HEMATOLOGY 1 - MIDTERMS

NORMOBLASTIC MATURATION
- pertaining to the maturation of RBCs
RED BLOOD CELLS
- aka Erythrocytes or Discocyte (Biconcave appearance in shape)
- Primary function: Carry oxygen from the lung to the tissues; and carry Carbon Dioxide from tissue to the lungs
- Secondary functions: Maintaining homeostasis, providing nutrition, (Prelims - Intro)
3 NOMENCLATURE IN NAMING RBC PRECURSORS:
A. Erythroblast Terminology (Europe)
B. Normoblastic Terminology (USA) - Descriptive of the appearance (General appearance)
C. Rubriblastic Terminology - Used by some since it parallels the WBC development nomenclature
** Normoblastic and Rubriblastic are commonly used; Rubriblastic terminology is only added by the American Society for Clinical
Pathology (ASCP) Organization because to denote the stages after the -blastic (refers to as primitive) stage which the maturation
stage of the cells (uses the suffix -cyte)
NOMENCLATURE FOR ERYTHROID PRECURSORS:
NORMOBLACTIC RUBRIBLASTIC ERYTHROBLASTIC
Pronormoblastic Rubriblast Proerythroblast
Basophilic Normoblast Prorubricyte Basophilic Erythroblast
Polychromatic Rubricyte Polychromic Erythroblast
(Polychromatophilic) Normoblast
Orthochromic Normoblastic Metarubricyte Orthochromic Erythroblast
Reticulocyte Reticulocyte Reticulocyte
Erythrocyte Erythrocyte Erythrocyte
**Peripheral smear is not used to identify these cells but rather bone marrow aspirate (not readily available in laboratory setting -
invasive procedure)
GUIDELINES TO IDENTIFY MATURATION OF RBC:
A. Decrease in Cell Size - as the cells mature there must a progressive decrease on the cell size (Pronormoblast > Erythrocyte)
B. Decrease in Cytoplasmic Basophilia - as the cells mature there must a progressive decrease on the cytoplasmic basophilia (From a
very dark blue color which will fade into a pink or red - color of mature erythrocyte)
**Cytoplamic basophilia - appearance of the cytoplasm is blue; presence of cell organelles particularly the ribosomes
C. Chromatin should be condensed as cells matures
** Chromatin is seen inside of the nucleus; made up of DNA, histones and other proteins - as the cell mature the chromatin will be
condensed or will clump just before the nucleus is excluded out of the cell
** As the chromatin condenses the diameter of the nucleus will become smaller
D. Nucleus - Round to oval shape
** Nucleus will be become very round just before extrusion

MATURATION PROCESS
ERYTHROID PROGENITORS
1. BFU-E (Burst Forming Unit - Erythroid) - 1st Erythroid; Forming Cell; 1 week to mature, earliest erythroid-committed cell
**use of KIT Ligands, IL-3, TPO, G-CSF convert BFU-E to CFU-E
2. CFU-E (Colony Forming Unit - Erythroid) - More receptors for EPO (Erythropoietin); 1 week also towards becoming the
pronormoblast
- 6 Days - Precursors to mature
- 18-21 Days - To produce a RBC (from multipotential stem cells to erythrocyte)

MATURATION SEQUENCE
**Heterochromatin - takes up the Basic Dye, Condense portion, Inactive portion of of chromatin
Euchromatin - cannot take up the Basic Dye, Active portion of the chromatin
Parachromatin - Light stained portion of the chromatin (more appreciated if the chromatin is already very condensed)

** High NC (Nucleus-Cytoplasm) Ratio means that the nucleus takes most part of the cell than the cytoplasm
** Pink color associated with hemoglobin production
** Blue color associated with cytoplasmic basophilia (present organelles especially ribosomes)
1. PRONORMOBLAST (Rubriblast/ Proerythroblast)
Nucleus: High (8:1 - Nucleus: Cytoplasm), Round to oval with 1 or 2 nucleoli
Cytoplasm: Deeply basophilic (Blue) - due to ribosomes, Golgi Apparatus- Light Stained structure beside nucleus
Division: Mitosis (2 Pro-normoblast)
Location: Bone Marrow
Size: 18-20 um
Cellular Activity: Production of proteins and enzymes are produced in the cell that is needed for iron uptake and protoporphyrin
synthesis; globin production begins
**Heme - protoporphyrin (synthesized in the cell) + iron
Length of time in this stage: More than 24 hours
**Mistaken for myeloblast - Less cytoplasmic basophilia and fine lacy chromatin pattern

2. BASOPHILIC NORMOBLAST (Prorubricyte/ Basophilic Erythroblast)

Nucleus: Ratio (6:1), Chromatin begins to condense, staining reaction is deep purple red
** Because of the condensation of chromatin the parachromatin area becomes larger
Cytoplasm: Deep Blue (Basophilic)
Division: Mitosis - 4 Daughter Cells
Location: Bone Marrow
Size: 16 um
Cellular Activity: Detectable hemoglobin synthesis occurrence; still with the presence of organelles blocking hemoglobin production
Length of time in this stage: Slightly more than 24 hours
**No more nucleoli - end of the basophilic normoblast stage
3. POLYCHROMATIC NORMOBLAST (Rubricyte/ Polychromatic Erythroblast)
Nucleus: Ratio (4:1), Chromatin condensation, No nucleoli
Cytoplasm: 1st stage redness is associated with hemoglobin and concurrent decrease of RNA. Mixed pink and blue (Murky gray-blue
or muddy gray-blue) - polychromatic
Division: Mitosis (Last stage capable) For each rubricyte: 2 daughter cells (8 Daughter Cells)
**as the chromatin condenses (3rd stage) which indicates a smaller nucleus which is not anymore capable of mitosis
Location: Bone Marrow
Size: 13 um
Cellular Activity: Hemoglobin synthesis is increasing and the accumulation is visible in the cytoplasm (redness of cell); organelles are
still present
Length of time in this stage: 24 hours
4. ORTHOCHROMIC NORMOBLAST (Metarubricyte/ Orthohromatophilic Normoblast)
Nucleus: Pyknotic (Dense mass of degenerated chromatin) completely condensed or nearly so. Low NC ratio (1:2) BLUE BLACK
SPHERE NUCLEUS IS EXTRUDED AT THE END OF THIS PHASE
Cytoplasm: Pink-Orange Color (nearly complete hemoglobin production) - nearly same as the eosin stain
Division: NONE
Location: Bone Marrow
Size: 8 um
Cellular Activity: Continuation of hemoglobin production, nucleus is ejected (contractions)
**Use of cytoplasmic projection (nucleus will go to the pseudopod-like projection and will be later on pinched off from the cell)
Macrophages (particulary spleenic macrophages) will eat the pinched off pseudopod
Howell-Jolly Bodies - RBC Inclusion flaw (not all the nucleus is extruded because it didnt fully enter the projection)
Length if time in this stage: Approximately 48 hours
5. RETICULOCYTE (Diffusely Basophilic Erythrocyte, Polychromatophilic Erythrocyte)
Nucleus: NONE
Cytoplasm: Predominantly the color of hemoglobin (Salmon Pink/ Red Color)
Division: NONE
Location: In Bone Marrow for 1-3 days; then Peripheral Blood for 1 day (Normal Range: 0.5-1.5%)
**Reticulocyte is a good index of your erythropoiesis or bone marrow activity - compensated or uncompensated anemia
**When to use Reticulocyte term of Polychromatic Erythrocyte term? - based on what was the stain used
Polychromatophilic - combination of red (production of hemoglobin) and blue color (ribosomes)
Wright Stain (uses fixative - not anymore living state of the stain) - Polychromatophilic Erythrocyte term used
Supravital Stain (staining a living state cell) - Reticulocyte term used (Brilliant Cresyl Blue or New Methylene Blue) 15-30 minutes -
presence of reticulin (aggregated RNA remnants) - better than Wright Stain (can be more differentiated)
Size: 8 um
Cellular Activity: Completes the hemoglobin production and endoribonuclease digest the ribosomes
Length of time in this days: 2 Days
6. ERYTHROCYTES
Nucleus: NONE
Cytoplasm: Mature cells are biconcave disc - Salmon-pink in color when stained with a central area (concavity) - Central Pallor (1/3 of
the cell size)
Division: NONE
Location and Length: Active approximately 120 days in the circulation
Size: 7-8 um
Cellular Activity: Delivers oxygen to tissues, releases it, and returns to the lung to be re-oxygenated
** can use Radioactive Chromium (51Cr) - To assess RBC Survival
Biconcave Disc - appearance is important to deliver its function (hemoglobin is in the middle - it would be pushed to the membrane
of the cell - easier delivery of oxygen) and also flexible
**Human Leukocyte Antigen (HLA) - Surface antigens - present in nucleated cells (but also present in Red Blood Cells because RBC
was nucleated in the past)
HLA of RBCs: Bennet - Goodspeed Antigen (BGAG)
** How many mature RBCs are formed from a single rubriblast?: 16 RBCs
Last stage capable of Mitosis?: Polychromatic Normoblast
Last nucleated stage?: Orthochromic Normoblast

