HANDBOOK OF COVID 19

Editor

Brig A S Menon

Associate Editors

Gp Capt TVSVGK Tilak Wg Cdr Rohit Vashisht

ARMED FORCES MEDICAL COLLEGE PUNE

Note from Editors

The science of COVID 19 is evolving. Readers are advised to refer to suggested reading for updates

Published by

Armed Forces Medical College, Pune

Cover Design Aditi Menon

This book is a training manual for officers of the Armed Forces Medical Services and is NOT for sale

FOREWARD

The world has been in the grip of COVID Pandemic since end of 2019. Such has been the impact of the disease, that the very behaviour of humankind has been altered with respect to awareness, hygiene, travel and working atmosphere etc. Despite the nationwide approach of lockdowns and restrictions, cases and mortality due to the disease continue to occur.

There has been an exponential growth in the literature on various aspects of the disease and the pandemic. A search on Pubmed with the terms ‘COVID/Therapy’ revealed more than seventeen thousand articles. Multiple agencies are issuing guidelines on all aspects of the disease. There is a need to distil the knowledge and make it available to Health Care Workers of the Armed Forces. The ‘Handbook of COVID-19’ is an attempt to make available information that is relevant in a concise manner.

I compliment the authors as well as the editorial team for the effort taken in putting together the Handbook of COVID-19.

PREFACE

1. Ever since the onset of COVID pandemic, the Armed Forces Medical College has been actively involved in the nation’s response to tackling the pandemic. AFMC was identified as a nodal centre for mentoring of institutions in setting up RT-PCR laboratories. Faculty from the college have been co-opted in state task force and have conducted online training for HCW’s of Armed Forces and civil alike. Hospitals affiliated to AFMC have participated in clinical trials on patients with COVID. Faculty, residents, nursing and paramedical staff were deployed at various ‘BROWNFIELD’ and ‘GREENFIELD’ COVID hospitals across the country during the first and second wave. The faculty has been actively involved in research about disease prediction models, epidemiology, immune response and have numerous publications in this regard.

2. It is but natural that the vast experience of AFMC in handling COVID-19, both administrative as well as clinical, would be useful in bringing about the handbook. The chapters in handbook are designed to provide the reader a quick reference without burdening them with details. The various departments of AFMC have endeavoured to bring forth latest information as available, however as the editor’s note states that the knowledge is accruing and readers should refer to suggested reading at end of each chapter for updates.

3. I compliment the authors as well as the editorial team for the effort taken in bringing about the Handbook of COVID-19 at short notice.

Best Wishes Jai hind

Sr No.

CONTENTS Chapter

Genealogy of COVID 19 Pandemic…………………………….. Coronavirus and SARS CoV2……………………………………… Immune response and Immunopathogenesis ……………………… Laboratory Diagnosis of COVID 19………………………………. Role of Radiodiagnosis in Management of COVID-19…………… Management of Mild COVID 19………………………………….. Management of Moderate and Severe COVID 19………………… Oxygen therapy, ventilation & ICU management…………………. COVID 19 in Children…………………………………………….. Complications of COVID 19……………………………………… Vaccines……………………………………………………………. Impact of COVID 19 Pandemic on mental health………………… Public Health challenges in COVID 19 Pandemic………………… Challenges in establishing COVID Care Hospital…………………

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109

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LIST OF CONTRIBUTORS

Surg Cmde KM Adhikari

Professor & Head Dept of Pediatrics

Maj Varun Anand

Clinical tutor

Dept of Radiodiagnosis

Col A T Atal

Assistant Prof

Dept of Internal Medicine

Surg Cdr Kavita Bala Anand

Assoc Prof

Dept of Microbiology

Surg Capt V Bhaskar

Professor

Dept of Community Medicine

Surg Capt Saurabh Bobdey

Professor

Dept of Community Medicine

Surg Cmde K Chatterjee

Professor & Head Dept of Psychiatry

Col Neeraj Garg

Assoc Professor

Dept of Hospital Administration

Col Nikahat Jahan

Professor

Dept of Anaesthesiology& Critical Care

Gp Capt BM John

Professor

Dept of Pediatrics

Lt Col S Karade

Assoc Prof

Dept of Microbiology

Brig SK Kaushik

Professor & Head

Dept of Community Medicine

Col Vikas Marwah

Professor & Head

Dept of Respiratory Medicine

Brig A S Menon

Professor & Head

Dept of Internal Medicine

Surg Lt Cdr Kranthi K Nethi

Resident

Dept of Hospital Administration

Maj Neelesh Patel

Clinical tutor

Dept of Hospital Administration

Col S K Patnaik

Professor & Offg HOD

Dept of Hospital Administration

Maj Deepu K Peter

Clinical tutor

Dept of Respiratory Medicine

Col Jyoti Prakash

Professor

Dept of Psychiatry

Col Ravinder Sahdev

Assoc Prof

Dept of Radiodiagnosis

Maj Apoorv Saxena

Clinical tutor Dept ofPediatrics

Air Cmde Arijit Sen

Professor & Head Dept of Pathology

Col Kiran Sheshadri

Professor

Dept of Anaesthesiology & Critical Care

Brig Rangraj Setlur

Professor & Head

Dept of Anaesthesiology & Critical Care

Air Cmde SP Singh

Professor & Head Dept of Microbiology

Gp Capt TVSVGK Tilak

Professor

Dept of Internal Medicine

Wg Cdr Rohit Vashisht

Assistant Prof

Dept of Internal Medicine

Surg Lt Cdr Shrinath V

Resident

Dept of Respiratory Medicine

Lt Col AK Yadav

Assoc Prof

Dept of Community Medicine

Maj Naveen Yadav

Clinical tutor

Dept of Internal Medicine

Lt Col Y Uday

Assoc Professor

Dept of Internal Medicine

1. Genealogy of the Pandemic

Lt Col AK Yadav, Surg Capt Saurabh Bobdey, Surg Capt V Bhaskar, Brig SK Kaushik

Introduction

COVID-19 (Coronavirus Disease-2019) pandemic is the worst of its kind faced by humanity after the ‘Spanish Flu’. COVID-19 is an acute viral disease caused by infection with Severe Acute Respiratory Syndrome – Corona Virus-2 (SARS- CoV-2), a newly emerged zoonotic infectious disease. As the disease spread across the globe, WHO declared COVID-19 as a Public Health Emergency of International Concern (PHEIC) on 30 January 2020 and a pandemic on 11 March 2020.

