A patient has a pre-operative coagulation screen and is found to have the following results:
Test Result (s) Normal Range (s)
Prothrombin time PT 13 Normal Range 11-15 secs
Activated Partial
thromboplastin time APTT
58 Normal range 33-42 secs
Critically discuss the pathophysiology of the potential underlying conditions that these results
depict, explaining how the laboratory data inform your analysis. Propose an appropriate
hospital laboratory strategy to determine a precise diagnosis for this patient, ensuring that
you explain the principles and expected results of relevant further laboratory tests.
Intrinsic Coagulation Pathway Abnormality Patient Investigation:
pathophysiology and diagnostics
Blood contains essential cellular components for many processes such as the delivery of
oxygen to tissues for respiration. Fluid losses can be life-threatening, so preventative
measures such as primary and secondary haemostasis mechanisms are in place (Hoffbrand et
Steensma, 2020). These include platelets, coagulation factors, vascular mechanisms, and
proteins like prothrombin. A series of factors function to form a cascade (Hoffbrand et
Steensma, 2020). The efficiency of this process is tested through the conversion of
prothrombin to thrombin by analysing the prothrombin time (PT). This investigates the
efficiency of factors VII, X, V, and II in the extrinsic coagulation pathway. A further method for
testing factors XI, XII, IX, or VIII in the intrinsic pathway is the Activated Partial thromboplastin
time (APTT) (Hoffbrand et Steensma, 2020). Both procedures measure the common
coagulation pathway too. The patients’ results indicate an abnormality in the intrinsic
pathway. This could be due to coagulation factor deficiencies or inherited bleeding disorders
such as Haemophilia A, and von Willebrand disease.
In response to injury in primary haemostasis, vascular constriction, platelet plug formation
through adhesion and aggregation, and presence of blood coagulation factors increases
(Hoffbrand et Steensma, 2020). Platelets adhere to the endothelial basement membrane, and
aggregation is stimulated by Thromboxane A2 and Adenosine diphosphate (ADP), produced
from platelet contractions at the site of injury. A large multimeric protein: von Willebrand
factor (vWF) also attaches to platelet glycoprotein Ib (GPIb) and collagen, stimulating platelet
structural changes, forming the ‘platelet plug’ (Hoffbrand et Steensma, 2020). Furthermore,
Thromboxane A2 reacts with prostacyclin produced from endothelial cells to regulate the
level of calcium ions (Ca2+) released from the platelet for activation of secondary haemostasis
(Hoffbrand et Steensma, 2020).
In secondary haemostasis, the loose platelet clot formed is stabilised further by the
conversion of fibrinogen to fibrin monomers to produce a cross-linked fibrin mesh after a
cascade of enzymatic reactions (Chaudhry et al, 2020). The intrinsic pathway begins through
activation of factor XII through exposure to endothelial collagen, which becomes activated
and acts as a catalyst to activate factor XI to factor XIa (Chaudhry et al, 2020). XIa activates
factor IX to factor IXA, which is catalysed by factor VIIIa to convert factor X into factor Xa.
Alternatively, the extrinsic pathway begins by the release of tissue factor (TF) from epithelial
cells at the site of injury, activated by vWF (Chaudhry et al, 2020). TF forms a complex with
factor VII to provide thromboplastin and form factor VIIa, which activates factor X into factor
Xa, where both intrinsic and extrinsic pathways become one in the common pathway. Factor
Xa is stimulated by factor Va to convert prothrombin to thrombin, which converts fibrinogen
to fibrin for further stabilisation of the clot (Hoffbrand et Steensma, 2020). This process is
illustrated in figure 1.
Figure 1:
Clotting pathways cascade
Note. Adapted from Hoffbrand’s essential haematology eighth edition, by A. Victor Hoffbrand and David P. Steensma, 2020.
