MLS 113B | LABORATORY

HEMATOLOGY 2 PRELIM |2ND SEM | 22-23

3

TOPICS ◆ In cases of dengue, most of the deaths result from

1 Platelet Count internal hemorrhage due to a very low platelet
count. Antibodies are produced against the platelets
2 Automated Platelet Count
and endothelium themselves, which causes the
3 Bleeding Time
]
destruction of platelets, even megakaryocytes and
PLATELET COUNT the endothelium. In this condition, the platelets are
➔ Number of platelets in 1L or 1uL too low to compensate for the destruction of the
endothelium.
Manual count is difficult:
➔ Platelets are capable of Reference Range
1. Platelets adhere to attaching themselves to objects, ADULT & PEDIATRICS 150-450 x 10^3 uL (x 10^9/L)
foreign objects and to such as the glass tubes, and (NORMAL)
each other have the tendency to clump. THROMBOCYTOSIS >450 x 10^9/L
Hence, they cannot be counted THROMBOCYTOPENIA <150 x 10^9/L
exactly.
➔ They may be mislooked due to NOTE
their very small size. They ➔ A platelet count of less than 150 x 109/L does not always
2. Very small cannot be distinguished necessarily mean that there is an immediate need for
because they do NOT have transfusion. This is still a case-to-case basis.
nucleus ◆ 10-20 x 10^9/L = need for transfusion
3. Easy to disintegrate ◆ 50 up x 10^9/L = does not necessarily need
➔ Residues from the stain which transfusion, as long as the WBC count is getting
4. Hard to differentiate were left unfiltered or water higher and the clinical features of the patient is not
from debris contaminants may be that ill
mistakenly identified as
platelets DIRECT PLATELET COUNT
➔ There are 2 ways to do platelet count
Detects: ➔ Platelets are counted using a hemacytometer (counting
➔ Inflammation, trauma, essential chamber), and manual dilutions made by calibrated,
thrombocythemia automated pipettes and diluents (commercially prepared
➔ ↑ higher platelet count than the or laboratory prepared).
1. Thrombocytosis normal)
◆ In ET, there is a very high MANUAL CELL COUNT
platelet count, which reaches
almost 800-1,000
2. Thrombocytopenia ➔ Drug treatment, bruising,
uncontrolled bleeding

THROMBOCYTOSIS
➔ Increased platelet count
➔ Signals inflammation or trauma
➔ Essential Thrombocythemia: extremely high an
uncontrolled platelet production (life-threatening)

THROMBOCYTOPENIA
➔ Decreased platelet count
NOTE
➔ Common consequence of drug treatment (may be
➔ The Neubauer counting chamber is still used when
life-threatening).
performing manual platelet count
◆ Note that platelets are crucial in hemostasis and
➔ When performing manual platelet count (direct method),
platelet plug formation in cases of wounds or
count the cells at the 5 small central squares (the same as
injuries. Failure to seal these “openings” in the body
the RBCs)
would result to the introduction of foreign materials
◆ same counting pattern as the manual RBC count
and allow subsequent infections
(Left to right; right to left)
➔ Bruising and hemorrhage are results of the decreased
platelet count

1 | MLS 113B
 MLS 113B | LABORATORY

HEMATOLOGY 2 PRELIM |2ND SEM | 22-23

DIRECT (MANUAL) PLATELET COUNT Calculation

➔ Hemocytometer
➔ Microscopes
➔ Blood diluting pipettes
◆ RBC thoma pipette is used in platelet count (color of
the bead in the RBC pipette is red)
➔ Diluting fluids (Rees-Ecker fluid)

Components of Rees-Ecker Fluid

◆Trisodium Citrate
◆Brilliant Cresyl Blue (thins the platelet) ➔ Platelets are counted in a hemocytometer as in RBC and
◆40% Formalin WBCs
◆Distilled water
A. DIRECT MANUAL COUNT (REES-ECKER’S)
NOTE ➔ Rinse the RBC pipet first with Rees and Ecker’s diluting
➔ The same method is used when performing platelet count. fluid by aspirating and dispensing the diluting fluid.
The counting chamber is charged on both chambers - the ➔ Aspirate blood to 0.5 mark of the RBC pipet.
upper and lower chamber. ➔ Aspirate the diluting fluid until the meniscus reaches the
➔ The 5 central squares from the upper and lower chambers 101 mark
will be counted, then, get the average. ➔ Shake the pipet (using electric pipette shaker) for 3-5
minutes to mix the contents. Discard 3-4 drops.
➔ Charge the hemacytometer and place it into the petri
dish with moist gauze or cotton for at least 3-10 minutes
(this is to allow the cells to settle).
➔ Place the hemacytometer on the microscope stage
carefully and securely.
➔ Use the Low Power Objective to bring the ruled area into
focus.
◆ Check if the ruled area can already be identified
using LPO, and since the microscope is parfocal,
shift to the HPO with only little manipulation.
➔ Rotate the High Power Objective into position and
carefully focus with the adjustment knob until the lines
are clear.
➔ Reduce the light by adjusting the diaphragm.

