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Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE
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Hematology Handbook
Reference &
Automation Methods FIRST EDITION
Mohammed H. Saiem Aldahr, BSc (MT), MSc, PhD
Assistant Professor, Department of Laboratory Technology
Vice Dean for Clinical Affairs.
Vice Dean for postgraduate studies and scientific research. Faculty of Applied Medical Sciences, King Abdulaziz University
Jeddah KSA
Hashem A. Abu-Hurirah, BSc (MT), MSc, PhD fellow
Application Manager Of Hematology Analyzer
Application Manager Of Fluorescent Flow Cytometry Urine Analyzer
PhD Fellow, Faculty of Science
Faculty Of Allied Medical Technology Assistant Research King Abdulaziz University, Jeddah, KSA
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Edited by:
Bilal Khalaf, BSc Engineering, MBA
20 years of experience in Business Development,
Operational Excellence (LEAN/6 Sigma), Marketing,
Event Management and Training in Distribution,
Healthcare, Education, Hardware and Software
Industries.
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To my late father, mother, wife and
children, Heba, Osama, Ibrahim and
Yousef for their support and
encouragement.
Hashem
Jeddah 2011
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Preface Technology is on the rise and growing daily demands
generate the need for instruments of this nature in the laboratory.
They constitute a giant leap forward in the laboratory field, given
the possibility of undertaking tests in a reliable, accurate, and
precise manner, and delivering results in a shorter time period and
under better control.
The advantages of automation are numerous. Technology is
continuously advancing to meet new developments in the field and
to reduce turnaround times, allowing tests to be reliable, accurate,
and precise, while maintaining quality.
This work is done to give a user whole idea about the high
end technology used in automated analyzer and reference methods
to understand and be aware about the difference between automated
principles and reference methodology.
This handbook can be as a key to know the reference
methods in laboratories, for medical applied students, laboratory
technicians, technologist, medical technology students and
laboratories trainees as well.
This project is harvest of more than twenty years of
educational experience in hematology field, in addition to more
than fifteen years of study and field experience in hematology as
student, technologist, sales, then marketing, application and
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training in hematology field with different hematology companies
merged to give useful and comprehensive information for the users
of this hand book.
The authors of this Hematology Handbook Reference &
Automated Methods hope that the publication of this handbook will
help technologists, technicians, and laboratories trainees in their
assessment to be reference for blood counting procedures. This
handbook has been compiled to help aid those involved to work in
the field of medical laboratories, to skillfully assess the procedures
and methodology which are used in hematology department and
analyzers.
INTENDED AUDIENCE
This text is designed for medical applied students,
laboratory technicians, technologist, medical technology students
and laboratory trainees to get excellent idea about the high end
technology used in automated analyzer and reference methods to
understand and be aware of the differences between automated
principles and reference methodology.
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ACKNOWLEDGEMENTS
I would like to acknowledge the work of Mr. Bilal Khalaf
for his significant contribution in the design, editing and layout of
the Hematology Handbook Reference & Automated Methods.
I would also like to thank the entire Hematology
Department team at King Abdulaziz University Hospital and Mr.
Waleed Nasruddin for his scientific support in this project.
Many thanks for my friends and colleagues Mohammed
Moghrabi, Baha Bodyab, Mohammed Damati, Khaldoon
Kasrawee & Jana Stark. Special thanks are due to Ms. Vicky
Medina for her excellent secretarial help.
Hashem
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Contents
Preface …..…………………………..…………………….. IV
1. Introduction …..…………………………………………..………..... 9
2. The Blood …..…………………………………….………………..... 11
3. Blood Collection.…..………………………………….……………… 14
3.1. Capillary Blood…..…………..…………………………………. 14
3.2. Venous Blood …..……………………………………………….. 15
3.3. Use Of Anticoagulants...………………….................…............. 17
4. Blood Cell Counting …..…………………………….....……………… 19
4.1. Red Blood Cell Counting.……………….………………..….... 19
4.2. White Blood Cells (WBCs) Counting.…….……..………...... 28
4.3. Platelets Count…………………………….………….….…….. 41
4.4. Reticulocytes Count.….……………………..………............. 44
5. Automated Nucleated RBC Analysis.……………..........………….. 48
6. Blood Film Examination.………………………………..…….……… 49
6.1. Preparation Of A Thin Blood Film..………………………….. 49
6.2. Preparation Of A Thick Blood Film.…………...................... 50
6.3. Staining Of Thin Blood Film With Wright’s Stain…............ 50
6.4. Automated Digital Morphology…………………………... … 53
6.5. Test For Malaria……………………………………………....…. 54
6.6. Test For Leishmania…………………………………………….. 55
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7. Hemoglobin Determination……………..……………………………. 57
7.1. Cyanmethemoglobin Method…………………………………. 57
7.2. Hemoglobinometer……………………………………………. 62
8. Hematocrit……………………………………………………………. 64
9. Erythrocyte Sedimentation Rate (ESR)…………………………..… 67
10. Sickle Cell Test…………………………………………………….……. 72
11. Coagulation……………………………………………………….…..… 74
11.1. Bleeding Time……………………………………………………. 74
11.2. Prothrombin Time…………………………………..…………… 77
11.3. Activated Partial Thromboplastin Time……..………………. 79
11.4. New Method Used In Semi-Automated & Full
Automated Methods…………………………….………………….. 82
12. ABO And Rh Grouping……………………………..…………….… 89
13. Anti-Human Globulin (AHG) …………………………………….… 103
14. Atlas Of Hematology…………………………………..…………..… 115
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Introduction Hematology Handbook
Reference & Automation
Methods
Hematology is defined as the study of blood (blood forming
tissues) and its components. Because of its critical role in
maintaining life, blood has been referred to by some as “the river of
life”. Additionally, because routine examination of the blood by
means of the complete blood count (CBC) is the most widely
performed test in the clinical laboratory and has also been referred
to as “a window on the body”.
Blood is one of the most complex organ systems in the
human body. The key parts that make up the hematological system
are the blood, bone marrow, spleen, and lymph system. In the adult,
blood consists of approximately 55% plasma (liquid component)
and 45% formed elements including: erythrocytes (red blood cells –
RBC), Leukocytes (white blood cells – WBC), and (thrombocytes -
platelets). Blood makes up about 7% of the human body weight.
Blood is formed from hematopoietic stem cells in the bone marrow.
Blood, as a whole, is responsible for the most important functions
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of life, such as the transport of metabolic components, nutrients,
hormones, gas exchange, the immune defense, and coagulation.
The science of hematology literally is part of the evaluation
of all disease states. Blood, the fluid that nourishes and cleanses the
body, has been at the center of interest and investigation from early
humans to the field of modern science it has become today.
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2. Blood Cells and Platelets
The blood is composed of three types of formed elements:
erythrocytes (red blood cells), leukocytes (white blood cells)
and platelets (thrombocytes).
Erythrocytes: also called red blood cells (RBCs) are the
body's most numerous blood cells. Average normal values of
RBCs in the blood is about 4.60 x 106/μL (4.60 million/ μL) for
female and 5.20 x 106/μL (5.20 million/μL) for male. RBCs
play a major role in oxygen and carbon dioxide transportation
through the circulation, as every cell contains approximately
280 million hemoglobin molecules and each molecule is
attached with four iron atoms, which in turn combine with
oxygen molecules by means of RBCs passing through the lung.
Following this, RBCs travel through the circulation to the
tissues where iron atoms release the attached oxygen into
interstitial fluid and hemoglobin molecules take up carbon
dioxide back to the lungs. This cycle keeps on repeating to
maintain the body function and free the circulation from the
harmful waste.
Leukocytes: known as white blood cells (WBCs) and their
function is defined in body defense mechanism against foreign
agents and infections. Average normal range in the blood is
between 4,500 to 11,000 cells per μL. This value may be
reported as 4.5-11.0 x 103/μL or k/μL (k = thousand). Unlike
RBCs, WBCs are differentiated in different types. Most WBCs
are filled with tiny grains and are called granulocytes (gran =
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grain). The normal range for granulocytes is 1.8-8.5 x 103/μL of
blood. These granulocytes are subdivided into the following:
Neutrophils: 50%-70% of total WBCs or about 1.8-7.7 x 10
3/μL
Eosinophils: up to 5% of total WBCs or about 0.0-0.450 x
103/μL
Basophils: up to 2% of total WBCs or about 0.0-0.2 x 10
3/μL
Another cell lineage is known as lymphocytes and
monocytes which are classified as a granulocyte (non-
granulocytic type of cells) as follows:
Lymphocytes: 20%-47% of total WBCs or about 1.0 - 4.8 x 10
3/μL
Monocytes: 3%-10% of total WBCs or about 0.0 - 0.8 x 10
3/μL
WBCs fight infection; some surround and destroy debris
and foreign invaders, while others produce antigen/antibody
reactions. An antigen is any substance (foreign or part of the
body) which causes stimulation of antibodies production;
subsequently, these antibodies neutralize antigens and such
phenomenon is known as immunological humeral response.
Important Considerations:
The average adult circulation contains five liters of blood (roughly 5.28 quarts).
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Blood completes the entire systemic circuit – from the left side of heart through the body to the right side of the heart –
in 90 seconds.
Every cubic millimeter of blood contains five millions of
RBCs.
RBCs survive about 4 months and neutrophils survive about 6 hours in the blood stream.
Platelets: Known as (thrombocytes), they are cell fragments
that travel in the bloodstream. The normal range for platelets
count is 140 - 440 x 103/μL. Platelets prevent blood and fluid
loss by clumping together to begin the coagulation process. A
blood clot is formed when sticky platelets become covered with
fibrin (a plasma protein that holds the blood clot together).
Each of the formed elements of blood will be covered in
more details in the following sections.
Hematopoiesis: The Formation of Blood Cells
All blood cells begin as undifferentiated stem cells capable
of reproducing themselves. Generations of cells eventually
differentiate into cell lines that will mature to produce
erythrocytes, leukocytes, and platelets. Stem cells in bone
marrow continuously proliferate – usually at a steady state to
maintain a constant population of mature blood cells. A
disruption in this process can lead to serious illnesses.
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As a group, immature cells are large. As they age and
mature, they become smaller and change in their reaction to the
dyes used to stain them for identification.
Role of the Spleen: Located beneath the diaphragm and
behind the stomach, the spleen is an intricate filter that receives
5% of the total blood volume each minute. In the embryo, the
spleen is a blood-forming center; it loses this function as the
fetus matures.
In the spleen, RBCs and WBCs are "inspected" by
specialized WBCs called “macrophages” (disease fighting cells)
which act to filter blood. Old or damaged cells are removed;
salvageable cells may be "pitted", that is, unwanted particles are
removed without destroying the cells.
3. Blood Collection
3.1 Capillary Blood
Capillary blood is obtained from the tip of a finger in adults
and from the great toe or the heel in infants. The area where the
blood is to be collected must be cleaned with 70% alcohol, dry
with sterile gauze, and puncture the skin with a sterile
disposable blood lancet that is designed to penetrate no deeper
than 2mm. Use a sterile gauze to wipe out the area for blood
collection since it will alter the composition of blood
specimens. If blood collection is difficult, warm or allow it to
remain in hanging position for some time.
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Advantages:
a. Capillary blood can be obtained with ease.
b. Capillary blood is the preferred material for making
peripheral blood smear.
Disadvantages:
a. Only a small specimen can be obtained and repeated
examinations require new specimens.
b. Blood in micro-tubes frequently haemolyses, resulting
in interferences with most laboratory tests.
c. Test results on capillary blood cannot be compared with
test results in venous blood.
d. The finger is not only sensitive but difficult to
adequately sterilize in the time usually available. In
patients with lowered resistance to infection, a specimen
taken from the finger by capillary is much likely to lead
to infection than one taken from the veins.
e. Red and white cell counts and enumeration of platelets
and Reticulocytes should not be performed on capillary
blood because of the difficulty in standardizing capillary
blood flow.
3.2 Venous Blood
Venous blood is mandatory for most tests that
require anticoagulation or larger volume of blood, plasma or
serum that can be provided by capillary blood. Although
other veins can be chosen, venous blood is usually obtained
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from one of the capital fossa veins. The vein is congested
by placing a tourniquet on the upper arm and tightens it
sufficiently to retain venous blood and prevent it from
return. Following this, the arm must be disinfected
thoroughly using (70% Ethyl Alcohol), once the blood is
started to rush out from the vein into the collecting tube,
loosen the tourniquet and obtain blood by gentle suction.
At the end, use sterile gauze to apply over the puncture site.
Remove the needle from the syringe, and quickly transfer
blood into a test tube which may or may not contain
anticoagulant. If an anticoagulant has been added, mix
blood gently by inverting the stoppered tube several times.
Advantages:
a. Multiple and repeated examinations can be performed
on the same specimen.
b. Aliquots of the specimen may be frozen for future
reference.
c. There is no variation in blood values, if specimens are
collected from different veins.
Disadvantages:
a. The venous method is somewhat lengthy procedure that
requires more preparation than the capillary method.
b. Prolonged stasis produced by the tourniquet must be
avoided, because it results in haemoconcentration.
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3.3 Use of Anticoagulants
Anticoagulant:
Anticoagulant is a substance that prevents coagulation;
(stops blood from clotting). A group of pharmaceuticals
called anticoagulants can be used in vivo as a medication for
thrombotic disorders. Some chemical compounds are used
in vitro such as; Ethylenediamine Tetra-Acetic (EDTA),
Trisodium Citrate, Ammonium Oxalate, Potassium Oxalate
and Heparin.
a. Ethylenediamine Tetra-Acetic Acid (EDTA)
It is a sodium or potassium salt of Ethylenediamine
Tetra-Acetic Acid which prevents the blood from
clotting by removing and binding calcium. EDTA can
be used for both hematology and some chemistry tests.
It is considered an excellent anticoagulant and it is
widely used. The amount of EDTA required for 10cc or
less of blood is 10mg of the dry powder or 0.1cc of a
10% solution. The EDTA in the test tube is not
necessarily to be evaporated; it may be used in liquid
form. The following Table discussed the time of blood
collected in EDTA.
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Table 3.1: Approximate keeping time for blood anti-
coagulated with EDTA
Test Keeping Time
White Blood Cell Count 24 hr
Red Blood Cell Count 24 hr
Hemoglobin Determination 1 hr
Stained Red Cell Examination 1 hr
Sedimentation Rate 2 hr
Hematocrit Reading 24 hr
Reticulocyte Count 1 hr
Red Blood Cell Indices 3 hr
Platelets Count 1 hr
Blood Grouping 48 hr
Differential White Cell Count 1hr
b. Trisodium Citrate
Trisodium citrate prevents the blood from clotting by
removing and binding calcium. The ratio of blood to
sodium citrate solution is 9:1 i.e. for 9ml blood add 1ml
sodium citrate solution for coagulation tests 4:1 for ESR
estimation.
c. Mixture of Ammonium Oxalate and Potassium
Oxalate
This mixture of oxalate salts prevents the blood from
clotting by removing and precipitating calcium. This
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anticoagulant should not be used in chemistry tests such
as Urea and Potassium.
d. Heparin
Heparin prevents the blood from clotting by neutralizing
thrombin. It is used at a concentration of 10-20 IU/ ml
of blood. Heparin is the best anticoagulant to use for
osmotic fragility tests. However, heparinized blood
should not be used for making blood films.
4. Blood Cell Counting:
RBC count and indices (MCV, MCH, MCHC)
4.1 : (RBC) Count Reference and Automated Methods
Reference Method
Measurement of the total number of circulatory RBCs is
important because the number of RBCs and the amount
of hemoglobin, they contain, must remain within certain
limits to maintain good health.
The main function of the red cell is to carry oxygen from
the lungs to the body tissue and to transfer carbon
dioxide from the tissue to the lungs. The process is
achieved by the (hemoglobin) found in the red cells that
combine easily with oxygen and carbon dioxide.
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Equipment:
a. 10ul automatic pipette or Red cell pipette (Figure 4.1).
