SEMINAR PRESENTATION

ON

WHITE BLOOD CELLS

PREPARED BY

AILAKO JOSEPH BOLU

FPA/ST/21/2-0785

SUBMITTED TO

THE DEPARTMENT OF SCIENCE TECHNOLOGY

SCHOOL OF SCIENCE AND COMPUTER STUDIES

IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE

AWARD OF NATIONAL DIPLOMA

 INTRODUCTION

White blood cells (WBCs) classification is an important step because it can assist

haematologists in the diagnosing several blood disorders, such as leukaemia, some

immunological disorders, and certain types of cancer. The analysis procedure can be done by

automatic and manual approaches to count and classify WBC. Manual classification of WBC

has many medical difficulties, including error in the accuracy of results due to sampling

errors and statistical probabilities and poor sensitivity, specificity and predictive values [1].

Furthermore, some automatic approaches in the laboratories have used instruments, such as

flow cytometry and automatic counting machine to detect and classify WBC. These

instruments do not make use of image processing techniques, and they can count and classify

WBCs quantitatively not qualitatively [2]. Therefore, it is necessary to design an automatic

system which includes image processing, signal processing, pattern recognition or deep

learning techniques to provide a qualitative and quantitative evaluation, precise results and

rapid processing. An automated classification of the WBC type system consists of six steps,

as shown in Fig.1: 1) image acquisition, 2) image pre-processing, 3) segmentation, 4) features

extraction and representations, 5) cell classification, and 6) the evaluation process.

Image Image Cell

Evaluation Cell Feature

Process

Fig. 1 Steps of automated classification of white blood cells [3].

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The Main types of WBC are: Granulocytes, Monocytes and Lymphocytes. There are seven

sub-types developed from these types. Granulocytes can be classified into Band neutrophils,

Basophils or Eosinophils. Monocytes can be classified into Macrophages or Dendritic cells,

and Lymphocytes can be classified into B-lymphocytes or Lymphocytes (as shown in Fig.3)

[1,2]. An overview of the structure of WBC, WBC types and their characteristics from [3,4]

is given in Section 1.1 below.

Structure of WBC

WBCs are produced from the bone marrow and found in the blood and lymphatic system. A

WBC has a nucleus, which often large and lobed, and it helps to distinguish WBC from the

other blood cells. Each WBC structure consists of a nucleus, cytoplasm and cell wall [1], as

shown in Fig. 2.

`Fig. 2 Diagram of WBC structure (Eosinophil cell example) consists of cell wall, a

nucleus and cytoplasm [4].

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High white blood cell count

If a person’s body is producing more white blood cells than it should be, doctors call this

leucocytosis.

A high white blood cell count may indicate the following medical conditions:

 allergic responses, such as due to an asthma attack

 those that may cause cells to die, such as burns, heart attack, and trauma

 inflammatory conditions, such as rheumatoid arthritis, inflammatory bowel disease, or

vasculitis

 infections, such as with bacteria, viruses, fungi, or parasites

 leukaemia

Surgical procedures that cause cells to die can also cause a high white blood cell count.

Low white blood cell count

If a person’s body is producing fewer white blood cells than it should be, doctors call

this leukopenia.

Conditions that can cause leukopenia include:

 autoimmune conditions such as lupus and HIV

 bone marrow damage, such as from chemotherapy, radiation therapy, or exposure to

toxins

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  bone marrow disorders

 leukaemia

 lymphoma

 sepsis, which is a severe type of infection

 vitamin B-12 deficiencies.

Common Treatments for White Blood Cell Disorders

Treatment for white blood cell disorders vary based on the diagnosis and severity of the

condition. Treatment ranges from:

 Taking vitamins.

 Taking antibiotics.

 Surgery to replace or repair bone marrow.

 Blood transfusion.

 Stem cell transplant.

