HEMATOLOGY
LECTURE 01 ORIENTATION AND FUNCTIONS OF BLOOD
Introduction
HEMOGLOBIN 101
FUNCTION OF BLOOD
- Hemoglobin
- How is hemoglobin synthesized/catabolized
- How is hemoglobin used for the transport of oxygen
DYSHEMOGLOBINEMIA
- Dyshemoglobin compounds that are significant in blood
diseases
- Carboxyhemoglobin, methemoglobin, sulfhemoglobin
IRON KENETICS
- How iron is being recycled in the body
- How iron is stored in the body
- Different tests related to iron used in the diagnosis of
certain blood disorders (ei. Anemia)
- Serum iron, serum ferritin, total iron binding capacity
METHODS OF HEMOGLOBIN DETERMINATION
- Gravimetric
- Chemical
- Gasometric
- Colorimetric
HEMOGLOBIN CATABOLISM (BREAKDOWN OF HEMOGLOBIN)
- Breakdown of globin chains - composed of amino acids
that are being recycled
- Breakdown of heme (NOTE: only protoporphyrin IX is
removed from the body)
OXYHEMOGLOBIN DISSOCIATION CURVE
- Diseases/conditions that will shift the dissociation curve
- Significance: provides an idea on how the primary function
of hemoglobin is being utilized by the body – to transport
oxygen and carbon dioxide
SPECIAL HEMATOLOGIC TESTS
- Tests that are not routinely used in hematology and some
of the tests are already obsolete
- Test are still important because it may be requested for
rare blood dyscrasias
Learning Objectives on Hematology Lecture Series Part 1:
1. Explain the different functions of blood.
2. Discuss the characteristics and synthesis of hemoglobin
and the clinical significance of iron kinetics.
3. Discuss the properties and clinical significance of the
different dyshemoglobins.
4. Apply the principles and procedures of the different
methods for hemoglobin determination.
5. Explain the principles of the oxyhemoglobin dissociation
curve and the catabolism of hemoglobin.
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6. Enumerate and discuss the different special hematologic
tests.
Seminar Topics (Week 2)
CYTOCHEMICAL STAINS IN HEMATOLOGY
- Used in tertiary laboratory
- Expensive tests
- Proper training is required
- Used for the diagnosis of leukemias and anemias (ei.
Myeloperoxidase, Leukocyte Alkaline Phosphatase,
Esterase, Sudan Black B, etc.)
AUTOMATION IN HEMATOLOGY
5 basic principles of automation
 Current Impedance
 Electrical impedance
 Cytochemical differentiation
 Light Scatter
 Flow Cytometry (combination of the previous principles)
- Common automation principles in hematology
- Discuss how each principle determine the number of cells
in a blood sample, hematocrit, hemoglobin and
differentiation of white blood cells.
COAGULATION AND HEMOSTASIS
- Coagulation Cascade
- Test used in coagulation
SPECIAL TOPICS
VERIFICATION OF HEMATOLOGY ANALYZERS
Delivered at the PAMET Scientific Conference in Pangasinan
Importance: Acc. to ISO 15189 (guideline for the accreditation of
clinical laboratories) and
CLSI – dictates the verification of the hematology analyzers
QUALITY CONTROL PLANNING AND SIGMA METRICS IN THE
HEMATOLOGY LABORATORY
Delivered at the PAMET Scientific Conference in Pangasinan and
Baguio/Benguet, Mt. Province Chapters
Learning objectives on Hematology Series Part 2:
1. Discuss the principles of testing and clinical significance of the
different cytochemical stains used in hematology
2. Explain the principles of automation in Hematology
3. Discuss the coagulation cascade
4. Explain how automated analyzers in hematology are verified
5. Discuss quality control planning and then apply sigma metrics
as a quality control tool in hematology (protect your dignity and
license in the future)
Hematology 1 1
 FUNCTIONS OF BLOOD
Blood is a specialized connective tissue – it originates from the
parenchyma cells, in the embro and the fetus.
It contains formed elements in an extra cellular matrix (ECM).
GAS TRANSPORT
- Transport O2 from the lung to the different parts of the body
and to collect CO2 to bring it out of the system
- During respiration CO2 is produced → respiration will
happen while tissues will need oxygen
ACID/BASE LEVELS
- Maintains acid/ base levels by hemoglobin (has a buffer
system in itself maintains acidity and alkalinity by way of
maintaining pH levels)
- Normal pH of blood: 7.35 – 7.45 (Slightly alkaline)
- Bloods cells has the affinity for the acid dye because the
acidic component of blood cell is alkaline
- pH changes based on respiration, amount of oxygen in the
blood and acid.
- Hemoglobin → brings O2 to the tissues → CO2 goes
inside the blood
Note: CO2 does not need red blood cells to be transported
but only water (plasma is made up of 92% water). It is
oxygen that needs hemoglobin to be transported because it
is non-water soluble. If CO2 is dissolved in water, it
produces carbonic acid which decreases the pH = increase
acidity.
BLOOD CLOTTING
Mediated by platelets as well as the coagulation factors present in
plasma.
BODY TEMPERATURE
In the environment, if the temperature is hot, the temperature inside
the body is much hotter
- Since the blood is constantly going around the body
(revolving), the heat inside the body and body organs is
brought to the superficial areas of the body (i.e., arms,
legs, face, torso)
- The warm blood inside the body that continuously
circulates around is carried outside the body (i.e., skin) →
perspiration (due to condensation) maintains the
temperature inside and outside the body.
OSMOTIC PRESSURE
Also known as oncotic pressure
- We are talking about the function of albumin and certain
proteins in the blood to maintain osmotic/oncotic pressure.
- Why water does not immediately enter the blood circulation
(e.g., when drinking water)? This is because of osmotic
pressure.
- The maintenance of the protein component (albumin) of
the blood maintains osmotic pressure. Basically, this
amount of solute is maintained by this amount of solvent.
- If the amount of albumin decreases, the amount of water
also decreases, and vice versa. But if the amount of
albumin is normal, water will not simply enter the blood.

IMMUNE RESPONSE
WBCs are made to combat infectious agents by way of immune
response.
2 types of lymphocytes in the body:
1. B-cells: involved in the production of antibodies and also
involved in humoral immunity.
2. T-cells: responsible for phagocytosis (direct cell killing) and
also involved in cellular immunity.
- Neutrophils are involved in bacterial infection because
they engulf bacterial cells. Neutrophils can only survive for
9-12 days because we are at constant exposure to
infectious agents.
- On a daily average, more than 3,000 types of bacteria,
fungal cells and viruses are breathed in every time we
breathe.
- Neutrophils are suicidal meaning if these cells have
engulfed a bacterial cell, it will release lytic agents within
and lysing itself together with the bacterial cells and
eventually turns into pus cells (manifestation of anti-
inflammation/infection).
- Pus cells are blood cells that have destroyed their
cytoplasm with no distinct morphological characteristic but
contains granules.
- Eosinophils are involved in parasitic infections.
Eosinophils have the Major Basic Protein (MBP) which is
responsible for anti-parasitic activity, killing the parasites.
- Any type of parasitic infection, the eosinophils increase in
number.
- If in case the infectious load of parasite is low, this is
because of the presence or increase of eosinophils.

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 LECTURE 01 Hemoglobin 101

hemoglobin  Salt bridges will bind the globin chains

 Major component of red blood cells together
 Blood can carry very little oxygen through plasma, because  Lead to more O2 deposited to your
oxygen is nonwatery soluble, thus it requires hemoglobin to Hgb
be transported.  O2 down, 2,3, - DPG also down
o Other References: 2-3% of oxygen is found in 
plasma. 97% of oxygen in blood is bound to HEMOGLOBIN SYNTHESIS
hemoglobin
o Sir AJ: 0.3% plasma 99.7% hemoglobin  Mitochondrion
 Hemoglobin is required to carry oxygen around  65% - developing erythroblasts
 If the body had to depend upon dissolved oxygen in the  35% - reticulocytes
plasma to supply oxygen to the cells, the heart would have  Derived from succinyl CoA + glycine
to pump 140 liters per minute – instead of 4 liters per  Remnants of heme production
minute, leading to an increased cardiac output o FEP
o Decreased RBC  Decreased Hemoglobin  o Ferritin
Decreased Oxygen  Increased Cardiac Output  Main function: O2 transport
(one of the complications of anemia) o 1.34 mL of O2/g Hgb
 Each red blood cells can carry about 1 million molecules of o 401 mL of O2/g iron
oxygen Heme synthesis:
 Arterial Blood (oxygenated blood): Hemoglobin is 97% - Protoporphyrin IX
saturated with oxygen when it leaves the lungs - Iron
 Venous Blood (Deoxygenated blood): Under resting o 90% of the iron that we use is recycled iron
conditions, hemoglobin is about 75% saturated with oxygen o 10% diet
when it returns to the heart Two pathways:
o Increased physical activity increases metabolic  Globin: via the ribosomes
activity in tissues, leading to increased  Heme: via the synthesis between the cytosol (cytoplasm)
respiration to provide more oxygen to the tissues and mitochondria
 Blood through perfusion is able to collect oxygen from the o 2 components in a developing RBC where heme
lungs is being produced
 Blood cells are made up of two components  Succinyl CoA
o Hemoglobin: solution inside the cells  Glycine
o Membrane: holds or encases the hemoglobin

Video 2 | Hemoglobin 101

• Iron is important for affinity.
• Without iron, there will be no O2 affinity.
• What regulates the affinity?
o 2,3, - DPG
 Transient resident (pag kailangan,
anjan sya WOW NAOL)
o Not because iron is present, doesn’t mean that it
will automatically bind to O2.
o Salt bridge: responsible for binding the globin
chains
o (+) 2,3, - DPG >> salt bridges will be broken s
down = lesser affinity of O2 to Hgb
 Ex. Increased physical activity
 Increased need for O2 by the tissues
 Needs more O2 because your muscles
are undergoing metabolism and
respiration
 2,3, - DPG will have to be produced by
Luebering Rapoport shunt of the
Embden Meyerhof pathway
 After release of 2,3, - DPG, O2 will be
released from the Hgb [It will hold the
globin chains to release the O2]
 After resting >> lower respiration >>
lower body temperature >> O2 will now
stabilize b/n blood and the tissues
 2,3, - DPG goes down