The red cell diluting pipette contains a red bead in the
bulb and has ten divisions on the capillary end, with
points 0.5 and 1.0 numbered. If the blood
is drawn to the 0.5 mark and the pipette filled with
diluting fluid, the resultant dilution is 0.5:100 or 1:200,
the dilution used in routine counting. One volume of
diluting fluid remains in the stem does not enter the
bulb, is blown out first the counting chamber filled and
therefore does not dilute the blood.
b. Counting Chamber (Haemocytometer).
The most commonly used method employs the
Improved Neubauer counting chamber (Figure 4.2).
There are two Chambers per Haemocytometer,
each consists of nine large squares and measuring about
1mm2 as demonstrated in (Figure 4.2).
Reagent:
Diluting Fluid
Trisodium citrate 3g
Concentrated formaldehyde (37%) 1ml
Distilled water 100ml
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Procedure:
1. Add 10ul of the patient whole blood sample to 2ml of
red cell solution. If red cell pipette is used, draw blood
to 0.5 marks and complete with red cell solution to 101
marks.
2. Mix well the diluted blood (The dilution of blood is
1:200).
3. Discard the first 5 drops before charging the counting
chamber, if red cell pipette is used.
4. Fill the counting chamber and be careful not to overfill
beyond the ruled area, and check if air bubbles present.
5. Place the chamber on the stage of the microscope and
use 40X objectives.
6. Count the cells in five squares of red cell count area (R)
as shown in the figure 4.3.
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Figure 4.1: Explanation of dilution of blood in red
and white blood cells counting pipettes.
Figure 4.2: Improved Neubauer haemocytometer
counting chamber ruling.
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Figure 4.3: Demonstrates the Counting chamber rulings and
dimensions. Haemocytometer of counting chamber has two ruled
areas etched on its surface, each consisting of a 3mm square
divided into nine large squares (W) and each measure 1mm2.
Center square, which is used for red cell counting, is subdivided
into 25 smaller squares (R), each occupying an area of 0.04 mm2.
Red cells in the five squares are enumerated. The depth of the
chamber is 0.1mm and the four large corner squares are subdivided
into 16 smaller squares and are used for leucocyte counting. Each
measures 1mm2 in area.
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Figure 4.4: Improved Neubauer ruling for one counting chamber.
White cell count is done on the four large corner squares (1, 2, 3
and 4) of each of two counting chambers.
Calculation:
Each “R” section (Fig. 4.2) has an area of 0.04mm2 and depth of
0.1mm. The volume of 1 “R” is found as follows:
Area of 1 “R” x Depth of 1 “R” = Vol. of 1 “R”
0.04mm2 x 0.1mm = 0.004mm
3
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Vol. Correction Factor = = 50
Vol. Correction Factor =
Number of “R” sections x Vol. of 1 “R” = total Vol.
5 x 00.004 = 0.02mm3
Vol. Desired
Vol. Used
1.0mm3
0.02mm3
Red Cell Count = No. of cells in 5 “R” x dil. factor x Vol.
Correction (Per cu.mm)
= Number of cells x 200 x 50
= Number of cells x 10000
Expected Range
Males 4.5-5.5 x 106/cu.mm
Females 4.0-5.0 x 106/cu.mm
Children 4.2-5.2 x 106/cu.mm
Red Cell Indices:
i. Mean Cell Volume (MCV)
The mean cell volume is the volume of average erythrocyte.
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MCV =
Normal range: 76-96 fl
PCV x 10
RBC Count in Million
ii. Mean Cell Hemoglobin (MCH)
This is a calculation of the ratio of hemoglobin to the
erythrocyte count.
The formula for the calculation is:
MCH = Hb (g/dL)/RBC (x 106/μL) x 10
iii. Mean Cell Hemoglobin Concentration (MCHC)
This formula is the calculation of the ratio of hemoglobin to
hematocrit that indicates the concentration of hemoglobin in the
average red cell. The calculation is:
MCHC = [Hb (g/dL)/Hct (%)] x 100
Automated Method
Direct Current (DC) Technology:
Also it is known as Impedance counting or Coulter Principle
(Electrical Sensing Zone Method) has become the accepted
"Reference Method" throughout the world for particle size
analysis and is the recommended limit test for particulate matter
in large-volume parenteral solutions.
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This method of sizing and counting particles is based on
measurable changes in electrical resistance produced by
nonconductive particles suspended in an electrolyte.
A small opening (aperture) between electrodes is the sensing
zone through which suspended particles pass. In the sensing
zone each particle displaces its own volume of electrolyte
(Figure 4.5).
Volume displaced is measured as a voltage pulse; the height of
each pulse being proportional to the volume of the particle.
The quantity of suspension drawn through the aperture is
precisely controlled to allow the system to count and size
particles for an exact reproducible volume. Several thousand
particles per second are individually counted and sized with
great accuracy. This method is independent of particle shape,
color and density.
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Figure 4.5: Explanation of Direct current small
opening (aperture).
4.2 White Blood Cell (WBC) Count
Reference Method
Measurement of the total number of circulating white blood
cells (WBCs) is an important procedure in the diagnosis and
prognosis of the disease process. The main function of
leucocytes is to fight infection by phagocytosis and
production of antibodies.
Since leucocytes are affected by so many diseases, the
leucocyte count serves as a useful guide to the severity of
the disease process. The increase of white blood cells above
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10x109/L is called leucocytosis and the decrease of white
blood cells below 4x109/L is called leucopenia.
Reagents
Diluting Fluid:
Acetic Acid (Glacial) 4ml
Distilled Water 200ml
Aqueous methylene blue (0.3gm %) 20 drops
Procedure
1. Add 50ul of whole blood sample to 0.95ml of diluting
fluid into a labeled test tube. If you use white cell
pipette is being used (Fig. 4.3), draw blood to 0.5 marks
and continue with the diluting fluid to 11 marks.
2. Shake well (The dilution of the blood is 1 in 20).
3. Prepare the counting chamber and attach the cover glass
by pressing it carefully into place.
4. Discard first 4 drops if you are using white cell pipette.
5. Fill the counting chamber (take care not to overfill
beyond the ruled area).
6. Allow the cells to settle for 3 minutes.
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Vol. correction = = 2.5
7. Place the chamber on the stage of the microscope, use
the 10X objective, and reduce the light by lowering the
condenser.
8. Count WBC in 4 big (W) squares at the corners of the
counting chamber (see Fig IV.2).
Making the Calculations
Each “W” section (Fig. IV.2) has an area of and depth of
0.1mm.
The volume of “W” = 1mm2 x 0.1 = 0.1 cu.mm
The volume = number of “W” x volume of “W”
1 cu.mm
0.4 cu.mm
Number of WBCs = Number of cells in 4 “W” (0.4 cu.mm)
x dilution
Fac. x correction factor
Number of WBCs = Number of cells in 4 “W” (0.4 cu.mm)
x 20 x 2.5
Expected Range
3 - 10 x 103/cu.mm
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Common Sources of Error
a. Failure to have required blood volume.
b. Failure to mix well.
c. Failure to discard the first 4 drops.
d. Failure to properly charge the counting chamber.
The least Frequent Sources of Error are
a. Inaccurate pipette or counting chamber.
b. Moist or unclean pipette.
c. Excessive pressure in finger when obtaining the blood.
d. Excessive or insufficient dilution of the fluid.
e. Slowness in manipulation, thus, allowing the blood to
clot.
f. Air bubbles in the pipettes.
g. Air bubbles in the counting chamber.
h. Presence of yeast or other contaminants in the diluting
fluid.
i. Presence of many nucleated red cells causing a high
white cell count.
j. Miss counting or calculations.
Correction for Nucleated Red Cells
In some anemia, such as thalassaemia and erythroblastosis
fetalis, many nucleated red cells may be found in the blood.
Since these nucleated red cells are not dissolved by the
white cell diluting fluid, they are counted as white cells.
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Corrected WBC = Uncorrected WBC x
This would result in an erroneously high white cell count.
Consequently, the count must be corrected.
If large numbers of nucleated cells are found in the stained
red cell examination, correction of the white cell counts
must be done as shown below:
100
100 + A
Where:
100 = White cells counted in the differential white cell count.
A = Number of nucleated red cells counted while counting the
100 white cells of the differential count.
Automated Method
There are more than one way to count WBCs with different
Techniques, these techniques are established and applied by
different companies.
a. Using Radio Frequency (RF) and Direct Current (DC) to
enumerate and identify WBC populations. The RF
method detects and sizes lyse-treated cells based on
density and nuclear size, whereas the DC method sizes the
entire cell, nucleus, and cytoplasm. Cells pulses detected
by RF and DC methods are then displayed as a 3-
dimensional WBC scatter gram which contains
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information about the distribution of lymphocytes,
monocytes and granulocytes. Separate histograms are
generated for basophil and eosinophil populations.
b. The best way to get the maximum accuracy is counting of
WBCs by different channels using of multichannel method
especially in abnormal samples.
Counting of total WBCs differential by using:
1. Size /volume of cells
DC detection method
Laser light scatter
2. Internal structure (density or complexity), e.g. granulation
Flow cytometry with laser light scatter
3. Nucleic acid content (RNA and DNA)
Fluorescence Flow Cytometry with laser light scatter
By using this method, the Size of blood cells is not relevant
in the Differential counting channel. Debris do not interfere,
also cells without nucleic acids such as mature Erythrocytes
are not stained, no interference by lyse-resistant RBC, and
non-cellular particles such as air bubbles are not detected.
(Figure 4.7)
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Figure 4.6: Explanation of optical system (Laser light scatter)
Figure 4.7: Explanation of side fluorescence, side scattered
and forward scattered light in laser flow cytometry
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c. Volume Conductivity and Light Scatter (VCS), performs
a five-part differential consisting of monocyte,
lymphocyte, granulocyte, eosinophil and basophil.
Established WBC differential technology uses three
measurements: individual cell volume, high-frequency
conductivity and laser-light scatter as explained below:
Volume Analysis - Electronic leukocyte volume analysis
uses a low-frequency current measure volume of the
WBCs as they impede the current when they pass through
the aperture.
Conductivity - Cell walls act as conductors to high-
frequency current. As the current passes through the
walls and through each cell interior, it detects
differences in the insulating properties of cell
components.
It characterizes the nuclear and granular constituents
and the chemical composition of the cell interior.
Scatter - Leukocytes are hydrodynamically focused and
passed in a steady stream through a sensing zone on
which a laser light is focused. As each cell passes
through the sensing zone of the flow cell, it scatters and
reflects the focused light which is detected by a
photodetector. The patterns of scatter are measure at
various angles (forward scatter at 180 degrees. and right
angle scatter at 90 degrees). Scattered light provides
information about cell structure, shape and reflectivity.
The characteristics are used to differentiate the various
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types of WBCs and to produce scatter plots with a five-
part differential.
d. By using of optical flow cytometry, cytochemistry and
light scattering and staining with peroxidase technologies
to enumerate and identify cells.
WBCs are fixed with formaldehyde and stained with
peroxidase in the peroxidase reaction chamber. The high
heat in the chamber lyses platelets and RBCs and causes
the WBCs to be fixed and dehydrated. Forward-angle
light scatter and tungsten light optics are used to measure
WBC size and peroxidase activity. Myeloperoxidase is a
granulocyte enzyme marker that is present in varying
degrees in neutrophils, eosinophils and monocytes but is
absent from basophils, lymphocytes and blasts. A
specific basophil count is determined separately in the
basophil/lobularity chamber. Whole blood is exposed to
an acid buffer that selectively lyses all cells except
basophils. The resulting particles are sorted and
quantified by measuring the forward angle and light
scattering properties. An additional category of cells is
reported as LUC (large unstained cells). This category
reflects atypical lymphocytes or blasts.
e. System of Multiangle Polarized Scatter Separation
(MAPSS) flow cytometry with hydrodynamic focusing of
the cell stream. The leukocyte differential is
accomplished by light scatter. It features three
independent measurements and focused flow impedance.
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Multidimensional light scatter and fluorescent detection
are used as well.
DIFFERENTIAL WBC AUTOMATED COUNTERS
3-Parts differential counting machines
Small automated machine with 8, 12, 16 or 18 parameters
screening machine for small, satellite laboratories,
emergency rooms, etc. some manufacturers count
monocyte, lymphocyte and mixed (granulocyte;
neutrophile, basophile and esinophile), but the others count
lymphocyte, neutrophile, and mixed (monocyte, basophile
and esinophile) to get an idea for the patient's status (viral or
bacterial infection).
5 Parts differential counting machines
Medium and large fully automated machine with up to 80
parameters, consider as a diagnostic machine for hospitals.
Immature granulocyte (IG):
Immature granulocyte (IG) which includes: myelocyte,
promyelocyte and metamyelocyte, recently has been
considered as the 6th
part differential in differential
counting.
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Figure 4.8: Explanation of the 5 part differential WBCs
find in differential BC count and band cells
Neutrophilic Segmented Cell
Size: medium
Nucleus: broken up into segments
Cytoplasm: contains small pink or
brownish granules
Comments: May be confused with
a neutrophilic band cell. When in
doubt, call cell a neutrophilic
segmented cell.
When found: 55 to 75% in normal
blood; increased in appendicitis,
pneumonia.
Neutrophilic Band Cell
Size: medium
Nucleus: shaped like a band
Cytoplasm: contains small pink or
brownish granules
Comments: May be confused with
neutrophilic segmented cell. When
in doubt, call cell a neutrophilc
segmented cell.
When found: 2 to 6% in normal
blood; increased in appendicitis,
pneumonia.
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Eosinophilic Segmented Cell
Size: medium
Nucleus: usually has 2 lobes or
segments
Cytoplasm: contains large red
granules
Comments: Eosinophilic
segmented cell has large red
granules whereas neutorphilic
segmented cell as small pink or
brownish granules
When found: 1 to 3% I normal
blood: increased in asthma, hay
fever, etc.
Basophilic Segmented Cell
Size: small or medium
Nucleus: usually indistinct; appears
buried under large purple or purplish-
black granules
Cytoplasm: contains large purple or
purplish-black granules
Comments: Easily identified by the
large purple or purplish-back
granules scattered throug hout the
cell.
When found: 0 to 1% in normal
blood.
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Lymphocyte
Size: small, medium, or large
Nucleus: closely knit and usually
round
Cytoplasm: light blue; may
contain a few reddish granules,
cytoplasm may be sparse and even
absent in some small lymphocytes
Comments: Large lymphocyte
may be confused with monocyte.
Nucleus of large lymphocyte is
closely knit and usually round.
Nucleus of monocyte is spongy
and sprawling.
When found: 20 to 35% in normal
blood; increased in infectious
monocleusis, lymphocytic
leukemia, and many other
diseases.
Monocyte
Size: large
Nucleus: spongy and sprawling
Cytoplasm: light grey; may contain
very tiny reddish granules
Comments: Monocyte may be
confused with large lymphocyte.
Nucleus of monocyte is sponsry and
sprawling. Nucleus of lymphocyte is
closely knit and usually round
When found: 2 to 6% in normal
blood; increase in tuberculosis and
monocyte leukemia.
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4.3 Platelet Counts
Reference Method
Platelets are the smallest elements in the blood. These cells are
non-nucleated round or oval shaped cells. Platelets activity is
necessary for blood clotting therefore, deficiency in these cells
will lead to prolonged bleeding time. The life span of a platelet
is approximately 5-7 days. Abnormally increased number of
platelets occurs in cancer, splenectomy, iron deficiency anemia,
and cirrhosis, while abnormally decreased number of platelets
occurs in idiopathic thrombocytopenic purpura (ITP),
pneumonia, allergic conditions, infections and toxic effects of
certain drugs.
Reagents
Diluting fluid (1% Ammonium Oxalate)
Ammonium oxalate 1g
Distilled water 100ml
Procedure
a. Pipette 0.95ml of diluting fluid into a labeled test tube.
b. Add 50ul (0.05ml) of the patient whole blood sample.
c. Shake well. The dilution of the blood is (1) in (20).
d. Fill one side of a Neubauer counting chamber and allow
the platelets to settle for 10-20 minutes in a moist petri-
dish.
e. Count the same area as for red cell count.