ERYTHROPOIESIS AND ERYTHROCYTE PHYSIOLOGY

ERYTHROCYTES
- After 18-21 Days, 16 Red Blood Cells will be formed from 1 Pronormoblast
- Development is confined mainly on the Bone Marrow
- Survival of RBC 120 Days
- Main Function - Carry oxygen to the tissue from the lungs and carry Carbon dioxide from the tissue to the lungs
- Main Component of Mature Erythrocyte: HEMOGLOBIN (Respiratory protein of the RBCs)

ANTON VAN LEEUWENHOEK

- 1673: Used the improved microscope and gave the first real description of the blood as SMALL ROUND GLOBULES
- Not studied thoroughly in the past, but today even the slightest alterations in the complicated feature (ex. size, color, shape) can be
associated with numerous disorders (usually life-threatening)

ERYTHRON
- Considered as a functioning unit
- Involves the Erythrocytes and its precursors
- Can deliver oxygen via hemoglobin in the RBCs
- Removal of CO2 waste products
**Erythron - Entirety of the blood (involves the precursors which are found in the bone marrow so it assesses the entirety of the
blood) while RBC Mass pertains only to the RBCs found in the circulation
RBC
- Life Span (120 Days) can be asses by Radioactive Chromium (51Cr)
- It only lasts for 120 days (Senescent RBCs) because it has no organelles especially the nucleus so it cannot replenish the enzymes
and Mitochondria for proteins and Hemoglobin are not synthesized
ERYTHROCYTE PRODUCTION
(**Review Erythropoiesis)
BFU-E (Erythroid Burst Forming Unit) - Earliest Erythroid-Committed cell
**use of KIT Ligands, IL-3 (Inteleukin 3), TPO (Thrombopoietin), G-CSF (Granulocyte Colony Stimulating Factor) convert BFU-E to CFU-
E
CFU-E (Colony Forming Unit - Erythroid) - More receptors for EPO (Erythropoietin)

**Erythropoiesis is a constant process

Why do Males have higher Red Blood Cell values than Females?
- Not necessarily because of Menstruation
**- Hormones in Males: Testosterone (Stimulant for Erythropoiesis) while Hormones in Females: Estrogen (Inhibits Erythropoiesis)
Males who secretes less testosterone have lower value of Hemoglobin (1-2 g/dL less than the normal results of Hemoglobin values)

STIMULATORS
1. ERYTHROPOIETIN (EPO) - Produces mainly in the Kidney particularly in the Peritubular Cells
- Can cross the placenta
- First human Hematopoietic Growth Factor identified
**EPO Gene is located in Chromosome 7
2. HYPOXIA - High altitudes; Can also induce Erythropoiesis. Hypoxia will signal the body cells that the body needs oxygen so the
Bone Marrow needs to release more RBCs
3. HORMONES - Growth Hormone, Thyroid Hormone, Steroid Hormone, Insulin, Monocytes, Macrophages
**Monocytes and Macrophage ndividually they can stimulate Erythropoiesis, but when combined with other cells they may inhibit
Erythropoiesis (regulatory mechanism)
4. T-CELLS - Just like the Monocyte and Macrophages when combined with other cells may inhibit Erythropoiesis (ex. Red Cell Aplasia
and Leukemic states)
 INHIBITORS (To regulate the process in the body)
1. MONOCYTES, MACROPHAGE, AND T-CELLS - Combined with other cells may inhibit
Erythropoiesis
2. VIRUSES
3. INTERFERONS - Signaling proteins and triggered by the cells attack by the virus
4. UREMIC TOXINS IN RENAL DISEASE - When the Kidney is affected the production of Erythropoietin is also affected
5. ALCOHOL - Particularly affecting the Liver which have a vital role to Erythropoiesis

PRODUCTION SITES
- Bone Marrow specifically:
- Flatbones
- Proximal Ends of the Long Bones
**Iliac Crest - safest spot to collect sample for Bone Marrow Biopsy

ERYTHROCYTE
MATURATION

RED BLOOD CELL/

ERYTHROCYTES
- Circular/ Spherical
particle (Term: Biconcave
Disk)
- Homogenous disks of
nearly uniform sizes
- Henrys: 6-8 um; Previous
information: 7-8 um
- But in normal blood
there is an allowance (still normal): small as 5.5 um to large as 9.5 um
** Less than 5.5 um - Microcytes; More than 9.5 um - Macrocytes
COLOR
- Center is pale
**The red color of RBC is indicated by Hemoglobin, the center is where they are concentrated
-Chromic - pertaining to Hemoglobin content
Normochromic Normal Normal
Hypochromic Decreased Hemoglobin Increased Central Pallor
Hyperchromic Increased Hemoglobin Decreased Central Pallor
**Anisochromasia - Unequal distribution of Hemoglobin in RBCs
- Able to see Normo, Hypo, and Hyperchromic RBCs in a Peripheral Blood Smear (PBS)

- Seen in Anemia caused by Iron Deficiency

- Normally seen in patients undergoing blood transfusion - mixture of healthy and normal RBCs to the patients RBCs

HYPOCHROMIA GRADING
1+ Area of the central pallor is one-half of cell diameter
2+ Area of pallor us two-thirds of cell diameter
3+ Area of pallor is three-quarters
4+ Thin rim of hemoglobin (Less than 50 g/dL)
Normal Central Pallor size: 1/3 of the RBC
 HYPOCHROMIA
- Usual partner is MICROCYTIC (usually termed as Microcytic-Hypochromic Anemia)
- Hypochromia is normally seen in Iron-Deficiency Anemia (IDA) and Thalassemia

POLYCHROMATOPHILIA / POLYCHROMASIA
- Usually appearing when using Wright stain
- Blue-Gray tint because of presence of RNA Remnant (more appeciated with the use of Brilliant
Cresyl Blue or BCB) - causing bluish tinge of the cells
Increased Polychromatia
- can be also termed as Reticulocytosis
- seen in marked hemolysis and acute blood loss
**Reticulocytes are normal: 0.5-1.5%, beyond that range would indicate Reticulocytosis
- Termed as Shift-Reticulocytes
**Remember the Reticulocytic maturation stage, the reticulocytes it will stay in the bone marrow for 1-3 days and in the peripheral
blood smear for 1 day. For stress environment (ex. Acute blood loss) the bone marrow is forced to release the reticulocyte in less
than a day - it will be shifted from the bone marrow to the circulation (PREMATURE RELEASE OF RETICULOCYTES) known as Shift-
Reticulocytes
- Reticulocytes are sometimes referred to as Polychromatophilic Erythrocytes especially in the Peripheral Blood Smear because of
the use of Wright stain used to stain PBS (Primaquine - Modified Wright Stain)
- Alteration of color of the cell cytoplasm it will indicated IMMATURE CELLS

