COVID-19; Updates on Pathophysiology and Laboratory Investigations

Dr. Abdulsalam Al-Ani
Consultant Haematopathologist
College of Medicine, University of Anbar, IRAQ
COVID-19;
Updates on
Pathophysiology and
Laboratory Investigations
A Short Review

• Pathophysiology of SARS-CoV-2 Infection.
This require a further work to elucidate.
 Direct Viral Infection.
 Immune Response in COVID-19.
 COVID-19 Induced Coagulopathy.
• Diagnostic and Prognostic Laboratory Parameters.
• Impact of Laboratory investigations.
OBJECTIVES
Dr. Abdulsalam Al-Ani, COVID-19; Updates on Pathophysiology and Laboratory Investigations #AIMCO2020

OBJECTIVES
• Pathophysiology of SARS-CoV-2 Infection.
This require a further work to elucidate.
 Direct Viral Infection.
 Immune Response in COVID-19.
 COVID-19 Induced Coagulopathy.
• Diagnostic and Prognostic Laboratory Parameters.
• Impact of Laboratory investigations.

Poland G. A. et al, The Lancet, Oct, 2020. DOI: 10.1016/S0140-6736(20)32137-1
Viruses of the Coronaviridae family
have a single strand, RNA structure
with 26 to 32 kilobases length.
Same as other, severe acute
respiratory syndrome coronavirus
SARS-CoV, SARS-CoV-2 uses for
angiotensin-converting enzyme 2
(ACE2) as its S-protein main receptor,
which is broadly expressed in
vascular endothelium, respiratory
epithelium, alveolar monocytes, and
macrophages.
Su S. et al, Trends Microbiol. 2016;24:490–502

 The SARS-CoV-2 has structural differences from
other coronavirus, in its surface proteins that enable
stronger binding to the ACE 2 receptor.
 Greater efficiency at invading host cells.
 Also it has greater affinity (or bonding) for the
upper respiratory tract and conjunctiva
 Thus it can conduct airways more easily.
Muge Cevik et al. BMJ, Oct 2020;371:bmj.m3862

The virus binds to ACE 2
receptor in synergy with the
host’s transmembrane
serine protease 2
(TMPRSS2), which is
expressed in the airway
epithelial and vascular
endothelial cells.
This leads to membrane
fusion and releases the
viral genome into the
cytoplasm.
The Virus replicate leading
to viral assembly,
maturation, with the release
of the virus.
Muge Cevik et al. BMJ, Oct 2020;371:bmj.m3862

Marcello C. and Agnello L. Diagnosis 7,4: doi; 10.1515/dx-2020-0057, 2020.

Neutrophi
l
Neutrophi
l
Pluripotent SC

TMPRSS2;
Transmembrane
Serine Protease
Tay, M.Z. et al. Nat Rev Immunol; 20, 363–374,
2020.
When SARS-CoV-2 infects cells
expressing the surface ACE2
receptors and Transmembrane
Serine Protease2 (TMPRSS2), the
active replication and release of
the virus cause the host cell to
undergo pyroptosis and release
damage-associated molecules.

MCP; Monocyte chemoattractant protein MIP ; Macrophage inflammatory protein
IP-10; IF gamma-
induced protein 10
2020.
Infected cells are recognised by
neighboring cells, endothelial cells
and alveolar macrophages, triggering
the generation of pro-inflammatory
cytokines and chemokines.
These proteins attract monocytes,
macrophages and T cells to this site,
promoting further inflammation.

2020.
In a healthy immune response,
the initial inflammation attracts
virus-specific T cells which can
eliminate the infected cells before
the virus spreads.
Neutralizing antibodies in these
individuals can block viral
infection, and alveolar
macrophages recognize
neutralized viruses and apoptotic
cells and clear them by
phagocytosis.

G-CSF, granulocyte
colony-
stimulating factor
TNF, tumour necrosis
factor
2020.
In a defective immune response,
this may lead to further
accumulation of immune cells,
causing overproduction of pro-
inflammatory cytokines, which
eventually damages the lung
tissue.
The resulting cytokine storm
circulates to other organs, leading
to multi-organ damage.

