Haematological AnalyserAuthor: Ivana Marinović

Mentor: dr. Matija Milanič

Academic year 2016/2017

Abstract

Blood is a flowing tissue, which enables many processes in our body. With counting blood cells and discovering abnormalities in cells, we can recognize many diseases before they are life-threatening and cure them on time. To test large amount of blood samples, we need to have some devices to do it instead of examination of each sample under the microscope. In this seminar, we will briefly repeat what we learned in high school about blood and blood cells. Then, we will get to know physics behind blood count devices based on Coulter principle and fluorescence, learn, how they work and see, where are possibilities for development in the future.

Contents

1. Introduction.........................................................................................................................2

2. Blood...................................................................................................................................3

2.1. Plasma..........................................................................................................................3

2.2. Blood cells....................................................................................................................3

2.2.1. Erythrocytes..........................................................................................................3

2.2.2. Leukocytes............................................................................................................4

2.2.3. Thrombocytes.......................................................................................................4

2.3. Complete blood count (CBC).......................................................................................4

3. Haematological analyser......................................................................................................5

3.1. Development................................................................................................................5

3.2. Coulter counter.............................................................................................................5

3.2.1. Maxwell’s mixture theory.....................................................................................5

3.2.2. Electrical circuit approximation............................................................................5

3.2.3. Coulter counter......................................................................................................6

3.3. Flow cytometry............................................................................................................7

3.3.1. Fluorescence.........................................................................................................8

3.3.2. Elastic light scattering...........................................................................................8

3.3.3. Fluorescence spectroscopy....................................................................................9

3.4. Future improvements and methods............................................................................11

4. Conclusion.........................................................................................................................11

1. Introduction

Blood is the most important fluid in human body. It takes care of our immune system, it feeds our cells and gives them oxygen for breathing. Many conditions can be recognized trough blood examination. Our blood is made of plasma and blood cells. There are few blood cell types and each of them has its own task. Because many diseases are recognized and confirmed trough blood examination, there is a huge amount of tests that has to be done as quickly as possible. Counting cells manually with the use of microscope is reliable but time consuming, so there has been a need to automatize procedure. Scientists have developed devices called haematological analysers, that distinguish blood cell types and count how many cells of each type is in the sample. First haematological analyser was invented by Coulter in 1953 [1]. Newer analysers work on a fluorescence principle. These methods enabled fast and accessible blood count. One sample can be tested in less than 30 seconds. This field is rapidly

progressing in many directions (in vivo, portable devices, faster analyses) and it is impossible to tell, which will prevail in the future.

2. Blood

Blood is liquid tissue that flows through each part of human body. There are approximately 5 litres of this fluid in a body. Blood carries nutrients to cells and takes away their waste. It contains immune cells that fight infections, red blood cells that carry oxygen and carbon dioxide and platelets, which allows blood to clot. Blood circulates around body through blood vessels. Heart pumps the blood around the body with rate of 5 litres per minute. Blood is made of 44% of plasma and 56% of blood cells. The structure of blood is illustrated on image 1.

Image 1: Components of blood [1]

2.1. PlasmaBlood plasma is made mostly of water and dissolved proteins, electrolytes, hormones, gases and products of cell metabolism.

2.2. Blood cellsThere are three major groups of blood cells: white blood cells called leukocytes, red blood cells also called erythrocytes and platelets. Their functions are immunological response, transport of gases and hemostasis. All of them are formed in bone marrow from precursor cells.

2.2.1. Erythrocytes They are so called red blood cells. Their name tells us these cells are responsible for the colour of blood. They represent the majority of blood cells and 25 percent of all cells in body[2]. Shape of erythrocytes is very characteristic biconcave shape. These cells also don’t have nucleus so they have more space for haemoglobin. Their task is to transport oxygen and in smaller part CO2. Every second 2-3 million of them are produced and they live approximately 120 days. Then they decompose in spleen. They are also membrane molecules carrier. If blood transfusion gives us an incompatible blood type, which is based on presence and absence of specific antigens on red blood cells, erythrocytes clump together.