Decreased coagulation factors in the intrinsic pathway will lead to bleeding disorders as the
formation of a clot will be slower and weaker due to inhibition by factor deficiency in the
cascade. This will prolong the APTT. Mixing tests should be conducted to investigate
potentially decreased coagulation factors (Kershaw et Orellana, 2013). This involves
combining patient plasma with normal plasma and repeating the PT and APTT. If the results
normalise, it evidences a clotting factor deficiency (Kershaw et Orellana, 2013). Further
specific factor assays should be performed where patient plasma is tested against specific
factors (Riley et al, 2017). The results are carefully analysed to identify any interferences by
inhibitors like antibodies (Kershaw et Orellana, 2013). A full blood count to see the platelet
count should also be done to rule out any anaemias or platelet abnormalities and non-
accidental injury. Mixing tests are advantageous as they are cheaper and less time-consuming
compared to other investigations and aid patient management options.
The most common hereditary clotting factor deficiency is Haemophilia A, affecting factor VIII
(Mingot-Castellano et al, 2017). It is an X-linked recessive condition that arises from different
mutations in the large factor VIII gene, like inversions where there is a break at the end of the
X chromosome leading to severe haemophilia A (Mingot-Castellano et al, 2017). The factor
VIII protein has a triplicated region A1A2A3 and a duplicated region C1C2, with a heavily
glycosylated B domain that is removed when factor VIII is activated by thrombin. This
structure is essential for its function (Hoffbrand et Steensma, 2020). The mutation causes
absence or low levels of functioning factor VIII. This impacts the intrinsic pathway as factor
VIII cannot convert to VIIIa, which converts factor IXa into factor X. There may also be
inhibition from autoantibodies preventing factor VIII receptor attachment, like in acquired
haemophilia (Yousphi et al, 2019). This deficiency or inhibition increases clotting time and
explains why the patients’ APTT could be prolonged. To test for haemophilia A, a one-stage
factor VIII coagulant activity assay should be done, and further chromogenic factor VIII assays
can be done if the patient is suspected of having severe haemophilia A (Rodgers et Duncan,
2017). If positive, patient will have low functional factor VIII levels.
Von Willebrand disease is an autosomal dominant condition with mutations on the vWF gene
on chromosome 12 (Bharati et Prashanth, 2011). This impacts the production of functioning
vWF, slowing platelet interaction for clot initiation in primary haemostasis, activation of TF,
and stabilisation of clotting factor VIII in the intrinsic cascade (Bharati et Prashanth, 2011).
These changes cause the multimeric structure and its capability for structural modification to
not function as required, resulting in slower platelet aggregation and clotting. If the patient
had this condition, the PT time will eventually become slightly prolonged due to less activation
of TF at the site of injury (Peyvandi et al, 2011). To test for this condition, an FVIII coagulant
activity assay should be conducted. If vWF is defective or absent, the results for the assay will
be low due to reduced stimulation of factor VIII (Rodgers et Duncan, 2017). The von
Willebrand factor activity and latex assay should be done, which will show reduced vWF levels
in plasma (Rodgers et Duncan, 2017). This provides a differential diagnosis to Haemophilia A
Overall, the patients’ PT of 13 seconds is within the reference range and normal, indicating
the common and extrinsic pathway is functioning as expected, comparatively the APTT is
higher by 16 seconds than the upper range of the reference range, indicating that the intrinsic
pathway is abnormal. Haemophilia A is indicated but tests for coagulation factor deficiencies,
and Von Willebrand disease which also impact the interconnected reaction cascade should
be conducted. A definitive diagnosis should be investigated before surgery to prevent blood
loss.
Feedback (grade 65%):
• No clear headings or sections – see assignment brief.
• No clear Introduction.
There is information on the coagulation cascade and the clotting screen.
• Pathophysiology and diagnosis mixed up rather.
Quite a lot on haemostasis -consider a broader range of causes.
Some good points noted here.
• Diagnosis, main points covered see point to consider on script. (tests for diagnosis?
