Hematology_Handbookfor Personal Use

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.

http://en.wikipedia.org/wiki/Blood_coagulation

http://en.wikipedia.org/wiki/Blood


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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.


37

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.


38

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.


blood; increased in appendicitis,

pneumonia.


39

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.


blood.


41

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.


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


blood; increase in tuberculosis and

monocyte leukemia.


41

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


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.


42

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


43

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


44

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.


45

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


46

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


47

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.


48

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


49

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).


51

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.


51

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.


52

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).


53

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.


54

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.


55

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).


56

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


57

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


58

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


59

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.


61

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).


61

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.

http://en.wikipedia.org/wiki/Chemical_compound


62

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.


63

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).


64

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.


65

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.


66

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


67

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


68

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).


69

Reagents

Trisodium Citrate 3.8%

Trisododium Citrate anhydrous 3.8g


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).


71

Figure 9.1

Reference values

Sex N.R.

Male 1 – 10mm/hr

Female 3 – 15mm/hr

Tem

per

atu

re


71

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.


72

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


Always should be freshly prepared before use.


73

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.


74

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.


75

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


76

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.


77

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.


78

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.


79

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.


81

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.


81

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).


82

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


83

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.


84

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


85

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.


86

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.


87

- 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.


88

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.


89

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.


91

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


92

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.


93

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.


94

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.


95

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.


96

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.


97

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).


98

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.


99

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.


111

Figure 12.3: Illustrates the different agglutination reaction

strength between Ag & Ab either for forward

and reverse grouping.


111

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


112

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.


113

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.


114

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.


115

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.


116

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.


117

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.


118

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.


119

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


111

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.


111

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.


112

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.


113

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.


114

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.


115

Atlas of Peripheral

Blood Smears


116

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


117

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


118

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


119

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


121

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


121

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.


122

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


123

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.


124

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.


125

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


126

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.


127

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


128

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


129

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


131

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


131

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.


132

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


133

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


134

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


135

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)


136

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).


137

Appendix I


138

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.

http://onlinelibrary.wiley.com/doi/10.1111/clh.2002.24.issue-6/issuetoc

http://www.cellavision.com/

http://www.abbottdiagnostics.com/

http://www.beckman.com/products/applications/partChar/%20CoulterPrinciple_dcr.asp

http://www.beckman.com/products/applications/partChar/%20CoulterPrinciple_dcr.asp


139

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.

http://www.nlm.nih.gov/medlineplus/ency/article/003344.htm/


141

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

Hematology Handbook Reference & Automation Methods- First Edition

141

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


142

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


143

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


146

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"


147

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


149

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


152

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


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Note:


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