pcr lab operating manual
TRANSCRIPT
Province Government
Ministry of Social Development
BHAKTAPUR HOSPITAL
Bagmati Province
Bhaktapur
PCR Lab
Operating
Manual
2
Message from the PCR Focal Person
In the field of laboratory medicine, teamwork of paramedical staffs and experts in various
branches of medicine is of paramount importance. Bhaktapur Hospital has undergone
tremendous change in the last six months with advanced technologies and well established
molecular lab is one of them.
I would like to acknowledge Medical Superintendent Dr Sumitra Gautam and Province lab
acting director Mr Narayan Karki also all the dedicated staffs of PCR lab for the devotion and
generous cooperation in development of healthy ambience. With so many overwhelming
responses, what comes to my mind is that “Success comes to those who work hard and devote
their whole life to make a dream come true.”
The input of my staffs has helped to refresh and energize to produce an outstanding result of
molecular lab. The commitment towards the work has brought tremendous enlightenment in both
personal and professional life.
The overwhelming response has been my reward over the years and in my continuous endeavor
to contribute for significant changes and updates. I heartily thank Sujata, Saruna, Renu, Manish
and Rabin for their continuous effort to execute elegant PCR service to Bhaktapur Hospital and
patient.
Sunita Neupane
Focal Person for COVID 19
3
Contents Message from Medical Superintendent ........................................ Error! Bookmark not defined.
List of Acronyms ........................................................................ Error! Bookmark not defined.
1. Introduction .........................................................................................................................4
2. Aims and Objectives ............................................................................................................7
3. Facility design ......................................................................................................................8
4. Equipments, Reagents and Consumables ..............................................................................9
4.1 Equipments ...................................................................................................................... 10
4.2 Reagents .......................................................................................................................... 10
4.3 Consumables ................................................................................................................... 10
4.4 Personnel ......................................................................................................................... 11
5. Staff Details ....................................................................................................................... 12
6. Job Descriptions ................................................................... Error! Bookmark not defined.
7. Workflow ........................................................................................................................... 13
a) Sample Collection .......................................................................................................... 13
b) Sample Processing .......................................................................................................... 14
- RNA Extraction .............................................................................................................. 17
- Master Mix ..................................................................................................................... 21
- Template addition ........................................................................................................... 21
- PCR amplification .......................................................................................................... 24
- PCR analysis .................................................................................................................. 24
c) Reporting ....................................................................................................................... 25
8. Occupational health and Safety Instructions ....................................................................... 31
a) Hospital Waste Management: ......................................................................................... 32
b) Hospital Infection Control: ............................................................................................. 34
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Introduction
Coronaviruses are a large family of viruses that mostly cause respiratory illness in humans.
Recently identified corona viruses caused illness like Middle East Respiratory Syndrome
(MERS), Severe Acute Respiratory Syndrome (SARS) and, most recently, Coronavirus
Disease19 (COVID - 19). The virus (which was initially called the novel Coronavirus 2019) has
been named SARS- CoV2.
WHO declared COVID-19 outbreak a Public Health Emergency of International Concern on 30th
January 2020 after initial infection seen in Wuhan, China in December 2019. On 11th March,
WHO declared the COVID-19 outbreak as “pandemic”. As of 8th December, 2020, the world has
witnessed 68,117,810 coronavirus cases so far and 1,554,160 deaths. Nepal is also not spared
with the dreary outbreak. The first case in Nepal was reported on 23rd January, 2020. Nepal went
into lockdown for 4 whole months in order to contain the pandemic. But ever since the
withdrawal of the prohibitory orders, the number of new cases in Nepal has been soaring high
with 4-digit number every passing day. (Add latest new case)
1. Test of Corona virus (2019-nCoV)
The gold standard test for SARS-CoV-2 is real-time, reverse transcriptase PCR (rRT-PCR).
Other tests/methods available are antibody test, antigen test, ELISA, CLIA etc.
Polymerase Chain Reaction (PCR) is a laboratory technique used to make multiple copies of a
segment of DNA. PCR is very precise and used to amplify a specific DNA target from a mixture
of DNA molecules. A PCR reaction consists of 3 steps; denaturation, annealing and extension.
