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)
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
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 RBC’s, 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 can’t
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
Total WBC Count / cumm = = 3500 / cumm = 3.5 x 109/L
9
Reference Range
Adults : 4.5 – 11.0 x 109 /L
Six years: 4.5 – 12.0 x 109 /L
One year: 6.0 – 14.0 x 109 /L
Newborn: 9.0 – 30.0 x 109 /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 WBC’s.
Hemacytometer Squares
5- Presence of clumped WBC’s.
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 RBC’s, 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 RBC’s.
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 (mm²)
Where:
N= Total number of red cells counted in the counting
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 WBC’s 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 anemia’s,
spherocytosis, hypochromic anemia’s, 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 anemia’s, 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 anemia’s 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 anemia’s
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 can’t 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
didn’t 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 patient’s 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
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