Table?)
• Summary present -findings considered.
• References used, correct style. Presentation needs heading and a logical layout as in
the assignment brief. Difficult to follow when mixed up.
Well written though.
Question 2
A 32-year-old teacher noticed a loss of vision in one eye. Her GP referred her to a neurologist
who found no obvious abnormalities in her retina and diagnosed inflammation of the optic
nerve. However, she had a family history of multiple sclerosis, and a magnetic resonance
imaging (MRI) brain scan was ordered. The results of the brain scan showed multiple lesions
in the white matter of the brain, and she was informed that she had a high probability of
developing multiple sclerosis. Further laboratory test for oligoclonal bands using isoelectric
focussing with immunoblotting showed the presence of oligoclonal bands in the CSF, but no
bands were seen in the accompanying serum sample.
Critically evaluate the laboratory data provided for this patient and discuss the
pathophysiology of the underlying condition that these results depict. Include in your answer
explanation of the key laboratory tests that would be used to confirm diagnosis, usefulness
of these tests and potential differential diagnosis that may be observed.
Multiple sclerosis patient investigation: pathophysiology and diagnostics
Autoimmune conditions such as Multiple sclerosis (MS) occur when there is disruption
between regulatory and effector immune responses, leading to defective; control and
elimination of self-reactive lymphocytes (Rosenblum et al, 2015). MS is a chronic
inflammatory demyelinating condition affecting the central nervous system (CNS). It can arise
from a combination of environmental factors such as hormones like estrogen and infection
but also genetic factors (Loma et Heyman, 2011). It is hypothesized defective regulation for
lymphocyte activation because of genetic polymorphisms, leads to the breakdown of central
and peripheral tolerance, and environmental factors triggers self-reactive lymphocyte
activation (Rosenblum et al, 2015). The patient has a genetic predisposition for MS and is
presenting with symptoms like unilateral visual loss. Other MS symptoms include weakness,
dyscoordination, sensory loss, and more. The progression of the disease can lead to severe
disability (Loma et Heyman, 2011). This investigation will explore MS pathophysiology, testing
methods, and differential diagnoses.
The exact pathophysiology of MS is unclear but genetically, the three human leukocyte
antigen (HLA) class II alleles; DRB5*0101, DRB1*1501 and DQB1*0602 are strongly in linkage
disequilibrium for MS (Sospedra et al, 2006). T cells use various disease-associated HLA class
II molecules as restriction elements, where any changes can lead to abnormalities (Sospedra
et al, 2006). A genetic analysis would not be useful for confirmation of MS as other
autoimmune diseases arise from HLA-association genes. Findings from immunological studies
on animal models like the experimental autoimmune encephalomyelitis (EAE) outline the
mechanisms for destruction as; inflammation leading to CNS tissue damage, demyelination,
axonal damage or loss, and gliosis (Constantinescu et al, 2011). A breakdown of central and
peripheral tolerance occurs, where T regulatory (Tregs) cells against self-antigens undergo
central tolerance induction in the thymus, and self-reactive T cells are removed by clonal
deletion. If self-reactive T cells escape the thymus, they need a second costimulatory signal
for activation against self-antigens as part of peripheral tolerance. It is hypothesised that
molecular mimicry may occur, where peptides from pathogens mimic those of a CNS antigen,
triggering autoimmunity (Libbey et al, 2007). In MS, self-reactive T cells stimulate B cells to
produce autoantibodies and cross the blood-brain barrier from the bloodstream. T cells then
interact with antigen-presenting cells (APC), where pathogen-associated molecules have
bound to toll- like receptors to release cytokines interleukin (IL)-12, IL-23, and IL-4 (Loma et
Heyman, 2011). This acts as the costimulatory signal.
