Determination of the Age of

Bloodstains
Presenters:

Emerald Blaides
Alana Ingram
Daniel Khouri
Sherray Parkinson
Nikitia Whyte
Affiliation: University of the West Indies
 PROJECT STATEMENT
To be able to determine the timeline of events
at a crime scene by utilizing the change in
miRNA levels in bloodstains as they age. This
project should be completed by the year 2020.

Determination of the Age of Bloodstains

Affiliation: University of the West Indies
 PROJECT GOAL
To determine the levels of miRNA present in the
bloodstains at varying times in an effort to
establish a database for determining the age of
bloodstains for the Forensic Science
Community.
Determination of the Age of Bloodstains
Affiliation: University of the West Indies
 PROJECT OBJECTIVES
By July 2020:

 To determine the levels of miRNA-16 and miRNA-

451 in the blood at varying times within 28 days.

 To determine the effects of temperature on the

analysis of miRNA-16 and miRNA-451.

Determination of the Age of Bloodstains

Affiliation: University of the West Indies
 INDICATORS FOR EACH
OBJECTIVE
By July 2020

Objective 1:

 miRNA-16 and miRNA-451 levels assessed and compared.

Objective 2:

 Effect of the change in temperature on miRNA analyzed.

Determination of the Age of Bloodstains

Affiliation: University of the West Indies
 MAIN STEPS
 1 Seeking Approval

 2 Laboratory Preparations

 3 Laboratory Analysis

 4 Data Management

 5 Reporting
Determination of the Age of Bloodstains
Affiliation: University of the West Indies
WORK BREAKDOWN STRUCTURE
 1. 1 SEEKING APPROVAL

1.1 Obtain ethical approval by research ethics committee………start as soon as possible

1.1.1 University of the West Indies, Mona and Ministry of Health….starts with 1.1

1.2 Awarding of Funding…………………………………………………starts after 1.1.1 finished.

1.3 Transfer of Funds……………………………………………………….starts after 1.2 is finished.

Determination of the Age of Bloodstains

Affiliation: University of the West Indies
 2 LABORATORY PREPARATION
 2.1 Purchase human whole blood from National Blood Bank……..starts after 1.2 is finished.

 2.2 Purchase reagents (e.g. QIAzol Lysis Reagent) and equipment…..starts with 2.1

 2.3 Calibrate all instruments that are to be used ( e.g. Mx3000P PCR System)..…..starts after
2.2 is finished

 2.4 Recruit and train staff to ensure competency…………….......................................starts

after 2.3 is finished.
 3 LABORATORY ANALYSIS
 3.1 Age bloodstains for miRNA analysis at 25 °C in 50% humidity…………starts after 2
is finished.
 3.2 Isolate leukocytes……………………………………………………………….starts after 3.1 is
finished.
 3.3 Extract and Purify RNA………………………………………………………....starts after 3.2 is
finished.
 3.4 Create cDNA for the PCR……………………………………………………starts after 3.3 is
finished.
 3.5 A serial of six bloodstains aged…………………………………………..starts after 3.4 is
finished
 4 DATA MANAGEMENT
 4.1 Record Data obtained from PCR Analysis……………………………………………...starts with 3.4.

 4.2 Enter data into excel……………………………………………………..…starts after 4.1 is finished.

 4.3. Perform data cleaning to ensure that there are no data entry errors……….starts with a two week delay

after 4.2

 4.4 Establish data access controls…………………………………………………….…….starts with 4.2

Determination of the Age of Bloodstains

Affiliation: University of the West Indies
 5 REPORTING
 5.1 Submit progress report to the funders on a trimonthly basis
…………………………starts after 4.2 with a delay of 3 months.