Haemoglobin is protein which is found in erythrocytes. It task is to transport oxygen. It is made of four polypeptide chains (globin), each chain surrounds one red pigment molecule called hem, which contains an iron ion, where one oxygen molecule is bonded. When haemoglobin bonds oxygen in lungs, it forms oxyhemoglobin. It carries oxygen to cells and releases oxygen, this form of haemoglobin is called deoxyhaemoglobin. Some molecules bind carbon dioxide with forming carbaminohaemoglobin.

2.2.2. Leukocytes They protect body from microorganisms and form antibodies. They are divided into subgroups by nucleus types: monocytes, lymphocytes and granulocytes, which also divide into three groups.

2.2.3. Thrombocytes Or platelets. They are sacs of cytoplasm which transport important factors for blood coagulation and blood vessel clothing.

2.3. Complete blood count (CBC)Complete blood count is a name for blood cell count. It contains information about number of blood cells, concentration of haemoglobin and parameters important for recognition of some conditions.

Red blood cell count also contains parameters such as average size of RBC (MCV), amount of space RBC’s take in blood (haematocrit), and amount (MCH) and concentration (MCHC) of haemoglobin in red blood cell.

What CBC normal values are is shown in table below on image 2.

Image 2: Complete blood count normal values [3]

3. Haematological analyser

Haematological analysers are devices that recognize and count blood cells. Modern automated analysers are based on optical methods or impedance methods, or combination of both. Basic analysers distinguish between blood cell types, but more advanced ones can difference between subgroups of types.

3.1. DevelopmentFirst blood count was done with the use of microscope with cell recognition and counting performed manually. This method was time consuming and unreliable. More advanced method appeared at the end of 19th century, when predecessor of automated blood cell counters was invented. It was based on a measurement of light loss caused by scattering or absorption on diluted blood. This method could be only used in red blood cell count, but it wasn’t very reliable because there was no method developed yet to precisely determine light loss. When first photodetectors were made, that problem was solved. But the trouble were variations in cell size, shape and amount of haemoglobin in them. The solution was flow device that isolated cells in small droplets. In ninety seventies method was upgraded to fluorescence based flow cytometry with the use of laser.

While optical method was developing, new method based on newly discovered Coulter effect introduced in 1953. [5] It was based on low electrical conductivity of blood cells compared to saline solution. [4] This effect will be introduced in the following paragraphs.

3.2. Coulter counter3.2.1. Maxwell’s mixture theory

Blood cells in a fluid can be described as suspension of spherical particles in a medium. Complex permittivity of the mixture ͠εmix is given by:

~ε mix=~εm

1+2 Φ~f CM

1−Φ~f CM

(1)

where ~f CM=~ε p−~εm

~ε p+2~εm, ͠εp complex permittivity of a particle, εm complex permittivity of the

medium and Φ volume fraction. Because cell isn’t homogenous solid object, we must treat it as it is homogenous object with thin membrane. Complex cell permittivity is given by

~ε p=~ε mem

( RR−d )

3

+2( ~εi−~εmem~εi+2 ~ε mem

)( R

R−d )3

−(~εi−~εmem

~εi+2~εmem ) (2)

where ~ε memis complex membrane permittivity, ~ε i complex cytoplasm permittivity R radius of the cell and d thickness of the membrane. [7]

3.2.2. Electrical circuit approximation For simplified analysis we approximate our system as equivalent electrical circuit. Cell is made of a cytoplasm, which is represented by resistor, and conductor, which represents cell membrane (Image 3). In this approximation we ignore membrane reactance and cytoplasm capacitance.

