Hematology Pre-laboratory

Christian Dominic Rosales, MD

3 April 2019
LEVEL 2
PATHOLOGY

OUTLINE
REFERENCES .......................................................................................................... 1
RED CELL COUNTING............................................................................................. 1
HEMOCYTOMETER .............................................................................................. 1
DIFFERENT TYPES OF COUNTING CHAMBER ................................................. 1
NEUBAUER’S SLIDE ............................................................................................. 1
COUNTING RULE .................................................................................................. 1
ERYTHROCYTE COUNT (FROM 2020 TRANS) .................................................. 3
NOTES ................................................................................................................... 3
WBC DIFFERENTIAL COUNTING ........................................................................... 4
OBJECTIVES ......................................................................................................... 4
PRINCIPLE ............................................................................................................. 4
METHOD ................................................................................................................ 4
CALCULATION ...................................................................................................... 4
PERIPHERAL SMEAR (THE BASICS) .................................................................... 4
INTRODUCTION .................................................................................................... 4
TECHNIQUE .......................................................................................................... 4
CHARACTERISTICS OF AN IDEAL SMEAR ........................................................ 4
CAUSES OF UNACCEPTABLE SMEARS ............................................................. 5
PERFORMING A MANUAL LEUKOCYTE DIFFERENTIAL COUNT ..................... 5
OPTIMAL AREA OF THE BLOOD SMEAR ........................................................... 5
OBSERVING DIRECTION ..................................................................................... 5
• The four corner squares are further divided into sixteen
PLATELETS – INDIRECT COUNTING .................................................................. 5 smaller squares and are used for WBC counting.
o Central square is divided into 25 medium-sized squares
and are divided by a triple line
REFERENCES o The medium-sized squares are further divided into 16
small squares (tiny)
• Doc Rosales’ PPT o The four corner and central squares are used for platelet
• Batch Astra 2020 Trans and RBC count.

RED CELL COUNTING

HEMOCYTOMETER
• Hemo: blood
• Cyto: cell
• Meter: measurement/counter
o Thus, it is an apparatus used to count the blood cells
§ Can also be used in sperm, CSF and synovial fluid

Components
• It includes:
o Neubauer’s slide
o Cover slip
o Diluting pipette COUNTING RULE
o Cover glass
• Do not count cells touching the:
DIFFERENT TYPES OF COUNTING CHAMBER o Bottom line
o Right line
• Ordinary Neubauer counting chamber § This is to avoid double counting
• Improved Neubauer counting chamber
• Levy’s counting chamber
• Fuch’s Rosenthal chamber

NEUBAUER’S SLIDE
• Thick glass slide with two ruling area on center
• Ruled areas are separated by H-shaped gutter/trough
• Beyond the 2 vertical arms of trough there are two raised
shoulder (ridge) which support the cover glass
• Lining is coated by shining metal or rhodium

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 PATHOLOGY: Hematology Pre-laboratory

Thoma Pipette
• Consists of a graduated capillary tube, mixing bulb with glass
bead and aspirating tube
• Parts:
o Stem
o Bulb
o Rubber tube
• Thoma pipette are:
o WBC pipette
o RBC pipette

Differences Between RBC and WBC Pipette

Features RBC pipette WBC pipette
Color of
It has a red bead It has a white bead
bead
It has graduations It has graduations
Markings
up to mark 101 up to mark 11
Bulb Size of bulb is Size of bulb is
appearance larger smaller

Notes from 2020 Trans:

