course material

1. ASC_5_HCI Human Haematology And Clinical Immunology
Answer:
Introduction
Blood found in humans as well as other species transports waste products of metabolism
aside from the cells while also delivering requisite nutrients around the body as well
as oxygen. Blood cells dissolved in plasma or serum make up that very same substance.
Plasma is the major medium for excretion product and transportation mostly has been
major amount of liquid or water, which is 92% volume as well as encompasses proteins,
glucose, essential minerals, hormones, also carbon dioxide, as well as blood cells (Bain,
2022). Plasma in blood encompasses around 55% of the fluid in body. Albumin is the
major abundant protein inside the plasma, and the main function is to regulate blood's
colloidal osmotic pressure. Red blood cells (also known as RBCs or even erythrocytes),
white blood cells (also known as WBCs or rather leukocytes), as well as platelets (also
referred to as thrombocytes) are the common blood cells (Garraud and Tissot, 2018).
Red blood cells are by far the most numerous cells throughout vertebrate blood. Those
same encompass haemoglobin, an iron-containing protein that helps carries oxygen whilst
also reversibly binding as well as massively increasing the solubilization throughout blood
(Pillai et al., 2020). Haemoglobin, an iron-containing oxygen transporting metalloprotein
found among almost every vertebrates' red blood cells or erythrocytes and some
invertebrates' tissues. RBCs contain haemoglobin, which transports oxygen around the
body towards the remainder of the body which is the tissues (NCBI, 2014). It then
discharges the oxygen, allowing aerobic energy system or respiration to focus
on providing energy towards that same organism's activities and functions during the
procedure termed as metabolic activities or metabolism. In each and every 100 millilitres of
blood in a normal adult would have 12 - 20 grammes of haemoglobin. In adults of
haemoglobin up to four oxygen molecules could be coupled or carried by the mammalian
haemoglobin protein. Haemoglobin is also engaged inside the travel of many other particles:
it transports concerning 20 to 25 % of bodies natural respiration, carbon dioxide as
carbamino haemoglobin, wherein the CO2 is bonded towards the heme protein (Costanzo,
2007). There are multiple haemoglobin genes in humans: HBA1, HBA2, as well as HBB
coded for haemoglobin A, the much more common protein in red blood cells found in adults.
The genes HBA1 as well as HBA2, which are also on chromosome 16 as well as nearer to one

2. another, codes for the haemoglobin subunits alpha 1 as well as alpha 2 (Uniprot, 2022). The
HBB gene found on chromosome 11 codes for the haemoglobin subunit beta. In
haemoglobins, the amino acid sequences of globin proteins differ substantially among
species. To increasing human evolution path length among species, the above distinctions
become much more noticeable. Many multiple subunit of the globular proteins, including
haemoglobin, have the quaternary structure. In haemoglobin, the significant proportion of
amino acid residues shape alpha helices, which are linked up by shorter form of non-helical
sections. The helical segments of all this protein are stabilised by hydrogen bonds, that also
induce attractions within in the particle, starting to cause every polypeptide chain towards
and fold as well as deform into a specific pattern and shape (Marengo-Rowe,
2006). Haemoglobin’s quaternary structure is made up of four subunits arranged in an
approximately tetrahedral pattern. The haemoglobin molecule is organized into four
globular polypeptide subunits throughout most vertebrate species. A protein sequence is
strongly connected to a non-protein prostheses heme group within every subunit. Inside a
globin fold configuration, for every protein sequence is organised into such a series of
alpha-helix functional sections that are associated around each other.
A blood type is categorization of blood determined by the presence as well as lack of
antibodies but also hereditary antigenic materials on the cell surface of RBCs. Based upon
that blood type system, those same antigens can be proteins, carbohydrates, glycoproteins,
or glycolipids (NHS, 2020). Most of the antigens could also be placed on the cell surface of
many other cell types in different body tissue. Almost all of the antigens present on the
surface of RBCs could come from the same allele or even different version of the very same
genotype as well as establish a blood type group system. Two antigens as well as two
antibodies reported in blood plasma make up the ABO blood group framework, an Antigen
A as well as antigen B are the respective two forms of the antigens. Antibody A as well as
the antibody B are the antibodies are the two forms of antigens (FDA, 2021). Antigens are
observed on RBCs, as well as antibodies have been found inside the serum. All humans
could be divided into four categories based on their blood antigen properties: those who
have antigen A are known to be group A), those who have antigen B are classified as group
B), those who are both the known antigen A as well as B are categorised in group AB, as well
as those with neither the antigen A nor B are grouped in AB , or group O (Xu et al.,
2019). Antibodies identified in conjunction within these antigens were: Antigen A
present with the antibody B, Antigen B present with the antibody A, Antigen AB with the
lack of no antibodies, as well as Antigen nil or group O with the antibody A are all examples
of antigens with antibodies. To 50 antigens, the Rh system, with Rh implying Rhesus is the
second greatest blood-group structure in living person. The D antigen is by far the most
essential Rh antigen which is the more likely to elicit an immune reaction of the present five
major Rh antigens (Redcrossblood, 2021). Anti-D IgG as well as IgM antibodies have all
been rarely generated by the sensitization towards the environmental stimulants, as such
anti-D antibodies have been rarely found in D-negative community members. Even so, a
sensitising occasion, such as the fetomaternal transfusion of the blood from that of a foetus
or baby present during pregnancy or even during a blood transfusion to D positive RBCs,

