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FOREWORD

Myanmar has signed Asia Pacific Leaders Malaria Alliance (APLMA) declaration to

eliminate malaria by 2030 in 9 East Asia Summit in 2014 along with other Greater

Mekong Sub region countries. The National Malaria Control Porgramme is committed

to pursue malaria elimination by 2030. “National Malaria Strategic Plan (NSP) for

Intensifying Malaria Control and Accelerating Progress towards Malaria Elimination

2016-2020” and “National Plan for Malaria Elimination in Myanmar 2016-2030” have

been developed.

Myanmar has made significant progress in reducing the malaria incidence by 49% in 2015

in comparison to 2012. The control programme has embarked into elimination

aiming to eliminate P. falciparum by 2025 and malaria by 2030. Malaria control and

elimination depends on parasitological diagnosis by microscopy or RDT to guide

individual treatment. Ensuring universal quality assured malaria microscopy is a priority

for the programme as we move towards malaria elimination. This will also reduce the

over-use of malaria medicines- the Artemisinin-based Combination Therapies and reduce

drug-resistance selection pressure on parasites. Malaria microscopy is a gold standard

malaria diagnostic test which is currently deployed in the hospitals under the Department

of Medical Services and some Public Health Centers under the Department of Public

Health.

National Malaria Control Programme and National Health Laboratory have developed

the Standard Operating Procedures (SOPs) for malaria microscopy with a technical

support from WHO and URC-ADB project. This describes in detail the pre-analytic, analytic

and post-analytic procedures for malaria microscopy services. The SOPs aim to provide

the guidance to the malaria microscopists for performing the malaria microscopy in

uniform, consistent and reliable manner that ensures the accuracy of the malaria

microscopy procedures performed. The programme will distribute the SOPs to all

microscopy centers (including private sectors) in the country to ensure the use of

quality assured malaria microscopy procedures and services are offered effectively and

efficiently.

I would fully endorse the malaria microscopy SOPs and ensure its full implementation.

Dr. Thar Tun Kyaw

Director General

Department of Public Health

Ministry of Health and Sports

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ACKNOWLEDGEMENTS

The SOP on malaria microscopy for every hospital in Myanmar has been revised by WHO and

URC-ADB project under the guidance of the National Health Laboratory (NHL) and National

Malaria Control Programme (NMCP), Ministry of Health and Sports, the Republic of Union of

Myanmar.

Version 1 was prepared by URC-CAP Malaria Project with the support of Dr. Khin Mon Mon,

former National Malaria Control Programme Manager in 2015. During 2016, “Malaria

Surveillance and Quality Assurance System” project (URC-ADB project) was launched with the

funding support from the Asian Development Bank (ADB). On 22nd July 2016 stakeholder

meeting, H.E Union Minister for Health and Sports delivered speech on malaria laboratory

quality assurance system that is essential for the malaria elimination by the year 2020. This

project along with WHO provided technical support to NMCP to develop “Quality Assurance

and Quality Control Manual” and “Standard Operation Procedures for Malaria Microscopy”.

National Malaria Control Programme would like to express heartfelt thanks to ADB Team

leader, Gerard Servais, ADB Public Health Specialist, Dr. Kyi Thar, URC Senior Vice President,

Dr. Neeraj Kak and Team Leader of URC-ADB project, Dr. Kheang Soy Ty for playing important

roles in providing technical advises throughout the knowledge product development.

Moreover, series of reviewing and fruitful technical advice were provided by Prof. Htay Htay

Tin, Deputy Director General (NHL), Dr. Win Thein, Director (Laboratory) and Dr. Thi Thi Htoon,

Head of parasitology section (NHL). Special thanks to Dr. Badri Thapa, Scientist Malaria Control

(WHO) for providing draft documents and proofreading. Moreover, contributors would like to

offer their sincere gratitude to Dr. Thandar Lwin, Deputy Director General (Disease Control),

Dr. Aung Thi, Deputy Director (Malaria) and Dr. Zaw Lin, Deputy Director (Dengue & Filaria) for

final proofreading. Acknowledge and thanks to Malaria Regional Officers, VBDC Team Leaders,

Ms. Thiri San, Chief Medical Technologist, NMCP, Medical Technologists and Grade 1

Laboratory Technicians from NMCP, Dr. Myat Kyaw, Deputy Team Leader (URC-ADB project)

and Dr. Mie Mie Han, Laboratory QA Specialist (URC-ADB project) and URC-ADB project team

for providing helpful discussion and supportive participation. Last, but not the least, National

Malaria Control Programme would not have completed this document with the support from

the partners and contributors.

National Malaria Control Program

Department of Public Health

Ministry of Health and Sports

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Acronyms

ADB - Asian Development Bank

APLMA - Asia Pacific Leaders Malaria Alliance

BHS - Basic Health Staff

ECA - External Competency Assessment

EQA - External Quality Assurance

EQAS - External Quality Assessment Scheme

IQC - Internal Quality Control

Lab - Laboratory

LHV - Lady Health Visitor

MLT - Malaria Laboratory Technician

MM - Malaria microscopy

MOHS - Ministry of Health and Sports

MP - Malaria Parasite

MRL - Malaria Reference Laboratory

NCA - National Competence Assessment

NGOs - Non government organizations

NHL - National Health Laboratory

NMCP - National Malaria Control Programme

NMRL - National Malaria Reference Laboratory

NRL - National Reference Laboratory

NSP - National Malaria Strategic Plan

OTSS - Outreach Training and Supportive Supervision

PPE - Personal Protective Equipment

QA - Quality Assurance

QC - Quality Control

QMS - Quality Management System

RDT - Rapid Diagnostic Test

RHC - Rural Health Centre

S&E - Supplies & Equipment

S/R - State/Region

SH - Station Hospital

SOP - Standard Operating Procedures

TP - True Positive

TN - True Negative

TMO - Township Medical Officer

URC - University Research Co., LLC

VBDC - Vector Borne Disease Control

WHO - World Health Organization

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Contents

No Subject SOP No Page No

OVERVIEW OF SOP

PREPARATION OF MICROSCOPE, MALARIA SLIDES AND STAINING

1 Use, care and maintenance of microscope NMCP-MM-SOP-01 3-7

2 Cleaning and storing of glass slides NMCP-MM-SOP-02 9-11

3 Preparation of Giemsa stock solution NMCP-MM-SOP-03 13-16

4 Collection and preparation of blood films for malaria

microscopy, labelling of malaria blood films

NMCP-MM-SOP-04 17-21

5 Staining of malaria blood films with Giemsa NMCP-MM-SOP-05 23-29

EXAMINATION OF MALARIA SLIDES UNDER MICROSCOPE

6 Examining thick and thin malaria blood films NMCP-MM-SOP-06 31-37

7 Recording and reporting of malaria results NMCP-MM-SOP-07 39-41

CROSS-CHECKING OF MALARIA SLIDES

8 Cross-checking of malaria blood films NMCP-MM-SOP-08 43-51

INFECTION CONTROL AND WASTE MANAGEMENT

9 Instruction for laboratory aspect of infection

prevention and control (IPC)