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With a strong history of 115 years, Bhaktapur hospital has been providing access to modern
medicine to the people since decades. At the dawn of pandemic, when only government hospitals
were rendering services to COVID patients, Bhaktapur Hospital was also one of the frontliners.
Bhaktapur Hospital initiated sample collection (Nasopharyngeal / Oropharyngeal) and blood
collection for RDT antibody from Chaitra 10, 2077 (23rd March, 2020). The sample volume
increased drastically so to provide easy access to needy people, it established the molecular lab
for diagnosis of SARS-CoV-2 from Kartik 27, 2077 (12th November 2020). Since its venture into
molecular diagnosis, it has been able to provide service uninterrupted 12 hours a day. To
increase its efficiency and accessibility, sample collection and report dispatch time allocated
from 9:00am to 1:00pm, Sunday to Friday. Validation of COVID-19 RT PCR from NPHL was
obtained on 5th Mangsir, 2077. Besides, proficiency testing samples are obtained from NPHL
and other QC samples from PPHL, Dhulikhel on a monthly basis.
As of 2077/8/26, Bhaktapur Hospital COVID PCR lab has processed 812 samples collected in
the hospital premises itself.
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Table 1: Total tests performed per day
Date Total Samples Positive
2077-07-26 14 5
2077-07-27 27 10
2077-07-28 25 11
2077-07-30 21 8
2077-08-01 9 3
2077-08-02 33 10
2077-08-03 33 10
2077-08-04 36 14
2077-08-05 45 11
2077-08-07 38 11
2077-08-08 43 11
2077-08-09 34 4
2077-08-10 29 8
2077-08-11 28 8
2077-08-12 37 12
2077-08-14 54 14
2077-08-15 37 8
2077-08-16 28 9
2077-08-17 33 7
2077-08-18 25 9
2077-08-19 30 10
2077-08-21 26 4
2077-08-22 26 7
2077-08-23 28 8
2077-08-24 24 4
2077-08-25 21 8
2077-08-26 28 5
Total 812 229
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Figure 1: Total number of PCR tests performed per day
2. Aims and Objectives
The main objective of this manual is to provide guidance to the staffs of COVID-19 lab. It states
the guidelines regarding collection of samples, physical infrastructures, workflow development,
quality assurance and communication of the result.
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2725
21
9
33 3336
45
38
43
34
29 28
37
54
37
28
33
25
30
26 2628
2421
28
5
10 118
3
10 10
1411 11 11
4
8 8
1214
8 97
9 10
47 8
4
85
2077
-07
-26
2077
-07
-27
2077
-07
-28
2077
-07
-29
2077
-07
-30
2077
-07
-31
2077
-08
-01
2077
-08
-02
2077
-08
-03
2077
-08
-04
2077
-08
-05
2077
-08
-06
2077
-08
-07
2077
-08
-08
2077
-08
-09
2077
-08
-10
2077
-08
-11
2077
-08
-12
2077
-08
-13
2077
-08
-14
2077
-08
-15
2077
-08
-16
2077
-08
-17
2077
-08
-18
2077
-08
-19
2077
-08
-20
2077
-08
-21
2077
-08
-22
2077
-08
-23
2077
-08
-24
2077
-08
-25
2077
-08
-26
Total Samples Positive
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3. Facility design
Laboratory has adequate space and designed in a way to prevent contamination of reactions with
amplified products from previous assays. It has 5 separate rooms allocated:
a) Donning room
b) Extraction room
c) Master Mix preparation and template addition
d) Amplification and product detection
e) Office room
There is a unidirectional workflow to reduce the opportunity of contamination to occur.
- No materials from extraction room should be taken into master mix preparation room.
- No supplies from amplification and product detection room should be taken into
extraction or master mix preparation room.
- A passing door is available in each room to pass the extract or the master mix product.
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4. Progress and planning of Bhaktapur Hospital for testing COVID-19
After establishment of well-equipped molecular lab at Bhaktapur Hospital, it has been rendering
PCR service uninterrupted.
Sample transport
In the initial days when the nation went into lockdown, sample collection facility was made
available in the hospital. There was a dreadful situation when everyone feared of infection.
During those days, samples were sent to NPHL, TUTH, Dhulikhel hospital, Provincial Public
Health Laboratory Dhulikhel and Sukraraj Tropical & Infectious Diseases Hospital. After the
establishment of molecular lab in the hospital’s own premises, all samples are processed here
itself.
