HEMATOCRIT DETERMINATION 4.

Determine the level of packed RBCs using a

microhematocrit reader. Do not include buffy
Hematocrit coat in reading haematocrit because it will
give false positive result.
- the volume of packed red blood cells (PRBCs) after
centrifugation of a blood sample Note: estimation of Hgb and RBC is possible on the basis of
- known as packed cell volume (PCV) or erythrocyte the Hct value under normal circumstances
volume fraction (EVF)
- reported in percent (%), cell volume percent (CV%) or 1% haematocrit = 0.34 gm % hemoglobin
volume percent (vol%)
- one of the simplest, most accurate, valuable tests in = 107,000 RBC/mm^3
haematological investigation
- more useful than RBC count in detecting cases of
anemia
- RBC indices can be computed manually from RBC
count values, Hgb levels, and Hct level.

Materials needed

 EDTA blood
 Microhematocrit capillary tube (blue/red)
 Disposable sterile blood lancet
 Wintrobe tube Other Methods
 Microhematrocrit centrifuge
 Wintrobe Method
 Sealing clay
- This method utilizes a Wintrobe tube with
 Microhematocrit reader
two calibrations, 0 to 10 (top to bottom)
Methods which is used for erythrocyte rate (ESR) and
10 to 0 (bottom to top), which is used for
 Adam’s Microhematocrit Method (most commonly haematocrit
used)
1. Fill around ¾ of the capillary tube with blood.
If the blood is from a skin puncture, use
heparinized (red) capillary tube. A non-
heparinized (blue) tube us used if blood
collected with anticoagulant
2. Seal one end of the capillary tube with

- Anticoagulant of choice is double oxalate

Procedure:
sealing clay (about 3mm). press it slightly at
the clay. 1. Fill Wintrobe tube with blood using Pasteur pipette.
3. Centrifuge the blood at 10,000 rpm for 4-5 Insert the pipette well and slowly raise it up to avoid
minutes using a microhematocrit centrifuge. bubbles
2. Centrifuge tube at 3000 rpm for 30 minutes
3. Read the volume of packed RBCs
4. Compute for the Hct level using the formula:

 Haden’s Modification

- The anticoagulant is 1.1% sodium oxalate in

distilled water.
- Uses a calibrated tube

Procedure:

1. Place 1 mL of 1.1% sodium oxalate in the tube

2. Add 5 mL of blood. Mix well
3. Centrifuge the mixture for 20 minutes at 3,000 rpm
4. Read the volume of packed RBCs

 Van Allen Method

- Anticoagulant is 1.6% sodium oxalate in
distilled water.
- Uses a tube with bulb and calibration of 1 to
10 cm or 10 to 100 mm

Procedure:

1. Fill the tube with blood up to the 10th mark

2. Dilute the blood with the diluting fluid up to the bulb
about half full
3. Seal the tube and centrifuge with shaft end down at
2500 rpm for 15 to 30 minutes
4. Read volume of packed RBCs

Note: each unit of division is equal to 1%

 Sanford-Magath
- Anticoagulant is 1.3% sodium oxalate, the
tube is calibrated at 1mm per division
- The tube is about 5 inches long with a
funnel-like mouth
Procedure:
1. Place 1 mL of anticoagulant into the tube
2. Add 5mL go benou blood and mix
3. Centrifuge mixture at high speed for 15 minutes
- It is the measure of the distance and speed of fall of
RBCs in the plasma in a tube of a standard bore and
length after standing perpendicularly
- Most important factor influencing ESR is the action of
plasma proteins

 Bray’s Method
- Anticoagulant is heparin and a Bray’s tube is
used.
- This tube is calibrated on both sides similar
to the Winstrobe tube
- The calibration is from 10-50 mm. each
division is 1mm and the capacity is 5 mL
Procedure:
1. Fill the tube with heparinized blood
2. Let the tube stand at a vertical position for one hour
3. Read the volume of RBCs from the lower right side
calibration
Stages of ESR
1. Initial period of aggregation of rouleaux formation – 10
minutes
2. Period of fast settling – 40 minutes
3. Final period of packing – 10 minutes
Total of 60 minutes / 1 hour
Importance of ESR
 It is used as an index of the presence of an active
infection
 It measures the suspension stability of RBCs

Automated Method

 Coulter counter
 Autoanalyzer

 It indicates abnormal concentration of fibrinogen,

globulin, and other plasma proteins

Materials Needed

 Unclotted blood
 Wintrobe tube
 Timer
 Test Tube Rack
 Pasteur pipette / syringe with cannula
 Westergen tube
 Westergen rack
ERYTHROCYTE SEDIMENTATION RATE