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The platelets will appear as small refractile bodies under the
x 40 objective under microscope.
Calculation
Platelet Count = No. of cells in 5 “R” x dil. Fac x fac Vol. Corr (per
cu.mm) (Figure IV.2):
= No. of cells x 20 x 50
Expected Range
150 - 400 x 103/cu.mm
Automated Method
The four main procedures for platelet counting are: manual
phase contrast microscopy, impedance, optical light
scatter/fluorescence and flow cytometry. Early methods to
enumerate platelets were inaccurate and irreproducible. The
manual count is still recognized as the gold standard or reference
method, and until very recently the calibration of platelet counts
by the manufacturers of automated cell counters and quality
control material was performed by this method, impedance
counts still have limitations as cell size analysis cannot
discriminate platelets from other similar-sized particles. More
recently, light scatter or fluorescence methods have been
introduced for automated platelets counting (Fluorescence-
optical: PLT-o = RNA content), fluorescent stain is used to
detect the residual amount of RNA (Figure 4.10), but there are
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still occasional cases where an accurate platelets count remains a
challenge. Thus, there has been interest in the development of an
improved reference procedure to enable optimization of
automated platelet counting (Figure 4.10).
Monoclonal antibodies to platelet cell surface antigens
conjugated to a suitable fluorophore is costly method, permits
the possible implementation of a new reference method to
calibrate cell counters, assign values to calibrators, and to obtain
a direct platelets count on a variety of pathological samples. In
future, analyzers may introduce additional platelet parameters; a
reliable method to quantify immature or reticulated platelets
would be useful.
Figure 4.9: Explanations one of the procedures used in
PLT counting by using Tungsten Lamp to detect light
absorbance and light scatter
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Figure 4.10: Explanations the detection the residual amount of
RNA in platelets
4.4 Reticulocytes Count
Reticulocytes are juvenile red cells that contain fine, deep violet
granules (remnants of the ribosomes and the ribonucleic acid
present in the precursor cell) arranged in a network.
Principle
Reticulocytes are immature red cells that pass into the blood
stream from the bone marrow. The number of reticulocytes
in the blood indicates the degree of activity of the bone
marrow. The number increases when the marrow is very
active.
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X 100
Reference Method
Reagent
(Saturated Solution of Brilliant Cresyl Blue)
Brilliant cresyl blue 1.0g
Trisodium citrate 0.4g
Sodium chloride solution (0.85%) 100ml
Procedure
a. Filter a small amount of the cresyl blue solution into a
test tube.
b. Add equal quantity of venous blood collected in EDTA
di-potassium salt.
c. Mix gently and leave for 10 minutes at 37°C or 15
minutes at room temperature.
d. Shake the tube and make a thin smear of the mixture.
e. Examine the smear using the oil-immersion objective in
microscope.
f. Examine at least 1000 red cells.
g. Count the total number of red cells and the number of
reticulocytes.
h. Calculate the percent of reticulocytes as follows:
Number of reticulocytes
1000
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Note: In order to decrease the microscopic field and thus make
it easier to count the cells, place a piece of paper containing a
“Window” in the eyepiece of the microscope as shown in this
(Figure 4.11).
Figure 4.11
Reference values
Adults & children 0.2 - 2.0%
Infants 2 - 6%
Conditions accompanied with abnormal reticulocytes count as seen
in the following Table 4.1.
Reticulocytosis Reticulocytopenia
Hereditary spherocytic anaemia A plastic anemia
Sickle cell anemia Pernicious anemia
Thalassaemia
Paroxysmal noctoral Hb-Uria
Acquired autoimmune Hem-anemia
Acute posthemorrhagic anemia
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Automated Reticulocyte Analysis
Can be measured by three different methods
a. Fluorescence based: Size and RNA/DNA content of
cells: (Reticulocyte channel)
The residual amount of RNA is measured by using
fluorescent stain to prevent RBC derbies, crystals or
protein interferences.
b. The exceptional capability of Volume Conductivity
and Light Scatter (VCS) Technology in which
simultaneous measurements of cell-by-cell
characteristics are determined using the three
distinct energy probes of volume, conductivity and
laser light scatter is used to interrogate red cells with
the same thoroughness that has made VCS
technology the right choice for more laboratories
than any other. Red cells are evaluated not only in
terms of size, using the DC technology, but also by
the degree of opacity and log light scatter. The Log
Light Scatter differentiates the reticulocytes from
mature red cells. Immature reticulocytes with more
RNA scatter more light and appear further to the
right in the reticulocytes population in the
scattergram.
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c. Nucleic acid dye Oxazine 750 is used to stain
isovolumetrically sphered cells then the analysis by
the laser optical assembly.
Additional Reticulocytes parameters
All Parameters determined for RBCs are separately analysed
also for Reticulocytes (MCVr, CHCMr, RDWr, HDWr,
(CHr), CHDWr), immature reticulocytes Fraction, (IRF), low
fraction reticulocytes (LFF), medium fraction reticulocytes
(MFR) high fraction reticulocytes (HFR) and mean
reticulocytes volume (MRV).
Reticulocyte haemoglobin (RET-He) or haemoglobin content
of reticulocytes (CHr) has been shown to be a sensitive direct
measurement (monitor EPO and iron therapies).
5. Automated Nucleated RBC analysis
Measurements were performed in the „extended lyse mode‟ because erythrocytes in newborns are relatively
resistant to the lysis agents, uses a semiconductor laser.
The cell membranes of NRBC and mature erythrocytes
are lysed, while the WBC become permeable, allowing
quick influx of the dye, but remain intact. The nuclear
material of NRBC and WBC is stained with a
polymethine-based. Thus two clear populations emerge
on the basis of differences in fluorescence intensity.
Using forward scatter light signals, volumetric
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differences between nucleated cells and ghosts permit
their clear distinction.
Uses an argon laser in combination with two fluorescence channels to measure NRBC. The reagent
lyses the membrane of NRBC and mature erythrocytes,
while WBC are left intact. The fluorochrome propidium
iodide binds to the nuclear DNA of the NRBC and
WBC. The combination of cell size and fluorescence
intensity permits the separation of NRBC from the
leucocyte population. The absolute NRBC count and
theproportion of NRBC per 100 WBC are reported.
6. Blood Film Examination
6.1 Preparation of a Thin Blood Film
a. Collect a drop about 3-4 mm in diameter at one end of
the slide.
b. Hold the slide with one hand and place the edge of the
spreader just in front of the drop of the blood using the
other hand.
c. Draw the spreader back until it touches the drop of
blood.
d. Let the blood run along the edge of the spreader.
e. Push the spreader to the end of the slide with a smooth
movement (all the blood should be used up before
reaching the end).
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f. Check if the blood film is satisfactory by the following:
- There should be no lines extending across or down
through the film.
- The film must be smooth at the end, not ragged and
lined.
- The film must not be too long.
- The film must not be too thick.
- The film must not contain holes because a greasy
slide has been used.
6.2 Preparation of a Thick Blood Film
a. Collect 3 drops of blood about 3-4 mm in diameter at
the centre of the slide.
b. Quickly join the three drops of blood using the corner of
the spreader, and spread them to make an even, thick
film. The blood should not be excessively stirred but
can be spread in a circular or rectangular form with 3-6
movements.
6.3 Staining of Thin blood Film with Wright’s stain
The stained thin blood films can be used to study the
morphology of RBCs, WBCs and platelets, besides the
WBCs differential count.
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Principle
Wright‟s stain is a methyl alcohol solution of an acid dye
and a basic dye. The acid is known as eosin, it is red in
color. The basic is known as methylene blue, it is blue in
color. In the staining process, a buffer solution is used to
control the acid-base balance of the stain.
Reagents
a. Wright‟s stain solution Eosine 0.3% (W/V)
Methylene blue
b. Phosphate Buffer (pH 6.8) Na2HPO4
KH2PO4
Procedure
1. Prepare a thin blood film.
2. Fix the film by absolute methyl alcohol for 2-3 minutes.
The smear will change from red to light brown.
3. Completely cover the slide with stain. After 3 minutes,
add an equal volume of buffer. Blow by using hair dryer
gently to ensure uniform mixing. A green metallic
sheen will appear.
4. After an additional 5 minutes rinse thoroughly with tap
water. Wipe the back of the slide to remove all traces of
stain. Air dry the slide and add a drop of immersion oil.
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5. Use the X40 & X100 objectives in the microscope to
examine the blood film for cells por-mpohology and for
WBCs differential (Figure 6.1).
Figure 6.1: Method of examining the blood smear for the
differential white cell count.
When a blood smear is made, the large cells tend to
accumulate on the edge of the smear, whereas the small
cells on the middle of the smear, it would not be a true
representative sample of the patient‟s cells.
Therefore, the smear is examined by following the path of
the arrow shown above (Fig 6.1).
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Figure 6.2: Explanation of the monolayer area of the blood film
where WBCs and platelet numbers are estimated and cell
morphology is examined.
6.4 : Digital Morphology
After slide staining digital morphology system will read the
slide and reported back for approval, automatic cell location
and pre-classification improves resource utilization, the
quality of results and employee satisfaction. One particular labor benefit is that highly skilled medical technologists are
able to spend more time on difficult cases that require
careful analysis and assessment. The ability to archive
images enables hospitals to look at previous cell images of
patients in case of relapse.
An additional service is the ability to create digital slides.
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Although primarily designed for hematology smears, the
digitization function is also applicable to other types of
samples, for educational purposes.
Advantages of automated image analysis as compared to
manual analysis
Increased productivity (speed and cost-efficiency).
Quality assurance (competence assurance and standardized
results).
Ability to track information.
Improved conditions for sample assessment by enhanced communication with other information systems and
instruments in the laboratory, giving the user more access to
information.
Improved cooperation within and between laboratories (transfer of digital images to experts outside the
laboratories).
Improved ergonomics (eyes, neck, and back).
6.5 Test for Malaria
The most commonly used technique for blood examination
of malaria is stained blood films. Giemsa stain (3% Giemsa
solution in buffered distilled water, pH 7.2) is usually used
to stain the films. For routine malaria microscopy, a thin
and a thick film are made on the same slide.
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In examining thin blood films for malaria, infected red
blood cells and the parasites inside the cells might be
identified. In stained thick blood films, the red blood cells
are lysed. Thus, diagnosis is based on the appearance of the
parasite. In thick films, organisms tend to be more compact
and denser than in thin films (Figure 6.3 A, B and C).
6.6 Test for Leishmania
The parasites are most easily obtained in slit-skin smears
from the nodular edge of the sore, which is held between
finger and thumb to cause blanching. Using a small scalpel
blade, an incision a few millimeters along is made through
the intact epidermis into the dermis and the exposed surface
is scraped to obtain tissue juice and cells. Smears are
stained with Giemsa or another equivalent stain and
examined directly. Smears that contain blood, pus, or
epithelial debris are unusable.
Smears are stained with Giemsa stain and examined under
oil-immersion (see illustration below). The pH of the buffer
saline used in the Giemsa stain should be 6.8 for
Leishmania (Figure 6.3 D).
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Figure 6.3
A. Malaria B. Malaria Vivax
C. Malaria plasmodium falciparum D. Leishmania promastigotes
The parasite density is graded according to the following
(Table 6.1).
Grade Average parasite density
6+ >100 parasites/field
5+ 10-100 parasites/field
4+ 1-10 parasites/10 field
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3= 1-10 parasites/10 field
2+ 1-10 parasites/100 fields
1+ 1-10 parasites/1000 fields
0 0 parasites/1000 fields
7. Hemoglobin Determination
7.1 Cyanmethemoglobin Method
Hemoglobin is the main component of red cells. It is
composed of two pairs of globin chains and a hem
compound which contains iron. The main function of
hemoglobin is to carry oxygen (O2) from the lungs to the
body tissue cells and to transfer carbon dioxide (CO2) from
the tissue cells to the lungs. The oxygen combining
capacity of the blood is directly proportional to the
hemoglobin concentration rather than to the RBCs count.
The hemoglobin determination is useful to screen and
measure for the severity of anemia and to follow its
response to treatment.
Principle
The basis of the method is dilution of blood in a solution
containing potassium cyanide and potassium ferricyanide.
Hemoglobin, Methemoglobin and Carboxyhemoglobin are
all converted to Cyanmethemoglobin. The absorbance of the
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solution is then measured in a photoelectric colorimeter or
spectrophotometer at a wavelength of 540nm.
Sample
Blood may be taken directly from a finger (or heel)
puncture without use of anticoagulant or may be collected
in a test tube (EDTA, K3) as an anticoagulant.
Reagents
a. Drabkin‟s Reagent
- Potassium Ferricyanide K3Fe (CN)6 = 0.2g
- Potassium Cyanide KCN = 0.05g
- Sodium Bicarbonate NaHCO3 = 1.0g
Procedure
One reagent blank per series is required.
Table 7.1: Illustrates the Cyanmethemoglobin Method for
Hemoglobin Determination
Reagent Blank Standard Test
(sample)
Drabkin‟s solution 2.5 ml 2.5 ml 2.5 ml
Hemoglobin standard - 10 ul -
Sample (blood) - - 10 ul
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Cb g / dl = x Cs
Mix thoroughly all blood specimens to be tested by repeated inversion immediately before testing.
Mix and let stand for approximately 5 minutes to ensure
complete reaction.
Read using spectrophotometer at a wavelength of 540nm
against blank.
Calculation
Ab
As
Cb = The concentration of Hemoglobin in a given sample
Ab = The absorbance of the sample
As = The absorbance of the standard
Cs = The concentration of the standard
Hemoglobin g/dl x 0.62 = Hemoglobin (Fe) mmol/L.
Example
First try to adjust the spectrophotometer machine to read
(0.00) absorbance against a blank in all of the following
examples.
a. Absorption Mode
For determination of Hemoglobin in an unknown
sample, the following results were obtained.
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Cb (g / dl) = x Cs
= x 15 = 11.3
Concentration (Cs) of Hemoglobin standard = 15g/dl
Reading (As) of Hemoglobin standard = 0.45
Reading (Ab) of unknown (sample or control) = 0.34
Ab
As
Concentration of 0.34
unknown (g/dL) 0.45
b. Concentration Mode
o Press mode selector in the spectrophotometer until
the concentration lid is lit. Place the standard
solution in sample compartment.
o Using the Concentration/Factor Adjust Control, set
the concentration value of the standard (15g/dl) on
the digital display.
o Insert the samples in the sample compartment and
read results in the concentration unit.
c. Factor Mode
If the factor is already known for the test, usually enter
this value by:
o Pressing the mode until the factor lid is lit.
o Using the Concentration/Factor Adjust Control to set
the display to the desired factor value (33).
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Cb = x Cs
Factor (F) = = = 3
o Insert the samples in the sample compartment and
read results directly in concentration unit.
Calculation of the Factor
Ab
As
Cs 15
As 0.45
Cb = F x Ab = 33 x 0.34
= 1.3mg/dl
Reference values
Adult Male 13.5 – 18g/dl
Adult Female 12 – 16g/dl
Newborn 16 – 20g/dl
Cyanide is toxic chemical compound, the new automated
methods using the same principle but in different reagents
instead of Drabkin‟s Reagent (e.g sodium laurryl sulfate or
organic quaternary ammonium salt with sodium chloride).
Standard
Cyanmethermoglobin standard: The concentration is usually
given as g/ dl.
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Note: Cyanide is a well known as a lethal chemical;
therefore, reasonable care must be exercised in handling this
solution.
7.2 : Hemoglobinometer (CYNOX – I)
Cynox – I Hemoglobinometer is a photometer designed to
provide accurate hemoglobin determinations using a 1.251
dilution with a readout in g/dl or mmol/L.
Method
a. Place the on-off power switch in either the up or the
down position to activate the instrument. Allow one
minute for warm up.
b. Adjust the zero control after inserting reference cuvette
into the hemoglobinometer to obtain a display reading
of 0.0.
c. Open the cover and insert a calibration reference slide
into the slide compartment matching the white dots.