Report Dispatch
We use MIDAS Software for report dispatch. Hardcopies are made available to the patients.
However, SMS services and email services are on process for the same.
Laboratory Information System
LIS System currently in use at Bhaktapur Hospital is MIDAS. It gives all the records of test done
in Bhaktapur hospital.
Logistic Management
PCR Machine (BIO-RAD) available: 1
Biosafety Cabinet Level-II (Quest Scientific Technologies): 1
Laminar Hood (Quest Scientific Technologies): 2
Centrifuges (Remi): 2
Serological water bath: 1
Refrigerator: 3
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5. Equipments, Reagents and Consumables
4.1 Equipments
Quality assurance of all the equipments used in PCR is to be done lately. A list of equipments
used is shown below:
1) Thermocycler (BIO-RAD)
2) Centrifuges
3) Biosafety cabinet
4) Laminar flow cabinet
5) Pipette (Accumax)
6) Temperature dependent instruments: Refrigerators
User Log and Temperature log of every instrument is to be maintained daily.
4.2 Reagents
- All reagents are to be maintained contamination free.
- All reagents should be clearly labeled with name, expiration date, relevant safety information.
-Opening date, expiration date and initials of individual should be mentioned on each reagent.
- Reagents from different lot numbers should not be exchanged.
- A copy of manufacturer’s specification and procedures should be kept in SOP binder.
- Logbooks should be maintained for all reagents and kits including product name, manufacturer,
product number, lot number, associated dates of receipt, preparation, open, expiration etc.
4.3 Consumables
Disposable materials used in PCR analysis include pipette tips, eppendorf tubes, PCR tubes and
gloves.
- Special tips should be used for PCR analysis including barrier tips and aerosol resistant
tips.
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- Laboratories should use polypropylene tubes that are lot-certified, RNase-free, DNase-
free and pyrogen-free.
- The size and style of PCR tubes should be as per recommended by manufacturer for
thermocycler.
- Disposable powder free gloves should be available in each section of laboratory.
- Gloves should be interchanged before leaving and entering each section of laboratory.
4.4 Personnel
- Adequate trained staff is required to perform the tests.
- Analysts should have adequate qualifications.
- Personnel responsible for verification must have at least Master’s degree.
-Proper orientation should be given by a trained staff.
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6. Staff Details
S.N. Name Designation
1 Dr. Pramila Vaidhya Consultant Pathologist
2 Sunita Neupane Focal Person
3 Saruna Prajapati Medical Lab Technologist
4 Sujata Baidya Medical Lab Technologist
5 Renu Baga Lab Technician
6 Manish Prajapati Lab Technician
7 Rabin Koju Lab Assistant
8 Dipa Thapa Attendant
Photograph 1: PCR lab team
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7. Workflow
The workflow in a PCR lab is as follows:
a) Sample Collection
b) Sample Processing
- RNA Extraction
- Master mix
- Template addition
- PCR amplification
- PCR analysis
c) Reporting
a) Sample Collection
Respiratory samples are recommended for real time PCR for diagnosis of
COVID-19
Samples should be collected with synthetic swabs with plastic shaft. Do
NOT use cotton swabs with wooden sticks)
Swabs must be collected in leak proof container with VTM1.
Specimen must be correctly labelled and accompanied with correctly filled
sample referral form.
Specimen that can be delivered promptly to the laboratory can be stored and shipped
at 2-8°C for up to 72 hours.
1 When VTM is not available, 0.9% NaCl (2ml) in screw capped vials
can be used. (References: https://jcm.asm.org/content/jcm/58/6/e00590-
20.full.pdf
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3295134/)
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Note: Consent has been taken from patient before taking the photo for sample
collection.
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b) Sample Processing
- RNA Extraction
Nucleic acid of virus is enclosed inside protective capsid and envelop so for PCR analysis
nucleic acid is to be extracted from the virus. It can be done by various methods; heat, enzymatic
or microbeads.
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- This is the process where the laboratory personnel are the most vulnerable so it
should be performed in a Class II biosafety cabinet with negative pressure.
- Use 0.5% hypochlorite followed by 70% ethanol to disinfect the work surface. Do not
use bleach for cleaning pipettes.