 5.2 Submit progress reports to UWI , Mona and The Ministry of

Health………...starts with 5.1

Determination of the Age of Bloodstains

Affiliation: University of the West Indies
 MILESTONES
Seeking Approval
 Submission of proposal to ethics committee by March 20,
2019

 Ethical Approval by June 03, 2019

 Awarding of Funding and Transfer of Funding by June 04,

2019
(2 week delay for transfer of funds)
 MILESTONES
Laboratory Analysis
 Begin aging first batch of bloodstains (30 samples at
250C- starts immediately (July 17, 2019)
 Isolation of leukocytes from dried bloodstains- start
by July 17, 2019
 Extract and purify RNA – start by July 17, 2019
 cDNA Synthesis and PCR – start by July 17, 2019

Determination of the Age of Bloodstains

Affiliation: University of the West Indies
 MILESTONES

Laboratory Analysis Cont’d

 PCR Analysis starts by July 18, 2019 (Delay of 28 days for completion

of each bloodstain)

 Repeat of process with second batch of bloodstains (30 samples) at

250C starts by August 14, 2019

Determination of the Age of Bloodstains

Affiliation: University of the West Indies
 MILESTONES

Data Management

 Record data obtained from PCR Analysis starts by July 18,

2019

 Enter data into excel- starts by July 19, 2019

 Proof data – starts July 20, 2019

Determination of the Age of Bloodstains
Affiliation: University of the West Indies
 MILESTONES

Reporting

 Progress Reports – starts by October 2019

(3 month intervals)

Determination of the Age of Bloodstains

Affiliation: University of the West Indies
PERT
CHART
GANTT CHART

GANTT CHART
 TEAM MEMBERS
Name Expertise Roles Responsibility

Emerald Blaides ● Serologist Project Director ● Plan, direct and coordinates

● Previous publications the overall project
on miRNA biogenesis ● Problem Solution
(miRNA16 and ● Establish work plan
miRNA451) ● Promotion and facilitation of
team effort to achieve
objectives

Alana Ingram ● Molecular Biologist Project Manager ● Activity and resource

● Specializes in planning
molecular ● Organizing and motivating
techniques with a project team
expertise in research ● Controlling time
design and project management
manager ● Cost estimating and
developing the budget
● Analyzing and management
project risk
● Managing reports and
necessary documentation
 TEAM MEMBERS
Name Expertise Roles Responsibilities
Nikitia Whyte ● Immunologist Laboratory Manager ● Establishing preparation
● certification in procedures
medical ● Developing and implementing
technology and analytical procedures
laboratory ● Train laboratory personnel
analysis ● Evaluating laboratory
information
● Maintain Laboratory equipment

Daniel Khouri ● Statistician Statistics and Data Manager ● Assist with data entry, as
● CPFA needed
certification ● Identify problems with data
● Experience in quality and communicate
Research Data issues with program staff and
analysis supervisor
● Assist with regular data review
and cleanup to maintain overall
data integrity

Sherray Parkinson ● Administration Recruitment Manager • Recruit laboratory personnel

Project ● Contribute to development of
Management policies and procedures
● expertise in ● Participate in the revision of the
Human relations budget
 TEAM MEMBERS
Name Expertise Roles Responsibilities
TBD* Data Assistants Data Entry Clerk ● Prepares source data for computer
entry by compiling and sorting
information; establishing entry
priorities.
● Maintains data entry requirements
by following data program
techniques and procedures
● Secures information by completing
data base backups.

Determination of the Age of Bloodstains

Affiliation: University of the West Indies
 COMMUNICATIONS
Type Means Frequency Topics Responsibilities/Co
ntact Manager
Project Team Face-to-Face Fortnightly ● Project Project Manager
Meetings Dialogue update
● Monitoring
process
• Problem
resolution

Network and Face-to-Face Bimonthly (Every ● Project Project Director and

Contact Meetings Dialogue 2 months) update and Project Manager
(Stakeholders) Monitoring
process

Funding Meetings Video Conference Quarterly ● Project update Project Directors and
● Budget Project Manager
Evaluation

Determination of the Age of Bloodstains

Affiliation: University of the West Indies
 REPORTING
Type Frequency Topics Recipients Format

Progress report Quarterly Updates on Network Internal and

implementation institutions and funder format
progress funder

Scientific report Mid-way project Update on Network Funder format

scientific results institutions and
funder

Dissemination End of Project Recommendatio Academic Internal Format

report ns and future institutions,
initiatives funders, network
institutions

Determination of the Age of Bloodstains

Affiliation: University of the West Indies
BUDGET USD
BUDGET USDCONT’D
BUDGET USD CONT’D

