Al-Azhar University- Gaza

Faculty of Applied Medical Sciences

Laboratory Medicine Department










Practical
Hematology
Manual #1


Prepared by: Ashraf Shaqalaih BSc(MT), MSc(MT), CLS(H), CLSp(H)
Clinical Laboratory Specialist in Hematology
Clinical Immunohematologist Technologist (Lic#238, State of
California, USA)





















Anticoagulants Used In The Hematology Laboratory
Anticoagulants are defined as substances which prevent
blood clotting / coagulation, and allow separation of the blood into
cellular and liquid (plasma) components. Generally plasma
contains coagulation factors. The three anticoagulants commonly
used in hematology laboratory are:
1] Ethylene Di-Amine Tetra-Acetic Acid (EDTA):
EDTA can be found in three salt forms:
1- Tri-Potassium EDTA
2- Di-Sodium EDTA
3- Di-Lithium EDTA
Also, EDTA can be crystalline or liquid. Liquid EDTA tubes
requires specific filling volume to avoid dilution effect. So, blood
: anticoagulant ratio must be maintained (this is applicable to all
anticoagulants). EDTA is also known as Versene or Sequestrene.
EDTA acts by chelating / removing ionized calcium (calcium is
required for blood to clot, so when it is removed blood will not clot).
Generally tri-Potassium EDTA is better than di-Sodium EDTA
and di-Lithium EDTA.



Always, be sure to mix blood with anticoagulant in a manner that guarantee
proper complete mixing, by gentle repeated inversion of the tube, in figure of 8
inversion for at least 20 times, do not shake or use vigorous inversion, since this
may cause hemolysis, and disintegration of cells, and the final effect will be
erroneous low results for cellular components of blood, which are our
hematology laboratory interest.

ANTICOAGULANTS
Used in Hematology
Laboratory

EDTA is the most commonly used anticoagulant in the
hematology laboratory, and is the anticoagulant of choice for the
CBC.
Excess EDTA (i.e. more EDTA, you fill less blood volume,
so EDTA is in excess), causes shrinkage of RBCs, causing
falsely / erroneously reduced hematocrit (HCT), and subsequent
increase in MCHC and decrease in MCV (MCV and MCHC are
RBC indices that will be studied later). Platelets are also affected,
they will swell and subsequently disintegrate, causing
erroneously high platelet count, since platelets will be disintegrated
into more than one fragment, each fragment will be counted as
one platelet (for example if one platelet will be disintegrated into 4
fragments, the 4 fragments will be counted as 4 platelets, but
actually they represent one platelet, causing erroneously high
platelet count).
From the previous discussion we conclude that correct ratio of
blood to anticoagulant is very important, to rule out these in vitro
effects.
EDTA can induce platelet aggregation and clumping, causing
falsely decreased platelet count, because these platelet clumps will
not be counted as platelets, they may counted as red blood cells
(causing low platelet count and high red blood cells counts). This
technical problem can be solved by (1) repeated measurements,
(2) extraction of new sample and repeat measurements, (3)
study the automated cell histograms, and (4) by visualizing blood
film, looking for these platelet clumps. Also, Aggregated and
clumped platelets interferes with WBC counting zone in

automated hematology counters that use electrical impedance
technology.
2] Sodium Citrate
Is the anticoagulant of choice for coagulation and platelet
function tests, also is used for ESR (erythrocyte sedimentation rate
test). It acts by precipitating calcium, thus it will not be available for
clotting process. It came in a liquid form, as 3.8% tri-sodium
citrate. For coagulation testing, the ratio of 9 volumes of blood to
one volume of anticoagulant (9 volumes blood:1 volume
anticoagulant) is very critical (very important), as variation from
this ratio may cause errors. For ESR (4) volumes of blood to one
volume of anticoagulant is used (4 : 1).Generally, this
anticoagulant is not suitable for routine hematology testing. From
this we conclude that sodium citrate acts as anticoagulant and as
diluent (as in the case of ESR). Because of its dilution effect it cant
be used for CBC.
3] Heparin
Heparin is an acid mucopolysaccharide, it acts by
complexing with anti-thrombin to prevent blood clotting
(antithrombin is one of the natural/physiological inhibitors of blood
coagulation, which is found in vivo, this will be studied later in
coagulation and hemostasis modules). It is not suitable for blood
films staining, since it gives too blue coloration to the
background, when films are stained with Romanovsky stains,
also, heparin may cause leukocyte and platelet clumping , this is
why heparin is not suitable for routine hematology tests. It is the
preferred anticoagulant for osmotic fragility test ( a special
hematology procedure, that will be studied in this course). Heparin
also is used in capillary tubes for spun hematocrit (HCT) (heparin

cover the entire capillary tube glass), these capillary tubes are also
called microhematocrit capillary tubes. Heparin is also used for
L.E. cell preparation (L.E.= Lupus Erythromatosus).
Heparin is found in basophil and mast cell granules.
Heparin is used therapeutically as an in vivo anticoagulant.

Anticoagulants commonly Used in the Hematology Laboratory and their use:

No. Anticoagulant Hematology Laboratory Use Universal Color Code
1 EDTA Routine Hematology Procedures. Lavender, Pink
2 Sodium citrate Coagulation , Platelets Tests, ESR. Blue
3 Heparin Osmotic Fragility, Spun Hematocrit Green, Brown





















I am a tube containing
specified volume of
anticoagulant, please only
fill me with the correct
required volume of blood
from the patient, do not
attempt to overfill or under
fill me, your results may be
negatively affected, so help
me and help your self.