- Vortexing should be done within the cabinet.
- Extracted RNA should be stored at -20˚C.
Photograph 2: Extraction Room
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- Master Mix
This is the cleanest step in PCR and should be performed in laminar flow hood. The
amplification reagents are prepared just before the template addition.
- Fluorescent probes are sensitive to light so put off the lights while preparing master
mix.
- Gloves should be changed each time upon entering this area.
- Template addition
It is done in a separate cabinet where the RNA extract is added to the master mix reagent.
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Photograph 3: Template Addition
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- PCR amplification
It should be done in a room separate from master mix area.
- PCR analysis
Analysis is done as per the protocol of PCR reagent used.
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c) Reporting
Reporting of the results is based on the protocol of the PCR reagents used. Most of the reagents
detect N gene, E gene, RdRP gene and ORF 1ab. A panel of combination of these genes are
analyzed to report the results.
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Figure 2: Flow Chart for releasing report*
*Reference: Interim Guidelines for SARS-CoV-2 PCR Laboratories in National Public Health
Laboratory Network Nepal
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Photograph 4: PCR reporting
1) Storage retention and disposal of specimen
Ensuring quality of result: QC results are documented with every run.
a) Internal QC Materials
- PCR Positive Control: to verify that extraction, PCR master mix preparation and
reagent preparation were done correctly.
- Negative Control: to verify that no contaminating nucleic acid has been introduced
into the master mix or into samples during processing.
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- No Template Control: to verify that no contaminating nucleic acid has been
introduced into the master mix.
b) External Quality Assurance
Participation in Validation program and Proficiency Testing program under NEQAS
conducted by National Public Health Laboratory (NPHL).
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Record Keeping
8. Occupational health and Safety Instructions
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a) Hospital Waste Management:
Every hospital should take appropriate authorization from competent authorities in waste
management. Detailed plans should be made out for waste management detailing like
segregation, collection, transportation, treatment, disposal, safety precautions and training of
staff within the ambit of waste management handling rules.
WHO estimates:
85% of hospital waste is non-hazardous
10% is infectious
5% is non-infectious
The responsibility of segregation should be with the generator of biomedical waste i.e. doctors,
nurses, technicians etc. (Medical and Paramedical personnel).
The segregation should be done as per the categories applicable mentioned in the rules.
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Figure 3: Effectiveness of Disinfectants on various agents
Appropriate Chemical Disinfectant for COVID 19
Chlorine-based product
70-90% alcohol
Hydrogen peroxide ≥0.5%
Things to be considered while selecting decontaminants
concentration
Resistance
compatibility with other materials present
surface being decontaminated
hazards to humans, animals, and the environment associated with the disinfectant.
Ineffective Disinfection can provide a false sense of security and spread
contamination.
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Preparation of Disinfectants
a. Calculation of liquid sodium hypochlorite concentration:
Formula 1
[% liquid hypochlorite / % desire liquid hypochlorite] -1 = Total part of water for each part of
sodium hypochlorite
E.g. [5% liquid hypochlorite / 0.5 % liquid hypochlorite] -1 = Total 9 part of water for each part
of sodium hypochlorite
Therefore, one must dissolve one part of sodium hypochlorite and 9 part of water in order to
prepare 0.5% sodium hypochlorite out of 5%.
Formula 2
C1*V1 = C2 * V2
Where, C= concentration and V= Volume
If you want to prepare a 0.5% out of 5% chlorine solution
C1 (available) =5% and C2 (desire) =0.5%
V1 is the volume of the chlorine solution to be added to prepare a 0.5% chlorine solution
V2 is the amount of water to be added to prepare a 0.5% chlorine solution.
C1*V1=C2*V2
5% * V1= 0.5% * 1000ml
V1= 0.5%* 1000ml / 5%
V1= 100ml
Therefore, the amount of chlorine solution added to prepare 1000ml of 0.5% chlorine solution
will be 100ml and water is 900ml
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B. Calculation of chlorine solutions from calcium hypochlorite
[% chlorine desired ∕ % chlorine in hypochlorite powder or granules] × 1 000 = grams of calcium
hypochlorite powder for each liter of water.
