The major function of erythrocytes is to transport oxygen and carbon dioxide in the blood. Such transport is vital for the delivery of oxygen from the lungs to respiring cells, where it is removed from the body. Erythrocytes have a high capacity for carrying oxygen and carbon dioxide because they contain in their cytoplasm two proteins; Haemoglobin and carbonic anhydrase. Haemoglobin binds, and thus transports, oxygen and carbon dioxide, whereas carbonic anhydrase is essential for the transport of carbon dioxide only.
HemoCue is a portable haemoglobinometer where any blood source can be used such as blood from a capillary, vein or an artery. The cuvette will collect the exact amount of blood and mix it with the reagents. Results appear on a screen displaying the erythrocyte count.
The total volume of blood in a normal healthy adult is approximately 5.5 litres. About 3 litres will consist of plasma and 2.5 litres will consist of erythrocytes. Leukocytes and platelets are also included. The fractional contribution of erythrocytes to the blood volume is called the haematocrit:
''The haematocrit (Ht or HCT) or packed cell volume (PCV) is the proportion of blood volume that is occupied by red blood cells. It is normally about 48% for men and 38% for women.'' (Purves, William K., 2004)
The haematocrit can be affected by factors such as erythrocyte size and number, however, in mammals; haematocrit is usually independent of body size.
A haemocytometer can be used for:
'Counting of blood cells...specially designed slides have chambers of known depth with an etched grid on the bottom chamber.'' (Willey, J.M., 2008)
Fig. 1: Neubauer counting chamber (haemocytometer)
The aims of this experiment were to see and distinguish between the formed elements of blood using the light microscope and to make blood cell counts of the red blood cells (erythrocytes) in a blood sample.
Methods and Materials
One change that occurred, that was not stated in the methods was that the blood that was used for the experiment was disposed of by washing it down the sink. Also heparinised capillary tubes were used.
All other methods and procedures were followed as stated in the practical booklet.
For full method, please refer to 4LFS0029, Human physiology, practical booklet, pp 14-17.
Results
Fig. 2: The given reference ranges for a rat, human male and human female
Determination of red cell count (RCC)
From the Neubauer haemocytometer, the following red cell counts were counted:
Table 1: Results of the Neubauer haemocytometer
Position of square in the central block
Number of red blood cells
Top left
137
Top right
133
Bottom left
139
Bottom right
142
Centre
136
Total = 687
The number of erythrocytes in one litre of blood = Number of cells x 1010
= 687 x 1010
= 6.87 x 1012/litre blood
Comparing this value with the standard human haematological indices, the value that has been calculated is approximately double the lower end of the standard haematological indices for the human male and female. However, comparing the value to the standard rat haematological indices, it lays closer to the lower end, although it is still within range.
Determination of haemoglobin (Hb)
The determination of haemoglobin was carried out via the 'Hemocue haemoglobinometer'.
The results obtained for haemoglobin was a figure of 171g/litre.
Comparing this value with the standard values for human blood, it is evident that the value is within the range for the human male; however it lies outside of the ranges for a human female. For the range of values for a rat, the value of 171g/litre is exactly 10g/litre over the maximum range.
Determination of haematocrit (Hct)
The percentage of a blood sample that was occupied by the erythrocytes after centrifugation in a heparinised capillary tube was 49%. Converting this percentage to SI units:
Percentage
100
49
100
0.49
For human males and females this value lies within both of their ranges. The value also lies within the normal range for a rat; however, it lies towards the higher end.
Mean corpuscular Haemoglobin (MCH)
MCH is the haemoglobin content of a single cell measured in absolute units instead of reference to an arbitrary Hb content and cell count. The following formula was used to determine MCH:
MCH = Hb/RCC (where Hb and RCC are in SI units)
= 171/6.87 x 1012
= 2.48 x 10-11
= 24.8 x 10-12
= 25pg
The MCH value does not lie within either the human male or female values, it is too low. It is, however, within the range for the rat values, towards the lower end.
Mean Cell Volume (MCV)
The size of an erythrocyte can be calculated from the following formula:
Mean cell volume = PCV/RCC (when PCV and RCC are in SI units)
= 0.49/6.87 x 1012
= 7.13 x 10-14
= 71.3 x 10-15
= 71fl
The figure of 71fl does not lie within the human male or female ranges, it does, however lie within the rat range.
Mean Corpuscular Haemoglobin Concentration (MCHC)
The MCHC is a measure of the average haemoglobin carried by a litre of blood. It can be calculated from the following formula:
MCHC = Hb/Hct
= 171/0.49
= 348.98
This value lies within the range for both human males and females and it also lies within the range for rats.
Differential White Cell Count
Microscope was set at x40 magnification. The following white blood cells were observed and their sizes were noted.