NMCP-MM-SOP-09 53-59

10 Medical laboratory waste management instruction NMCP-MM-SOP-10 61-63

SUPERVISORY VISIT

11 Supervisory visits to laboratories providing malaria

microscopy services

NMCP-MM-SOP-11 65-72

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1

OVERVIEW OF SOP 1. PURPOSE AND SCOPE

These SOPs provide an overview of the processes and procedures relevant to providing quality assured malaria microscopy services.

This procedure will be updated and modified as and when necessary with approval of the National Malaria Program Manager and National Health Laboratory.

All procedures specified herein are mandatory for all malaria microscopists working for Malaria Microscopy in public and private health facilities.

2. PRINCIPLE

Microscopy using Giemsa-stained blood films remains the gold standard procedure for malaria diagnosis in the National Malaria Control Programme. Therefore a quality assurance (QA) system for malaria microscopy is needed to ensure that there are good practices related to sample collection, staining, examination, recording and reporting and cross-checking of results both in the central and peripheral diagnostic facilities. SOPs will serve a reference document to follow by the microscopists that brings uniformity and consistency to perform the malaria microscopy. Training and supervision of the microscopists and laboratories are also important aspects of ensuring quality microscopy services.

3. DOCUMENT

Standard Operating Procedures (SOPs) of the different procedures for malaria microscopy.

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2

Cross- checking of malaria blood films

FLOWCHART DESCRIPTION OF ACTIVITY

NMCP-MM-SOP-01

NMCP-MM-SOP-02 NMCP-MM-SOP-03 NMCP-MM-SOP-04

NMCP-MM-SOP-05

NMCP-MM-SOP-06

NMCP-MM-SOP-07 NMCP-MM-SOP-08

NMCP-MM-SOP-09

NMCP-MM-SOP-10

NMCP-MM-SOP-11

Preparation of Giemsa stock solution

Collection and preparation of blood films for malaria microscopy

Labeling of malaria blood films

Staining of malaria blood films with Giemsa

Cleaning and storing of glass slides

Examining thick and thin malaria blood films

Recording and reporting of malaria results

Supervisory visits to laboratories providing malaria microscopy services

Instruction for Laboratory aspect of Infection Prevention and Control

Medical Laboratory waste management instruction

Use, Care and Maintenance of Microscope

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3 SOP 1: Use, Care and Maintenance of Microscope

National Malaria Control Program Department of Public Health Ministry of Health and Sports

Republic of the Union of Myanmar

Document Control No:NMCP-MM-SOP-01

Version - 2

Giemsa Malaria Microscopy Date of issue: 15 September 2017

Title: Use, Care and Maintenance of Microscope

Total Page : 5

1. PURPOSE AND SCOPE

This SOP describes the general procedures for the use, care and maintenance of microscopes in laboratories where malaria microscopy is performed.

2. SUPPLIES, MATERIALS AND EQUIPMENT

• compound microscope fitted with paired 10x oculars (eye pieces), 10x, 40x, and 100x (oil immersion) objectives and a mechanical stage

• dust cover • lens paper • commercial microscope cleaning solution, and • soft cloth

3. TRANSPORTING AND MOVING MICROSCOPES

• Transport in original container with the packing materials to prevent from moving within the container.

• If original packaging is not available, use a specifically designed microscope transport box supplied by the manufacturer or a fabricated container with foam or similar packing materials to prevent the microscope from moving within the container during transport.

• During transport, protect it from excessive movement or vibration; pack it so that it does not move around in the vehicle and cannot fall from the top of other items being transported.

• Carry a microscope with hands, one supporting the base and the other holding the arm.

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4 SOP 1: Use, Care and Maintenance of Microscope

4. PLACEMENT

• Place it on a flat, level, firm bench, free from vibration. At high magnification, very small movements of the bench will cause large movements in the image being viewed by the microscopist.

• Place it in a position or bench with sufficient leg space for the microscopist; preferably use height-adjustable chairs

• Do not place the microscope in front of a brightly lit window. Place it in front of a wall or a darkened window.

5. MICROSCOPE SET UP

Follow the manufacturer’s directions for optimal set-up of the optical system and general use.

• Adjust the iris aperture to the setting recommended by the manufacturer to achieve maximum depth of field.

• If the manufacturer provides an iris diaphragm setting on the condenser, set this to the 100x objective lens setting.

• Use the following method if the eyepiece of the microscope can be removed: raise the condenser set the lamp to “low” select the 40x objective close the iris diaphragm remove one eyepiece Look down the tube, then adjust the

condenser until the edge of the diaphragm is in sharp focus without showing an outer fringe ring in green or in red.

open the iris diaphragm until the objective lens completely fills with light the shape of the light is usually octagonal; when the points of the octagon

touch the outside of the objective lens, open the iris diaphragm until the light appears circular

replace the eyepiece. • An alternative method, which does not require removal of the eyepieces, is to set

the microscope into Kohler illumination: ▪ Close the iris diaphragm on the condenser. ▪ Close the light source at the base of the microscope, where the light is

reflected up to the condenser. ▪ Turn the light up as bright as possible. ▪ Put a stained blood smear on the stage, add immersion oil, and use the 100x

oil immersion objective lens.

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5 SOP 1: Use, Care and Maintenance of Microscope

▪ Look through the microscope oculars, and focus the condenser up and down until the edge of the field is sharp.

