic anemias
Reticulocytosis / Polychromasia
Normal maturation time for leukocytes
1 day
Production of leukocytes
50 x 10^9/L/day
What makes up a reticulum?
RNA remnants
- reticulum is made up of RNA remnants not the nucleus. Reticulocytes are already anucleated
Decreased reticulocyte count
Reticulocytopenia
True / False
Using a wright stain, you can see the reticulum of the reticulocytes
False. Only supravital stain can stain the reticulum
Normal values for adult and newborn reticulocyte count
Adult: 0.5-1.5%
Newborns: 1.8-5.8% (by 1st two weeks)
Name of reticulocyte when wright stain is used
Polychromatophilic erythrocyte / diffusely basophilic erythrocyte
Hereditary membrane defect concerning membrane structure
- Hereditary spherocytosis
- Hereditory elliptocytosis
- Hereditary ovalocytosis
Most preferred supravital stain for reticulocytes
New Methylene Blue
- consists of sodium oxalate and sodium chloride
Alternative supravital stain for reticulocytes when new methylene blue is unavailable
Brilliant cresyl blue
- Composed of so. citrate and sod. chloride
True / False:
Reticulocytes are not counted in the total RBC count
False. Reticulocytes are counted in the total RBC count
Give the formula for the computation of reticulocytes using standard light microscope
Retic%= (No. of retics. observed / 1,000 RBCs) *100
The total magnification of OIO
1000x
Minimum no. of RBCs counted in the small square (B) of the calibrated miller disk method
112 RBCs
Give the formula for the computation of reticulocytes using the calibrated miller disk method
Retic%= (No. of retics. in square A / No. of retics in square B*9 ) *100
The most rapid accurate and precise method for reticulocyte count
Flow cytometry
Method of counting retics. wherein retics are counted on the basis of optical scatter of fluorescence
Flow cytometry
Degree of difference in the counting results of reticulocytes
Within 20%
Sysmex R-3500 uses what specific supravital fluorescent stain for reticulocytes?
Auramine-o
True/ False
A reticulocyte is considered as a reticulocyte and an erythrocyte in the retic. count using the light microscope method
True
Kasma yung retic sa 1,000 RBCs na immeasure.
Sum of the middle-fluorescence and high-fluorescence ratios
Immature Reticulocyte Fraction (IRF)
- middle-high fluorescence = early indication of erythropoiesis (youngest= higher RNA content = more fluorescence)
IRF and ARF value of chronic renal disease
Low
- kidneys produce EPO. chronic renal disease halts the production of EPO (kasi nga destroyed na siya), therefore there is NO stimulus in the BM to produce RBCs
Another name for the corrected reticulocyte count
Reticulocyte index / Hematocrit Correction / Poor Man’s Bone Marrow Aspirate
When to consider using a corrected reticulocyte count?
When RBC values are low
in order to quantitate retics, you must also count the RBC
Pyruvate kinase deficiency is the most common form of what anemia
Hereditary nonspherocytic hemolytic anemia
Possible PBS finding of PK deficiency
Acanthocytes & ecchinocytes
Deficiency related to emblem meyerhof pathway
PK deficiency
Recommended screening test for PK deficiency
PK fluorescent spot test
Confirmatory test for PK deficiency
Quantitative PK assay
Pathway which aerobically converts glucose to pentose and produces NADPH
Hexose monophosphate shunt
RBC parameters that are elevated in people living at higher altitudes
RBC count, hematocrit, hemoglobin
1g Hb/dL at 2 km or
2g hb/dL at 3 km
Type of G6PD deficiency associated with the fava beans
Type II G6PD Deficiency (specifically the G6PD-Mediterranean variant)
Shift to the left oxygen dissociation curve
Increased Affinity
^pH = ^O2 affinity
Primary molecule responsible for the release of O2 to the tissues
2,3-BPG
Fill in the blanks:
Oxygen dissociation curve: SHIFT to the RIGHT
pH: _
pCO2: _
2,3-BPG: _
Temperature: _
pH: dec.
pCO2: inc.
2,3-BPG: inc.
Temperature: inc.