POLYCHROMASIA GRADING
Percentage of Red Cells that are Polychromatophilic
Slight 1%
1+ 3%
2+ 5%
3+ 10%
4+ >11%

HYPERCHROMASIA
**NO GRADING FOR HYPERCHROMIA (Increased MCHC, Increase HGB content - due to a decrease or reduced surface area to
volume ratio)
- forced increase because space is reduced

SPHEROCYTES

- Hyperchromia
- No Central Pallor due to increased thickness

SICKLE RED CELLS

- Seen in patients with Sickle Cell Anemia (SCA)

- Do not show normal central pallor because the morphology is altered
- Patients with SCA resistant to Plasmodium (Resistant to Malaria)

HEMOGLOBIN CC AND HEMOGLOBIN SC CRYSTALS

Hemoglobin CC Crystals Hemobin SC Crystals
- Genotypes of Hemoglobin
- Hemoglobin is concentrated within a crystal (not having the normal concavity )
- HGB CC Crystals: Parallel Hexagonal Crystals
- HGB SC Crystals: Washington Monument
**Big RBCs with no central pallor that are seen in the end of the feathery-edge of the PBS is not considered hyperchromia (only in
increased MCHC) but rather just an ARTIFACT
SIZE
- Microcyte
- Macrocyte
- Normocyte
ANISOCYTOSIS
- Has variation of cell sizes in PBS
**MCV - not only focus on the cell diameter/size but also the cell volume
MCV - Has more meaning if regarded as cell volume than cell diameter
NORMOCYTIC RBCs
- MCV: 80-100 fL (Femtoliter)
- Normocytic cells can be found in pathologic conditions such as Acute Blood Loss, Aplastic Anemia, and Hemolytic Anemia
MACROCYTES
- 8 mm or larger in diameter
- MCV is greater than 100 fL

Evaluate Macrocytic Cells for:

A. Shape (Round vs Oval)
B. Color (Red vs Blue)
C. Pallor (If present)
D. Presence or Absence of RBC Inclusions
Why are Macrocytes seen in the circulation?
**Remember Maturation Stage: No mitotic division if there is no nucleus (presence of DNA)
Lack of Vitamin B12 and Folate it will interfere proper DNA synthesis which will cause the nucleus to be abnormal which halts
Mitosis
- For every Mitosis the RBCs size decreases - 6-8 um, if lacking of Vit B12 and Folate - LARGE RBCs will be released to the circulation
(did not undergo proper Mitosis)
ACCELERATED ERYTHROPOIESIS
- Ending in a premature release of Reticulocytes which will be termed as Shift-Reticulocytes **refer to notes above
- Reticulocytes are bigger than mature Erythrocytes (considered as Macrocytes)
Membrane Cholesterol and Lecithin are Increased:
- Cholesterol and Lecithin are important for the cell membrane of RBCs when altered (increased) it will cause enlarged RBCs which is
particularly seen in OBSTUCTIVE LIVER DISEASE
- Lacking of Vitamin B12 and Folate causes MEGALOBLASTIC ANEMIA
**Post-Splenectomy - Spleen is not present to filter out flawed RBCs (ex. Macrocytes)
Chemotherapy - alter proper DNA synthesis
MICROCYTES
- Diameter less than 7 um
- MCV is less than 80 fL
- Any defect impairing Hemoglobin synthesis results in Microcytic-Hypochromic RBCs
**The maturation of the nucleus is more abundant than the cytoplasm
- Decrease in Hemoglobin synthesis results in an increase cellular division and consequentially small cells
Causes:
- Ineffective Iron Utilization, Absorption, or Release
- Decreased or ineffective Globin Synthesis

** TAILS - Thalassemic Conditions, Anemia of Chronic Disease, Iron Deficiency, Lead Poisoning, Sideroblastic Anemia
Common: Thalassemia and IDA - Microcytic-Hypochromic Anemia

RBC COLOR VARIATION

- Correlates with MCHC (NV: 32-36%)
RBC SIZE VARIATIONS
- Anisocytosis (alterations in the size of the RBCs)
- Correlate with MCV and RDW (Red Cell Distribution Width)
**RDW is the machine parameter for Anisocytosis in automation (*marked anisocytosis)
RDW Normal Values: 11.5% - 14.5%
SPHEROCYTOSIS
-RESULTS: Increased MCHC (reduced surface area to volume ratio), Decreased MCV to Normal
- The MCV is in the normal range though many of the cells have smaller diameter, their volume is not decrease because they are
thicker than normal
**MCV is not only about the diameter but also the volume of the RBCs - Spherocytes are very thick which can be accounted for the
volume so NORMAL range is still possible
MICROSPHEROCYTES
- are usually seen in burnt patients; Decreased MCHC and Decrease MCV
NUCLEATED RED BLOOD CELL
- Usually Polychromatophilic Normoblast
- Seen in the extreme demand of the marrow (compensation for loss of blood supply), Extramedullary Hematopoiesis (exhaustion of
fat reserves), Marrow Replacement
- Seen in patients with Hemolytic Disease of the Newborn (HDN)(Mother is Rh -) and Thalassemia Major
SHAPE AND STRUCTURE
- Poikilocytosis (variation of shape)
- Abnormalities in shape see RBC anomalies
- Decreased ESR - no rouleaux formation due to altered morphology of the RBCs
Poikilocytosis Secondary to Developmental Macrocytosis
- another type of Poikilocytosis
- mature cytoplasm but immature nucleus

ERYTHROPOIESIS AND ERYTHROCYTE PHYSIOLOGY

REVIEW
- Mature RBC has no nucleus and mitochondria
- Main component is hemoglobin
SHAPE AND DEFORMABILITY
- Important
- Discoid = Discocyte
- Biconcave appearance for maximizing Surface Area : Volume Ration
- Allow cell flexibility or deformability
- Allows to adjust in masculature
- Altered ratio (prone to lysis or fragmentation)
- E.G. Spheroid Shape: Membrane loss (Decrease surface area) or Increase uptake of cations and water (Increase volume)

MEMBRANE COMPOSITION AND STRUCTURE

Functions:
*PPT
A. Separate Intracellular and Extracellular Fluid environment of the plasma
B. Allows ion and nutrient passage
C. Allows cell to deform when required
*FROM MAAM PLONG
D. Act as supporting skeletal system for surface antigen and receptors
MEMBRANE COMPONENT
- Lipids and Protein
- Different inside and outside
- Allow selective passage of molecules

LIPIDS
- Phospholipid Bilayer is nearly 50% of the membrane
- PHOSPHOLIPIDS
- External: PHOSPHATIDYLCHOLINE, GLYCOLIPIDS, SPHINGOMYELINS
- Internal: PHOSPHATIDYLETHANOLAMINE, PHOSPHATIDYLINOSITOL, PHOSPHATIDYLSERINE

UNESTERIFIED CHOLESTEROL
- For membrane fluidity and permeability to maintain SA:Volume Ratio
- (There is plasma in the plasma and cholesterol in the membrane) Lipid exchange happens: 98% in Membrane is Unesterified while
70% of Plasma Cholesterol is Esterified
- Cholesterol content of the membrane depends on the Concentration of Plasma Cholesterol, Bile Acids, and Activity of the enzyme
Lecithin-Cholesterol Acyltransferase (LCAT)