- Refers to the speed of the settling or RBCs in

anticoagulated blood

 Rubber aspirator
Wintrobe-Landsberg Method
- Used in majority of cases and is quite accurate
- Advantages include outweighing of few drawbacks
- Uses the Wintrobe tube, calibrated on two sides (0-
10, 10-0
- Commercially available
Procedure:
1. Fill the Wintrobe tube with blood with a Pasteur
pipette or cannula attached to a syringe
2. Place the tube in a vertical position on a rack
3. After letting the tube stand for one hour, record the
ESR in milimeters
Westergren Method
- Most sensitive for ESR determination
- Can be used for the serial study of chronic diseases
like tuberculosis, carcinoma, etc.
- Has a smaller bore
Procedure:
1. Fill the Westergen tube with blood using a rubber
aspirator
2. Let the tube stand vertically on a Westergen rack
3. Record the ESR in millimetres after 1 hour
Let the blood go up by capillary action and let it stand for 1
hour. Blood should be in the 0 mark, if it does and is
irreversible, cancel the whole processes.
Other Methods of ESR Determination
A. Macro Methods
 Graphic or Cutter – anticoagulant of choice is 3%
sodium citrate, uses Cutler tube which has a 5
mL capacity; graduation of 0-50 mm
Procedure:
1. Add 0.8 mL of blood to 0.5 mL of 3% sodium citrate
2. Close the tube with paraffin-coated cork, then mix
3. Allow the tube to stand for 1 hour, observing every
five minutes
4. Compare results with normal values in the
sedimentation chart
 Linzenmeier method – anticoagulant of choice is
3% sodium citrate, uses Linzenmeier tube which
is 65 mm in length, 5 mm in diameter, and has a
capacity of 1 mL (with a mark of18 mm)
Procedure:
1. Add 0.8 mL of blood of 0.02 mL of 3% sodium citrate
2. Mix and pour the mixture into the tube up to the 1 mL
mark.
3. Allow the tube to stand in an upright position until the
RBCs settle at the 18mm mark
4. Note the time for this event to take place. Record in
minutes
B. Micro Methods
  Micro Laundau (a modification of
Linzenmeier-Raunert) – anticoagulant is 5%
sodium citrate, that uses a Micro-Landau
tube which is calibrated 0-50 mm and has
two graduation marks, one at 12.5 mm and
another at 62.5 mm, with a small bulb similar
to RBC and WBC pipettes
Procedure:
1. Draw 5% sodium citrate by turning the top-screw to
the left until the upper meniscus of the solution
reaches the lower mark A
2. Draw up the blood until the height of the liquid
reaches the upper meniscus of graduation B
3. Clean the tip of the tube with ether, then continue to
draw up the mixture into the bulb by turning the top-
screw to the left until the lower meniscus of the blood
column ends just a few millimetres below the lower
opening of the bulb. Do not draw the blood into the
bulb completely
4. Shake the tube carefully
5. Force the blood up and down twice very slowly by
turning the screw to the left then to the right
6. Set the tube vertically on a rack and read the ESR at
the end of one hour
 Smith Micro – used for infants and when
venipuncture children may not be practiced
The automated ESR system is a fully automated
instrument for ESR determination. One mL of blood is
collected from an evacuated tube, containing liquid
sodium citrate. The tube is then placed in the Ves-
Matic analyser where it is automatically mixed,
allowed to settle, and read. Results are comparable to
the Westergren method, and it takes only 22 minutes
to finish.
Mini-Ves = four samples at one time
Ves-Matic = 20 samples at one time, prints results
Ves-Matic 60 – 60 samples at one time, prints results,
identifies sample by a barcode reader
Clinical Significance of ESR
- The ESR is a non-specific test. It is raised in a wide
range of infectious, inflammatory, degenerative, and
malignant conditions associated with changes in
plasma proteins, particularly increases in fibrinogen,
immunoglobulins, and C-reactive protein
- The ESR is also affected by other factors like anemia,
pregnancy, hemoglobinopathies, hemoconcentration,
and treatment with anti-inflammatory drugs

Procedure:

1. Fill the special pipette with 5% sodium citrate and

expel 0.04 mL
2. Draw capillary blood with the same pipette. Three
successive batches of 0.1 mL are collected and
expelled into the tube containing the citrate. Thorough
shaking is necessary to ensure adequate mixing and
prevent coagulation
3. The blood is transferred to the special sedimentation
tube using a capillary pipette and the test is
completed in the usual manner

