A Haematological Investigation:

The use of Coagulation Assays in the Early Diagnosis of Bleeding

Disorders
Student No. 3021879
[Word Count – 1,373]

ABSTRACT
Patient Y has been suffering from fatigue, menorrhagia and frequent epistaxis. From the full blood
count (FBC) and coagulation assays, Patient Y most likely has microcytic, hypochromic anaemia and
Von Willebrand’s disease, but further tests are required to confirm the working diagnosis. The
coagulation assays such as prothrombin time (PT), Activated partial thromboplastin time (aPTT) and
Thrombin time (TT) were conducted to investigate clot formation. The results revealed a prolonged
aPTT and a normal platelet count, which was indicative of an array of disorders but was narrowed
down to the most likely diagnosis; Von Willebrand’s disease. The patient’s clinical symptom of
menorrhagia affect 37% of women in the adolescent population and epistaxis has a prevalence of
10-12%, which emphasizes the clinical relevance of this study (Marajó Dal Secchi, M., et al. 2009).
Improved understanding in both bleeding disorders and methods of screening to diagnose the
diseases will continue to have a beneficial effect on patients.

INTRODUCTION
Haemostasis is the process by which a clot (primarily composed of platelets and fibrin) forms at the
site of injury to stop bleeding, whilst maintaining blood flow (Gale, A J. 2010). It can be divided into 2
processes; known as primary haemostasis, the initial adhesion and platelet plug formation, and
secondary haemostasis, the activation of the coagulation cascade to deposit fibrin around the
platelet plug (Periayah, M H., et al. 2017). Synthesised from fibrinogen, fibrin is a strong, insoluble
protein that is organised into long fibrous strands and is weaved around a clot to increase its stability
(Britannica,2018). It is important to note that the primary and secondary haemostatic processes are
mechanistically linked and occur concurrently to form a stable clot (thrombus) (Gale, A J. 2010).
Primary haemostasis, as shown in Fig.1, is dependent on interactions between von Willebrand factor
(vWF) and the following receptors (GPIb complex, GPVI, IIb3, 21 and fibrinogen) (Ogedegbe, H
O. 2002). Whereas secondary haemostasis uses several clotting factors (Figure 2), which interact
within the extrinsic, intrinsic or common pathway (Figure 3) (Palta, S., et al. 2014). The haemostatic
pathways are tightly regulated for survival and an imbalance can lead to bleeding disorders (Zaidi, A
and Green, L. 2022). These disorders can be congenital (inherited at birth) or acquired throughout
one’s lifetime (Anon, NIH. 2023). They are often caused by thrombocytopenia (low platelet count),
low platelet function and defects in coagulation factors (Goodman, D M., et al 2012).
A way in which these disorders are investigated is through a series of coagulation assays; PT, TT,
aPTT (Raber, M N. 1990). PT is used to assess the extrinsic pathway and identify deficiencies in
coagulation factors (VII, X, V, II and fibrinogen) (Tripodi, A., et al. 2007). TT assess the common
pathway, measuring the formation of fibrin through thrombin action in the coagulation cascade
(Saint-remy, J. 2006). aPTT is used to analyse the coagulation factors in the intrinsic and common
pathways such as factors XII, XI, IX, VIII, II, V, X and fibrinogen (Zimring, J C. 2009).
This report analyses the use of the coagulation assays in the early diagnosis of bleeding disorders.

Figure 1. The steps in primary haemostasis (Clemetson, K J. 1999), which demonstrate vWF, from
exposed endothelium, binding with surface platelet receptors to form a platelet plug– shown in (c),
aggregation.

Figure 2. Clotting factors involved in the coagulation cascade (Palta, S., et al. 2014).
Figure 3. Demonstrates the coagulation cascade and how the clotting factors from Fig.2. interact to
form a thrombus (Pocock, G and Richards, C D. 2013).
METHODS
This report’s protocol was based on the coagulation assays, using standard methodology by Winter,
W E (et al. 2017) and Raber, M N (1990).

The assays were performed in duplicate to ensure higher precision and performed at 37°C to mimic
the physiological conditions of the human body. We determined the endpoint by comparing our test
tubes to standard ones on display, which may have produced variable results. However, the use of
platelet poor plasma (PPP) eliminated the interference of platelets during the clotting process.
Platelets contain various surface proteins and components that could affect the coagulation cascade;
therefore, PPP is used to provide a more accurate assessment of the plasma's coagulation factors.

