Hematology : Hb,Hct.

ESR
Hesty Utami,M.Clin Pharm.,Apt
Erythrocyte/RBC
Main function : carry O
2
from
the lungs to the tissue and
transport CO
2
back to the
lungs
Produced in the bone marrow
The life span of erythrocytes in
circulation is 120 days
Erythrocytes are removed by
the reticuloendothelial system
that includes phagocytic cells
in circulation and in the spleen,
liver, lymph nodes, bone
marrow, lungs

HEMOGLOBIN
Found in red cell

Consist of:
Heme + Globin
Heme : Protoporfirin +
Fe2+
Globin: 4 chain: 2 , 2


function : oksigen
transport
Hemoglobin (Hb)
The primary component of erythrocytes
Hb determination is preferred to RBC because it more
directly reflexes the oxygen transport capability of blood
Hb values are varied among population

Men : 13.6-17.2 g/dL
women : 12-15 g/dL
pregnant : 11-12 g/dL
children : 11-16 g/dL
Hemoglobin
Hb shows diurnal variation with the highest value in the
morning ( 8 am) and the lowest in the evening ( 8 pm)
Hb values are slightly lower in older men and women
Hb values are approximately 1 g/dL lower in blacks than
whites
Heavy smokers have higher Hb values than nonsmokers
Living in high altitude will increase Hb values
When fully saturated, each gram of Hb holds 1.34 mL O2
Erythrocyte mass of adult contains 600 g of Hb can
carry 800 mL of O2
HEMOGLOBIN
Why get tested?
The test is used to:
measure the severity of anemia (too few red blood cells)
or polycythemia (too many red blood cells),
monitor the response to treatment of anemia or
polycythemias, and
help make decisions about blood transfusions if the
anemia is severe
Interpretation of results
Increased Hb values :
1. dehydration,
2. excess production of red blood cells in the bone
marrow,
3. severe lung disease, or
4. several other conditions.

Decreased Hb values
1. iron deficiency or deficiencies in essential vitamins of
other elements, such as B12, folate, B6,
2. inherited hemoglobin defects, such as sickle cell
anemia or thalassemia,
3. cirrhosis of the liver (during which the liver becomes
scarred),
4. excessive bleeding,
5. excessive destruction of red blood cells,
6. Kidney disease,
7. bone marrow failure or aplastic anemia, or
8. cancers that affect the bone marrow.

Hb derivatives
Hb deoxyhemoglobin
HbO
2
Oxyhemoglobin
HbCO Carboxyhemoglobin
SHb Sulfhemoglobin
SHbCO Carboxysulfhemoglobin
Hbi Methemoglobin
HbCN Cyanmethemoglobin
Method of Hb Determination
1. Lakmus paper method
Material required : lakmus paper, blood sample
Procedure :



Leave it at room temperature until dry (5 minutes)

Compare the color of blood sample visually with standard
color. Each standard color represent certain Hb value

blood
Lakmus paper
Method of Hb Determination
2. Cyanmethemoglobin method (recommended by WHO)
Blood + drabkin solution [K3 Fe (CN)6, KCN]

Cyanmethemoglobin (HbCN)

Absorbance of sample solution is measured in
spectrophotometer at 540 nm

Compare the absorbance of sample
with standard drabkin solution


Advantages :
Precise ( 2%)
Convenience
Readily available
Stable standard

Disadvantages :
Not all forms of Hb can
Be measured, particularly
SHb
Turbidity due to abnormal
plasma protein, hyperlipemia,
large numbers of leukocytes,
fatty droplets absorbance
Method of Hb Determination
3. Indirect cyanmethemoglobin
Modified cyanmethemoglobin method by using dried blood sample
instead of liquid blood
Can delay testing after sampling since dried blood can be stored up
to 1 month
Pipet blood sample on the surface of paper
And allow it dry

Send to lab/store until testing

Immerse dried sample in drabkin solution and undergo
cyanmethemoglobin method
Method of Hb Determination
4. Sahli method
Principle; Hb (blood) + HCl acid hematin (brown)
the colour formed is compared with standard visually
Sample : capillary blood + anticoagulant
Equipment : hemoglobinometer sahli
Advantages : simple procedure, portable equipment
Disadvantages : subjective result, less precise (10%),
lack of standard solution
To convert the result from sahli method to
cyanmethemoglobin method X 1.1

Digital
hemoglobinometer
Hematocrit (packed cell volume)
Hct : the percentage of red blood cells volume to the
whole blood volume
A measure of both the number and the size of red blood
cell
Hct reflects the concentration of red blood cell, not the
total of red blood cell mass
Hct value :
Male 40-54% (0.4-0.54)
Female 37-47% (0.37-0.47)
Hematocrit
Hct test is used to evaluate :
Anemia
Polycythemia
Response to treatment of
anemia and polycythemia
Decision to undergo blood
transfusion and the
effectiveness of those
transfusions
Principle of Hct test
Blood + anticoagulant