Image 3: Electrical circuit approximation of a cell [5]

For a cell positioned between two planar electrodes electrical components are:

For medium:

Rm= 1

σm(1−3 Φ2 )G f

(3)

Cm=ε∞G f (4)

Cell components:

Cmembrane=9Φ R Cmembrane .0

4Gf (5)

Ri=4( 1

2σm+

1σ i )

9 ΦGf

(6)

Where σm is suspending medium conductivity, σi cytoplasm conductivity and Gf is geometric constant, for ideal parallel electrode simply the ratio of electrode area to gap. Specific

membrane capacitance is Cmembrane, 0=εmem

d and limiting high-frequency permittivity of

suspension ε ∞≃ εm(1−3Φεm−εi

2 εm+εi). [7]

Typical values of dielectrical properties of cell are: εo = 8.854×10−12 F m-1, d = 5nm, εm = 80 εo, σm = 1.6 S m-1, εmem = 11.0 εo, σmem = 10 S m-1, εi = 60 εo, σi = 0.6 S m-1 [8]

This simplified circuit model is good enough for single-cell impedance measurements. For more advanced systems, where resistance of membrane and capacitance of cytoplasm vary too much use of complete circuit model is required, where all electrical properties of cell must be considered.

3.2.3. Coulter counter First cell counter was made in 1956 [1]. It measured the change in resistance as particle passed between two isolated chambers filled with fluid. Two electrodes are positioned at each side of an opening. Through the opening flows electrical current. As particle passes through the opening, it induces change in resistance which is noticed as a change in current and calculated with simple Ohm’s law. Simple Coulter counter is shown on image 4. The cell size can be calculated with equation 5. Analysis of Coulter counter was made in 1970 by Deblois and Bean [7].

Image 4: Coulter counter illustration

Resistance of a tube filled with electrolyte with diameter Dt, length Lt and resistivity ρm is

calculated by: Rt=4 ρm Lt

π Dt2 .

They calculated the resistance of the mixture:

Rmix=4 ρm Lt

π Dt2 (1+

d p3

Dt2 Lt

) (7)

The change of resistance caused by particle is then: ∆ R=4 ρm d p

3

π Dt4 , where dp stands for cell

diameter. [5] Because cell size varies on a large scale, the change they make in resistance is even larger because in this equation we have a cube of cell diameter. Suspending medium resistivity and tube diameter is the same for each cell, so they don’t affect the change in resistance.

The problem with the use of Coulter counter is that the opening must be big enough that larger cells can go through, which makes it possible for two smaller cells to clump and pass by together and be falsely recognized as one large cell. To reduce this problem, our sample must contain additives to dilute solution. [6]

3.3. Flow cytometry In flow cytometry, cells must flow through device one by one. Our blood cells are different shapes and various diameters, so there must be a method to line them up. System which arranges cells into line is called fluidic system. It works on a principle called hydrodynamic focusing (image 5). It has two fluids flowing in it. In the middle of the system we put our sample, around our sample flows sheet fluid which flows under slightly larger pressure, which makes it flow faster. As this fluid flows, it narrows the centre fluid flow and creates single cell flow. Thickness of the sample is manipulated with adjusting pressure on sample fluid. If our flow is laminar, these fluids don’t mix. [7] Commonly used sheath fluid is simple saline solution. [8]

Image 5: Hydrodinamic focusing [7]

When cells are lined up, they pass through one or more laser beams. Flow cytometer can use fluorescence spectroscopy, elastic scattering spectroscopy or combination of both, which is the most frequent. [7]

3.3.1. Fluorescence Fluorescence spectroscopy is, as its name says, based on fluorescence phenomenon. This process occurs in some chemical compounds, called fluorophores or fluorescent dyes. These substances emit light of longer wavelengths after they were excited by light of shorter wavelengths. When molecule absorbs energy, it reaches higher excited states (Image 6). When molecule is excited to higher vibrational levels, it loses this excess of energy quickly and goes to the lowest vibrational level of the excited state. Most molecules in higher than second excited states go through internal conversation and cross to the highest vibrational level of lower exited state which has the same energy. Then they lose energy again until they reach lowest vibrational level of first excited state. Now they emit fluorescence light to reach the ground state. [9]

Image 6: Energy level transitions illustration [9]

3.3.2. Elastic light scattering Mie or forward scattering occurs when light is scattered on particles with similar size to the wavelength of incident light.

