HEMA311 01

HEMATOLOGY 1 LABORATORY TERM

WEEK 10: ERYTHROCYTE SEDIMENTATION RATE, SMEAR PREPARATION, AND OSMOTIC FRAGILITY TESTING

ERYTHROCYTE SEDIMENTATION RATE MECHANICAL / TECHNICAL FACTORS

• A tilt of 3 degrees can cause errors up to 30%

• The rack holding the tubes should not be subject to any
movement of vibrations
• Large changes in temperature – Increased temp =
Increased ESR

• Non-specific measurement to detect and monitor an

inflammatory response to tissue injury in which there is a
change in the plasma concentration of acute phase
proteins. CLINICAL SIGNIFICANCE
• Allows blood to sit in a vertical position for a period of time
(1 hour), and the distance (in mm) that the red calls fall is • The ESR represents a nonspecific response to tissue
the ESR. damage and inflammation and denotes the presence of
• Reported in mm/hour disease, but not its severity
• Affected by 3 factors: red cells, plasma compositions, and • Elevated ESR – pregnancy (after 3rd month), acute and
mechanical/technical factors chronic infections, rheumatic fever, rheumatoid arthritis,
myocardial infarction, nephrosis, acute hepatitis,
RED BLOOD CELL FACTORS menstruation, tuberculosis, macroglobulinemia,
cryoglobulinemia, hypothyroidism, and hyperthyroidism
• Red cells repel each other because of their net negative • Decreased ESR – polycythemia, congestive heart
charge failure, hypofibrinogenemia, presence of red cell
• In disease states, plasma protein concentration changes, abnormalities (poikilocytosis, spherocytes, and sickle
causing a reduction in the negative charge of the RBCs cells)
and consequent formation of rouleaux.
• This leads to a larger mass and an increased WINTROBE AND LANDSBERG METHOD
sedimentation velocity.
• The larger the particle, the faster its rate of fall
• Macrocytes settle more rapidly than microcytes
• RBCs with shape alterations (sickle cells and
spherocytes) are unable to form rouleaux – ESR is
decreased
• Anisocytosis and poikilocytosis – reduced ability of
RBCs to form large aggregates – ESR is decreased • Specimen: EDTA anticoagulated blood
• Severe anemia – Low numbers of RBCs, aggregation
and rouleaux formation are increased – ESR is increased Procedure:

PLASMA FACTORS 1. Mix the blood

2. With a long stem pipette, fill the
• Single most important factor determining the ESR wintrobe tube up to 0 mark (no air
• Rouleaux and aggregation are controlled primarily by bubbles)
levels of acute phase proteins (fibrinogen, alpha-1 3. Place the tube in a vertical position on
globulin, alpha-2 globulin), increasing as these three the rack for 60 minutes
plasma protein levels are increased in the plasma 4. Record the level of sedimented ESR
from the scale on the left side (red) of
the tube. Read downward.

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 WESTERGREN METHOD BLOOD SMEAR PREPARATION

BLOOD SMEARS

• Preparations that, after staining, allow the technician to

perform a differential white blood cell count and
morphological study
• EDTA anticoagulated blood
• Cover glass smears – more even distribution of white
cells than wedge smear
• Specimen: Citrated blood (0.109M trisodium citrate)
• Wedge smears
• Spun smears
Procedure:
1. Mix the blood • Buffy coat smears – for differential counting of patients
2. Place the Westergren tube into the blood sample, making with <1.0x10^9/L wbc count
sure that the blood reaches the 0 mark on the tube • Thick blood smears – blood parasites
3. Place in a rack for 60 minutes
4. Read the graduations downward

REFERENCE VALUES

• Westergren ESR
▪ Women: 0-15 mm/hr.
▪ Men: 0-10 mm/hr.
▪ Children: 0-10 mm/hr.
• Wintrobe and Landsberg ESR
WEDGER SMEAR
▪ Women: 0-20 mm/hr.
▪ Men: 0-9 mm/hr.

STAGES OF ESR

1. Initial rouleaux formation/lag phase (10 minutes) –

sedimentation rate is slight
2. Rapid settling of RBCs/Rapid packing of cells/
decantation (40 minutes) – sedimentation is more rapid
1. Obtain a clean glass slide, a spreader slide, EDTA blood,
and constant
and a plain \microhematocrit tube
3. Final sedimentation of RBCs (10 minutes) –
2. Fill the microhematocrit tube and carefully place a small
sedimentation rate is slow because of the accumulation
drop of blood in the middle of the slide, approximately 1
of RBCs at the bottom of the tube
cm from the labelled end (if using blood from finger or
heel, careful not to touch the skin with the slide)
Westergren and Wintrobe tube
3. Place the slide on a flat table top
4. With the thumb and index finger of the left hand, hold the
• Standard/Original Westergren two edges of the slide.
• 300.5 mm long (± 0.5 mm), tube bore 2.65 mm (± 0.15 5. With the right hand, hold the spreader slide with the
mm) thumb on the edge of one side and the other four fingers
• Wintrobe and Landsberg on the edge of the other side
• 115 mm long, tube bore 3 mm 6. Place the end of the spreader slide slightly in front of the
drop of blood
SOURCES OF ERROR 7. There should be an approximate 30-40 degrees angle
between the two slides
• If concentration of EDTA is greater than recommended, 8. Draw the spreader slide back toward the drop of blood.
the ESR will be falsely low. The blood will begin to spread to the edge of the spreader
• If the ESR stands for more than 60 minutes, the results slide.
will be falsely elevated. 9. Keeping the angle, push the spreader slide rapidly over
• If it is timed for less than 60 minutes, ESR will be low. the entire length of the slide
• Marked increase in temperature = Increased ESR
• Marked decrease in temperature = Decreased ESR
• Tilting increases sedimentation rate
• Bubbles cause invalid results
• Fibrin clots invalidate results