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*Start with Succinyl CoA
*Heme synthesis starts and ends inside the mitochondria
*Most enzymes are found in the cytoplasm
 Yellow part: mitochondria
 White part: cytoplasm
 Protoporphyrin IX will bind to Iron in the presence of
Ferrochelatase to form HEME
 Heme goes out of the cytoplasm >> looks for globin chains
>> form Hgb
 Succinyl CoA + Glycine >> dALA goes outside and P.H.UCs
>> Protoporphyrinogen III
 1 RBC can carry 1 million molecules of Hgb
o 1 molecule of Hgb carries 4 molecules of O2
o Ex. If 250 000 molecules x 4 = 1M O2
 If NO iron: Protoporphyrin IX
 Bone marrow will produce RBC as long as there is hypoxia
(detected by the kidneys – production of EPO)
 ↑ EPO = RBC produced
 What if no iron? Ex. Iron-deficiency anemia
o No iron = produce FEP (Free Erythrocyte
Protoporphyrin)
o FEP = an analyte being determined among iron-
deficiency anemia patients
o ↑ FEP = no iron; diagnostic of IDA
 What if iron overload?
o Sideroblastic anemia
o Low EPO = iron overload
o Ferritin = storage iron; goes back to the liver
(stored there for future use)
• 1.34 mL of O2/g Hgb
o Collect 100 mL of arterial blood
o Contains 15g of Hgb
o Hgb concentration is 15g/dL
o 15g/100mL
o 15 x 1.34 = 20mL of O2 is present per 100 mL of
blood
PORPHYRIN RING
 At the core of the molecule is porphyrin ring which holds an
iron atom.
 An iron containing porphyrin is termed a heme.
 This iron atom is the site of oxygen binding.
 Should always be in the Ferrous state
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 Oxidized Ferric state = produce hemiglobin/methemoglobin
1 hemoglobin molecule is made up of 4 globin chains
 2 beta
 2 alpha
*binding is always Beta 2 alpha 1 ; Beta 1 alpha 2
Video 3 | Hemoglobin 101
IRON METABOLISM
Storage iron – liver
 Ferritin
o Iron + apoferritin
 Hemosiderin
o Non water soluble
Iron absorption
 Duodenum and jejunum
o Intestinal mucosal cells will oxidise it to its ferric
state
o Mediated by hephaestin (protein on the
basolaminal enterocyte membrane of the GIT
paryicularly the small intestine – responsible for
oxidizing iron as it exits the enterocyte and into the
blood stream)
o Once it is oxidized, the iron is now ready for
plasma transport carried by a specific protein
known as apotransferrin.
o Once the iron binds, the molecule is now known
as transferrin.
o Apotransferrin binds to molecules of ferric iron.
Iron recycling
 Salvage iron from the dying cells
 Largest percentage of recycled iron: from RBCs; senscent
RBCs ingested by macrophages in the spleen
 Not yet senescent RBCs: due to increased hemolytic
reactions coming from either pathologic or physiologic
reactions
 Iron from Hgb has to be recycled
 Hgb is first degraded with the iron >> ferritin
 In the blood vessels, the macrophages posses ferroportin in
their membrances that allow macrophages to be iron
exporters so that they can be salvaged into usable iron for
use by other cells.
o Mediated by a substance produced by the liver
= Hepcidin
o Responsible for regulating the absorption of iron
o ↑ absorption from the GIT may lead to iron
overload
 Life-threatening
 Liver damage
 Heart disease
 Diabetes
o Regulator molecule that will determine the activity
of ferroportin (responsible for the absorption of
iron)
o Hephaestin will be involved in absorption of iron
 When needed, iron is eventually
released from the mucosal cells and
then binds to transferrin developing
RBCs through ferric transferrin
membrane receptors
Hematology 1 4
 • After releasing iron into the
bone marrow, the transferrin
receptor will complex then
move back to the cell
membrane and return to the
plasma.
• Only the excess iron will be
incorporated into the
transferrin and return to the
liver.
o Important: regulatory mechanism b/n the
absorption of iron in the body
 iron transport
o Hephaestin
 65-68% of total body iron is found in HGB as hemoglobin
iron
 18% - form of ferritin and hemosiderin found in the liver
 10% - myoglobilin iron in muscles
o Peroxidase
o Catalase
o Cytochrome
o Riboflavin enzymes
 Iron is important for the functionality of hemoglobin
*transferrin is measured indirectly by TIBC
*test for tranferrin not always readily available
 Several blood disorders that may detedted with the use of
iron panel tests
1. SERUM IRON
 measure of iron content in serum
 affected by diurnal variations
 Iron intake and acute phase responses will actually be
affected by diurnal variation
 Limited utility in assessment of overall iron status
 Still used in the computation of transferrin saturation
 Decreased:
 IDA – anemia because of deficiency of
iron
 ACD – iron in the bodyis sufficient but it
is stored in the form of ferritin or
hemosiderin; not releaased in the serum
• Compensatory mechanism of
our in cases of inflammatory
reactions or infectious
diseases
• In the (+) of infectious
diseases in our body, will
trigger our body to restrict the
release of iron. Stored in the
form of ferritin or hemosiderin.
• Therefore, there will be an
apparent decrease in the
serum iron because it is not
released by the body to deter
the growth of mcgs.
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Hemochromatosis
 Disease which causes your body to absorb too much iron
from the food that you eat
 Too much iron can be dangerous >> life-threatening
because it can be stored
Sideroblastic anemia
 Result of a deffect in an X-linked recessive gene
 ALAS 2: makes an enzyme which is essential in the
production of heme (O2 carrying portion of the Hgb
molecule)
 Ferrochelatase: absense of ALAS 2 will
make a deficiency in an enzyme
responsible for produxn of heme
 Decreased O2 carrying capacity of the
Hgb molecule
 Serum iron: decreased
 Because most of the iron being supplied
to the BM for erythrocyte production is
not being taken in
 Iron return back to the liver or in the
plasma
 Charactrized by the presence of iron granules present
around the mitochondria of precurser cells of red cells;
preipheral membance of RBCs
 Thalassemia: decreased globin synthesis
PCT
 Group of acquired and familial disorders in which the activity
of the heme synthetic enzyme uroporphyrinogen
decarboxylase is deficient
 Same with SDA; missing enzyme that is responsible for
binding or synthesis of protoporphyrin IX
 Less amount of protoporphyrin IX that is
supposed to bind iron
 Eventually, decrease synthesized heme
 Increase in serum iron because it is not
being used
• Vitamin B12 deficiency
 Inappropriate of RBC due to problems with normal nuclear
maturation
Hematology 1 5
 2. SERUM FERRITIN Hemoglobin compounds

 Good indicator of the total body iron stores • Oxyhemoglobin – type of hemoglobin that is bound to
 Major storage form of iron • Carboxyhemoglobin
 Low levels: diagnostic of IDA o Cherry Red
 <100ug/L does not always exlude iron-deficiency o Increased affinity for O2
 Feritin: acute phase reactant that can be increased in o Level in smokers: 5-15% (0.5% in non-smokers)
inflammatory states (ex. Liver disease, malignancies) o 10% - impairs judgement
 Elevated: iron overload o Greater than 40% - fatal
 Transferrin Saturation: test required for iron overload o Tests (absorption band: 576 nm)
 Only decreased in cases of IDA  Gas chromatography
 Palmer’s
3. Transferrin Iron Saturation Percentage  Sunderman’s
 Usually computed as the percent of transferrins carrying  Dithionate
capacity that is saturated with iron • Methemoglobin
 For every molecule of transferrin, only 1/3 is occupied by o Chocolate brown
iron. 2/3 a reserve for more iron to bind o Remedies
 Formula: serum iron/TIBC x 100  Methemoglobin reductase
 Decreased Saturation system/diaphorase
 IDA  Glutathione reductase system
 ACD o Absorption band: 630 nm using KCN
 Any type of inflammation o Methemoglobinemia
 Useful test for the assessmemt of high ferritin  Acquired – exposure to oxidants
 High ferritin below 45% especially among women or below (antimalarial drugs, sulfonamides,
50% among men is more likely secondary to another cause aniline dyes, nitrate-rich food stuff)
 Inherited – NADH mthemoglobin
4. Total Iron Binding Capacity (TIBC) reductase system def
Indirect measure of transferrin levels  Inherited Hgb M amino acid substitutes
Transferrin levels are increased in the setting of ion
deficiency as well as in pregnancy, also in cases of estrogen
administration
 Decreases
 Inflammation
 Chronic liver disease
 Malnutrition
 Absence of iron >> transferrin is increased
 Because it is the body’s compensatory
mechanism for the lack of iron
5. Hemoglobin
 Mostly anemia, therefore decreased
 *Hemochromatosis, PCT, African Siderosis

COLLATED BY: ♣ ♠ ♥ ♦ Hematology 1 6

LECTURE 02 DYSHEMOGLOBIN: hemoglobin derivatives and their associated disorders
HEMOGLOBIN
• Normal hemoglobin is produced with no problems
regarding globin chain synthesis nor substitution of
amino acids within globin chain or structural problems
with the globin chain
• Thalassemia: problems in globin chain synthesis
• Hemoglobinopathies: problem in amino acids or
structure of globin chain
OXYHEMOGLOBIN AND DEOXYHEMOGLOBIN
• Oxyhemoglobin – with oxygen; oxygen is being
transported by hemoglobin as O2 is non-water soluble,
meaning it needs a transport molecule
• Deoxyhemoglobin – hgb with no oxygen
CARBAMINOHEMOGLOBIN
• hgb with carbon dioxide
• carbon dioxide that first binds with an amino group, then
to hemoglobin
• co2 cannot bind to hgb without amino group
• hgb can only transport 5% on Carbon Dioxide, the
remaining 95% is transported by plasma and the water
present in RBC
• Plasma can only transport 3% of Oxygen
• Carbon Dioxide is water soluble so it doesn’t need
hemoglobin, it can easily dissolve in the plasma
PATHOLOGIC HEMOGLOBIN COMPOUNDS
CARBOXYHEMOGLOBIN
• Hemoglobin derivative that binds to carbon monoxide
• Normally found in blood at less than 1% of Total
Hemoglobin
• Hemoglobin has 200-250 times greater affinity to
Carbon Monoxide (CO) than Oxygen (O2)
• Carbon monoxide is an colorless, odorless gas that
when encountered even in small percentages can
prevent the oxygen transport of many hemoglobin
molecules. It can lead to asphyxiation
Symptoms and Physical Findings
• The skin may turn bright cherry red with increasing
levels of carboxyhemoglobin
• Exposure and Effect
10% impairs judgement; some may undergo
dizziness or fainting
>40% Fatal
50-70% Asphyxiation
Laboratory Findings and Correlation with Disease
• Level in smokers: 5-15%
• Non-smokers: 0-5%
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o Rural areas have lesser levels of carbon
monoxide
• One spot test
o Procedure:
 Hemolyze 0.5 mL of whole blood with
20 mL distilled water
 Add 1mL of NaOH, 1.0 mol/L
o Result:
 Brown blood: Normal
 Light Cherry Red Blood: >20%
Carboxyhemoglobin
• Quantitation of CO (absorption band: 576 nm)
o Gas chromatography
o Spectrophotometry
• Other Tests
o Palmer’s
o Sunderman’s
o Dithionite
Effects of Treatment on Laboratory Results
• It requires administration of high levels of Oxygen
• Hyperbaric Oxygen Chamber
o Patients are exposed to 600x oxygen as
compared to the normal environment;
o Oxygen plasma can be maximized from 0.3%
to 3%
• As long as there is no brain damage, carbon monoxide
poisoning has no long-term effect as long as the patient
receives proper and prompt treatment
METHEMOGLOBIN
METHEMOGLOBIN (Hemiglobin or Hi)
 Produced when the iron component of blood is oxidized
from the ferrous state to the ferric state
 Normally, 0.5 - 3% of hgb in blood is converted to
methemoglobin daily
 NADH-methemoglobin reductase (diaphorase) enzyme
is present in erythrocytes to counter the normal
production of methemoglobin
METHEMOGLOBIN REDUCTION
 Requires a properly functioning Embden-Meyerhof
pathway to produce NADH, which is the main source for
electron donation in the reduction process
 The electron is transferred from NADH to the
Methemoglobin Reductase and then to an erythrocyte
cytochrome before being used in the actual heme
reduction
Hb+3 ----------------------------------------------------------------- Hb+2
(NADH NAD)  e-  Met Hb Reductase  Cytochrome
Hematology 1 7