RESULTS

Test Time (seconds) Mean Total Total

(per test) Group Mean Class Mean

28 38 17 17 25.000
6 6 10 10 08.000
PT 19 19 18 18 18.500 15.150 12.480
12 12 10 10 11.000
12 15 15 11 13.250
193 193 140 140 166.50
300 300 300 300 300.00
APTT 135 135 81 81 108.00 194.65 150.82
155 155 105 105 130.00
300 280 255 240 268.75
2 2 4 4 3.0000
12 12 16 16 14.000
TT 9 9 4 4 6.5000 10.100 13.660
16 16 10 10 13.000
16 15 12 13 14.000
Table 1. Coagulation assay results for PT, aPPT, TT, which range from 0-300 seconds. The mean was
calculated for each test, the group’s average and the entire class average (which was used as the
standard result).
Figure 4. Ryan-Joiner (RJ) normality test run for PT results from the group data. P-value is less than
0.05 (<0.010) so the null hypothesis is rejected; the data is not normally distributed.

Figure 5. RJ normality test run for PT results from the class data. P-value is less than 0.05 (<0.010)
so the null hypothesis is rejected; the data is not normally distributed.
Figure 6. Demonstrates Mann-Whitney U Test results for PT, comparing the group and class means.
P value of 0.133 is greater than 0.05 so we accept the null hypothesis (population mean in group 1 =
group 2)

Figure 7. Ryan-Joiner normality test run for TT results from the group data. P-value is greater than
0.05 (>0.100) so the null hypothesis is accepted; the data is normally distributed.
Figure 8. Ryan-Joiner normality test run for TT results from the class data. P-value is less than 0.05
(<0.010) so the null hypothesis is rejected; the data is not normally distributed. Difference in
distribution between group and class data means that the Mann-Whitney test is still most
appropriate.

Figure 9. Shows a screenshot of the Mann-Whitney U Test for TT results. P value of 0.174 is greater
than 0.05 so we accept the null hypothesis (population mean in group 1 = group 2).
Figure 10. Ryan-Joiner normality test run for aPTT results from the group data. P-value is greater
than 0.05 (>0.100) so the null hypothesis is accepted; the data is normally distributed.
Figure 11. Ryan-Joiner normality test run for aPTT results from the class data. P-value is less than
0.05 (<0.010) so the null hypothesis is rejected; the data is not normally distributed.

Figure 12. Shows a screenshot of the Mann-Whitney U Test for aPTT results. P value of 0.043 is less
than 0.05 so we reject the null hypothesis (population mean in group 1 ≠ group 2).
The analysis of the results was completed using Minitab, version 21.1.0. A Ryan-Joiner (RJ) test for
distribution normality was the most suitable for our sample sizes (ranging between 3-300) and was
conducted to determine the subsequent tests. Consequently, the Mann-Whitney U Test was used to
compare non-normally distributed data sets (Fig.4-6) / or those with a difference in normality (Fig.7-
12). From the results, we can confirm the PT and TT results were accurate, as the hypothesis was
accepted. However, the aPTT results led to the rejection of the null hypothesis, as shown in Figure
12.

DISCUSSION

The coagulation assay results produced a long aPTT time, which is a measure of the intrinsic pathway
(Raber, M N. 1990) and could be linked to the following disorders: Von Willebrand’s Disease,
Haemophilia A, Haemophilia B, Factor XI deficiency, Factor XII deficiency, Prekallikrein (PK)
deficiency, and HMWK deficiency.

Haemophilia A is an inherited disease that effects factor VIII (FVIII) (Doncel, S S., et al 2023), which is
a glycoprotein that functions as a co-factor in the activation of FX (Bhopale, G M., et al. 2003). It is X-
linked recessive, so males are mostly affected (1 in 5000 to 10 000) and females are often carriers of
the disease (Doncel, S S., et al 2023). Clinical presentations are often excessive bleeding after minor
trauma/surgery and bleeding within joints/muscles (Mehta, P., et al. 2022). Similar to Haemophilia
A, Haemophilia B is a FIX genetic disorder that primarily effects men (X-linked recessive) and
presents with almost indistinguishable symptoms to the former (Miller, C H. 2021). However further
tests such as genetic screening using PCR of the F8 gene (Haemophilia A) and the F9 gene
(Haemophilia B) (Medlineplus, 2020) or a factor FVIII/FIX assay should differentiate between the two
and confirm the diagnosis (Mehta, P., et al. 2022). Decreasing levels of FVIII and FIX correlate to the
severity of the condition (White, G C., et al. 2001)

