INTRODUCTION

Blood is a red syrupy fluid that is thicker than water. It carries out the important functions of transporting oxygen and getting rid of carbon-dioxide and other waste products as well as transporting nutrients to our cells. And it also plays a crucial function in immune system and in maintaining a relatively constant body temperature. Blood is a tissue that is composed of many different kinds of components. Blood is composed of cells, cellular fragments and fluid. And these are red blood cells, white blood cells, platelets and plasma.

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Red blood cells (erythrocytes) are cells with no nuclei. They appear relatively large under microscope. These cells are similar to the primitive prokaryotic cells of bacteria. Red blood cells transport oxygen from the lungs to all of the living tissues of the body and carry away carbon-dioxide. ‘’The red blood cells are produced continuously in the bone marrow from stem cells at a rate of about 2-3 million cells per second’’ (O’Neil, 2007).

White blood cells (leucocytes) are much larger than red blood cells, but it is able to change its shape so it can squeeze through little gaps. White blood cells contain nucleus and has colourless cytoplasm. They are involved in protection from bacteria, fungi, parasites and viruses. These cells are formed in the white bone marrow of the long bones. White blood cells have five types (each cell has a task in helping the body fight infections):

Granulocytes:

Neutrophils (engulf bacteria) Eosinophils (antihistamine properties) Basophils (produce histamine and heparin)

Agranulocytes:

Monocytes (engulf bacteria) Lymphocytes (produce antibodies)

(Fullick, A., 1994, p.142)

Platelets (thrombocytes) are cell parts with no nuclei. It releases blood clotting chemicals at the site of wounds. Platelets are fragments of a larger cell called megakaryocyte, which stays in the bone marrow after it differentiates and matures from the stem cell. The platelets circulate body after leaving the bone marrow. Platelets contain high concentrations of 5-Ht (G-protein linked receptors), which is not synthesized inside the platelets but is actively absorbed from the circulation by specific uptake mechanisms. 5-HT is stored in secretor granules and discharged during platelet aggregation in reaction to endothelial damage. 5-HT causes a direct local constrictor effect as well as an increase in platelet aggregation, thereby encourages haemostasis. Haemostasis is a vital defence mechanism aimed at minimizing blood loss during injury.

There are billions of living blood cells in human body that are floating in liquid called plasma. The plasma is a mixture of water, sugar, fat, protein, potassium, calcium salts and many other chemicals that help for clotting when necessary to stop bleeding. More than 92% of plasma is water. As the heart pumps blood to cells throughout the body, plasma brings nourishment to them and also removes the waste products of metabolism.

The layers of blood cells can be seen under the microscope, once it is centrifuged. This machine spins the blood around so fast that it separates the red blood cells from the white blood cells and from platelets. In this process the red blood cells sink to the bottom as they are heavier and have more solid parts. Therefore the plasma remains right at the top, as it is lighter.

(Genetic science learning centre, 2008)

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When clotted blood is centrifuged to separate the cellular components a fluid is formed, called serum. The difference between plasma and serum is that the latter lacks fibrinogen and some of the coagulation factors. Fibrinogen is an important blood protein made in the liver and it is involved in clotting. Fibrinogen is transformed into fibrin by an enzyme called thrombin. And this enzyme is essential to coagulation. Coagulation is to prevent excessive blood loss. A clot is composed of two elements, which are platelets and fibrin. Blood coagulation takes place due to the sequential conversion of series of inactive proteins into catalytically active protease enzymes. Anticoagulants correspond to important therapeutic agents in the management of thromboembolic disease. (Blann, 2007; Lewis, 2006).

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The main purpose of the Haematology Laboratory is to provide assistance in the diagnosis, monitoring and treatment of patients. To accomplish this, the components of the blood are analysed. This is almost always achieved in custom-designed equipment. All the blood tubes and forms must be fully labelled to avoid any possible fatal error. Caution needs to be taken when dealing with the blood samples and all laboratory rules must be applied.