PROTEINS
- Bound to lipids
- Classification: Peripheral and Integral Proteins (Differ by extraction method and location)
PERIPHERAL PROTEINS
- Seen in the cytoplasmic Side - responsible for the biconcave appearance of the RBC, for shape and deformability
2 SKELETAL PROTEINS:
- Spectrin (Bands 1 & 2)
- Actin (Band 5)
- Abnormal Spectrin have been found in Hereditary Elliptocytosis (RBC wil be more oval in shape because of the abnormality) and
Spherocytosis
INTEGRAL PROTEINS
- With Sialic Acid (Zeta Potential) - REPELING PROPERTY OF RBCS WITH EACH OTHER
PRINCIPAL INTEGRAL PROTEIN: Glycoprotein designated Band 3 - Inorganic Anion Transport Protein
**Decrease Zeta Potential is seen in altered plasma protein thereby increasing the ESR (because of rouleaux formation)
OTHER IMPORTANT MEMBRANE PROTEIN
- Na+/K+-ATPase Ion Pumps
(Increase Sodium uptake = LYSIS; Increase Potassium uptake = CRENATION/SHRINKAGE)
- Ca2+, Mg2+-ATPase Enzyme Systems
(Abnormality in Calcium and Magnesium exchange = TOO MUCH RIGGIDITY/DECREASE DEFORMABILITY)
- Membrane Proteins act as receptors for Transferrin (stored formed of Iron) and Erythropoietin (EPO)
- Antigenic Determinants (Blood Type) - Different Red Cell antigens
HEMOGLOBIN VISCOSITY
- Normal: Low Viscosity (to ensure fluidity)
- Less Deformability due to:
A. Water Loss
B. Polymerization of the HB will form HGB S
C. Precipitated HB will form Heinz Bodies (RBC Inclusions)
D. Crystallization of HB will form HGB C (Crystal)
ENERGY METABOLISM
- ensure the survivability of RBC
2 Sites prone to Oxidation:
**OXIDIXATION IS ALWAYS TOXIC and needs to be counteracted because it cannot deliver oxygen because it will convert Ferrous Iron
to Ferric Iron
A. Iron Atom in the Heme Ring - Acquired or Hereditary
B. Sulfhydryl Group of the Globin - Causes HB Precipitation (form Heinz Bodies - oxidized RBCs)

SOURCES OF ENERGY
1. GLUCOSE
- RBC can also Galactose, Fructose, Mannose (permeable to the membrane)
- Membrane is impermeable to Disaccharides (ex. Sucrose and Lactose)
**NO MITOCHONDRIA so metabolism is by:
- Anaerobic Glycolysis (Embden-Meyerhof Pathway - EMP) - 90%-95%
- HMS/PPS - 5%-10%
**BOX 9-1 ERYTHROCYTE METABOLIC PROCESSES REQUIRING ENERGY
- Maintenance of intracellular cationic electrochemical gradients
- Maintenance of membrane phospholipids
- Maintenance of skeletal protein plasticity
- Maintenance of functional ferrous hemoglobin
- Protection of cell proteins from oxidative denaturation
- Initiation and maintenance of glycolysis
- Synthesis of glutathione
- Mediation of nucleotide salvage reactions
Energy Production: ANAEROBIC GLYCOLYSIS
A. GLUCOSE
- Enters RBC passively through the transmembrane protein GLUT-1
- Required by anaerobic glycolysis (EMP) to generate ATP

EMBDEN MEYERHOF PATHWAY

- same anaerobic glycolysis of other cells but the difference is that this will produce:
A. 2,3-DIPHOSPHOGLYCERATE (2,3-DPG) (coming from the Rapoport Leubering Shuttle) which will assist in the delivery of the oxygen
B. ATP
- Maintain shape and deformability (By Phosphorylation of Spectrin and Chelation of Calcium)
- For active transports
- Assists in modulating amount of 2,3-DPG
C. NAD+ (oxidized form) and NADH (reduced form)
D. GLUCOSE IS CATABOLIZED TO PYRUVATE (PYRUVIC ACID)
- Consumes 2 ATPs per Glucose molecule
- Generates 4 ATPs per Glucose molecule
- Net gain of 2 ATPs

EMBDEN MEYERHOF PATHWAY

**REFER TO THE PHOTOCOPY
**If no reaction noted, only conversion is happening
1st PHASE
1. GLUCOSE (through GLUT-1)
Hexokinase - Glucose Phosphorylation
2. GLUCOSE-6-PHOSPHATE
Glucose-6-Phosphate Isomerase - Isomerization (CONSUMES 1 ATP)
3. FRUCTOSE-6-PHOSPHATE
6-Phosphofructokinase - Diphosphorylation (CONSUMES 1 ATP)
4. FRUCTOSE-1,6-BIPHOSPHATE
Cleaved by Fructose-Biphosphate Aldolase
5. GLYCERALDEHYDE-3-PHOSPHATE
**HEXOKINASE AND 6-PFK CONSUMES 2 ATP
-Limit the rate of Glycolysis
2nd PHASE
6. GLYCERALDEHYDE-3-PHOSPHATE
Glyceraldehyde-3-Phosphate Dehydrogenase - Oxidation
7. 1,3-BISPHOSPHOGLYCERATE
Phosphoglycerate Kinase - Diphosphorylation
8. 3-PHOSPHOGLYCERATE + 2 ATP

3rd PHASE
9. 3-PHOSPHOGLYCERATE
Phosphoglycerate Mutase - Isomerization
10. 2-PHOSPHGLYCERATE
Phosphopyruvate Hydratase (Enolase) - Conversion only
11. PHOSPHOENOLPYRUVATE (PEP)
Pyruvate Kinase - Will split off the Phoshate in PEP forming:
12. PYRUVATE + 2 ATP
**PYRUVATE can either be diffused in the RBC or will become a substrate for LDH
Lactate Dehydrogenase
13. LACTATE + NAD+
**Regeneration of oxidized NAD from reduced form

GLYCOLYSIS DIVERSION PATHWAYS (SHUNTS)

1. AEROBIC GLYCOLYSIS - 5-10%
2. METHEMOGLOBIN REDUCTASE PATHWAY
3. RAPOPORT-LUEBERING PATHWAY

HEXOSE MONOPHOSPHATE SHUNT

- AKA PENTOSE PHOSPATE SHUNT
- Provides reducing potential of the cell (counteract the oxidizing agent by providing a reducing agent)
- NADPH (reduced NAD)
- G-6-P generated in the first step of EMP is catabolized to 6-Phosphogluconate rather than passing through the EMP by enzyme
G6PD (Deficiency is the most common enzyme Deficiency) - Oxidation of RBCs which will cause lysis
- Detoxifies H2O2 (Arises from O2 reduction in the cells aqueous environment) which oxidizes and destroys Heme Iron, Proteins,
Lipids, and Thiol Groups
- To generate reduced Glutathione (Provides protection)
GSH = REDUCED GLUTATHIONE - principal reducing agent of the cell
GSSG = OXIDIZED GLUTATHIONE

** PHOTOCOPY
** As GLUCOSE-6-PHOSPHATE DIVERTED TO HEXOSE MONOPHOSPHATE PATHWAY with G6PD
1. NADP - oxidized NAD
2. NADPH - reduced NAD Which will convert:
3. GSSG - oxidized glutathione
Glutathione Reductase - NADPH Reduces GSSG to:
4. GSH Which will detoxify:
5. H2O2
Glutanthione Peroxidase - GSH Reduces H2O2 to:
7. H2O + O2
**As the GSH detoxify H2O2 it will turn back to its oxidized state (GSSG)
8. GLUCOSE-6-PHOSPHATE
Glucose-6-Phosphate Dehydrogenase
9. 6-PHOSPHOGLUCONATE
6-Phosphogluconate Dehydrogenase
10. RIBULOSE-5-PHOSPHATE