Determination of the Age of Bloodstains Affiliation: University of the West Indies

 BUDGET JUSTIFICATION
Project/Research Personnel
Project Director, MSc, PhD
 Dr. Blaides will serve as a principal Research, she will commit 3.6 person months (40%
effort). She is an expertise in miRNA Biogenesis. She will plan and organize this project,
and performed scientific examinations of the project results. Total salary requested for
the Project Director is $53,923 USD which is calculated from a base salary of $13480.75
USD.
Project Manager, PhD
 Dr. Ingram will contribute 6 person months (70% effort). Dr. Ingram specializes in
Molecular Genetic Research techniques, with methodological expertise in qualitative
research design, conduct, and textual data analysis. Dr. Ingram will oversee the
qualitative research component of this project. Total salary requested for the Project
Manager is $49 000 which is calculated from a base salary of $12, 250 USD.

Determination of the Age of Bloodstains

Affiliation: University of the West Indies
 BUDGET JUSTIFICATION
Laboratory Manager BSc, MSc.
 Ms. Whyte will commit 9.6 person months (80% effort). a certified
American Medical Technologists (AMT). Ms.Whyte will oversees
laboratory safety and ensures that the laboratory remains in
compliance with all local, state and federal regulations. Total
salary requested for the Laboratory Manager is $37,828. 40 USD
which is calculated from a base salary of $9,457.10 USD.

Statistics and Data Manager, CPA, MA

 Mr. Khouri will contribute12 person months (100% effort) , CPFA
certification. He will oversee all the data entry and data analysis of
the project. Total salary requested for the Statistics and Data
Manager is $28, 219.96 USD which is calculated from a base salary
of $7054.99 USD.
Determination of the Age of Bloodstains
Affiliation: University of the West Indies
 BUDGET JUSTIFICATION
Recruitment Manager MSc.
 Ms. Parkinson will contribute 9 person months (75% effort). Masters in
Human Resource Management and public relations. She will train and
employ personnel to assist with the project. Total salary requested for the
Recruitment Manager is $23,652 which is calculated from a base salary
of $5913 USD.

Data Entry Clerk

 This individual with will contribute 100% of their time to the project. He/She
will enter the enter and clean data. Total salary requested Research is
$17,670 which is calculated from a base salary of $4417.66 USD.

Determination of the Age of Bloodstains

Affiliation: University of the West Indies
 BUDGET JUSTIFICATION
A. Key Personnel
 To be selected. The Senior Research Analyst will commit 13 person months (100 % effort) to this
project and will primarily be responsible for the laboratory analysis of the bloodstains,
interpretation of the resultant data and coordination of all aspects within the lab. Total salary
requested for the Senior Research Analyst I is $39,000 which is calculated from a base salary of
$3000 USD.

 To be selected. The Senior Lab Technician will commit 13 person months (100% effort) to this
project and will primarily be responsible for operating and maintaining the laboratory equipment,
supervising the assistant lab technicians and assisting the Senior Research Analyst. The total salary
requested for the Senior Lab Technician is $32,500 USD which is calculated from a base salary of
$2500 USD.
 BUDGET JUSTIFICATION
B. Assistant Personnel
 To be selected. The Assistant Research Analyst will commit 13 person months (100 % effort)
to this project and will primarily be responsible for the analysis of the results, maintaining
the workspace and preparing for the experiments. Total salary requested for the Assistant
Research Analyst I is $26,000 which is calculated from a base salary of $ 2000 USD.

 To be selected. The Assistant Lab Technician will commit 13 person months (100 % effort) to
this project and will be primarily responsible for the maintaining the workspace and
preparing for the experiments. The total salary requested for the Assistant Lab Technician is
$24,000 USD which is calculated from a base salary of $1900 USD.


