COVER LETTER

To, Date:-15th Sep 2021

The Chairman,
Institutional Ethics Committee,

ILBS, New Delhi

Subject: Proposal for ethical clearance

Respected Sir/Madam,

I, Dr.Sumit Garg, here by submitting my research protocol titled ‘Role of bone marrow examination in
Hemophagocytic lymphohistiocytosis diagnosis in liver disease patients’ for approval by institute ethics
committee(IEC).

Since this is retrospective study, so I would request you to consider waiving off the patient information sheet
and the consent form.

All the required enclosures are attached along with.

Thanking you

Yours sincerely,

Dr Sumit Garg
Senior Resident (PDCC)
Dept of Hematology
Institute of Liver and Biliary Sciences,
New Delhi
 Curriculum Vitae (CV)

Dr Sumit Garg
M.B.B.S., M.D. Pathology (PGIMER), PDCC Neuropathology (ICP, PGIMER)
Contact: 9675011227, dr.sumitgrg@gmail.com
Senior Resident- PDCC, Department of Hematology, Institute of Liver & Biliary Sciences, Vasant Kunj,
New Delhi, Pincode -110070.

Educational Qualifications:

Degree (Period) Institution (University/Board)

PDCC Indian College of Pathologist (ICP)
Neuropathology Postgraduate Institute of Medical Education and Research,
(2017-2018) Chandigarh
MD Pathology Postgraduate Institute of Medical Education and Research,
(2015- 2017) Chandigarh
M.B.B.S Lala Lajpat Rai Medical College, Meerut
(2006-2012) (C.C.S. University, Meerut, U.P.)

Work Experience (starting from Post-graduation)

Post held Total Period Institution

From To
Junior resident (Pathology) 1st Jan 2015 31th Dec 2017 PGIMER, Chandigarh
Senior resident 1st Jan 2018 31st Dec 2018 PGIMER, Chandigarh
(Histopathology along with
Neuropathology PDCC)
Senior resident 1st Jan 2019 31st Dec 2019 PGIMER, Chandigarh
(Cytopathology)
Senior resident 1st Jan 2020 30th Jun 2020 PGIMER, Chandigarh
(Immunopathology)
Senior Resident (Hematopath) 1st July 2020 15th Jan 2021 PGIMER, Chandigarh

Achievements and Awards:

● Participated as speaker at 49th Annual conference of Indian Academy of Cytologists (CYTOCON

2019), PGIMER Chandigarh.
● Participated as faculty and conducted muscle biopsy processing workshop at 4th annual conference
of Neuropathology society of india (NPSICON-2019), PGIMER Chandigarh.
● Got first prize in oral presentation at 4th annual conference of Neuropathology society of india
(NPSICON-2019), PGIMER Chandigarh.
● Got first prize in poster presentation at 2nd annual conference of Neuropathology society of india
(NPSICON-2017), NIMHANS, Bangalore.

Additional qualifications such as membership of Scientific Societies:

● Life Member of Neuropathology society of India (NPSI)

 ● Life Member of Indian Association of Pathologists and Microbiologists (IAPM, membership id:
OL4545)

Research experience and works under publication

1. Completed thesis during Post Graduation under the guidance of Prof. Rakesh K. Vasishta, Head of
the Department of Histopathology titled “Spectrum of Adult Inflammatory Myopathies in a tertiary
health care center from North India”.
2. Primary Dural Acute Lymphoblastic Leukemia/ Lymphoma Masquerading Meningioma: Report of a
Case and Review of Literature
3. Histopathological spectrum of spinal lesion at a tertiary health care centre from North India

Publications
1. Garg S, Rohilla M, Srinivasan R, Bal A, Das A, Dey P, et al. Fine-Needle aspiration diagnosis of
lymphoma based on cytomorphology alone: How accurate is it? - A cyto-histopathology correlative
study. J Cytol 2021;38:164-70.
2. Digital Intraosseous Epidermoid Inclusion Cyst of the Distal Phalanx. Saikia A, Garg S, Dey P
DiagnCytopathol. 2016 Dec;44(12):1130-31
3. Karthigeyan M, Malik P, Garg S, Radotra BD, Salunke P, Sunil N, Kumar N. Intracranial Low-
Grade Fibromyxoid Sarcoma with Atypical Radiology. World Neurosurg. 2019 Feb;122:500-504.
 Research Project by the Principal Investigator (PI) to
Institutional Ethics Committee (IEC)