▪ Use the adjustment screws on the condenser to move the circle of light to the center of the field if necessary

▪ Turn the light down and open the iris diaphragm and the light source before use.

• Adjust the inter-pupillary distance. • Adjust the diopter.

6. Use of the microscope

• Be familiar with the parts of the microscope and their function. • Turn on the light source, and adjust the optimum light setting to ensure the correct

level of brightness by turning or sliding the brightness adjustment knob at the base.

• Rotate the low power objective into position. • Remove the eyepiece, look down the body tube and adjust the mirror and

diaphragm setting so that light is reflected up the tube and a circle of evenly illuminated light is visible in the field of view.

• View the specimen with the 10x objective, then with the 40x and then with the 100x oil immersion objective.

a. Prevention of damage to the 40x objectives

• Remove immersion oil that comes into contact with the 40x objective. Because the 40x lens is located next to the 100x lens and because it is also a long lens, it is easy for it to come into contact with immersion oil accidentally. The 40x objective is not sealed against oil penetration, and any immersion oil left in contact with this objective will penetrate the lens and be deposited on the inside of the lower lens.

• Only the manufacturer can repair, after dismantling the objective lens. • Lower the stage before removing a slide. • After use, immediately clean the immersion oil from the objective with lens paper

and commercial lens cleaner. • If not done, the oil may thicken and harden over time and affect the optical

performance of the lens. • To protect the 100x objective lens, the objective should be rotated up when the

microscope is not in use.

b. Daily maintenance “Do’s”

• Inspect for damage or malfunction. • Record any damage or malfunction in the daily microscopy maintenance sheet. • Clean the parts of the microscope with a clean cloth. • Clean the objectives after each day’s work. • Ensure that immersion oil residues are removed.

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6 SOP 1: Use, Care and Maintenance of Microscope

• Clean the objectives with lens cleaning tissue only.

• Cover microscope with a dust cover when not in use.

• Turn off the power and unplug the microscope at the end of the day, to protect it

from a power surge.

c. Daily maintenance “Don’ts”

• Do not clean any part of the microscope with xylene, which will damage the

microscope and is toxic.

• Never clean lenses with alcohol, ordinary tissues, cleaning paper, toilet paper,

cotton wool or hand towels, which will scratch the lens surface.

• Do not leave lens ports uncovered; use the port cover or sealing tape.

d. Storage

• In humid places, store the microscope in dry conditions when not in use to prevent

fungal growth on glass surfaces.

• Keep in a “warm cupboard” with a constant temperature and low humidity.

• Store lens and prism heads in airtight boxes with desiccant.

e. Repairs

• Staff can replace a broken bulb or replace a blown fuse by strictly following the

manufacturer’s instructions.

• Other repairs must be performed by a qualified service engineer or technician.

• Do not exchange lenses and ports with those of other microscopes.

• Routine servicing, such as realignment of optics, replacement of lens and

maintenance and lubrication of the stage, should be performed by a qualified

service technician.

• Record all repairs on the microscope maintenance sheet.

f. Precautions and safety

• Secure and stabilize the power source and connections to the microscope to avoid

risk of electric shock.

• Do not expose the microscope and electrical connections to water.

• Care should be taken to prevent damage to the eyes from exposure to the high

light-intensity halogen lamp.

• Use the microscope ergonomically to prevent back and neck strain.

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7 SOP 1: Use, Care and Maintenance of Microscope

7. Microscope Maintenance Chart (Attachment-1)

8. REFERENCES – WHO Collaborating Center for Malaria Diagnosis 9. DOCUMENT HISTORY

Date Version Comments Responsible Person January

2017

2 Add use and repair of microscope and

format change; flowchart introduced

Add the chart for microscope maintenance

Medical Technologist and

Laboratory Technicians

Grade I, NMCP

Page 5 of 5

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9 SOP 2: Cleaning and storing of glass slides

National Malaria Control Program

Department of Public Health

Ministry of Health and Sports

Republic of the Union of Myanmar

Document Control No:

NMCP-MM-SOP-02

Version – 2

Giemsa Malaria Microscopy Date of issue: 15 September 2017

Title:Cleaning and Storing of glass slides

Total Page: 3

1. PURPOSE AND SCOPE This SOP describes the procedure for cleaning and storing glass slides that are to be used for preparing blood films for routine malaria microscopy.

2. PRINCIPLE It is important to ascertain that the slides to be used are clean and scratch-free, because dirty and scratched slides can lead to poorly prepared blood films that can compromise the quality and integrity of the diagnosis.

3. SUPPLIES AND MATERIALS a. Glass slides, 25 x 75 mm, preferably with one frosted end for labeling and with ground

edges, and must be of superior quality b. Two (2) basins, medium sized c. Dishwashing or liquid detergent d. Washing cloth or sponges e. Clean water f. Lint-free towels and paper g. Slide boxes (made of hard plastic) h. Latex disposal gloves (powder- free) i. Marking pen j. Silica gel

4. SAFETY PRECAUTION

Wear gloves to prevent accidental cuts or injury from the glass slides during washing.

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10 SOP 2: Cleaning and storing of glass slides

5. PROCEDURE

FLOWCHART DESCRIPTION OF ACTIVITY

Soak slides for at least 1-2 hours in water mixed with a mild dishwashing or liquid detergent.

With a washing cloth or sponge, clean both

sides of each slide. Rinse the slides individually in clean water

to remove all traces of detergent. Dry each slide thoroughly with a lint-free

towel. Handling the dried slides by their edges

only, wrap them in clean lint-free paper (by 10’s or 20’s), and return them in their original cardboard boxes, or in plastic slide boxes, each secured with a rubber band

If the slides will not be used immediately,

put some silica gel inside the box to prevent fungal growths.

With a marker pen, label the boxes cover

with the date, box number (out of the total) and number of slides per box as in the example below, and document in a record or logbook: 1. Example:

Store the boxes containing the cleaned slides inside a cabinet.