 Crista / Hellige-Vollmer

C. Automated Methods
 Automated ESR system by Vega Biomedical

ERYTHROCYTE METABOLISM AND MEMBRANE
STRUCTURE AND FUNCTION
- The RBC is the primary blood cell, circulating at 5
million RBCs per microliter of blood on average.
- It is anucleated and biconcave and has an average
volume of 90 fL
- The cytoplasm provides abundant hemoglobin, a
complex of globin, protoporphyrin, and iron that
transports elemental oxygen from the lung capillaries
to the capillaries of organs and tissues.
- Hemoglobin, plasma proteins, and additional RBC
proteins also transport molecular carbon dioxide and
bicarbonate from tissues to lungs
- Hemoglobin is composed of four globin molecules,
each supporting one heme molecule. Each heme
molecule contains a molecule of iron.
- The biconcave RBC shape supports deformation or
flexibility, enabling the circulating cell to pass
smoothly through capillaries, where it readily
exchanges oxygen and carbon dioxide while
contacting the vessel wall.
- RBCs are produced through erythrocytic
(normoblastic) maturation in bone marrow tissue.
- The nucleus while present in maturing marrow
normoblasts, become extruded or rejected as the cell
passes from the bone marrow to peripheral blood.
- Cytoplasmic ribosomes and mitochondria disappear
24 to 48 hours after bone marrow release, eliminating
the cell’s ability to produce proteins or support
oxidative metabolism
- ATP is produced within the cytoplasm through
anaerobic glycolysis (Embden-Meyerhof
pathway/EMP) for the lifetime of the cell
- ATP drives mechanisms that slow the destruction of
protein and iron by environmental peroxides and
superoxide anions, maintaining hemoglobon’s
function and membrane integrity
- Oxidation, however, eventually takes a toll, limiting
the RBC circulating life span to 120 days, whereupon
the cell becomes disassembled into its reusable
components globin, iron, and the phospholipids and
proteins of the cell membrane while the
protoporyphrin ring is excreted as bilirubin.
- RBC energy production, the protective mechanisms
that preserve structure, function, deformability and
maintenance of the cell membrane – form the basis
for understanding RBC disorders (anemia)
Energy Production and Anaerobic Glycolysis
- mature RBC relies on anaerobic glycolysis for its
energy – lack of mitochondria
- glucose enters RBC with no energy expenditure via
the transmembrane protein Glut-1
- erythrocyte metabolic processes requiring energy:
 intracellular cationic gradient maintenance
 cytoskeletal protein deformability
 prevention of the peroxidation of proteins
and lipids
 maintaining cytoplasmic cationic
electrochemical gradients
Pathways of Hemoglobin
 Embden-Meyerhof Pathway/EMP
- anaerobic EMP metabolizes glucose to
pyruvate, consuming two ATP molecules
 - EMP subsequently generates four ATP
molecules per glucose molecule, a net gain
of two.
 Hexose-Monophosphate Pathway (HMP)
- Aerobically converts glucose to pentose and
generates NADPH
- NADPH reduces glutathione, which reduces
peroxides and protects proteins, lipids, and
heme iron from oxidation.
 Methemoglobin Reductase Pathway
- Converts ferric heme iron (valence 3+ iron,
methemoglobin) to reduced ferrous (valence
2+ form) which bind oxygen
 Rapaport-Luebering Pathway
- Generates 2,3 BPG and enhances oxygen
delivery to tissues
RBC Membrane Deformability
- Excess surface-to-volume ratio enables RBCs to
stretch undamaged up to 2.5 times their resting
diameter as they pass through narrow capillaries and
through splenic pores 2 um in diameter (RBC
deformability)
- Depends on RBC geometry and cytoplasmic (Hgb)
viscosity
RBC Membrane Lipids
- RBC membrane is a lipid bilayer whose hydrophobic
components are sequestered from aqueous plasma
and cytoplasm
- The phospholipid membrane provides
semipermeable barrier separating plasma from
cytoplasm and maintaining an osmotic differential
- Phospholipids are asymmetrically distributed
o Phosphatidylcholine and sphingomyelin
predominate in outer layer
o Phosphatidylserine (PS) and
phosphatidylethanolamine in inner layer
- Distribution of these four phospholipids is energy
dependent, relying on a number of membrane
associated enzymes termed flippases, floppases, and
scramblases, for their positions
- When phospholipid distribution is disrupted as in
sickle cell anemia and thalassemia or in RBCs that
have reached the 120 day life span, PS redistributes
to the outer layer
- Membrane phospholipids and cholesterol may also
redistribute laterally so that the RBC membrane may
respond to stresses and deform as they pass through
a narrow passage
- In liver disease with low bile salt concentration,
membrane cholesterol concentration becomes
reduced. As a result, the more elastic cell membrane
shows a “target cell” (codocyte) microscopically
- Acanthocytosis (spur cells) and target cells (Hgb
concentration in the center of the RBC and around the
periphery to resemble a bull’s eye) are associated
with abnormalities in the concentration and
distribution of membrane cholesterol and
phospholipids
- Glycolipids (sugar bearing lipids) may bear copies of
carbohydrate-based blood group antigens of the ABH
and the Lewis blood group systems
- Any disruption in transport protein function changes
the osmotic tension of the cytoplasm, which leads to
rise in viscosity and loss of deformability
- RBC membrane cholesterol is replenished from the
plasma
RBC Membrane Proteins
- any change affecting adhesion proteins permits RBCs
to adhere to one another and to the vessel walls
promoting fragmentation (vesiculation) reducing
membrane flexibility, shortening the RBC life span
- signal transduction, a process whereby signalling
receptors bind plasma ligands and trigger activation of
intracellular signalling proteins which then initiate
various dependent cellular activities
 Transmembrane proteins
a
 Serve as transport and adhesion sites
and signalling receptors
 Channel ions, water, and glucose and
anchor cell membrane receptors
 They also provide the vertical support
connecting the lipid bilayer to the
underlying cytoskeleton to maintain
membrane integrity
- The shape and flexibility of the RBC are
essential to its function depend on the
cytoskeleton
 Cytoskeleton is derived from a group of
peripheral proteins on the interior of the
lipid membrane
- The major structural proteins are a and B-
spectrin which are bound together and
connected to transmembrane proteins by
 ankyrin, actin, protein 4.1, adducing,
thropomodulin, dematin, and band 3
- Cytoskeletal proteins provide horizontal support
for the membrane
- RBC cytoplasm K+ concentration is higher than
plasma K+ whereas Na+ and Ca+
concentrations are lower
 Disequilibria are maintained by
membrane enzymes K+ ATPase, Na+
ATPase, and Ca+ ATPase.
 Pumpl failure leads to Na+ and water
influx, cell swelling, and lysis
 Spectrin – major cytoskeletal protein forming a lattice
at the cytoplasmic surface of the cell membrane,
providing lateral support to the membrane and thus
maintaining its shape What are Needed:
o Abnormalities account for hereditary
spherocytosis, ovalocytosis, and
pyropoikilocytosis
 Hereditary spherocytosis arises
from defects in proteins that provide
vertical support for the membrane
 Hereditary elliptocytosis is due
defects in cytoskeletal proteins that
provide horizontal support for the
membrane
 Glycophorin A – transmembrane or integral
membrane protein
o Abnormalities in the horizontal and vertical
linkages of the transmembrane and Staining Jar/DIP method
cytoskeletal RBC membrane proteins may
be seen as shape changes
DIFFERENTIAL WHITE BLOOD CELL COUNT
- the linear representation of the percentage of the
various types of leukocytes in the peripheral or
venous blood, known as the hemogram
- the determination of the percentage of each type of
WBCs in the peripheral blood Differential Counting
- consists of the enumeration of the relative proportion
of the various types of WBCs as seen as stained
blood smears
Steps in Making a Differential Count
1. Making the blood smear
2. Staining the blood smear
3. Counting the cells
4. Reporting the results
Fixation of Blood Smears
- To preserve the cell morphology, films must be fixed
ASAP after they have dried
- It is important to prevent contact with water before
fixation is complete
- Methyl alcohol (methanol) is the chose, although ethyl
alcohol (absolute alcohol) can be used
- Methylated spirit (95% ethanol) must not be used as it
contains water
- To fix films, place them in a covered staining jar in
tray containing the alcohol for 2-3 minutes
- In humid climates, it might be necessary to replace
the methanol 2-3 times per day, the old portions can
be used for storing clean slides
 Blood smear
 Methanol
 Eosin, methylene blue
 Compound light microscope
 Buffer solution pH 7.2/aged distilled water
 Differential counter
 Cedar wood oil
 Xylol
 Xylol-alcohol
1. Dip in solution with fixative (methanol) for 30 seconds
2. Dip in solution 2 (eosin, acidic dye) for 6 seconds
3. Dip in solution 3 (methylene blue, basic dye) for 4
seconds
4. Dip in buffer solution / aged distilled water for 45
seconds
5. Air dry
1. Prepare a stained blood smear
2. Place one drop of cedar oil on the feathery edge of
the stained blood smear
3. Examine the smear using LPO of the microscope.
Focus on area where the RBCs are not too
overlapping or too scanty
 4. Shift to OIO. Using the strip differential method, count broken into segments but still connected by a fine
100 WBCs while differentiating them. strand
- Cytoplasm contains small pinkish granules
- NV of relative count: 50-70% (CU)
- NV of absolute count 2,300-8,100 / cu. Mm

Counting the Cells

1. Strip Differential: all the cells are counted in the

longitudinal strip that is, from the head to the tail of
the smear
2. Exaggerated battlement: the count starts at one edge Neutrophilic Band (stab / staff)
of the smear and counting all the cells, advancing
- Younger form of neutrophil with C, S, U, or horseshoe
inward to 1/3 of the width of the smear, then on the
shaped nucleus
line parallel to the edge, then out of the edge, then
- Nucleus is continuous, no cut or division
along the edge
- NV of relative count: 0-5% (CU)
3. Two-field Meander method: the count is made by
- NV of absolute count: 0-600/cu. mm.
dividing the smear into two fields and proceeds as
exaggerated battlement method
4. Four-field Meander method: the count is made by
dividing the smear into four fields and proceeds as
exaggerated battlement method

Lymphocyte
Goal: 100
WBCs - Nucleus is compact or intact and usually round
- Cytoplasm is light blue and scanty
Neutrophilic - NV of relative count: 18-42% (CU)
Segmenter - NV of absolute count: 800-4,800/cu. mm.