Red blood cells contain a special protein called Haemoglobin (Hb), which is responsible for making red blood cells red. Hb is a protein that is premeditated to carry oxygen from the lungs to the tissues; this is where the oxygen is given up to take part in respiration. The standard range of Hb in blood differs between sexual categories. Hb values for women are lower due to menstruating and higher for men. But even when the women are in post-menopausal period, the Hb levels are still lower than age-matched men. This is because the latter produce testosterone to stimulate red cell production. ‘’ The red blood cell count (RCC): Normal range in men 4.32 to 5.66x 1012/L, in women 3.88 to 4.99?1012/L.’’ (Blann, 2007. p.8).

The amount of Hb in blood could be measured in following conversion to azidmethaemoglobin by the addition of sodium nitrate and sodium azide. This is a method employed by one portable haemoglobinometer (HemoCue, Clandon Scientific Ltd.)- This instrument only measures Hb. It takes up measurements at two wavelengths, which are 570nm and 880nm to compensation for turbidity. The reading of the haemoglobin content of the sample is measured in gram/litre blood. A modification of this instrument permits accurate measurements down to 0.1 g/L so that it is also suitable for measurement of dilute solutions of haemoglobin, e.g. haemoglobin in fluid salvaged during surgery, plasma or urine. (Bain, 2002, p. 18).

Haematocrit is known as packed cell volume (PVC) or Hct, Crit. This is a count to calculate the whole blood that is taken up by all the blood cells. Haematocrit is measured in percentage and can be converted to SI units by diving into 100.

The red blood cell indices are mean cell volume (MCV), mean cell haemoglobin (MCH) and mean cell haemoglobin concentration (MCHC). MCV is the size of the average (mean) red blood cell. MCV can be calculated by; PCV?RCC. MCH reports the average amount (mass) of haemoglobin in the average cell. The size of the cell is not taken into account. MCH can be calculated by; Hb?RCC. MCHC is the average concentration of haemoglobin inside the average size cell. MCHC can be calculated by Hb?Hct.

Aim is to study rat blood sample given to us. Therefore count the visible red blood cells under microscope at high power using Neubauer Chamber. Following this count of red blood cells calculations will be applied in which it will determine the haematology of the provided rat blood sample.

MATERIALS & METHODS

Method carried out is exactly the same as in the practical schedule. The practical booklet is called ‘Level 1, Biosciences Programme; Human Physiology, 1LFS0029 ’. The practical carried out is on pp. 29-32 and called HP6- Haematology’. No amendments have been made.

RESULTS

Table 1:

Comparing the values obtained from rat blood samples to the rat reference ranges

Parameter Values obtained from rat blood samplesReference ranges for rats Difference between the standard and obtained results Red blood cell count (RBC/RCC)8.64?1012/l6.76-9.75?10/lWithin the range Determination of haemoglobin (Hb)178 g/l115-161 g/lHigher by 17g/l of the maximum range value Haematocrit (Hct/PVC)0.49 l/l0.38-0.51l/lWithin the range Mean cell volume (MCV)57 fl69-85 flLower by 12 fl of the minimum range value Mean cell haemoglobin (MCH)20.6 pg24-30 pgLower by 3.4 pg of the minimum range value Mean cell haemoglobin concentration (MCHC)363.27 g/l320-360 g/lHigher by 3.27 g/l of the maximum range value

Table 1 is showing values that have been gained from rat blood sample during the experiment and comparing it with the rat reference range. The difference has been recorded above (Table 1). The standard values and all the units are included in the table.

As seen in Table 1; some values obtained are not within the rat reference range. These values will be discussed in the discussion. To obtain red blood cell indices (MCV, MCH and MCHC), series of calculations have been made; these calculations are shown step by step in the calculations section. Reference range values were taken from ‘Haematology Proforma’ booklet given during the experiment.

Table 2:

Comparing the values obtained for rat blood samples to human (female and male) reference ranges.