(Glass slides should be free of dirt, oil or fingerprints).
d. Close the cover and compare display reading against
value provided with the calibration of reference
standard. (Standard reading should agree within ± 0.5
units).
e. Dispense 5ml of Cynox – 1 reagent or Drabkin reagent
into a clean dry test tube.
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f. Draw blood until the 0.02ml end to end capillary is
completely filled. Wipe off excess blood on the outside
very carefully.
g. Drop the capillary into the reagent, cover the end of the
test tube and shake horizontally until no blood is left in
capillary.
h. Transfer the resultant solution to a clean dry cuvette
bringing the level to approximately ¾ full. Clean the
outside of the cuvette.
i. Place the cuvette containing diluted blood sample into
the cuvette compartment.
j. Close the cover and obtain the hemoglobin value from
the digital display.
Automated Method
In cell blood counters the principle is same but potassium
cyanide almost is not still used. Separate Hemoglobin
channel ensure reliable results to prevent interference of
Leukocytes is minimized by means of a cyanide-free
reagent (Figure 7.1).
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Figure 7.1: Hemoglobin measurement channel in
Automated Method.
8. Haematocrit (Packed Cell Volume, PCV)
This is one of the simplest, most accurate and most valuable of
all hematological investigations.
By means of haematocrit, hemoglobin and red cell count the
absolute indices can be calculated.
High speed micro haematocrit centrifuge has become
commercially available; it provides a centrifugal force of about
12,000 g which gives a constant packed cell volume after a 3-
minute centrifugation.
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Reference Method
a. Capillary or venous blood is drawn into 75mm long;
1.5mm bore heparinized capillary tubes from finger,
lobe of the ear or the heel (infants).
b. Seal the capillary tube at the end with a commercially
available sealing compound or by heating the end of the
tube over a spirit lamp.
c. Place the capillary tubes in the numbered slots in the
centrifuge head, making sure that the number of the slot
corresponds with the specimen number.
The end of the sealed tube should point outward away
from the centre.
d. Centrifuge for 3 minutes.
e. After centrifugation the tubes will show 3 different
layers:
i. Top : a column of plasma
ii. Middle : a thin layer of white blood cells.
iii. Bottom : a column of red blood cells.
f. Hold the tube against the scale of the reading device so
that the bottom of the column of red cells is aligned with
the horizontal zero line.
g. Move the tube across the scale until the line marked as
1.0 passes through the top of the plasma column. Make
sure that the tube is vertical.
h. The line that passes the top of the column of red cells
gives the Haematocrit value.
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Automated Method
Generation of electrical pulses (Hct). Passage of a cell
through the of an impedance counter or through the bean of
light in a light scattering instrument lead to the generation
of an electrical impulse.
Results
Before the introduction of international system of unite (SI)
units, the haematocrit (erythrocyte volume fraction =
packed cell volume, PCV) was reported as a percentage
rather than a decimal fraction e.g. PCV = 45%.
No. of pulses = count of
particle of particle
Average pulse
Height = volume
Summation of height = PCV
Par
ticl
e
pulse
heig
ht
aperture
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In using International System of unit (SI), the results
become PCV = 0.45 (Vol. Fraction).
Expected Range
Sex N.R.
Male 0.4-0.54
Female 0.4-0.54
Children (5) years 0.38-0.44
Infants (3 months) 0.35-0.40
Newborn 0.44-0.64
9. Erythrocyte Sedimentation Rate (ESR)
This is based on the fact that inflammatory processes cause
an alteration in blood proteins, resulting in aggregation of
red cells, which makes RBCs heavier and more likely to fall
rapidly when placed in a special vertical tube. The faster
the sedimentation rate, the higher the ESR value.
ESR is a non-specific test, because abnormal results
indicate a pathological state rather than a functional
disturbance.
Westergren Method
The Westergren method requires collecting 2 ml of venous
blood into a tube containing 0 .5 ml of sodium citrate. It should
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be stored no longer than 2 hours at room temperature or 6 hours
at 4°C. The blood is drawn into a Westergren-Katz tube to the
200 mm mark. The tube is placed in a rack in a strictly vertical
position for 1 hour at room temperature, at which time the
distance from the lowest point of the surface meniscus to the
upper limit of the red cell sediment is measured. The distance of
fall of erythrocytes, expressed as millimeters in 1 hour, is the
ESR.
Wintrobe Method
The Wintrobe method is performed similarly except that the
Wintrobe tube is smaller in diameter than the Westergren tube
and only 100 mm long. EDTA anticoagulated blood without
extra diluent is drawn into the tube, and the rate of fall of red
blood cells is measured in millimeters after 1 hour. The shorter
column makes this method less sensitive than the Westergren
method because the maximal possible abnormal value is lower.
However, this method is more practical for demonstration
purposes.
Principle
The red cells settle to the bottom of a long graded tube held in a
vertical position leaving a layer of plasma above. The height of
the column of plasma after 1 hour indicates the sedimentation
rate of the erythrocytes (red cells).
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Reagents
Trisodium Citrate 3.8%
Trisododium Citrate anhydrous 3.8g
Distilled water 100ml
Mix and keep in the refrigerator
Procedure
1. Add 1.6ml of whole blood or collected with K2 EDTA
to 0.4ml of the trisodium citrate solution.
2. Mix and draw the citrated blood into the westergren
tube up to the 0-mark.
3. Place the tube in the Westergren stand in an upright
position.
4. Wait for one hour.
5. Read the height of the plasma in mm starting from the
0-mark.
The ESR increases with temperature above 23°C. Use the
following chart for correction of ESR reading (Figure 9.1).
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Figure 9.1
Reference values
Sex N.R.
Male 1 – 10mm/hr
Female 3 – 15mm/hr
Tem
per
atu
re
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The following table demonstrates some conditions accompanied
by increased in the ESR level
Rheumatic fever Multiple myeloma Anemia
Rheumatic arthritis Nephrosis Leukemia
Coronary thrombosis Metallic poisoning Pregnancy
Pneumonia Syphilis Menstruation
Nephritis Tuberculosis Agranulocytosis
Cancer
Sources of Error in the Sedimentation Rate
a. Unclean sedimentation rate tubes: Dirt, water, alcohol,
ether, etc., will cause hemolysis and decrease the
sedimentation rate.
b. Partially clotted blood will decrease the sedimentation
rate.
c. Old blood: the blood must be fresh; therefore it must be
used within 2 hours after withdrawal. As the blood
stands, the red cells becomes more spherical and thus
less inclined to assume rouleau formation. This
decrease in rouleau formation decreases the
sedimentation rate.
d. Failure to mix blood: The test tube containing the blood
should be completely inverted 10 to 12 times before
filling the sedimentation rate tube.
e. Use of cold unmixed blood.
f. Air bubbles in the column of blood.
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g. Inclined sedimentation rate tube. The sedimentation rate
tube must be placed in an exact vertical position.
10. Sickle Cell Test
The purpose of this test is to detect sickle cell disorder (Anemia
or Triat). Sickle cell anemia is caused by an abnormal form of
hemoglobin known as hemoglobin-S, which tends to precipitate
in such a way that the red cell takes the sickling shape.
Principle
One drop of blood is mixed with one drop of a sodium
metabisulfite reagent on a slide. If the red cells contain
abnormal hemoglobin, they will become sickle-shaped (or
half-moon shape). The reagent sodium metabisulfite
removes oxygen from the cells, allowing sickling to take
place.
Reagents
Sodium metabisulfite 2% Aqueous Solution:
Sodium metabisulfite (Na2S2O5) 0.2g
Distilled water 10ml
Always should be freshly prepared before use.
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Procedure
a. Place a small drop of capillary blood in the centre of a
slide.
b. Add an equal drop of the fresh sodium metabisulfite
solution.
c. Mix carefully and cover with a cover slide, make sure
that no air bubbles form. Seal with Vaseline or DPX.
d. Place the slide in a wet chamber.
e. Examine under the microscope after 15 minutes using
the X40 objective. If the result is negative, re-examine
after one hour, then after a further hour and after 24
hours.
f. The test is negative if the red cells remain round.
g. The test is positive if the cells become sickle-shaped, or
banana-shaped, often with spikes.
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11. Coagulation
11.1 Bleeding Time
The bleeding time (BT) is the interval between formation of
a controlled, standard wound and the time the wound stops
bleeding. The BT is prolonged in disorders of platelet
function or capillary integrity.
Specimen
Free flowing capillary blood from the extensor (volar)
surface of the forearm is collected on clean filter paper.
Patient must avoid aspirin or acetyl-salicylic acid containing
drugs for one week prior to test.
Materials and Equipment
1. Simplate or Surgicutt (ET lancet device)
2. Sphygmomanorneter
3. Interval timer (stop watch)
4. Filter paper
5. Butterfly bandage
Procedure
1. Select a site on the extensor (valor) surface of the patient‟s
forearm distal, to the antecubital fossa. Avoid surface veins,
scars, and bruises. If the forearm is hairy, lightly shave the
area. Clean with alcohol and allow air-drying.
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2. Place the sphygmomanometer cuff on the upper arm and
inflate to 40 mm Hg. The time between inflation of the cuff
and incision should be 30-60 seconds. Ensure the pressure
remains constant throughout the procedure.
3. Remove the bleeding time lancet from the pack and prepare
for incision according to the manufacturer‟s instructions.
There is always an “arming” device or “safety” to be
activated before triggering.
4. Place the device firmly on the selected site on the forearm
but do not press down. The incision should be made parallel
to the fold of the elbow, across the tong axis of the arm. The
length and depth of the lancet incision must be uniform
from patient to patient. For this reason, no pressure is
applied at the time of triggering the device.
5. Depress the trigger and simultaneously start the timer.
Remove the bleeding time lancet about 1 second after
triggering.
6. At 30 seconds, blot the flow of blood with the edge of the
filter paper. Do not touch the edge of the wound with the
paper.
7. Continue to blot every 30 seconds „until blood no longer
stains the paper. Stop the timer.
8. Remove the sphygmomanometer cuff, clean the arm, and
apply a butterfly bandage across the incision, drawing the
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edges of the wound together. Advise the patient to keep the
bandage in place for 24 hours.
Reporting Results
1. Record results to the nearest 0.5 minutes.
2. Normal range is 2.5-9.5 minutes.
Clinical Significance
Prolonged in thrombocytopenia when the platelet count is below 100 X 10
9\l.
Prolonged after aspirin ingestion.
Prolonged in qualitative platelet disorders such as Glanzmann‟s thrombasthenia, storage pool disorder, or
aspirin- like syndrome.
The BT is routinely performed as a pre-surgical screen.
Limitations
The bleeding time (BT) is actually a qualitative test
indicating the presence or absence of abnormality. A
prolonged BT test must be followed up with platelet count
and aggregometry.
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11.2 Prothrombin Time (PT)
Manual Method
The Prothrombin Time (PT) is the time required for plasma
to clot after tissue thromboplastin and optimal amount of
calcium chloride have been added. The PT is a measure of
extrinsic clotting pathway.
Principle
One stage Prothrombin Time (PT)
The PT is the time required to form a fibrin clot when
plasma is added to thromboplastin calcium mixture. The test
is a measure of the extrinsic pathway of coagulation
involving factors II, V, VII, IX and X (as well as
fibrinogen).
Tissue thromboplastin activates factor VII, proceeds
through the cascade, ultimately generating thrombin. The
thrombin thus form converts fibrinogen to fibrin. The rate of
fibrin formation therefore depends on the level of factor II,
V, VII, and X, and fibrinogen, and thus measures the overall
activity of these factors.
The test is used as screening procedure to indicate possible
factor deficiency of extrinsic pathway.
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The PT test is sensitive to vitamin K dependent factors of
the extrinsic pathway (factors II, VII, and X) and therefore
is used as a means of monitoring oral anticoagulant therapy.
Procedure
1. Place a small amount of patient plasmas and controls to be
tested into tubes and Incubate in 37°C water bath for 5
minutes.
2. Place 0.2 ml of thromboplastin reagent into a series of tube
(2 X‟ the number of tests to be performed), and incubate in
37°C water bath for 3 minutes.
3. Forcibly deliver 0.1 ml of plasma to be tested into a tube
containing the pre-warmed thromboplastin reagent, and
simultaneously start a stopwatch.
4. Then begin swirling the tube in the water bath until 10
seconds have elapsed. Quickly remove tube from the water
bath and wipe off.
5. Gently tilt the tube back and forth and stop the stopwatch at
the earliest appearance of fibrin strands.
6. Report PT to the nearest tenth of a second.
7. Duplicate determinations should be done on each specimen
and the results should agree within 0.6 seconds.
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Interpretation
Normal Prothrombin Time (PT) is 12 to 14 seconds
The preferred method to reports the patient‟s PT in seconds
to compare it with normal control time also reported in
seconds.
11.3 Activated Partial Thromboplastin Time (aPTT)
Principle
The time required for plasma to clot after a measured
amount of phospholipids extract from brain or lung called
partial thromboplastin and CaCI is added. The interval is
prolonged when any coagulation factor of the intrinsic or
common pathway is deficient. The test is not sensitive to
moderate reductions in levels of fibrinogen.
Manual Method
1. Test plasma collected in sodium citrate (3.2% or 3.9%) as
an anticoagulant from whole blood, assayed or frozen less
than four hours after collection.
2. Control plasma, reconstituted according to manufacturer‟s
instructions.
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3. Partial thromboplastin reagent, reconstituted according to
manufacturer‟s directions. Reconstitute only enough for one
day‟s run and store at room temperature.
4. 0.025 M CaCI.
5. Water incubator set at 37 +/-0.5°C.
6. 0.1 ml and 0.2 ml pipits, tips, 12X75 mm glass tubes.
7. Interval timer.
Procedure
1. Place enough 0.025 M CaCI for the entire test run into a test
tube and incubate at 37°C for at least five minutes. Reagent
may be incubated for several hours provided there is no
evaporation.
2. For each test or control plasma to be assayed, pipette 0.1 ml
partial thromboplastin reagent into a labeled test tube and
place in water bath for three minutes.
3. Add 0.1 ml test or control plasma into the tube with partial
thromboplastin, mix and incubate for exactly three minutes.
4. When reagent and plasmas are correctly warmed, forcibly
add 0.1 ml CaCl to the mixture and simultaneously start the
timer.
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5. Immediately begin swirling the tube in the water bath until
20 seconds have elapsed. Remove the tube from the bath
and wipe.
6. Gently tilt the tube back and forth. Stop the timer at the first
appearance of fibrin in the plasma. Record the results to the
nearest tenth of a second.
7. Perform steps 2-6 for each determination.
8. Duplicate determinations are required for each test or
control plasma and results must agree within seconds. The
average of two determinations is the actual lest result.
Expected Results
Normal control is between 25-35 seconds.
Significance
1. Prolonged in congenital or acquired single or multiple
intrinsic or common factors deficiency.
2. Used to test for effectiveness of heparin therapy.
3. Prolonged in deficiency of factors (II, V, VIII, IX, X, XI,
XII, HMWK, kallikrein, or when fibrinogen levels are
below 100 mg/dl).
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4. Prolonged in circulating inhibitor, vitamin K deficiency,
intestinal mal-absorption, liver disease, or obstructive
jaundice.
11.4 New Methods: Semi-Automated and Full Automated
Techniques
Automated Methods
Optical or Spectro-photometric Principles
a. Photo-optical principle
Optical systems are based on the notion that clot formation
induces change in the plasma‟s optical density. As the clot
is formed, there are changes in the optical characteristics
from the initial reading of the plasma/reagents. These
changes are monitored and used to derive the time taken for
a particular degree of change to occur.
b. Nephelometric principle
The nephelometric principle is employed by some systems.
In coagulation assays, a monochromatic laser light source is
transmitted for example, by fiber optics. The light
dispersion readings are made possible by a sensor that may
be installed at 90 or 180 degrees from the light path,
depending on the particular system, which then measures
scattered light at an angle or records the change in light
transmission. When the light reaches insoluble complexes
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such as fibrin fibers, it disperses in forward scattered angles
(180 degrees) and lateral scattered angles (90 degrees). The
chronometer stops when the amount of scattered light or
transmitted light reaches a specific predetermined level.