- Nu
cle
us
is Monocytes

- Nucleus is spongy
and sprawling with
brain-like convolutions
 - Cytoplasm is gray. Vacuoles are sometimes present.
- NV of relative count: 2-11% (CU)
- NV of absolute count: 450-1,300/cu. mm.
Eosinophil
- Nucleus is usually bilobed
- Contains large, coarse, reddish, or orange granules
- NV of relative count: 1-3% (CU)
- NV of absolute count: 0-400/cu. mm.
Various Stains for Peripheral Blood Film:
Basophil
- Nucleus is usually indistinct and obscured by the
granules
- Cytoplasm contains large purplish-black or dark blue
granules
- NV of relative count: 0-2% (CU)
- NV of absolute count: 0-100/cu. mm
Staining of Blood Smears
- The microscopic study of stained, peripheral blood
smear constitutes the most important part of routine
haematological examination
- Cytochemical stains are essential for the identification
of hematopoietic cells
- The most commonly used stains are polychrome
stains, those belonging to the Romanowsky group
- A polychrome stain is a stain of many colors and the
original polychrome stain was discovered by
Romanowsky
- Polychrome methylene blue and eosin stains are the
outgrowth of the original time-consuming
Romanowsky method and are widely used
- They stain differently most normally and abnormal
structures in the blood
- Intravital stain is used to stain the tissue by a dye
which is introduced into a living organism and which,
by virtue of selective attraction to certain tissues, will
stain these tissues.
- Supravital stain is used to stain and inspect living
cells which have been removed from the body. It
enables the cells to remain alive and mobile, but it
does not stain the nucleus or cytoplasm. It does stain
significant structures in cytoplasm (ex. Reticulocyte
count)
 Romanowsky Stain
o Employed for staining blood films
o Combinations have two essential ingredients
(i.e., methylene blue and eosin or azure)
o Most are prepared in methyl alcohol to
combine fixation and staining
o Includes Giemsa and Wright’s
 Giemsa stain is recommended and
most reliable procedure, excellent
for staining thin and thick blood
films (inclusion bodies and
intracellular parasites as well as for
staining WBCs)
 It is composed of eosin
and azure blue, methylene
blue in methanol and
glycerin. The eosin
component stains the
parasite nucleus red while
the methylene blue
 component stains the Other Methods of Staining
cytoplasm blue.
 Wright’s stain is a histologic stain 1. Staining dish method involves placing the blood
that facilitates the differentiation of smear in a rack positioned on a dish.
blood cell types. 2. Automated method
 It is classically a mixture of a. Hemastainer automatic slide stainer
eosin azures and oxidized - Freshly prepared staining solutions are
methylene blue dyes. used daily or every 4-8 hours during
 It is used primarily to stain operations
PBS, urine samples, and b. Hema-Tek 1000 Slide stainer
bone marrow aspirates - Bottles of the Stain-Pak (stain, buffer,
which are examined under and rinse solutions) are opened by
light microscope making a small hole and a cannula is
o Panoptic stains – consists of Romanowsky inserted into each solution.
- A pump tube set is installed to transport
and another dye to improve cytoplasmic
each solution
granules
- Tubing 1 = stain solution; Tubing 2 =
 Examples: Wright’s-Giemsa,
buffer solution; Tubing 3 = rinse solution
Jenner-Giemsa, May-Grunwald-
c. Hema-Tek 2000 Slide Stainer
Giemsa
- Stainer employs the same principle as
 Methylene blue
Hema-Tek 1000.
o Basic dye
- The innovation is the improved staining
o Has affinity for acidic component of the cell
system through the use of new pumps
(nucleus)
and volume controls.
 Eosin/azure - The operator can electronically adjust the
o Acidic dye stain, buffer, and rinse solution
o Has affinity for basic component of the cell
(cytoplasm) Stations in Automated Method

Station 1 – methanol (500mL)

Station 2 – Wright’s or Wright’s-Giemsa stain (500mL)

Station 3 – stain buffer mixture, Wright’s or Wright’s-Giemsa

(80 mL), phosphate buffer (420 mL)

Station 4 – deionized water (1000 mL)

Station 5 – phosphate buffer (500 mL)

Station 6 – warm air

Staining of Two-coverglass
Methods of Classification of Cells in Differential Count
1. Schilling hemogram
- In this method, all leukocytes are grouped
according to maturity of cells into
 Granulocytes – neutrophils,
eosinophils, basophils
 Non-granulocytes – lymphocyte and
monocytes
- The PMNs are further classified according to
myelocytes, metamyelocytes, bands or
stabs, segmenters
2. Arneth’s classification
- The PMNs are classified according to the
number of lobes which their nuclei possess.
The more lobes, the older the cells.
 Class I – with lobe or indented
nucleus (5%)
 Class II – 2 lobes (35%)
 Class III – 3 lobes (41%)
 Class IV – 4 lobes (17%)
 Class V – oldest with 5 lobes (2%)
Under the traditional unit, the results in differential leukocyte
count are reported in percentage.
Under the SI unit, the proportion of each type of cell is reported
as a decimal fraction and is called leukocyte type number
fraction.
 Regenerative shift to the left – if predominating cells
are younger forms with the presence of myelocytes
and metamyelocytes and increase in band cells and it
is accompanied by a high leukocyte count.
 Degenerative shift to the left – if predominating cells
are younger forms, with an increase in band cells but
without myelocytes and metamyelocytes and it is
accompanied by low WBC count.
Shifting Processes
 Shift to the left – if there is an increase in younger
forms of WBCs particularly classes I and II; seen in
pyogenic infections
 Shift to right – if there is an increase in older forms of
leukocytes particularly classes IV and V; seen in
megaloblastic anemia and in convalescence
3. Haden’s classification
- Classifies the neutrophil according to the
presence of filaments.
- These neutrophils whose lobes are
connected by thin filaments are classified as
filamented, while those that are not
connected by filaments are grouped under
non filamented cells
 Filamented cells = 60%
 Non-filamented cells = 7%
 Eosinophils = 3%
 Basophils = 1%
 Lymphocytes = 21%
 Monocytes = 8%
Automated Differential Count
Two general principles:
1. Digital image processing – a uniformly made and
stained blood film is placed on a microscope slide,
which is driven a motor. A computer controls the
movement, scanning the slide and stopping it when
leukocytes are in the field. The optical images are
 then recorded by television camera, analyzed by  Acid slides and acid distilled water
computer and converted to digital form  Unclean slides
2. Flow through system – this system analyze the cells  Evaporation of the stain
suspended in a liquid. In photo-optical system,  Incorrect buffer pH
measurement of light scattering and f light absorption  Imperfect polychroming of the stain
are made while the cells are being counted  Incomplete reaction of the staining fluid
 Error of the operator
Overstained Smears