Magnitude of the problem

World As of 30 July 2021, there have been approximately 19.4 crores COVID-19 cases and nearly 41.6 lakh deaths worldwide. As per published literature, it is believed that the virus originated from the seafood market of Wuhan city of Hubei province, China. However, it was soon brought under control in China by strict lockdown in the affected areas. Nevertheless, the virus rapidly spread across the globe, causing high morbidity and mortality in many countries. A few of the worst affected countries are the USA, United Kingdom, Italy, Brazil, Russia, France and India. However, in varying proportions,this deadly disease has affected almost the whole world.

India On 30 January 2020, the first case was confirmed in Kerala’s Thrissur district in a student who had returned home for a vacation from Wuhan University in China. In February 2020, very few cases were reported from India. However, in March 2020, COVID cases started rising in many states, and therefore, to suppress transmission, the Union government declared a countrywide lockdown on 24 March 20. In early Aug-Sep 2020, India experienced a surge of cases and crossed the five million mark on 16 September 20 (‘first wave’). There was a steady decline in the number of cases reported from the end of September 2020. However, from the middle of February 2021, a massive upsurge started (‘second wave’), with the number of cases and deaths

reported daily showing a very steep climb (Fig1.1). During the second wave, more than four lakh new cases and 4000 deaths were reported per day. COVID cases started declining from mid-May, but India continues to simmer with almost forty thousand new cases and more than 500 deaths per day till 30 July 2021.

Fig 1.1 Graph of occurrence of new COVID cases in India Agent factors

a) Morphology: The agent is a positive sense, single-stranded, enveloped RNA virus belonging to the family Coronaviridae. The word Corona means crown. It is so named because the virus has a shape of a crown with small bulbar projections formed by the viral spike (s) peplomers

b) Reservoir: Human being is the only reservoir for SARS-CoV-2

c) Source of Infection: The source of infection is an infected human being who discharges the virus into the environment in respiratory droplets.

d) Mode of entry: The virus gains access into the human body primarily through the respiratory route. It establishes infection through “spike protein” on its surface, which combines with the “ACE receptors” present in the respiratory tract of human beings.

Num bers per day

e) Infectivity: The virus is highly infectious, especially in a congested environment. An infected person can transmit the virus to others before the onset of symptoms and in the early course of illness. Peak viral shedding seems to occur at the time of symptom onset and declines thereafter.

f) Pathogenicity: Though the infectivity is high, the pathogenicity is low, as a large percentage of infected persons remain asymptomatic.

g) Survival in nature: The COVID-19 virus is a very fragile organism and does not survive for more than a few hours outside the human body. However, studies have shown that the virus can survive on artificial surfaces in the form of moist droplets for up to a few days. A study evaluating the duration of the viability of the virus on objects and surfaces showed that SARS-CoV-2 could be found on plastic and stainless steel for up to 2-3 days, cardboard for up to 1 day, and similarly virus remains viable on commonly touched items such as computer mouse, keyboard, handrails, etc.

h) Susceptibility: The virus is quite susceptible to natural agents as bright sunlight, heating, and drying. Direct bright sunlight and total drying can inactivate the virus in a few hours while heating at 50OC for 10 minutes will also inactivate it. The virus is susceptible to chemical agents, with 1% sodium hypochlorite, 60% alcohol, and scrubbing with ordinary soap deactivating it within a few seconds.

j) Mutant Strains: Mutations arise as a natural by-product of viral replication and hence are bound to happen with the passage of time. RNA viruses typically have higher mutation rates than DNA viruses. Coronaviruses, however, make fewer mutations than most RNA viruses because they encode an enzyme that corrects some of the errors made during replication. In most cases, the fate of a newly arising mutation is determined by natural selection. Those that confer a competitive advantage for viral replication, transmission, or escape from immunity will increase in frequency. Those that reduce viral fitness tend to disappear from the population of circulating viruses. Multiple variants of the SARS-CoV-2 virus have emerged, which are quite different from the

original Wuhan virus. For ease and uniformity, WHO recommends that the nomenclature of the variants be based on Greek alphabets like alpha, beta, gamma, delta, etc.

Host Factors

(a) Age: Children less than ten years of age seem to be less susceptible. Persons more than sixty years tend to have more severe disease and have a higher risk of mortality than younger ones.

(b) Sex: The occurrence of cases shows a slightly higher preponderance among males, but this may be due to more reporting of symptoms and consequent testing rather than due to actual biological differences.

(c) Pre-existing diseases: Persons with co-morbidities (Diabetes, Chronic Lung Disease, Obesity, cardiovascular disease, diseases associated with immunosuppression like cancers) tend to have more severe diseases and a higher risk of mortality.

(d) Occupation: Healthcare workers are at a particularly high risk of being infected. Similarly, other frontline workers such as surveillance workers, police, etc. are at a higher risk

(e) Genetic & racial Factors: The role of genetic factors is not clear. Environmental Factors

(a) Climate and seasons: So far, no evidence has emerged indicating the role of climate or seasons in the transmission of COVID-19

(b) Overcrowding: The single most important social factor, which determines the transmission of COVID-19, is overcrowding. It is primarily a disease of ‘closely huddled humans’. Whenever a large number of humans crowd together, COVID transmission is likely to be intense.

Transmission dynamics

(a) Infective material: The infective material is primarily the oro-

pharyngeal secretions of an infected person.

(b) Modes of transmission: Transmission of the disease can broadly occur by direct transmission i.e. when a susceptible individual comes in close contact with an infected people through infected secretions such as saliva or respiratory secretions or due to indirect transmission i.e. involving contact of a susceptible host with a contaminated object or surface. A detailed brief of various modes of transmission is as follows

(i) Droplet Transmission Transmission of SARS-CoV-2 can occur through direct, indirect, or close contact with infected people through saliva and respiratory secretions or their droplets, which are expelled when an infected person coughs, sneezes, talks, or sings. In these circumstances, droplets that include viruses can reach the mouth, nose or eyes of a susceptible person and can result in infection.