METHEMOGLOBINEMIA
 Elevated erythrocyte methemoglobin concentrations due
to inability to adequately reduce methemoglobin
 Can cause cyanosis
 Clinical Manifestations: few and generally mild
 Rarely inherited; there are five inherited Hemoglobin M
variants, that cause methemoglobinemia
 Usually acquired
 Treatment: Patients respond to therapeutic doses of
methylene blue (except patients with Hb M disorder).
o Cyanotic  Normal Skin Color
Inherited Methemoglobinemia
 Inherited NADH-methemoglobin reductase enzyme
deficiency or Diaphorase Deficiency
 The deficiency does not adequately reduce
methemoglobin even with sufficient NADH levels
produced by Embden-Meyerhof pathway
 Patients respond to Methylene Blue Treatment
Acquired Methemoglobinemia
 May be caused by ingested or absorbed substances
o Antimalarial drugs
o Sulfonamides
o Drugs of abuse
o Aniline dyes (fresh dye on shoes)
o Nitrate-rich water and foods
o Common aromatic chemicals
 Patients also respond to methylene blue treatment
Inherited Hemoglobin M Methemoglobinemia
 There are five Hb M variants caused by amino acid
substitutions in the globin chains
 The amino acid substitutions in globin chains directly
affect the heme group, causing it to enter the ferric or
oxidized state
 Patients do not respond to methylene blue
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 Treatment may not be necessary since there are no
other clinical abnormalities than cyanotic appearance
Laboratory Findings and Correlation with Disease
Peripheral Blood Film
 Denatured hemoglobin demonstrates Heinz bodies due
to the ingested toxins
 Require special stains
Diaphorase Enzyme Screening Test
 Rapid
 Specific enzyme assays for use with prepare
hemolysates are also available
Methemoglobin Quantitation
 Absorbance at 630 to 635 nm
 Potassium cyanide (KCN) is added to a prepared
hemolysate which is converts methemoglobin to
cyanmethemoglobin (does not absorb at 632nm)
 The concentration of methemoglobin is proportional to
the amount of cyanmethemoglobin produced in the
reaction
 Results
o Abnormal: >1.5 % methemoglobin
o Methemoglobin due to toxic substances: 10 to
< 70%
o Untreated diaphorase deficiency: 15-30%
Other Findings
 Patients with methemoglobinemia can cause a slight
compensatory erythrocytosis and a slight left shit in the
O2 dissociation curve. Erythrocyte survival is normal
Remedies
 Methemoglobin reductase system/diaphorase
 Glutathione reductase system
 Vitamin E (alpahatocopherol) is an antioxidant which
prevents the oxidation of iron from ferrous to ferric state
 Injection of methylene blue reducing agent
o Methylene blue donated electron to the
enzyme NADPH-methemoglobin reductase ,
leading to the reduction of Methylene blue to
Leukomethylene blue
o Leukomethylene blue nonenzymatically reduce
methemoglobin
Blue family of Kentucky
 In US, they are cyanotic, and they are injected with
methylene blue reducing agent due to their system
deficiency
Hematology 1 8
 SULFHEMOGLOBIN
 Oxidation derivative of hemoglobin that cannot carry
oxygen
 Exposure to certain drugs and chemicals
o Acetanilid, phenacetin, sulfonamides
 Sulfur containing drugs that may lead
to binding of sulfur to hemoglobin
 Sulfur has the same affinity with oxygen but once sulfur
has bound to hgb, oxygen cannot bind to hgb anymore,
leading to cyanosis
 Once sulfhemoglobin is formed it cannot be converted
back to a functional hemoglobin
 IrreversibleA
 Mauve-lavender blood
 Absorption band: 618 nm

Symptoms and Physical Findings

 Excessive sulfhemoglbin concentration hardly exceed
20% in the blood
 Not life threatening and is generally benign
 Cyanosis is the only significant effect

Laboratory Findings and Correlation with Disease

 Sulfhemoglobin quantitation
o Examines hemolysate for a distinct, broad
increase in the absorption curve in the range of
600 to 620 nm
 Treatment
o Removal of the offending agent

COLLATED BY: ♣ ♠ ♥ ♦ Hematology 1 9

 LECTURE 03 Methods of Hemoglobin Determination

Gravimetric colorimetric
• Crude method Visual Methods
• Used for mass blood donations – because of simplicity of
the test ACID HEMATIN METHOD
o Basis of donor’s acceptance to donate
 Sinks or stays in the middle:
QUALIFIED
 Floats: DEFFERED

PROCEDURE
1. Place 500mL Copper Sulphate (CuSO4) Solution in a 1L
beaker Comparator
2. Adjust the CuSO4 by adding water until its specific gravity blocks
is 1.050, 1.052 or 1.053
3. Place a drop a patient’s blood  observe if it floats or
sinks

RESULTS
FLOAT: SG is less than the CuSO4
SINK: SG is greater than the CuSO4
STAYS IN THE MIDDLE: equal SG

Specific Gravity Corresponding HgB conc PROCEDURE:

1.050 - 1. Add acid hematin to the hemometer tube (0.1 N HCl) up to
1.052 - the 2-mark
1.053 12.5 g/dL 2. Add 20 uL blood
1.055 13.5 g/dL 3. Mix with glass stirrer or applicator stick
4. Place the tube in the hemometer set
Note: The SG of the sample is directly proportional to the HgB 5. Add drops of distilled water – for every drop, stir the soln
content of the blood until the color of the solution is the same as the comparator
block (endpoint)
Gasometric 6. Check the level of the soln – this is now the level of HgB
• Indirect measure for HgB ALKALI HEMATIN METHOD
o measure O2 to determine the level of HgB
o 1.34 mL 02/g HgB • Same procedure as Acid hematin
o Difference: reagent is 1 N Sodium hydroxide
• Specimen: arterial blood
o The sample should be ~97% oxygenated • Standardization method: Gibbson/Harisson method

Example: 100 mL of blood with 20.1 mL of oxygen Photometric Method (Medical)

20.1 𝑚𝑚𝑚𝑚 • Direct HgB determination
= 𝟏𝟏𝟏𝟏 𝒈𝒈 𝒐𝒐𝒐𝒐 𝑯𝑯𝑯𝑯𝑯𝑯⁄𝟏𝟏𝟏𝟏𝟏𝟏 𝒎𝒎𝒎𝒎 𝒐𝒐𝒐𝒐 𝒃𝒃𝒃𝒃𝒃𝒃𝒃𝒃𝒃𝒃 • Reference method
1.34
• Principle is also used by hematology analyzers
Chemical o Cyanmethemoglobin reagent  dilute blood 
• Indirect measure lyse red cells  read spectrophotometrically
o Measures iron to determine the level of HgB
o ICSH: 3.47 mg Fe/g HgB DRABKIN’S METHOD/CYANMETHEMOGLOBIN METHOD
 ICSH – International Committee for • Most common manual method
standardization in Hematology • Drabkins reagent
• Involves lysing of blood cells  measure iron in the o Potassium ferricyanide + Potassium cyanide +
hemolysate sodium bicarbonate + surfactant
 Cyanide is slowly replaced by Sodium
lauryl sulfate
• Cyanide is hazardous

PRINCIPLE: Drabkins reagent converts hemoglobin to

methemoglobin  cyanmethemoglobin (orange in color; Abs: 540
nm in spectrophotometer)

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 [RODAKS]
1. Create a standard curve, using a commercially available
Note:
• The absorbance of cyanmethemoglobin is directly
proportional to the hemoglobin concentration.
• Sulfhemoglobin can’t measured by this method since it
is not converted to cyanmethemoglobin
PROCEDURE
[Sir Aj’s]
1. 5 ml of Drabkin’s reagent is placed in a tube
2. Dispense 20 uL of blood
3. Mix  incubate for 15 mins at 37OC
4. Read against a standard
b. Transfer the dilutions to cuvettes. Set the
wavelength on the spectrophotometer to 540 nm
and use the blank to set to 100% transmittance.
c. Using semilogarithmic paper, plot percent
transmittance on the y-axis and the hemoglobin
concentration on the x-axis. The hemoglobin
concentrations of the control and patient samples
can be read from this standard curve
d. A standard curve should be set up with each new
lot of reagents. It also should be checked when
alterations are made to the spectrophotometer
2. Controls should be run with each batch of samples.
Commercial controls are available
3. Using the patient’s whole blood anticoagulated with EDTA
or heparin or blood from a capillary puncture, make a 1:251
cyanmethemoglobin standard.
a. When a standard containing 80 mg/dL of
hemoglobin is used, the following dilutions should
be made:
dilution by adding 0.02 mL (20 mL) of blood to 5 mL of
cyanmethemoglobin reagent. The pipette should be rinsed
thoroughly with the reagent to ensure that no blood
remains.
4. Cover and mix well by inversion or use a vortex mixer. Let
stand for 10 minutes at room temperature to allow full
conversion of hemoglobin to cyanmethemoglobin
5. Transfer all of the solutions to cuvettes. Set the
spectrophotometer to 100% transmittance at the
wavelength of 540 nm, using cyanmethemoglobin reagent
as a blank
6. Using a matched cuvette, continue reading the %
transmittance of the patient samples and record the values
7. Determine the hemoglobin concentration of the control
samples and the patient samples from the standard curve

SOURCES OF ERROR IN DRABKIN’S METHOD

Effect Resolution
Drabkin’s reagent is sensitive to light Store in a brown/amber bottle or in a dark
place
High WBC count (>20x 109/L) Centrifuge reagent-sample solution and
Turbidity  false high result
High platelet count (>700 x 109/L) supernatant measured
Lipemia Corrected by adding 0.01 ml of patient’s
Turbidity  false high result plasma to 5 mL cyanmethemoglobin  used
as reagent blank
Presence of Hb S and Hb C Make 1:2 dilution with water and multiplying
Resistant to hemolysis  turbidity
the results from the standard curve by 2
Presence of abnormal globulins in patients Add 0.1 g of potassium carbonate to
with plasma cell myeloma or Waldenstorm cyanmethemoglobin reagent
macroglobulinemia
Globulins may precipitate in the reagent
Note: Commercial cyanmethemoglobin
reagent are now modified to contain potassium
carbonate