Causes:

 Too thick smears

 Insufficient washing
 Too prolonged staining time
 Excessive alkalinity of the stain, buffer or water

Appearance of Cells:

 Erythrocyte stains blue or green

 Cytoplasm of the lymphocytes become gray or
lavender
 Granules of neutrophils are intensely overstained
 Eosinophilic granules become deep gray or blue

Understained Smears

Causes:

 Too thin smears

 Excessive washing of the smears
 Excess acidity of the stain, buffer, or water

Appearance of Cells:

 Nuclear chromatin is stained pale blue rather than

vivid blue
 Erythrocyte stains bright red or orange rather than
pink
 Eosinophilic granules stain brilliant red

Precipitated Stain Between Cells

Causes:

 Unclean slide or coverglass

 Faulty washing because of failure to hold the slide
horizontally and to float off the scum
 Permitting dust to settle on the film

Poor Staining

 Alkaline slides and alkaline distilled water


Hemoglobin Metabolism (HEMA LEC 6TH WEEK)
Heme Structure
 Heme consists of a ring of carbon, hydrogen and
nitrogen atoms called protoporphyrin IX, with a
central atom of divalent ferrous iron
 Each of the four heme groups is positioned in a
pocket of the polypeptide chain near the
surface of the hemoglobin molecule
 The ferrous iron in each heme molecule
reversibly combines with one oxygen molecule
 When the ferrous irons are oxidized to the ferric
state, they no longer can bind oxygen.
 Oxidized hemoglobin is also called
methemoglobin
 Four identical heme groups, each consisting of a
protoporphyrin ring and ferrous iron
 Four globin (polypeptide) chains
o Alpha chains have 141 amino chains
o Beta chains and delta chains have 146
amino acids
 The amino acid sequence of the globin chain
determines the type of hemoglobin, normal
adult hemoglobin consists of two alpha and two
nonalpha chains in pairs
Complete Hemoglobin Molecule
 The hemoglobin molecule can be described by
its primary, secondary, tertiary, and quaternary
protein structures
 Primary structure refers to the amino acid
sequence of the polypeptide chains
 Secondary structure refers to chain
arrangements in helices and non-helices
 Tertiary structure refers to the arrangement of
the helices into a pretzel like configuration
 Quaternary structure is also known as tetramer
which describes the complex hemoglobin
molecule. The globin chains dissociate into
alphaBeta dimmers
 Globin chain amino acids in the cleft are
hydrophobic, whereas amino acids on the
outside are hydrophilic, which renders the
molecule water soluble.
 This arrangement also helps iron remain in its
divalent ferrous form regardless of whether it is
oxygenated (carrying an oxygen molecule) or
deoxygenated (not carrying an oxygen
molecule)
 The complete hemoglobin molecule is spherical,
has four heme groups attached to four
polypeptide chains, and may carry up to four
molecules of oxygen
Hemoglobin synthesis
 65% hemoglobin synthesis occurs in immature
nRBCs
 35% hemoglobin synthesis occurs in
reticulocytes. Heme synthesis occurs in the
mitochondria of normoblasts and is dependent
on glycine, succinyl, coenzyme A, aminolevulinic
acid synthase (aminolevulinate synthase), and
vitamin B6 (pyridoxine)