(ii) Airborne Transmission Airborne transmission is defined as the spread of an infectious agent caused by the dissemination of aerosols that remain infectious when suspended in air over long distances and time. Airborne transmission of SARS-CoV-2 can occur during medical procedures that generate aerosols. Epidemiologists and scientists have been evaluating SARS-CoV-2 spread through aerosols, particularly in indoor settings with poor ventilation. Evidence suggests that the virus spreads through airborne transmission especially in closed areas with poor ventilation (places of worship, marriage halls, lecture halls, theatres, restaurants etc.), and situations where human beings are congested in a small space (religious ceremonies, marriages, business meetings, lectures, festivals, political rallies, etc.).

(iii) Fomite Transmission Respiratory secretions or droplets expelled by infected individuals can contaminate surfaces and objects, creating fomites. Viable SARS-CoV-2 virus and/or RNA

detected by RT-PCR can be found on those surfaces for periods ranging from hours to days, depending on the ambient environment (including temperature and humidity) and the type of surface. Transmission may also occur indirectly through touching surfaces/ objects contaminated with virus from an infected person (e.g. stethoscope or thermometer), followed by touching the mouth, nose, or eyes. Despite evidence of SARS-CoV-2 contamination and survival of the virus on surfaces, there has been no report that demonstrates conclusively direct fomite transmission. People exposed to potentially infectious surfaces often also have close contact with the infectious person, making the distinction between respiratory droplet and fomite transmission difficult to differentiate.

(iv) Other modes of Transmission SARS-CoV-2 RNA has also been detected in other biological samples, including the urine and faeces. However, there have been no published reports of transmission of SARS-CoV-2 through faeces or urine. Current evidence suggests that humans infected with SARS-CoV-2 can infect other mammals, including dogs, cats, and farmed mink. However, it remains unclear if these infected mammals pose a significant risk for transmission to humans.

(c) Incubation period: The usual incubation period is 5 to 7 days (range 1-14 days) and studies have shown that more than 97% of cases experience symptoms within eleven days of contacting the virus.

(d) Period of communicability: The usual period of communicability ranges from 2 days before the onset of symptoms to 5-7 days after the onset. The infected person can shed the virus as much as twenty eight days or more following entry of the organism into the body. Depending upon whether the infected person develops symptoms, the potential to transmit the disease is as follows:

(i) COVID 19 cases They are the most important source of infection and transmit the virus during the entire phase of symptomatic illness.

(ii) Pre-symptomatic infected persons These are infected persons who will develop symptoms as against asymptomatic infected persons. These persons have an important role in transmitting the infection, as they transmit the infection two days before the onset of symptoms.

(iii) Asymptomatic infection These are persons who are infected but will never develop symptoms. These persons may transmit the infection; however, their role in the extent of transmission is debatable.

Conclusion

COVID-19 is an acute infectious disease caused by SARS-CoV-2. It has spread

rapidly across the world and has affected all continents and countries. The

person is infective in asymptomatic and pre-symptomatic phase of disease,

which makes prevention and control of infection challenging. India has already

seen two waves of COVID 19, with the second wave more devastating than the

first. The future of the pandemic will depend on the ongoing evolution of

SARS-CoV-2 besides the behaviour of citizens, governmental efforts to respond

effectively including rapid vaccination and the extent to which the international

community can cooperate in these efforts.

Suggested Reading

1. Guo ZD, Wang ZY, Zhang SF, Li X, Li L, Li C, et al. Aerosol and surface distribution of Severe Acute Respiratory Syndrome Coronavirus 2 in hospital wards, Wuhan, China. Emerg Infect Dis. 2020; 26(7):1583-1591.

2. Lauring AS, Hodcroft EB. Genetic Variants of SARS-CoV-2—What Do They Mean? JAMA. 2021; 325(6):529–531.

3. WHO, scientific brief. Transmission of SARS-CoV-2: implications for infection prevention precautions. Available at https://www.who.int /news-

room/ commentaries/detail/transmission-of-sars-cov-2-implications-for- infection-prevention-precautions (Last visited on 13 Aug 2021)

2. Coronaviruses & SARS-CoV-2

Air Cmde SP Singh, Surg Cdr Kavita Bala Anand, Lt Col S Karade

Introduction

Coronaviruses were first discovered in the nasal washings of a male child in 1965 by Tyrrell et al and since then a number of coronaviruses have been discovered. Till the emergence of SARS-CoV in 2002 & 2003 in Guadong China, this group of virus was considered innocuous only causing mild illness in the immunocompetent individual. Ten years later in 2012, a highly pathogenic MERS-CoV emerged in the Middle Eastern countries. In Dec 2019, a cluster of cases of Pneumonia were reported from Wuhan, China. Next Generation sequencing technology led to the discovery of new human Coronavirus belonging to the Genus Beta coronavirus which was provisionally named as 2019 novel Coronavirus (2019-nCoV) and later named as the SARS- CoV-2 by the International Committee on Taxonomy of Viruses (ICTV) and disease caused by it was named as COVID 19.

Classification of Coronaviruses

Corona viruses belong to the family Corona viridae. They are divided into four genera: Alpha coronavirus, Beta coronavirus, Gamma corona virus and Delta corona virus.

Structure of Coronaviruses

Corona viruses are named after the crown like spikes on their surface as seen under Electron microscope. SARS-CoV-2 belongs to the genus beta corona virus, subgenus Sarbeco virus. It is approximately 80–200 nm in size. It contains about 30kb single stranded positive sense RNA. The 3’ end encodes for the structural proteins including spike (S), envelope (E), membrane (M) and nucleoprotein (N). The 5’ terminal end of this virus encodes for the non

The beta corona viruses are further divided into four

lineages A, B, C & D. The humans are affected by the lineage A-OC 43 &

HKU1, lineage B (SARS-CoV and SARS-CoV-2) and lineage C (MERS-CoV).

structural proteins polyprotein 1ab that gives rise to further 16 non structural proteins.(Fig 2.1 & 2.2).

SARS-CoV-2 contains 20 nm spike glycoproteins embedded in its envelope, these are club shaped and are called peplomers, these help the virus attach to host cells. The spike protein has two subunits S1 and S2. The S1 subunit harbours the receptor binding domain (RBD). Betacoronaviruses belonging to lineage A also harbour a hemagglutinin esterase on their surface.