• Dilution factors
o For cell counting
§ Blood is filled till mark 0.5 and fluid is then filled till mark RBC Pipette
101 or 11
§ Both are thoroughly mixed
• The numbers written on the chamber means that the spaced
between the chamber and the cover slip is 0.100 mm and that
the smallest square on the grid has an area of 0.0025 mm2
• Clean the Neubauer chamber and the cover slip with 70%
ethanol
• With the microscope, using a 4x objective (scanner), identify WBC Pipette
the nine main squares of the counting chamber delimited by
School Year 2017-2018
three lines each as shown in the following image
• Now change to 10x objective and focus on the 9 main squares
• The counting is performed in the area delimited by three lines
ong and 1• mm
Cells that touch the
- upper and left border
The counting are counted
is performed in the(black
area delimited
color) while the cells bythatthree
touchlines
the right and lower border are
C pipette not counted (gray - color)
Cells that touch the upper and left border are
WBC pipette counted (black color) while the cells that touch
t has a white the right and lower border are not counted (gray
bead color)
t has
graduations up
o mark 11
Size of bulb is
smaller

• In the lab, for right & lower side:

- In the lab, for right & lower side:

è Don’t count

è Count

• The smallest square

- has
Theansmallest
area of 0.0025
squaremm
2
has= an
eacharea
mainof 0.0025
2
square has an area ofmm
0.04
2
= mm
each(0.0025 x 16 = 0.04)
main square has an area of 0.04
ll mark 0.5 and mm2 (0.0025 x 16 = 0.04)
ed till mark 101 Erythrocyte count 2/6
- Is the number of erythrocytes per microliter of
ghly mixed blood
 PATHOLOGY: Hematology Pre-laboratory

Cover Slip • Red cell diluting fluid must be:

o Anti-coagulant anti-hemolytic
• Special cover glass with smooth surface and even thickness
o Anti-aggregation
• Thickness = 0.3, 0.4, 0.5 mm
o Anti-Rouleaux
• Length: 16x22 mm, 22x23 mm o Preserve RBC shape
• Diluting fluid
Principle o One of the following solutions may be used:
• Dilution of blood 1. Isotonic saline
• Sampling of diluted suspension into measured volume - 0.85% sodium chloride in distilled water
• Counting of cell in that volume - Using normal saline as a diluent:
§ Maintain the normal disk shape of the RBC
§ Prevents autoagglutination
Focusing 2. Hayam’s solution:
• 4X to see the general formation of slide - Sodium sulphate 10g
• 10X for WBC counting - Sodium Chloride 2g
• 40X for RBC/Platelet counting - Mercuric Chloride 0.25g
- Distilled Water 100mL
Calculation 3. Gower’s solution
- Sodium sulphate 12.5 g
𝑁 𝑥 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝐹𝑎𝑐𝑡𝑜𝑟 𝑥 𝐷𝑒𝑝𝑡ℎ 𝐹𝑎𝑐𝑡𝑜𝑟 - Glacial acetic acid 33.3 mL
𝐶𝑒𝑙𝑙 𝐶𝑜𝑢𝑛𝑡 =
𝐴𝑟𝑒𝑎 𝐶𝑜𝑢𝑛𝑡𝑒𝑑 - Distilled water 100mL
4. Citrate formalin solution
Notes from 2020 Trans: - Tri-sodium citrate
• Total RBC count - Formalin
o N x dilution factor x volume correction factor
• Procedure
• Where: o Preparation of the slide
o N = the total number of red cells counted in the counting § Step 1: Place the cover slip
chamber § Step 2: Dilute the sample 1:200
o Dilution 1:200 § Step 3: Load step
§ Dilution factor = 200 § Step 4: Count in the small 5 center squares for RBC
o Counted volume:
• Note:
§ Each counted square has a volume of 0.2 x 0..2 x 0.1 =
o In certain conditions, Hayam’s solution may cause
0.004
clumping of RBCs and rouleaux formations, while Gower’s
§ 5 squares volume = 5x 0.004 = 0.2 mm3
solution prevents these problems
o Total RBC Count: N x 200 x 50 = N x 10,000
• Sample:
o Whole blood using EDTA or heparin as anti-coagulant.
Source of Error Capillary blood may also be used.
Source of Error
False High Count False Low Count NOTES
Blood diluted with tissue
Improper mixing • The usage of syringe and blood tube is discouraged in the
fluid
laboratory to prevent injuries in inexperienced hands
Undue delay in counting of
Uneven distribution of cell • Please be guided accordingly.
cell
Error in pipetting Clumping of cell (AIHA)
Notes from 2020 Trans:
Error in calculation Uneven distribution of cell
• In certain conditions, such as polycythemia, the red blood cell
Blood taken from area of count may be extremely high, which makes it difficult to obtain
Faulty technique of counting
hemoconcentration an accurate count in this instance, make a larger dilution of
Yeast, dirt and leucocyte are Improperly standardized blood. For a 1:301 dilution, add 20uL of whole blood to 6.0mL
counted as RBC counting chamber of diluting fluid
• For patient who has severe anemia and in whom the RBC is
T/N: Microhematocrit determination was discussed during this low, make a 1:101 dilution by adding 20uL of whole blood to
part. Dili complete akong notes kay naa’y gi-sapawan si doc na 2.0mL of diluting fluid
part. Nag ingon na siya na iya daw i-e-mail ang procedure. I’ll • Make certain the pipettes, hemocytometers, and cover glass
share once available na. are free from dirt, lint and dried blood. Ensure that the diluting
fluid is free from blood and other contamination
ERYTHROCYTE COUNT (FROM 2020 TRANS) • RBC takes longer to perform than a WBC because of the
larger number of cells. Therefore, proceed as quickly as
• Is the number of erythrocytes per microliter of blood possible once the cells have settled. Drying of the dilution if
• Normal range the counting chamber causes inaccuracies in the final cell
o Male: 4.2 -5.4 x106 count
o Female: 3.6-5.0 x106 • The range of error for a manual RBC is general 10 to 20%
o Newborn: 5.5-6.5 x106
• Erythrocyte count increased in cases of polycythemia and
decreased in anemia
• Principle
o In order to facilitate RBCs count a specified volume of
blood is diluted with a specified volume of isotonic fluid
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 PATHOLOGY: Hematology Pre-laboratory