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 Lecture 04 Hemoglobin dissociation
Oxygen dissociation curve (01)
Other Blood Functions
1. Thermoregulation
QFR #1: How does the blood regulate the temperature of the body?
 The heat of the internal organs will perfuse in blood and will
later disseminate and distribute that heat throughout the
body, from the core to the surface and vice versa. By
changing the blood flow to the skin, the body can control
heat exchange at its surface with its surroundings
2. Immunologic function (WBCs)
3. Hemostasis
4. pH Buffer
Factors that affect Hemoglobin affinity for Oxygen
[Hematology 1- Module 3: Hemoglobin]
1. Partial pressure of oxygen
 If the oxygen dissociation curve shifts to the left, a patient with
arterial and venous PO2 levels in the reference intervals (80 to
100 mm Hg arterial and 30 to 50 mm Hg venous) will have a
higher percent oxygen saturation and a higher affinity for
oxygen than a patient for whom the curve is normal. With a shift
in the curve to the right, a lower oxygen affinity is seen.
2. pH of blood
 The pH of blood affects the shifting of the oxygen dissociation
curve either to the left or to the right. This is explained by the
Bohr effect which explains the lower affinity of hemoglobin for
oxygen due to increases in the partial pressure of carbon
dioxide (CO2) which eventually decreases the blood pH.
Whenever the human body undergoes increased cellular
respiration, such as in strenuous physical activities, there is also
an increase in metabolic activity within the tissues involved
resulting in the production of CO2 as a metabolic waste product.
To transport CO2 through the venous blood, it diffuses into the
red blood cells combining with water to form carbonic acid
(H2CO3). This reaction is facilitated by the enzyme, carbonic
anhydrase. The carbonic acid will then dissociate to release H+
and bicarbonate (HCO3-). The increase in H+ due to this
reaction decreases the blood pH as explained by the Bohr effect
COLLATED BY: ♣ ♠ ♥ ♦
Figure 1.0 The oxygen affinity of hemoglobin is depicted by the
Oxygen dissociation curve (ODC). The fractional saturation of
hemoglobin (y axis) is plotted against the concentration of oxygen
measured as the PO2 (x axis). At a pH of 7.4 and an oxygen tension
(PO2) of 26 mmHg, hemoglobin is 50% saturated with oxygen (red
line). The curve shifts in response to temperature, CO2, O2, 2,3-
BPG concentration, and pH. When the curve shifts left (light blue
line), there is increased affinity of Hb for O2. When the curve shifts
right (dark blue line), there is decreased affinity of Hb for O2
[McKenzie, 3rd ed]
SUMMARY
LEFT SHIFT [R] RIGHT SHIFT [T]
↑ O2 ↓ O2 (to tissues)
↑ pH (No HCO3 & H2CO3) ↓ pH
↓ CO2 ↑ CO2 (to blood)
↓ 2,3-BPG (No Release) ↑ 2,3-BPG
↓ H+ ↑ H+
↓ Temperature ↑ Temperature
[At resting phase] [↑ Metabolism = ↑ respiration]
3. 2,3-Biphosphoglycerate (2,3-BPG)
 . 2,3-BPG affects oxygen-binding affinity by binding in a
small central cavity of deoxygenated hemoglobin. This shifts
the equilibrium towards deoxy-hemoglobin. The presence of
acids leads to increased H+ and a reduction in pH. This
promotes formation of the deoxy form of hemoglobin,
shifting the oxygen dissociation curve to the right, promoting
oxygen release to actively respiring tissues. At high altitude,
when oxygen in the atmosphere is scarce because the air is
“thinner,” production of 2,3-BPG is greatly increased,
helping hemoglobin to release more of its bound oxygen. It
takes about 24 hours for 2,3-BPG levels to rise, and over
longer periods of time, the levels continue to increase as part
of the acclimation effect. The 2,3-BPG production is one
reason that athletes train at high altitudes to increase their
aerobic capacity. Interestingly, 2,3-BPG does not bind to
fetal hemoglobin. This results in tighter binding of oxygen
relative to maternal hemoglobin, giving the developing fetus
better access to oxygen from the mother's bloodstream.
4. Temperature
 Tissues of the human body that are actively undergoing high
metabolic rates produce excess amounts of thermal energy
as a waste product. Through heat transfer, the thermal
energy diffuses to the blood in nearby capillaries. When the
thermal energy reaches the blood stream, it increases the
average kinetic energy of the molecules within the plasma
thereby resulting to higher temperature. Higher temperature
in the blood is interpreted by the body that cells are working
harder thus requiring more oxygen to keep them going. In
response to higher temperature, the Hb decrease its affinity
for oxygen to facilitate delivery to the tissues. Thus,
increased temperature in the blood shifts the oxygen
dissociation curve to the right such as when tissues are
actively engaged in physical activity, these tissues would
require and eventually receive more O2.
Hematology 1 12
5. Carbon dioxide
 CO2 is a waste product of cellular respiration. As discussed earlier, CO2 diffuses from the tissues to red cells to form H2CO3 which dissociates
to H+ and HCO3- resulting to a shift of the curve to the right. Once the red cells reach the lungs, O2 will diffuse into the deoxygenated Hb
resulting to release of H+ that will combine with HCO3- to form H2CO3 which in turn will dissociate to water and CO2 where the latter will
diffuse out of the cells and eventually expelled by the lungs. This phenomenon is explained by the Haldane effect which describes the binding
of oxygen to Hb which promotes the release of CO2

2,3 BPG Binding to Hemoglobin

PRINCIPLE:

Oxygen interacts weakly with heme iron, and the two

can dissociate easily. As O2 is released by hemoglobin
in the tissues, the heme pockets narrow and restrict
entry of O2, and the space between the β-chains
widens and 2,3-BPG binds again in the central cavity.
Thus, as 2,3-BPG concentration increases, the T
configuration of hemoglobin is favored and the
oxygen affinity decreases. [McKenzie, 3rd ed]

Oxyhemoglobin VS Deoxy-hemoglobin

Oxyhemoglobin [R] Deoxy-hemoglobin [T]

 Tense or “T” state
 Relaxed or R state
 2,3 BPG causes a conformational change in hemoglobin shape
 O2 can freely enter in the body
because it replaces the salt bridges connecting the globin chains.
 Hemoglobin tetramer is fully oxygenated
 2,3 BPG is ONLY produced when there is increased need for O2
 NO 2,3 BPG (Instead, salt bridges hold α1β1 and α2β2)

NOTE: The tense form incorporates one 2,3-bisphosphoglycerate (2,3-BPG) molecule, bound between the β-globin chains with salt bridges. It is unable to
transport oxygen. As hemoglobin binds oxygen molecules, the α1β1 and α2β2 dimers rotate 15º relative to each other as a result of the change in hydrophobic
interactions at the α1β2 contact point, disruption of salt bridges, and release of 2, 3-BP [Rodak,5th]

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 Hemoglobin as a Blood Buffer

OPTIMAL blood pH : 7.40

 <7.4 : acidosis
 >7.4 : alkalosis
NORMAL pH range: 7.35- 7.45
Buffering System

PRINIPLE :The effect of pH on hemoglobin–oxygen affinity is known as the Bohr effect, an

example of the acid–base equilibrium of hemoglobin that is one of the most important buffer
systems of the body. A molecule of hemoglobin can accept H+ when it releases a molecule of
oxygen. Deoxyhemoglobin accepts and holds the H+ better than oxyhemoglobin. In the tissues,
the H+ concentration is higher because of the presence of lactic acid and CO2. When blood
reaches the tissues, hemoglobin’s affinity for oxygen is decreased by the high H+ concentration,
thereby permitting the more efficient unloading of oxygen at these sites. [McKenzie, 3rd ed]

QFR #2: How does the buffer system work ?

i. During respiration CO2 is produced.
ii. CO2 diffuses in the blood cells and plasma (water)
iii. [CO2 + H2O] : Forms carbonic acid (H₂CO3) in the presence of carbonic
anhydrase [enzyme]
iv. H2CO3 produced in increased amounts will decrease blood pH.
v. Blood will have increased acidity
vi. [HCO3- + H+] : In order to maintain blood pH, some H2CO3 is further converted
into bicarbonates and hydronium ions in the same presence of carbonic
anhydrase [enzyme]
vii. Bicarbonates (HCO3-) will act as the buffering system
viii. HCO3- and H2CO3 will be dissociated in the lungs as H2O and CO2
ix. H2O will be reabsorbed in the system
x. CO2 will be expelled during respiration

Note: CO2 does not require hemoglobin to be transported out of the body. It only needs H2O
because it is water soluble. .
 95% of CO2 is transported by the water component of blood and dissolved in the plasma
 99.7% of O2 is transported by Hgb.

CHECKPOINT
OXYGEN CONCENTRATION is the same through all levels of the earth at 20% to 21%.
However, the partial pressure of O2 is dependent on elevation.
 ↑↑ Altitude = ↓ partial pressure = O2 will diffuse harder
 ↓↓ Altitude (sea level) = ↑ partial pressure = O2 will diffuse easier

 INHALED O2 will be decreased by 4% to 5%. Hence, EXHALED O2 will be around 16% to 17%.