Proposal Title:“Role of bone marrow examination in Hemophagocytic lymphohistiocytosis

diagnosis in liver disease patients”

Name, Designation Address, Telephone, Fax Signature

& Qualifications and Email Id

Principal Dr.Sumit Garg ILBS, Vasant Kunj, New Delhi

Investigator SR PDCC Hepato Hematology, 9675011227
Department of Hematology
MBBS MD dr.sumitgrg@gmail.com
Co-Principal Dr Aniruddha P. Dayama ILBS, Vasant Kunj, New Delhi
Investigator Assistant Professor 9650373043
Department of Hematology dayama@gmail.com
MBBS MD, DM
Co-Principal Dr. Chhagan Bihari ILBS, Vasant Kunj, New Delhi
Investigator Additional Professor, Department 9990526503
of Pathology drcbsharma@gmail.com
MBBS MD
Co-Principal
Investigator

Sponsor Information:

1. Indian a) Government Central State Institutional

b) Private

2. International Government Private Others

3. Industry National Multinational

Contact Address of Sponsor:

Total Budget: Nil (Retrospective observational study)

Section1: Project title: “Role of bone marrow examination in Hemophagocytic

lymphohistiocytosis diagnosis in liver disease patients”

Section 2:Broad Subject: Clinical Hematology

Section 3: Duration: 4 months

Section 4: Total cost & FE Component: Nil (Retrospective study)

Section 5: Funding agency to which applying: NA

Section 6: Project Category: Cross Sectional Study

Section 7A: Collaborating Investigators/Institutions (Indian/International): None

7B: If any collaborators mention details:

Section 8:Project summary (in 250 words maximum)

Hemophagocytic lymphohistiocytosis (HLH) is a severehyperinflammatory syndrome induced by

aberrantly activatedmacrophages and cytotoxic T cells. [1]HLH has been traditionally divided into
a primary (genetic) form,which typically manifests in children with documented
geneticabnormalities of the cytotoxic function of NK cells and T cells,and secondary (acquired)
form that tends to occur at older ages in thesetting of an associated condition, such as infection
and malignancy,without an identifiable genetic abnormality. [2,3]

The diagnosis of HLH in adults is based onthe HLH-2004 diagnostic criteria in conjunction with
clinicaljudgment and the patient’s history. According to revised HLH-2004 criteria, the diagnosis
of HLH can be established if Criterion 1 or2 is fulfilled. [4-6]
1. A molecular diagnosis consistent with HLH
2. Diagnostic criteria for HLH fulfilled (5 of the 8 criteria below)
a. Fever
b. Splenomegaly
c. Cytopenias (affecting ≥2 of 3 lineages in the peripheral blood)
i. Hemoglobin <90 g/L (hemoglobin <100 g/L in infants ,4 wks.),
ii. Platelets <100X109/L
iii. Neutrophils <1.0 X 109/L
d. Hypertriglyceridemia and/or hypofibrinogenemia
i. Fasting triglycerides ≥3.0 mmol/L (i.e., ≥265 mg/dL)
ii. Fibrinogen ≤1.5 g/L
e. Hemophagocytosis in bone marrow or spleen or lymph nodes. Noevidence of
malignancy.
f. Low or no NK cell activity (according to local laboratory reference)
g. Ferritin ≥500 mg/L
h. sCD25 (i.e., soluble IL-2 receptor) ≥2400 U/mL

HLH in underlying liver disease setting is very difficult to diagnose as both have similar finding in
respect of fever, organomegaly, cytopenias, hyperferritinemia and elevated enzymes level.
[7]
Early recognition and prompt treatment of HLH is associated with improved clinical outcome.
E. Gras et al. proposed the significance of granulocytic phagocytosis and nucleated erythroid
cell phagocytosis over the RBCs and platelet phagocytosis in bone marrow for diagnosis of
HLH.[8]

So, here we proposed a retrospective study to determine, the diagnostic accuracy of bone
marrow examination in a suspected case of HLH in a known case of primary liver disease
patient by correlating the hemophagocytosis of different cell type in bone marrow. In addition, to
find out the relevance of other parameters of HLH-2004 diagnostic criteria in liver disease
patients.
 Section 9: Subject Keywords: Hemophagocytic lymphohistiocytosis (HLH), Hepatic disease,
Bone marrow, Hemophagocytosis

Section 10: State of knowledge (National and International)

- International Status:

International literature over HLH in Liver disease patients and its diagnosis by bone marrow
evaluation is very limited.