Fig 2.1 Structure of SARS-CoV-2

Fig 2.2 Genome of SARS-CoV-2

UTR (Untranslated region) ORF (Open Reading Frame)

Origin of SARS-CoV-2

Corona viruses have been found in large number of domestic and wild mammals and birds, suggesting that birds and bats are the natural reservoirs of the virus. Corona viruses also have potential for interspecies transmission which can also cause zoonotic outbreaks. The genomic characterization of the SARS- CoV-2 from Wuhan cluster carried out by various study groups was done using next generation sequencing of samples from bronchoalveolar lavage fluid and cultured isolates. These studies showed that SARS-CoV-2 was related (with 88% identity) to two bat-derived severe acute respiratory syndrome (SARS)- like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21 and more distant from SARS- CoV (about 79%) and MERS-CoV (about 50%). Zhou et al. demonstrated that the novel virus has 96.2% similarity to a bat SARS-related Coronavirus (SARSr-CoV; RaTG13). The S1 protein, is phylogenetically closer to pangolin-CoV than RaTG13 and the RBD region within the S1 has been found to be conserved between Pangolin CoV and SARS-CoV2. The origins of the SARS-CoV-2 however still remain a matter of debate.

Virus entry into host cells

The host cellular receptor for SARS-CoV-2 is ACE-2 receptor (Angiotensin convertase enzyme 2). I

bytransmembrane protease, serine 2

SARS-CoV-2. TMPRSS2 primes the spike protein domain by cleaving the S1/S2 site, which leads to fusion of the virus to the respiratory epithelial cells by binding to the ACE 2 receptors. The receptor for SARS-CoV is also ACE-2 receptor. MERS-CoV binds to the Dipeptydyl peptidase 4 (DPP4).

Variants

All viruses, including SARS-CoV-2, undergo changes in their genomic structure over time. These heritable changes in the nucleotide sequence of the genome are called mutation. Most mutations have little to no impact on the virus’ properties. However, some of the mutations in the virus may provide survival advantage. The original SARS-CoV-2 isolated in 2019, in Wuhan,

Infection starts when the viral spike protein attaches to its complementary

host cell receptor.

nitial spike protein priming

(TMPRSS2) is essential for entry of

China has undergone variety of changes in different structural and functional genes due to natural selection process or immune pressure.

A new SARS-CoV-2 variant may have different mutations that can affect transmissibility, antigenicity, or virulence. WHO has defined a SARS-CoV-2 variant of concern (VoC) as one that has propensity of increased transmissibility, alteration in clinical presentation as compared to the original. Rise in infections due to VoC is a threat as it decreases the effectiveness of various public health measures such as vaccine outreach, therapeutics and diagnostics concern. Currently SARS-CoV-2 Delta variant, first reported from India, is considered an important VoC that has spread to over 100 countries till now. In addition, Variant of interest (VoI) is another term used for those variants that have caused community transmission or cluster of cases. WHO recognized VoI and VoC are enumerated in Table 2.1 and Table2.2

Table 2.1 WHO labelled SARS-CoV-2 Variant of concern. Picture Courtesy: WHO; Tracking SARS-CoV-2 variants (who.int)

Table 2.2 WHO labelled SARS-CoV-2 Variant of interest. Picture Courtesy: WHO; Tracking SARS-CoV-2 variants (who.int)

Suggested Reading

1. Tyrrell DAJ, Bynoe ML. Cultivation of a novel type of common cold virus in organ cultures. BMJ. 1965; 1:1467e1470.

2. Fields Virology

Nat Rev Microbiol.2019; 17:

4. Zhong NS., Zheng BJ, Li YM, Poon LLM, Xie ZH, Chan KH et al.

Epidemiology and cause of severe acute respiratory syndrome (SARS) in

Guangdong, People’s Republic of China. Lancet.2003;362:1353–1358

5. DrostenC, GuntherS, PreiserW, van den WerfS, BrodtH, Becker S et al. Identification of a novel coronavirus in patients with severe acute

respiratory syndrome. N. Engl. J. Med.2003; 348:1967–1976 .

6. Naming the coronavirus disease (COVID-19) and the virus that causes

it”. https://www.who.int/emergencies/diseases/novel-coronavirus- 2019/technical-guidance/naming-the-coronavirus-disease-(covid-2019)- and-the-virus-that-causes-it (Last visited 13 Aug 2021)

Masters, P. S. & Perlman, S. in

Vol. 2 (Eds Knipe, D. M.

& Howley, P. M.) 825–858 (Lippincott Williams & Wilkins, 2013).

Cui, J., Li, F. & Shi, ZL. Origin and evolution of pathogenic

coronaviruses.

181–192.

Research. 28: 35–112

Sturman LS, Holmes KV (1983-01-01). Lauffer MA, Maramorosch K

(eds.).

The molecular biology of coronaviruses.Advances in Virus

8. Gadsby NJ, Templeton KE. Coronaviruses.In: Caroll Karen C, Pfaller MA, Landry ML, McAdam AJ, Patel R. editors. Manual of Clinical

Microbiology. 12thed. Vol. 2. Washington DC: ASM Press; Chapter 92.

9. Lu R, Zhao X, Li J, NiuP, YangB, WuH, Wang W et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet. 2020;395 :

565-574.

3. Immune response and Immunopathogenesis Air Cmde Arijit Sen

The initial entry and response to the virus

The first event is the interaction of the COVID-19 virus and human cell takes place through the Angiotensin Converting Enzyme 2 Receptor(ACE2 R). The spike proteins of the virus attaches to the

the upper respiratory pathway. In the lower respiratory tract it attaches to type II

goblet and mucociliary cells of

pneumocytes in the alveoli, macrophages and the dendritic cells. It also

manages to penetrate the endothelial lining cells. All the mentioned cells are

rich in ACE2 receptors. Cellular serine protease TMPRSS2 is used by the virus

to initiate this binding. Further the virus particle enters the cell by endocytosis.

After entry of the virus into the epithelial and endothelial cells, it starts

multiplying and eventually causes pyroptosis. This is a special type of pro-

inflammatory apoptosis. This recruits macrophages which release IL-6

(interleukin), IL10 and TNFα (Tumor Necrosis Alpha). Other pro-inflammatory

cytokines are released too IL-2, IL-2R, IL-7, IL-8 and MIP1A (Macrophage

inflammatory protein 1). The more the severe infection the more the number of

macrophages and the more the amount of cytokines released. IL6 also

suppresses T Cell function. On binding of the virus the ACE2 is released from

the surface of the epithelial surface into the airway surface liquid. This is

brought about by the metallopeptidase. Metallopeptidase too processes the

activation of the membrane form of interleukin-6 (IL6) receptor to soluble form.