Page 2 of 3

1. Soak slides in water with detergent for 1-2 hours.

2. Clean slides with cloth or sponge.

3. Rinse slides in clean water.

4. Dry slides thoroughly.

5. Wrap slides by 10’s or 20’s, in clean paper and place inside box.

6. For long storage, place silica gel inside box.

7. Label box and document procedure In a record or logbook

05 Aug 2015

8. Store boxes inside the cabinet.

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11 SOP 2: Cleaning and storing of glass slides

6. PROCEDURE NOTES a. Recycling of glass slides must NOT be done unless there is a shortage or stock out. b. For newly purchased slides, ascertain that they are clean, scratch-free and greaseless.

If they are, this SOP may be skipped. c. Discard chipped or scratched slides. d. Use cleaned slides following the “first in, first out” rule.

7. REFERENCES WHO Malaria Microscopy Quality Assurance Manual. Version 1. 2009

8. DOCUMENT HISTORY

Date Version Comments Responsible Person

Oct 2014 1 SOP introduced and formatted NMCP

Feb 2016 2 Minor revision and format change;

flowchart introduced

NMCP

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13 SOP 3: Preparation of Giemsa stock solution

National Malaria Control Program

Department of Public Health

Ministry of Health and Sports

Republic of the Union of Myanmar

Document Control No:

NMCP-MM-SOP-03

Version – 2

Giemsa Malaria Microscopy Date of issue: 15 September 2017

Title:Preparation of Giemsa Stock Solution

Total Page: 4

1. PURPOSE AND SCOPE This SOP describes the procedure for preparing stock solution of Giemsa which is used for routine staining of malaria blood films.

2. PRINCIPLE Giemsa is the standard stain used for staining blood films for malaria diagnosis. It is commercially available as a ready to use product, but the quality varies according to source. To ensure quality, Giemsa is best made up at the Zonal Production Center and distributed to the different facilities providing malaria microscopy services.

3. SUPPLIES, MATERIALS AND EQUIPMENT a. Giemsa powder/stain, 3.8g (preferably BSC grade -Biological Stain Commission, to

ensure a very good, standard quality of product) b. Methanol, pure, 250 Ml c. Glycerol, pure, 250 mL d. Solid glass beads, 3-5 mm in diameter, 50-100 pieces e. Spatula or measuring spoon f. Weighing paper g. Graduated cylinder h. Glass or plastic funnel i. Screw-capped, dark or amber glass bottle, clean and dry, 500 mL capacity (if one is not

available, a chemically clean and dry, clear hard glass or polyethylene bottle of suitable size may be used, but should be fitted with a brown paper wrapping).

j. Analytical balance, weighing in divisions of 0.01 g 4. SAFETY PRECAUTION

a. Methanol and Giemsa stain are highly flammable and toxic if inhaled or swallowed. Avoid contact and inhalation. Universal safety precautions, including use of relevant Personal Protective Equipment (PPE), e.g., gloves and laboratory coat, must be practiced.

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14 SOP 3: Preparation of Giemsa stock solution

5. PROCEDURE

FLOWCHART DESCRIPTION OF ACTIVITY

Place about 50 glass beads into a dark or amber bottle.

Weigh 3.8 g of stain powder on an analytical balance, and pour into bottle containing the beads.

Measure 250 mL methanol in a graduated cylinder and pour gently into the bottle.

Allow the stain powder to sink through the methanol until it settles to the bottom.

Stopper the bottle tightly then shake it with circular motion for 2-3 minutes to dissolve the stain.

Measure 250 mL glycerol in a graduated cylinder and pour gently into the bottle.

Stopper the bottle tightly before repeating the shaking process.

Continue the shaking for 3–5 minutes for about 3-5 times during the first day.

Repeat the shaking every day for 2-3 days until the stain is completely mixed.

Page 2 of 4

1. Place 50 beads in a dark bottle.

2. Add 3.8 g Giemsa powder.

3. Add 250 mL methanol.

4. Allow stain to settle at the bottom.

5. Stopper bottle and shake for 2-3 minutes with circular motion.

6. Add 250 mL glycerol.

7. Stopper bottle and repeat shaking as above.

8. Shake bottle as in above 3–5 times on first day.

9. Repeat shaking as in above for 2-3 days.

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15 SOP 3: Preparation of Giemsa stock solution

FLOWCHART DESCRIPTION OF ACTIVITY

Assign batch number for each stock solution prepared. Label the bottle as in example below:

Batch #2015-01 stands for the year prepared and stock or lot number.

Store the bottle tightly stoppered to prevent absorption of water vapour from the air, and in a cool place away from direct sunlight.

6. PROCEDURE NOTES

a. Do NOT shake, or filter the stock Giemsa bottle before use to avoid re-suspending the precipitates which settle on blood films during staining and obscure important details during microscopy.

b. Do NOT contaminate the stock Giemsa solution with water. Keep a small amount in a small bottle for daily use to avoid contaminating the whole stock.

c. Do NOT return unused or left-over stain to the stock bottle or to the bottle containing the working solution; stain once out of the bottle must be used quickly or discarded.

d. A single batch of Giemsa stain must not be made up for use, or re-use, throughout the day, or longer. Giemsa stain quickly absorbs water vapor in the air; and when diluted with de-ionized, distilled or any form of water, it rapidly loses its staining properties so that slides stain poorly after just a short time. The iridescent scum on the surface of made-up Giemsa stain adheres easily to the blood film making examination difficult.

e. If a clear stock bottle is used, wrap it with a thick dark paper to avoid light penetration.