**REVIEW: HEXOSE MONOPHOSPHATE PATHWAY

- Glucose-6-Phosphate Dehydrogenase (G6PD) is the only means of generating NADPH for Glutathione Reduction
- HMP protects Hemoglobin, Sulfhydryl-containing enzymes and membrane thiols
- G6PD Deficiency is the most common inherited RBC enzyme deficiency worldwide

METHEMOGLOBIN REDUCTASE PATHWAY

- METHEMOGLOBIN - Hemoglobin oxidized with Iron (From Ferrous/Fe2+ to Ferric/Fe3+) due to exposure to O2 and H2O2 so no
oxygen delivery
- The reduction of this Hemoglobin variant by NADPH is more efficient in the presence of Methemoglobin Reductase (Cytochrome-
B5-Reductase) *to hasten the reaction conversion of Methemoglobin to Hemoglobin
- Enzyme accounts for 65% Methemoglobin-Reducing Capacity within RBC

**PHOTOCOPY
1. H+ FROM NADH with the help of
Methemoglobin Reductase (serves as intermediate electron carrier)
2. WILL REDUCE OXIDIZED Fe3+ to Fe2+ (METHEMOGLOBIN TO HEMOGLOBIN)

RAPOPORT-LUEBERING PATHWAY
- 2,3-BIPHOSPHOCLYCERATE (2,3-BPG) (AKA 2,3-DIPHOSPHOGLYCERATE/2,3-DPG) regulates O2 delivery to tissues by competing with
O2 in the heme for the O2-Binding Site of Hemoglobin
- When bound with Heme, O2 will be released

**Competitive si 2,3-BPG HAHAHA

RAPOPORT-LUEBERING PATHWAY
1. 1,3-BIPHOSPHOGLYCERATE
Biphosphoglycerate Mutase
2. 2,3-BIPHOSPHOGLYCERATE
**The diversion sacrifices production of 2 ATP. Further loss of 2 ATP at the level of PK, because fewer PEP are formed so no ATP will
be formed from this diversion (ATP Deficit)
Biposphoglycerate Phosphatase
3. 3-PHOSPHOGLYCERATE
**Two ATPs to generate 1,3-BPG, but no production of four ATPs

HEMOGLOBIN SYNTHESIS
- Components
- Globin Synthesis
- Heme Synthesis
- Hemoglobin Degradation
- Hemoglobin Function (Hemoglobin Variants and Derivatives)
1862 - Felix Seyler identified this respiratory protein and proved that this was the true coloring matter of blood
**Hemoglobin-single most common organic molecule present in vertebrates and first protein that is described using X-Ray
Crystallography
COMPONENTS OF HEMOGLOBIN
- 2,3-Diphosphoglycerate
- Globin Chains (present as Dimers or 2 Different Polypeptide Chains)
- Protoporphyrin IX (Nitrogenous Substance)
- Iron
**1 HB Molecule = 4 P9 Molecules and 4 Iron Atoms (the Iron will bind to P9 and the combination will form HEME)

GLOBIN CHAINS
**The Embryonic Globin chains are only found in the first 3 Months of life
** Named in Greek because of the position or designation of the Amino Acid (Globin Chain is a sequence of Amino Acids with
particular positioning)
- Consist of varied sequences of Amino Acids
**4 Globin Chains - 2 -chains and 2 -chains forming 22 (Hemoglobin A or A1 - most
common HB in adults)
**4 Iron Atoms inserted in 4 Protoporphyrin IX Molecules

NORMAL ADULT HEMOGLOBIN - 574 AMINO ACIDS

** - 141 AMINO ACIDS x 2 = 282 ; - 146 x 2 = 292
282 + 292 = 574 AMINO ACIDS

Greek Designation Greek Name # of Amino Acids Comments

Alpha 141
Beta 146
Gamma 146 Differs from Beta chain by 36
Amino Acids
 Delta 146 Differs from Beta chain by 10
Amino Acids

Epsilon 146 Embryonic only

Zeta 146 Embryonic only

2,3-DIPHOSPHOGLYCERATE
Produced in the the Embden-Meyerhof Pathway specifically the Rapoport-Luebering Shunt (Will compete in the Oxygen-Binding site
of heme)

O2

O2
O2 O2
2,3-DPG

O2 O2
O2 O2

PROTOPORPHYRIN IX
- Nitrogenous substance synthesized in the RBC specifically in the MITOCHONDRIA and CYTOPLASM OF
NUCLEATED RBCs (IMMATURE RBCs because hemoglobin synthesis will stop before RBC is released in the
circulation - Physiologic) - From the Cytoplasm back into the Mitochondria
IRON
- Will always combine with Protoporphyrin IX; inserted at the center of Protoporphyrin IX
HEMOGLOBIN VARIANTS VS DERIVATIVES
Variants Derivatives
- Particular in Globin Chains (any alteration - Alteration on the Heme Portion
of the Globin Chains will cause different
Hemoglobin Variants)

NORMAL HEMOGLOBIN VARIANTS

HEMOGLOBIN DEFECTS
A. STRUCTURAL DEFECTS
- Qualitative
- It is seen in Hemoglobinopathies (problem in the amino acid sequence)
- The problem is 1 or more substitution of Amino Acids
B. SYNTHETIC DEFECTS
- Quantitative
- Thalassemias (addition or deletion of amino acids - problem in the synthesis of globin chains)
- Decreased or No Production of 1 or More Globin Chain/s
GENETIC CODING FOR GLOBIN CHAINS
A. CHROMOSOME 11
- , , ,
B. CHROMOSOME 16
- ,
GLOBIN CHAIN STRUCTURE

**Same as other protein synthesis in the body

4 STEPS: TRANSCRIPTION, PROCESSING, TRANSLATION, TRANSFER
Primary - Parallel chain having Amino Acids; Secondary - Helix; Tertiary - Bending and Coiling, Quaternary -
Forming of Tetramer
HEME SYNTHESIS
- From Mitochondria to the Cytoplasm, then back to the
Mitochondria
SUCCINYL CoA + GLYCINE (occurs in the Mitochondria) =
HEME
TO REMEMBER:
**At the start - condensation of Succinyl CoA and Glycine
**(presence of Aminolevulinic Acid (ALA) synthase) it
would produce Delta-Amino Levulinic Acid (Mark for:
MITOCHONDRIA)
** (presence of Porphobilinogen Synthase) it will create
Porphobilinogen (Mark for: CYTOPLASM)
**Inside the Mitochondria in Protoporphyrinogen IX
(REMEMBER UNTIL HEME)
IRON METABOLISM
SOURCES
A. Diet- (10%) Stomach;
- Abosorbed in: Duodenum & Jejunum
- Fe2+ and Fe3+ but only the reduced form is absorbed
B. Dead RBCs (recycling) MAJOR SOURCE - But decrease in chronic, acute blood loss
IRON STORAGE
INTRACELLULAR SPACES OF BONE MARROW AND LIVER
A. Ferritin (Fe2+ + Apoferritin)
B. Hemosiderin - no Apoferritin

IRON TRANSFER
1. Transferrin - Transports 2 Atoms of Iron at once
2. Sideroblast - Nucleated RBC with Ferritin (IMMATURE RBC)
3. Siderocyte - Mature RBC with Ferritin (MATURE RBC)
**Can only be appreciated with Prussian Blue Stain
**If not used for Heme Synthesis