ParameterValues obtained from rats blood samplesReference ranges for male (with the units)Difference between the standard and obtained resultsReference ranges for female (with the units)Difference between the standard and obtained results Red blood cell count (RBC/RCC)8.64?1012/l3.5-6.0?10/lHigher by 2.64?1012/l of the maximum range value3.5-5.5?10/lHigher by 3.14?10/l of the maximum range value Determination of haemoglobin (Hb)178 g/l135-180 g/lWithin the range115-165 g/lHigher by 13g/l of the maximum range value Haematocrit (Hct/PVC)0.49 l/l0.35-0.55 l/lWithin the range0.3-0.5 l/lWithin the range Mean cell volume (MCV)57 fl80-96 flLower by 23 fl of the minimum range value80-96 flLower by 23 fl of the minimum range value Mean cell haemoglobin (MCH)20.6 pg27-32 pgLower by 6.4 pg of the minimum range value27-32 pgLower by 6.4 pg of the minimum range value Mean cell haemoglobin concentration (MCHC)363.27 g/l320-360 g/lHigher by 3.27 g/l of the maximum range value320-360 g/lHigher by 3.27 g/l of the maximum range value

Table 2 is showing values that have been gained from rat blood sample during the experiment and comparing it with the human (male and female) reference range. The difference has been recorded above (Table 2). The standard values and all the units are included in the table.

As seen in Table 2; some values obtained are not within the rat reference range; this was expected as these values are compared to the human reference range values. Values are different between sexual categories, apart from the values for red blood cell indices (MCV, MCH and MCHC). These values will be discussed in the discussion. To obtain red blood cell indices (MCV, MCH and MCHC), series of calculations have been made; these calculations are shown step by step in the calculations section. Reference range values were taken from ‘Haematology Proforma’ booklet given during the experiment.

Calculations

Red cell count (RCC)

The number of erythrocytes counted in 80 small sequences on the Neubauer haemocytometer is 864. Multiplying this value by 1010 will give the number of erythrocytes in one litre of blood: 864?1010 = 8.64x 1012 /l

This RCC value is higher than an average value of standard human haematological indices. The standard value of human haematological indices for male is 3.5-6.0?1012/l and for female is 3.5-5.5?1012. The RCC value is within the average value for standard rat haematological indices. The standard value of rat haematological indices is 6.76-9.75?1012/l.

Haematocrit (PCV)

The percentage of a blood sample occupied by erythrocytes after centrifugation in a capillary tube is 49%.

Hct= (distance of red blood cell collected in the centrifuge tube ? distance of total blood composition) x 100

This value can be converted into SI units, dividing by 100: 49(%) ? 100= 0.49 l/

The PVC value is within the normal range of value for human males and females. The standard value of PVC for male is 0.35-0.55 l/l and for female is 0.30-0.50 l/l. The PVC value is also within the normal range of values for rats. The standard value of PVC for rat is 0.38-0.51 l/l.

Haemoglobin (Hb) content of a blood sample using ‘Hemocue’ haemoglobinmeter

The recorded Hb content is 178 g/l of blood. The value recorded for Hb content of blood is within the standard value for human blood. The standard value of Hb content for human blood for male is 135-180g/l and for female is 115/165g/l. The value recorded for Hb content of blood is above the standard value for rats. The standard value of Hb content for rat blood is 115-161g/l.

Mean Cell Volume (MCV)

MCV = PCV?RCC

= n x1012 x1000= y x1015

MCV is in 1015 so therefore this value needs to be multiplied by 1000.

0.49?8.64= 0.057?1012

0.057?1000=57 x1015

MCV= 57fl

The MCV value is lower than the normal range of values for human males and females. The standard values of MCV for human male and female are 80-96fl. The MCV value is lower than the normal range of values for rats. The standard value of MCV for rats is 69-85fl.

Mean Cell Haemoglobin (MCH)

MCH= Hb?RCC

178?8.64= 20.60pg (2d.p.)

MCH= 20.6 pg

The MCH value is lower than the normal range of values for human male and females. The standard value of MCH for human male and female are 27-32pg. The MCH value is lower than the normal range of values for rats. The standard value of MCH for rats is 24-30pg.

Mean Corpuscular Haemoglobin Concentration (MCHC)

MCHC= Hb?Hct

178?0.49= 363.27g/l (2d.p.)

