Khezia Chalwe

Student ID: 202098

Group 1: Dr Wong
Human Biology
Physiological Practical 3 - Blood: Practical Haematology

The aim of this practical is to

1.
2.
3.

Use blood typing techniques to determine the ABO and Rh blood group of blood samples
Use a haemocytometer to determine the red blood count in a sample of blood
Obtain a measurement of Packed Cell Volume (PVC) from a sample of fractionated blood and use the PVC
to calculate the Mean Cell Volume of the red cells. (1)

Methods
Experiment 1: Blood typing
The method for this experiment is as described on page 72 of the Human Biology lab manual
Experiment 2: Red Cell Count
The method for this experiment is as de scribed on page 74-75 of the Human Biology lab manual
Experiment 3: Packed Cell Volume PCV
The method for this experiment is as described on page 76 of the Human Biology lab manual
Results
Experiment 1: Blood typing
This experiment involved the blood typing of samples to determine their ABO blood group and whether they
are Rhesus positive or negative. The results for the experiment were compiled from the results of different
students in the laboratory on Monday 12 May 2014, and only the results from sample D8054468 were my own.
Agglutination of Different Samples of Blood in the Presence of Various Antisera
Sample

Presence of agglutination

Blood Type

Anti-A

Anti-B

Anti-D

D8054468

O+

D4963181

B+

D8148725

O+

D4913662

A-

D8052526

B-

D9140495

A+

D8053303

O+

D9294804

O+

D9052526

B-

Khezia Chalwe
Student ID: 202098

Group 1: Dr Wong
Human Biology

D9205119

O-

D9148445

A+

Statistical Analysis
Pr(A)
Pr(B)
Pr(O)
Pr(Rh+)
Pr(Rh-)
Mode
Pr(O+)

0.275
0.275
0.45
0.64
0.36
O+
0.36

In the results, the presence of agglutination, and therefore the presence of a specific antigen, was indicated by a +
sign and the absence of agglutination was represented by a - sign i.e. for sample D8054468, there was no
agglutination for anti-A and anti-B antiserum, therefore antigens A and B were absent on the red cells of that
particular sample, meaning that it was ABO blood type O. However, sample D8054468 did agglutinate in the
presence of anti-D antiserum, testing positive for the Rhesus factor, therefore the sample was O+. The statistical
analysis comes from simple probability calculations i.e. of the 11 samples, 3 were ABO blood type A (both Rh
positive and negative) and so the probability of a blood sample, in the set of results, being ABO blood type A was
0.275 or 25.7%. The most common blood group was O+ with 36% of the samples being of that blood type (Pr(O+)
was 0.36).
Experiment 2: Red Cell Count
This experiment involved using a Neubauer counting chamber to determine the RCC of a sample of blood with a
known dilution. The more squares counted, the greater the accuracy so I counted 96 small squares instead of the
minimum 80 squares.

Red Cell Count in the Squares of a Neubauer Counting Chamber

Square (1 square contains 16 small squares)
Cell Count
1

122

88

92

120

113

120
Total

655

5.458 1012

Red Cell Count

L
Average Cell Count square

109.17 13.86

Cells per

Khezia Chalwe
Student ID: 202098

Group 1: Dr Wong
Human Biology

Calculations:
Red Cell Count =

number of cells counted

dilution 106
Number of small squares volume of small square ( L )

Red Cell Count =

655
200 10 6
1
96 (
)
4000

Red Cell Count =

5.458 1012

cells per L

Each square contains 16 small squares, so for the first 16 squares had 122 RBCs, the second 16 squares had 88
RBCs and so on. On average there were 109.17 squares (with a standard deviation of 13.86) per square (

655 cells
6 squares

). To calculate the RCC we were given the volume of each small square (which was 1/4000 l)

and the dilution factor was 1:200. The resulting RCC was 5.458 1012 cells per L, which falls in within
two standard deviations of the average Red Cell Count for women. (1)
Experiment 3: Packed Cell Volume - PCV
Haematocrit Reading in a Fractionated Sample of Blood
Pack Cell Volume (L/L)

0.42

The reading of the PCV was taken off of a haematocrit reader, using a sample of fractionated blood in a capillary
tube.
Calculations:
Mean Cell Volume = Packed Cell Volume/Red Cell Count
12

Mean Cell Volume = 0.42 /

5.458 10

Mean Cell Volume = 7.698

10

Mean Cell Volume = 76.95

fL

14

L/L

The blood samples used for the calculation of PCV and the calculation of RCC are from the same person (I asked
Rachel Berry), so it is possible to use the two results to calculate a Mean Cell Volume. The MCV calculated falls
within two standard deviation of the average adult MCV (86 10 fL). The PCV also falls within two standard
deviation of the average for an adult woman (0.42 0.05)(1)
Discussion

In experiment 1, we were trying to assign a blood type to a sample of blood. The blood was supplied by the Hobart
blood bank. On the surface of every RBC there are specific antigens that are sometimes referred to as isoantigens.
The different types of isoantigens allow for the categorization of different blood groups. There are over 100
isoantigens, which define 30 different blood groups, which can be accounted for in through cross typing of blood.
Two blood groups are of particular importance; the ABO and Rhesus blood group. In ABO blood typing, blood
plasma contains antibodies for each ABO antigen that is not present on the surface of the RBC. Anti-A and anti-B
antibodies react strongly with the RBCs bearing the antigen of the same label (anti- A forms a complex with
antigen A), which in turn stimulated plasma proteins to adhere to the RBCs and causes them to agglutinate. This
agglutination reaction is the basis for ABO blood typing in the laboratory and in the experiment.