7. QUALITY CONTROL AND DOCUMENTATION

a. Perform quality control check for every new batch/lot of stock solution prepared. b. On logbook, record information as in the following example:

Page 3 of 4

Giemsa Stock Solution Batch No.: 2015-01

Date prepared: 5 Aug 2015

10. Assign batch number and label bottle of stock Giemsa.

11. Store Giemsa stock bottles in cool, dark area.

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16 SOP 3: Preparation of Giemsa stock solution

Date of stock

preparation

Batch number

Number of

bottles prepared

Volumeper

bottle (mL)

Date of QC

pH of stain

Remarks on the

quality of stain

Action taken

Name and signature of

staff who performed

QC 5 Aug 2015

2015-001

5 50 5 Aug 2015

7.4 Good/ poor

Adjusted pH to 7.2

8. REFERENCES Basic Malaria Microscopy. Part I. Learner’s Guide, Second Edition. 2010

WHO Malaria Microscopy Quality Assurance Manual. Version 1. 2009

9. DOCUMENT HISTORY

Date Version Comments Responsible Person

Oct 2014 1 SOP introduced and formatted NMCP

Feb 2016 2 Minor revision and format change;

flowchart introduced

NMCP

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17 SOP 4: Collection and preparation of blood films for malaria microscopy, labelling of malaria blood films

National Malaria Control Program

Department of Public Health

Ministry of Health and Sports

Republic of the Union of Myanmar

Document Control No:

NMCP-MM-SOP-04

Version – 2

Giemsa Malaria Microscopy Date of issue: 15 September 2017

Title: Collection of Finger- Prick Blood and

Preparation of Thick and Thin Blood Films Labeling of Malaria Blood Films

Total Page: 5

1. PURPOSE AND SCOPE

This SOP describes the preparation of thick and thin blood films using finger prick blood for malaria diagnosis by light microscopy and also describes the proper and correct labeling of malaria blood films

2. PRINCIPLE

Examination of blood films by microscopy is a basic technique, which remains the gold standard for the diagnosis of malaria. Blood films for malaria diagnosis are best prepared from capillary blood obtained by finger prick. Good quality blood films are essential to establish accurate diagnosis.

Proper and correct labeling of malaria blood films is important to ensure that the sample and the data correspond to the patient. The integrity of the diagnosis may be compromised by unlabeled or wrongly-labeled blood films.

3. SUPPLIES AND MATERIALS a. Glass slides, 25 x 75 mm, with one frosted end for labeling, preferably with ground

edges and must be of good quality b. 70% ethyl alcohol or alcohol swabs c. Sterile lancets, one per patient d. Dry cotton e. Latex protective gloves (powder free) f. Sharps container g. Slide tray or box and cover to dry slides horizontally protected from dust and flies h. Drying rack i. Record forms (Malaria microscopy registration form) j. Lead pencil or permanent marker pen

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18 SOP 4: Collection and preparation of blood films for malaria microscopy, labelling of malaria blood films

4. SAFETY PRECAUTIONS a. Wear gloves before starting the blood collection and when handling slides to avoid

leaving behind oil on the slide that may interfere with the smear preparation b. Always use a new lancet for each patient. NEVER re-use lancets

5. PROCEDURE (1)Collection of Finger- Prick Blood and Preparation of Thick and Thin Blood Films

FLOWCHART DESCRIPTION OF ACTIVITY Illustration

Prepare a glass slide that has been pre-cleaned and the other materials needed for blood collection.

Select the 3rd or 4th finger from the thumb (or big toe for infants, not the heel; do not use thumb, for either children or adults). Hold the patient’s hand, palm facing upwards, and clean the selected finger with 70% ethanol or alcohol swab. Use firm strokes to remove dirt and oils from the ball of the finger and to stimulate blood circulation. Let the alcohol dry from the finger.

Prick the finger (or big toe) with a lancet. Use a new, sterile lancet for every patient.

Apply gentle pressure to the finger (or toe) and express the first drop of blood. Wipe the first drop of blood with dry cotton, making sure that no cotton strands remain on the finger that might stick to the blood.

Make both thick and thin blood films (for one patient) on the frosted side of the same slide.

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1. Prepare a pre-cleaneglass slide and other

materials needed.

2-3. Clean the 3rd or 4th finger from the thumb with 70% ethanol or alcohol swab. Let the finger dry.

4. Prick finger with a new, Sterile lancet.

5-6. Express and wipe first drop of blood with dry cotton.

7. Make the thick and thin blood films on the same slide.

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19 SOP 4: Collection and preparation of blood films for malaria microscopy, labelling of malaria blood films

FLOWCHART DESCRIPTION OF ACTIVITY Illustration

Apply further gentle pressure to express more blood, and collect two or three drops on the slide similar to these , about 1 cm away from the drop intended for the thin film.

Apply gentle pressure to the finger and by touching the slide to the blood, collect a single small drop of blood about this size on the middle of the slide. This is for the thin film.

Wipe the remaining blood off the finger with a clean, dry cotton.

Do not delay between applying and spreading the drops. Prepare the blood films with the slide lying on a flat surface.

- To prepare the thin film, place the edge of a clean “spreader” slide at a 45o angle in front of the blood drop intended for the thin film.

- Slowly pull the “spreader” back until it touches the drop of blood and it spreads along the edge of the “spreader”.

- Rapidly push the “spreader” forward (away from the center) in a smooth, continuous motion, until the spreader leaves the bloody part of the slide leaving a “feathery” end for the thin film.

Page 3 of 5

8. Collect 2-3 more small drops of blood, and use them to make the thick film.

9. Touch the blood with the slide to collect a small drop of blood, and use it to make the thin film.

10. Wipe clean the blood from the finger.

11. Place the slide with the blood facing up on a flat surface.

12-14. Using a clean "spreader" slide, make the thin film first by pushing forward the one drop of blood in a smooth, continuous motion.

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20 SOP 4: Collection and preparation of blood films for malaria microscopy, labelling of malaria blood films

FLOWCHART DESCRIPTION OF ACTIVITY Illustration

With the corner of the same “spreader” used for making the thin film, make the thick film by swirling the 3 drops of blood together forming a circle of about 1.2 cm diameter in size similar to this

Using a permanent marker or lead pencil, label the frosted end of the slide with the patient’s unique identifier (name/ ID number, sex, age and date of collection).

After preparation of the thin and thick blood films, allow them to air dry in a horizontal position on a slide tray. If a rapid drying is required, dry films with low heat from a hair drier for few seconds. Do not place the slides too close to the drier as the films might be heat fixed.

(2) Labeling of Malaria Blood Films

Page 4 of 5

FLOWCHART DESCRIPTION OF ACTIVITY

Label the slide immediately after the blood film preparation to avoid mix ups especially if there are more than one slides to prepare.

Verify the details against the test request form to ensure that they are correct and correspond to the sample or patient.