WBC Types and Sub-types

The nuclei of WBC have different shapes, texture and sizes and might present one or more

lobes based on the reaction of their specific granules with a staining process as shown in

Fig.3. The most useful shape, size and texture information for cell segmentation and

classification comes from the nuclei of the WBCs [2]. To provide a brief review and

perspective, the features and functions of types and sub-types of WBCs and information

about WBC nuclei shape are explained as follows.

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• Granulocytes are phagocytes, which have the ability to ingest viruses, bacteria and

other parasites. They have visible granules or grains in their cytoplasm and have

large elongated or lobed nuclei. The diameter of cell measures approximately from

(12 – 20) μ, and their nucleoli cannot be seen. They account for approximately 60%

of our WBCs. The sub-types of granulocytes are: neutrophil, basophil and eosinophil

[5].

 Neutrophils are a part of the innate immune system and an essential line of

defence against bacteria. The shape of nucleus is like a “U” or a curled rod

prior to segmentation. They are also known as “band neutrophils”. The

diameter usually ranges between (10–18). The cytoplasm is moderate to

abundant with a few non-specific granules. Neutrophils account for

approximately (1% – 3%) of the peripheral WBCs. The diameter of a

segmented neutrophil cell usually ranges between (9 – 16). They have a

multi-lobed nucleus (three or four lobes normally), and these lobes may

overlap or twist [6]. The number of lobes can increase according to the cell

age. For example, a hyper segmented neutrophil cell has seven lobes in

mature stage. The intra-cellular granules are visible in the cytoplasm

(Giemsa-stained, high magnification) [5].

 Basophils secrete anticoagulant substances and antibodies that have the

ability to fight against hypersensitivity reactions in the blood stream. They

are the smallest circulating granulocytes. The basophilic granules in this cell

are large and very numerous, so they often mask the nucleus. The nucleus is

often bilobed or unsegmented and it is rarely separated into three or four

lobes. The average diameter ranges between approximately (10 – 15) μ.

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 Eosinophils have the ability to release toxins from their granules for killing

pathogens, such as parasites and worms. They are easily recognized in

stained smears by their large granules. The nucleus of the eosinophil has

often two lobes connected by a band of nuclear material. The diameter

usually ranges between (9 –15). They account between (1% –4%) of the

peripheral WBCs [2, 5].

 Monocytes stimulate osteoclasts cells, which have the ability to dissolve

bone. They are the largest type of WBCs. Their average diameter ranges

from (10-30) and are often referred to as scavenger cells or phagocytes.

They only contain one nucleus which is rarely or barely lobed. The nucleus

shape in monocytes is often bend-shaped (horseshoe) or kidney-shaped

(reform). Two types of cells can be developed from monocyte cell:

macrophages and dendritic cells [5].

 Macrophages are phagocyte cells which eat any type of dead cell in the

body. They are larger and live longer than neutrophils and have a large-size

single nucleus that is often kidney-shaped. They are also able to act as

antigen-presenting cells.

 Dendritic cells aid the development of antigen immunity. The shape of the

nucleus is small and round shaped, which as the cell matures, turns into a

large nucleus with an irregular star shape and cytoplasmic protrusions

(dendrites) [7].

 Lymphocytes are described according to size and cytoplasmic granularity

and can have a small or large nucleus depending on the maturation stage.

Small lymphocytes are well-known, and the diameter of a small nucleus

ranges from (6 –9) μ, while the diameter of a large nucleus is approximately

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 (10 – 15) μ. It contains just one nucleus which is rarely or barely lobed

[6,7]. The shape of the nucleus is slightly oval or round and stained dark.

Pathologists cannot easily distinguish T-cells and B-cells using traditional

light or electron microscopes. They always use an optical microscope to

distinguish between them.

 B lymphocytes (B-cells) produce antibodies and proteins that connect to

infected microbes or cells of the body and differentiate into a plasma cell in

immature stage. They are made in the bone marrow. They have oval nuclei.