Rayleigh and Tindall scattering is light scattered in all directions. Rayleigh scattering is light scattered from molecules while Tindall scattering is caused from small particles in colloidal suspension. [10] Intensity of light scattered by Rayleigh scattering is calculated with:

I=I 08 π4 N α2

λ4 R2 (1+cos2θ ) (6)

Forward scattering is correlated with cell size and side scattering is proportional to the cell granularity. [11] Directions of light scattered by both scatterings is shown on image 7.

Image 7: Direction of waves scattered by different scatterings [10]

3.3.3. Fluorescence spectroscopy Most of the modern cell counters are based on fluorescence phenomenon. To measure fluorescence, we must dye our sample with fluorescent dyes that are bonded on speciffic antibodies. Most commonly used are FITC (fluorescein isothiocyanate; emitted wavelength 520nm) and PE (phycoerythrin; fluorescence wavelength 576 nm), both can be excited by 488nm argon-ion laser. Each cell has specific proteins on the cell membrane called antigens which bind with explicit type of antibodies. [12] Then our sample must be illuminated to

absorb light. First we measure forward scatter, which is proportional to cell size. To gain information of cell granularity and internal complexity, we measure side scattered light, which is separated from the fluorescent light with dichroic mirror. [13] If we have more fluorescent antibodies, we divide fluorescent light with dichroic mirrors and measure each wavelength separately. Then, emitted light is measured by photomultipliers (Image 9). When we measure fluorescence, we measure events. When the cell passes laser and emits fluorescence, we detect it and count (Image 8). [7]

Image 8: Fluorescence detection [12]

Image 9: illustration of flow cytometer [13]

Cell type differentiation is made with plots, where intensity of forward scattering is plotted against side scattered cells. Cells, which emitted fluorescence of specific wavelength are coloured (Image 10).

Image 10: Fluorescence of two different scatterings plotted against each other [12]

Coulter counters (Image 12) are cheaper and smaller devices, which makes them easy to move. But they don’t give us complete information of blood cells because they don’t distinguish leukocyte types. Flow cytometers (Image 11) are larger and more expensive, but they are quick and reliable.

Image 11: Flow cytometer [14] Image 12: Coulter counter [15]

3.4. Future improvements and methodsEven though modern devices work at impressive speed, quicker and more advanced techniques are needed. One possible method is development of contrast agent like quantum dots. New algorithms for reducing number of false results are being developed. Their task is to detect when result could be false and require morphologic review.

There is also need for smaller portable devices as current ones are quite large machines and as such are not portable. This is possible because of improvements science has offered such as smaller optical components and smaller and stronger electronic parts and more efficient software.

New improvements have been made in image based analysers. This method started as manual count with microscope, but now method is automatized which makes it quicker and cheaper. This method requires smaller devices, which are portable. Technique is based on in-line holographic imaging technique which uses partially coherent light to record cell shadows on sensor. Cell information is gained through recovery of phase information of each cell. New

devices using simple image analysis for blood count were also recently developed. They use simple sample preparation and automated image analysis on small blood volumes which is useful technique for portable cell counters.

In-vivo counters are researched because of the need of noninvasive techniques of blood count. This method could improve possibility of detecting rare abnormal cells. Current flow devices cannot be adapted for in-vivo test because the flow in blood vessels is slower, some cells are quicker than other which makes it difficult to perform blood count. Scientists are working on application of optical, ultrasonographic, photothermic and photoacoustic methods for in-vivo blood count. [4]

4. ConclusionBlood is very important tissue in our body. It contains information about many conditions. It is quite simple to detect them through blood count. Blood count consists of number of blood cells, their volume and concentration. To perform blood count are currently available two main methods: Coulter counter and flow cytometry. Coulter counter is based on electrical properties of cells. Flow cytometry is based on light absorption and scattering. In future, there are many options for further development of this field. Currently are being researched methods to count blood in vivo, make the count faster, cheaper and more accessible, to develop smaller portable devices and so on. Future will tell, which is the best method.

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