Using a permanent marker or lead pencil, write the following information on the frosted end of the slide: Name of patient or ID number, sex, age, date of collection Example:

15. With the corner of a “spreader, make the thick film by swirling together the 3 drops of blood to form a circle.

16. Label the slide with the patient's name/ ID, sex, age and date of collection.

17. Air dry the films in a horizontal position.

1. Label the slide immediately

2. Check the patient information on the test request form.

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3. On the frosted end, write the patient’s name/ID number, sex and age of patient and date of

collection.

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21 SOP 4: Collection and preparation of blood films for malaria microscopy, labelling of malaria blood films

6. REFERENCES Basic Malaria Microscopy. Part I. Learner’s Guide, Second Edition. 2010

WHO Malaria Microscopy Quality Assurance Manual. Version 1. 2009

7. DOCUMENT HISTORY

Date Version Comments Responsible Person

Oct 2014 1 SOP introduced and formatted NMCP

Feb 2016 2 New, separate SOP introduced; minor revision and format change; flowchart introduced; blood collection photos change

NMCP

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23 SOP 5: Staining of malaria blood films with Giemsa

National Malaria Control Program

Department of Public Health

Ministry of Health and Sports

Republic of the Union of Myanmar

Document Control No:

NMCP-MM-SOP-05

Version – 2

Giemsa Malaria Microscopy Date of issue: 15 September 2017

Title: Staining of malaria blood films with Giemsa

Total Page: 7

1. PURPOSE AND SCOPE

In order to prepare staining of malaria blood films with Giemsa, there are three steps to follow: First, prepare the buffered water to pH of 7.2 for use in the preparation of Giemsa stain solution. Second, prepare the working solution of Giemsa stain for routine staining of malaria blood film. Then, malaria blood films are staining with Giemsa stain.

2. PRINCIPLE Freshly-prepared working solution of Giemsa, made from well-prepared stock and diluted with water buffered to pH 7.2 is recommended to achieve optimal staining quality of malaria blood films. Giemsa stock solution prepared for the national programme is standardized to minimize the need for frequent adjustment of SOPs for staining.

Freshly-prepared working solution of Giemsa, made from well-prepared stock and diluted with water buffered to pH 7.2 is recommended to achieve optimal staining quality of malaria blood films. Giemsa stock solution procured for the national programme is standardized to minimize the need for frequent adjustments to staining SOPs.

A properly stained blood film is critical for malaria diagnosis especially for precise identification of malaria species. The use of Giemsa stain is the recommended and most reliable procedure for staining thick and thin blood films. Giemsa solution is composed of eosin and methylene blue (azure). The eosin component stains the parasite nucleus red, while the methylene component stains the cytoplasm blue. The thin film is fixed with methanol. Dehemoglobinization of the thick film and staining takes place at the same time during the process. An ideal pH of 7.2 is required to demonstrate stippling of the parasites to allow proper species identification.

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24 SOP 5: Staining of malaria blood films with Giemsa

3. SUPPLIES AND MATERIALS

1. Commercial phosphate buffer tablets for 1 L of water (pH 7.2) 2. Commercial distilled or deionized bottled water, 1 L 3. Graduated cylinder, 1 L capacity 4. Conical flask or beaker, 1 L capacity 5. Measuring cylinder, clean, 10ml, 50ml & 100mL capacity 6. Coplin jar for stain immersion 7. Methanol, absolute 8. Gloves, latex, disposable 9. Pasteur pipette, 3ml, Brand® 10. Drying rack 11. Staining platform or tray 12. Timer

4. SAFETY PRECAUTIONS Methanol (methyl alcohol) is highly toxic and flammable if inhaled or swallowed; it can cause blindness and even death if swallowed in any quantity. Avoid contact and inhalation. Universal safety precautions, including use of relevant Personal Protective Equipment (PPE) - gloves and laboratory coat – must be practiced.

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25 SOP 5: Staining of malaria blood films with Giemsa

5. PROCEDURE

(1) Procedure of preparation of buffer solution

FLOWCHART DESCRIPTION OF ACTIVITY

1. Measure 1 L of distilled or deionized water into a flask or beaker.

2. Add the phosphate buffer tablet.

3. Mix by gentle swirling until the tablet is completely dissolved. Should be tested with pH paper to get correct pH

4. Label the bottle of buffer solution as below:

5. Use buffered water solution with pH 7.2 for preparing working solution of Giemsa stain.

Remark: If buffer tablet is not available, deionized water with pH 7 – 7.2 can be used.

Page 3 of 7

1. Pour 1 L of distilled or de-ionized water into flask or beaker.

2. Add 1 buffer tablet.

Buffer solution pH 7.2 Date prepared: 05 Aug 2015

3. Mix solution by swirling.

4. Label the bottle of buffered water solution.

5. Use buffered water solution pH7.2 to prepare Giemsa working

solution.

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26 SOP 5: Staining of malaria blood films with Giemsa

(2) Preparation of Giemsa Working Solution

FLOWCHART DESCRIPTION OF ACTIVITY

Preparation of 10% Giemsa working solution

Preparation of fresh 10% Giemsa working solution from stock solution for rapid method of staining a few number of slides. Each slide with blood film needs approximately 3 mL of stain to cover it.

1. In a clean container or test tube, add previously prepared 1 mL buffered water, pH 7.2.

2. Using a Pasteur pipette, add 3 drops of

Giemsa stock solution previously transferred into a small container. Do NOT get this from the big bottle containing the stock to avoid contaminating it.

3. Prepare the Giemsa working solution just before staining the blood film(s), and use it within a maximum of 15 minutes after preparation.

4. Discard any excess stain.

Preparation of 3% Giemsa working solution

Preparation of fresh 3% Giemsa working solution from stock solution for slow staining of a batch of slides (20 - 100 slides).

1. In a clean measuring cylinder, add 97 mL buffered water, pH 7.2 that has been previously prepared.

2. Using measuring cylinder, measure 3 mL of

Giemsa stock solution previously transferred into a small container. Do NOT get directly from the big bottle containing the stock to avoid contaminating it.

3. Prepare the Giemsa working solution just

before staining the blood film(s), and use it within a maximum of 15 minutes after preparation.