They have a low fractal dimension and smooth cell surface. Pathologists

incubated the slides with Giemsa stain.

 T lymphocytes (T-cells) produce proteins called cytokines which help to

direct the response of other cells. They have circular nuclei and a wrinkled

cell surface. They are stained dark blue [6, 7].

Fig. 3 White blood cell taxonomy from bone marrow, including three main

types (Granulocytes, Monocytes and Lymphocytes) and seven sub-types

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 (Neutrophils, Basophils, Eosinophils, Macrophages, Dendritic, B-lymphocytes

and T-lymphocytes) [3].

PROPERTIES OF WBCs

1. Diapedesis is the process by which the WBCs squeeze through the narrow blood vessels.

2. Amoeboid Movement Neutrophils, monocytes and lymphocytes show amebic movement

characterized by protrusion of the cytoplasm and change in the shape.

3. Chemotaxis is the attraction of WBCs towards the injured tissues by the chemical

substances released at the site of injury.

4. Phagocytosis Neutrophils and monocytes engulf the foreign bodies by means of

phagocytosis.

Genesis of the White Blood Cells T

he granulocytes and monocytes are formed only in the bone marrow. Lymphocytes and

plasma cells are produced mainly in the various lymphogenous tissues—especially the lymph

glands, spleen, thymus, tonsils, and various pockets of lymphoid tissue elsewhere in the body,

such as in the bone marrow and in so-called Peyer’s patches underneath the epithelium in the

gut wall. The white blood cells formed in the bone marrow are stored within the marrow until

they are needed in the circulatory system.

Life Span of the White Blood Cells

The life of the granulocytes after being released from the bone marrow is normally 4 to 8

hours circulating in the blood and another 4 to 5 days in tissues where they are needed. In

times of serious tissue infection, this total life span is often shortened to only a few hours

because the granulocytes proceed even more rapidly to the infected area, perform their

functions, and, in the process, are themselves destroyed. The monocytes also have a short

transit time, 10 to 20 hours in the blood, before wandering through the capillary membranes

into the tissues. Once in the tissues, they swell to much larger sizes to become tissue

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macrophages, and, in this form, can live for months unless destroyed while performing

phagocytic functions. Lymphocytes enter the circulatory system continually, along with

drainage of lymph from the lymph nodes and other lymphoid tissue. After a few hours, they

pass out of the blood back into the tissues by diapedesis. Then, still later, they re-enter the

lymph and return to the blood again and again; thus, there is continual circulation of

lymphocytes through the body. The lymphocytes have life spans of weeks or months; this life

span depends on the body’s need for these cells.

Phagocytosis

The most important function of the neutrophils and macrophages is phagocytosis, which

means cellular ingestion of the offending agent. Phagocytes must be selective of the material

that is phagocytized; otherwise, normal cells and structures of the body might be ingested.

Whether phagocytosis will occur depends especially on three selective procedures. First,

most natural structures in the tissues have smooth surfaces, which resist phagocytosis. But if

the surface is rough, the likelihood of phagocytosis is increased. Second, most natural

substances of the body have protective protein coats that repel the phagocytes. Conversely,

most dead tissues and foreign particles have no protective coats, which makes them subject to

phagocytosis. Third, the immune system of the body develops antibodies against infectious

agents such as bacteria. The antibodies then adhere to the bacterial membranes and thereby

make the bacteria especially susceptible to phagocytosis. To do this, the antibody molecule

also combines with the C3 product of the complement cascade, which is an additional part of

the immune system The C3 molecules, in turn, attach to receptors on the phagocyte

membrane, thus initiating phagocytosis. This selection and phagocytosis process is called

opsonization.