WBC DIFFERENTIAL COUNTING PERIPHERAL SMEAR (THE BASICS)

OBJECTIVES • A properly prepared blood smear is essential to accurate
assessment of cellular morphology
• To accurately count WBC in chamber of the Neubauer • The wedge smear is the most convenient and commonly
hemocytometer used technique for making PBS (peripheral blood smear)
• To perform reliable dilution of blood cells
• To calculate the number of cells/µL
INTRODUCTION
PRINCIPLE • There are three types of blood smears:
o The cover glass smear
• Whole blood collected in EDTA is diluted according to the o The wedge smear***
type of cell count obtained o The spun smear
• The diluted blood suspension is then placed in a chamber and • There are two additional types of blood smear used for
the cell counted specific purposes
• The count is multiplied by dilution and reported as number of o Buffy coat smear for WBCs <1.0x109/L
cells per microliter (µL) of whole blood o Thick blood smears for blood parasites
• Specimen
METHOD o EDTA blood within 2 to 3 hours and collected to the mark
on tube
• Put the cover slip or glass slip on top of grid area in the
§ For our exercise, we use the heparinized blood in
chamber
capillary tubes as conveniently provided
• Dilute your sample:
• Factors that can affect the outcome of the preparation:
o 1:20 for WBC count o Excessive amount of anticoagulant to the specimen
o 1:200 for RBC count and platelets
o Longstanding old blood sample
• Loud your sample into the metal platform in the chamber o Warm testing environment
• Count the cells in the 4 large squares for WBC
• Calculate the number of cells counted / µL
TECHNIQUE
• Dilution of whole blood sample:
o Diluents
§ Acetic acid; or,
§ Distilled H2O
o Purpose
§ Dilute the amount of WBC to be able to count it
§ NR WBC: 4.3-10.8 x 103 / µL
• To lyse the RBC and platelets (the diluent lyses also the WBC
but takes longer time)
• The hemocytometer contains 2 Neubauer counting chamber