The difference between light scattered or transmitted before
and after the clot formation is normally proportional to the
amount of fibrin formed.
c. Chromogenic principle
This is based on the use of a color-specific generating
substance known as chromophore, of which para-
nitroaniline (pNA) is the most common. It has a maximum
absorbance at 405 nm. The principle of chromogenic testing
resides in adherence of pNA to synthetic substrates. pNA is
attached to a series of amino acids that mimics the target
sequence of the activated coagulation factors needed to be
determined. The coagulation protein cleaves the
chromogenic substrate at a specific site between a defined
amino acid sequences and releases the pNA.
The intensity of the yellow color is proportional to the
amount of pNA released. This is can be measured by photo
detection at 405 nm wavelength. As more pNA is cleaved
and freed, the absorbance capacity of the sample increases,
which leads to greater change in the solution‟s optical
density.
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d. Immunological principle
Latex microparticles coated with a specific antibody are
generally used against the analyte (antigen) being measured.
A beam of monochromatic light goes through a latex
microparticle suspension. When the wavelength is greater
than the suspension particle diameter, the particles absorb a
small amount of light. Yet, when the specific antigen-coated
latex microparticles come in contact with the antigen
present in the solution, they adhere to the antibody, forming
links between the particles, which produces agglutination.
When the particles‟ diameter approaches the wavelength of
the monochromatic light beam, a greater amount of light is
absorbed. This increase in light absorbance is proportional
to the agglutination, which, in turn, is proportional to the
amount of the antigen present in the sample. This type of
technology is available in more sophisticated coagulation
analysers introduced in the market in the 1990s. Usually
time-consuming standard immunological assays can be
performed in minutes when using any of these automated
tools.
Advantages of Automation in a Coagulation Laboratory
1. Improves the capacity and flexibility of professional time
spent.
2. Improves test reproduction. In the past, manual
coagulation tests were inaccurate, with variation
coefficients greater than 20%; the semi-automatic
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equipment provided greater accuracy in coagulation
testing. However, with manual dispatch of samples and
reagents, testing has to be done in duplicate. With totally
automated equipment accuracy improved, attaining
variation coefficients of less than 5%, and even 1% for
some tests.
3. Reduces cost in samples and reagents.
4. Facilitates data storage and recovery systems by means of
computer programs.
5. Allows automatic replay of results when mistakes are
made in the first run.
6. Offers the possibility of running different tests using a
single sample.
7. Permits sampling from a closed tube, which improves
safety and efficiency in coagulation tests. This reduces, to
a great extent, the possibility of exposing the operator to
sprays or patient sample spills, or mistakes in labeling.
8. Provides capacity to dilute samples, calibrators, and
controls. The equipment can be programmed for
additional dilutions if the initial results escape the
method‟s linearity. It can also automatically carry out
other tests without the operator‟s intervention if clinically
indicated or because of initial run results.
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9. Most analyzers include alarm systems that warn the
operator of excess in pre-established readings, which may
identify equipment problem (e.g. small amount of
reagent, temperature failure, low sample level, and quality
control errors).
The different methodological types available have advantages
and disadvantages that should be known and understood in
order to guarantee precision and validity of test results. It is
important to consider that laboratories are responsible for
trustworthy results. A laboratory‟s main concern is to select
the coagulation equipment that will generate appropriate
results in spite of budget restraints. Such instruments demand
regular technical maintenance, permanent knowledge, and
system control, since a mistake or failure may decisively
influence a number of results. Control systems that guarantee
analytical confidence are therefore compulsory.
Many laboratories may be fortunate enough to be able to
evaluate equipment before purchasing. If this is not possible,
it is very important to obtain adequate information and advice
from a reference laboratory.
When evaluating new equipment before purchase, first
compare analyzers according to certain criteria such as:
- Equipment cost.
- Inactivity period and reliability.
- Repair response time.
- Ease of use.
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- Availability of adequate maintenance within an
appropriate timeframe.
- Validation process and throughput.
- Cost of disposable elements.
- Flexibility in using reagents from other manufacturers.
- Possibility of adding new tests protocols.
- Training courses and continuous training support.
Table 11.1: Explains the advantages and disadvantages of different
detection methods in defining parameters.
Method Advantages Disadvantages
Mechanical No interference due to
physical characteristics such
as lipemia or hemolysis.
Use of small sample
volumes.
Whole blood can be used to
eliminate centrifugation
step.
Impossible to observe
graphics of clot formation.
May present problems of
endopoint detection in
some samples with low
fibrinogen.
Photo-optic Possibility of graphics on
clot formation.
Optical checks for
hemolysis/lipemia/icterus on
some optical systems.
Interference due to
lipemia, hemolysis,
hyperbilirubinemia, or
protein increase on some
systems.
Some systems may
present difficulties with
clot detection when using
some completely
transparent reagents.
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Cont’d.:
Method Advantages Disadvantages
Very short coagulation
periods may go
undetected owing to delay
prior to initiation of
monitoring.
Nephelometric Measurement of residual
amount of Ag-Ab reaction.
Limited number of
available tests.
Expensive costs of
reagents.
Chromogenic Fully specific assays may be
easier.
Additional parameters not
suitable for measurement by
clot detection may be
possible.
Increases the list of possible
tests.
Possible improvements in
precision compared to clot
based analyses.
Limited by the
instrument‟s wavelength.
Requires large test
volumes for positive cost-
benefit ratio.
Cost of instrument and
reagents.
Immunological Fully automated analyzer
reduces time for the test.
Increases the number of
possible tests.
Limited number of tests
available.
Cost of instruments.
Cost of reagents.
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12. ABO and Rh-Grouping
12.1: History
Landsteiner had discovered the ABO Blood Group System
in 1901. He and five co-workers began mixing each other‟s
red blood cells and serum together and accidentally
performed the first forward and reverse ABO groupings.
Landsteiner's rule: If an antigen (Ag) is present on patient's
red blood cells, the corresponding antibody (Ab) will be
absent in the patient's plasma, under „normal conditions‟.
12.2: Blood Group System
Is a series of antigens exhibiting similar serological and
physiological characteristics, and inherited according to a
specific pattern.
12.2.1: Importance of ABO
There are two Principles
1. Almost all normal healthy individuals above 3-6
months of age have “naturally occurring antibodies” to
the ABO antigens that they lack (Table 12.1). These
antibodies (Abs) termed naturally occurring because
they were thought to arise without antigenic
stimulation.
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Table 12.1: Illustrates the different blood groups and the Ags
present in each, also demonstrates the Abs which present in each
blood group.
Antibody Present Antigen
Missing
Antigen Present ABO Group
Anti-B B A A
Anti-A A B B
Anti-A&B A and B NONE O
None None A and B AB
2. These “naturally occurring” Abs are mostly IgM class.
This means that, they are Abs capable of agglutinating
saline/low protein suspended red cell without
enhancement and may activate complement cascade.
Therefore, ABO compatibility between donor and
recipient is crucial since these strong, naturally
occurring A and B antibodies are IgM and can readily
activate complement and cause agglutination. If ABO
antibodies react with antigens in vivo, the result is
acute hemolysis and possibly death.
12.2.2: Indications for ABO grouping
ABO grouping is required for all of the following
individuals: blood donors, transfusion recipients, prenatal
patients, newborns and paternity testing.
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12.3: ABO Typing
ABO typing involves both antigen typing and antibody
detection. The antigen typing is referred to as the forward
typing (cell) and the antibody detection is the reverse typing
(serum).
1. Forward typing: determines antigens (Ags) on patient's
or donor's cells.
- Cells are tested with the different types of antisera
reagents such as anti-A, anti-B, (and in the case of
donor cells anti-AB).
2. Reverse typing: determines antibodies (Abs) in
patient‟s or donor's serum or plasma is also known as
back typing or serum confirmation.
- Serum tested with reagent A1 cells and B cells.
12.4: Principle and Applications
The ABO system is the most clinically significant blood
group system for transfusion practice, because it is the only
blood group system in which antibodies are consistently and
predictably present in the serum of normal individuals
whose red cells lack the antigen. ABO compatibility
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between donor and recipient is the foundation upon which
all other pre-transfusion testing rests.
The D (Rho) antigen is consider as one of the most
immunogenic, after A and B antigens, the most important
red cell antigen in transfusion practice due to its potent
antigenicity. Unlike the ABO system, however, individuals
who lack the D antigen do not consistently and predictably
have anti-D in their serum.
The ABO and Rh are determined for all patients who are
candidates for transfusion, for all blood donors, for all
prenatal patients, and for all potential organ recipients and
donors.
Methodology
1: Slide Method
The slide method is less satisfactory than the tube method.
Agglutination is rapid on that slide; this method is useful
when only one or two samples of blood are to be tested.
Grouping anti-sera should be obtained ready for use from a
known source and should be stored according to the
recommendations of the manufacturer. They must be kept
free from bacterial growth to avoid non-specific false-
positive results.
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Procedure
1. Put one drop of the patient‟s whole blood to each
labeled square on a special slide (A, B, AB and D).
2. Add one drop of each grouping anti-sera (anti-A, anti-B,
anti-AB and anti-D), respectively.
3. Mix the blood and the anti-sera in each square with a
wooden-stick.
4. The results may be read within 1-5 minutes.
5. Presence of agglutination means a positive reaction.
6. Doubtful presence or absence of agglutination may be
checked by viewing under the microscope.
2: Tube Method
4-5 ml clotted blood from an adult or a 2 ml clotted blood
from a pediatric patient should be collected for ABO
reverse grouping and the same volume of blood in EDTA
tube or citrate phosphate dextrose adenine (CPDA) (CPDA-
1) for forward grouping. ABO forward grouping may also
be done on a sample from a segment on a donor unit.
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Reagent and Equipment
Reagent (Anti-sera) Anti-A, Anti-B and Anti-AB, Anti-D
Reagent (Cells) A1, A2 and B Cells
Tubes 12 x 75 mm and test tube rack
Marking pen
Disposal pipettes
Physiological saline
Centrifuge
Lighted agglutination viewer
Preparation of Washed Cell Suspensions
2-5% Red Blood Cell Concentration in Saline
Between 2-5% cell suspension provides optimum antigen concentration in the tube method for red blood cells typing.
Washing Red Blood Cells
The aim of washing the red blood cells is to remove plasma,
which contains substance that may interfere with antigen-
antibody reaction. The following may be in the plasma and
may interfere with testing:
Interfering proteins such as Wharton's jelly that is seen in newborn cord blood, cold-acting autoimmune
antibodies, and increased levels of immunoglobulins
that may cause either agglutination or rouleaux
formation.
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Fibrinogen can result in fibrin strands forming that makes grading reactions difficult.
Procedure
1. Place 1 to 3 drops of blood in the tube
2. Aim the tip of the saline bottle towards the center of the
tube and forcibly squeeze saline into the tube.
3. Fill the tube 3/4 full of saline (there will be less splattering
in the centrifuge) (Fig. 12.1).
4. Centrifuge for 60 seconds in calibrated centrifuge to pull
most of cells into a button in the bottom of the tube.
5. Decant the saline completely.
6. Shake the tube to re-suspend cell button before washing the
cells again. It will depend on the procedure being done as to
how many washing steps are going to be done.
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Figure 12.1: Shows the Red Blood
Cells Suspension in the Test Tube
Washing Procedure.
Figure 12.2a: Demonstrates the
different types of anti-sera (Anti-
B, Anti-B, Anti-Ab and Anti-D).
Figure 12.2b: Shows labeling
of different tubes for reverse
grouping.
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ABO Blood Group
Blood Grouping Procedure
Confirm that patient information on the sample matches the
information on the worksheet.
1. Centrifuge the samples and remove the serum to a labeled
tube.
2. Label 8 tubes with the first 3 or 4 letters of the patient‟s last
name/or first 3 or 4 digits of identification number and line
them up in test tube rack.
3. Add 2-3 drops of the patient's cells to the washed cells tube,
and prepare a washed suspension of 2-5%. Add 2 drops of
patient's serum to both A1 and B tubes.
4. Add one drop of reagent anti-A to the A tube, one drop of
reagent anti-B to the B tube, and one drop of reagent anti-A,
B to the A, B tube (Figure. 12.2a).
5. Add one drop of anti-D reagent to the D tube.
6. Add one drop of patient washed cells to the following tubes:
A tube B tube AB tube D tube.
7. Add one drop of reagent A1 cells to the tube which has been
labeled as A1 tube, and one drop of B cells to the tube which
has been labeled as B tube (Figure. 12.2b).
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8. Shake all 6 tubes gently to mix.
9. Centrifuge tubes in the centrifuge for the calibrated time for
saline suspended cells.
10. Gently re-suspend each cell button individually and
examine for hemolysis or agglutination with the aid of the
lighted agglutination viewer. Grade results negative (0) to
positive (4+). Immediately record the results in the
appropriate spaces on the worksheet.
11. Discard all materials in the isolation trash containers.
Results
Positive Agglutination (+):
The red cells form one or more clumps with a clear
supernatant fluid.
Negative Agglutination (0):
The red cells re-suspend easily without any visible
clumps.
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Grading System:
Table 12.2: Explains the different types of agglutination
reaction between Ag & Ab in blood grouping. Solid
clumping indicated by 4+ and no agglutination indicated by
(0) as seen in figure 12.3.
4+ Solid clump.
3+ Several large clumps.
2+ Small to medium sized clumps; clear background.
1+ Small clumps; cloudy background.
0 Negative: the red cells resuspend easily without any
visible clumps.
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Figure 12.3: Illustrates the different agglutination reaction
strength between Ag & Ab either for forward
and reverse grouping.
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Interpretation
The following Tables (12.3a): Demonstrates the reaction
between Ag & Ab in forward grouping using patient‟s RBC
with anti-sera. Positive with anti-A (4+) indicated that the
patient‟s blood grouping is A. Positive with anti-B (4+)
indicated that the patient‟s blood is group B.
ABO Blood
Group
Patient Red Cells Tested with Known Anti-sera
Forward Grouping
Anti-A Anti-B Anti-A,B
A 4+ 0 4+
B 0 4+ 4+
O 0 0 0
AB 4+ 4+ 4+
+ = Agglutination (graded 1+ to 4+); 0 = No agglutination
(12.3b): Demonstrates the reaction between patient‟s serum and
known cells in the reverse grouping positivity indicated the
presence of Ag & Ab reaction (4+) and negativity by (0), there
is no Ag & Ab reaction.
ABO Blood
Group
Patient Serum Tested with Known Reagent Cells
Reverse Grouping
A Cells B Cells
A 0 4+
B 4+ 0
O 4+ 4+
AB 0 0
+ = Agglutination (graded 1+ to 4+); 0 = No agglutination or hemolysis
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Technical Errors:
The first step in resolving a discrepancy is a careful repeat
of the entire test procedure, paying careful attention to
details possible pitfalls are list below:
a. Partial drying of the slide in cell groupings may be
misinterpreted as agglutination.
b. Over centrifugation or under centrifugation may result
in false positive or false negative readings respectively.
c. Rough dislodgement of the centrifuged cell button may
disrupt small agglutination and result in false negative
reading.
d. The cell-serum mixture may be accidentally heated,
resulting in false negative reading.
e. The use of improper concentration of cells or old cells
may lead to a false negative reading.
f. Failure to observe haemolysis will result in false
negative readings.
g. Dirty glassware will stimulate clumping and result in a
false positive reading.
h. The specimen may be identified incorrectly.
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13. Anti-Human Globulin (AHG)
Types of AHG
There are two different types of anti-human globulin test
(AHG):
1. Indirect of anti-human globulin test known as (IDAT,
IYT) test which formed in vitro.
2. Direct anti-human globulin test known as (DAT) test
which formed in vivo.
The direct anti-human globulin test does not require the
incubation phase, because the antigen- antibody complexes
have formed in vivo.
For indirect anti-human globulin test (in vitro) antigen-
antibody reactions the following is required;
1. Incubate red cells with anti-sera to allow time for
antibody molecules attachment to red cells antigen.
2. Perform three times saline washing to remove free
globulin molecules.
3. Add anti-human globulin reagent to red cell
agglutinates.