RINGED SIDEROBLAST AND SIDEROCYTES

**Arrowed is Sideroblast because Nucleated (Immature)
Others are Siderocytes

**REVIEW

**LIFE OF IRON:
1. Transferrin will carry 2 atoms of Iron into the cell membrane of the RBC, which has receptors for Transferrin,
the RBC membrane will invaginate the Transferrin with Iron present and will form a vacuole containing the 2
substances
2. Iron has two fates in the RBC: Used for Heme synthesis or only Stored
3. HEME SYNTHESIS - Iron should be reduced to Fe2+ (Ferrous Iron) and will bind to the center of Proporphyrin
IX and will cause the formation of Heme which will be released in the cytoplasm to combine with Globin
(produced by the Polyribosomes) which will for Hemoglobin
MYOGLOBIN
- Heme pigment of striated muscle
- Still needs Amino Acids, Iron, and Proporphyrin IX
- Made up of 1 polypeptide chain & 1 heme molecule
- Myoglobin has high affinity for Oxygen
OXYGEN DISSOCIATION CURVE:
- Myoglobin = Hyperbolic Curve
- Hemoglobin = Sigmoid Curve (Normal Curve)

HEMOGLOBIN DEGRADATION
- Aged Erythrocytes
- Blood Vessels and Spleen (Reticuloendothelial System / RES)

**Recycled -
Iron and
Globin;
Porphyrin -
Excreted

FACTORS
AFFECTING
AFFINITY OF

HEMOGLOBIN TO OXYGEN
A. Blood Temperature (Increased Blood Temp. will cause the release of Oxygen)
B. Blood pH (Bohr Effect - relation of Blood pH and Oxygen Affinity)
**ex. Increase Hydrogen Ions will cause an Acidic Environment, the blood is used as a buffer to create
Homeostasis. The hydrogen ion will be attached to the heme, but the oxygen will be released from the heme
which will create a neutral environment
C. 2,3-Diphosphoglycerate - Delivery of oxygen (In Blood Transfusion: The longer storage of blood (In-vitro) will
cause the loss of the native 2,3-DPG so hindi kaagad makarelease ng oxygen but can be normal after proper
metabolic activity sa EMP)
D. Blood CO2 (Haldane Effect - relationship of CO2 and O2 Affinity) - Increase CO2 = Increase release of O2
E. Hemoglobin Variants (Altered Globin Chains) - Attachment or release of O2
F. Amount of Hemoglobin F (Increased Oxygen Affinity because it assures that there is enough oxygen for the
fetus)
**HBF is normal for Adults but decreased

FACTORS:
- Amount of Fetal Hemoglobin
**HbF has slightly higher O2 affinity causing shift to the LEFT
- Abnormal Hemoglobin Variants
SHIFT TO THE RIGHT (Let Go of O2) - CADET (Carbon Dioxide, Acidic - decrease pH, 2,3-DPG, Exercise and
Temperature) (ex. Renal Failure)
Increased: Temperature, 2,3-Diphosphoglycerate, CO2
Decreased: pH (**CO2 will bind to heme releasing O2)
SHIFT TO THE LEFT (Hold On to O2) (ex. Hyperventilation)
Increased: pH
Decreased: Temperature, 2,3-Diphosphoglycerate, CO2
(**O2 will bind to heme and CO2 will be released)
OXYGEN DISSOCIATION CURVE
Oxygen Pressure - concentration of O2 in the environment
Oxygen Saturation - amount of O2 already bound to Hemoglobin
**HEMOGLOBIN CURVE - Sigmoid or Normal Curve, P50 Value =
50% of Oxygen is already saturated reached when it is
approximately 27mmHg/26.6mmHg
**DEOXYGENATED HGB - lower affinity to O2 but if ever kahit 1
molecule of O2 will go to the HB it will eventually be saturated (and it invite other O2 molecules)
SIGMOID CURVE:
**Increase Oxygen Tension = Increase Oxygen Affinity AND VICE VERSA - ex. Lungs and Tissues (Lungs - Oxygen
Molecules that require uptake = Increase Oxygen Affinity; Tissues - Decrease Oxygen Tension + Acid
Metabolites: release O2 because Decrease Oxygen Affinity
**LEFT SHIFT - Increased O2 Affinity, Less than 27mmHg but 50% O2 Saturated
**RIGHT SHIFT - Decreased O2 Affinity, Greater than 27mmHg for 50% O2 Saturated

**MYOGLOBIN - Greater
affinity for O2 than
Hemoglobin; Less than
20mmHg for 50% O2 Saturated
HEMOGLOBIN FUNCTION
 - Not only provides O2 for tissues but also removes CO2 from the tissues and
transports it to the lungs for expiration but also is a BUFFER for the acid base
balance in the body

HEMOGLOBIN DERIVATIVES (Problem: Heme Portion - all are non-functional but only 1 is irreversible)
A. METHEMOGLOBIN
B. SULFHEMOGLOBIN
C. CARBOXYHEMOGLOBIN
METHEMOGLOBIN
- Used to create Cyanmethemoglobin
- Aka HEMIGLOBIN (Hi) which causes nonbinding of Heme to Oxygen
- Iron oxidized to Fe3+ (cannot take up O2)
- NADH-Methemoglobin Reductase (Diaphorase) in RBC that counteract Hi
- Methemoglobin Reductase Pathway - daily the body synthesizes 0.5 - 3%

METHEMOGLOBINEMIA
- Beyond 3% Methemoglobin
- Methemoglobin not reduced
- will cause Cyanosis (Bluish appearance of skin)
- will be resolve by the administration of Methylene Blue

INHERITED METHEMOGLOBIN
- NADH-Methemoglobin Reductase Deficiency / Diaphorase Deficiency
Or
- Inherited as an Autosomal Recessive Trait (2 parents need to carry the trait)
Treatment:
- Methylene Blue
- Ascorbic Acid

**PICTURE: Methemoglobin - CHOCOLATE BROWN color of Blood in

compare to Normal
**OXYHEMOGLOBIN: Bright Red - Arterial Blood
REDUCED HEMOGLOBIN: Purplish Red - Venous Blood

ACQUIRED METHEMOGLOBIN (more common)

- Ingestion or Absorption
- Antimalarial Drugs
- Sulfonamides
 - Drug Abuse
- Aniline Dyes
- Nitates

INHERITED HEMOGLOBIN-M METHEMOGLOBINEMIA

- 5 HbM Variants from 1 HB Derivative

- Amino Acid Substitution
- Heme enter the oxidized state (cannot carry O2)
- DOES NOT respond to Methylene Blue
- Treatment not necessary

SYMPTOMS AND PHYSICAL FINDING

- If Methemoglobin exceeds 10% - Cyanosis
- 35% - Hypoxia which may lead to Tachycardia and Dizziness / Headache
- >60% - Rare and Fatal

SULFHEMOGLOBIN
- Caused by oxidation
- Acquired condition
- Hemoglobin + Hydrogen Sulfide (H2S) = Sulfhemoglobin
- IRREVERSIBLE
- Generally benign
- 0- 2.2% normal in blood
- Oxidized + partially denatured Hgb during oxidative hemolysis
- MAUVE-LAVENDER BLOOD
- Causes: Phenacetin, Acetanilide, Sulfonamides (Analgesics and Anti-Pyretics)

CARBOXYHEMOGLOBIN
- Hemoglobin + CO = Carboxyhemoglobin
- Can attach with Sulfhemoglobin = CARBOXYSULFHEMOGLOBIN
- 200 or 210x Affinity to CARBON MONOXIDE leading to Asphyxiation (the state of not being able to breathe
and CarboxyHB used for suicide - no color, no odor, and painless)
- CHERRY RED COLOR and Reversible
- Chief sources: Tobacco Smoking, Gasoline Motors, Illuminating Gas, Gas Heaters, Defective Stoves