HEMOCYTOMETRY



I mproved Neubauer Hemacytometer

Hemacytometry
Hemacytometry means the use of the hemacytometer counting
chamber to count blood cells (to count WBC, RBC, and Platelets, as
will as, counting cells in other body fluids, e.g. CSF and semen
analysis). Hemacytometer is a counting chamber device made of
heavy glass with strict specifications, it resemble a glass slide.
Also, the hemacytometer have a special glass slide manufactured to

strict specifications, it is very thick and non-flexible. There are many
types of hemacytometers, in which they differ in rulings, but the
commonest and the easiest one is the Improved Neubauer
Chamber, bright line type. When viewing the hemacytometer
from the top (figure below), it has 2 raised platforms surrounded by
depressions on three sides, each raised platform has a ruled
counting area marked off by precise lines etched into the glass.
The raised areas and depression form H letter, this H has two
coverglass supports on each side which are exactly 0.1 mm higher
than the raised platforms. The coverglass is placed on top of the
coverglass supports so it covers both ruled areas. The depth
between the bottom of the ruled area and the coverglass is
exactly 0.1 mm. So, coverglass function is to confines the fluid and
regulates the depth of the fluid to be applied.














Figure1: Top view of the hemacytometer









Figure 2: Coverglass position on the hemacytometer
COVERGLASS
COUNTING
CHAMBER
COVERGLASS SUPPORTS
AL AZHAR
H-SHAPED DEPRESSION
PLATFORM WITH
RULED AREA
COVERGLASS
COVERGLASS SUPPORTS
AL AZHAR
COUNTING
CHAMBER
COVERGLASS
CENTER PLATFORM
COVERGLASS
SUPPORT

Hemacytometer Counting Areas
Hemacytometer has 2 identical ruled counting areas, each
composed of etched area consists of a large square, with a
diameter of 3 mm. This large square is subdivided to 9 small
squares, each with a diameter of 1 mm. So, each 1mm square
can accommodate a volume of 1 mm x 1mm x 0.1 mm (depth) =
0.1 mm (cubic millimeter). WBC cells are counted in the entire
9 squares. The central square is further subdivided into 25 smaller
squares each with a diameter of 0.2 mm, so the volume
accommodated within this square will be 0.2 mm x 0.2 mm x 0.1
mm(depth) = 0.004 mm (cubic millimeter). Red blood cells are
counted in the large central square (1 from 9 squares), in which only
the four corner squares and the center square (look figure 3 , in
which R denotes for red blood cells). Platelets are counted in the
entire large center squares (the 25 small squares).

Figure 3 - Red Blood Cells Counting Area

Using The Hemacytometer
1- Position a clean, dust free, coverslip so it covers the ruled
counting areas of a clean hemacytometer.
2- Fill the hemacytometer with the fluid containing cells to be
counted, by touching the tip of the capillary tube or
micropipette tip to the point where the coverslip and raised
platform meet on one side, the fluid will drawn under the
coverslip and over the counting area by capillary action, this
requires about 10 l.
3- Repeat on opposite side of the chamber.
4- The chamber must not be overfilled or underfilled, if accurate
results are needed!.
5- Place the hemacytometer on the microscope stage, so one of
the ruled counting areas is aligned directly above the light
source (condenser); rotate the low power objective (x10) into
place; using the coarse focus knob, move the low power
objective very near the coverslip; rotate coarse focus knob to
increase the distance between the low power objective (X10)
and the hemacytometer until etched/ruled lines come into
focus; all nine large squares must be viewable; very carefully,
rotate the high power objective (X40) into place, with the aid of
fine focus knob, adjust the focus until the etched lines come
into focus, you can now carefully move the hemacytometer by
using the mechanical stage, so that the ruled area on the other
side can be viewed.
The Counting Pattern
Either left to right or right to left counting pattern can be used
( fig.4); but with the insurance that each cell is counted only
once, to accomplish this, cells that touch the right boundary lines or
the bottom boundary lines are not counted, because they will be

counted with the other squares (look figure). After cells are counted
on one side, the hemacytometer is moved and the cells are
counted on the other side. Results for each side are recorded,
then are totaled and the average is calculated.







Figure 4 Counting Pattern


Figure 5: Cells touching the right and bottom boundaries are not counted
Calculating The Cell Counts
1st. The total number of cells per cubic millimeter of sample can be
calculated from:
1. The average number of cells counted.
2. The ruled areas contain an exact volume of diluted sample.
3. The dilution of the sample.
2nd. The hemacytometer Formula:
N x D (mm) x DF
= C/mm
A (mm )
Cells
touching
right, and
bottom
boundaries
are not
counted!
Begin

Where:
A- C/mm = number of cells/ mm
B- N= Total number of cells counted in the counting
chamber.
C- D (mm) = Depth factor in mm
D- DF = Dilution Factor
E- A (mm) = Area counted (mm)
1. The dilution factor is determined by the blood dilution
made by you as a laboratory technologist..
2. The depth factor is always = 10 (1/0.1).
3. The area counted will vary for each type of cell count and
is calculated using the dimensions of the ruled area.
Comments:
Although some specialists still considers hemacytometry
is the standard method of cell counting, but its C.V. is high, which
indicates impression and sometimes inaccuracy, especially when
counting red blood cells . In cases of leukopenia (low WBC count,
below normal ranges ), still hemacytometry the method of choice for
cell counting.
























WBC (Leukocyte) Manual Counting

Principle:
Blood sample is mixed and diluted with weak concentration of
hydrochloric acid (HCl), or acetic acid (in specified known volumes).
Weak acids will lyse red blood cells, and will darken WBCs to
facilitate counting by the hemacytometer.
Manual WBC counting is used in cases of very low WBC
count (leukopenia) with automated hematology cell counters, and
when automated cell counters are not available.
Sample:
EDTA anticoagulated whole venous blood.
Reagent and Supplies To Prepare Diluting Fluid:
1- Volumetric Flask 100 cc.
2- Serological pipettes.
3- Concentrated HCL
4- Glacial Acetic Acid
Preparation of Diluting Fluid:
Diluting fluid is either:
1% hydrochloric acid in distilled water ( 1 ml Conc. HCL + 99
ml Dist. water).
2% Acetic Acid in distilled water ( Turks solution) (2 ml
glacial acetic acid
+ 98 ml distilled water).
Glassware, Apparatus, Equipment :
1- Neubauer improved hemacytometer.
2- Clean cover slip slide (especially made for the hemacytometer).
3- Automatic micropipette (20 l, 380 l are the required
volumes).
4- Gauze 10 x 10 cm