In the experiment, the blood samples that showed agglutination in the presence of anti-A antiserum had A antigens
on their red blood cells and the blood was ABO blood type A. The blood samples that showed agglutination in the

Khezia Chalwe
Student ID: 202098

Group 1: Dr Wong
Human Biology

presence of anti-B antiserum had B antigens on their red blood cells and the blood was ABO blood type B. The
blood samples which showed no agglutination to anti-A and anti-B antiserum had no A or B antigens present on
their red blood cells and the blood was ABO blood type O. according to the Australian Red Cross Blood Service,
49% of Australians have type O blood (2)and it is the most common. This was reflected in the results as blood type
O was the most common blood type out of the samples, with 45% of the samples being type O. another
observation is that none of the blood samples agglutinated in the presence of both Anti-A and Anti-B (did not have
both A and B antigens present) therefore none of the samples were ABO blood type AB. This is a reflection of the
fact that AB is not a common blood type in Australia; only 3% of Australians display the blood type.(2) A larger
sample size would provide results that better reflect the probability of having blood type A and B in Australian
society; 10% of Australians have B blood type and 38% of Australians have type A blood as opposed to the 27.5%
of samples in the experiment.
The Rh blood group is so named because the Rh antigen, called Rh factor, was first found in the blood of the
Rhesus monkey. The blood samples that agglutinated in the presence of Anti-D tested positive for the presence of
the Rhesus factor, thus were Rh blood typed Rh+. The samples that did not agglutinate were type Rh-. Normally,
blood plasma does not contain antiRh antibodies. If an Rh- person receives an Rh+ blood transfusion, however,
the immune system starts to make antiRh antibodies that will remain in the blood. If a second transfusion of Rh+
blood is given later, the previously formed antiRh antibodies will cause agglutination and haemolysis of the RBCs
in the donated blood, and the reaction may be fatal.
In blood transfusion, blood typing is of vital importance, so that a safe match is made. A safe match is one where
no agglutination occurs. Where agglutination occurs transfusion would be unsafe and the match is not compatible;
blood received in a transfusion must have the same antigens as yours, otherwise the antibodies in your plasma will
destroy the donor blood cells. This is called a transfusion reaction, and it occurs immediately when incompatible
blood is transfused and can be fatal. So it is important that before transfusion occurs, that blood type is correctly
determined. ABO type O- is a universal donor as it is compatible with any blood type (due to the lack of antigens
on the RBCs) and type AB+ is a universal recipient of blood transfusions, as people with the blood type AB+ can
receive any type of blood (due to the lack of antibodies in the plasma).(3)
In experiment 2, we were trying to obtain a Red Cell Count from a sample of blood. The RCC obtained (
5.458 1012 cells per L) is within two standard deviations (95% of the population falls within two standard
deviations) of the average RCC for a woman, (4.8 1) x 1012 cells per L, (1) so the results are credible, as the
blood sample came from a woman. The major limitation in this experiment was the small sample size; if the
experiment were to be repeated, counting more than 6 squares would produce more accurate results. Formal citrate
was used to fix the blood sample so that it would not undergo coagulation and the blood cells could be counted and
seen clearly. Another limitation was the quantitative measure used in this procedure; we counted the red cells by
eye. It was possible to count a red cell more that once, although we eliminated as much of this as we could by only
counting within certain parameters, and at times the red cells would move. The method used is very prone to
human error and requires a lot of attention, so nowadays RCC is obtained electronically, leaving less room for
error.
In experiment 3, the aim was to determine the Packed Cell Volume of a sample of blood, and then to use that PCV
to calculate a Mean Cell Volume. When blood is spun in a centrifuge, the different components split into 3 separate
phases; a plasma phase and haemotocrit phase separated by a buffy coat. Plasma makes approx. 55% of the total
blood volume and contains plasma proteins. The buffy coat, the phase between the plasma and the packed cell
volume, is composed of platelets and white blood cells. The haematocrit, also called the packed cell volume,
contains the red blood cells. Generally the PCV makes up 45 % of the total blood volume and the results fall
within two standard deviations of the average PCV for a woman; the average PCV is 0.42 0.05L/L and my
reading was 0.42 L/L. because the blood used in experiments 2 and 3 are from the same person, we were able to
calculate a MCV. The MCV calculated (76.95
fL) falls within two standard deviations of the average adult
MCV (86 10 fL). Mean Cell Volume can then be used to diagnose anaemia; a low MCV can be indicative of a
microcytic anaemia like thalassemia, whilst a high MCV could signify a macrocytic anaemia. (3) Anaemia is a
condition in which the oxygencarrying capacity of blood is reduced, and the fact that the MCV falls in a normal
range indicates that the owner of the blood is unlikely to suffer from anaemia.
Conclusion
The experiments provided an insight into the processes behind transfusion, blood typing and many other laboratory
tests, and a better understanding of red blood cells and their role.

References
4. 1.

Cooley M, Chapman J. Unit Manual for Human Biology. Hobart 2014.

Khezia Chalwe
Student ID: 202098

Group 1: Dr Wong
Human Biology

5. 2.
Donateblood.com.au. Blood types | Australian Red Cross Blood
Service 2014 [cited 2014
26 May]. Available from:
http://www.donateblood.com.au/about-blood/types.
6. 3.
Tortora G DB, editor. Principles of anatomy & physiology. . 13 ed.
Hoboken, NJ: Wiley.