Study by Dong et al describes the clinical and laboratory characteristics of hemophagocytic

lymphohistiocytosis after definite diagnosis of liver failure and to provide clues forearly diagnosis
and treatment of HLH in patients with liver failure. [9] Pancytopenia, persistent high-grade fever and
splenomegaly was common finding in all the patient at the time of diagnosis of liver failure and
suspected HLH. Bone marrow biopsy revealed the evidence of hemophagocytosis (histiocytosis,
100%). Further examination for HLH revealed the most frequent laboratory abnormalities are
increased ferritin levels (>500 ug/L, 90.91%), increased hypertriglyceridemia (>265mg/dL,
45.45%), and decreased hypofbrinogenemia (<1.5g/L, 81.82%). Soluble IL-2 receptor (CD25)
levels were increased in 88.89% patients, and NK-cell activity was absent or reduced in 33.33%.

- National status:

National literature is also limited to describe the HLH in liver disease patients. A few studies are
described below.

Jagtap et al includes six patients,which were diagnosed as secondary HLH. All patients were
presented with history of deep jaundice and high-gradefever with pancytopenia and splenomegaly.
Underlying diagnosis was viral infectionin 4 and probable viral infection in remaining two. Bone
marrow hemophagocytosiswas present in 3 cases. Median duration to reach diagnosis of HLH
afterhospitalization was 14 (range 2 to 30) days. They concluded that HLH is one of the rare
causes of prolonged fever, deep jaundice with pancytopenia and splenomegaly, it is essential to
consider HLH as differential diagnosis of ALF. [7]

Chandra et al study the hemophagocytosis on bone marrow aspirates, its causes, clinical
correlation and associated features on bone marrow examination and to observe if there is any
difference on bone marrow examination between HLH and non HLH cases showing
hemophagocytosis.They concluded that higher grade of hemophagocytosis (2+ to 3+) in cases
with HLH may help in establishing an early differentiation of HLH from non HLH cases. [10]

References

1. Rosado FG, Kim AS. Hemophagocytic lymphohistiocytosis: an update on diagnosis and

pathogenesis. Am J Clin Pathol. 2013;139(6):713-27.
2. George MR. Hemophagocytic lymphohistiocytosis: review of etiologies and management. J
Blood Med. 2014;5:69-86.
3. Al-Samkari H, Berliner N. Hemophagocytic Lymphohistiocytosis. Annu Rev Pathol.
 2018;13:27-49.
4. Henter JI, Horne A, Aricó M, et al. HLH-2004: Diagnostic and therapeutic guidelines for
hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer. 2007;48(2):124-31.
5. Lehmberg K, Ehl S. Diagnostic evaluation of patients with suspected haemophagocytic
lymphohistiocytosis. Br J Haematol. 2013;160(3):275-87.
6. La Rosée P, Horne A, Hines M, et al. Recommendations for the management of
hemophagocytic lymphohistiocytosis in adults. Blood. 2019 Jun 6;133(23):2465-2477.
7. Jagtap NS, MithunG, RajeshPN, et al. Hemophagocyticlymphohistiocytosis masquerading
as acute liver failure: A single center experience(2017),
http://dx.doi.org/10.1016/j.jceh.2017.01.119.
8. Gars E, Purington N, Scott G, et al. Bone marrow histomorphological criteria can accurately
diagnose hemophagocytic lymphohistiocytosis. Haematologica. 2018;103(10):1635-1641.
9. Dong J, Xie F, Jia L et al. Clinical characteristics of liver failure with hemophagocytic
lymphohistiocytosis. Scientific Reports. 9.https://doi.org/10.1038/s41598-019-43909-w.
10. Chandra H, Chandra S, Kaushik R, Bhat N, Shrivastava V. Hemophagocytosis on bone
marrow aspirate cytology: single center experience in north himalayan region of India. Ann
Med Health Sci Res. 2014;4(5):692-696.