Parasher A, Postgrad Med J. doi:10.1136/postgradmedj-2020-138577 (modified image)
CXCL-10; C-X-C; chemokine ligand 10,
IFN; interferon, IL; interleukin, MCP-1;
monocyte chemoattractant protein-1,
MIP-1α; macrophage inflammatory
protein-1α, SARS-CoV-2, severe acute
respiratory syndrome coronavirus-2,
TNF-α; tumour necrosis factor-α, G-
CSF, granulocyte colony-stimulating
factor, GM-CSF, granulocyte-
macrophage colony-stimulating factor.
About 80%
of all
patients
1
2 3

Mechanism of Coagulopathy
 The novel coronavirus, SARS-CoV-2, activates the
thrombotic
process in addition to the inflammatory reaction.
 The disease it causes is associated with hypoxia, an
increase in
inflammatory cytokines (storm) and coagulation disorders,
by endothelial injury, with predisposition to thrombus
formation.
3 main mechanisms play as procoagulants
predisposing for the coagulopathy;
1. Severe and prolonged hypoxemia.
2. High incidence of cytokine storms.
3. Local pulmonary endothelial injury.
B. Marchandot et al, J. Clin. Med. 2020, 9, 1651:
1-17

Serebrovska Z.O., et al. Acta Pharmacol Sin, Oct, 2020. https://doi.org/10.1038/s41401-
020-00554-8
1. Hypoxia-Inducible Factors.
HIF; are nuclear transcriptional factors essential for genes that
mediate cellular and tissue homeostatic responses to altered
oxygenation.
Cells adapt to hypoxia through the dynamic equilibrium
between ACE-1 and ACE-2, under the control of HIF-1 and
HIF-2.
Hypoxia is a primary feature and main cause of mortality
in patients with severe COVID-19, almost all disease
stages.

1
2
3
4
5
C. S. Lim et al, J. OF V. Surgery, 58, 1; 219-230,
2013
Effect of Hypoxia-inducible Factors
Pathway

2020.
2. Inflammatory Effects
Inflammation is the triggering factor for thrombogenesis.
Cytokines and chemokines have been associated with an
important role in immunity and immunopathology during viral
infections.
In SARS-CoV-2 infection;
1- Immune response with the surge of cytokines and
inflammatory mediators, leading to activation of pro-coagulant
pathways.
2- On other hand, cytokines impaired the natural coagulation
pathways and shut down of fibrinolysis.

Perico, L. et al. Nat Rev Nephrol (2020). https://doi.org/10.1038/s41581-020-00357-4
Tay, M.Z. et al. Nat Rev Immunol; 20, 363–374, 2020.
3. Endothelial Injury
Endothelial injury, can up-regulate tissue factor expression
and further drive a pro-thrombotic state.
Endothelial cell activation/damage with collagen exposure
tigering the activation of (TF), promoting hypercoagulation
may be explain the high thrombotic burden observed.
Disseminated Intravascular Coagulation (DIC) develops as an
advanced complication of sever infection and sepsis, which is
the causative factor of high mortality rate.

Kumar V., Abbas A.K. & Aster J.C. Robbins and Cotran Pathologic Basis of Disease. 9th ed.: Saunders Elsevier.

COVID-19 Coagulopathy vs DIC
DIC, is a generalized consumptive process with
microangiopathic hemolytic process, while COVID-19 is a localized
process mostly to the lung alveoli.
1. Mild thrombocytopenia.
2. Prothrombin time (PT) not always elevated.
3. Normal activated partial thromboplastin time (aPTT).
4. No microangiopathic hemolytic process.
Iba T. et al, J Thromb Haemost. 2020;18:2103–2109.

Laboratory Parameters
Laboratory medicine has a crucial role for the
appropriate COVID-19 management from the early
recognition to the assessment of disease severity.
Also for the prediction risk of evolution towards
severe disease, characterised by the impairment of several
organs and tissues.
Marcello C. and Agnello L. Diagnosis 7,4: 10.1515/dx-2020-0057, 2020.

1- Viral Detection
 Samples are collected from the upper respiratory tract via
nasopharyngeal and oropharyngeal swabs and from the lower
respiratory tract via expectorated sputum and bronchoalveolar
lavage (for mechanically ventilated patients).
 The sample examined by polymerase chain reaction (PCR)
 The PCR results usually show positivity after 2–8 days and can
remain detectable up to 25–50 days after the onset of symptoms
and longer in the stool.
 The sensitivity of these tests is not very high, approximately 53.3%
had positive oropharyngeal swabs, and about 71% with sputum
samples.
Parasher A, Postgrad Med J. doi:10.1136/postgradmedj-2020-138577
Peeling R. W. et al, Lancet Infect Dis; 20: e245–49, Sept. 2020

2- Rapid Antigen Test
 Rapid antigen detection tests are simple and can give results in
less than 30 min.
 Although it offers an effective means in community settings, it
shows suboptimal sensitivity to be recommended for clinical
diagnosis.