➔ Count platelets in 5 of the small squares using L-R, R-L

counting patterns and record the results
➔ Repeat the procedure on the other side of the
hemacytometer. The total number of the platelets

2 | MLS 113B
 MLS 113B | LABORATORY

HEMATOLOGY 2 PRELIM |2ND SEM | 22-23

counted on each side should agree with each other by ± ➔ Count the number of platelets in the 25 small squares in
10. the center square of the grid.
➔ Average the results from two sides of the hemacytometer. ◆ Note that in the tube method, platelets from 25
➔ Calculate the platelet count using the formula. small squares are counted. This means that all of the
platelets found in the whole central square are to be
counted.
Platelet count/uL= average # of platelets counted X 200 X
➔ Count on each side of the chamber and the difference
10 X 5
should not be not more than 10%. Get the average count.
Platelets/L= platelets/uL X 10^6
➔ Calculate the platelet count by using the formula:

Normal Reference Range

ADULT & PEDIATRICS 150-450 x 10^3/uL (x 10^9/L)

B. DIRECT MANUAL COUNT (TUBE METHOD)

➔ Mix 20 uL of a well-mixed blood into 1,980 uL of 1%
ammonium oxalate in a small test tube. Dilution is 1:100
(20/2000).
◆ Named as tube method because dilutions are made
in the test tube, and the thoma pipette is no longer
used. Normal Reference Range
◆ In this method, 1% ammonium oxalate is used as the ADULT & PEDIATRICS 150-450 x 10^3/uL (x 10^9/L)
diluting fluid
➔ Mix the dilution thoroughly and charge the chamber. TAKE NOTE
◆ The Straw pipette is used to aspirate the mixture ➔ The accuracy of the manual platelet count should be
and charge the chambers verified by performing a platelet estimate on a
➔ Place the charged hemacytometer in a moist chamber for Wright-stained peripheral blood film made from the
15 minutes to allow the platelets to settle. same specimen.
➔ Platelets are counted using the 40X objective lens (400X
total magnification). INDIRECT PLATELET COUNT
➔ Platelets are counted in their relationship to red cells on a
Distinguish between “ghost cells” fixed-stained smear.
➔ Ghost cells → remnants of RBCs ➔ This method is NOT RELIABLE because the results
◆ Lysed or disintegrated RBCs depend upon the distribution of platelets and on the red
◆ These remnants are not as round as platelets cell count.
(irregularly-shaped)
◆ Should not be counted as platelets INDIRECT PLATELET COUNT (Fonio’s Method)
◆ Can sometimes have hair-like structures (resembles ➔ Perform manual RBC count (using hemacytometer) using
pili in bacteria) the blood sample of the patient.
➔ The platelets have a diameter of 2 to 4 mm and appear ➔ Next, choose which finger will be used as the source of
round or oval, displaying a light purple sheen when blood sample.
phase-contrast microscopy is used. ➔ Disinfect the puncture site.
➔ The shape and color help distinguish the platelets from ➔ Place a drop of 14%MgSO4 over the disinfected area.
highly refractile dirt and debris. “Ghost” RBCs often are ➔ Puncture through the drop of MgSO4 to a depth of 3mm.
seen in the background ➔ Transfer a drop of blood-MgSO4 mixture on a glass slide.
➔ Make a smear. Dry and stain with Wright’s stain.
➔ Examine the Wright’s-stained smear under Oil
Immersion Objective.
➔ Perform RBC count on the stained smear. Per field, count
the RBCs and platelets.
➔ Compute the platelet count using the formula below and
record the result.