5- Glass/Plastic tubes- (12x75 mm).
6- Handy tally counter.
7- Conventional light microscope.
Procedure:
1- Mix the blood sample gently but thoroughly by inversion,
manually or by mechanical rocking mixer.
2- Pipette 0.38 ml (380 l) of diluting fluid into a 12x75 mm tube.
3- Pipette 0.02 ml (20 l) of well mixed blood to be counted
and wipe the tip with gauze into the tube containing diluting fluid
and mix the tube.
4- Let the tube stand for 2-3 minutes to ensure complete RBC
lyses, then mix well.
5- Prepare the clean hemacytometer and cover it with the
designed coverslip.
6- Load one side of the hemacytometer with the aid of a
capillary tube or micropipette, do not attempt to overload or
underload the hemacytometer.
7- Allow the hemacytometer to sit for several minutes to allow the
WBCs to settle in the counting chamber, to avoid drying effect,
place the loaded hemacytometer in a covered Petri dish with a
moist gauze, until counting.
8- Place the hemacytometer in the microscope stage.
9- Focus with x10 objective lens (low power), with lowering the
condenser.
10-The WBCs are counted in the 9 corner large squares, with the aid
of hand tally counter.
11-Follow the counting pattern shown in the figure below. During
counting, do not count cells that touch the right or bottom boundaries
to ensure unduplicated counting.












12- The total counted WBCs in the 9 squares are added together.


Fig. WBCs are counted in the 9 hemacytometer squares
If the number of cells in a square varies from any other
square by more than 9 cells, the count must be repeated,
because this represents an uneven distribution of cells, which is
may be caused by improper mixing of the dilution or improperly
filled hemacytometer.
Calculations:
N x D (mm) x DF
Total WBC Count =
A (mm)

Begin

Where:
N = Total WBC counted by the counting chamber.
Depth factor in mm = 10
DF = Dilution Factor = 20
A (mm) = Area counted = 3 mm x 3 mm = 9 mm
So,
N x 10 mm x 20
Total WBC Count =
9 mm
Example:

20

19 18

21

14 16

19

16 15


N= 20 +19 +18 +21 +14 +16 +19 +16 +15 = Tallied 158 Counted WBC Cell

158 x 10 x 20
Total WBC Count / cumm =

= 3500 / cumm = 3.5 x 10
9
/L
9

Reference Range
Adults : 4.5 11.0 x 10
9
/L
Six years: 4.5 12.0 x 10
9
/L
One year: 6.0 14.0 x 10
9
/L
Newborn: 9.0 30.0 x 10
9
/L
WBC count varies according to age but not to sex.
Sources of Error:
1- Contaminated diluting fluid.
2- Incorrect dilution.
3- Uncalibrated Micropipettes.
4- Uneven distribution of WBCs.
Hemacytometer Squares

5- Presence of clumped WBCs.
6- Unclean hemacytometer or cover slips.
7- Presence of air bubbles.
8- Incompletely filled hemacytometer.
9- Over flow.
10- Presence of debris.
11- Drying of the dilution in the hemacytometer.









































1 ml of gentian violet can be added to the diluent to color the
white blood cells, thus counting will be easier.
In leukopenia ( decreased WBC count), with a total
WBC count below 2500/cumm, the blood is diluted 1:10,
whereas in leukocytosis (increased count), the dilution
may be 1:100 or even 1:200.

RBC Manual Count
Principle:
A specified volume of blood is diluted with a specified volume
of isotonic fluid. This isotonic diluting fluid will not lyse RBCs, and
will facilitate counting with the aid of the hemacytometer.
Sample:
EDTA anticoagulated whole venous blood.
Diluting Fluid:
Isotonic saline:0.85% sodium chloride (NaCl) in distilled
water.
OR
10 ml of 40% Formalin made up to 1 liter with 32 g/l Tri-
sodium Citrate.
OR
6.25 g of crystalline Sodium Sulfate. Transfer to 100 cc
volumetric flask, and add approximately 50 cc distilled water.
Then add 16.7 ml of Glacial Acetic Acid. Finally add distilled
water up to the mark.
Apparatus and Equipment:
1- Micropipette 20 l is the desired volume.
2- Serological Pipette, 5ml.
3- Handy Tally counter.
4- Improved Neubauer counting chamber with the cover slips.
5- Conventional light microscope.
Procedure:
1- Pipette 4.0 ml of diluting fluid into a tube.
2- Pipette 20 l of will mixed anticoagulated whole blood to the
tube.
3- Mix continuously for 2-3 minutes.
4- Load the cleaned hemacytometer.

5- Place the hemacytometer on the microscope stage, lower the
condenser.
6- Focus with x10 objective lens on the large central square.
This square is ruled into 25 small squares, each of which is
further divided into 16 smaller squares, of the 25 squares, only
the four corner squares, and one middle square are used to
count RBCs.
7- Switch to x40 objective lens, and start counting in the five
designated squares.
Calculations:
N x Dilution Factor x Depth Factor
Total RBC Count =
Area Counted (mm)
Where:
N= Total number of red cells counted in the counting
chamber.
Dil. Factor = 0.02 : 4 = 2 : 400 = 1:200, Dilution Factor = 200.
Depth Factor = 10
Area Counted = 0.2 x 0.2 x 5 = 0.2 mm
So,
N x 200 x 10
Total RBC count = = N x 10,000
0.2
Normal Reference Range:
Males : 4.6 6.2 x 10
12
/L
Females : 4.2 5.4 x 10
12
/L
Children: 4.5 5.1 x 10
12
/L

Sources of Error:
Same as WBC manual Counting, refer to WBC manual
Counting.