Oxygen dissociation CURVE (02)

PRINCIPLE: The oxygen dissociation curve illustrates the relationship between oxygen
saturation of hemoglobin and the partial pressure of oxygen.
 In the Normal (N) hemoglobin-oxygen dissociation curve, P50 is the partial
pressure of oxygen (O2) needed for 50% O2 saturation of hemoglobin.
 In the Left-shifted (L) curve with reduced P50, it can be caused by decreases
in 2,3-bisphosphoglycerate (2,3-BPG) (e.g., multiple transfusion of stored
blood), H+ ions (raised pH), partial pressure of carbon dioxide (PCO2), and/or
body temperature. A left shifted curve is also seen with hemoglobin F and
hemoglobin variants that have increased oxygen affinity and in alkalosis.
 In the Right-shifted (R) curve with increased P50 can be caused by
elevations in 2,3-BPG (e.g., in response to hypoxic conditions such as in high
altitudes), H+ ions (lowered pH), PCO2, and/or temperature. A right-shifted
curve is also seen with pulmonary insufficiency, congestive heart failure, severe
anemia and in the presence of hemoglobin variants that have decreased
oxygen affinity.
[Hematology 1- Module 3: Hemoglobin]

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 OXYGEN TRANSPORT
MUST KNOW [Rodak 5th]
 The function of hemoglobin is to readily
bind oxygen molecules in the lung,
which requires high oxygen affinity; to
transport oxygen; and to efficiently
unload oxygen to the tissues, which
Main Hgb requires low oxygen affinity.
function in O2
transport  During oxygenation, each of the four
heme iron atoms in a hemoglobin
molecule can reversibly bind one
oxygen molecule.
Approximately 1.34 mL of oxygen is
bound by each gram of hemoglobin
 Affinity of hemoglobin for oxygen
 Reflects the amount of oxygen gas
dissolved in the blood. It primarily
PO2 / PaO2
measures the effectiveness of the lungs
Partial pressure of in pulling oxygen into the blood stream
Oxygen from the atmosphere.
 NORMAL: 27 mm Hg results in
50% oxygen saturation
 LEFT SHIFT : < 27 mm Hg
 RIGHT SHIFT : >27 mm Hg
 The amount of oxygen needed to
P50
saturate 50% of hemoglobin
Oxygen
 Plots the percent oxygen saturation of
Dissociation
hemoglobin versus the PO2
Curve (ODC)
 The curve is sigmoidal, which indicates
low hemoglobin affinity for oxygen at
low oxygen tension and high affinity for
Sigmoidal
oxygen at high oxygen tension.
o At the plateau (75%) O2
dissociation will be more rapid
 The reference interval for arterial
oxygen saturation is 96% to 100%.
 LEFT SHIFT : a patient with arterial and
venous PO2 levels in the reference
Arterial oxygen intervals (80 to 100 mm Hg arterial and
saturation 30 to 50 mm Hg venous) will have a
higher percent oxygen saturation and
a higher affinity for oxygen than a
patient for whom the curve is normal.
 RIGHT SHIFT: Lower oxygen affinity
 A shift in the curve due to a change in
pH (or hydrogen ion concentration) ‘
 It facilitates the ability of hemoglobin to
exchange oxygen and carbon dioxide
Bohr effect
(CO2)
o ↑↑CO2 and ↑↑ H+ ion
concentrations (lower pH)
decrease oxygen saturation
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o ↓↓CO2 and ↓↓ H+ ion
concentrations (higher pH)
increase oxygen saturation
 The local fluid flow through the capillary
network and extracellular spaces of
living tissue. It is characterized as the
Perfusion
volumetric flow rate per volume of
tissue.
o Ex. Obtaining O2 from the lungs
CONDITIONS THAT PRODUCE A LEFT SHIFT [R]
 Lowered body temperature due to external causes.
 Multiple transfusions of stored blood with depleted 2,3-BPG;
alkalosis
 Presence of hemoglobin variants with a high affinity for
oxygen.
 Shift to the left indicates a decreased MCV (microcytic).
[Ciulla]
CONDITIONS THAT PRODUCE A RIGHT SHIFT [R]
 Increased body temperature
 Acidosis
 Presence of hemoglobin variants with a low affinity for
oxygen
 Increased 2,3-BPG concentration in response to hypoxic
conditions, such as :
 high altitude
 pulmonary insufficiency
 congestive heart failure
 severe anemia
 Shift to the right indicates an increased MCV (macrocytic).
[Ciulla]
MOVEMENT OF O2 FROM BLOOD TO TISSUE
 O2 from blood can be transferred into the tissues within the
ff conditions:
o PaO2 of tissue should be lower than blood
 NOTE: PO2 of blood is p50 = 50% Hgb
saturation.
 Normal range is p50 to p100
o Diffusion: movement of solute from higher to a
lesser concentration.
o Example:
i. Physical activity induces a right shift.
ii. Increased respiration will be observed
as more O2 is used.
iii. Hence, PaO2 in the muscles will be
decreased.
iv. Diffusion will now occur.
v. CO2 will move to the blood stream and
will be converted to H2CO3 and later
into HCO3. These byproducts will be
transferred into the lungs
vi. HCO3 and H2CO3 is converted to CO2
and H2O
vii. H2O is absorbed ; CO2 is expelled
Hematology 1 15
SAMPLE SCENARIOS 4. Which of the following is associated with a "shift to the left"
1. PHYSICAL ACTIVITY in the oxygen dissociation curve of hemoglobin [CIULLA]
a. Decreased pH and elevated temperature
o Increased unloading O2 into the tissues
b. Decreased oxygen affinity
o ↓ PaO2
c. Decreased oxygen release
o ↓ SpO2 d. Presence of 2,3-bisphosphoglycerate (2,3-BPG)
o Results to a hypoxic tissue
5. A "shift to the left," when used to describe a cell
2. >1,500m ABOVE SEA LEVEL [Baguio] population, refers to: [CIULLA]
o PaO2 is 100 mmHg a. Increased cells in the blood due to a redistribution of
o PaO2 in lungs: 40 mmHg blood pools
o Inhalation of O2 at this altitude will result to a decreased b. An increase in immature blood cells following release
PaO2. From 100 mmHg it will be diffused into 80 mmHg of bone marrow pool
o Compensatory mechanism: ↑ RBC production ↑ O2 c. A cell production "hiatus" or gap
o DID YOU KNOW: d. A higher percentage of lymphocytes than neutrophils
Living at places such as Baguio increases your
hematocrit level by 2 to 3 points. 6. In the normal hemoglobin-oxygen dissociation curve, what
Congestion is commonly observed when travelling percentage of oxygen is released to the tissues when PO2
from high altitude places to the lowlands. It is averages 40 mmHg? [BOC]
commonly manifested through “rosy cheeks” hihi a. 75%
3. SEA LEVEL b. 25%
o PaO2 is 140 mmHg c. 100%
o PaO2 in lungs: 40 mmHg d. 50%
o Diffusion of O2 in the lungs will be from a high to a low
concentration 7. What would the hemoglobin-oxygen dissociation curve
o Inhalation of O2 at this altitude will result to a decreased depict in a patient exhibiting clinical signs of alkalosis?
PaO2. From 140 mmHg it will be diffused into 120 mmHg [BOC]
a. normal
4. >10,000 m ABOVE SEA LEVEL [Mt. Everest] b. shift to the left
o PaO2 is 40 mmHg c. shift to the right
o PaO2 in lungs: 40 mmHg d. none of the above
o Inhalation of O2 at this altitude will result to a decreased
PaO2. From 40 mmHg it will be diffused into 20 mmHg 8. What would the hemoglobin-oxygen dissociation curve
o Thin O2 levels cause hypoxic situations. It can initially be depict in a patient exhibiting clinical signs of acidosis?
manifested through fainting. [BOC]
a. normal
NOTE: b. shift to the left
A decrease in the PO2 is due to the admixture of other forms of c. shift to the right
gases d. none of the above

REVIEW QUESTIONS 9. One of the most important buffer systems of the body is
[Rodak, Ciulla, BOC, McKenzie, Module ] the: [McKenzie]
1. Which of the following forms of hemoglobin molecule has a. chloride shift
the lowest affinity for oxygen? [Rodak] b. Bohr effect
a. Tense c. heme–heme interaction
b. Relaxed d. ODC

2. Using the normal hemoglobin-oxygen dissociation curve in
Figure 1 for reference, predict the position of the curve when
there is a decrease in pH. [Rodak]
a. Shifted to the right of normal with decreased oxygen
affinity
b. Shifted to the left of normal with increased oxygen
affinity
c. Shifted to the right of normal with increased oxygen
affinity
d. Shifted to the left of normal with decreased oxygen
affinity
3. How does hyperventilation affect the behavior of
hemoglobin in response to the imbalance in the levels of
oxygen and carbon dioxide in the body? [Module, QFR]
10. When the H+ concentration in blood increases, the oxygen
affinity of hemoglobin [McKenzie]
a. Increases
b. is unaffected
c. decreases
d. cannot be measured
11. During exercise, the oxygen affinity of hemoglobin is
[McKenzie]
a. Increased in males but not females
b. decreased due to production of heat and lactic acid
c. unaffected in those who are physically fit
d. affected only if the duration is more than 1 hour

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12. The oxygen dissociation curve in a case of chronic
carboxyhemoglobin poisoning would show [McKenzie]
a. a shift to the right
b. b . a shift to the left
c. c . a normal curve
d. d . decreased oxygen affinity

13. An aerobics instructor just finished an hour of instruction.

Blood is drawn from her for a research study, and the oxygen
dissociation is measured. What would you expect to find?
[McKenzie]
a. shift to the left
b. a shift to the right
c. no shift
d. an increased oxygen affinity

14. In the lungs, a hemoglobin molecule takes up two oxygen

molecules. What effect will this have on the hemoglobin
molecule? [McKenzie]s
a. It will increase oxygen affinity.
b. It will narrow the heme pockets blocking entry of
additional oxygen.
c. The hemoglobin molecule will take on the tense
structure.
d. The center cavity will expand, and 2,3-BPG will enter

Answer Key:
1.a 2.a 3. 4.c 5.b 6.b 7.b
8.c 9.b 10.c 11.b 12.b 13.b 14.a
RATIONALIZATION
Question # 3 : Hyperventilation causes acute hypocapnia resulting
in cerebral vasoconstriction, hypocalcemia, a shift in the oxygen–
hemoglobin dissociation curve, and reduced oxygen delivery.
Symptoms include lightheadedness, dizziness, faintness,
paresthesias, and altered consciousness. Acute hyperventilation
will result in an acute respiratory alkalosis and cause a left shift in
the oxyhemoglobin dissociation curve

Question # 4 : A "shift to the left" in the oxygen dissociation

curve of hemoglobin means that a higher percentage of
hemoglobin will retain more of its oxygen at a given pressure.
Thus affinity will be greater and oxygen delivery will be reduced.
[CIULLA]

Question # 5 : A "shift to the left" means an increase in immature

neutrophilic cells in the blood caused by bone marrow release of
cells in response to infection or tissue damage. A redistribution of
the blood pools because of emotional or physical stimuli is
characterized by an increased WBC count without a left shift. A
cell "hiatus" refers to a population of cells in which there is a gap
in the normal maturation sequence. A cell hiatus is most often
seen in acute leukemia, in which there are many blasts and a few
mature cells but no intermediate stages. [CIULLA]