 Globin synthesis occurs in the ribosomes and it
is controlled on chromosome 16 for alpha
chains and chromosome 11 for all other chains
 Each globin chain binds to a heme molecule in
the cytoplasm of the immature RBC
Hemoglobin/Erythrocyte Breakdown
Intravascular hemolysis (10%)
- It occurs when hemoglobin breaks down in the
blood and free hemoglobin is released into the
plasma
- Free hemoglobin binds to haptoglobin (major
free hemoglobin transport protein), hemopexin
and albumin. And all of them are phagocytized
by the liver macrophages
- Laboratory: Increased plasma hemoglobin,
serum bilirubin, serum LD and urine
urobilinogen; hemoglobinuria and
hemosiderinuria present, decreased serum
haptoglobin.
Extravascular hemolysis (90%)
- Occurs when senescent or old RBC are
phagocytized by macrophages in the liber or
spleen
- Protoporphyrin ring metabolized to bilirubin
and urobilinogen excreted in urine and feces
- Globin chains are recycled in the amino acid
pool for protein synthesis
- Iron binds to transferrin and is transported to
bone marrow for the production of new RBC
Hemoglobin and Iron
 Most iron in the body is in hemoglobin and
must be in the ferrous state (Fe²+) to be used.
Fe2+ binds to oxygen for transport to lungs and
body tissues
 We can determine or detect extravascular or
the presence of the transferrin. Transferrin fully
saturated or we can also measure storage iron
in tissues and bone using the following
laboratory assays
 Ferric iron (Fe³+) is not able to bind to
hemoglobin, but does bind to transferring
 Iron is an essential mineral and is not produced
by the body
o Serum iron measures the amount of
Fe3+ bound to transferring
o Total iron-binding capacity (TIBC)
measures the total amount of iron that
transferrin can bind when fully
saturated
o Serum ferritin is an indirect
measurement of storage iron in tissue
and bone marrow
Types of Hemoglobin
 Hgb F (Fetal Hemoglobin)
o Two alpha and two gamma-globin
chains
o Functions in a reduced oxygen
environment Predominates at birth
(80%)
o Gamma chain production switches over
to chain production and is complete by
6 months of age
o We can detect the presence of the fetal
hemoglobin in the laboratory through:
 Alkali denaturation test
Kleihauer-Berke acid elution
(Hgb F is resistant to
denaturation or elution)
 Column chromatography
 Radial immunodiffusion
 Column chromatography and
Radial immuno diffusion are
 usually done in Clinical section
in Chemistry and serology
o A compensatory hemoglobin and can be
increased in homozygous and
hemoglobinopathies and beta-thalassemia
major
 Adult
o Hgb A
 2 alpha and 2 beta-globin chains
 Subdivided into glycosylated
fractions
 A1c fraction reflects glucose levels
in the blood
 It monitors individuals with
diabetes mellitus Blood is examined
after 3 months in order for us to
reflect the glucose levels in the
blood and to indicate if the patient
has diabetes mellitus
o Hgb A2
 2 alpha and 2 delta-globin chains
o Reference range for adults is 97% Hb A, 2%
Hb A2 and 1% Hb F
Different Forms of Hemoglobin
 Oxyhemoglobin - with Fe²+ O2 seen in arterial
circulation
 Deoxyhemoglobin with Fe2+ but no 0₂ seen in
venous circulation
 Carboxyhemoglobin
o With Fe2+ and carbon monoxide (CO)
o Has 200x affinity for CO than O2, so CO
is carried instead of O2
o Result in death but is reversible if given
pure O2 (gives red color to the blood)
 Sulfhemoglobin
o With S
o Cannot transport oxygen
o Seldom reaches fatal levels
o Caused by drugs and chemicals
o Irreversible Not measured by the
cyanmethemoglobin method
 Methemoglobin
o With Fe³+
o Cannot transport O2
o Increased levels cause cyanosis anemia
o We can see an increase levels in
cyanotic and anemic patients
Hemoglobin
 Cyanmethemoglobin
o Reference method for hemoglobin assay
o Principle
 Lysing agent (cyanmethemoglobin
reagent) frees hemoglobin from
the RBCs. Free hemoglobin
combines with the potassium
ferricyanide (cyanmethemoglobin
reagent), converts hemoglobin
iron from ferric state to form
methemoglobin, then combines
with pigment
cyanmethemoglobin. Measured
potassium cyanide to form the
stable using spectrophotometer)
 The cyanmethemoglobin color
intensity is proportional to
hemoglobin concentration, is
measured at 540 nm
spectrophotometrically and
compared with a standard
 Other instruments used sodium
lauryl sulfate (SLS) to convert
hemoglobin to
SLSmethemoglobin. This method
does not generate toxic wastes
Function of Hemoglobin
 When the oxygen tension in arterial blood is
high (about 95mmHg), the hemoglobin
molecule is about 95% saturated with oxygen.
This conformation of the hemoglobin molecule
in the oxygenated form is termed the relaxed
(R) state
 In this condition, the faces of hemoglobin
dimers have moved apart to bind oxygen.
However, in the veins and tissues, the oxygen
tension is lower. The hemoglobin molecule
picks up and binds oxygen while in the capillary
system of the lungs
 As the hemoglobin travels through the tissue
capillaries, in which the oxygen concentration is
decreased, it releases this oxygen to the tissues.
The deoxygenated form of hemoglobin is called
the tensed (T) state (lowest affinity for oxygen)
Oxygen Dissociation Curve
  The oxygen affinity is the ability of hemoglobin stages, iron in myoglobin
to bind or release oxygen. Expressed in terms of (muscles), and cytochromes (in
oxygen tension at which hemoglobin is 50% all cells)
saturated with oxygen  Storage Compartment (25%) -
 The relationship between oxygen tension and the iron that is not currently
hemoglobin saturation with oxygen is described functioning but is available
by the oxygen dissociation curve when needed. The major
 If we're going to plot the oxygen dissociation sources of this stored iron
curve, it may have sigmoidal shape (S) and the (ferritin and hemosiderin) are
steep is at 50% the macrophages and
o Right shift decreases oxygen affinity, hepatocytes, but every cell,
more oxygen release to the tissues except mature red blood cells,
o High 2,3 biphosphoglycerate level or stores some iron
increased body temperature; decreased  Transport Compartment (10%)
body pH - the iron that is in transit from
 Left shift increases oxygen affinity, less oxygen one body site to another in the
release to the tissues plasma and transferrin carries
 Low 2,3 biphosphoglycerate level or decreased iron in its ferric form
body temperature; increased body pH

Introduction to Iron

 Iron
o Critical for transport and use of oxygen
that the body conserves and recycles it
o Does not have a mechanism for its
active excretion
o Most essential trace element
o Free radical production by iron ions
severely damages cells and thus
demands regulation. The body adjusts
its iron levels by intestinal absorption,
depending on need. Iron is distributed
into three compartments
o The largest percentage of body iron,  Iron chemistry
nearly 65% of it, is held within o The metabolic functions of Iron depend
hemoglobin in red blood cells of various on its ability to change its valence state
stages while about 25% of body iron is from reduced ferrous (Fe+2) Iron to the
in storage, mostly within macrophages oxidized ferric (Fe+3) state
and hepatocytes. o Thus, it is involved in oxidation and
o The remaining 10% is divided among reduction reactions such as the electron
muscles, the plasma, the cytochromes transport within mitochondrial
of cells, and various iron-containing cytochromes
enzymes within cells. o In cells, ferrous iron can react with
o Three compartments peroxide via the Fenton reaction,
 Functional compartment - the forming highly reactive oxygen
largest percentage of body iron, molecules
nearly 65% of it, is held within o The resulting hydroxyl radical (OH), also
hemoglobin in RBCs of various known as a free radical, is especially
 reactive as a short lived but potent production in response to body
oxidizing agent, able to damage iron status and regulates
proteins, lipids, and nucleic acids transfer of iron from the
 Iron Kinetics enterocyte into the plasma
o Systemic body iron regulation  The mechanism by which the
 The total amount of iron hepatocytes are able to sense
available to all body cells, iron levels and produce
systemic body iron, is regulated hepcidin is highly complex, with
by absorption into the body multiple stimulatory pathways
because there is no mechanism likely involved
for excretion
 In the lumen of the small
intestine, ferrous iron is carried
across the luminal side of the
enterocyte by divalent metal
transporter 1 (DMT 1)
o Ferroportin
 Once iron has been absorbed
into enterocytes, it requires
another transporter,
ferroportin, to carry it across
the basolaminal enterocyte
membrane into the
bloodstream, thus truly
absorbing it into the body
 Is the only known protein that
exports iron across cell
membranes
 When the body has adequate
stores of iron, the hepatocytes
sense that and will increase
production of hepcidin, a
protein able to bind to
ferroportin (transports iron out
of macrophages, hepatocytes,
and enterocytes), leading to its
inactivation
 As a result, iron absorption into
the body decreases
 When the body iron begins to
drop, the liver senses that
change and decreases hepcidin
production
 As a result, ferroportin is once
again active and able to
transport iron into the blood.
Thus, homeostasis of iron is
maintained by modest
fluctuations in liver hepcidin
 (SMAD) complex, able to
migrate to the nucleus
and upregulate
hepcidine gene
expression
- When BMPR binds to HJV, there is initial
transduction. Together with second messenger
they will now be able to migrate to the nucleus
then they will be upregulate the hepcidine gene
expression