Hemoglobinometry
Hemoglobin Determination
Decrease in hemoglobin concentration beyond established
normal ranges for age and sex is called anemia, whereas
increase in hemoglobin concentration beyond established normal
ranges for age , sex, and geographical distribution is called
polycythemia. So that, for correct diagnosis it is important to
determine accurately and precisely hemoglobin concentration.
Many methods are available for the determination of hemoglobin,
but among them the relevant, and the recommended one is the
Modified Drabkins Method. ICSH (International Committee for
Standardization in Hematology) consider this method as the
reference method for hemoglobin determination.
Drabkins solution contains the following:-
1- Potassium Ferricyanide
2- Potassium Cyanide.
3- Non- ionic Detergent
4- Dihydrogen Potassium Phosphate.
Well mixed EDTA anticoagulated blood is diluted in Drabkins
solution; non-ionic detergent will lyse the red cells to (1) liberate
hemoglobin, and to (2) decrease the turbidity caused by red cell
membrane fragments by dissolving them. Then, hemoglobin is
oxidized and converted to methemoglobin (Hi) by potassium
ferricyanide, this step is accelerated by the dihydrogen potassium
phosphate, and requires approximately 3 minutes for total
conversion. Potassium cyanide will provide cyanide ions to form
cyanomethemoglobin (HiCN), which have a broad spectrum of
absorption at 540 nm. The absorption can then be compared
with a hemoglobin standard with a known hemoglobin

concentration, and by applying Beers law extract the hemoglobin
concentration of the unknown (i.e. the patient).

















































Hemoglobin + Potassium Ferricyanide Methemoglobin (Hi)

Methemoglobin + Potassium Cyanide Cyanomethemoglobin

Hemoglobin Concentration Determination
Modified Drabkins Method
Principle:
Whole blood is diluted in a solution containing Potassium
Ferricyanide and Potassium Cyanide. Hemoglobin will be
oxidized by the action of Potassium Ferricyanide to form
Methemoglobin (Hemiglobin, Hi). Potassium Cyanide will provide
Cyanide ions to form Cyanomethemoglobin (HiCN). This solution
can be measured spectrophotometrically and compared to known
hemoglobin standards. This procedure is applicable in diagnosing
and monitoring therapy in cases of hemoglobin deficiency anemias.
Sample: EDTA anticoagulated venous whole blood .
Apparatus:
1- Brown bottle- 1 liter
2- Volumetric flask 1 liter
3- Balance
4- Spectrophotometer adjusted at 540 nm
5- Spectrophotometer Cuvettes
6- Glass or plastic centrifuge tubes
7- Micropipette (adjusted at 20 l)
8- 5 ml volumetric pipette or graduated pipette (or you can use
bottle top dispenser)
Reagents:
1- Potassium Ferricyanide K3Fe (CN)6 0.200 g (200 mg)
2- Potassium Cyanide (KCN) 0.050 g (50 mg)
3- Dihydrogen Potassium Phosphate KH2PO4 0.140 g (140 mg)
4- Distilled Water (laboratory grade 1)
5- Non-Ionic Detergent:
- Sterox S.E. 0.5 ml
or - Trinton X-100 1.0 ml

or - Quolac Nic 218 1.0 ml
6- Standard Cyanomethemoglobin solution/ solutions.
Working Drabkins Solution Preparation:
Add the reagents to the volumetric flask and add distilled
water up to the mark (avoid bubble formation), with continuous
mixing as you are adding the distilled water. Transfer to a
stoppered brown bottle, and label with name, date of preparation,
and the name of the technologist who prepared the solution.
Ready made commercial preparations are available in
the market, just dilution with distilled water is required,
such that preparations are for example available from
Randox company.
Procedure:
1- Add 20 l of whole anticoagulated blood to 5 ml of Drabkins
solution.
2- Mix well.
3- Allow the mixture to stand at room temperature for at least 3
minutes.
4- Measure the absorbance at 540 nm against a diluent Drabkins
solution (blank).
5- Measure the absorbance of the standard HiCN solution in the
same manner.
6- Extract the unknown hemoglobin concentration, using the
following equation:


Abs. of Unknown
Unknown Hb concentration = x Conc. Of STD
Abs. of STD

Or read directly from the hemoglobin standard curve. See below,
how to prepare the hemoglobin standard curve.

Hemoglobin Standard Curve Preparation:
A standard curve must be made each time new Drabkins
solution is prepared. A commercially prepared standard kit with
hemoglobin concentrations of 20, 15, 10, 5 g/dl is available, read
each corresponding absorbance, and plot the results on a linear
graph paper (absorbance versus Hb concentration). Or, you can
use a stock standard solution of 20 g/d, and dilute it to various Hb
concentrations with Drabkins solution.

Absorbance

0.3

0.2

0.1


5 10 15 20 Hb-Concentration- g/dl

Hemoglobin Standard Curve

Notes:
1- Drabkins method is the recommended method by the ICSH.
2- Drabkins solution should be clear and have a pH of 7.0 to 7.4,
discard if turbid.
3- The Drabkins solution is the blank, and should read zero (0)
absorption.
4- Take care when preparing the solution, as cyanide is fatal,
and toxic, although the amount of cyanide in the prepared solution
is less than the human lethal dose.
5- Do not expose the solution to acids, because cyanide will be
released.
6- Keep the solution in a dark bottle at room temperature, but
discard after a month.