This in turn activates the signal transducer and activator of transcription factor 3

(STAT3) via gp130. The STAT3 in turn activates pro-inflammatory nuclear

factor kappa beta (NF-kB). There is thickening of the interstitium over the next

few days. The alveolar lining permeability increases leading to pulmonary

oedema and subsequently formation of hyaline membrane. Neutrophils and

monocytes also pour into the alveolar space (Fig3.1).

Immune response to infection

The immune response to any infection can be divided as follows:- Innate Immunity

Adaptive Immunity

Humoral Immunity (B Cell Response) Cell Mediated Immunity (T Cell response)

a) Innate Immunity

During routine viral infection the innate immunity works with Pattern

Recognition Receptors (PRR) such as TLR7 (Toll Like Receptor) and TLR8.

This further downstream activates production of antiviral interferons and

chemokines which recruit immune cells like lymphocytes and natural killer

cells. It has been found, that COVID-19 does not induce any such immune

response through the innate immunity. This suggests that unlike other

Coronaviruses, COVID-19 does not elicit adequate immune response

through the innate immunity pathway to induce an adequate adaptive

immunity. This allows the virus to continue to multiply inside the

pneumocytes.

b) Adaptive Immune response

(i) Humoral Immunity (B Cells)

In the early phase of infection there is rise of IgM and IgA, which do not

appear to be protective. In 7-14 days the IgG appears against the spike

protein. The CD4+ T Helper cells present in the follicular centre induce

the naive B lymphocytes to progress towards plasma cells and produce

these antibodies. Memory B cells also get formed in response. This is

supposed to be the neutralizing antibody. They peak by 50 to 60 days post

infection and fade by around 10 months. The memory B cells generated

in the process can again produce the IgG antibody in cases of re-infection

and be protective. Two IgG antibodies have been identified which are

effective against the receptor binding domain (RBD) of the COVID-19

virus and can be effective in preventing of binding of the virus to the

ACE2 receptors and hence are protective. The short lived existence of the

neutralizing antibodies has raised a concern against protection to re-

infection of vaccine induced protection. The memory B cells and the long

lived plasma cells will play a role in generating anti-RBD antibodies

during reinfection, this needs to be further studied. In addition T Cell

memory may help in producing cytotoxic T cell response. The

convalescent plasma therapy did not hold ground and has been shown to

have failed to have been protective in a controlled trial. The use of

monoclonal antibody has shown some benefit when used for mild

infection in OPD patients.

(ii) Cell Mediated Immunity (T Cells)

CD4 + T cells (T Helper Cells) reactive against spike protein have been

detected in most of the patients infected with COVID-19. However it has

also been found in one third of non-infected normal population,

suggesting that these CD4+ cells likely developed due to seasonal

Coronavirus and are of cross reacting nature with other viruses of the

Corona family. Likely this protects the children and the young adults who

are frequently exposed to seasonal virus. This is in contrast to the

antibodies which are specific only to COVID-19.

CD8+ T cells (Cytotoxic T Cells) response in COVID is quite

heterogeneous. In mild COVID disease, the CD8+ T cell responds with

activation and clonal expansion. This results in formation of effector and

terminally differentiated T cells and also memory T cells. There number

is normal or increased. This leads to increased production of Interferon γ,

IL-2, CD107a (Cluster Differentiation), TNF α and GZMB (Granozyme

B) which have cytokine, chemokine and cytoxic function against the

virus. The co-inhibitory signals like PD1 (Programmed Cell Death

Protein 1), CD38, CD39, TIM3 (T Cell Immunoglobulin and Mucin

Domain) also are reduced in expression. Whereas in moderate to severe

COVID infection the activation factors IFNγ, IL-2, CD107a, TNFα,

GZMB, Perforins, CCL 3 & 4(chemokine ligand) and IL1β are all normal

or reduced and also the co-inhibitory signals are grossly increased

resulting in altered T cell function and exhaustion (Fig2.2).

Patients may have lymphopenia, leucopenia of leucocytosis, but the first is

most common. Lymphopenia is a poor prognostic factor in COVID infection

and correlates well with severity of the infection and levels of IL6 and IL 8

production. Both CD4+ (Thelper1 and Tregs) and CD8+ T cells reduce in

number. CD8+ (Cytotoxic T Cells) reveal abnormal function and

exhaustion. NK Cells and monocytes too reveal reduced number and

abnormal function in moderate and severe COVID -19 infection. Neutrophil

to lymphocyte ratio (NLR) is calculated for prognostication. Moderate

infection usually has a NLR of 4.8 ( +/- 3.5), whereas in severe infection a

NLR of 20.7 to 24.1 is usually seen. A NLR of 3.3 is taken as a cut off for

poor prognosis.

Cytokine Storm

Cytokines are a large family of small proteins. They are important in cell

signaling and play a role as immune-modulators. The family includes

interferons, interleukins, chemokines, lymphokines and tumor necrosis factor.

Cytokine storm is an unregulated host immune response in auto-amplifying

production of cytokines. Adaptive immunity induced by the CD4 (Th1) is the

first response in SARS COV-2 infection like in any other viral infection.

However a dysregulated and excessive adaptive immune response may be

disastrous. Various Cytokines have been found to be grossly raised in severe

COVID-19 patients getting admitted to the ICU as compared to the mild and

moderate illness. They have been found to have raised levels of granulocyte-

macrophage colony-stimulating factor (GM-CSF), interferon gamma-induced

protein 10 (IP10), monocyte chemoattractant protein-1 (MCP-1), macrophage

inflammatory protein 1 alpha (MIP1A), TNFα , IL-1βbeta, IL-8 and IL-6. The

pathway initiating cytokine storm and its effect is illustrated in (Fig3.3). High

levels of IL-6 is damaging on alveolar epithelium and the endothelium leading

to severe diffuse alveolar damage. There is multi-organ damage affecting the

primarily the lungs but also the GIT, brain, heart, liver and the eyes. IL-6 along

with other pleotropic cytokines raise the levels of other acute phase reactants

like C reactive protein (CRP), Ferritin, complement and pro-coagulant factors.

The prognostic markers used to monitor moderate and severe COVID infection

are IL-6 (normal level-5-15pg/ml), Ferritin (normal levels in males 24-336

micrograms per litre and for females 11-307 micrograms per litre), CRP

(normal levels < 3mg/L), D-dimer (normal levels < 250ng/ml), Procalcitonin

(normal levels 0.05ng/ml) and Troponin I ( Normal levels 0.04ng/ml).