3 | MLS 113B
 MLS 113B | LABORATORY

HEMATOLOGY 2 PRELIM |2ND SEM | 22-23

➔ The number of platelets per 1000 RBC is counted REAGENTS

➔ In counting, there’s no exact number of field needed as ➔ an isotonic electrolyte solution that dilutes
long as you count up to 1000 RBC whole blood samples
➔ stabilizes cell membranes for accurate
Normal Reference Range Reagent 1: counting and sizing
ADULT & PEDIATRICS 250 000 –500 000/uL or 250 –500 Diluent ➔ conducts aperture current
x 10^9/L ➔ rinses instrument components between
analyses, and
AUTOMATED PLATELET COUNT ➔ prevents duplicative cell counts by using
the sweep-flow process.
➔ lyses red blood cells for white blood cell
count and hemoglobin measurement.
Reagent 2: ➔ Caution: Eye irritant. Avoid contact with
Lytic Agent skin and eyes. Avoid breathing gas.
◆ Contact with acid liberates
poisonous gas
➔ AcT Rinse Shutdown Diluent
➔ prevents protein buildup that occurs in and
Reagent 3 around the apertures.
➔ Caution: Avoid eye and skin contact. Do not
ingest.

➔ If the probe is loose or bent, do not run the instrument.

Call your Beckman Coulter Representative/ engineer.

Automation using Coulter AcT diff2 Analyzer

➔ The COULTER AcT diff2 Series Analyzer is a quantitative,
automated hematology analyzer and leukocyte differential
counter for In Vitro Diagnostic Use in clinical laboratories.

Coulter Principle:
➔ Electrical impedance where whole blood is passed
between two electrodes through an aperture so narrow
Reference ranges
that only one cell can pass through at a time.
➔ The impedance changes as a cell passes through. The ➔ Coulter’s normal range study derived the following
change in impedance is proportional to cell volume, reference ranges (below) for the Act diff2 Series Analyzer/
resulting in a cell count and measure of volume. You may use these ranges, or establish your own. If you
use Coulter’s ranges, write “same in the last column of the
following chart.
Specimen Collection and Handling
➔ Handle blood as a potential biohazard capable of
transmitting infection. Always wear protective gloves and
lab coat when processing specimens.
➔ Draw specimen in into a lavender-top Vacutainer tube
containing K2EDTA. Thoroughly mix blood with EDTA
anticoagulant. If hemolysis or small clots are observed,
discard specimen.
➔ Do not test samples that are incorrectly filled or that are
clotted. If hemolysis or small clots are observed, discard
specimen.
➔ Analyze venous blood samples within 24 hours of
collection. Do not refrigerate samples for platelet and
differential counts.

4 | MLS 113B
 MLS 113B | LABORATORY

HEMATOLOGY 2 PRELIM |2ND SEM | 22-23

Reporting results RBCs may be big enough to be counted as WBC instead as

➔ Results are reported to the ordering physician through LIS RBC.
interface. Critically abnormal results are reported verbally ➔ 2. Very small RBCs: May be counted as platelets instead as
to the clinician and documented on the patient report RBCs
➔ 3. Hemolysed sample: Destroyed RBCs will not be detected
Panic/Alert value procedures (obviously)
➔ 4. Clotted sample: RBCs are lost in the clots.

Result: RBC (falsely increased):

Causes:
➔ 1. High WBC count: This happens when patients could be
anemic, high WBC count (>100x10^9/L) can change the
RBC count.
➔ 2. Giant platelets: May be counted as RBCs instead as
platelets

Interfering Substances
LIMITATIONS OF THE PROCEDURE
➔ K2EDTA is the recommended anticoagulant. ➔ Very high WBC count
➔ K3EDTA and Na2EDTA are also acceptable. Use of other ➔ Severe lipemia
anticoagulants can yield misleading results. Hgb ➔ Certain unusual RBC abnormalities that
resist lysing
Interfering Substances ➔ Anything that increases the turbidity of the
sample such as elevated levels of
➔ Certain unusual RBC abnormalities that triglycerides
resist lysing ➔ Very high WBC count
➔ Nucleated RBCs ➔ High concentration of very large platelets
WBC ➔ Fragmented WBCs MCV ➔ Agglutinated RBCs
➔ Any unlysed particles greater than 35 fL ➔ RBC fragments that fall below the 36-fL
➔ Very large or aggregated platelets as when threshold
anticoagulated with oxalate or heparin ➔ Rigid RBCs
➔ Very high WBC count ➔ Very small red blood cells near the upper
RBC ➔ High concentration of very large platelets threshold
➔ Agglutinated RBCs ➔ Cell fragments
➔ RBCs smaller than 36 fL Plt ➔ Clumped platelets as with oxalate or
heparin
Result: WBC (falsely decreased) ➔ Platelet fragments or cellular debris near
Causes: the lower platelet threshold
➔ 1. Agglutination of WBCs: WBCs will be too big to be ➔ Known factors that interfere with the
counted as WBCs. Hct parameters used for its computation
➔ 2. Clotted sample: WBCs will be too big to be counted as ◆ RBC
WBCs. ◆ MCV
➔ Known factors that interfere with the
Result: WBC (falsely increased) MCH parameters used for its computation
Causes: ◆ Hgb
➔ 1. Aggregation of platelets (sticking to each other): ◆ RBC
Clumped platelets may be big enough to be counted as ➔ Known factors that interfere with the
WBC. parameters used for its computation
➔ 2. Very large platelets: May be counted as WBC MCHC ◆ Hgb
➔ 3. Nucleated RBCs: May be counted as WBC ◆ RBC
➔ 4. Unlysed RBCs: May be counted as WBC ◆ MCV
➔ Known factors that affect the WBC count,
Result: RBC (falsely decreased): LY, MO, GR such as high triglycerides, that can affect
Causes: lysing
➔ 1.Cold agglutinins (make the cells to stick to each other
when sample is refrigerated or in room temp): clumped