These cytokines activate T helper (Th) cell (CD4+ T cells) differentiation into Th1, Th2, or Th17,
which produce proinflammatory cytokines; interferon-gamma (IFNy), and tumour necrosis
factor-alpha (TNF-a) (Ghasemi et al, 2017). Th2 differentiation is suppressed by IFNy and TNFa to reduce the production of anti-inflammatory cytokines and Decreasing IL-10, IL- 35, and
transforming growth factor-beta (TGFb) levels, forming multifocal zones of inflammation
(Ghasemi et al, 2017). This inflammation leads to tissue damage. The CD4+ T cells also cause
demyelination by acting against the myelin basic protein, a myelin oligodendrocyte
glycoprotein alongside macrophagic and autoantibody destruction (Criste et al, 2014). Axonal
damage and loss occur due to primary inflammatory demyelination. These reactions explain
the symptoms commonly experienced by patients as nerve signals are disrupted (Criste et al,
2014).
Gliosis occurs when glial cells are produced as a response mechanism to trauma and form
scars around the myelin sheath (Dharmarajan et al, 2017). Retinal astrocytes, which reside in
the nerve fibre, form scars around the optic nerve in response to demyelination, disrupting
signals to the retina (Dharmarajan et al, 2017). This explains the patients’ unilateral vision
loss. This scarring is seen as lesions in the white matter of the brain in a magnetic resonance
imaging (MRI) scan, as presented for the patient. MRI is a useful technique used to visualise
white matter, microstructural changes in myelin, and the cortex’s neuroaxonal integrity
(Granberg et al, 2017). MS affects both the white and grey matter of the break but lesions in
the white matter indicate the early stages of the disease (Granberg et al, 2017).
The laboratory findings are useful as the 2017 McDonald criteria can be applied. This sets out
the clinical findings required for confirmation of MS, like the requirement that other
conditions have been tested for and ruled out, alongside guidelines for lesion spread
observation in the CNS as seen in the MRI (Thompson et al, 2018). A diagnosis of MS can be
made based on Cerebrospinal fluid (CSF) specific oligoclonal bands (OCB) if the other criteria
are met in the patient, such as the lesions evidencing spread within the brain. The patient was
tested for OCB using isoelectric focussing with immunoblotting. The isoelectric point of
protein separation is measured using gel electrophoresis (Deisenhammer et al, 2019). The
separated proteins are added to a gel, where an indirect assay is performed, with a secondary
antibody that binds to any primary antibodies present. Binding produces a colorimetric
measurable positive reaction (Deisenhammer et al, 2019). The presence of oligoclonal bands
(OCB) in CSF and the absence of OCB in serum are strongly indicative of MS, reflecting a local
B-cell response due to inflammation within the CNS (Deisenhammer et al, 2019). This is
commonly observed in autoimmune conditions. The paired serum sample demonstrates the
blood-brain barrier hasn’t been breached as the serum is a sterile site. OCBs are found in more
than 94% of MS patients, therefore are a good indicator of disease but differential testing for
other inflammatory neurological diseases such as neuromyelitis Optica (NMO) must be
conducted before confirmation as OCB are not MS specific (Deisenhammer et al, 2019). The
differentiation of MS using OCB in comparison to other autoimmune conditions falls from
94% to approximately 61% (Deisenhammer et al, 2019). For a differential diagnosis, other
specific biomarkers can be measured, such as antibodies against aquaporin 4, found on
astrocytes and elevated in diseases like NMO (Deisenhammer et al, 2019).
Overall, the patients MRI is showing lesions as a result of gliosis forming scars around
demyelinated axons because of inflammation and damage from defective Tregs and
autoantibodies in the white matter, indicating early MS. This scarring has started occurring
on the optic nerve, explaining the patients’ vision loss in one eye. The presence of OCB in CSF,
and not serum, show that the blood-brain barrier hasn’t been broken and that an
autoimmune response to inflammation within the CNS is present. Testing for other rare
diseases must be carried out before confirmation of MS can be done for this patient. A
diagnosis can have profound impacts on the patient as she is a teacher and may ne