Inflammation:

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Role of Neutrophils and Macrophages Inflammation When tissue injury occurs, whether

caused by bacteria, trauma, chemicals, heat, or any other phenomenon, multiple substances

are released by the injured tissues and cause dramatic secondary changes in the surrounding

uninjured tissues. This entire complex of tissue changes is called inflammation. Inflammation

is characterized by

(1) vasodilation of the local blood vessels, with consequent excess local blood flow;

(2) increased permeability of the capillaries, allowing leakage of large quantities of fluid into

the interstitial spaces;

(3) often clotting of the fluid in the interstitial spaces because of excessive amounts of

fibrinogen and other proteins leaking from the capillaries;

(4) migration of large numbers of granulocytes and monocytes into the tissue; and

(5) swelling of the tissue cells. Some of the many tissue products that cause these reactions

are histamine, bradykinin, serotonin, prostaglandins, several different reaction products of the

complement system, reaction products of the blood clotting system, and multiple substances

called lymphokines that are released by sensitized T cells. Several of these substances

strongly activate the macrophage system, and within a few hours, the macrophages begin to

devour the destroyed tissues. But at times, the macrophages also further injure the still-living

tissue cells.

The Leukaemia’s

Uncontrolled production of white blood cells can be caused by cancerous mutation of a

xylogenous or lymphogenous cell. This causes leukaemia, which is usually characterized by

greatly increased numbers of abnormal white blood cells in the circulating blood.

Types of Leukemia.

Leukaemia’s are divided into two general types:

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 1- lymphocytic leukaemia’s: The lymphocytic leukaemia’s are caused by cancerous

production of lymphoid cells, usually beginning in a lymph node or other lymphocytic

tissue and spreading to other areas of the body.

2- xylogenous leukaemia’s: The xylogenous leukaemia, begins by cancerous production

of young xylogenous cells in the bone marrow and then spreads throughout the body

so that white blood cells are produced in many extra medullary tissues—especially in

the lymph nodes, spleen, and liver. In xylogenous leukaemia, the cancerous process

occasionally produces partially differentiated cells, resulting in what might be called

neutrophilic leukaemia, eosinophilic leukaemia, basophilic leukaemia, or monocytes

leukaemia. More frequently, however, the leukaemia cells are bizarre and

undifferentiated and not identical to any of the normal white blood cells. Usually, the

more undifferentiated the cell, the more acute is the leukaemia, often leading to death

within a few months if untreated. With some of the more differentiated cells, the

process can be chronic, sometimes developing slowly over 10 to 20 years. Leukemic

cells, especially the very undifferentiated cells, are usually non-functional for

providing the normal protection against infection.

White Blood Cell Measurements

The Cell-Dyn gives three basic WBC measurements, i.e. the WIC, WOC and the reported

white blood cell count. With each sample analysis, the WIC and WOC are measured and the

values compared. A flag will be displayed if the difference between the two counts exceeds a

predetermined value. The WOC is the primary value reported as the white blood cell count.

Differences can be due to resistant red cells, nucleated red cells or fragile white blood cells.

Nucleated red cells will be included in the total white cell count of the WIC, while they will

not be included in the WOC count. Fragile white cells will cause a false low white cell count

in the WIC, as the lytic reagent can also damage them, while they will still be counted in the

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WOE. Lytic-resistant red cells will cause interference in the WOC, leading to a large

percentage of the WOC count located in the stroma region. The comparison of the two

methods allows the instrument to identify and report these abnormalities in an attempt to give

an accurate white blood cell count.

White Cell Impedance Counting (WIC)

For determination of the WIC, a dilution of the sample is made with diluent (LIN 9923 1-0 I)

and WICIHGB Lyse (LIN 99431-0 I). The latter reagent lyses the red cells and strips the

cytoplasm from the white blood cells, leaving only the white blood cell nuclei to be counted,

using the aperture impedance method. In order to obtain an absolute cell count, the precise

volume of blood that passes through the aperture during the count cycle is known. A

volumetric metering process is used to ensure that a precise volume of sample is analysed.