5 | MLS 113B
 MLS 113B | LABORATORY

HEMATOLOGY 2 PRELIM |2ND SEM | 22-23

Result: Hemoglobin (falsely increased) BLEEDING TIME

Causes: ➔ First functional platelet evaluation test
➔ 1. Lipemic samples: Increased in turbidity also increases ➔ Introduced by W.W. Duke in 1910
the spectrophotometer reading for hemoglobin. ➔ Used to detect defects in primary hemostasis
➔ 2. High WBC count: Causes turbidity in the sample. The ➔ Used as a screening test for vascular disorders as well as
more turbid the sample, the higher the spectrophotometer platelet function test
reading for hemoglobin.
➔ 3. Hemolysed sample: free hemoglobin (hemoglobin in the PRINCIPLE
plasma, outside RBCs) will be increased. ➔ A standardised incision is made on the volar surface of the
➔ 4. Carboxyhemoglobin: Carboxyhemoglobin is difficult to forearm.
transform into cyanmethemoglobin which increases the ➔ The time the incision bleeds is measured.
spectrophotometric reading for hemoglobin ➔ Cessation of bleeding indicates the formation of
hemostatic plug.
Result: Hemoglobin (falsely decreased) ➔ Depends on:
Causes: 1. The adequate number of platelets
➔ 1. Clotted sample: Less RBC will be available, leading to less 2. Ability of the platelets to adhere to the
RBCs lysed, which also leads to less hemoglobin freed and subendothelium.
tested.
➔ 2. Sulfhemoglobin: RBCs with sulfhemoglobin are difficult
to lyse.

MCV
➔ 1. Is INCREASED due to Cold agglutinins (refrigerated
sample) which causes decreased RBC count. Decreased
RBC count leads to increased MCV.
➔ 2. Is INCREASED due to high WBC counts (provided that
there is TRUE decreased RBC count). Analyzers are
sensitive to leukocytosis over 100x10^9/L.
➔ ***take note: Normal WBC count will not affect MCV.
MCV will be affected only by increased or decreased RBC
count.
➔ Remember the formula: MCV= Hct/RBC count x 10 Which
means any changes in hct and rbc count will also cause
changes MCV

Result: MCH (increased)

Causes:
➔ 1. Cold agglutinins (refrigerated sample) which causes
decreased RBC count. Decreased RBC count leads to
increased MCH.
➔ 2. Lipemic sample (increases hemoglobin which leads to
increased MCH).
METHODS
➔ 3. Carboxyhemoglobin (abnormal hemoglobin) causes
➔ Standard template method
increased MCH.
➔ Duke's method
➔ Ivy’s method
Result: MCH (decreased)
➔ Copley and Lalitch method
Cause:
➔ 1. High MCV and falsely low hemoglobin
TAKE NOTE
➔ Remember the formula: MCH= Hb/RBC count x 10 Which
➔ Drug intake that may cause falsely abnormal bleeding
means any changes in Hb and RBC count can also change
time:
MCH
1. Aspirin
2. Aspirin-containing compounds
3. Blood thinners (heparin or coumadin)

6 | MLS 113B
 MLS 113B | LABORATORY

HEMATOLOGY 2 PRELIM |2ND SEM | 22-23

DUKE’S METHOD away from the fold of the arm)