7- All types of hemoglobin which include hemoglobin,
oxyhemoglobin, carboxy hemoglobin, methemoglobin are
measured by this method, only Sulfhemoglobin (S-Hb) is not
measured by Drabkins method..
8- Sulfhemoglobin is found at increased amounts in cases of
Clostridium septicemia, as a result of drug intake, and in severe
cases of constipation. At increased amounts blood sample may
color as lavender to green.
9- Increased methemoglobin concentration occurs as a result of
inherited conditions or more commonly as acquired as a result of
drug intake or exposure to chemicals.
10-Turbidity due to leukocytosis , lipemia, or high protein levels as
seen in para proteinemias ( e. g. multiple myeloma ) may
interfere and cause erroneously high hemoglobin concentration.
Prepare sample blank to overcome erroneous readings).
11-Try as possible as you can to obtain venous whole blood.
12- Use automatic micropipettes, but not Sahli pipettes, to reduce
technical errors.
13- Panic values for hemoglobin is less than 6.0 g/dl.
14- Hemoglobin concentration is unrelated to patient eating status.
15- Blood obtained from heavy smokers requires 3 minutes more
incubation time for full conversion of hemoglobin to
methemoglobin.
16- Do not expose Drabkins solution and standards to light, and
sunlight.
17- People living at high attitudes tend to have polycythemic
hemoglobin concentrations, because of the low oxygen tension
at high attitudes, causing tissue hypoxia, so that body
compensate and adapt for this by increasing hemoglobin.
18- Pregnant females tend to have lower hemoglobin

concentrations than normal for their age, because their
fetuses compete with them for iron, vitamins and other
essential substances for hemoglobin synthesis, this is why
pregnant females are of great needs for iron and vitamin
supplements during their pregnancies.

Preparation Of Sample Blank
Turbidity can cause erroneously increased hemoglobin
concentration due to increased light scattering, to overcome this
you can prepare a sample blank. To prepare a sample blank use
the formula:

5 ml
N =
(1- Hct)

Where N = ml of Drabkins to be added to 20 l of patient plasma.

So, when you have a turbid sample, take a portion from it, and
centrifuge it, take 20 l of its turbid plasma and add it to the
calculated Drabkins solution amount according to the above
formula . This is considered the zero blank, adjust the
spectrophotometer absorbance reading to zero, then read the
absorbance of the patient hemoglobin according to the procedure
above. The sample blank will correct the erroneous hemoglobin
result caused by turbidity.

Example:
If a patient has a hematocrit of 50, then:

5 ml 5
N = = = 10 ml
(1- 0.50) 0.50


So, the sample blank is prepared by adding 20 l of patients
plasma to 10 ml Drabkins solution.





Reference Hemoglobin Concentration Ranges:
Males : 13.5 18.0 g/dl
Females: 12.0 16.0 g/dl
Newborn: 16.5 19.5 g/dl
Children : 11.2 16.5 g/dl (varies with age)





































Hemoglobin concentration is expressed in g/dl (gram per deciliter)

Spun Microhematocrit

Principle
Hematocrit is the ratio of the total volume of RBCs to that of
whole blood expressed as percentage(%) (whole blood = total
volume of cells + plasma). The second synonym for hematocrit is
PCV (Packed Cell Volume). The procedure is easy to perform, whole
blood is centrifuged in a narrow tube (capillary tube), cellular
elements will be separated from the plasma, after centrifugation
blood will be separated into 3 layers : (1) Bottom layer contains
packed RBCs, (2) Middle layer contains WBCs and Platelets (on
top of RBCs), (3) Upper plasma layer. The hematocrit value is
determined by comparing the volume of RBCs to the total volume of
the whole blood sample, it is usually reported as a %.







Sample:
EDTA anticoagulated whole venous blood (correct volume
is highly required. When EDTA is in excess, cell shrinkage
occurs, and as a result a falsely low hematocrit is obtained, with
corresponding increase in MCHC, and decrease in MCV).
Heparin
Or directly from a finger prick, to a heparin coated capillary
tube.
Apparatus and Materials:
1- Microhematocrit centrifuge.
2- Modeling clay (seal material).
3- Capillary tubes (7 cm long, 1mm diameter)
4- Hematocrit measuring device reader or a conventional ruler.
Procedure:
1- Fill the capillary tube with blood by capillary attraction. Either
from free flowing finger punctured by a sterile lancet/ or from a
well mixed anticoagulated whole venous blood (this requires only
few microliters of blood).
2- Seal with the modeling clay the empty end of the capillary tube.
3- Place and position the capillary tube in the radial grooves of
the microhematocrit centrifuge with the sealed end away from the
center (pointed toward the outside).
4- Centrifuge for 5 minutes at 12000 g, so that additional
centrifugation does not pack the red blood cells more.
5- The height of the RBC column, and the total column should
be measured with the aid of a ruler in cm and mm, then divide
the RBC column height over the total column height (total height
= RBC column + buffy coat + plasma column), or simply with
the aid of a special HCT reader device.
6- Express the results in percentage (%).




















Reference intervals:-
Males : 40 - 53%
Females : 37 - 47%
Newborns: 51 - 60%
Children : 34 - 49%
Notes:
1- Higher values than the reference intervals is called
polycythemia.
2- Lower values than the reference intervals is called anemia.
3- In cases of very high HCT, additional centrifugation for
5 minutes is recommended to reduce plasma trapping. In general
the higher the hematocrit, the greater the centrifugal force
required.
4- Adequate centrifugation time and speed are important for
accurate hematocrit.
5- Cells should be packed so that additional centrifugation does
not alter or reduce HCT reading.
Buffy coat is the layer where WBCs and Platelets are collected
to, after centrifuging a whole blood sample, this is the middle whitish-tan
colored layer.
Buffy coat layer will contain all nucleated cells, including the nucleated
red cells, which are not normally found in the peripheral blood, but are
seen in pathological conditions. Also, all abnormal cells, including
leukemic cells are found in this layer, i.e. the buffy coat layer.
Red Blood Cell Layer
Buffy Coat Layer
Plasma Layer

6- Plasma trapping is slightly more in macrocytic anemias,
spherocytosis, hypochromic anemias, and in sickle cell anemia.
7- Errors may occur as a result of:
Inadequate mixing of the blood.
Improper reading of the column lengths.
Inclusion of buffy coat height with RBC column height (in
leukocytosis or in thrombocytosis, the buffy coat column height
will be increased).
Plasma trapping is still one of the causes of erroneously
increased HCT results.
Hemolysis of blood sample (due to improper collection,
delay in processing) will cause erroneously decreased HCT.
8- Increased anticoagulant to red cell ratio (short EDTA
sample), will cause red cell shrinkage and the hematocrit will be
erroneously decreased.
Clinical Significance:
HCT is used to detect anemias, polycythemias, hemodilution,
hemo-concentration, and also is used in the laboratory to calculate
the MCV, and the MCHC manually.