1. Place a drop of blood, about 2-3 mm in diameter

approximately 1 cm from one end of slide
2. Place the slide on a flat surface, and hold the other end
between your left thumb and forefinger
3. With your right hand, place the smooth clean edge of a
second (spreader) slide on the specimen slide, just in front of
• Each chamber contains: the blood drop
o 4 counting squares 4. Hold the spreader slide at a 30° - 45° angle, and draw it back
§ Each contains 16 squares against the drop of blood
o 100 RBC = 10 Platelets = 1 WBC 5. Allow the blood to spread almost to the edges of the slide
• Choose 90° lines, count only the cells that on those lines 6. Push the spread forward with one light, smooth moderate
(example L-shape), apply it to all squares for maximum speed. A thin film of blood in the shape of tongue.
accuracy 7. Label one edge with patient name, laboratory ID and date
8. The slides should be rapidly air dried by waving the slides or
CALCULATION using an electrical fan.
Cells 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑖𝑛 1 𝑙𝑎𝑟𝑔𝑒 𝑠𝑞𝑢𝑎𝑟𝑒 𝑥 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
= CHARACTERISTICS OF AN IDEAL SMEAR
µL 𝑉𝑜𝑙𝑢𝑚𝑒 𝑓𝑎𝑐𝑡𝑜𝑟 (0.1)

• Dilution factor = reciprocal of dilution (20)

• Volume factor = (Width x Length x Height) = 0.1
• Number of cells / mm3 = N x 50

Example:
• If total number of WBCs in 4 squares is 120
• Then the number of WBCs in 1 mm3 = 120 x 50 = 6000 Well-made PBS

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 PATHOLOGY: Hematology Pre-laboratory

1. Thick at one end, thinning out to a smooth, rounded, OPTIMAL AREA OF THE BLOOD SMEAR
feathery edge, not bullet-shaped
2. Should occupy 2/3 of the total slide area.
3. Should not touch any edge of the slide.
4. Lateral edges of the smear should be visible.

OBSERVING DIRECTION

• Observe one field and record the number WBC according to

the different type then turn to another field in the snake-like
direction
• Avoid repeating or missing some cells

Technique
• These counts are done in the same area as WBC and platelet
Gallery of Poorly-Made Smears estimates with the red cells barely touching
• This takes place under x100 (oil) using the zigzag method
CAUSES OF UNACCEPTABLE SMEARS • Count 100 WBCs including all cell lines from immature to
mature
1. Drop of blood too large or too small • Report results as recorded
2. Spreader slide pushed across the slide in a jerky manner.
3. Failure to keep the entire edge of the spreader slide against
the slide while making the smear.
4. Failure to keep the spreader slide at a 30° angle with the
slide.
5. Failure to push the spreader slide completely across the
slide.
6. Irregular spread with ridge and long tail: edge of spreader
dirty or chipped; dusty slide.
7. Holes in film: slide contaminated with fat or grease.
8. Cellular degenerative changes: delay in fixing, inadequate
fixing time or methanol contaminated with water.

PERFORMING A MANUAL LEUKOCYTE DIFFERENTIAL

COUNT
• White blood cells
1. Check for even distribution and estimate the number PLATELETS – INDIRECT COUNTING
present (also, look for any gross abnormalities present on
the smear) 1. Estimate number of platelets present per OIF in 10 OIF and
2. Perform the differential count. take an average.
3. Examine for morphologic abnormalities. 2. Multiply the average by 20,000.

Range of Reference:
• Platelets per oil immersion field (OIF)
1. <8 platelets/OIF = decreased
2. 8 to 20 platelets/OIF = adequate
3. >20 platelets/OIF = increased

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 PATHOLOGY: Hematology Pre-laboratory

Indirect Platelet Counting Formula:

𝑇𝑜𝑡𝑎𝑙 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑃𝑙𝑎𝑡𝑒𝑙𝑒𝑡𝑠 𝑖𝑛 10 𝑂𝐼𝐹

𝑥 20,000 = 𝑁 𝑚𝑚N
10

Platelet Clump in PBS

(Not the same as the slide of doc’s, but familiar J)

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