MCHC= 363.27 g/l

The MCHC value is higher than the normal range of values for human male and female. The standard value of MCHC for human male and female are 320-360g/l. The MCHC value is higher than the normal range of values for rats. The standard value of MCHC for rats is 320-360g/l. DISCUSSIO

The value obtained from rat blood sample for red cell count (RCC) is within the range in rat reference range. The value obtained for Hb is 8.64?1012/l. When this value is compared to the human reference range, it is higher. RCC differs between the sexual categories, reference range for human male is between 3.5-6.0?1012/l and for human female is 3.5-5.5?1012/l. So therefore the RCC value obtained for rat blood sample is closer to the human male reference range than female reference range. The standard RCC of rat blood is much higher than human’s standard RCC. So the results obtained for RCC is accurate.

The value obtained from rat blood sample for determination of haemoglobin (Hb) is higher than the expected value. The value obtained for Hb is 178 g/l/. So therefore the obtained value is 17g/l higher than the maximum reference range for rats. This could be because of an operator error whilst filling round the chamber, or while removing the excess blood and place in haemoglobinmeter. The other source of error could be rat blood sample used. It might have had different Hb values from the rest.

When this value is compared to the reference range of human values, noticed that obtained values are closer to the standard Hb of human values. Hb values differ between sexual categories; reference range for human male is between 135-180 g/l and for human female is115-165 g/l. So therefore the values obtained from rat blood sample are within the range in reference range value of human male and for female it is 13 g/l higher from the maximum reference range of female.

The value obtained from rat blood sample for determination of haematocrit (PVC) is within the range in rat reference range. The value obtained for PVC is 0.49 l/l. This value has been converted to SI units from the percentage number. It has been converted by dividing the percentage number into 100. This value is also compared with the human reference range, both for male and female. The reference range value for human male is 0.35-0.55 l/l and for human female is 0.3-0.5 l/l. the value obtained for PVC from rat blood sample is within the range of human male and female reference range a value.

Mean cell volume (MCV) value is obtained by the values already obtained for PCV and RCC. So therefore MCV value varies due to these values. The value obtained from rat blood sample for MCV is lower by 12fl of the minimum range value. The value obtained for MCV is 57fl; this value has been gained after a series of calculations. The value of MCV is lower than the reference range for rats (69-85fl). This could be because the PVC value obtained is lower than the expected or the RCC value is bigger than the expected value. As the RCC value increases the MCV value decreases and as PCV increases the MCV value increases.

When the value obtained from rat blood sample for MCV is compared to the reference range of human values (in this case the values are the same for male and female), it is lower by 23fl from the minimum value.

Mean cell haemoglobin (MCH) value is obtained by the values already obtained for Hb and RCC. So therefore MCH value varies due to these values. The value obtained from rat blood sample for MCH is lower by 3.4pg of the minimum range value. The value obtained for MCH is 20.6pg, where as the reference range value for rat is 24-30pg. This could have been because the Hb value obtained is low for the calculation, but the Hb value is already higher than the reference range value. And RCC is within the range so the expected value for MCH was higher than the reference range. This inaccurate value could be a result of the RCC value being close to the maximum value. If this value was a bit lower, the MCH value would be in the range or closer to the reference range of rat.

When the value obtained from rat blood sample for MCHC is compared to the reference range of human values (in this case male and female values are the same), it is lower by 6.4pg of the minimum range value.

Mean cell haemoglobin concentration (MCHC) value is obtained by the values already obtained for Hb and Hct. So therefore MCHC value varies due to these values. The reference range numbers for rat and human (male and female) values are all the same with each other. The value obtained from rat blood sample for MCHC is lower by 3.27 g/l of the maximum range value (for human and rat). As the Hb value increase, the MCHC values increases too and as the Hct value increases, the MCHC value decreases.

Hct value is already within the range, so therefore Hb value is the in accurate result. Hb value needs to be the nearest to the maximum value, so that the MCHC value would be in the range.

The values calculated for red blood cell indices (MCV, MCH and MCHC) from rat blood sample are not within the range. So this means that the results obtained are not accurate. The values obtained for RCC and PVC/Hct from rat blood sample is within the range of rat reference range. But Hb value is higher than the maximum range value. This could be because of an operator error in poor pipetting technique or poor counting technique

Strong dilution of sample could result in inadequate results. As seen on the Table1 RCC and PVC/Hct values are very close to the maximum of reference range value, and Hb value is higher than the maximum of reference range value. So therefore the sample might not have been diluted properly, take for granted that the sample was stronger than how it should have been.

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