Conditions like Prekallikrein (PK) deficiency, HMW-K disorder and FXI deficiency all present with an
elevated aPTT but are often asymptomatic (Yasin, H., et al. 2020) (NICE guidelines, 2024) (Dahah, Y.,
et al. 2022). Genetic screening of the mutation on the KlKb1 gene (PK deficiency) (Riano, I., et al.
2021), using Next-generation sequencing (NGS) to analyse the entire coding region may increase the
likelihood of the diagnosis (NICE guidelines, 2024). Furthermore, PCR can be used to screen for the
genetic abnormalities on the F11 gene (FXI deficiency) (Soliman, M., et al. 2023) and on the KNG1
gene at nucleotide position 587 (HMW-K disorder) (Cheung, P P., et al. 1993). However, with all
attempts to genetically screen for a disease, challenges arise due to more than one mutation or
mutations at different loci. For example, Jing Yang (et al. 2020) identified a variant on the KGN1 gene
at nucleotide position 1456 for HMW-K disorder.

Another suspected condition is FXII deficiency, which is distinct from the other diseases as it does
not cause bleeding defects (Chaudry, L A., et al. 2019). Clinical presentations are a prolonged aPTT
but further tests– such as a mixing study, in which a 1:1 ration of the patient’s plasma is mixed with
normal plasma and PT, aPTT, TT assays are performed– are recommend for diagnosis (Fernandes, H
D., et al. 2018). However, as patient Y does present with symptoms of bleeding, this is less likely to
be the cause of her condition.

Whereas Von Wilebrand’s disease (vWD) is the most common inherited bleeding disorder, caused by
a defect in the von Willebrand factor (vWF) (Bharati, K P., et al. 2011). vWF binds platelets to
exposed subendothelial collagen, which is the initial step in platelet plug formation at the site of
injury (Kaur, V., et al. 2024). It is classified into 3 types: Type.1, Type.2, Type.3, of which Type 2
consists of 4 subtypes (Imm, N. 2016). Therefore, clinical presentations often vary between mild to
moderate mucocutaneous bleeding– such as epistaxis, gingival bleeding, postpartum bleeding or
menorrhagia (Kaur, V., et al. 2024). Further tests such as a vWF antigen immunoassay, Ristocetin
cofactor assay, genetic testing (real-time PCR) and vWF multimer analysis via gel electrophoresis are
recommended. (Ng, C., et al. 2015) (Kaur, V., et al. 2024) (Torres, R., et al 2009).

Patient Y presents with a low MCV, MCH, haematocrit and haemoglobin (Hb) levels. From the FBC it
is most likely that patient Y has microcytic, hypochromic anaemia. This may be a secondary condition
to patient Y’s bleeding defect, as the excessive loss of blood with each menstrual cycle can cause
iron deficiency (Chauhry, H S et al. 2019). A peripheral blood smear in which the morphology of RBCs
appears small, with central pallor will confirm this (Massey, A C., et al. 1992). Furthermore, the
patient has a normal platelet count (300x103 µL) according to the FBC report. This means that the
bleeding disorder is less likely linked to thrombocytopenia. Patient Y has “menorrhagia and frequent
epistaxis” which is a common clinical presentation of vWD. Furthermore, the coagulation assay
results produced a normal PT and TT but a prolonged aPTT which may also be seen in vWD when
FVIII levels are reduced (James, P D., er al. 2011).
The diagnosis was based on a prolonged aPTT results which had a low accuracy (when compared to
the standard, fig.12) and a low precision (Table.1) but still produced a result which could be
distinguished from the norm values between 30-40s (aPTT). Future investigations would benefit in
increasing sample sizes for a higher precision and automating the methodology to eliminate human
error, and thereby increasing the accuracy. To conclude, vWD is the most likely diagnosis and whilst
the other conditions are less likely, they cannot be ruled out until further tests are complete.
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