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 LECTURE 06 Special hematological tests
Erythrocyte sedimentation rate
• Erythrocyte Sedimentation Rate (ESR) – is a non-specific
indicator of inflammation.
3 FACTORS AFFECTING ESR
RBC
o dependent on how the RBCs will be able to form rouleaux or to
pack or to be attached to one another making them heavier. In
a simple analogy, it is like stacking several coins and letting
them fall in a bucket of water.
 Coins that are stacked together will settle faster than
those that are not attached to each other/separated.
 In terms of the RBC, the zeta potential which results
from the negatively charged sialic acid groups on the
red cell membrane is responsible for affecting the
results of ESR particularly the rouleaux formation of
RBC
 A decrease in the zeta potential of RBC will make them
near negative, near neutral, or near positive in terms of
their electroneutrality on the plasma membrane.
 Changes on the electroneutrality of RBCs will allow
them to attach to one another and form rouleaux.
 Factors that affect the zeta potential: Fibrinogen and
Gamma-globulins = ↓ decrease the zeta potential of
RBCs = ↑ rouleaux formation = ↑ ESR
PLASMA
o components in plasma that would affect ESR.
 Factors in plasma that affect ESR: Presence of acute
phase proteins (e.g., beta-2-macroglobulin, alpha-1-
antitrypsin, and others) which are responsible for
decreasing the viscosity of plasma.
 The role of plasma in ESR is very important because
the viscosity or the lesser viscosity of plasma will affect
the rest.
 ↑ viscosity of plasma will deter sedimentation of blood
cells while ↓ viscosity of plasma (less viscous) will allow
rapid sedimentation of red cells
 Presence of acute phase proteins in the plasma
especially in cases of inflammation → decrease the
viscosity of plasma → faster sedimentation of RBCs
 The presence of high levels of albumin or
hyperalbuminemia → increase the viscosity of
plasma → slow sedimentation of RBCs
TECHNICAL
 Shaking or vibrations on the table = ↑ ESR
 Tilting of the tube = ↑ ESR
 Dilution factors = ↑ or ↓ ESR
WINTROBE
 The common anticoagulant is double oxalate
 The Wintrobe method uses the Wintrobe tube.
MICROMETHOD (LANDAU)
 Often used for infants
 Uses sodium citrate as the specimen
 The ESR must be setup within 4 hours of collection of
blood, otherwise it may cause variations in the test result
STAGES OF ERYTHROCYTE SEDIMENTATION
o Rouleaux formation – first 10 minutes
o Sedimentation of RBCs in plasma – next 40 minutes
o Packing/settling of RBCs – last 10 minutes
CLINICAL SIGNIFICANCE:
INCREASED
 Pregnancy
 Anemia among females
 Macrocytosis
 Technical errors (e.g., tilted ESR tubes)
 Seen in elevated fibrinogen and gamma-globulin levels
in cases of infection and inflammation
 Malignancy
 Acute myocardial infarction
 Multiple myeloma
 Waldenstrom’s macroglobulinemia
DECREASED
 Extremely high WBC count
 RBC anomalies:
 Spherocytosis
 Acanthocytosis
 Microcytosis
 Some of these cells will not allow rouleaux
formation to take place
 May result in dilution problems (e.g., inadequate mixing
of the sample and anticoagulant)
 May also be seen in hyperalbuminemia,
hypofibrinogenemia, hypogammaglobulinemia, and
certain dysproteinemias.
Zeta sedimentation ratio
o Measurement of the ease with which the RBC are able to
sediment under standardized cycle of compaction and
dispersion stress with the use of zetafuge.
 Zetafuge – a centrifuge which thins capillary tubes in a
vertical position in 4 x 45 sec. cycles

METHODS:
WESTERGREN/MODIFIED WESTERGREN/DISPETTE
 The usual specimen used for these methods is sodium
citrate treated blood
 For the modified Westergren method the anticoagulant
used is modified by adding 2mL of EDTA + 0.5mL
either sodium citrate or NSS before diluting it in
blood.
 The common tube used is the Westergren tube for the
 Zetacrit
Westergren method with a total length of 300mm, while
 ZSR = Hct/Zct
the graduations is only 200mm. The internal bore of the
tube is 2.5mm ± 0.15mm.

COLLATED BY: ♣ ♠ ♥ ♦ Hematology 1 18

  Correction for anemia (patient with anemia will drastically
affect ESR whether or not there is inflammation will most
likely be high)
 NV: 41-54%
 Advantages:
o Easier to fill capillary tubes compared to ESR
o Less sample needed
o Unaffected by anemia
Reticulocyte count
o Measure of the bone marrow’s effective erythropoietic activity
o NV:
o Adults – 0.5 -2.5%
o Infant – 2- 6%
 in most laboratories, retic count is expressed as the
Relative Retic count (%), the percentage of
reticulocyte count in relation to the total red blood
cell count of the patient
 ↑ reticulocyte count may indicate hemolysis, in
some follow up samples it may result to response
to treatment for IDA
METHODS:
GLASS SLIDE METHOD (Stains are used)
1. Dry/Schilling
 Brilliant Cresyl Blue or the reference stain - new
methylene blue N (NMBN) to dry on a surface of
the glass slide→ once the stain is dry on the glass
slide, put one drop of blood and overlay a coverslip
→ incubate for 10-15 mins under a wet preparation
(covered in a petri dish) → view under the
microscope (10 fields HPF/OIF)
2. Wet (create a blood smear out of the solution)
 Stain is still wet when supravital staining with
NMBN/BCB
 Adding equal amounts of blood with NMBN/BCB →
incubate for 5 mins (inside the clinical lab
incubation is 10-15 mins at 37⁰C for better uptake
of the stain) → place one drop of the mixture of
blood and stain in a glass slide → let it dry→
examine under the microscope
 Note: supravital – staining while the cells are living;
not yet fixed
CALIBRATED MILLER DISK
o Reticulocyte (%) = Total Retic count in square A ×
100/Total RBC in square B × 9
COLLATED BY: ♣ ♠ ♥ ♦
COMPUTATIONS:
RELATIVE RETICULOCYTE COUNT
 commonly expressed in the laboratory
# 𝑜𝑜𝑜𝑜 𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐
𝑅𝑅𝑅𝑅𝑅𝑅 = × 100
1000 𝑅𝑅𝑅𝑅𝑅𝑅
ABSOLUTE RETICULOCYTE COUNT
 the actual count of reticulocytes in the sample
𝐴𝐴𝐴𝐴𝐴𝐴 = 𝑅𝑅𝑅𝑅𝑅𝑅 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 × 𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅 𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 (𝑖𝑖𝑖𝑖 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑)
CORRECTED RETICULOCYTE COUNT
 also known as reticulocyte index or hematocrit correction –
RI/HC
 Done when the patient has ↓ hematocrit
𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅 𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 × 𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞 𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝐻𝐻𝐻𝐻𝐻𝐻
𝐶𝐶𝐶𝐶𝐶𝐶 =
𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝐻𝐻𝐻𝐻𝐻𝐻 [0.45]
RETICULOCYTE PRODUCTION INDEX
 Also known as the second correction/Shift correction
𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐
𝑅𝑅𝑅𝑅𝑅𝑅 =
𝑚𝑚𝑚𝑚𝑡𝑡𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑖𝑖𝑖𝑖 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 𝑜𝑜𝑜𝑜 𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟
𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 𝑜𝑜𝑜𝑜 𝑡𝑡ℎ𝑒𝑒 𝐻𝐻𝐻𝐻𝐻𝐻
 Hct = 0.35- 0.39 – maturation time = 1.5 days
 Hct = 0.25- 0.34 – maturation time = 2 days
 Hct = 0.15- 0.24 – maturation time = 2.5 days
 Hct = < 0.15 – maturation time = 3 days
SOURCES OF ERROR
o Recent blood transfusion
o Proper specimen handling, preparation and transport (viability
of RBCs are very important; fresh samples should be collected
and should be processed/prepared right away)
o Red cell inclusions mistaken for reticulocytes
 Heinz bodies (usually prepared via supravital staining)
 Howell-Jolly bodies
 Pappenheimer bodies
Hematology 1 19
 LE CELL PREPARATION
• Detect presence of LE cell for diagnosis of SLE
• LE Cell
o Neutrophil that engulfed antibody-coated nucleus of another cell due to the production of antinuclear antibodies.
o Discovered by Malcolm Hargraves, Helen Richmond and Robert Morton (1948).
 This ANA or LE factor which is present in serum actually destroys the nuclear protein and neutrophils eventually will
phagocytose the antibody coated nuclear material
• Smears
LE Cell

a. Zimmer & Hargraves

i. Macerate the blood clots using
mortar and pestle and the wire sieve • Homogenous nuclear material that are presumed
ii. Allow the clotted blood to pass to have been antibody coated already which are now
through the sieve by slowly phagocytosed by nuclear material of a neutrophil
macerating it • LE cell has already been lysed so it is more
iii. the maceration will actually homogenous
introduce the trauma to the WBCs if • often mistaken as TART CELLS
Blood clot preparation the ANA is present, this will trigger
the production of antibodies  and Tart cell
then eventually the ANA will coat
some of the nuclear materials of the
WBCs  the triggered neutrophils
will then phagocytose this antibody
coated nuclear material

b. Magath & Winkle

• Heterogenous nucleus
• Tart cells are not always clinically significant but then
it is seen in cases of infections or inflammations
• There is just an adjacent cells near another nuclear
material

LE cell: phagocytosed
Tart cell: no phagocytosis
Rotary/ Zinkam & Conley
Defibrinated method
Slide method Snapper & Nathan
Capillary tube method Mudick

• Serologic
DNP – use of latex beads that have been coated of deoxyribonucleic proteins. If agglutination happens then it is usually interpreted as a
positive test for the presence of ANA or the LE cell

COLLATED BY: ♣ ♠ ♥ ♦ Hematology 1 20

 ANTI-NUCLEAR ANTIBODY TEST

Detects ANA in patients adhering to reagent test cells forming distinct fluorescence pattern
Types of substrates
• Rat liver
• Multiblock substrates (combination of mouse stomach + rat liver + rat kidney)
 Standard substrate before
• Hep-2: increased sensitivity
• Substrate being used now
• Cultured cells of laryngeal squamous cell carcinoma
• they are usually available commercially in the form of prefixed on glass slide  drop the blood of the Px
on the glass slide where Hep 2 was prefixed  Interpret

Interpretation
• Qualitative
POSITIVE/ negative
• Quantitative
o Last dilution with a (+) result is the titer
o Reporting:
 -/+
 Titer
 Type of substrate
IF-ANA  Pattern of fluorescence

Homogenous: - Characterized by total nuclear fluorescence due to antibodies that are derive
against the nucleoprotein
- Common in SLE, drug induced-SLE
Peripheral - Fluorescence occurs at the edges of nucleus in a shaggy appearance, the anti-
dna antibodies cause this pattern
- Common in SLE, and systemic sclerosis
Speckled - Result from antibodies that are directed against different nuclear antigens.
- They are often seen in SLE, mixed connective tissue diseases, systemic
sclerosis, Sjogren syndrome, polymyositis
Nucleolar - Results from antibody directed against a specific RNA configuration of the
nucleolus or an antibody that is specific for proteins that are necessary for
maturation of nucleolar RNA
- Systemic sclerosis, pyomyositis

Additional notes:
 IF-ANA was introduced by George Friou (1957)
 One of the widely used test for the diagnosis of not only SLE but most connective tissue diseases such as SLE,
polymyositis, dermatomyositis, Sjogren syndrome, lupoid hepatitis, rheumatoid arthritis