If you direct the capillary tube towards the microhematocrit centrifuge center, the
sealed material will be removed, and at the end of centrifugation you will find an empty
capillary tube, blood will go out from the tube!!
Nowadays, Hct is supplied by the widely used automated hematology analyzers. But
this Hct is calculated rather than measured, these analyzers are not equipped with
centrifuges, Hct is calculated from the MCV, and RBC count, by using the following formula:

MCV x RBC
Hct=
10
The sealed end of the capillary tube should be directed to the outside.
The microhematocrit should be read at the top of red cell layer not at the top of
the buffy coat.















































There are two types of capillary tubes, red banded and blue banded capillary
tubes. The red banded are heparin coated, and use it when doing finger
prick hematocrit. Blue banded capillary tubes are plain, and use it with
EDTA blood samples.
Manual hematocrit is slightly more than the calculated automated
hematocrit, because of the trapped plasma which will be included with
manual hematocrit, and excluded with automated hematocrit (because it is
calculated not measured).

Red Blood Cell Indices

Red blood indices are calculated parameters which determine
red blood cell size, hemoglobin content of red cells, and
hemoglobin concentration of red cells. These parameters are
useful in classifying anemias into microcytic, normocytic, or
macrocytic; and hypochromic or normochromic. These parameters
are calculated from total red cell count, hematocrit and hemoglobin.
1- MCV
Mean Cell (Corpuscular) Volume, is the average volume of
red cells. This parameter is useful in classifying anemias
into: Microcytic, normocytic, and macrocytic. MCV is calculated
from the hematocrit (HCT), and the Red Blood Cells Count (RBC
count).


HCT
MCV = x 10
RBC


The results of MCV are expressed in femtoliters (fl). 1 fl = 1 x
10
-15
L.
In automated hematology analyzers measure (not
calculating) MCV from the area under the RBC histogram, and
then calculating the HCT from MCV and Total RBC count.
MCV Normal Range:
80 96 fl
If results are less than 80 fl, the red cells are said to be
Microcytic:
a. Slight Microcytosis 75-79 fl
b. Moderate Microcytosis 70-74 fl
c. Marked Microcytosis <70 fl

If results are within 80-96 fl, the red cells are said to be
Normocytic.
If results are higher than 96 fl, the red cells are said to be
Macrocytic:
a. Slight Macrocytosis 96-105 fl
b. Moderate Macrocytosis 106-110 fl
c. Marked Macrocytosis > 110 fl
2- MCH
Mean Cell Hemoglobin, is the hemoglobin content in the
average red blood cell, or in other words, the average weight
of hemoglobin per RBC. It is calculated from the hemoglobin
concentration (Hb), and the total RBC count.


Hb g/dl
MCH = x 10
RBC

Results of MCH are expressed in picograms (pg). 1 pg = 1 g = 10
-
12
g.
MCH Normal Range:
27 32 pg
Macrocytic red cells have higher MCH, because they are
larger and contain more hemoglobin.
Microcytic red cells have lower MCH, because they are
smaller and contain less hemoglobin.
3- MCHC
Mean Cell Hemoglobin Concentration, is the average
hemoglobin concentration in 100 cc red blood cells. It
indicates the average weight of hemoglobin as compared to
the cell size. It correlates with the degree of hemoglobinization of

the red cells on the peripheral blood film. MCHC is calculated from
the hematocrit and hemoglobin.


Hb g/dl
MCHC = x 100
HCT

OR




Results of MCHC are expressed in percentage (%) or gm/dl.
Normal Range:
32 36 g/dl (%)
If results are within this range, it is said that red cells are
Normochromic.
If results are less than normal, red cells are said to be
Hypochromic, which is seen in microcytic hypochromic anemias
e.g. iron deficiency anemia.
Notes:
The only highly comparable red cell parameter between
automated cell counters and manual hematology tests is the
MCHC, because MCHC needs hemoglobin, and hematocrit in order
to calculate it , which are easy to perform manually with high
reproducibility and accuracy.
Red cells cant accommodate more than 37 g/dl of
hemoglobin, which is seen only in cases associated with
spherocytosis. Macrocytic anemias have normal MCHC. If you have
a case with high MCHC, and you checked the blood film and you
didnt find spherocytes, this may indicate an error in hemoglobin
MCH in picograms
MCHC =
MCV in femtoliters

and/or Hct. Since Hct is a calculated parameter, it is derived from
RBC and MCV, so may also indicate an error in RBC count and / or
MCV. Solutions to resolve this error include: retesting the
specimen, perform a spun microhematocrit, performing a manual
hemoglobin determination, and checking the quality control and other
patients results.
























Hematology Automated Analyzers nowadays can perform all of the
following:
1- Count RBC
2- Measure hemoglobin spectrophotometrically.
3- Directly measure MCV, from the area under the RBC histogram.
4- Calculate Hematocrit, which is derived from MCV and RBC count.
5- Calculate MCH, which is derived from Hb, and RBC count.
6- Calculate MCHC, which is derived from Hct, and Hb.

Preparation of Blood Films

Principle:
Blood film enables us to evaluate WBC, RBC, and PLT
morphology, also, allows us to perform differential WBC count,
furthermore estimation of WBC and platelets counts can be done on
blood films. Blood films are made on glass microscopic slides.
Sample:
Finger stick blood or EDTA anticoagulated venous whole
blood may be used. Films of peripheral blood must be made
immediately. Films may be made from EDTA anticoagulated
blood as long as two to three hours after collection. All specimens
should be free of clots.
Procedure:
1- Use clean standard size glass slides (3 inch x 1 inch =
7.5 cm x 2.5 cm), wiped from dust just immediately before use.
2- Place a small drop of well mixed anticoagulated whole
blood, in the center line of the slide, about 1.5 to 2 cm from one
end, with the aid of a capillary tube.
3- Immediately, without delay, with the aid of a second
clean slide with uniform smooth edges (spreader slide), with
a 30 40 degrees angle, move back so blood drop will spread
along the edge of the spreader slide, when this occurs, spread,
or smear the film by a quick, unhazizating, uniform forward
motion of the spreader.