E.g. [0.5% chlorine desired ∕ 35% in hypochlorite powder] × 1 000 = 0.0143 × 1 000 = 14.3
Therefore, you must dissolve 14.3 grams of calcium hypochlorite powder in each liter of water
used to make a 0.5% chlorine solution.
WEAR PROPER PPE BEFORE
STARTING Disinfection PROCESS!!!
DISINFECTION OF BIOSAFETY CABINET
Wet sponge/ tissue paper with 0.5% hypochlorite and Wipe down all surfaces.
Again wipe it with 70% ethanol.
Run in-built UV Disinfection programme for 15 minutes.
LABORATORY CLOTHING DISINFECTION
Laboratory clothing for reuse (e.g. scrubs) should be washed with hot water/ detergent solution.
– soaked in 0.05% hypochlorite for 30mins (preferably overnight) for Disinfection
OR
Autoclave at 121 ◦C, 15psi for 15 minutes and then washed through laundry
DISINFECTION OF PAPER
Any kinds of documents accompanying samples are sprayed with 70% alcohol.
Ensure key information is captured on laboratory tracking forms/in database prior to
spraying
DISINFECTION OF SURFACES AND FLOORS
Floor surfaces should be cleaned with 0.5-1% hypochlorite solution.
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If required, cleaning with detergent should precede hypochlorite cleaning. (Lab areas
with high inflow of staff)
Other surfaces and door handles should be cleaned with detergent and 70% ethanol.
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b) Hospital Infection Control:
Each hospital should have the following organization for prevention and control of Hospital
Acquired Infections (HAI):
1) Infection Control Committee:
The committee formulates policies for infection control. Head of hospital should be the
chairman. Members are representative of medical/surgical (HOD), nursing, engineering,
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administrative, domestic, pharmacy. Committee should meet every three months or
earlier if required.
2) Function:
Policymaking regarding:
a) Provision of adequate buildings, equipments, isolation facilities etc.
b) Ventilation of operation theatre
c) Standardization of procedures for operation theatre, wards, housekeeping, kitchen,
laundry,
d) Preparation of manuals for procedures like preparative skin preparation, IV infusion
and catheterization, lumbar puncture, wound dressing.
e) Formulation of disinfection policy
f) Antibiotic policy for rational use of antibiotic in therapy andprophylaxis
g) Implementation of biomedical waste management rules in hospital
3) Infection Control team:
The team carries out day-to-day measures for control of infection. Infection Control Officer
is usually microbiologist.
Function:
a) Surveillance of infection to give baseline information of the level of endemic infection in
hospital.
b) Investigation of outbreaks of infection including source of infection with epidemiological
typing
c) Controlling the outbreak by rectifying technical lapses if any
d) Monitoring of procedures
e) Monitoring of hospital staff carriers.
f) Training of staffs
g) Ensure implementation of Universal Precautions in hospital
Universal Precautions
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Basic Principles:
- Consider all patients as potentially infective
- Use appropriate barrier precautions routinely (gloves, masks, aprons and eye protection)
Procedures:
- Hands should be washed before and after all patients or specimen contact.
- Blood of all patients should be handled as potentially infectious.
- Gloves should be worn for potential contact with blood and body fluids.
- Used syringes should be immediately placed in impermeable container.
- Protective eyewear and mask should be worn if splatter with blood or body fluids is
possible (e.g. bronchoscopy, oral surgery)
- Gowns should be worn when splash with blood or body fluid anticipated.
- Masks should be worn when handling patients of TB and respiratory organisms
Protection against blood borne infections (HBV and HIV)
- Mechanical pipetting should be used.
- Spills should be immediately decontaminated.
- Gloves should be worn during handling of blood and blood products.
- All open wounds should be covered with water tight dressing
- Hands should be washed with soap andwater immediately after exposure to the
specimens.
- Work surfaces should be non-penetrative. Use 1% hypochlorite to decontaminate
surfaces.
- Specimens should be decontaminated with 1% hypochlorite before disposal.
Spillage or surface contaminated by blood or blood products:
- Spillage should be covered with absorbent material after which disinfectant (1%
hypochlorite/bleaching powder 14g/dl) should be poured over it and left 20 minutes.
Waste disposal to be dealt separately.
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- Tissues, organs or limbs removed during surgery should be incinerated or buried deep
with bleaching powder or lime.