4. Centrifuge, to accelerate agglutination by bringing cells
closer together.
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5. Examine for agglutination to interpret test as positive or
negative.
6. Grade agglutination to determine the strength of the
reaction.
7. Add antibody coated red blood cells to those with
negative reaction, to checks for neutralization of anti-
human sera by free globulin molecules (Coombs control
cells are D-positive red blood cells that are coated with
anti-D).
Indirect AHG Test Applications
There are many applications for this test including;
1. Detection of incomplete antibodies to potential donor
red cells (compatibility testing) or to screening cell
(antibody screen) in serum.
2. Identification of antibody specificity using a panel of
red cells with known antigen proteins.
3. Determination of red cell phenotype using known anti-
sera for example (Kell typing, Du testing).
4. Titration of incomplete antibodies.
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Principle and Applications of (DAT)
Direct anti-human globulin test is used to detect in vivo
sensitization of red blood cells. Clinical conditions that can
result in vivo coating of red blood cells with antibody or
complement or both are;
1. Hemolytic disease of the new born (HDN).
2. Hemolytic transfusion reaction (HTR).
3. Auto-immune and drug-induced hemolytic anemia.
Direct anti-human globulin Testing Panel (DAT)
Initial (DAT) should include both anti-IgG and anti-C3d
reagents or poly-specific (anti-IgG-C3d) reagent.
A DAT panel using poly-specific and mono-specific
reagents characterizes the type of protein sensitization that
has occurred in vivo.
A DAT panel is useful in determining whether the patient‟s
red cells are coated with antibody, complement, or both.
Procedure
1. Prepare a 2-5% cell suspension in normal saline for the cells
to be tested.
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2. Add 2 drops of the prepared cell suspension to a tube
marked “test” and 2 drops to a tube marked “auto control‟.
3. Wash cells 3 times by filling both tubes with fresh normal
saline and centrifuging according to calibration of
centrifuge for cells washing. Decant supernatant completely
after each washing (Cord blood specimens must be washed
a minimum of six times).
4. After the third washing, add two drops of Coombs anti-
human globulin (AHG) reagent to the dry cell button in the
tube marked “test”. Do not re-suspend the cells in the „test”
tube with saline before adding the Coombs serum or
reagent.
5. Add two drops of saline to the tube marked auto control.
6. Re-suspend the cell buttons in both tubes with gentle
shaking and centrifuge according to calibration of
centrifuge for the antihuman globulin (AHG) technique.
7. Read macroscopically by holding the tubes to a light source
and gently a agitating them. Examine for agglutination.
Read microscopically only if results are macroscopically
doubtful.
8. Cared the reaction from 4+ to 0.
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Antibody Detection and Identification
Purpose
The purpose of the antibody screen is to detect red blood
cell antibodies other than anti-A or anti-B. These antibodies
are called “unexpected” because only 0.3 to 2 % of the
general population have positive antibody screen.
Once an unexpected antibody is detected, antibody
identification studies are performed to determine the
antibodies specificity and clinical significance.
All red blood cell antibodies are significance if they cause
shortened survival of antigen positive red blood cells. For
example, anti-D is a clinically significant antibody, because
it will bind to D-positive red blood cells, resulting in
immune destruction or hemolysis. Therefore, proper
detection and identification of red blood cell antibodies is
important for the selection of appropriate blood for
transfusion and in the investigation of hemolytic disease of
the new born and immune hemolytic anemia.
Antibody screening test involve testing patient‟s serum
against two or three reagent red blood cell samples called
screening cells.
Screening cells are commercially prepared group O cell
suspensions obtained from individual donors that are
phenotype for the most commonly encountered and
clinically important red blood cell antigens.
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Group O cells are used so that naturally occurring anti-A or
anti-B will not interfere with detection of unexpected
antibodies. The cells are selected so that the following
antigens are present on at least one of the cell sample:
D, C, E, c, e, M, N, S, s, P, Lea, Le
b, K, k, Fy
a, Fyb, and Jk
b
An anti-gram listing the antigen makeup of each cell
provided with each lot of screening cells issued from a
manufacture (Fig. 13.3). It is important that the lot number
on the screening cells matches the lot number printed-on the
anti-gram because antigen make up will vary with each lot.
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Figure 13.3 Explanation of an anti-gram listing the antigen
makeup of each cell provided with each lot of screening cells
issued from a manufacture
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Screening Cells
The screening cells are available in three different forms:
1. A single vial of no more than two donors pooled
together in one vial.
2. Two vials each with different donors.
3. Three vials representing three different donors.
Two or three cells screening sets are required for detection
of antibodies in pre-transfusion testing.
Auto-logous Control
Autologous control is considered as part of the Ab
screening; it can be performed in parallel with the Ab screen
and involves testing the patient‟s serum against the patient‟s
red blood cells. A positive auto-logous control is an
abnormal finding and usually means that patient has a
positive direct anti-globulin test (DAT).
Procedure
1. Label three test tubes as 1, 2 and 3.
2. Add 2 drops of patient‟s serum to each tube.
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3. To tube No. 1, add two drops of screening cells No. 1.
To tube No. 2, add two drops of screening cells No. 2.
To tube No. 3, add two drops of screening cells No. 3.
4. Spin; read macroscopically this is considered as immediate
spin (IS).
5. Incubate at 37°C for 30 – 60 minutes
6. Spin, read macroscopically, if no agglutination, go to next
step.
7. Wash all the tubes 3 times with normal saline.
8. Add to each tube 2 drops of Anti-human globulin (AHG)
reagent.
9. Spin, read macroscopically and microscopically. Any
agglutination or haemolysis detected at any step, means
indirect Coomb‟s is positive.
10. If the test was negative then a control cell (Check Cell)
must be added and re-centrifuge all the tubes.
11. Read all the tubes macroscopically and microscopically
observe for agglutination. If there is positive agglutination
that indicates the final reading is valid. However, if the
results were negative indicates that the test results were
invalid and the test must be repeated again.
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Interpretation
Evaluation of the antibody screening and auto-logous control
results can provide clues and give direction for the
identification and resolution of the Ab or Abs. The investigator
should consider the following questions.
In what phase (s) did the reaction (s) occur?
- Low temperature: Abs of the IgM class will react best at
low temperatures and are capable of causing agglutination
of saline-suspended red blood cells (immediate spin reading
(IS). Of the commonly encountered Abs are, anti-N, -I. and
-P (IgM). Abs of the IgG class (warm Abs) will react best at
the AHG phase (37°C). The most common Abs are: anti-Rh
and ante- kell.
Is the auto-logous control negative or positive?
- A positive Ab screen and a negative auto-logous control
indicate an allo-antibodv has been detected. A positive
auto-logus control may indicate the presence of auto-
antibodies, antibody to medication or it may idiopathic, if
the patient has been recently transfused, the positive auto-
logous control may be due to alloantibody coating
circulating donor red blood cells.
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Did more than one Ab screening cell react?
- If the patient has multiple Abs, when the Abs corresponding
Ag is found on more than one screening cell, or when the
patient‟s serum contains an autoantibody, more than one
screening cell will be positive.
Did the Ab screening cells react at the same strength and
phase?
- A single Ab specificity should be suspected when all cells
react at the same phase and strength. Multiple Abs is most
likely when cells react at different phases and strengths and
auto-antibodies are suspected when the auto-logous control
is positive.
Coomb’s Cells (C.C)
Control cell used when AHG test is negative. To confirm
that washing has been adequate and the anti-globulin
reagent is reactive. They can be used to ensure that AHG
test with negative results are not false negative because of
inactivation of the AHG reagent. When AHG test is
negative, they should be free AHG reagent in the test tube.
When the CC is added, the free AHG in the test should
cause agglutination of the sensitized RBCs.
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Preparation of Control Cell
A group O Rh positive donor‟s sample is mixed with 1:10
dilution of reagent anti-D and allowed to incubate. After
sensitization of RBC, they are washed with saline and
suspended to a 50% RBC suspension. These sensitized
RBCs then used to confirm the anti-IgG activity of the
AHG.
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Atlas of Peripheral
Blood Smears
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Figure XIII. 1 Normal Erythrocyte
Normal erythrocyte:
Normal erythrocyte Is a mature non-nucleated red cell
appearing as a biconcave disc. It should stain pink to red
with a central pallor occupying 1/3 the diameter of the
cell with a Wright-Giemsa stain.
Figure XIII. 2 Echinocytes
Echinocytes (Burr Cells, Crenated Cells)
Echinocytes are normochromic red cells with blunt short. projections, which are evenly distributed over the surface
of the red blood cell.
Echinocytes are commonly seen in:
• Renal disease
• Liver disease
Figure XIII. 3 Macrocytes
Macrocytes
Macrocytes are large red cells with a high mean
corpuscular volume (MCV), usually greater than 100 fl. Their hemoglobin concentration is normal. They may be
oval or round.
Macrocytes are commonly seen in:
• Normal newborn • Drug associated macrocytosis (e.g., anticonvulsants,
antidepressants, sulpha, chemotherapeutic agents,
estrogen and antiretroviral agents)
• Folate deficiency • BI2 deficiency
• Dyserythropoiesis
• Liver disease
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Figure XIII.4 Microcytes
Figure XIII.5 Ovalocytes and elliptocytes
Microcytes
Microcytes are smaller than normal red cells with a MCV
less than 75 fl in children less than 5 years of age and less
than 80 fl in children over 5 years of age. Microcytosis is
usually associated with hypochromia. Microcytic hypochromic cells are commonly seen in:
• Iron deficiency anemia, Lead poisoning
Ovalocytes (Elliptocytes)
Ovalocytes and elliptocytes are red cells that are elongated with blunt ends and parallel sides. The term
ovalocyte is interchangeable with the term elliptocyte.
Their names are descriptive of their appearance.
A small number of elliptocytes are seen in the normal
peripheral smear. elliptocytes are commonly seen in:
• Hereditary elliptocytosis • Renal and liver diseases
• Vitamin B12 deficiency
• Myelodysplasia
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Figure XIII.6 Reticulocytes
Polychromatophilic Red Cells (Reticulocytes)
A polychromatophilic red cell is a non-nucleated red cell
precursor slightly larger than the mature red cell (8-10
microns in diameter). It contains RNA in addition To the
hemoglobin and stains gray blue or pale purple with Wright-Giemsa stain. It has a deep blue granular or
filamentous structure when supravitally stained.
Reticulocytes are seen in:
• Hemolytic anemias
• Blood loss anemias
• Recovering anemia
Figure XIII.7 Sickle Cells
Figure XIII.8 Spherocytes
Sickle Cells (Drepanocytes)
Sickle cells are red cells with two pointed ends which are
in the shape of a crescent or sickle. This is due to the polymerization of deoxygenated hemoglobin S causing
changes to the red blood cell making it less deformable
and much more rigid.
Sickle cells are usually seen in:
• Sickle cell anemia
• Hemoglobin SC
Spherocytes
Spherocytes are dense, staining spherical red cells with normal or slightly reduced MCV without any central
pallor. Spherocytes are commonly found in:
• Hereditary spherocytosis
• Autoimmune hemolytic anemia (warm antibody type) • Post transfusion
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Figure XIII.9 Stomatocytes
Stomatocytes
Stomatocytes are red cells with a central clear opening
appearing like a mouth, hence the name stoma, meaning
mouth. Stomatocytes are commonly seen in:
• Hereditary Stomatocytosis
• Liver disease
Figure XIII. 20 Target Cells
Target Cells (Codocytes)
Target cells have a central hemoglobinized area within
the surrounding area of pallor. These morphological
features give these red cells the appearance of a sombrero
or a bull‟s eye. Target cells are larger than normal cells with excess cell membrane. target cells are commonly
seen in:
• Hemoglobin C • Sickle cell disease
• Hemoglobin E
• Hemoglobin H disease • Thalassemias
• Iron deficiency anemia
• Liver disease
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Figure XIII. 21 Teardrop Cells
Teardrop Cells (Dacrocytes)
Red cells in the shape of a teardrop or a pear with a
single short or long, blunted or rounded end are called
teardrop cells.
Teardrop cells are commonly seen in:
• Osteopetrosis • Myelofibrosis
• Bone marrow infiltrated with hematological or non-
hematological malignancies
• Iron deficiency anemia • Pernicious anemia
• Anemia of renal disease
Figure XIII.22 Basophilic Stippling
Basophilic Stippling
Basophilic stippling is a collection of fine or coarse
granules in the red cells. Clinically insignificant, fine stippling is often seen in reticulocytes. Coarse stippling is
seen in clinically significant conditions with impaired
hemoglobin synthesis and is a result of accumulation of
abnormal aggregates of ribosomes and polyribosomes.
Basophilic stippling is commonly seen in:
• Lead poisoning • Iron deficiency anemia
• Thalassemia
• Refractory anemia
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Figure XIII.23 Hemoglobin C crystals
Hemoglobin C crystals:
Hemoglobin C crystals are dense rhomboid, tetragonal or
rod-shaped structures within red cells. They often distort
the red cell and project beyond its rim.
Hemoglobin C crystals are commonly seen in:
• Hgb CC
• Hgb SC
Figure XIII.24 Heinz Bodies
Heinz Bodies
Heinz bodies are multiple blue-purple inclusions attached
to the inner surface of the red cell membrane. They are not visible in Wright-Giemsa-stained blood
films, but are visible in supravitally stained smears.
Heinz bodies are precipitated normal or unstable
hemoglobin usually secondary to oxidant stress.
Heinz bodies are commonly seen in:
• G6PD deficiency
• Unstable hemoglobins • Congenital Heinz body (bite cell) anemias
Figure XIII.25 Howell-Jolly Bodies
Howell-Jolly Bodies
Howell-Jolly bodies are small round bodies composed of
DNA, about 1 μm in diameter, usually single and in the periphery of a red cell. They are readily visible on the
Wright-Giemsa-stained smear. The spleen is responsible
for the removal of nuclear material in the red cells, so in
absence of a functional spleen, nuclear material is removed ineffectively.
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Howell-Jolly bodies are seen in:
• Post splenectomy
• Functional asplenia
• Anatomical absence of spleen
Miscellaneous Abnormalities
F IGURE XIII.26
AGGLUTINATION
Agglutination
Red cell agglutination occurs when red blood cells clump in irregular masses. Agglutination is secondary to cold
agglutinins, most commonly an igm antibody.
Red cell aggutination is most commonly seen in: • Viral infections (e.g., influenza, parainfluenza)
• Lymphoproliferative disorders
• Plasma cell dyscrasias
F IGURE XIII.27
ROULEAUX
Rouleaux
Rouleaux formation is a common artifact in the thick area of any blood film.
True Rouleaux is seen in the thin part of the blood smear.
There are four or more red cells organized in a linear arrangement like a stack of coins. The central pallor is
generally apparent.
True rouleaux formation is due to increased amounts of plasma proteins primarily fibrinogen and globulins.
Rouleaux are commonly seen in:
• Infections • Inflammation
• Monoclonal gammopathies
• Neoplastic diseases
• AIHA warm antibody disease
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Hemolytic Anemias in Pediatrics
Figure XIII.28 Hereditary elliptocytosis
Hereditary elliptocytosis
Hereditary elliptocytosis (HE) is a common and mild
anemia due to a structural defect of the erythrocyte
membrane protein, spectrin. It is common in individuals
of African and Mediterranean descent.
Approximately 85% to 90% of patients have only
morphological
evidence of HE. The remainders of patients have a hemolytic anemia of varying severity. Spherocytic HE
and stomatocytic HE (Melanesian or Southeast Asian
Ovalocytosis) are described. Hereditary
Pyropoikilocytosis is a related disorder.
F IGURE XIII.29
HEREDITARY
STOMATOCYTOSIS
Hereditary stomatocytosis
Hereditary stomatocytosis (HST) is a mild autosomal dominant hemolytic anemia. There is an inherited
abnormality in erythrocyte cation permeability, leading to
abnormal erythrocyte hydration. The most common
defect is in the red cell membrane protein, stomatin.