Notes:
Before preparing the films, you must check that blood
samples are free from clots, and this is done with two wooden
applicator sticks. If clots are present the specimen is
unsatisfactory.
Films can be labeled with patients name and /or Lab. No. on
the thick end of the film itself, after being dried, by using a pencil.
With anemia (low Hct, reduced viscosity), the spreading
angle should be greater, to avoid running off the slide.
With polycythemia (high Hct, increased viscosity),the
spreading angle should be less, to avoid short, too thick films.
With large blood drops, increase the spreading angle.
With small blood drops, decrease the spreading angle.
If the anemia is too severe, let the blood specimen
settle, so that blood is divided into two layers, plasma layer
and red cell layer, then discard part of the plasma layer, then

mix the blood specimen, by doing this you have increased the
viscosity of blood, by this you will be able to prepare a nice blood
film.


















































DO NOT ATTEMPT TO CENTRIFUGE TO DISCARD
PLASMA, THIS MAY DISTORT AND DISINTEGRATE THE
CELLS, WHICH ARE OUR INTEREST!

Staining Blood Films
With Romanovsky Stains

Blood films are stained so that morphology of blood
cells become more easily viewed, identified, and evaluated. In
addition, blood films may be examined for the presence of blood
parasites (Malaria, Trypanosoma, Babesia). Furthermore, stained
blood films can provide important information about a patients
health, they may lead to a diagnosis or verify a diagnosis, or
they may rule out a diagnosis. Evaluation of stained blood films
also may lead to the decision of performing other hematology
special blood stain procedures in order to identify specific
cell components.
As soon blood films are air dried , it is best to stain them as
soon as possible. Blood films are stained with one of the
Romanovsky stains, which are universally used for staining blood
films. There remarkable property is creating distinctions in shades of
staining granules differentially and this is dependent on two
staining components: Azure B (the basic dye) and Eosin Y ( the
acidic dye). Other factors which affects the staining results include :
1) Staining time, 2) Ratio of Azure B to Eosin Y, 3) pH of the
staining solution . Azure B will stain the acidic cell Components
(e.g. nucleus, because it contains nucleic acids; basophilic
granules also take the Azure B staining because they contain
heparin, which is acidic in origin), while Eosin Y will stain the
alkaline basic components ( e.g. Eosinophilic granules in
eosinophils, because these granules contain spermine derivatives,
which are basic in origin). Red cells have affinity for acidic Eosin Y
dye, because it contain hemoglobin which is basic in origin.



Romanovsky stains include:
Giemsa Stain
Wrights Stain
Leishman Stain
May-Grnwald Stain
The widely and popular used Romanovsky stains are:
Leishman Stain
Wrights Stain
Leishman Stain Procedure:
1- Let the films be air dried.
2- Put the films on a staining trough rack.
3- Flood the slides with the stain.
4- After 2 minutes ( or more, if the stain in newly
prepared), add double volume of water, and blow to mix the
stain with water, until a shiny layer is seen.
5- After 5-7 minutes, wash with a stream of water.
6- Wipe the back of the slides with gauze.
7- Set the films in upright position on a filter paper to dry .
8- Read the blood films microscopically.

















If delay in staining blood films may occur, fix the films in absolute
methanol, for 1-2 minutes, but do not stain the slides until completely
dried.

Romanovsky Stain Blood Cell Characteristics
No. Cell Structure Staining characteristics
1 Red cells Red or pinkish red
2 Nuclei of all cell types Purple/violet
3 Lymphocyte cytoplasm Blue
4 Monocyte cytoplasm Grayish blue
5 Platelets cytoplasm Light blue
6 Neutrophilic granules Violet-pink
7 Eosinophilic granules Orange-red
8 Basophilic granules Purplish black/ Deep blue
9 Platelets granules Purple

Sources Of Errors In Staining
1- Stain Precipitate: May obscure cell details, and may cause
confusion with inclusion bodies. Filter the stain before use.
2- pH of the buffer or water:
Too acidic pH causes too pinkish slides.
Too basic pH causes too bluish slides.
3- Improper stain timing may result in faded staining or altered
colors:
Too long staining time causes too blue slides
(overstaining).
Too short staining time causes too red slides.
4- Forced drying may alter color intensities and/or distort cell
morphology.
5- Non-stain related errors:
1st- EDTA causes crenation of the cells after blood collection.
2nd- Severely anemic blood samples causes slower drying
(before staining) due to excessive plasma.

3rd- Old blood specimens may cause disintegration in WBCs
and decrease in their numbers.
4th- Collection of blood in heparin causes blue staining of
RBCs with bluish background, which makes heparin
unsatisfactory for routine hematology testing, also heparin
induces platelet aggregation and clumping , with
subsequent erroneous platelet count with automated counters.






































Always filter the stain before each use, to eliminate stain
precipitates.
Automatic stainers are available in the market, in which slides are
moved automatically and they are stained as they move.

WBC Differential Count

Principle:
Testing a Romanovsky stained blood films in order to
determine and assess the percentage of various classes of
WBCs present, and to assess red blood cells and platelets
morphology.
Increased or decreased normal WBCs class/ subpopulation
counts or the presence of immature precursors of WBCs or
RBCs in the peripheral blood film are of diagnostic importance
in various inflammatory and disease states.
Morphological red blood cells abnormality are important in
various anemias (spherocytes, sickle cells, acanthocytes, burr
cells, microcytes, macrocytes, target cells, .. etc).
Platelet morphology, distribution, and size abnormality are
suggesting a platelet disorder.
Sample:
EDTA anticoagulated whole venous blood film, bone
marrow film, and body fluid sediments (e.g. CSF).
Reagents, Supplies, and Apparatus:
1- Differential Tally Counter.
2- Conventional Binocular Microscope.
3- Oil immersion.
4- Well Stained Blood Film/s.
Procedure:
1- Focus the film under x10 lens, and scan the film to check cell
distribution.
2- Add a drop of oil, and move to the x100 oil immersion lens.
3- Choose a suitable area, where cells are evenly distributed
without appreciable overlapping- the monolayer cell zone.