 Functions and locations of proteins involved in  Iron transport

body iron sensing and hepcidin production o Ferrous iron is exported from the
enterocyte into the blood is ferrous and
Protein Function
must be converted to the ferric form for
Hemochromatosis A protein that is bound transport in the blood
(HFE) to transferrin receptor o Hephaestin, a protein on the
1 (TfR1) until released
basolaminal enterocyte membrane, is
by the binding of
able to oxidize iron as it exits the
transferrin to TfR1
enterocyte
o Once oxidized, the iron is ready for
Transferrin A hepatocyte plasma transport, carried by a specific
receptor 2 transferrin receptor protein, apotransferrin (ApoTf)
that is able to bind o Once iron binds, the molecule is known
freed HFE to initiate an as transferrin (Tf). Apotransferrin binds
internal cell signal for two molecules of ferric iron
hepcidin production  Cellular iron absorption and disposition
o Individual cells adjust the number of
transferrin receptors on their surface to
Bone morphogenic The ligand secreted by
regulate the amount of iron they
protein (BMP) macrophages that
absorb; receptor numbers rise when
initiates signal
transduction when it the cell needs additional iron but
binds to its receptor in decrease when iron in cell is adequate
a cell membrane o Truncated soluble transferrin receptors
Bone morphogenic A common membrane are also shed into the plasma in
protein receptor receptor initiating proportion to their number of cells
(BMPR) signal transduction o Cells store iron as ferritin when they
within a cell when its have an excess. Iron can be released
ligand (BMP) binds from ferritin when needed by
degradation of protein by lysosomes
o Partially degraded ferritin can be
Hemojuvelin (HJV) A coreceptor acting
detected in cells as stainable
with BMPR for signal
hemosiderin
transduction
o Ferritin is secreted in to the plasma by
macrophages in proportion to the
Sons of mothers A second messenger of amount of iron that is in storage
against signal transduction
decapentaplegic activated by BMPR HJV
 o Ferritin is elevated in plasma by the
acute phase response, unrelated to the
amounts of stored iron
 Iron recycling
o When cells die, their iron is recycled.
Multiple mechanisms salvage iron from
dying cells. The largest percentage of
recycled iron comes from red blood
cells
o Senescent (aging) red blood cells are
ingested by macrophages in the spleen
(remember culling and splitting). The
hemoglobin is degraded, with the iron
being held by the macrophages as
ferritin
o Like enterocytes, macrophages possess
ferroportin in their membranes. This
allows macrophages to be iron
exporters so that the salvaged iron can
be used by other cells
o The exported iron is bound to plasma
apotransferrin, just as if it were newly
absorbed from the intestine
o Haptoglobin and hemopexin are plasma
proteins able to salvage free
hemoglobin or heme, respectively,
preventing them from urinary loss at
the glomerulus and returning the iron
to the liver. (Actually, iron is not
secreted in the urine but in some
disorders like protic syndrome - loss of
transferrin results to increase loss iron
in the urine)
o There are iron disorders of iron
metabolism like iron deficiency
(reduction in the rate of hemoglobin
synthesis and erythropoiesis leading
also to a disease called iron deficiency
anemia) and iron overload (disorders
associated with this are acquired or
hereditary diseases, hemosiderosis, and
hemochromatosis significant tissue
destruction occurs)
o Like macrophages, hepatocytes are
important to iron salvage. They also
possess ferroportin so that the salvaged
iron can be exported to transferrin and
ultimately to other body cells
Dietary Iron, Bioavailability, and Demand
 Under normal circumstances, the only source of
iron for the body is from the diet
 Foods containing high levels of iron include red
meats, legumes, and dark green leafy
vegetables, cereals Although some foods may
be high in iron, that iron may not be readily
absorbed and thus is not bioavailable
 Iron can be absorbed as either ionic iron or
nonionic iron in the form of heme. Ionic iron
must be in the ferrous (Fe+2) form for
absorption into the enterocyte via the luminal
membrane carrier, divalent metal transporter
(DMT1)
 However, most dietary iron is ferric, especially
from plant sources. As a result, it is not readily
absorbed. Furthermore, other dietary
compounds can bind iron and inhibit its
absorption. These include oxalates, phytates,
phosphates, and calcium
 Release from these binders and reduction to
the ferrous form are enhanced by gastric acid,
acidic foods (e.g., citrus), and an enterocyte
luminal membrane protein, duodenal
cytochrome B (DcytB)
 Thus, although the U.S. diet contains on the
order of 10-20mg of iron/day, only 1-2mg is
absorbed
 This amount is adequate for most men, but
menstruating women, pregnant and lactating
women, and growing children usually need
additional iron supplementation to meet their
increased need for iron
 Heme with its bound iron is more readily
absorbed. than ionic iron. Thus meat, with
heme in both myoglobin of muscle and
hemoglobin of blood, is the most bioavailable
source of dietary iron
Laboratory Assessment of Body Iron Status
 Disease occurs when body iron levels are either
too low or too high
 The tests used to assess body iron status are
able to detect both conditions
 They include the traditional or classic iron
studies: serum iron (SI), total iron-binding
capacity (TIBC), percent transferrin saturation,
Prussian blue staining of tissues, ferritin assays,
 soluble transferrin receptor (STfR), and RBCs
hemoglobin content of reticulocytes The results  Serum iron (SI)
of these measured parameters can be o Serum iron can be measured
combined to calculate an sTfR/log ferritin ratio colorimetrically using any of several
or graph a Thomas plot reagents such as ferrozine The iron is
 Finally, zinc protoporphyrin is another assay first released from transferrin by acid,
with special application in sideroblastic anemia and then the reagent is allowed to react
 Diagnostically, the tests can be organized to with the freed iron, forming a colored
assess each of the iron compartments as complex that can be detected
indicated here: spectrophotometrically
o The serum iron level has limited utility
on its own because of its high within-
Laboratory Typical Adult Diagnostic Use day and between-day variability; it also
Assay Male Compartment increases after recent ingestion of iron-
Reference Assessed containing foods and supplements
Interval o To avoid the apparent diurnal variation,
Serum Iron 50-160 µg/dL Indicator of the standard practice has been to
Level available collect the specimen fasting and early in
transport iron the morning when levels are expected
to be highest
o A diurnal variation in hepcidin has been
Serum 250-300 µg/dL Indirect