Cells that exit the aperture tend to swirl around and can re-enter the sensing zone. To prevent

this, and thus the cells from being counted twice, a von Behrens Plate is located in the WIC

counting chamber. The WIC is also corrected for coincidence passage loss. Coincidence

passage loss is a reduction in the count due to the fact that two or more cells can pass through

the aperture simultaneously. This will lead to the generation of a single pulse with high

amplitude and increased pulse area, giving the impression that only one large cell has passed

through. The coincidence passage loss can be predicted statistically and can be corrected.

White Cell Optical Counting (WOC)

For determination of the WOC, a dilution of the sample is made with the sheath reagent (LIN

99321-01). The cellular integrity of the white cells, in the sheath fluid, is maintained, but the

basophils change slightly due to their hygroscopic nature. A measured volume of this dilution

is injected into the sheath stream. The cells are aligned in single file as they pass through the

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WOC flow cell. The WOC flow cell is an optically clear quartz chamber and the light source

is a vertically polarised Helium Neon laser.

CONCLUSION

White blood cells are an important component in the human blood. All white blood cells

have nuclei, which distinguishes them from the other blood cells and also between white

blood cell types and subtypes themselves. In this Chapter, white blood cell types and their

structure are reviewed. An automatic white blood cell classification system, including image

acquisition, pre-processing, segmentation and feature extraction is presented in this Chapter.

Different automatic techniques that have been used for feature extraction and classification

of white blood cells, from 2005 to state-of-the-art, are also reviewed. Despite the large

amount of work that has been undertaken in this field, segmentation, feature extraction and

classifications of white blood cells is still challenging, particularly in the presence of non-

uniform illumination, low resolution of images, and different shape, size and phase of

maturation of cells. The challenges still faced by an automated classification system are also

summarised by this chapter.

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 REFERENCE

Adjouadi M, Zong N, Ayala M. Multidimensional pattern recognition and classification of

white blood cells using support vector machines. Particle & Particle Systems

Characterization. 2005;22(2):107-18.

Al-Dulaimi K, Chandran V, Banks J, Tomeo-Reyes I, Nguyen K , editors. Classification of

White Blood Cells Using Bispectral Invariant Features of Nuclei Shape . International

Conference on Digital Image Computing: Techniques and Applications (DICTA);

2018:IEEE

Al-Dulaimi K, Tomeo-Reyes I, Banks J, Chandran V, editors. White blood cell nuclei

segmentation using level set methods and geometric active contours. International

Conference on Digital Image Computing: Techniques and Applications (DICTA); 2016:

1-7; IEEE.

Bishop CM. Pattern Recognition and Machine Learning: Material: Springer; 2006.

Blumenreich MS. The white blood cell and differential count. Clinical Methods: The History,

Physical, and Laboratory Examinations [Internet] 3rd ed Boston: Butterworths. 1990.

Cseke I, editor A fast segmentation scheme for white blood cell images. Conference C:

Image, Speech and Signal Analysis, Pattern Recognition;1992;Vol III: IEEE.

Clark VL, Kruse JA. Clinical methods: the history, physical, and laboratory examinations.

Jama. 1990; 264(21):2808-9.

15
Carleton HM, Drury RAB, Wallington EA. Carleton's histological technique: Oxford

University Press, USA; 1980.

Cellavision-Database, “Cellavision – leading the way in digital cell morphology,”

CellaVision AB, 2011. Available: http://www.cellavision.com/

Dorini LB, Minetto R, Leite NJ, editors. White blood cell segmentation using morphological

operators and scale-space analysis. XX Brazilian Symposium on Computer Graphics and

Image Processing (SIBGRAPI); 2007: IEEE.

Kemal J. Laboratory manual and review on clinical pathology. OMICS Group. 2014.

Standring S. Gray's anatomy e-book: the anatomical basis of clinical practice: Elsevier Health

Sciences; 2015.

Wadsworth-Center, “White blood cell images,” New York State Department of Health.

[Online]. Available: http://www.wadsworth.org/

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