Fig. 3: Drawings of the white blood cells observed under microscope
Discussion
Table 1 in the results section shows the results that were obtained from the Neubauer haemocytometer. A total of 687 red blood cells were counted from the five chambers. It can therefore be calculated that in one litre of the rat's blood there were 6.87 x 1012/litre blood of erythrocytes present. This figure fits well into the reference ranges for the rat; however it is also evident from the figure that the amount of erythrocytes in one litre of the rat's blood is towards the lower end of the ranges. The sample of blood that was given from the rat shows that for any human male or female, the rat will always have double the amount of erythrocytes of the lower end of the human ranges.
A figure of 171g/litre was obtained for the haemoglobin (Hb) value. This figure does not lie within either of the rat nor the human female reference ranges; it does, however, lie in the reference range of the human male. The maximum amount of haemoglobin that is normal for a rat is 161 g/litre, and the figure of 171 g/litre is over the maximum. A high amount of haemoglobin could result in a condition called iron overload. This high amount of haemoglobin could be due to the rat's diet;
''Experimental chronic iron overload was produced in rats by feeding them a chow diet'' (A J Dabbagh, T Mannion, S M lynch and B Frie)
The above statement shows that iron overload can be produced due to what diet a rat has. The same statement could apply to a human female as the maximum normal range of haemoglobin is 165 g/litre. This could possibly be eliminated by having a healthier diet.
After centrifugation of the rat's blood, it was determined that the haematocrit value of the rat's blood was 0.49l/l. This value fits into the rat's reference range and also into both the human male and female reference ranges. This value is, however, towards the higher end of the reference ranges in both rats and human females.
''Higher than normal haematocrit levels can be seen in people living at high altitudes. Dehydration produces a falsely high haematocrit that disappears when proper fluid balance is restored.'' (Harrison's Principles of Internal Medicine, McGraw-Hill, edited by Eugene Braunwald, et. al., 2001.)
From the above, it could be concluded that the high haematocrit value for the rat is due to dehydration and its habitat. However, gender and age are also contributors to high haematocrit values, another factor to consider.
The value of 25pg calculated for the Mean Corpuscular Haemoglobin (MCH) does not lie within the reference ranges of neither the human male nor the human female as it is too low. Concerning the rat's blood sample it did fit within the reference range, towards the lower end of the range. The mean corpuscular haemoglobin represents the weight of haemoglobin in an average erythrocyte. Lower values of mean corpuscular haemoglobin are usually diagnosed in certain types of anaemia such as microcytic and normocytic anaemia.
The size of an erythrocyte was calculated from the mean cell volume (MCV). It was found from the results that the mean cell volume of the sample of rat's blood has 71fl of blood. This value does not match the reference ranges of the human male or female but it is within the reference range of the mouse. If 71fl was the value obtained from a human or female this would be significantly lower than the minimum range of 80 and thus they would be diagnosed with microcytosis which results in Excess EDTA which is hypertonic and can cause cellular dehydration which in turn decreases the mean cell volume and increases the mean corpuscular haemoglobin concentration.
The mean corpuscular haemoglobin concentration is a measure of the average haemoglobin carried by a litre of blood. The value that was obtained for this calculation was 348.98 which lie well within all the reference ranges of the rat, the human male and female. The reference ranges for the rat, human male and female were of the same boundaries. Elevated MCHC is associated with Spherocytosis
''Spherocytosis is an auto-hemolytic anemia, characterized by the production of erythrocytes, that are sphere-shaped, rather than bi-concave disk shaped.'' Robert S. Hillman (2005).
Normal MCHC is associated with pernicious anaemia which is caused by the inability to absorb vitamin B12.
References - remember to put in alphabetical order
Maton, Anthea; Jean Hopkins, Charles William McLaughlin, Susan Johnson, Maryanna Quon Warner, David LaHart, Jill D. Wright (1993). Human Biology and Health. Englewood Cliffs, New Jersey, USA: Prentice Hall.
Cindy L. Stanfield, 4th edition Principles of human physiology, benjamin cummings, international edition 2011, page 437
Purves, William K.; David Sadava, Gordon H. Orians, H. Craig Heller (2004). Life: The Science of Biology (7th ed.). Sunderland, Mass: Sinauer Associates. pp. 954.
Willey, J.M., Sherwood, L.M. & Woolverton, C.J. (2008). Microbiology. (7th ed.). McGraw-Hill. P 128
Biochem J. 1994 June 15; 300(pt3):799-803 the effect of iron overload on rat plasma and liver oxidant status in vivo. (A J Dabbagh, T Mannion, S M lynch and B Frie)
Harrison's Principles of Internal Medicine, McGraw-Hill, edited by Eugene Braunwald, et. al., 2001
Robert S. Hillman; Kenneth A. Ault; Henry M. Rinder (2005). Hematology in clinical practice: a guide to diagnosis and management. McGraw-Hill Professional. pp. 146-150