Organ Involvement

The organs affected by the COVID-19 Virus.

(a) Lungs

Diffuse alveolar damage (DAD) is the most prominent pathology in the

lung. These results as a result of destruction of type 2 pneumocytes and the

endothelial cells by the virus. DAD leads to exudation of fibrin rich fluid into

the alveoli and formation of hyaline membrane. Evidence of microvasulature

thrombosis, fibrinous organizing pneumonia. Pulmonary embolism has also

been identified. Further details of findings in the lung have been brought out

under the autopsy section.

(b) Heart

Pre-existing coronary artery disease (CAD) may get complicated with

plaque rupture causing acute coronary even. However in absence of CAD an

event mimicking myocardial infarction can occur due to inadequate oxygen

supply, Cytokine storm may lead to myocarditis in absence of viral affection of

the myocytes. Intra-mycocardial thrombosis is another important finding. In

later stages of illness heart failure may produce raised levels of Troponin and

brain-type natriuretic factor (BNP)

The virus infects the glomerular tufts, podocytes and the tubular

epithelium due to presence of ACE2 receptors. Patients present with acute

kidney injury with microvasculature thrombosis and acute tubular necrosis.

Hypoperfusion of the kidney is also a part of cytokine storm.

(d) Brain

Brain stem and the cerebral cortex have ACE2 receptors. Some patients

may present with meningitis and encephalitis. Cytokine storm may also produce

inflammation in the brain. Arterial thrombosis though rare can produce a

ischemic stroke. Olfactory nerve involvement may produce anosmia.

(e) Gastrointestinal Tract

The lower GI tract is rich in ACE 2 receptors. Patients may produce GI

symptoms in the form of loss of appetite, vomiting, abdominal pain and

diarrhea.

(f) Skin

Patients having skin manifestation have an erythematous patch. They do

not correlate with severity of disease. Vesiculo-bullous eruptions like in cases of

chicken pox also have been seen. The manifestations are mainly immune

mediated and and due to microangiopathy.

(g) Eye

As the cornea, sclera and the epithelium of the eyelid have ACE2

receptors. Patients present with conjunctivitis.

Hypercoagulable State in COVID 19

The patients of COVID-19 virus infection have been seen to present with hypercoagulable state and thrombosis inspite of anticoagulant therapy. The whole phenomenon has been called COVID associated Coagulopathy. The pathogenesis is still being studied. It is explained by the activation of the Virchow’s Triad of thrombus formation in patients infected by the COVID-19 virus. First pillar of the triad is endothelial injury. Endothelial injury in SARS COV-2 infection is due to the direct invasion of the virus into these cells using the Angiotensin Converting Enzyme (ACE) 2 receptors found on the surface of these cells. Transmission Electron Microscopy (TEM) has detected the presence of these viruses inside the endothelium, (Fig 3.4). The COVID19 spike protein also induces complement mediated injury of the endothelium. Endothelial injury has also been postulated to be induced by IL 6 storm (as brought out above) and neutrophil extracellular traps from chromatin released from dying neutrophils. All this leads to endotheliitis and microvascular inflammation. The second pillar of the triad is stasis and contributed by the immobilization of a patient on oxygen therapy. The third pillar of the Virchow’s triad is the hypercoagulable state. The changes seen in COVID-19 infection are Elevated levels of factor VIII (FVIII), fibrinogen, von-Willebrand factor (vWF), circulating

prothrombotic microparticles, neutrophil extracellular traps (NETs) and hyperviscosity (likely due to raised polyclonal gamma globulin levels). D-dimer a product of cross linked fibrinogen degradation is also raised in severe illness.

Though in severe COVID-19 disease, DIC (disseminated intravascular coagulation) does develop as a complication but the hypercoagulable state in COVID appears different from DIC. The major effect in DIC is bleeding

whereas in COVID it is thrombosis. Though marked increase in D-dimer in severe COVID and mild thrombocytopenia are similar to DIC but increased levels of fibrinogen and factor VIII levels are in contrast to DIC. Hence COVID-19 does not appear to be a case of consumption coagulopathy. So the hypercoagulable state in COVID is more likely to be like compensated DIC and unlike decompensated DIC where the patient is bleeding. Also usually the prothrombin time (PT), Activated partial thromboplastin time (APTT) and platelets are usually within normal range unlike in a decompensated DIC.

Thus the hypercoagulable state results in widespread venous thrombosis,

venous thromboembolism (VTE) and deep vein thrombosis in COVID-19 infection. The risk of VTE is about 3% in COVID-19 infection.

Cases of arterial thrombosis resulting in ischaemic stroke, thrombotic microangiopathy in the lungs and myocardial infarction also has been reported but cases are far and few. Finally it appears that we are dealing with situation of inflammatory thrombosis. The inflammatory cytokines Interleukin 1, 6 and 8 are produced by the inflammatory reaction induced in the respiratory pathway. The direct infection of the endothelial cells as brought out above too activates the endothelial cells resulting in release of von Willebrand Factor and Factor VIII. Also it leads to activation of platelets. All this promotes fibrin clot formation and thrombosis. Thrombosis in return induces inflammation. Thrombosis activates the endothelium through the PAR (Protease activated receptors). The endothelium produces C5A that activates the monocytes. The cross talk between inflammation and thrombosis is postulated to be the mechanism as this is how the two events are usually related. Thus as we see the whole process is initiated in the respiratory passage by epithelial and endothelial damage both of which carry ACE2 receptors. A local thrombo-inflammatory event in COVID-19 induces a generalized hypercoagulable and prothrombotic state which produces thrombosis in the micro-vasculature of the brain, kidneys and venous plexus of the prostate.