Section 11: Importance of the proposed project/ Justification for subject area:

Hemophagocytic lymphohistiocytosis (HLH) is a close differential diagnosis to acute liver

presentation. It is always difficult to make a confirm diagnosis of secondary HLH solely on the
basis of clinical and laboratory parameters. Both results into elevated liver enzymes, similar
presenting features (Fever, splenomegaly, cytopenias). HLH is a life-threateninghyperinflammatory
syndrome and early prompt treatment could be lifesaving. By evaluating our data and bone
marrow in suspected HLH cases with underlying liver disease, we can establish significance bone
marrow criteria for prompt diagnosis. In addition, we can establish cut off values for various
parameters in our set-up for making a diagnosis of secondary HLH in liver disease patients.

Section 12: Review of facilities and expertise available in institution

- Facilities:
1. Hematological parameters (Hb, TLC, Platelet count)
2. Biochemical parameter (S. Feritin, S. Triglycerides, S. Fibrinogen)
3. Radiology (For organomegaly)
4. Bone marrow examination
- Expertise:
1. This is a retrospective study. All data will be collected from institutional
database.
2. Bone marrow slides will be reviewed by hematopathologist.

Section 13: Study Design

(a) Aim and Objective –

- Primary objective:To analyze the applicability of HLH Criteria to such patients suspected to
have HLH in Liver disease
- Secondary objectives:
1. To analyze the significance of bone marrow hemophagocytosis in respect of
cell type and number of cellsphagocytized.
2. To determine significant cutoff values of other laboratory parameters for
making a diagnosis of HLH in Liver disease patients
 (b) Methodology:
- Study population – All clinically suspected cases of HLH who underwent bone marrow
examination.
- Study design – Cross sectional study
- Study period – January2010 to August 2021
- Sample size – All clinically suspected of HLH
- Intervention – It is a retrospective study no intervention is required
- Monitoring and assessment
- Adverse effects – NA (Retrospective study)
- Stopping rule - NA (Retrospective study)

(c) Expected outcome of the project:

1. Cases with final diagnosis of HLH show significantly more number of hemophagocytosis as
compared to cases with not meeting with final diagnosis of HLH.

2. Granulocytic phagocytosis and nucleated erythroid precursor phagocytosis is more significant

for making a diagnosis of HLH

Section 14: Inclusion and exclusion criteria for admission of patients in the study
- Inclusion criteria – Jan 2010 to August 2021, all cases with clinical suspicion of HLH and
underlying liver disease will be included in study population
- Exclusion criteria – Cases with incomplete data will be excluded

- STATISTICAL ANALYSIS:Statistical analysis will be done by using Statistical Package for

Social Sciences (SPSS), version 22.0. The categorical data analyzed by chi-square test and the
quantitative data by t-test/ Mann-Whitney or one-way ANOVA or Kruskal-Wallis test. A p-value of
< 0.05 will be considered significant.

Section 15: Work Plan with Time schedule of activities giving milestones
- Flowchart of work

Selection of cases from bone marrow database from Department of Hematology

Retrieval and evaluation of hemophagocytosis bone marrow of selected

Collection of other laboratory and radiological parameters from hospital data base

Application of Appropriate statistical test on collected data

Section 16: Permission from Drug Controller of India for use of drug / vaccine / device in
the study, wherever applicable. NA

Section 17: Safety of the proposed interventions, drugs, or vaccine to be tested including
results of relevant laboratory and animal toxicity and safety trials, and results of studies
carried out in humans.NA

Section 18: Description of plans to withdraw or withhold standard therapy in the course
of research. NA

Section 19: For research carrying more than minimal risk, an account of plans to provide
medical therapy for such risk or injury. NA

Section 20: Budget Estimates. NIL

Section 21: Other research projects with investigators:

If Yes
S. No. Title of Project Status (Recruiting/Not Yet Started/Terminated/no Funds)
1. Assessment of plasma cells ageing in acute on - Ongoing
chronic liver failure patients and its impact on myelopoiesis

Place: Signature & Designation of SI/Faculty Supervisor

Date:
 Data Collection Performa

Parameters to be collected
1. UHID
2. Date of bone marrow examination
3. Bone marrow number
4. Age
5. Sex

6. Clinical history
7. CBC parameters (Hb, TLC, Plt
count)
8. Biochemical parameter (S. Feritin, S.
Triglycerides, S. Fibrinogen)
9. Radiological findings for
organomegaly
10.Bone marrow examination for
hemophagocytosis
11. Treatment history

The above data will be collected by Dr Sumit Garg for his thesis entitled: ‘ Role of bone
marrow examination in Hemophagocytic lymphohistiocytosis diagnosis in liver
disease patients’

Dr Sumit Garg
PDCC-SR - Hematology