ADDITIONAL:
**NADP - HMS; Anabolic reaction
NAD - EMP, MetHb; Catabolic reactions
**Methemoglobin M Variants
- Methemoglobin M Saskatoon (63 His>Tyr)
- Methemoglobin M Hyde Park (92 His >Tyr)
- Methemoglobin M Boston (58 His>Tyr)
- Methemoglobin M Iwate (87 His>Tyr)
- Methemoglobin M Milwaukee (67 Val>Glu)
THE PORPHYRIAS
1. PRIMARY PORPHYRIAS
- Inherited deficiencies of enzymes
- Erythropoietic (BM)
2. SECONDARY PORPHYRIAS
- Acquired due to drugs/chemicals
- Hepatic (Liver)
**SEE TABLE 31-2 OF HENRYS FOR KEY FEATURES OF MAJOR PORPHYRIAS

MORPHOLOGIC EVALUATION OF ERYTHROCYTES

Importance
- Light microscopy, well stained, well made smear
- Examined in shape, size, distribution, concentration of hemoglobin and inclusions
- Suggests a particular disease, aids in diagnosis
DISTRIBUTION:
NORMAL:
- Even distribution
- Thin portion of the smear - RBCs are slightly separated
(X) Do not read in the Feathered Edge and Thick Area
ABNORMAL:
1. ROULEAUX
- Not separated with each other in short/long stacks resembling coins
- Happens when their biconcave surfaces are in apposition
- Hyper-proteinemia (Increase Gamma-Globulin), Multiple Myeloma (Increase Gamma-Globulin), Macroglobulinemia (Lengthy
Chains)
- Increase ESR
- Increased fibrinogen (Tissue Necrosis) - Long Stacks (Infection, Pregnancy, Macroalbuminemia)
**Spherocytes cant from Rouleaux; Rouleaux DO NOT form in the presence of Antibodies
2. AGGLUTINATION
- Cells are in clusters or masses when exposed to antibodies
- AUTOAGGLUTINATION - Normal or in disease state (Atypical Pneumonia or Hemolytic Anemia)
**Autoagglutination occurs when an individuals Red Cells agglutinates in his own plasma without Agglutinins
- COLD AGGLUTININ DISEASE - Clumping at below 25C (Enhances Autoantibodies Activity - Need to be warmed by rubbing between
hands or incubation)
- Increased MCV in Automated Machine (Artifactual) since clumps are counted as one - FALSE MCV - Peripheral Blood Smear needs
to be checked
**ADD NORMAL SALINE SOLUTION to differentiate: Rouleaux if Red Cells Separate

MORPHOLOGY
NORMAL:
- Biconcave disc (Discocyte)
- Uniform size, shape, and Hemoglobin concentrtion
- No inclusions
- Central pallor should not be more that 1/3 of the cell

POIKILOCYTOSIS
- General term for mature RBC that have other shape than the normal in stained smear
HEMOGLOBIN CONTENT
A. NORMOCHROMIC
- Clear central pallor
B. HYPOCHROMIC
- Decreased Hemoglobin - Increase central pallor
- Associated often with Microcytosis
- Iron-Deficiency Anemia, Sideroblastic Anemia, Thalassemia
C. HYPERCHROMIC
- Lack of central pallor
- Associated with Macrocytosis
- MCHC is elevated in True Hyperchromia
- Hemolytic Anemia, Hemolysis caused by burns
**Anisochromia
SIZE
- Correlate with MCV
A. NORMOCYTIC
- Normal MCV even there is minor population of smaller or larger cells
B. MACROCYTIC
- If diameter exceeds 8.5-9 um and MCV exceeds 100fL
- Low Vitamin B12 and Folate (Immature Nucleus), Alcoholism with or without Liver Disease, Cancer Chemo, Chronic Hemolytic
Anemia with Reticulocytosis, Myeloma, Immature Release of RBC (For Compensation)
C. MICROCYTIC
- Small RBC, occurs when MCV is below 80fL
**Marked Anisocytosis (Tested with Red Cell Distribution Width (RDW) - Automated)
CHARACTERISTIC OF IRON DEFICIENCY ANEMIA
A. Spherocytes - Lack of central pallor and appear to have increase Hemoglobin seen in Hemolytic Anemia, Hereditary Spherocytosis
B. Leptocyte - Thinner than normal and have a colorless center, smaller leptocytes are seen in Thalassemia, Hemoglobin C,
Steatorrhea (Malabsorption of Fats in Stool)
- (May be Normocytic or Microcytic)
**Resemble Codocytes (Difference: Leptocyte - Noct Completely Dettached Inner Membrane)

SHAPE VARIATION (POIKILOCYTOSIS)

POIKILOCYTES SECONDARY TO DEVELOPMENTAL MACROCYTOSIS
1. OVAL MACROCYTES
- Markedly increased MCV
- Vitamin B12 or Folate deficiency - nuclear maturation defect
- Mature Cytoplasm but Immature Nucleus
- Appear well filled of Hemoglobin due to increase thickness (Same as Spherocytes)
- Megaloblastic Anemia
- MEGALOCYTES ** >125 fL

POIKILOCYTES SECONDARY TO MEMBRANE ABNORMALITIES

2. STOMATOCYTES
- Show a mouth/ slit-like area of pallor
- Defect in Na-K Transport Ratio (Protein Stomatin)
- Hereditary Stomatocytosis - Mild Anemia, Rh Null Disease
- Alcoholism, Cirrhosis, Obstructive Liver Disease
- Increased Cellular Na+, Decreased K+
- Decrease Deformability = Not Flexible

3. OVALOCYTES
- Egg-like/ Oval Shaped; Wider than Elliptocyte
- Bipolar arrangement in Hemoglobin
- Reduction in Membrane Cholesterol
- Megaloblastic Anemia, Myelodysplasia

- Normal Central Palor

4. ELLIPTOCYTES
- Rod or Cigar-Shaped, Narrower than Ovalocytes
- Hereditary Elliptocytosis - defect in Cytoskeleton (25-90%
Elliptocytes in Blood Film)
- Decreased Skeletal Membrane Protein Band 4.1
- Increased Heat Sensitivity of Spectrin, Osmotic Fragility Test -
Normal, Decreased Lifespan
- Function is still normal

5. CRENATED RBC (ECHINOCYTES)

- Have blunt spicules (Sea Urchin)
- Evenly distributed over the surface
- Usually artifactual, Minimal Difference with Burr Cells
(Pathologic)
**Artifactual in the sense that blood has been long standing in
EDTA Tube, Glass Slides can also cause Alteration in Blood pH,
Blood Bag can also be a factor so it is normal to see this on the
PBS of Newly Transfused Patients
- Artifact in Air Drying
- Exposure to Hypertonic Solution

6. BURR CELLS
- Pathologic
- With Irregularly sized and unevenly spaced spicules
- Reversible spicules (In-Vitro)
- Uremia, Anemia associated with Renal Insufficiency
- 10-30 Scalloped Projections present in RBCs
- Patients with Increased BUN

7. ACANTHOCYTES
- With irregularly spaced (Thorn- like) projections (Spikes/
Spicules) Usually with bulbous end and varying width, cant
regain normal shape
- Abnormal ratio of Membrane Lecithin and Sphingomyelin
- Abetalipoproteinemia (Retina Degeneration and Steatorrhea)
- Alcoholic Cirrhosis with Hemolytic Anemia
- Malabsorption and Postsplenectomy States
- Hepatitis of the Newborn
- Pyruvate Kinase Deficiency

8. SPUR CELLS
- Found in severe Hemolytic Anemia associated with Cirrhosis
and in Metastatic Liver
- Cxd by Sharp Points
- 3-12 Spicules Present