Autopsy Studies

Limited knowledge exists as autopsies are restricted due to safety concerns. The lungs in all cases were heavy with evidence of consolidation and presence of intravascular thrombi on gross examination. On microscopy, exudative diffuse alveolar damage (DAD) and severe capillary congestion was

found in early disease. Tracheo-bronchtis and large vessel thrombi (Fig3.1b) and microthorombi in pulmonary vasculature was a common finding. Evidence of pulmonary embolism, alveolar haemorrhage and evidence of bronchopneumonia has also been documented in the lung. Beside this all cases showed evidence of Type II pneumocyte hyperplasia with atypia. The alveolar septae of the lung showed presence of thrombi with intra-alveolar extravasated RBCs and fibrin thrombi fibers. The bronchial lining too showed evidence of squamous metaplasia. The immunohistochemistry (IHC) revealed presence of viral spike proteins in the trachea-bronchial lining epithelium and the hyaline membrane in the alveoli. Electron Microscopy (EM) revealed presence of 67 nm electron dense particles in the cytoplasm of the pneumocytes. The understood mechanism of entry of the COVID-19 virus in the upper respiratory epithelium and the type 2 pneumocytes using the

Heart commonly showed presence of intra-myocardial thrombi on histology. No other specific

findings were detected like coronary thrombosis and myocarditis.

Among the other organs liver histology showed presence of peculiar basophilic structures in the sinusoids. Likely to nuclear remnants of degenerated cells. These could not be characterized even with IHC. Rest of the findings in the liver has been non-specific. Lymph nodes of most cases showed large transformed cells in the subcapsular and intra-parenchymal sinuses. These cells showed a vesicular nuclei, prominent nucleoli and moderate amphophilic cytoplasm and were positive for CD 20 a marker of B lymphocyte on IHC. Kidneys in most cases at autopsy showed and acute tubular necrosis and peri- tubular micro-angiopathy. Inspite of most patients who underwent autopsy were on anti-coagulant therapy still micro-thrombi were detected especially in the pulmonary vasculature. Gross and Histopathology Images of autopsy studies are not available in open access and are listed under further studies.

proteins of the virus as demonstrated by IHC in both these cells. Evidence of

ACE2 and transmembrane

protease serine 2 (TMPRSS2) is well confirmed by the presence of the spike

trachea-bronchitis and DAD is well explained by the preference of these cells

by the virus due to the expression of the ACE2 receptor.

Fig 3.1 Entry of SARS CoV2 and initial response

Fig 3.2 Cellular response to SARS CoV2

Fig 3.3 Pathway to cytokine storm

Fig 3.4 Pathogenesis of prothrombotic state in COVID 19

Acknowledgement

Fig3.1: COVID 19 Pathology Outlines (Open Access)

Fig 3.2 :Mortaz Esmaeil, Tabarsi Payam, Varahram Mohammad, Folkerts Gert, Adcock Ian M. The Immune Response and Immunopathology of COVID-19. Frontiers in Immunology. 2020;11:2037. (Open Access)

Fig3.3: Bhaskar Sonu, Sinha Akansha, Banach Maciej, Mittoo Shikha, Weissert Robert, Kass Joseph S., Rajagopal Santhosh, PaiAnupama R., Kutty Shelby. Cytokine Storm in COVID-19—Immunopathological Mechanisms, Clinical Considerations, and Therapeutic Approaches: The REPROGRAM Consortium Position Paper. Frontiers in Immunology. 2020;11:1648. (Open Access)

Fig 3.4:Luis Ortega‐Paz, DavideCapodanno, Gilles Montalescot, Dominick J. Angiolillo. Coronavirus Disease 2019–Associated Thrombosis and Coagulopathy: Review of the Pathophysiological Characteristics and Implications for Antithrombotic Management. J Am Heart Assoc. 2021;10:e019650. https://doi.org/10.1161/ JAHA. 120.019650 (Open Access)

Suggested Reading

2. 3.

The hypercoagulable state in COVID-19: Incidence, pathophysiology, and management.

115.

Uday Jain. Effect of COVID-19 on the Organs.Cureus. 2020: 12(8);e9540.

Alain C. Borczuk et al. COVID-19 pulmonary pathology: a multi- institutional autopsy cohort from Italy and New York City. Modern Pathology. 2020: 33; 2156-2168.

Menter Tet al. Postmortem examination of COVID-19 patients reveals diffuse alveolar damage with severe capillary congestion and variegated findings in lungs and other organs suggesting vascular dysfunction.Histopathology. 2020;77(2): 198.

Autopsy Findings in 32 Patients with COVID-19: A Single-Institution Experience. Sarah S. Elsoukkarya Maria Mostykaa Alicia Dillarda Diana R. Bermana Lucy X. Maa Amy Chadburnb Rhonda K. Yantissb Jose Jessurunb Surya V. Seshanb Alain C. Borczukb Steven P. Salvatore. Pathobiology. 2021;88 :56–68.

Mouhamed YazanAbou-Ismail, Akiva Diamond, Sargam

Kapoor, Yasmin Arafah and LalithaNayak. Thromb Res 2020; 194: 101–

4. Laboratory diagnosis of COVID 19

Lt Col S Karade, Air Cmde SP Singh Introduction

Laboratory plays a pivotal role in diagnosis, prognosis, and overall management of a patient of COVID-19. Several modalities of COVID-19 diagnosis are currently available, however reverse transcriptase based real-time polymerase chain reaction (RT-PCR) to detect viral genetic component in respiratory samples is considered gold standard for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. These tests need to be conducted in an ICMR approved molecular laboratory. A typical diagnostic cycle includes sample collection, nucleic acid extraction, PCR, interpretation, and generation of results. Commonly available ICMR or US-FDA approved COVID-19 diagnostic tests are summarized in Table 4.1

Table 4.1 Diagnostic tests for COVID-19

Name of the test

Sample requirement

Turn-around time and cost/test

Remarks

 Can handle 90-94 sample in one run, useful for medium and high-level BSL-2 laboratory

 Needs skilled manpower and sophisticated equipment

Generic real time RT-PCR test

OP/NP swab in VTM

5-8 hr 450 INR

 Useful in acute health care setting and triage situations

 User friendly

GeneXpert COVID Xpress

OP/NP swab in VTM

45 min 2000 INR

 Indigenous assay

 Useful for low and medium level

laboratories

 User friendly

Tru NAT (MolBio)

Respiratory sample in viral lysis buffer

< 1 hr 1000 INR

 Lateral flow assay-based detection of amplicons

 Does not require expensive Real-time PCR instrument

FELUDA (Tata group)

96% sensitivity and 98% specificity

45 min 450 INR

 Inexpensive test

 Positive test indicative of active infection

 Screening tool for high-risk groups

 Low sensitivity as compared to RT-PCR

Antigen detection

NP swab

20-30 min

test

250 INR

 Tool to assess the prevalence of the diseases in a specific area

 Not recommended for diagnosis by ICMR

IgG/IgM Antibody detection test

Serum

2-3 Hr 250 INR

Sample Collection

Preferred sample for molecular study of SARS-COV-2 infection includes throat swab or oropharyngeal swab (OP) and nasal swab or nasopharyngeal swab (NP). Both NP and OP swabs are collected using a nylon flocked swab in a viral transport medium (VTM) following appropriate biosafety precautions. Other effective but invasive samples include bronchoalveolar lavage and endo- tracheal aspirate collected in wide mouth sterile plastic containers. The sample should be collected ideally within 3 days of onset of symptom and no later than 7 days, preferably prior to initiation of antivirals. For children and uncooperative patient, saliva or buccal swab samples are useful, however the sensitivity is compromised. Good laboratory practices, abiding standard precautions and biosafety guidelines including PPE kit are essential while handling patient samples.