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Hemoglobinopathies
Figure XIII.30 Hereditary acanthocytosis
Hereditary acanthocytosis
Hereditary acanthocytosis is an autosomal recessive, mild
hemolytic anemia due to a defect in beta lipoprotein.
Figure XIII.31 Sickle hemoglobinopathies
Sickle hemoglobinopathies
Sickle hemoglobinopathies are most commonly present in people of African descent. In the United States, these
Pathologies are seen in African Americans, African
immigrants, Caribbean and Central Americans,
particularly from the Caribbean coast of Central America. In addition, sickle syndromes are seen in Middle Eastern,
Mediterranean and East Indian populations. Hemoglobin
S is a qualitative defect of the β globin chain of the
hemoglobin inwhich there is a substitution of amino acid valine for glutamic Acid leading to polymerization of
hemoglobin in the presence of hypoxemia.
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F IGURE XIII.32 DYSKERATOSIS
CONGENITA (DC)
Dyskeratosis Congenita (DC)
DC is a rare form of ectodermal dysplasia. The diagnostic triad consists of:
Fifty percent of patients with
DC develop aplastic anemia in the second decade of life
and 10% develop cancer in the third and fourth decades of life.
F IGURE XIII.33
MYELODYSPLASTIC
SYNDROME (MDS)
Myelodysplastic Syndrome (MDS)
MDS is usually associated with pancytopenia and
normocellular to hypocellular bone marrow. MDS is
characterized by megaloblastic and dyserythropoietic
erythropoiesis with maturational defect. Dysmyelopoiesis
is common with maturational aberrations in myeloid
cells. Megakaryopoiesis often is atypical.
The common causes of MDS are:
• Drugs, toxins and chemicals
• Radiation
• Preleukemia • Chromosomal syndromes of downs, other trisomies,
monosomy 7
• Refractory anemias
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Figure XIII.34 Microcytic Anemias
Microcytic Anemias:
Iron Deficiency Anemia (IDA) is the most common
anemia worldwide and commonly affects infants between
the ages of 9 months and 2 years, because of poor iron
intake. Chronic blood loss is the commonest cause of IDA in children over 2 years of age.
F IGURE XIII.35
LEAD POISONING
Lead Poisoning
Anemia of lead intoxication is largely caused by
inhibition of heme synthesis and inhibition of pyramidal
5' nucleosidase. Anemia of lead poisoning mimics IDA
but has a normal iron profile. Basophilic stippling in the
red blood cells and polychromatophilic cells are the
hallmarks of lead poisoning.
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Figure XIII.36 Neutrophil,Seg.
White Blood Cells (Leukocytes)
Neutrophil, Segmented (Segs)
The segmented neutrophil is the predominant white blood cell in the peripheral blood. It is 10 μm to 15 μm in
diameter with pale pink cytoplasm and specific fine
granules. Rare azurophilic (primary granules) are seen. The nucleus is lobulated (between 2 and 5 lobes) and the
lobes are connected by a thin filament.
F IGURE XIII.37
BAND NEUTROPHIL
Band Neutrophil
Band neutrophils constitute from 0% to 5% of the
nucleated cells under normal conditions in the peripheral blood. The band is round to oval in shape and 10μm to
18μm in diameter. The nucleus can be band like,
Sausage-shaped, S-, C- or U-shaped and may be twisted
and folded on itself. The cytoplasm is pale with specific granules in it. Increased numbers of bands appear in the
blood in a number of physiologic and pathologic states.
Bands are increased in the peripheral blood in the
following conditions:
• Severe infections--Sepsis/bacteremia
• Inflammation
• Stress
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F IGURE XIII.38
BASOPHILE
Basophile
In the normal physiological state there are very few (0%-
1%) basophils in the peripheral blood. All basophils,
from the basophilic myelocyte to the mature segmented
Basophil, are characterized by the presence of a moderate number of large coarse and densely stained
granules of varying sizes and shapes. The granules in the
Wright–Giemsa-stained preparation are blue-black;
Some may be purple to red.
Basophils are increased in the blood in:
• Myeloproliferative disorders (e.g., chronic Myelogenous leukemia)
• Hypersensitivity reactions
• Mastocytosis
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F IGURE XIII.39
EOSINOPHIL
Eosinophil
Eosinophils are distinct cells, about the size of a
neutrophile (10 μm -15 μm) with abundant cytoplasm filled with many large, coarse, orange-red granules which
are refractile because of their crystalline structure. About
80% of segmented eosinophils will have the classic two-lobe appearance. Only 1% to 8% of circulating
leukocytes are eosinophils.
Morphologically abnormal eosinophils are seen in:
• Myelodysplastic syndrome
• Megaloblastic anemias
Eosinophils are increased in the following conditions:
• Allergies
• Parasitic infestations
• Infections • Acute leukemia
• Myeloproliferative diseases
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F IGURE XIII.40
MONOCYTES
Monocytes
Monocytes are larger cells, 12 μm to 20 μm in diameter.
The majority of monocytes are round with smooth edges.
Usually, there is abundant gray to gray-blue cytoplasm
which may contain fine, evenly distributed granules and vacuoles. The nucleus is usually indented, the chromatin
is condensed and occasionally a small and inconspicuous
nucleolus is seen. Monocytes are seen in 1% to 5% of the
leukocytes in the peripheral smear. Monocytes are increased in the following conditions:
• Chronic infection (e.g., tuberculosis)
• Recovery from severe neutropenia in neoplastic or a plastic disorders
• Benign neutropenia
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F IGURE XIII.41
LYMPHOCYTES
Lymphocytes
Most lymphocytes seen on a blood smear are fairly
homogeneous. Lymphocytes are small, round- to avoid
shaped cells that range in size from 7 μm to 15 μm with
round to oval nuclei. Some normal lymphocytes are medium-sized due to an increase in the amount of
cytoplasm. The nucleus appears dense or coarse and
clumped with ridges of chromatin and parachromatin.
nucleoli, if present, are small and inconspicuous. The majority of lymphocytes have a scant amount of pale
blue to basophilic agranular cytoplasm.
F IGURE XIII.42
REACTIVE LYMPHO .
Reactive Lymphocytes
Reactive lymphocytes are notable due to their remarkable
heterogeneity. They tend to be large with abundant
Cytoplasm. They are indented by surrounding red cells, and they may have blue skirting (blue outline around the
Cytoplasm). Another descriptive term used is the “fried
egg” appearance. This cell corresponds to the Downey
Type II cell. Accompanying this type of reactive lymphocyte, plasmacytoid lymphocytes are frequently
seen. These lymphocytes have deeply basophilic
cytoplasm and resemble plasma cells. Their size varies
from small to moderate and may have one or more prominent nucleoli.
They correspond to Downey Type III cells. The Downey
Type I cell which is not observed as frequently is small with a slightly basophilic cytoplasm and an indented or
lobulated nucleus. Reactive lymphocytes are frequently
seen in children with viral diseases, but the condition
where reactive lymphocytes demonstrating all the downey type cells are seen is usually infectious
mononucleosis.
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F IGURE XIII.43
TOXIC VACUOLIZATION
Toxic Vacuolization
Large, clear areas in the cytoplasm of neutrophils
F IGURE XIII.44
HYPERSEGMENTED
NEUTROPHILS
Hypersegmented Neutrophils
Large hypersegmented neutrophils are a result of
megaloblastic hematopoiesis. In megaloblastic myelopoiesis, eosinophils and basophils are large and
also hypersegmented. To be considered hypersegmented,
Neutrophils should have 6 or more lobes. Megaloblastic
hematopoiesis is seen in:
• Vitamin B12 deficiency
• Folate deficiency
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F IGURE XIII.45
AUER RODS
Auer Rods
Auer rods are pink or red, rod-shaped cytoplasmic
inclusions seen in myeloid cells and occasionally in
monocytes. Auer rods are thought to be an abnormal
crystalline form of primary granules:
• Acute myeloid leukemia
• Myelodysplastic/pre-leukemic states.
An immature myeloid cell containing multiple Auer rods
clumped together is known as a faggot cell, and is seen in
acute promyelocytic leukemia.
Platelet Morphology:
Figure XIII.46 Normal Platelets
Platelets
Normal Platelets (Thrombocytes)
Platelets are small non-nucleated cells derived
from the cytoplasmic fragments of megakaryocytes and are variable in size. Normal-
sized platelets are 1.5 μm to 3 μm in diameter and
have fine purple-red granules aggregated at the
center or dispersed throughout the cytoplasm:
• Normal platelets are 1.5 μm to 3 μm in diameter
• Large platelets are 4 μm to 7 μm in diameter
• Giant platelets are greater than 7 μm in diameter
and may be 10 μm to 20 μm. • Small (micro) platelets are less than 1.5 μm in
diameter
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F IGURE XIII.47
LARGE PLATELETS
Large Platelets
Large platelets are usually 4μm to 7μm in
diameter.
Large platelets are commonly seen in:
• Reactive thrombocytosis
• Autoimmune thrombocytopenia
• Myeloproliferative,disorder,leukemoid reaction
• Myelodysplastic disorder • Neoplastic diseases: Acute
Megakaryocytic Leukemia (M7)
• Hereditary thrombocytopenias
F IGURE XIII.48
G IANT PLATELETS
Giant Platelets
Giant platelets are larger than 7μm and may be 10 μm to 20 μm in diameter. The periphery of the
giant platelet may be round or scalloped. The
cytoplasm may contain fine azurophilic granules
or the granules may fuse into Giant forms. Giant platelets are commonly seen in:
• Myelodysplastic disorder
• Hereditary
Thrombocytopenias, such as:
−− May-Hegglin anomaly −− Storage pool syndrome
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Figure XIII.49
Small Platelets
Small Platelets (Microthrombocytes)
Microplatelets are usually less than 1.5 μm in diameter and are not counted adequately by the
impedance blood cell counters, giving
spuriously low platelet counts. Microplatelets
are seen inWiskott Aldridge Syndrome
F IGURE XIII.50
PLATELET SATELLITISM
Platelet Satellitism
Platelets sometimes clump and adhere to
neutrophils
and more rarely to monocytes forming “platelet
rosettes,” which is known as platelet satellitism. Platelet
satellitism
is a cause of spurious thrombocytopenia because
the cellular aggregates are counted as leukocytes rather
than platelets.
Quantitative Disorders of platelets: • Thrombocytopenia
• Thrombocytosis
Common Causes of Thrombocytopenia: • Decreased production
Aplastic anemia
Acute leukemia
Viral infections • Parvovirus
• CMV
• Increased destruction
Immune thrombocytopenia • Idiopathic thrombocytopenic purpura (ITP)
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Figure XIII.51 Thrombocytosis
Thrombocytosis
Causes may include:
• Reactive thrombocytosis
−− Post infection
−− Inflammation
−− Chronic diseases −− Juvenile rheumatoid arthritis (JRA)
−− Collagen vascular diseases
−− Benign tumors: adenomas, lipomas
−− Ganglioneuroblastoma / neuroblastoma
Most of Reprinted Pictures from the DM96 machine Sysmex Middle East-
after written permission (May 2011).
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References
1. UNRWA,(1999): Manual on Basic Techniques for
UNRWA laboratory personnel, third edition).
2. Keijzer M. and Der Meer W., (2002): Automated Counting
Of Nucleated Red Blood Cells In Blood Samples Of
Newborns,Clinical & Laboratory Haematology (24), 343–
345.
3. Briggs C. et al, (1999): The Automated Nucleated Red Cell
Count On The Sysmex Xe-21oo™, Department of
Hematology, University College Hospital, Grafton Way,
London, U.K.
4. Murai M. et al, (1999): WBC Differential, Hematology,
Complete Blood Count (CBC), Leukemia, Peripheral Blood
Stem Cell (PBSC), Hematopoietic Progenitor Cell (HPC),
Sysmex Journal, (2 ), 2, in Japanese.
5. Buttarello, M. et al, (1996): Reticulocyte Quantification by
Coulter MAXM VCS (Volume, conductivity, light scatter)
technology, Clinical Chemistry (42):12,1930-1937.
6. www.cellavision.com
7. Wright D., (2010): Abbott Learning Guide, Abbott
Laboratories HM_09_39649/v2-3.
8. www.abbottdiagnostics.com/www.beckman.com/products/a
pplications/partChar/ CoulterPrinciple_dcr.asp.
9. Paterakis G. et al, (1998): Comparative Evaluation Of The
Erythroblast Count Generated by Three-Color Fluorescence
Flow Cytometry, The Abbott CELL-DYN® 4000
Hematology Analyzer, and microscopy, Int Journal of
Laboratory Hematology (4):64–70.
Hematology Handbook Reference & Automation Methods- FOR PERSONAL USE
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10. Briggs C. et al, (2007): Continuing Developments with the
Automated Platlet Count, Int Journal of Laboratory
Hematology (2):77-91.
11. www.nlm.nih.gov/medlineplus/ency/article/003344.htm\.
12. Harmening D., (2002): Hemtology And Fundamentals of
Hemostasis , F.A. Davis, fourth edition.
13. Harmening D., (2002): Modern Blood Banking and
Transfusion Practice, F.A. Davis, fourth edition.