4- Count the WBCs using tracking pattern.
5- Each cell identified should be immediately tallied as:
Neutrophil- segmented.
Neutrophil band
Lymphocyte
Monocyte
Eosinophil
Basophil
Immature cells: blast, promyelocyte, myelocyte, metamyelocyte,
promonocyte.
Variant atypical lymphocyte.
6- Morphological abnormalities of WBCs, RBCs and platelets
should be noted.
7- Nucleated red blood cell precursors (nucleated red blood cells-
NRBC) are not included in the differential count, but are counted per
100 WBCs, and if they are more than 10 NRBC/100 WBCs, a
corrected WBC count should be made (because as discussed
before that NRBCs are counted as WBCs, and will be included in
the total WBC count erroneously) by applying the following formula:


Uncorrected WBC X 100
Corrected WBC count =
NRBC + 100

NRBC is the number of NRBC seen per 100 WBC during
differential process.
8- Express the results as percentage for each cell class/
subpopulation.



! Count in the monolayer
zone

Most of abnormal / immature cells tend to be accumulated at the blood
film edges, do not forget to scan these areas. All nucleated red cells,
especially megaloblasts also tend to be accumulated at these edges.
Scan the blood film edges !!!

Method of Differential Counting Pattern- Tracking Pattern




Blood film made for WBC differential counting should be
evaluated for red cells. Red cells are evaluated for variation in
red cell volume/size, variation in shape, variation in staining
properties, alteration in distribution, presence of intracellular
inclusions and the presence of extracellular or intracellular parasites.
Blood film for WBC differential counting should be evaluated
for variation in platelet size (large, giant), presence of
megakaryocyte fragments, dwarf megakaryocytes, or
megakaryocytes. Also, platelet distribution(satellitism, aggregation,
clumping) which produce erroneous platelets count results with
impedance technology electronic counters, should be noted
(causing thrombocytopenia, and an increase in WBC count and/or
RBC count, and affects RBC and WBC histograms).
Blood film is evaluated for abnormal WBCs inclusions, toxic
granulation, Alder-Reilly granules , Chediak Higashi granules, Dhle
bodies, and toxic vacuolization. Immature WBC cells ( left shift)
should be included in the differential. Noting hypersegmentation (right
shift), and hyposegmentation (Pelger Huet anomaly, or
pseudopelger Huet cells). All of the above may indicate specific
disease process of acquired or inherited origin.


If total WBC count is high (more than 20 x 10
9
/L), a 200 or 300 cell
differential is advisable.

Normal Values for Differential WBC count in Adults:
Cell Type/ Population %Normal Differential Normal Absolute Count
*
x 10
9

Neutrophils- Segmented 50-70% 2.0-7.0 x 10
9
/L
Neutrophil- Band 0-10% 0.0-1.0 x 10
9
/L
Lymphocytes 15-45% 0.6-4.0 x 10
9
/L
Monocytes 0-10% 0.0-1.0 x 10
9
/L
Eosinophils 0-6% 0.2-0.7 x 10
9
/L
Basophils 0-1% 0.0-0.2 x 10
9
/L

*Absolute count is obtained by multiplying the % differential of
the cell type in concern by the total WBC count, obtained either by
manual or automated methods. automated CBC counters supply
us with this absolute count, for the main three cell types (i.e. the
neutrophils, the lymphocytes, and the monocytes). So, differential
count can be expressed as percentage and also as absolute count.
Automated Differential Counting
Nowadays, hematology laboratory is equipped with automated
hematology analyzers capable of automated differential counting,
which are more quicker, precise, and accurate than the manual
time consuming differential count, but this is true when the sample
contains normal cell populations. When the sample contains
abnormal or immature cell populations, your eyes are not
substituted. Although current laser cell counters identify abnormal
and immature cell populations in a sample, but this does not
substitute looking to a nice looking blood film to identify these
abnormal cell populations or subpopulation.
Clinical Significance Of Increased Normal WBC Counts:
Neutrophilia: Bacterial Infections, Inflammation , Stress, Chronic
Myelocytic Leukemia.
Lymphocytosis : Viral Infections, Whooping cough, Chronic
Lymphocytic Leukemia.

Monocytosis: Tuberculosis, Rheumatoid Arthritis, Pyrexia of
unknown origin.
Eosinophilia: Invasive parasite, Active Allergy, Myeloproliferative
diseases/disorders..
Basophilia: Ulcerative Colitis , Myeloproliferative Diseases,
Hyperlipidemia.
Blood Film WBC and Platelet Quantitative Estimation
Total WBC count can be quantitatively estimated from blood
films (under x 40 objective lens), also this estimation may be used
for quality control purposes, and as delta check for manual and
automated WBC counts, follow the table below for
estimating WBC count in blood films:
Number of WBC cells seen per x40 field Estimated total WBC Count
2 4 4,000 6,000 /cumm
4 6 6,000 10,000 /cumm
6 10 10,000 13,000 /cumm
10 13 13,000 20,000 /cumm

Also WBC count can be estimated quantitatively from blood film,
by the following formula : Average number of nucleated cells per
field at x 100 magnification = nucleated cell count x 10
9
/L.
Platelets can be estimated quantitatively from blood films, each
platelet seen under oil immersion lens approximately equals to
20,000 PLT/cumm. Normally, blood film from healthy individuals
usually shows 7-22 platelets per oil immersion field. When platelet
aggregates or clumps are present in the blood film, then platelet
estimation would be absolutely unreliable.





Differential Tally Counter