3- Antibody Detection (Serology Tests)
 IgM antibodies start to be detectable around 5–10 days after
onset of symptoms and rise rapidly.
 Seroconversion from IgM to IgG is typically within the first 3
weeks.
 Neutralising antibodies can be detected approximately 7 days
after onset of symptoms and rise steeply over the next 2
weeks.
 IgG titres (levels) usually remain for months or years.
WHO, Update: August 2020

Sethuraman N, Jeremiah SS & Ryo A. JAMA. 2020;323(22):2249–2251.
doi:10.1001/jama.2020.8259

Liani D. et al, The Lancet Haematology 2020; 7: e671-e678
Parasher A, Postgrad Med J. doi:10.1136/postgradmedj-2020-138577 Tan L. et al. The Nature, Target. Ther., vol. 5, no. 1, pp. 16–18, 2020
4- Haematological Parameters in CBC
 Haemoglobin concentration usually not affected.
 A normal or decreased white blood cell count can be observed.
 Lymphopenia found to be one of the commonest CBC parameters
abnormality.
 This may due to a direct target of viruses or Inflammatory cytokines
on lymphocytes or the lymphatic organs (thymus and spleen).
Elevated blood levels of metabolites may inhibit lymphocytes
proliferation.
 An elevated neutrophil-to-lymphocyte (N/L) ratio also reported.
 Other WBC differential counts usually show variable results.
 Mild thrombocytopenia can be present which correlates with the
severity.

Moderate disease Severe disease Critical disease
White blood cell count, 10 9 /L
5·05
(3·98–6·26)
5·89
(4·36–8·28)
9·33
(6·65–13·40)
Neutrophil count, 10 9 /L
3·22
(2·37–4·39)
4·19
(2·97–6·33)
8·08
(5·30–12·12)
Lymphocyte count, 10 9 /L
1·2
(0·90–1·59)
0·89
(0·59–1·36)
0·54
(0·30–0·91)
Neutrophil / lymphocyte ratio
2·67
(1·69–4·08)
4·71
(2·62–7·78)
16·02
(6·49–24·79)
Monocyte count, 10 9 /L
0·46
(0·35–0·61)
0·52
(0·32–0·64)
0·38
(0·20–0·54)
Eosinophil count, 10 9 /L
0·04
(0·01–0·10)
0·05
(0·01–0·11)
0·01
(0–0·03)
Basophil count, 10 9 /L
0·01
(0·01–0·03)
0·01
(0·01–0·03)
0·03
(0·01–0·03)
Haemoglobin, g/dL
122·00
(111·0–133·0)
118·00
(108·50–129·50)
115·00
(92·75–127·25)
Platelet count, 10 9 /L
198·0
(145·5–249·5)
227·0
(142·5–328·0)
105·0
(55·75–200·75)Liani D. et al, The Lancet Haematology 2020; 7: e671-e678

Tan L. et al. The Nature, Target. Ther., vol. 5, no. 1, pp. 16–18, 2020
5- Blood Film
 Presence of reactive lymphocytes was reported, but still needs more
confirmative results.
 Two lymphocyte subsets where observed; the large with abundant
cytoplasm with more darkly stained at the peripheral margins
(Hyperbasophilic) and the lymphoplasmacytoid forms.
 Other WBC lineage showed variable degrees of changes.
 As a complementary test, white blood cell (WBC) differential
fluorescence (WDF) scattergram shows atypical form. This consisted
of a discontinuous cluster of lymphocytes characterized by the
presence of more than four dots in the upper graduation of the
scattergram (‘sandglass’ aspect).

V. C. L. Chong, et al., Br. J. Haematol., 189.5;844, 2020 Al-Ani A., AMJ. doi.org/10.33091/AMJ.1001622020

Osman J. et al, British Journal of Haematology; 190 : 5; 718-722, 2020,
DOI: (10.1111/bjh.16943)
White blood cell (WBC)
differential fluorescence
(WDF) scattergram, displaying
a classification of WBCs
based on their morphology
and their intracellular
components.

Al-samkari H. et al. Blood, 136 (4): 489–500.2020Iba T. et al. J Thromb Haemost.18:2103–2109, 2020.
6- Coagulation Parameters
Thrombotic complications can developed in about; 5% and 18%
in noncritically ill and critically patients respectively.
The coagulation study is one of crucial tests for disease
progression.
 PT and PTT in most of the cases are normal or showed slight
prolongation.
 Elevated D-dimer and fibrinogen degradation product (FDPs) at initial
presentation was reported as a predictive of coagulation-associated
complications and associated with poor outcome.
 Nonsurvivors developed higher D‐dimer and FDP levels, with longer
PT and aPTT compared with survivors at admission
 Incidence of thrombocytopenia is relatively low. A low platelet count
is associated with increased risk of disease severity and mortality.