➔ Easy to perform simultaneously.
➔ Requires minimal equipment ➔ Stopwatch is started as soon as the
1. Alcohol bleeding startsin each wound.
2.Sterile lancet ➔ Using the edge of a filter paper
REQUIREMENTS (5mm wide and 1mm deep puncture) (whattman No:1), blot the blood
3. Stopwatch accumulated over the wound after
4. Filter paper 50 seconds.
5. Disinfectant ➔ Blot the site every 30 seconds.
➔ Clean the ear lobe or finger tip with ➔ Rotate the filter paper after 15
alcohol sponge seconds.
➔ Infants-heel of foot ➔ The time from which incision was
➔ Make a deep puncture with sterile made to the time at which the
lancet. PROCEDURE bleeding stops to stain the filter
➔ Start the stop watch paper is taken.
PROCEDURE ➔ Discard the glass slide ➔ The BP cuff is removed.
➔ Using filter paper blot the drop of ➔ The puncture wounds are cleaned
blood coming out from incision ➔ Sterile bandage applied
➔ When bleeding ceases stop the stop ➔ The average of the 3 bleeding time
watch. is taken .
➔ Count the number of drop on the filter ➔ Longer bleeding time could be
paper caused by puncture of superficial
➔ Multiply by 30 sec. veins
➔ Report the closest minute. ➔ If bleeding continues more than 15
➔ If the cut bleeds more than 10 min- apply pressure.
minutes, discontinue the test ➔ Repeat the bleeding time on other
• The ear lobule contain abundant arm.
subcutaneous tissue and is vascular. ➔ If the same result is recorded on the
ADVANTAGES • Flow of the blood is quite good other arm, report “greater than 15
min".
Normal bleeding time: 1-5 minutes ➔ Reports correlated with platelet
DISADVANTAGES • Difficult to get a standardized wound. count and finger prick smear.
• Least precision and accuracy.
• Standardized wound should be used. Reference range: 2-7 minutes
PRECAUTIONS • Light stain of blood should NOT be ➔ Standardised method
avoided. ADVANTAGES ➔ Bleeding time more accurate
• Time should be properly noted
Normal BT: 2-7 minutes
➔ Standardization of method
IVY’S METHOD
➔ Wound is of standard length and
1. BP cuff
QUALITY CONTROL depth
2. Disposable lancet
➔ Constant pressure of 40 mmHg is
REQUIREMENTS 3. Stop watch
maintained throughout the
4. Filter paper
procedure.
5. Alcohol
➔ Not a very reliable test.
➔ Clean the inner portion of the
➔ The puncture wound may close
forearm
before the cessation of bleeding.
➔ Place a BP cuff on the upper arm ,
➔ Improperly performed puncture.
inflate to 40 mm of mercury.
LIMITATIONS ➔ Prolonged Aspirin intake: 7-10 days
➔ Select an area on the volar surface
before the test
PROCEDURE which is devoid of veins
➔ Prolonged Incompletely dried
➔ Should be performed at room
alcohol
temperature.
➔ Prolonged Touching the filter paper
➔ A with a point of about 3mm /No.11
on the wound.
Bard Parkersurgical blade is taken.
➔ Three skin punctures 1mm deep
and 3 mm long are made (3 fingers

7 | MLS 113B
 MLS 113B | LABORATORY

HEMATOLOGY 2 PRELIM |2ND SEM | 22-23

STANDARD TEMPLATE METHOD COPLEY AND LALITCH METHOD

➔ More standardised method ➔ Clean the finger
➔ Uses a glass or plastic template ➔ Make a puncture wound 6mm deep
➔ Allows the lancet to make a cut-11 mm long and 1mm ➔ Immerse the wound in sterile
deep. physiological saline warmed to 37
PROCEDURE degrees C
➔ Leave it until there is no free flow of
blood.
➔ The BT is measured from the moment of
REQUIREMENTS the first bleeding on the wound to the
cessation of bleeding.

VARIABLES AFFECTING BLEEDING TIME

➔ Anemia prolongs the bleeding time.
➔ Patients with thrombocytopenia(<100×109 /L) will have
increased BT.
➔ Aspirin, penicillin, cephalothin prolongs BT
➔ Pediatric px and neonates
◆ smaller incisions are required.
◆ Pressure -20 mm of Hg

Clinical significance

➔ Thrombocytopenia
➔ Disorders in platelet function
◆ Thrombasthenia
◆ storage pool disease
◆ Bernard –Soulier syndrome
➔ Afibrinogenemia
Prolonged ➔ Severe hypofibrinogenemia
PROCEDURE
Bleeding ➔ Vascular disorders
Time in ➔ Aspirin
➔ Aplastic anaemia
➔ A/c leukemia
➔ Liver diseases
➔ Von Willebrand disease
➔ DIC
➔ Vascular abnormalities (Ehlers-Danlos
syndrome)
➔ Severe deficiency of factor V or XI

➔ Test is very sensitive and

ADVANTAGES reproducible
➔ Detects even minor alterations in
platelet function.

8 | MLS 113B