- inc=increase; dec=decrease
More 2,3-BPG
More oxygen released to the tissues
Binding of O2 to the hemoglobin which promotes the release of CO2
Haldane effect
4 constituents of the hemoglobin
- 2,3-BPG
- Globin
- Protoporphyrin IX
- Ferrous iron
Ferrous + Protoporphyrin
Heme molecule
Globin chains with only 141 amino acids
Alpha and Zeta
- the rest have 146 AAs
Difference of Gamma A and Gamma G
136th position in the AAs is Alanine (Gamma A) and Glycine (Gamma G)
The best, and widely used hemoglobin determination method
Cyanmethemoglobin
Reagent for cyanmethemoglobin method
Drabkin’s reagent
Drabkin reagent component which converts hemoglobin to methemoglobin
Potassium ferricyanide
*potassium cyanide converts methem. to cyanmeth.
Fastest hemoglobin in cellulose acetate electrophoresis
HbH
pH of buffer in cellulose acetate electrophoresis
Alkaline environment (8.4-8.6 pH)
Net charge of hemoglobin in cellulose acetate electrophoresis
Negative —> will go to the Anode (+)
Fastest hemoglobin found in normal individuals in cellulose acetate electrophoresis
HbA1
2nd most fastest in cellulose acetate electrophoresis
HbF
Method used to confirm variant hemoglobins and further differentiates Hb S, D, G and Hb C from Hb E, O(Arab), and C(Harlem)
Citrate Agar
pH buffer of citrate agar
Acidic environment (6.0-6.2 pH)
Slowest hemoglobins in electrophoresis
C, A2, E, C (Harlem) , O(Arab)
True / False
HbS, D and G migrate to the same area at the cellulose acetate electrophoresis
True
Complementary procedure to cellulose acetate Hb electrophoresis
Citrate Agar Electrophoresis
Site of heme synthesis
Mitochondria
Other name for ferroprotoporphyrin IX
Heme
Other name of ferrochelatase
Heme synthetase
Master regulatory hormone for systemic iron metabolism
Hepcidin
Enzyme that inactivates ferroportin
Hepcidin
Site of hepcidin production
Liver
Protein affected by increased hepcidin production
Ferroportin
Protein that transports iron from the tissues to the blood
Ferroportin
True/ False
Iron is normally excreted
False. Iron is recycled
Site of maximal iron absorption
Duodenum and upper jejunum
Enhances the reduction of ferric to ferrous form in intestinal absorption
Duodenal Cytochrome B (DcytB)
*acidic food and gastric acid
Reoxidizes ferrous into ferric
Hephaestin
Transport protein of iron in the blood
Apotransferrin
Abnormal form of stored iron (iron overload)
Hemosiderin
Binds free hemoglobin
Haptoglobin
Binds free heme
Hemopexin
Most sensitive test for IDA
Serum ferritin
Other name for hemochromatosis
Bronze diabetes
Increased tissue iron stores without tissue damage
Hemosiderosis
Increased tissue iron stores with tissue damage
Hemochromatosis
Site of globin synthesis
Ribosomes
Chromosome that dictates the production of Alpha and Zeta
Chromosome 16 – 141 chromosomes
a and z
Chromosome that dictates the production of Beta, Epsilon, Delta and Gama
Chromosome 11 – 146 chromosomes
Adult hemoglobins
HbA1
HbA2
HbF
BFU-E > CFU-E > Pronormoblast
Erythropoiesis
Range of mature RBCs from a single pronormoblast
8-32 (Rodak’s)
How many mature RBCs can be produced by a single pronormoblast
16
Basis of N:C ratio
Nucleus
- -> means that if the Nucleus increases, the ratio increases
- -> if cytoplasm increases, ratio decreases
How many heme groups and how many globin (polypeptides) molecules are in hemoglobin
4 HEME and 4 POLYPEPTIDE / GLOBIN GROUPS
Heme - mitochondia
Globin - _
Ribosomes
Which of the ff. forms of Hgb molecule has the LOWEST affinity for oxygen
a. Tensed
b. Relaxed
Tensed
Tensed–> No O2 (Low affinity for O2)
Relaxed –> With O2 (High affinity for O2)
Wavelengths for dyshemoglobins
- Carboxyhemoglobin - 540 nm
- Methemoglobin - 630 nm
- Sulfhemoglobin - 630 nm
Colors of dyshemoglobins
- Carboxyhemoglobin - cherry red
- Methemoglobin - chocolate brown
- Sulfhemoglobin - mauve lavender */ green
- ———–> Bursa of Fabricius of birds
What type of iron is absorbed in our intestines?
Ferrous (2+)