Respiratory specimen should be transported in triple layer packaging as early as possible from site of collection to diagnostic laboratory as per ICMR, New Delhi and WHO guidelines. If delay is, anticipated specimen can be stored at 4-8 degree Celsius for up to 5 days.

Reverse transcriptase based real-time PCR (RT-PCR)

PCR is a molecular tool for amplifying DNA/RNA targets in geometric progression. In SARS-CoV-2 real-time PCR test we amplify three or more genetic determinants of SARS-CoV-2 and detect these target amplicons using fluorescent probes in real-time. The assay also includes a human gene component as internal control. The typical process involves viral RNA extraction from respiratory specimen, master-mix preparation, addition of template RNA, amplification of target genes followed by detection of signal and analysis of the assay. The entire process is carried out in a biosafety level two molecular laboratory and takes 5-6 hr for a batch of 90-94 samples. The viral genetic targets commonly used includes combination of envelope gene (E) with RNA-dependent RNA polymerase (RDRP) or HKU open reading frame (HKU Orf 1b), or nucleoprotein (N) or S gene target. As SARS-CoV-2 undergoes mutations, newer variants are produced in infected individuals across the world. Use of multiple target genes improves sensitivity and help to detect such variants. The advantage of commercially available assay is low cost/test (approx. INR 450/test) and ability to handle bulk samples. However, time

constraint, requirement of trained staff, biosafety cabinets and sophisticated equipment limits its use in emergency setting.

Interpretation of RT-PCR results

A positive SARS-CoV-2 RT-PCR indicates qualitative detection of 2 or

more virus specific sequences in the given sample. Since prolonged viral

shedding upto 4-8 weeks can occur in a confirmed case of COVID 19, the test

does not aid in prognostication. A false negative test can occur due to poor

specimen collection, unsatisfactory specimen transport, specimen collection too

early or too late after onset of symptoms (> 1 week) and due to technical errors

(3). RT-PCR test if negative needs to be repeated if clinical suspicion is strong.

Cartridge based Nucleic acid amplification-based tests (NAAT)

Currently, Xpert Xpress SARS-CoV-2 (Cepheid) and TrueNatTM (Molbio) are the two Indian Council of Medical Research (ICMR) recommended cartridge-based nucleic acid amplification tests (CBNAAT) for qualitative detection of SARS-CoV-2 in respiratory samples. These are closed system and require minimum handling of specimen and poses minimum biothreat to laboratory personnel. The turn-around time is 45 min making its use suitable for emergency setting. However, a single CBNAAT system can handle only 4 specimens in one go.

Isothermal amplification Assay

ID NOW COVID-19 assay (Abbott) utilizes an isothermal nucleic acid

amplification technology for qualitative detection of genetic targets from the

SARS-CoV-2 viral RNA in direct nasal, naso-pharyngeal or oro-pharyngeal

samples. This platform is US FDA approved and commercially available as

point of care test as the turn-around time is just 5 min to 20 min depending upon

number of copies of target RNA present in the sample.

CRISPR/Cas9 based assay (FELUDA)

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) based rapid test was recently approved by ICMR and Director General

Drug controller of India. This test is indigenously developed for COVID-19 diagnosis by Institute of Genomics and Integrative Biology (CSIR-IGIB), New Delhi and marketed by TATA group. The test is named FELUDA, an acronym for ‘FNCas9 Editor Limited Uniform Detection Assay’, based on fictious detective character portrayed by renowned film director Satyajit Ray. The assay does not require costly Real-time PCR instrument and the SARS-CoV-2 genomic sequence can be detected by paper strip-based test. This assay takes 45 min and claimed to have limit of detection of as low as 10 copies of purified viral sequence.

Rapid antigen detection test (RAT)

These group of rapid diagnostic test (RDT) detects the presence of viral proteins (antigens) expressed by the SARS-CoV-2 in a sample from the respiratory tract of a person. If the target antigen is present in sufficient concentrations in the sample, it will bind to specific antibodies fixed to a paper strip enclosed in a plastic casing and generate a visually detectable signal in form of a band within 20-30 minutes. The antigen(s) detected are expressed only when the virus is actively replicating; therefore, such tests are best used to identify acute or early infection.

Antibody based tests

Detection of antibodies against various SARS-CoV-2 antigen in the serum sample of an individual helps us to determine evidence of past infection. Qualitative or quantitative estimation of antibodies against nucleoprotein or surface (S1 /S2) antigen can be done, based on Enzyme linked immune-sorbent assay (ELISA) or chemiluminescence assay (CLIA). These tests are useful in determining the seroprevalence of COVID-19 in given population. A positive antibody test does not necessarily mean immunity against SARS-CoV-2 infection as these may be non-neutralizing antibodies. Presently, quantitative antibody estimation test following vaccination is not recommended to determine level of protection from COVID-19.

Ancillary tests

These sets of haematological and biochemical tests are essential for clinical monitoring and prognostication of COVID-19 cases. These tests also

help in assessing the severity of disease. The detail of these tests is shown in Table 4.2.

Table 4.2 Ancillary test for management of case of COVID-19

Sample and type of vacutainer

Parameter measured

Remarks

EDTA blood, purple top vacutainer

Total blood counts

Essential for monitoring of neutrophilia, Lymphopenia, N:L ratio and thrombocytopenia:

Citrated, light-blue top vacutainer

D Dimer

Indicator of activation of coagulation system/DIC

EDTA blood, purple top vacutainer

Prothrombin time

Indicator of activation of coagulation system