Transferrin indicator of detected that may explain some of the
Level iron stores serum iron variability and may still
support the early morning phlebotomy
Transferrin 20%-55% Indirect practice
Saturation indicator of  Total iron-binding capacity (TIBC)
iron stores with o The amount of iron in plasma or serum
transport iron will be limited by the amount of
Serum Ferritin 40-400 ng/mL Indicator of transferrin that is available to carry it
Level iron stores o To assess this, transferrin is maximally
Bone marrow Normal iron Visual saturated by addition of excess ferric
or liver biopsy stores qualitative iron to the specimen
with Prussian visualized assessment of
o Any unbound iron is removed by
blue staining tissue iron
precipitation with magnesium
stores
Soluble 1.15-2.75 mg/L Indicator of carbonate powder
transferrin functional iron o Then the basic iron method as
receptor (sTfR) available in described above is performed on the
level cells absorbed serum, beginning with the
sTfR/log ferritin 0.63- 1.8 Indicator of release of the iron from transferrin
index functional iron o The amount of iron detected represents
available in all the binding sites available on
cells transferrin-that is, the total iron-binding
RBC zinc <80 µg/dL of Indicator of capacity (TIBC)
protoporphyrin RBCs functional iron o It is expressed as an iron value,
level available in
although it is actually an indirect
RBCs
measure of transferrin.
Hemoglobin 27-34 pg/cell Indicator of
content of functional iron  Percent transferrin saturation
reticulocytes available in o Since the TIBC represents the total
developing number of sites for iron binding and the
 SI represents the number bound with
iron, the degree to which the available
sites are occupied by iron can be
calculated
o The percent of transferrin saturated
with iron is calculated as:
SI/TIBC X 100% = % transferrin
saturation
It is important that both the SI and TIBC
be expressed in the same units
o A convenient rule of thumb evident
from the table is that about one third
(1/3) of transferrin is typically saturated
with iron
 Prussian blue staining
o Prussian blue is actually a chemical
compound with the formula Fe7(CN)18.
The compound forms during the staining
process, which uses acidic potassium
ferrocyanide as the reagent/stain
o The ferric iron in the tissue reacts with
the reagent, forming the Prussian blue
compound that is readily seen
microscopically as dark blue dots
o Tissues can be graded or scored
semiquantitatively by the amount of
stain that is observed
o Prussian blue stain is considered the gold
standard for assessment of body iron
o Staining is conducted routinely when
bone marrow or liver biopsies are taken
for other purposes
o Although ferric iron reacts with the
reagent, ferritin is not detected, likely
due to the intact protein cage
o However, hemosiderin stains readily
 Ferritin
o The level of serum ferritin has been
shown to correlate highly with stored
iron as indicated by Prussian blue stains
of bone marrow
o Increase in ferritin can be induced
without an increase in the amount of
systemic body iron. These rises may not
be outside the reference interval but still
high enough to elevate a patient's
ferritin above what it would otherwise
be
 Soluble transferrin receptor (stfr)
o Increase in the sTfR reflect either
increases in the amounts of TfR on
individual cells, as in iron deficiency, or
an increase in the number of cells each
with a normal number of TfRs
o The latter occurs during instances of
rapid erythropoiesis, such as a response
to hemolytic anemia
 Hemoglobin content of reticulocytes
o It is analogous to the mean cell
hemoglobin (MCH), but just for
reticulocytes
o Under normal conditions, the number
of circulating reticulocytes represents
the status of erythropoiesis in the prior
24-hour period; the amount of
hemoglobin in reticulocytes provides a
near real-time assessment of iron
available for hemoglobin production
 Soluble transferrin receptor/log ferritin
o Although ferritin and sTfR values alone
can point to iron deficiency, the ratio of
sTfR to ferritin or STfR to log ferritin
improves the identification of iron
deficiency when values are equivocal
o Because the sTfR rises in iron deficiency
and the ferritin (and its log) drops,
these ratios are especially useful when
one of the parameters has changed but
is not outside the reference interval
 Thomas plot
o Demonstrated that when the sTfR/log
ferritin is plotted against the
hemoglobin content of reticulocytes, a
four-quadrant plot results that can
improve the identification of iron
deficiency
o In instances where there is true iron
deficiency, the sTfR will rise and the
ferritin will drop so that the sTfR/log
ferritin will be high and the hemoglobin
content of reticulocytes will be low
o Patient results will plot to the lower
right quadrant
o In instances where the ferritin may be
falsely elevated by inflammation, the
sTfR/log ferritin will be normal despite
reduced availability of iron for
 hemoglobin production -- thus low a
hemoglobin content in reticulocytes
o In this instance, patient values will plot
to the lower left quadrant called
functional iron deficiency because the
systemic body stores are adequate but
not available for transport and use by
cells
o As iron deficiency develops, other cells
are starved before erythrocytes;
production of hemoglobin in
reticulocytes remains at a normal level
for as long as possible
o However, the body's other iron-starved
cells will increase sTfR production and
systemic iron stores of ferritin will be
depleted, thus elevating the sTfR/log
ferritin value
o These early iron-deficient patients’
 Zinc protoporphyrin
results will plot to the upper right
o Zinc protoporphyrin (ZPP) accumulates
quadrant called latent iron deficiency
o Plotting the ratio of soluble transferrin in red blood cells when iron is not
incorporated into heme and zinc binds
receptor to log ferritin (sTfR/log ferritin)
instead to protoporphyrin IX. It is easily
against the hemoglobin content of
detected by fluorescence
reticulocytes produces a graph with
o Although ZPP will rise during iron-
four quadrants
o Patients with values within the deficient erythropoiesis, the value of
this test is greatest when the activity of
reference intervals for each assay will
the ferrochelatase is impaired, as in
cluster in the upper left quadrant
lead poisoning
o Those with functional iron deficiency,
like the anemia of chronic
inflammation, will cluster at the lower
left. (low hemoglobin content of
reticulocytes, normal sTfR/log ferritin)
o Latent iron deficiency, before anemia
develops, will cluster to the upper right
with frank iron deficiency in the lower
right quadrant
o Latent iron deficiency - normal
hemoglobin content of reticulocytes,
increased sTfR/log ferritin)
o Iron deficiency - low hemoglobin
content of reticulocytes, increased
sTfR/log ferritin