3. could indeed cause D-negative individual people towards the production of IgG anti-D
antibodies.
Thrombin would be a serine protease molecule which is encrypted by that of the F2 gene
found inside the humans. Throughout the clotting of blood process, prothrombin
the coagulation factor II, is mostly proteolytically cleaved to produce thrombin (Cleveland
Clinic, 2022). Thrombin, throughout turn, functions as just a serine protease, converting
solubilized fibrinogen into the insoluble fibrin layers as well as catalysing a variety of many
other coagulation-related responses. The ABO blood group system is found on the concept
of the agglutination. It would be the most widely used technique for evaluating the
appearance as well as utter lack of the cellular antigens as well as their corresponding
antibodies inside the blood. Forward typing throughout blood typing refers to the
identification of antigen inside the donor's RBCs. Reverse typing, and from the other hand,
has been the identification of antibodies inside the donor's serum or plasma (Dean, 2005).
The Abo blood system's antigens are glycolipid-based, as well as the antibodies have
become mostly of the IgM type.
Type A, B, AB, as well as O phenotypes represent approximately the ABO blood system, that
would be used to recognize the nature of exterior antigen - antibody existing inside a
participant's blood. Whether the RBCs agglutinate, the commensurate antibody would have
to be lacking from the plasma proteins (Ewald, and Sumner, 2016). Only when antigens are
not found on the membranes of RBCs, antibodies would have to be available inside the
plasma proteins. Agglutination reactions take place seen between the exterior proteins that
are involved upon that RBCs exterior inside the ABO blood classi?cation system. The above
antigen or agglutinogen would be a surface protein. Antigens also seem to be primarily
glycolipids, which have been classified as either self-antigens and otherwise foreign-
antigens. Whereas if antigen seems to be distant, the immune response inside the body, as
well as the WBCs are activated to recognise these antigens. The WBCs whereupon begin to
produce antibodies against such an antigen. The prothrombin time (PT) would be a blood
calculation that evaluates how quickly blood clots. To verify for haemorrhagic issues, a
PT test could be used. PT can also be used to see if blood clot-prevention medication is
effective. An INR test is another name for a PT test., the INR (international normalised ratio)
is a technique of standardising PT testing results regardless of measurement technique. For
the blood coagulation, clotting components are essential (UCSF, 2019). Prothrombin, also
known as factor II, would be a clotting component secreted by the bodies liver. Prothrombin
as well as other coagulation factors require vitamin K to be produced. The PT test is
very important since it verifies for the presence of five distinct blood coagulation, factors
being I, II, V, VII, as well as X). The following factors prolong the PT test: warfarin, for
example, is a blood thinner, blood coagulation factors are at an all-time negligible, the
action of some of the coagulation factors keeps changing, some of the coagulation factors
are missing, inhibitors are other compounds which actually effect the coagulation factors, as
well as any use of coagulation factors has increased. An anomalous PT test is generally
triggered by the liver illness or disorder, as well as blood thinner therapeutic interventions.

4. Method
The following reaction to occur begins with the collection of specimens in the correct tube,
the main reason being collection in any wrong tube could alter the pathway and the
experimental procedure. As the test requires blood which are not coagulated the tube (light
blue) containing EDTA (Ethylenediaminetetraacetic acid) whose main function in these
procedures is to bind with the metallic ions for instance iron, magnesium, calcium as well as
lead. The EDTA acts as a chelating agent which hinders the formation of blood clot as well as
removes the presence of lead from the body. The EDTA tube contains the blood sample, in a
slide 5 circles were drawn and labelled as A,B, D1 and D2 and – (blank). ~50ul of antiserum
was added then one drop of blood was added and mixed with yellow tip then observed for
the formation of agglutination. The PT test was obtained in a sodium citrate tube and
centrifuged at 2000 rpm for 15 minutes. The samples are stored at -20 ºC for longer time, as
storage solution for the tests. The thromboplastin D was warmed at temperature of 37 ºC,
the tubes were labelled with ID number and 25ul of the plasma was added and warmed for
2 minutes. The reagent measuring 50ul was added to the plasma and the clotting formation
was observed. Carefully the reaction was carried to avoid contamination of any other
foreign sources to avoid formation of fibrinogen or substances.
AQ1. Antibodies for antigens A and B accumulate in plasma and alluded as anti-A and B sera.
Some of the antigens can be mixed together producing blood group AB (Palomar, 2014)
AQ2. The appearance of agglutination after the addition of the reactants within the given
normal time frame (Mycts, 2022).
AQ3. Assay done at room temperature or lower maintains the reactants, higher than that
weakens the reactions.
AQ4. The technique to identify the ABO blood group based on the presence of anti A or anti
B sera.
AQ5. Each person either has anti D negative or positive as they vary due Rhd protein; one is
developed during in womb, other later.
AQ6. The ABO blood grouping are A, B, AB, and O group, and with Rh divides it into
positive and negative types.
BQ1. The PT measures the time and rate at which blood coagulates in sample, and PTT
monitors an characteristics of coagulation of blood.
BQ2. Haemoglobin, lipids, bilirubin, and paraproteins, or any condition interferes with PT
test.