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INDEX
A
ABO and Rh-Grouping ............89, 97
ABO antibodies ................................. 90
ABO compatibility .......................90, 91
ABO grouping .................................. 90
ABO groupings ................................. 89
Acetic Acid. Glacial ......................... 29
Activated Partial Thromboplastin
time (aPTT ................................. 79
agglutination ... 84, 90, 93, 94, 98, 101,
102, 103, 104, 106, 111, 112, 113,
122
Agreen metallic sheen ...................... 51
AHG ..... 103, 104, 106, 111, 112, 113,
114
AHG reagent ................................... 113
allergic conditions ............................ 41
alloantibody .................................... 112
Ammonium oxalate .......................... 41
Ammonium Oxalate ............ 17, 18, 41
ammonium salt with sodium chloride
...................................................... 61
anaemia .......................................46, 57
antecubital fossa. .............................. 74
antibodies ..... 12, 28, 43, 89, 90, 91, 94
anticoagulation .................................. 15
anti-D reagent ................................... 97
antigen ....... 12, 84, 89, 91, 92, 94, 145,
150, 153
antigens (Ags) ................................... 91
antiglobulin ..................................... 106
anti-gram ......................................... 108
anti-human globulin ...... 103, 105, 106
aperture ...........................27, 28, 35, 66
Aqueous methylene blue .................. 29
as methylene blue ............................. 51
aspirin.......................................... 74, 76
autoantibody ................................... 113
auto-logous .................... 110, 112, 113
auto-logous control ......................... 112
Auto-logous Control ...................... 110
auto-logus ....................................... 112
B
Basophils .................................. 12, 128
bleeding time ......... 41, 74, 75, 76, 148
bleeding time (BT) ........................... 74
Blood Cell Counting..................... 7, 19
Blood Film .................................... 7, 49
bone marrow .. 9, 13, 44, 125, 151, 154
Brilliant cresyl blue .......................... 45
buffer ..................................... 36, 51, 55
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C
CaCI ............................................79, 80
Capillary blood ...........................14, 15
capital fossa veins ............................. 16
carbon dioxide ..................... 11, 19, 57
Carboxyhaemoglobin ....................... 57
CBC .............................................9, 138
compelet blood count ................... 9
CHCMr ............................................. 48
CHDWr ............................................. 48
Check Cell....................................... 111
CHr .................................................... 48
Chromogenic principle .................... 83
chronometer ...................................... 83
cirrhosis ............................................. 41
clotting ..... 17, 18, 19, 41, 77, 148, 154
common pathway .............................. 79
complete blood count ......................... 9
Coomb’s Cells ............................... 113
Coulter Principle ..... See Direct current
Counting Chamber............................ 20
CPDA (CPDA-1) .............................. 93
cresyl blue solution ........................... 45
cuvette .........................................62, 63
Cyanmethemoglobin......................... 57
Cynox ................................................ 62
D
D (Rho) antigen ................................ 92
damaged cells.................................... 14
DAT ... See Direct anti-human globulin
Testing Panel
DC ........................... See direct current
Digital morphology ......................... 53
dipotassium salt ................................ 45
Direct current .............................. 26, 28
Drabkin‟s Reagent ...................... 58, 61
E
EDTA ................ 17, 18, 45, 58, 69, 93
Eosine................................................ 51
Eosinophils .............................. 12, 129
EPO ................................................... 48
Erythrocyte Sedimentation Rate .. 8, 67
Erythrocytes ......................... See RBC
ESR .............................8, 18, 67, 69, 71
Ethyl Alcohol.................................... 16
Ethylenediamine Tetra-Acetic ....... See
EDTA
extrinsic clotting pathway ................ 77
extrinsic pathway ........................ 77, 78
F
factors II ............... 77, 78, 81, 147, 148
factors IX .......................................... 81
factors V............................................ 81
factors VIII ....................................... 81
factors X............................................ 81
factors XI .......................................... 81
factors XII ......................................... 81
ferricyanide ....................................... 57
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fibrin .. 13, 77, 78, 81, 83, 95, 147, 153,
154
fibrinogen ....... 77, 79, 81, 87, 122, 154
Fibrinogen ......................................... 95
filter paper ...................................74, 75
FIVE PART DIFF .......................... 37
five-part differential....................35, 36
Flow cytometry ................................. 33
fluorochrome propidium iodide ....... 49
formaldehyde ..............................20, 36
G
Giemsa stain............... 54, 55, 116, 118
Glanzmann‟s thrombasthenia ........... 76
H
Haematocrit ...................... 8, 18, 64, 65
haemoconcentration .......................... 16
Haemocytometer .........................20, 23
haemoglobin 19, 48, 57, 62, 63, 64, 72
Haemoglobin content of reticulocytes
(CHr) ............................................ 48
Haemoglobinometer ..................... 8, 62
haemoglobin-S .................................. 72
haemolyses ........................................ 15
HDWr ................................................ 48
Hematology ....... 1, 5, 6, 8, 9, 138, 139,
147, 149, 151, 153
Hematopoiesis ................................. 13
hemolysis71, 87, 90, 98, 101, 148, 154
Heparin ........................................17, 19
HMWK ............................................. 81
I
IDAT ............................................... 103
idiopathic thrombocytopenic purpura
(ITP) ............................................. 41
IgG ................................. 105, 112, 114
IgM .................................................... 90
Immature granulocyte (IG) ............ See
Immature granulocyte (IG)
Immature granulocyte (IG)" ......... 37
immature Reticulocyte Fraction ....... 48
immunological humeral response .... 12
Impedance counting ......................... 26
in vitro ............................................... 17
in vivo .......................................... 17, 90
infection 12, 15, 28, 37, 130, 136, 148,
149
Internal structure............................... 33
IYT .................................................. 103
K
kallikrein ........................................... 81
Keeping Time of EDTA Blood ........ 18
L
Landsteiner's rule .............................. 89
Latex microparticles ......................... 84
Leishmania.............................. 7, 55, 56
leucocytosis ...................................... 29
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Leukocytes ........................... See WBC
lungs ..................................... 11, 19, 57
lymph ................................. 9, 150, 151
Lymphocytes ................................... 12
lyse-resistant RBC ............................ 33
M
Malaria ....................................7, 54, 56
MCH ...........................................19, 26
MCHC ........................................19, 26
MCV .......................... 19, 25, 116, 118
MCVr ................................................ 48
Methaemoglobin ............................... 57
methyl alcohol .................................. 51
Monoclonal antibodies ..................... 43
Monocytes ................................12, 130
Multiple Abs ................................... 113
Myeloperoxidase .............................. 36
N
nephelometric principle .................... 82
Nephelometric principle .................. 82
neubauer counting chamber ............. 41
Neubauer counting chamber ............ 20
Neutrophils ..............................12, 132
Nucleated Red Cells ........................ 31
Nucleic acid content (RNA and DNA)
...................................................... 33
Nucleic acid dye Oxaz ...................... 48
O
oxygen ............ 11, 19, 57, 72, 148, 149
P
Packed Cell Volume, PCV ............. 64
para-nitroaniline (pNA) .................... 83
peroxidase ......................................... 36
phagocytosis ..................................... 28
Phosphate Buffer .............................. 51
photoelectric colorimeter ................. 58
Photo-optical principle .................... 82
plasma 9, 13, 15, 65, 68, 69, 77, 78, 79,
80, 81, 82, 89, 91, 94, 122, 131,
146, 147, 154
platelet ...... 41, 42, 43, 53, 74, 76, 134,
135, 149, 154
Platelet Counts ................................ 41
PLT-o = RNA content ...................... 42
pneumonia .................................. 38, 41
polymethine ...................................... 48
positive Ab screen .......................... 112
positive agglutination ..................... 111
positive antibody screen ................. 107
Potassium Cyanide ........................... 58
Potassium Ferricyanide .................... 58
Potassium Oxalate ...................... 17, 18
Prothrombin time .................... 8, 77, 79
puncture ................................ 14, 16, 58
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R
RDWr ................................................ 48
RET–HE ............................................ 48
Reticulocyte ............. 7, 15, 44, 48, 118
Reticulocyte haemoglobin ................ 48
Reticulocyte Volume (MRV) ........... 48
rouleau formation ............................. 71
rouleaux. Formation ......................... 94
S
screening cells ........ 107, 108, 110, 111
serum . 15, 89, 91, 92, 93, 97, 102, 145,
153
Sickle cell anaemia .....................46, 72
Sickle Cell Test ................................. 72
Simplate ............................................ 74
single Ab ......................................... 113
Sodium Bicarbonate ......................... 58
sodium laurryl sulfate ....................... 61
sodium metabisulfite ..................72, 73
spectrophotometer ......................58, 59
Sphygmomanorneter......................... 74
spleen ................... 9, 14, 121, 153, 154
Spleen ............................................... 14
splenectomy ............. 41, 121, 149, 153
Surgicutt ............................................ 74
T
thalassaemia ...................................... 31
the intrinsic ....................................... 79
the nodular edge of the sore ............. 55
thick film ..................................... 50, 54
thrombin. ..................................... 19, 77
thrombocytes .................... See platelets
thrombocytopenia ... 76, 134, 135, 148,
154
thromboplastin ................77, 78, 79, 80
tissue juice ........................................ 55
tourniquet .......................................... 16
Trisodium citrate .................. 18, 20, 45
Trisodium Citrate ................. 17, 18, 69
V
VCS ..................................... 35, 47, 138
venous blood .................15, 45, 65, 150
Venous Blood ............................... 7, 15
venous method .................................. 16
vitamin K .................................... 78, 82
W
Washed Cell Suspensions ............... 94
Wharton's jelly .................................. 94
Wright‟s stain ......................... 7, 50, 51
X
X100 objectives ................................ 52
X40 objectives .................................. 52
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GLOSSARY OF TERMS
anemia: Low red blood cell (RBC) count or hemoglobin in blood;
caused by blood loss or by impaired production or destruction of RBCs
antibody: A protein molecule in serum or body fluids that reacts with,
protects against, and helps destroy foreign or natural substances
(antigens)
antigen: Any substance, either foreign or natural, that stimulates
lymphocytes (white blood cells) to initiate an immune response;
includes bacteria, viruses, fungi, toxins, living and dead tissue, etc.
aplastic: Characterized by incomplete or defective development
basophil: A cell that stains specifically with basic dyes
-blast: A suffix indicating an immature cell
calibrator: A known quantity of an analyte used to establish a normal
curve
catalyst: A substance whose presence changes the velocity of a reaction
but does not form part of the final product of the reaction; the verb
form is catalyze
chemistry: In clinical testing, refers to the solutes dissolved in the
plasma such as uric acid, etc.
coefficient of variation (CV): A statistical term that indicates the
precision or reproducibility of a measurement; expressed as
percentages, CVs indicate the degree of small variations between the
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same tests run on the same sample (the smaller the number, the more
precise the instrument or test)
congestive heart failure (CHF): A collection of symptoms indicating
that the heart is unable to pump effectively; inadequate blood
circulation results in decreased oxygenation of tissues and
breathlessness
control: In chemistry, a known quantity of an analyte that is tested as if
it were an unknown to find out how well the instrument is performing
correlation coefficient (r): A value indicating accuracy; values closest
to 1.00 are best
-cyte or cyto-: Combining forms meaning cell
dialysis (also called hemodialysis): The process of removing toxins and
excess water from the blood; used in kidney failure
diapedesis: The process by which cells squeeze through the pores of a
membrane
diffusion: The movement of particles from an area of greater
concentration to an area of lesser concentration
ecchymosis: Bruising
electrolyte: Any substance that, in solution, becomes an ion and
conducts electricity; examples are sodium and potassium
-emia: Suffix that can be interpreted as "in the blood"
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end point: Term for a reaction that is measured at the end; for example,
a test converts all of a substance
present in a specimen into a chromogen – which has a specific color –
and then measures the color
enzymes: Proteins that catalyze chemical reactions; many enzymes are
present in one organ; enzymes appear in blood because they have a
natural function there or because disease in the tissue in which they
originate caused them to be dispersed via the blood
epistaxis: Nosebleed
erythro-: Prefix meaning red
erythroblast: Literally, an immature red blood cell
erythropoiesis: Red blood cell formation
ferritin: The primary form of storage iron
fibrin: A plasma protein that acts with other blood factors to create
blood clots
glucose: The sugar that is the energy source for all body cells
granulopoiesis: The formation of granulocytes
hema- or hemo-: Prefixes indicating blood
hematology: Literally, the study of blood; refers to the gross features of
blood such as cell counts, bleeding time, etc.
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hematopoiesis: Formation of blood cells
hemoglobin: The oxygen-carrying component of red blood cells;
composed of two pairs of protein chains called globin and four smaller
units called heme, which contain iron
hemolysis: Destruction of red blood cells, often by separation of
hemoglobin; caused by many substances or by freezing or heating
hemolytic-uremic syndrome (HUS): A sudden disorder that involves
thrombocytopenia, hemolysis, and acute renal failure; HUS primarily
occurs in infants and children following an infection but occasionally
occurs in adults; most patients recover, but some require renal dialysis
hemophilia: A bleeding disorder due to hereditary deficiencies in the
blood clotting factors
hemostasis: The process of stopping bleeding
hepat-: Prefix indicating the liver
hepatitis: An inflammation of the liver
histiocytes: Macrophages in the connective tissue; part of the
reticuloendothelial system
histogram: A graphic means of comparing magnitudes of frequencies
or numbers of items; usually shown in bar graphs or columns
homeostasis: The body's tendency to maintain a uniform or stable state
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hormones: Endocrine gland secretions that travel to and act on specific
target organs
hyper-: Prefix indicating abnormally increased values or activity
hypo-: Prefix indicating abnormally decreased values or activity
hypochromia: A decrease of hemoglobin in the red blood cells so that
they are abnormally pale
hypoxia: Abnormally low oxygen level
idiopathic: Occurring without known cause
idiopathic thrombocytopenic purpura (ITP): A chronic blood disorder
with no apparent cause (except in children when it may follow a viral
infection); the body develops an antibody directed against platelet
antigens, causing bleeding (petechiae, purpura, or mucosal bleeding)
that may be minimal or extensive; treatment options include steroids,
infusion of platelet concentrates, or splenectomy
interstitial fluid: Fluid surrounding cells; transports nutrients, gases,
and wastes between blood and cells
ion: An atom or molecule that carries either a positive (cation) or
negative (anion) electrical charge
-itis: Suffix indicating inflammation
lymphoid: Associated with lymph; refers to the lymphatic system and
to the fluid collected from tissues that flows through the lymph vessels
and is added to venous blood
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lyse: To break up to cause cells to disintegrate
lysin: An antibody that acts destructively on cells depending on the
antigen that stimulated its production
lysis: The destruction of red blood cells, bacteria, and other antigens by
a specific lysin
macro-: Prefix meaning large; for example, a macrocyte is a large cell
megalo-: Prefix meaning large; for example, a megalocyte is a large
cell
menorrhagia: Excessive menstrual bleeding
meta-: Prefix indicating after or behind; this prefix has the same
meaning as the prefix postmetabolism: Physical and chemical processes
by which a living organism breaks down complex substances into
simpler substances for nutritional use or disposal
microcyte: A red blood cell that is five microns or less in diameter
microcytic, hypochromic: Adjectives describing a form of anemia with
red blood cells that are small and pale
mye-: Prefix meaning bone marrow
myeloid: Associated with the bone marrow
neoplasm: Any new or abnormal growth
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nephr-: Prefix indicating the kidney
nephritis: Inflammation of the kidney
normo-: Prefix meaning normal or usual
normochromia: Indicating red blood cells having normal coloring
normocyte: A red blood cell that is normal in size, shape, and color
normocytic, normochromic: Adjectives describing normal cells with
normal coloring; used to indicate the status of red blood cells
-oma: Suffix indicating a tumor or neoplasm; for example, lymphoma
means tumors affecting the lymph system
-osis: Suffix indicating a condition; for example, thrombocytosis is a
condition affecting thrombocytes (platelets)
pancytopenia: A condition in which the numbers of all types of blood
cells are reduced
panel tests: Usually, a group of three to five tests involving one organ
system (heart, liver, kidneys, etc.) or having reference to one condition;
used to confirm a diagnosis, to monitor a condition or disease state, or
to modify therapy
-penia: Suffix indicating a decreased amount
petechiae: Pinpoint bleeding into the skin from broken blood vessels –
usually on arms or thighs – without trauma
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pH: means of indicating the acidity of body fluids; pH is measured on a
scale of 0 (highly acidic) to 14 (highly alkaline), with 7.0 as neutral;
normal range for blood pH is 7.35 to 7.45
phagocyte: A cell that ingests bacteria, foreign particles, and other cells
to protect the body
polycythemia: Literally, many cells in the blood; in this condition,
which is the opposite of anemia, the blood becomes highly viscous
(thick) and flows sluggishly
polymorphonuclear: Having various forms of nuclei
porphyria: A group of inherited conditions in which the production of
heme is deficient
pro-: Prefix indicating before or a precursor
profile: In chemistry, a group of tests that can be used to screen for an
abnormality that may not be readily detected in another way; often
involves 12 to 28 tests performed on a venipuncture sample
purpura: Hemorrhagic disease characterized by the escape of blood
into the tissues, under the skin, and through the mucous membranes;
results in spontaneous bruises and small red patches on the skin
rate reaction: Term referring to a measure of the rate of activity caused
by the presence of an analyte (the constituent or substance that is
analyzed); the more analyte in the specimen, the more activity occurs in
a specific period of time
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reference method: Method of comparing accuracy between two or more
testing systems or kits
reference range: Normal values; ranges of values for each assay for
people in a defined population
renal: Pertaining to the kidneys
reticul-: Prefix indicating a network
reticulocyte: A young red blood cell containing a network of basophilic
substances
rheumatoid arthritis: An incurable inflammatory disease of the joints
and connective tissue; an autoimmune disease
serology: The study of antigen-antibody reactions in blood samples (as
opposed to within the body)
serum: The fluid portion of the blood after fibrin and the formed
elements have been removed
splenectomy: Surgical removal of the spleen
systemic: Pertaining to or affecting the body as a whole
systemic lupus erythematosus (SLE): An incurable inflammatory
disease affecting many body systems; an autoimmune disease
thrombin: An enzyme that converts fibrinogen to fibrin
thrombo-: Prefix indicating blood clot
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thrombocytopenia: Reduced platelet count
thrombocytosis: Elevated platelet count
thrombotic thrombocytopenic purpura (TTP): An acute and potentially
fatal disorder, TTP involves fragmented RBCs, severe
thrombocytopenia, hemolysis, elevated reticulocyte count, fever, and
possible damage to many organs caused by lack of adequate blood
flow. Cause is unknown; without therapy (repeated administration of
large volumes of plasma), TTP is usually fatal
timed reaction: Term for test measurements such as blood coagulation
tests in which clotting times are calculated from reaction rates
transferrin: A blood protein synthesized by the liver; transferrin binds
to iron, transporting and releasing it to storage tissues (liver, bone
marrow, and spleen)
uremia: An excess of nitrogen waste in the blood
venipuncture: Blood taken from a vein