9. CODOCYTES
- Target Cells (Bulls Eye Appearance)
- AKA Mexican Hat Cell (HAHAHA)
- Have a central area of Hemoglobin surrounded by colorless
ring
- Increased Cholesterol and Phospholipid
**Ovalocytes if Cholesterol ONLY
- Excess of Surface Membrane to Volume Ratio
- Maybe acquired Hemoglobinopathies (SS, CC, DD, EE),
Thalassemia, Obstructive Liver Disease, Postsplenectomy
States, Iron Deficiency Anemia
**Maybe Artifactual (Fix with Methanol to Test whether Codocyte or Artifact)
- Decrease OFT

POIKILOCYTES SECONDARY TO TRAUMA

10. DACRYOCYTE
- Tear Drop Cell/ Pear Shaped Cells
- Squeezing a fragmentation during splenic passage
- If RBC with Inclusion (Difficult to Deform) - Small Opening
(Blood Vessel) > STRETCHED
- Found most notably in Myelofibrosis with Myeloid Metaplasia
- Beta-Thalassemia, Pernicious Anemia, Tuberculosis,
Metastisized tumor to the Marrow, Hypersplenism

11. SCHISTOCYTE/ SCHIZOCYTES

- Split cells / Helmet Cells (Ruptured Schistocytes)
- RBC Fragments occur due to damage by Fibrin, Altered
Vessel Walls, Prosthetic Heart Valves
- In Microangiopathic Hemolytic Anemia, ABO HDN
- Hemolytic Anemias - Burns, Prosthetic Implants, Renal
Transplants Rejections
**Disseminated Intravascular Coagulation and Thrombotic
Thrombocytopenic Purpura (Fibrin - Coagulation Process)

11A. KERATOCYTE
- A Schistocyte with 1 or more horn like projections
- A rare phenomenon associated with DIC (Fibrin Strands)
- A result of RBC being caught on a Fibrin Strand which cut it to
two, when this escapes, it may have a vacuole like area (Blister
Cell) - associated with G6PD
**Blister Cell: Ruptured Form:
Keratocyte or
Schizocytes (Longer Projections

11B. KNIZOCYTE
- Stomatocytes
- Pinched-Bottle Cells
- Hemolytic Anemia; Macrospherocytosis

12. MICROSHEROCYTES AND PYROPOIKILOCYTES

- Abnormality in Spectrin
- Small round cells occuring in severe burns
- Rare hereditary HA - Heat Sensitivity
- Fragment at 45C (Normal RBC fragment at 49C)
- MCV: <60fL

13. SEMILUNAR BODIES

- Half-Moon Cell, Crescent Cell
- Large, Pale-pink staining ghost of the RBC -
membrane remaining after the contents have been released
- Frequently seen in Malaria - Overt Hemolysis (Noticeable Hemolysis)

ABNORMAL HEMOGLOBIN
14. DREPANOCYTES
- Sickle Cells
- Crescent shaped due to the formation of Rod-like Polymers of HB
S (Deoxygenated Hemoglobin)
- Found in Sickle Cell Anemia, HB SC Disease
- Increased MCHC; Hyperchromia

**NOTE:
A. ABETALIPOPROTEINEMIA - Acanthocyte
B. CHRONIC RENAL DISEASE - Burr Cells
C. HEMOGLOBINOPATHIES/ THALASSEMIA - Codocyte
D. MICROANGIOPATHIC HEMOLYTIC ANEMIA - Schizocyte
E. MYELOID METAPLASIA - Dacryocyte
F. RH NULL DISEASE - Stomatocytes

INCLUSIONS DEVELOPMENTAL ORGANELLES

15. HOWELL-JOLLY BODIES
- Small rounded fragments if nucleus
- Positive reaction with Fuelgen - contains DNA
- Megaloblastic Anemia, Severe Hemolytic Process, Thalassemia, Accelerated Erythropoiesis
- Karyorrhexis
- Improper Extrusion of Nucleus
-Increased in Post Splenectomy

16. BASOPHILIC STIPPLING/ PUNCTATE BASOPHILIA

- Studded
- Fine or Coarse
- Stains Deep Blue-Purple in Wright Stain
- Smaller than H-J Bodies
- Aggregates (precipitation) of Ribosomes and RNA
Homogenous Distribution
- Deficiency of PYRIMIDINE-5-NUCLEOTIDASE (3rd Most
Common Enzyme Defficiency)
- Lead Poisoning (Coarse Granulation - Pathologic), Heavy Metal
Poisoning (Secondary Porphyria), Thalassemia

17. PAPPENHEIMER BODIES

- Small, Irregular Dark staining granules located in the periphery
- Positive with Prussian Blue (Sideroblast), Unused Iron Deposits
- Sideroblastic Anemia, MDS, Thalassemia, HA, Defective
Erythropoiesis

18. CABOT RINGS

- Thin like ring structure forming of 8 / Loops
- Stain a Red or Reddish Purple color
- Remnant of Microtubules of Mitotic Spindle
- Megaloblastic Anemina, Other Severe Anemia (Pernicious
Anemia), Lead Poisoning

ABNORMAL HEMOGLOBIN PRECIPITATION

19. HEINZ BODIES

- Oxidative
- Round refractile inclusion not visible in Wright stain - Use
SUPRAVITAL STAIN
- Pitted Golf Ball appearance if multiple
- Precipitated, Denatured Hemoglobin due to oxidative injury
(HMS - Deficiency of G6PD)
- Hereditary defects in HMS, G6PD Def,
Unstable HB, Splenectomized Px, Thalassemia
- Polychromatophilic
20. HEMOGLOBIN H INCLUSIONS
- Small, Greenish-Blue Inclusion Bodies
- Precipitated HB H
- Brilliant Cresyl Blue
- HB H Disease (a-Thalassemia)

21. HEMOGLOBIN CC CRYSTALS

- Angular crystals
- Hexagonal with Blunt Ends and Stain Darkly
- Homozygous C (HB CC) Disease

22. HEMOGLOBIN SC CRYSTALS

- Dark hued crystals, Distort the RBC membrane
- Cystalline projection is often straight with parallel sides and one
blunt, pointed protruding end
- Washington Monument Shape
-HB SC Disease

ABNORMAL HEME SYNTHESIS

23. RINGED SIDEROBLAST
- Nucleated RBC that contains nonheme Iron particles
- Excessive Iron overload in Mitochondria of Normoblast
- Due to defective Heme Synthesis
- Perls Prussian BLue
- Sideroblastic, MDS
INCLUSION FEULGEN SUPRAVITAL WRIGHT
Basophilic Staining Negative Positive Positive
Cabot Rings Negative Negative Positive
Howell-Jolly Bodies Positive Positive Positive
Polychromatophilia Negative Negative Positive
Reticulocytes Negative Positive Negative
Pappenheimer Negative Positive Positive
Bodies
Heinz Bodies Negative Positive Positive or
Negative
**Fuelgen Stain demonstrates the presence of DNA
**Supravital Stains (NMB or BCB) demostrate the presence of RNA
- Can be demonstrated with Crystal Violet Stain
PROTOZOAN INCLUSIONS
24. MALARIA
PLASMODIUM SPP - Transmitted to man by Anopheles mosquito
A. PLASMODIUM VIVAX
- Schuffners Dots
- Infects RBC - Enlarged
B. PLASMODIUM MALARIAE
- Ziemanns Dots
- Infects RBC - NOT Enlarged
C. PLASMODIUM FALCIPARUM
- Maurers Dots
- Small delicate ring forms, gametocytes are banana shaped
D. PLASMODIUM OVALE
- James Dots
- RBC is oval
**Sickle Cell anemia - Resistant to Falciparum
**G6PD Deficiency - Generally Malaria Resistant

25. BABESIA MICROTI

- Babesiosis
- Rare transmission to human by tick bites
- Resemble ring stages of malarial parasites
- Tiny rings 1-5 um
- Tetrad formation
- RBC not enlarged