Liani D. et al, The Lancet Haematology 2020; 7: e671-e678 (modified)
https://rebelem.com/covid-19-thrombosis-and-hemoglobin/
Disease Progression

Imazio M. et al, Heart 2020;106:1127-1131, BMJ.com
Manson J. J. et al, Lancet Rheumatol; 2: e594–602, 2020Lin Z. et al. J Infect. 2020;81(4):647-679.
7- Biomarkers;
Most important are; CRP, Ferritin, Procalcitonin (PCT) and Troponin I.
 A clear associations was found between elevated biomarkers, and
survival in COVID-19 patients.
 COVID-19 hyperinflammatory syndrome (COV-HI), which might
contribute to morbidity and mortality in sever disease.
 COV-HI; CRP>150 mg/l with doubling within 24hrs or Ferritin>1500
μg/L.
 Hyperferritinemia, was a common feature in sever disease.
 Increased CRP level correlates with higher serum ferritin. Both were
able to predict an increased risk of disease severity in patients with
COVID-19.

Lin Z. et al. J Infect. 2020;81(4):647-679.

Lippi G, Plebani M. Clin Chim Acta. 2020;505:190-191.
 ESR may be elevated, but it is usually showed a variable values
 Substantial increase of PCT would reflect bacterial coinfection in
those developing severe form. Nevertheless, the synthesis of this
biomarker is inhibited by interferon, which increases during viral
infections.
 Troponin I levels were significantly higher in those with severe
COVID-19 infection compared with those with non-severe disease.
 Its elevation was related to non-ischaemic myocardial injury.

Troponin elevation in COVID-19 can be related to non-ischaemic myocardial injury.

Huan H, et al; Emerg Microbes Infect,9:1, 1123-1130
8- Serum Cytokines
 Results showed that COVID-19 patients have higher serum level
of certain cytokines (TNF-α, IFN-γ, IL-2, IL-4, IL-6 and IL-10) than
control individuals.
 Within COVID-19 patients, serum IL-6 and IL-10 levels are
significantly higher in critical group than in moderate and severe
group.
 The levels of IL-10 is positively correlated with CRP amount.

The levels of cytokines ( Median & range) in COVID-19 patients and controls

The levels of cytokines ( Median & range) in covid-19 patients with different severity

9- Biochemical Parameters
 Elevated lactate dehydrogenase (LDH), found to be on the most
important biochemical changes at time of diagnosis.
 Assessment of renal and liver functions are essential for detection
of any organ damage.
 For prognostication purposes, increased values of LDH, aspartate
aminotransferase (AST, GOT) and alanine aminotransferase (ALT,
GPT), total bilirubin, together with decreased values of serum
albumin, have been found of value.
Marcello C. and Agnello L. Diagnosis 7,4: 10.1515/dx-2020-0057, 2020.
Lippi, G., & Plebani, M. Clinical Chemistry and Laboratory Medicine, 58(7), 1063-1069. 2020

Haematological
Lymphocytes Reduced
Platelets
Reduced
Increased
Neutrophils
Increased
Reduced
N/L ratio Increased
Eosinophils Reduced
Leukocytes
Reduced
Increased
Hemoglobin (Hb) Reduced
Coagulation
D-dimer Increased
Prothrombin time Increased
Silvia Toledo et al, Clin Chim Acta. 2020 Nov; 510: 170-1

Inflammatory
C-reactive protein, CRP Increase
Ferritin Increased
Procalcitonin Increased
Troponin Increased
Cytokines Increased
Biochemical (Cont.)
Alanine aminotransferase, ALT Increased
Aspartate aminotransferase, AST Increased
Urea & Creatinine Increased
Total bilirubin Increased
Urea & Creatinine Increased
Albumin Reduced
Creatine kinase, CK Increased
Biochemical
Lactate dehydrogenase, LDH Increased
Silvia Toledo et al, Clin Chim Acta. 2020 Nov; 510: 170-1

Impact of COVI-19
The COVID-19 indebtedness was so serious from the social and
economic aspects.
 Burden on the community.
Effect on the healthcare system and workers.
The Bulletin of RCPath, number 191, 2020

Impact on the Patients and Families
(Social and Economic)
 Avoidance of unnecessary laboratory tests is crucial.
 Precise selection of laboratory investigations, both, at the time of
diagnosis and follow up and a balance between the requested laboratory
tests and its importance.
 Reducing the patients’ burden and who take care of them and the
psychological stress needs to be taken into consideration when request
frequent tests.
 Associated financial costs also need to be aware of it.
 The pandemic necessitates reducing the test volumes to decrease the
laboratory workload.

Impact on Laboratory Workers
The pandemic has necessitated significant changes in how pathology
laboratories operate.
 Strict social distancing requirements have imposed limitations on
staff and workflows in all workspaces.
 Laboratory staff were affected from their pattern of life and family
time.
 Additional burden due to the use of personal protective equipment
requirements meant that some processes newly required use of
masks, gowns and goggles.
 The coronavirus pandemic has required significant changes to
workflow, sample collection and processing within the laboratory.

COVID-19; Updates on Pathophysiology and Laboratory Investigations

COVID-19; Updates on Pathophysiology and Laboratory Investigations

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