COLLATED BY: ♣ ♠ ♥ ♦ Hematology 1 21

 OSMOTIC FRAGILITY TEST
• Diagnosis of Hereditary Spherocytosis/ Hereditary Elliptocytosis
• Result of test may also be increased in hereditary stomatocytosis
• Principle
o Heparinized WB is added to decreasing concentration
of NaCl then incubated at RT or at 37ºC
• Incubated OFT
o 2nd method of OFT
o OFT that has been incubated at RT that will show normal
results are followed up with incubation
at 37ºC for 24 hrs.
o If the same results come out, the Px is interpreted as having normal OF
o Increase fragility: diagnostic of hereditary spherocytosis

• Prepare decreasing concentration of salt solutions (it depends on the procedure but usually it starts with 0.90 which is NSS)
• To the 0.9 NSS add several drops of distilled water to decrease the concentration of the NSS
• NSS is 0.90, you are going to add 1 drop of water to make it 0.75  2 drops on 0.65 tube  3 drops on 0.60, etc.
• The drop of distilled water will tend to decrease the concentration of salt in the solution.
• As we all know most cells survive or there is no osmosis that will happen if you are going to incubate blood in 0.9 NaCl but if you are going
to decrease the conc of NaCl, there will be a tendency for osmosis to happen because there will now be a movement of the solvent which is
water from an area of lesser conc. to greater conc.
o the water from the tubes will tend to go into the RBCsIf red blood cells are spherocytic, they cannot accommodate water anymore
 easily lyse

 When we are talking about OFT: it is about red blood cells that are not fragile, it means they are strong to invite more water
 The larger the amt. of the red cell membrane or the surface area in relation to the size of the cell, the more the fluid the cell is able
to absorb or invite before they are able to rupture

DECREASED  Reticulocytes
OFT  Certain types of anemia (cells in IDA, thalassemic cells, sickle cell anemia)
 After splenectomy when target cells are present
( liver disease, polycythemia vera, hemoglobinopathies)
Hemoglobinopathies: where you see target cells, so target cells are able to invite more
fluid because they are actually flat cells.
INCREASED  Spherocytosis
OFT  Elliptocytosis
 Stomatocytosis
- These cells are in their maximum capacity in terms of solvent volume within the RBCs.
That’s the reason why when you incubate them in LISS, they will easily lyse

COLLATED BY: ♣ ♠ ♥ ♦ Hematology 1 22

 SICKLE CELL STUDIES

• Sickle cells: cells that are products of the presence of Hemoglobin S

• Sickle cell disease
o Either Sickle Cell Anemia (SCA)/ Sickle Cell Trait (SCT)
Sickle Cell Anemia - A phenomenon whereby you inherit both hemoglobin S genes from both parents
(SCA) - Px suffering from SCA: 80-100% Hgb S or homozygous inheritance
- Sickling phenomenon may lead to infections, dehydrations and hemolytic anemia also the excruciating
pain that may be suffered by patients

Sickle Cell Trait - Inherited disorder leading to the production of hemoglobin S but the inheritance is only from 1 parent
(SCT) - 20-40% inheritance of Hgb S
- No health issues
- Patients who are resistant to malarial infections
- More difficult to diagnose

o Inherited disorder
o Production of Hgb

• Sickling phenomenon
o Only happens in temporary state of hypoxia just like when they are infected with the malarial parasite
 The malarial parasite will not be able to survive in this type of cells because they are devoid of oxygen as well as of
potassium.
 Under temporary state of hypoxia, the cells assumed sickling phenomenon (rigid shape or the sickle shape) which leads
to blockage of blood vessels causing excruciating pain among the patients
o HgbS: arises from an altered or mediated S gene. This differs compared to Hgb A1 by one amino acid substitution. Just a mutation of
the beta-globin chain which contain 146 amino acids  Hgb S production.
 This single amino acid substitution which is actually valine substituting glutamic acid in the 6th position of the 146 AA
chain of the beta globin chain of the hgb  Hgb S sickling phenomenon

Laboratory Methods
a. Scriver and Waugh: Sealed WB method Other sickling hemoglobin
Procedure  Hgb CHARLEM
i. In order for temporary state of hypoxia to be initiated  Hgb STRAVIS
ii. tie a rubber band around the finger that you are going to  Hgb CZINGUINCHOR
puncture until the finger becomes lightly cyanotic
iii. perform finger puncture
iv. place drop of blood on slide and make a blood smear
v. stain with Wright stain
vi. look for sickle cells

b. Daland and Castle: Na metabisulphite method

Direct smear method
Procedure
i. Do finger puncture
ii. Mix a drop of blood with a drop of Na metabisulphite
- Na metabisulphite: deep oxygenating reagent which
will remove the oxygen from blood
iii. Make a smear of the sample
iv. Stain with wright stain
v. Look for sickle cells

Sickle cells: diagnostic for Sickle Cell Anemia

Dithionite solubility test

- Not commonly use
Turbidimetric - Uses dithionite solubility reagent
- Increase turbidity due to the presence of Hgb S is interpreted as positive

COLLATED BY: ♣ ♠ ♥ ♦ Hematology 1 23

 HAM TEST
● Also known as Acid serum test / PNH test/ Serum lysis
test
● Discovered by Dr. Thomas Hale Ham
- He demonstrated that RBCs in PNH were lysed
by the complement when normal serum is
acidified or activated by alloantibodies
● A reliable test for the diagnosis of PNH
● PNH (Paroxysmal Nocturnal Hemoglobinuria)
- Acquired clonal disorder or the RBCs of the
patient are abnormally sensitive to lysis by
complement present in the plasma
- Increased complement sensitivity of RBC
membrane
PRINCIPLE OF KLEIHAUER-BETKE TEST
- Signs & symptoms:
➢ Hemoglobinuria during sleep
➢ Jaundice
➢ Hemosiderinuria
● (+) Result: Lysis of RBCs in acidified serum samples with
patient cell but is not present with normal cell
PRINCIPLE OF HAM TEST
● Exposure of patient red cells to normal acidified serum at
lytic pH (6.5 to 7.0)
● Sample: EDTA-treated blood
● Normal: RBCs do not lyse in LISS particularly a sugar
water solution which contain 9 to 10 ug sugar/100mL water
● If positive, confirm with Ham test
KLEIHAUER-BETKE TEST
● Measures fetal Hgb transferred from fetus to an Rh (-)
mother’s bloodstream or amount of fetomaternal
hemmorrhage
● Acid elution - mother’s cells only
- Pale staining
**Fetal cells: bright pink
● To determine dose of Rh Ig
- Prevent Rh antibodies
● Adult hemoglobin but NOT fetal hemoglobins are soluble in
a citrate buffer with pH 3.2 and will elute out of the RBCs
● After acid elution the ff. are seen
Maternal cells Pale staining
Fetal cells Bright pink
- Resistant to an acid
solution
● Used to determine the dose of Rhogam or Rh
immunoglobulin that will be given to the mother to prevent
the production of Rh antibodies
● Maternal blood is fixed on a slide w/ 80% ethanol
→treatment with citrate phosphate buffer → staining with
hematoxylin and eosin →fetal cells are stained; maternal
cells appear as light “O” cells → computation
● Computation:
𝑵𝑵𝑵𝑵. 𝒐𝒐𝒐𝒐 𝒇𝒇𝒇𝒇𝒇𝒇𝒇𝒇𝒇𝒇 𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄 𝑭𝑭𝑭𝑭𝑭𝑭𝑭𝑭𝑭𝑭 𝒃𝒃𝒃𝒃𝒃𝒃𝒃𝒃𝒃𝒃 𝒗𝒗𝒗𝒗𝒗𝒗𝒗𝒗𝒗𝒗𝒗𝒗
=
𝑵𝑵𝑵𝑵. 𝒐𝒐𝒐𝒐 𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂𝒂 𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄𝒄 𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴𝑴 𝒃𝒃𝒃𝒃𝒃𝒃𝒃𝒃𝒃𝒃 𝒗𝒗𝒗𝒗𝒗𝒗𝒗𝒗𝒗𝒗𝒗𝒗
● It is assumed that 1 fetal cells = 20 mL mother’s blood
volume

BONE MARROW STUDIES

CAUSES OF FALSE-POSITIVE RESULT IN HAM TEST
INDICATIONS
1. Use of old serum of the patient
● Evaluation of:
2. Recent transfusion of blood to the patient
- Specimen cellularity or types of cells present in
Note:
the bone marrow
● Verification method: Heated serum of the patient can also
- Marrow architecture or make-up and
be used at 56 degrees Celsius
cytochemistry of the bone marrow
● Heating the sample at 56 degrees Celsius inactivates the
● Utilized in chromosomal culture and determination of the
complement hence there will be no hemolysis both in the
cellular makeup of the components of the bone marrow
control and the PNH patient
● Disease/s that are diagnosed using this method:
SUGAR WATER SCREENING TEST - Leukemia
● Also a test for PNH
METHODS OF COLLECTION
● Whole blood is mixed with sugar water solution or low ionic
1. Aspiration
strength solution ➜ incubate at room temperature - Jamshidi needle: special needle where
percutaneous aspiration of a bone marrow is
Low ionic strength solution performed
● Promotes binding of complement components (C3) to RBC
surface