4. Discard any excess stain.

Page 4 of 7

1. In a cylinder, add 97 mL buffered water solution.

2. Add 3 mL of Giemsa stock.

3. Use stain within 15 minutes.

4. Discard excess stain.

1. In a test tube, add 1 mL buffered water solution.

2. Add 3 drops of Giemsa stock.

3. Use stain within 15 minutes.

4. Discard excess stain.

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27 SOP 5: Staining of malaria blood films with Giemsa

(3) Staining of malaria blood films with Giemsa (For 10%)

FLOWCHART DESCRIPTION OF ACTIVITY

1. Estimate the amount of 10% Giemsa working solution needed for the number of slides to be stained.

2. Using a Pasteur pipette, fix the thin film by

dropping absolute methanol into it. Avoid contact between the thick film and methanol, to avoid accidentally fixing the thick film.

3. Place the blood film in the drying rack.

Allow the slide to air-dry. 4. Place the slides to be stained with the

blood films facing up on the staining tray or staining platform.

5. Pour the stain gently on the slides until each is covered with stain, or gently pour the stain onto the top of the slide lying face upwards on the staining rack.

6. Stain the blood films for 10 minutes (8-15 min). Set the timer for 10 minutes.

7. Gently flush the stain off the slide by adding drops of clean water until all stain are washed away. Do not pour the stain directly off the slides, or the metallic green surface scum will stick to the film.

8. Place the slide in the drying rack, film side downwards, to drain and dry. Drying of the blood films can be hastened by using low heat from a slide dryer.

Page 5 of 7

1. Prepare 10% Giemsa solution.

2. Fix the thin film with a drop of methanol on it.

3. Let the thin film dry flat on a tray.

4. Place the slides to be stained with the blood films facing up on the staining

tray.

5. Pour stain slowly on the slides until the blood films are covered.

6. Set the timer to 10 minutes (8-15 mins) for the staining.

7. Gently flush all the stain from the slides by dropping clean water over it.

8. Allow the slides to air-dry, or use a slide dryer to rapidly dry

them.

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28 SOP 5: Staining of malaria blood films with Giemsa

Staining of malaria blood films with Giemsa (For 3%)

Page 6 of 7

FLOWCHART DESCRIPTION OF ACTIVITY

1. Estimate the amount of 3% Giemsa working solution needed for the number of slides to be stained. Prepare the stain according to SOP.

2. Using a Pasteur pipette, fix the thin film by

dropping absolute methanol into it. Avoid contact between the thick film and methanol, to avoid accidentally fixing the thick film.

3. Place the blood film in the drying rack.

Allow the slide to air-dry. 4. Place the slides to be stained with the

blood films facing up on the staining tray or staining platform.

5. Pour the stain gently on the slides until each is covered with stain, or gently pour the stain onto the top of the slide lying face upwards on the staining rack.

6. Stain the blood films for 40 minutes (30-45

mins). Set the timer for 40 minutes.

7. Gently flush the stain off the slide by adding drops of clean water until all stain are washed away. Do not pour the stain directly off the slides, or the metallic green surface scum will stick to the film.

8. Place the slide in the drying rack, film side

downwards, to drain and dry. Drying of the blood films can be hastened by using low heat from a slide dryer.

1. Prepare 3% Giemsa

2. Fix the thin film with a drop of methanol on it.

8. Allow the slides to air-dry, or use a slide dryer to rapidly

dry them.

3. Let the thin film dry flat on a tray.

4. Place the slides to be stained with the blood films facing up on the staining

tray.

5. Pour stain slowly on the slides until the blood films are covered.

6. Set the timer to 40 minutes (30-45 mins) for the staining.

7. Gently flush all the stain from the slides by dropping clean water over it.

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29 SOP 5: Staining of malaria blood films with Giemsa

6. REFERENCES

Basic Malaria Microscopy. Part I. Learner’s Guide, Second Edition. 2010 WHO Malaria Microscopy Quality Assurance Manual. Version 1. 2009 7. DOCUMENT HISTORY

Date Version Comments Responsible Person

Oct 2014 1 SOP introduced and formatted NMCP

Feb 2016 2 New, separate SOP introduced;

minor revision and format change;

flowchart introduced

NMCP

Page 7 of 7

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31 SOP 6: Examining thick and thin malaria blood films

National Malaria Control ProgramDepartment of Public Health Ministry of Health and Sports

Republic of the Union of Myanmar

Document Control No: NMCP-MM-SOP-06

Version – 2

Giemsa Malaria Microscopy

Date of issue: 15 September 2017

Title: Examining thick and thin malaria blood films

Total Page: 7

1. PURPOSE AND SCOPE

This SOP describes the procedure for proper examination, detection, identification and quantification of malaria parasites in Giemsa-stained blood films under light microscopy; and correctly recording and reporting results.

2. PRINCIPLE

In the thick film, the red blood cells (RBCs) are lysed and dehemoglobinized while the malaria parasites are left intact and concentrated allowing their proper detection and identification. In the thin film, when fixed with absolute methanol, the RBCs retain their original morphology, and if malaria parasites are present, become visible inside the cells. It is critical that malaria diagnosis is based on well-prepared thick and thin malaria blood films to ensure correct speciation and accurate estimation of parasite density. Detection and identification of the Plasmodium species and stages, and estimating parasitaemia is very important in the clinical management of patient, drug efficacy studies and malaria epidemiological surveys.

3. SUPPLIES, MATERIALS AND EQUIPMENT

a. Giemsa-stained blood film b. Non-drying Immersion Oil for microscopy (Type A) c. Compound microscope with 100x objective and 10x paired oculars d. Cell counter with multiple tallies (piano type) e. Calculator, handheld f. Slide tray g. Lens paper h. Malaria registry book or results record book i. Patient result form j. Pens

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32 SOP 6: Examining thick and thin malaria blood films

4. PROCEDURE

FLOWCHART DESCRIPTION OF ACTIVITY

4.1. Examining the thick film

1. Place the Giemsa-stained blood film to

be examined on the microscope stage. Position the thick film in line with the objective lens.

2. Switch on the microscope and adjust the

light source optimally by looking through the ocular and the x10 objective. (low power)

3. Place a drop of immersion oil on the

thick film and allow it to spread. To avoid cross contamination, ensure that the immersion oil applicator never touches the slide.

4. Scan the blood film for parasites and

blood elements. Select part of the film that is well stained and has evenly distributed white blood cells.