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 OSMOTIC FRAGILITY TEST

Principle:

• The osmotic fragility test is a measure of the ability of the

red cells to take up fluid without lysing. In this test, whole
blood is added to varying concentrations of sodium
chloride solution and allowed to incubate at room
temperature. The rate of hemolysis is then determined
through visual inspection of each saline concentration.

Glossary of Terms:

• Tonicity – the relative concentration of solutes dissolved

in a solution which determine the direction and extent of
diffusion.
A. Isotonic – solution with the same number of solute
particles as the cell
B. Hypotonic – solution with fewer solute particles per
liter of solution
C. Hypertonic – solution with higher solute particles per
liter of solution

WEDGER SMEAR • Target cell/ Codocytes/ Mexican hat cell

– red cell with peripheral rim of
hemoglobin surrounded by clear area
and central hemoglobinized area (bull’s
eye)
• Spherocyte – small, round, dense red
blood cells with no central pallor

Purpose of the test:

• This test is employed to diagnose conditions in which the

physical properties of red blood cells are altered. The
shape of the red blood cells is the primary factor affecting
the osmotic fragility test, which, in turn, depends on the
volume, surface area and functional state of the red blood
cell membrane. The larger the amount of surface area of
the red cell membrane, the more fluid the cell is capable
of absorbing before rupturing ( OFT), and is usually
observed in target cells. As the red cell takes in fluid
however, it becomes rounder. It therefore follows that the
spherocyte has the smallest surface area for its volume
and ruptures more quickly ( OFT).
• Increased osmotic fragility is found in hemolytic anemias
and hereditary spherocytosis. Decreased osmotic fragility
is seen in sickle cell anemia, iron-deficiency anemia,
thalassemia, polycythemia and conditions where target
cells are present.

Materials:

• 12 test tubes
• Test tube rack

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• 0.5% NaCl Reference value:
• Distilled water
• Sahli pipette • Initial hemolysis: 0.42-0.44% (Test tube #21 or 22)
• Heparinized blood • Complete hemolysis: 0.32-0.34% (Test tube #16 or 17)
• Dropper • Note: to get the value in percent, multiply the number of
the tube with initial and complete hemolysis by 0.02 (the
Procedure: amount of blood delivered in each test tube)

1. Fulfill the ff. as indicated by the table Correlations:

Test 0.5% NaCl Distilled Values • Decreased OFT: sickle cell anemia, iron-deficiency
Tube Water anemia, thalassemia, polycythemia vera, conditions
14 14 gtts 11 gtts 0.28 % where target cells are present, reticulocytes
• Increased OFT: hereditary spherocytosis, conditions
15 15 gtts 10 gtts 0.30 %
where spherocytes are present, older RBCs
16 16 gtts 9 gtts 0.32 %
17 17 gtts 8 gtts 0.34 % Lecturer’s notes:
18 18 gtts 7 gtts 0.36 % 1. Test tube number = Hypotonic (more drops of
19 19 gtts 6 gtts 0.38 % distilled H2O than drops of NaCl)
20 20 gtts 5 gtts 0.40 %
2. Interpret as follows
21 21 gtts 4 gtts 0.42 %
Test tube Osmotic Interpretation
22 22 gtts 3 gtts 0.44 % fragility
23 23 gtts 2 gtts 0.46 % Test tube # with LOWER the RBC doesn’t get
24 24 gtts 1 gtts 0.48 % first initial and OFT destroyed easily,
25 25 gtts 0 0.50 complete (Increase can resist lysis
hemolysis is capacity to even in hypotonic
2. Add 20 uL (0.02 mL) of blood in each of the test tubes LOWER than swell in solutions
using a Sahli pipette. Mix. reference hypotonic
3. Incubate at room temperature for 2 hours. solution)
4. Centrifuge for 5 minutes. Starting from test tube #25, Test tube # with HIGHER the RBC gets
identify the test tube number where the first initial and first initial and OFT destroyed easily,
complete hemolysis was observed. complete (Decrease gets lysed even
hemolysis is capacity to when exposed to
• Initial hemolysis: tinge of pinkness in the HIGHER than swell in isotonic solution
supernatant and some cells at the bottom of the tube reference hypotonic
• Complete hemolysis: clear, red supernatant with no solution)
microscopic cells at the bottom of the tube

Example:

Given:

• Initial hemolysis was first observed in test tube #23

• Complete hemolysis was first observed in test tube #19

• Reference value for initial hemolysis: test tube #21 and

22
• Reference value for complete hemolysis: test tube #16
and 17

Interpretation:

• Since the test tube with initial hemolysis (#23) is higher

than the normal for initial hemolysis (#21 or 22), and the
test tube with complete hemolysis (#19) is higher than
the normal for complete hemolysis (#16 or 17), the
osmotic fragility is said to be INCREASED.

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