Centrifuge

Blood cells will precipitate


Erythrocyte platelet+
(bottom) leukocyte


Technique to do Hct test
1. Macromethod of wintrobe
Venous blood +anticoagulant

Wintrobe tube

centrifuge 3000 rpm 30

Read the scale from bottom
Hct = L1/L2
L1 = the height of erythrocyte column, exluded buffy coat
L2 = the height of the whole blood specimen
Can be done after ESR test
2. Micromethod
Capillary blood is sucked into
microhematocrit tube

The tube should be filled to at least 5 cm

Centrifuge 16.000 rpm, 3-5

Measure the length of the blood column and the length of
erythrocyte column alone with a milimeter rule
Advantages : less centrifugation time, less sample required
Disadvantages : cannot identify buffy coat
Sources of error in Hct test
Inadequate duration and speed of centrifugation
inadequate centrifugal force will increase the trapped plasma &
other blood cellsfalsely elevated Hct (micromethod can reduce
this error)
inadequate duration some proportion of blood cells hasnt been
packed already
Prolonged tourniquet stasis
Inadequate amount of anticoagulant
Hct will be falsely decreased due to cell shrinkage (Hb and cell
count wont be affected)
Other errors : improper reading of the level of erythrocyte and
plasma, inclusion of buffy coat as part of red cell volume
Interpretation of Hct result
Increased hematocrit :
Dehydration resolved with adequate hydration
Polycythemia vera could be due to problem with bone
marrow
Early indication of dengue shock syndrome
Polycythemia secondary to chronic hipoxia i.e COPD
Living in high altitude
Interpretation of Hct result
Decreased hematocrit :
Indication of anemia
Further testing may be necessary to determine the exact
cause of anemia ( i.e deficiency of iron, folate, vit B12)
Recent bleeding
Liver cirrhosis
Bone marrow suppressant effect of drugs
Sickle cell anemia
Hemolysis
Pregnancy due to extra fluid in the blood
Erythrocyte Sedimentation Rate (ESR)
The rate at which erythrocytes settle to the bottom of a
test tube through the forces of gravity in a certain period
of time, usually 60 minutes
It is directly proportional to the weight of cell aggregates
and inversely to the surface area and plasma viscosity
(microcyte < macrocyte)
ESR is a sensitive but nonspecific (the earliest indicator
when other chemical and physical signs are normal)
Function of ESR test :
monitor inflammatory or malignant disease
Aid detection and diagnosis of occult disease i.e tb,
tissue necrosis, chronic infection

ESR is governed by the balance between pro-
sedimentation factors,mainly plasma protein
(fibrinogen, IgG, CRP) and factors resisteng
sedimentation (negative charge of erythrocyte called
zeta potential and ratio of surface area to volume)

When an inflammatory process occurs, the high
proportion of plasma proteins causes erythrocyte to
stick to each other to form roleaux

Roleaux will settle faster

sedimentation
Methods to determine ESR
Principle :
Anticoagulated blood is
placed in vertical tube

Erythrocytes will tend to fall
toward the bottom

The length of fall of the
column of erythrocytes in
60 minutes =ESR
ESR test
Westergren Wintrobe
Comparison of ESR method
Westergren Wintrobe
Anticoagulant Na sitrat 3.8%
(0.4 ml/1.6 ml blood)
Balanced oxalate
Dried EDTA
Dilution 5/4 -
Length of equipment 300 mm, pipet 110 mm, tube
Diameter 2.5 mm 2.5 mm
Scale 0-200 mm 0-100 mm
Normal value
Male
Female

0-10 mm/hr
0-15 mm/hr

0-10 mm/hr
0-20 mm/hr

Advantage easy Reliable
disadvantage If ESR>50 mm/hr, the
result is unreliable
More difficult
Westergren wintrobe
Wintrobe
Factors affecting ESR
Category Increased ESR Decreased ESR
Blood protein and
lipid
cholesterol,
fibrinogen, IgG,
bile salt,
fibrinogen. IgG,
hyperglycemia
Red blood cell Anemia,
macrocytosis
Polycythemia,
sickle cell,
thallasemia
White blood cell Leukimia lekocytosis
Drugs Procainamide, vit
A, theophylline
ACTH,
ethambutol,
cortisone
Factors affecting ESR
Category Increased ESR Decreased ESR
Clinical
condition
Acute bacterial infection,
DM, end stage renal
failure, gout, rheumatoid
arthritis, myocardial
infarction, malignancy,
pregnancy
Congestive heart
failure, newborn
baby
Specimens Refrigerated sample
not returned to room
temperature
Clotted blood
sample, delay
testing
Technique High room
temperature, tilted
ESR tube, vibration
Bubble in ESR
column, low room
temperature, narrow
ESR column