Determination of the Age of Bloodstains

Affiliation: University of the West Indies
 BUDGET JUSTIFICATION
C. Other Personnel
 To be selected. The Quality Control Analyst will commit 13 person months (100 % effort)
to this project and will be responsible for ensuring the quality of the raw material,
ensuring that lab cleanliness is maintained and that safety standards are upheld. The
total salary requested for the Quality Control Analyst is $6,500 which is calculated from a
base salary of $1000 USD.

 To be selected. The Data Entry Operator will commit 13 person months (100% effort) to
this project and will be responsible for the collection and entering of data in the
database as well as establishing data access controls. The total salary requested for the
Data Entry Operator is $ 23, 350 which is calculated from a base salary of $1950 USD.
 BUDGET JUSTIFICATION
C. Other Personnel Cont’d

 To be selected. The Calibration Technician will commit 6.5 person months (50%
effort) to this project and will be responsible for the maintenance, testing and
repair of all lab equipment. The total salary requested for the Calibration
Technician is $ 8 887.71USD which is calculated from a base salary of $ 1 367.34.
USD.

Determination of the Age of Bloodstains

Affiliation: University of the West Indies
 BUDGET JUSTIFICATION
D. Reagents

 Centrifuge tubes ($948.72 USD) will be used to centrifuge samples in order to separate
solids out of liquids.
 Human whole blood ($) will be used to analyze miRNA.

 White cotton balls ($ 91.08 USD) will be used as a medium to absorb and retain the
whole blood and other chemical reagents to be analyzed.

 Micro Pipette ($990 USD) this will be used to measure accurate volume of the liquid
after centrifugation.

 Micro pipette tips ($32.46 USD) this will be used to take up and dispense liquid solution
when using the micropipette.
 BUDGET JUSTIFICATION
D. Reagents Cont’d
 Laptops- Used laptops are crucial for the research as it allows for the digital
recording of our data using our internally generated data management systems.

 ABI 7500 Real-Time PCR supplied by a reputable brand such as Applied

Biosystems is another crucial equipment as it will be used to determine the levels
of miRNA present in the blood at varying times and conditions.

 The Midi 40 Compact Incubator is needed to age bloodstains in a control

medium at varying temperatures. Thermo Scientific is a reputable company
supplying thousands of laboratories worldwide.

Determination of the Age of Bloodstains Affiliation: University of the West Indies

 BUDGET JUSTIFICATION
D. Reagents Cont’d
 Plastic Petri dish ( $ 103.60) this will be used to store the blood soaked cotton that
will be used for analysis.

 Plastic Pipettes ($52.48 in total) this will be used to mix the QIAzol lysis reagent
when added to the leukocyte pellet.

 QIAzol lysis reagent (327 in total) will be added to the leukocyte pellet to allow
for lysis of the fatty tissue.

Determination of the Age of Bloodstains

Affiliation: University of the West Indies
 BUDGET JUSTIFICATION
D. Reagents Cont’d
 RNeasy miniElute cleaning kit ($1436 in total) will be used for the cleanup and
the concentration of miRNA enzymatic reactions.

 RNASE free water ($124 in total) will be used for the degradation of miRNA.

 RNeasy mini spin column ($1420 in total) will be used to clean up miRNA and
purify miRNA.

Determination of the Age of Bloodstains

Affiliation: University of the West Indies
 BUDGET JUSTIFICATION
D. Reagents Cont’d
 miScript SYBER Green PCR Kit, HS-RNU6-2 miScript Primer Assay, Hs-miR-16-1
miScript Primer Assay, and Hs-miR-451-1 miScript Primer Assay are all used to
perform the real-time PCRs in which the U6b is a normalization standard.

 70% Ethanol and chloroform needed for the total extraction and purification of
RNA

Determination of the Age of Bloodstains

Affiliation: University of the West Indies
 THANK YOU
For any quires please contact us

Nikitia Whyte Nikitia.whyte@gmail.com

Alana Ingram Alanam.ingram@gmail.com
Daniel Khouri dkhouri18@gmail.com
Sherray Parkison sherray_parkinson@yahoo.com
Emerald Blaides emerald.blaides@yahoo.com

Determination of the Age of Bloodstains Affiliation: University of the West Indies