COLLATED BY: ♣ ♠ ♥ ♦ Hematology 1 24

 Common sites of percutaneous
aspiration NITROBLUE TETRAZOLIUM TEST (NBT)
● aka “Neutrophil reduction test”
Adults ● Posterior iliac crest ● Test that distinguishes bacterial infection and CGD
● Spinous processes of the ribs
Bacterial Infection Reduction of NBT to formazan
● Vertebra
● Sternum CGD (Chronic No reduction of NBT to
- Not always used due Granulomatous Disease) formazan
to its location being ** Formazan - black dye
near to the heart
Metabolic defects of
neutrophils
Infants ● Head of the tibia
SPECIMEN
● Heparinized whole blood
2.
Biopsy
PROCEDURE
- Open trephine
● Involves making of smear of blood that was incubated with
- Sample of the core of the bone marrow is
NBT dye
obtained
- Usual site of collection: Posterior iliac crest / hip
bone Reduction ● Presence of black colored
METHODS OF EXAMINATION (NBT➜ Formazan) neutrophils
1. Smear Preparation
No reduction ● Normal neutrophils
Thin direct ● Aka “Imprint”
smear ● Usually done on glass Note: This test is not performed if the patient’s neutrophil count is
slides low. Hence, it is only performed if neutrophilia is present in the
● Usually stained with smear.
RED CELL ZINC PROTOPORPHYRIN TEST
- Wright’s stain
● Confirmatory diagnosis of Iron Deficiency Anemia
or;
(IDA)
- Hematoxylin &
Eosin ➢ IDA: there is decreased iron that would be taken
up by precursor cells of RBCs → meaning the
Particle smear ● The sample is crushed mature RBCs may have increased free
between two glass slides erythrocyte protoporphyrin → since there is no
● Either the aspirate or the iron we look for the free radicals scavenger zinc
biopsy bone marrow → there will be increased zinc free erythrocyte
collection method is used protoporphyrin present in the RBCs → presence
of zinc protoporphyrin will react with the
Concentrate ● Aka “Buffy coat smear” protofluor reagent which will be detected using
smear ● Sample is obtained from the protofluor-Z-Hematofluorometer
the buffy coat layer
➢ Result: Increased zinc protoporphyrin →
Note: In bone marrow studies, the most recommended or indicates IDA
best stain are: (1) Wright’s stain (smear) and (2) H & E ● Screening for lead poisoning
stain (histologic samples) ● Protoflour-Z-Hematofluorometer
2. Histologic - Uses Protoflour reagent
● Usually done for histologic examinations of bone
GEL ELECTROPHORESIS
marrow sample
● In Hgb electrophoresis, the red cell lysate is subjected to
● Fixative: Zenker’s
electrical fields under alkaline or acidic pH
● Stain: Hematoxylin & Eosin
● Alkaline gel → Cellulose acetate
CRITERIA THAT INDICATE PROPER COLLECTION OF BONE ● Acidic gel → Citrate agar
MARROW SAMPLES ● This can be carried out in a:
1. Presence of 1 to 3% of fat cells - Filter paper
- Absence of fats indicates dry tap or improper - Cellulose acetate membrane
marrow collection - Starch gel
2. Presence of 5 to 8% of nucleated RBCs - Citrate gel
- Agarose gel
COLLATED BY: ♣ ♠ ♥ ♦ Hematology 1 25
 ●
The separation of different Hgbs is dependent on the
Region Hemoglobin Present
charge of Hgb molecule
● Change in the amino acid composition of the globin chains Alkaline gel electrophoresis
will result in the alteration of the charge of Hgb molecule →
leading to a change in the speed of migration of the Hgb Top band (farthest from Hb H
molecule origin: H lane) Hb I
**fastest to migrate
● This test if for the diagnosis of different Hgb variant
METHODS J Lane Hb J
Hb Bart’s and Hb N are
pH between Hb J and Hb H lanes

1. Cellulose 8.2 to 8.6 ● Hgb is negatively A Lane Hb A

Acetate at an charged and will F Lane Hb F
alkaline pH migrate toward the
anode (+) S Lane Hb S
● Most common pH: 8.4 Hb D
Hb G
2. Citrate Agar 6.0 to 6.2 ● Confirmation for Hb Lepore
Electrophoresi variant hemoglobins
C Lane Hb C
s ● Differentiate
Hb E
- S from D and G Hb O
- C from E, O ARAB Hb A2
and CHARLEM Hb S/G hybrid

Carbonic anhydrase band Hb G2

(faint) Hb A2
**band nearest the origin Hb CS

Acid gel electrophoresis

- Used to differentiate Hgb S from Hgb D and G since in
alkaline gel Hgb S, D, G, are all found in S lane.

Top band (farthest from Hb C

origin)
C Lane

S Lane Hb S
Note: Hgb S is more clinically significant than Hgb D and Hgb G. Hb S/G hybrid
Hence, further test using citrate agar must be performed to separate Hb O and Hb H are between S
Hgb S from Hgb D and G. If Hgb S is still present it is indicative of the and A lanes
presence of Hgb S.
Note: Hgb S is the fastest in
acid gel electrophoresis as
compared to alkaline gel
electrophoresis

A Lane Hb A
Hb E
Hb A2
Hb D
Hb G
Hb Lepore
Hb J
Hb I
Hb N
Hb H
MIGRATION OF VARIOUS HEMOGLOBIN BANDS IN ALKALINE
GEL AND ACID GEL ELECTROPHORESIS F Lane Hb F
● Different hemoglobins will migrate at different speed Hb Hope
Hb Bart’s

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 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY ANION EXCHANGE MICROCHROMATOGRAPHY
 Weak cation exchange column system; a sample of RBC  Detection of hemoglobin A2 levels
lysate buffer is injected into the system → application of  Elevated levels indicate beta thalassemia
mobile phase (the hemoglobin variants can interact (homozygous/trait)
between the stationary phase and mobile phase) → the heading
eluted hemoglobin with different hemoglobin variant will be TEST FOR UNSTABLE HEMOGLOBIN
detected by light absorbance
 Unstable hemoglobins (globin mutations)
 The time required for the hemoglobin variants to elute is
o Hemoglobin variants with amino acid deletion or
referred to as retention time (for each hemoglobin variant
substitutions have weakened the binding forces
there is a specific retention time that would differentiate it
to maintain the hemoglobin structure
from the others)
 Mutations may lead to precipitation of
o Identification of more hemoglobin variants that
hemoglobin (decrease solubility →
were not detected by gel electrophoresis
precipitation→ decreases the survival
 Disadvantage
time of the RBC)
o Carry-over of specimen – when HPLC is used
o Inherited as autosomal dominant disorder
and the first specimen with sickle cell disease a
o May lead to anemia with reticulocytosis
small peak will be seen at the S window in the
o Mostly with mild compensated anemia/ mild
next specimen that can lead to diagnostic
reticulocytosis – may show sever chronic
confusion and the sample to be rerun or it may
hemolysis with splenomegaly and jaundice
also result to false positive result
 Some have severe hemolytic type of
anemia
Approximate Retention Times of Various Hemoglobins in o Example
HPLC Analysis  Hemoglobin H (homotetramer of beta
Approximate Hemoglobin globin chains), Hemoglobin Bart’s
Retention (homotetramers of gamma globin
Time (Min) chains), hemoglobin M, hemoglobin
0.7 (Peak 1) Acetylated Hb F, Hb H, Hb Bart’s, bilirubin Köln, Hemoglobin Zurich –
1.1 Hb F hemoglobins usually seen in α-
1.3 (Peak 2) Hb A1c, Hb Hope thalassemia
1.7 (Peak 3) Aged Hb A (Hb A1d), Hb J, Hb N, Hb I
2.5 Hb A, Hb S1c
ISOPROPANOL PRECIPITATION TEST
3.7 Hb A2, Hb E, Hb Lepore, Hb S1d
3.9-4.2 Hb D, Hb G  Detection of unstable hemoglobins
4.5 Hb S, Hb A20, Hb C1c  Normal: Hgb precipitation in a 17% isopropanol solution
Hb O Arab has a broad range from 4.5 to 5 after about 40 mins
min  Unstable hgbs begin to precipitate in 5 mins and show
4.6-4.7 Hb G2 heavy flocculation in 20 mins
4.9 Hb C (preceding the main peak is a small
peak, Hb C1d), HEAT STABILITY TEST
Hb S/G hybrid, Hb CS (three peaks: 2_3%)  Mix then heat hemolysate and buffer (TRIS buffer –
maintain buffer at pH 7.2) at 50 ⁰C
 Hydrophobic bond/ force are weakened → decrease
stability of hemoglobin molecule
 Under controlled conditions, unstable hemoglobins will
precipitate while stable hemoglobins will remain in the
solution
 Interpretation: unstable Hgbs precipitate within 60 mins and
heavy flocculation is seen at 120 mins, normal result show
minimal flocculation
 Sample used: EDTA treated whole blood

HEINZ BODY PREPARATION

 Most unstable hemoglobins may show precipitated
hemoglobins in RBCs and the most commonly
encountered manifestation is Heinz bodies
 Prevention of oxidation of hemoglobin – 2 aspects that
X axis – retention time (mins) should not be oxidized
Y axis – light absorbance (manifest the peak of the elution time of the 1. Iron in the heme molecule
different hemoglobins) 2. Sulfide group in the globin molecule - oxidation will result to
precipitated hemoglobins and will only occur when the
glycolytic enzymes were able to prevent the oxidation →
Heinz body formation (late sign of oxidative damage;
represents the degradation products of hemoglobin)

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  Usually seen in post splenectomy patients, chemical
poisoning, drug toxicity, G6PD deficiency BASOPHIL COUNT
 Specimen: heparinized/EDTA – treated whole blood COOPER & CRUICKSHANK
 Crystal violet/ methyl violet – supravital staining  Stain: Toluidine blue (metachromatic stain that stains the
 Acetylphenylhydrazine heparin component basophils) – heparin
 May lead to hemolytic anemia because it may hinder the  Basophil color: purple red
flexibility and deformability of the RBCs → RBC passing
through microvasculature = bite cells/ rupture of the RBC NEUTRAL RED STAIN
→hemolysis
 Supravital staining technique; stains the heparin and
 ↑ Heinz bodies in the circulation = Heinz body hemolytic
histamine components of basophils
anemia
 Basophil color: Red
HEMOGLOBIN H PREPARATION # 𝑜𝑜𝑜𝑜 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 /𝑚𝑚𝑚𝑚3 = 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 × 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓
 Detect presence of four β in α-thalassemia × 𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓
 Hgb H precipitates (multiple pale staining greenish blue
spherical inclusions) when exposed to BCB or NMBN; can
be mistaken as Howell jolly body/ reticulocytes
 Specimen: EDTA-treated blood
 3 drops of BCB + 3 drops of EDTA treated blood →
incubate for 2 hours at 37⁰C → create a smear→ air
dry→ examine under the microscope (OIO)
 In hemoglobin H disease about 10% of the cells or more
will develop inclusions
 .01%-1% may be seen in α-thalassemia trait and
myelodysplastic syndrome

ASCORBATE-CYANIDE SCREENING TEST

 Non-specific screening procedure for detecting deficiencies
in the pentose phosphate pathway (PPP)
 (+): G-6-PDH deficiencies
 Incubate blood with ascorbate-cyanide

LOBE CELL COUNT

EOSINOPHIL COUNT
 Use of special hemocytometer (Fuchs Rosenthal counting
chamber/ Speirs levy counting chamber)
 Diluting fluid: propylene glycol or distilled water
 Stain/Dye: eosin, flusin
 Acetone may be used to prevent the lysis of eosinophils
 Most common methods used are method by Pilot,
Friedman method and Randolph Method
 Clinical Significance:
Eosinophilia - found in parasitism and allergies
Eosinopenia – seen in hyperadrenalism (Cushing syndrome)

EOSINOPHIL DEPRESSION TEST/ THORN TEST

 Utilizes eosinophil count
 Antiquated test for adrenal function
 Collection of 2 blood samples; 1st collect Fasting Blood
Sample → after the first collection, 25mg of
adrenocorticotropic hormone (ACTH) will be administered
(IM) to the px→ after 4 hours, 2nd sample is collected →
perform eosinophil count on both samples
 Hyperadrenalism and hypoadrenalism may be distinguish
with the use of this test
 Normal result: 50% decrease in the original eosinophil
count
o No decrease in the original eosinophil count is
manifested by hypoadrenalism
o No decrease in the second sample -
hypoadrenalism/ Addison’s disease
o Drastic decrease of more than 50% in the second
sample – hyperadrenalism (Cushing’s syndrome)
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