5. BQ3. Liver disease, factor VII and vitamin K deficiency, also coagulation factor deficiency
could delay coagulation in samples.
BQ4. Heparin present in sample or contamination in samples along with thromboplastin
could alter results. Blood when not stored at correct temperature losses their function
mainly, coagulation.
BQ5. Due to sample storage at different temperature the prothrombin time gets delayed or
increases than usual time compared to other samples.
Result
The ABO blood grouping test for the three samples indicates; S1 is anti A negative thus the
presence of antigen A and anti D negative, S2 indicates anti B sera presence and anti D
negative sera thus the presence of B- blood group, and for S3 from the test indicated the
presence of the anti A and B sera along with anti D sera, which shows the presence of blood
group O positive (Table 1). The PT test for the different plasma and blood kept at different
temperature indicates the formation of blood and plasma coagulation ; -80 has clotting time
of 30 seconds whereas blood and plasma kept at 4º C coagulates at time of 25 sec and 19 sec
respectively.
Sample ID
Anti A
Anti B
Anti Rhd D1/D2
-
Blood group

6. S1
+
-
-
A-
S2
-
+
-

7. B-
S3
+
+
+
O+
Table 1: ABO blood grouping of sample 1, 2, and 3
Sample ID
PT

8. INR
Comment
Plasma -80
30 sec
6.07
Visible loss clotting factor
Plasma 4 C
25 sec
4.53
Almost loss of clotting factor

9. Blood 4 C
19 sec
2.49
Little loss of clotting factor
Table 2: PT test, indicating the presence of blood coagulation time after the addition of
thromboplastin D, also delay in PT as normal range is 11 to 13.5 seconds
References
Bain, B.J., 2022. Blood cells: a practical guide. John Wiley & Sons.
Cleveland Clinic, 2022. Prothrombin Time and INR. [online] Available at: [Accessed 3
January 2022].
Costanzo, L., 2007. Physiology : Linda S. Costanzo : Free Download, Borrow, and Streaming :
Internet Archive. [online] Internet Archive. Available at: [Accessed 3 January 2022].
Dean, L., 2005. The ABO blood group. [online] Ncbi.nlm.nih.gov. Available at: [Accessed 3
January 2022].
Ewald, D.R. and Sumner, S.C., 2016. Blood type biochemistry and human disease. Wiley
Interdisciplinary Reviews: Systems Biology and Medicine, 8(6), pp.517-535.
Garraud, O. and Tissot, J.D., 2018. Blood and blood components: from similarities to
differences. Frontiers in medicine, 5, p.84.
Marengo-Rowe, A.J., 2006, July. Structure-function relations of human hemoglobins. In
Baylor University Medical Center Proceedings (Vol. 19, No. 3, pp. 239-245). Taylor &
Francis.
Mycts, 2022. [online] Mycts.org. Available at: [Accessed 3 January 2022].
NCBI, 2014. Haemoglobin and iron: information for blood donors. [online] Ncbi.nlm.nih.gov.

10. Available at: [Accessed 3 January 2022].
NHS, 2020. Blood groups. [online] nhs.uk. Available at: [Accessed 3 January 2022].
Palomar, 2014. Human Blood: ABO Blood Types. [online] Www2.palomar.edu
Pillai, A.S., Chandler, S.A., Liu, Y., Signore, A.V., Cortez-Romero, C.R., Benesch, J.L.,
Laganowsky, A., Storz, J.F., Hochberg, G.K. and Thornton, J.W., 2020. Origin of complexity in
haemoglobin evolution. Nature, 581(7809), pp.480-485.
Redcrossblood, 2021. What Is The Rh Factor? Why Is It Important?. [online]
Redcrossblood.org. Available at: [Accessed 3 January 2022].
Redcrossblood, 2021. What Is The Rh Factor? Why Is It Important?. [online]
Redcrossblood.org. Available at: [Accessed 3 January 2022].
UCSF, 2019. Prothrombin Time (PT). [online] ucsfhealth.org. Available at: [Accessed 3
January 2022].
Uniprot, 2022. HBB - Hemoglobin subunit beta - Pan paniscus (Pygmy chimpanzee) - HBB
gene & protein. [online] Uniprot.org. Available at: [Accessed 3 January 2022].
Xu, X., Xu, F., Ying, Y., Hong, X., Liu, Y., Chen, S., He, J., Zhu, F. and Hu, W., 2019. ABO antigen
levels on platelets of normal and variant ABO blood group individuals. Platelets, 30(7),
pp.854-860.