5. Switch to x100 oil immersion objective

over the selected portion of the thick film. Raise the mechanical stage until the objective lens gently touches the immersion oil but not the slide.

6. Using fine adjustment, focus on the cell

elements and confirm that the portion of the film is acceptable for routine examination: 15-20 white blood cells per thick film field will give a satisfactory film thickness. Films with fewer white blood cells per field will require more extensive examination.

Page 2 of 7

1. Place Giemsa-stained blood smear on microscope

stage with the thick film under the objective lens.

2. Switch on the microscope and adjust light optimally.

3. Place a drop of oil immersion on the thick film.

4. Scan and select a well-stained and even portion of the blood film.

5. Switch to x100 oil immersion objective and allow the lens to touch

the oil.

6. Using the fine adjustment, focus on the blood film.

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33 SOP 6: Examining thick and thin malaria blood films

7. Examine the slide in a systematic manner.

Start at the top left end of film (marked with vertical green arrow on Figure 1 below) and begin at the periphery of the field, then move horizontally to the right, field by field.

8. When the other end of the film is reached,

move the slide a little bit downward, then to the left, field by field, and so forth (see illustration below). For efficient examination, continuously focus and refocus using the fine adjustment throughout examination of each field.

Alternatively, from the top left end of the film, move vertically downwards to the next adjacent fields. When the other end of the film is reached, move the slide to the right, then go upwards to the adjacent fields, and so forth.

Page 3 of 7

7. Start at the field on the top left end part of the film, then move the

slide to the right, field by field.

8. When the other end of the film is reached, move the slide

downward, then to the left, field by field, and so forth.

Figure 1. Examining the thick film

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34 SOP 6: Examining thick and thin malaria blood films

FLOWCHART DESCRIPTION OF ACTIVITY

4.2. Determining if a thick film is negative or positive for malaria parasites and identifying the species

1. Continue to examine the thick film

under oil immersion objective, moving from one field to the next, either horizontally or vertically, following the pattern shown in Figure 1. Use the fine adjustment for focusing.

2. A minimum of 100 fields must be

examined before the thick film is considered negative (no malaria parasites), but if time permits, the whole thick film should be scanned.

3. If parasites are observed, scan the

rest of the blood film to ensure that there is little possibility of a mixed infection being overlooked.

4. When doubtful with the parasite

species, or if mixed infections are suspected, carefully examine the thin smear under oil immersion objective. The thin film has the advantage of providing detailed parasite and RBC morphology over the thick film.

5. Identify all species present. Refer to

the WHO Bench Aids for the Diagnosis of Malaria for morphological confirmation of each species.

Page 4 of 7

1. Examine the thick film under oil immersion objective, field by field, horizontally or vertically.

2. Read a minimum of 100 fields before declaring a negative diagnosis

for malaria.

3. If parasites are found, scan the rest of the film to rule out

possible mixed infections.

4. Examine the thin film if doubtful of the species, or if mixed infection is

suspected.

5. Determine all species and stages observed.

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35 SOP 6: Examining thick and thin malaria blood films

4.3 Performing a parasite count on the thick film and calculating parasite density (parasitaemia)

1. If malaria parasites are present, count asexual forms (in either single or mixed species infections) as well as sexual (gametocyte) forms. If different species are observed, this should also be recorded.

2. Start counting in a field where

parasites and white cells are observed together. A typical field (at 100x magnification) should have approximately 12–15 white cells.

3. Using a piano type tally counter, count parasites and white cells simultaneously by clicking on the assigned key as parasites or white cells are observed.

4. After counting all the parasites and white cells in one field, move to the next field following the pattern of movement shown in Figure 1 (examining the thick film) and do the same counting procedure and so on.

5. Stop the count by following these rules:

8. Count all parasites and white cells in the final field even if the white cell count exceeds 200 or 500. Record actual number of parasites and white cells counted in an appropriate worksheet.

Page 5 of 7

If after 200 white cells, 100 or more parasites have been counted, stop the count and record the results in terms of number of parasites per 200 white cells.

If after 200 white cells, only 99 or less parasites have been counted, continue the count until 500 white cells, then stop and record the results in terms of number of parasites per 500 white cells.

1. Determine presence of malaria parasites including

stages and species.

2. Starting at top left most part of the smear, look for a typical field with

both parasites and white cells. Preparation of Giemsa stock solution

3. Click the assigned key on the tally counter for each parasite or white

cell observed.

4. After counting all the parasites and white cells in one field, move to the next field and do the same counting

procedure and so on.

5. Depending on the number of parasites observed, stop the count

after 200 or 500 white cells.

6. Count all parasites and white cells in the final field.

7. Record actual number of parasites and white cells counted.

8. Calculate the parasite density.

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36 SOP 6: Examining thick and thin malaria blood films

FLOWCHART DESCRIPTION OF ACTIVITY

Parasites µL blood =

No of parasites counted x 8000 WBCs/µL

No. of WBCs counted

When counting is completed, calculate the parasite density using an arbitrary white cell count of 8000/µL. Use the patient’s actual white cell count if available. Use the following formula in the calculation:

FLOWCHART DESCRIPTION OF ACTIVITY 4.4 Examining the thin film for confirmation of species and mixed infections

1. If doubtful of the parasite species or suspicious of mixed infections after examining the thick film, move the x100 oil immersion over the middle of the thin film.

2. Place a drop of immersion oil on the

middle of the thin film.

3. Rack the mechanical stage up until the objective lens touches the immersion oil, but not the slide.

4. Perform examination at the feathery end/edge of the thin, following the pattern of movement shown in Figure 2. Move along the edge of the film, then move the slide inwards by one field, returning in a lateral movement and so on.

5. Continue examining the thin film until the presence and species of malaria parasites have been confirmed.

Page 6 of 7

1. To confirm species and mixed infections, move the objective

over the thin film to examine it.

3. Touch the objectives on the oil and focus on the film.

2. Place a drop of oil on the center of the film.

4. Read on the thin or feathery edge of the film, moving from one field to the

next, horizontally or vertically.

Figure 2. Examining the thin

5. Scan the film until all the species have been confirmed.

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37 SOP 6: Examining thick and thin malaria blood films

6. REFERENCES Basic Malaria Microscopy. Part I. Learner’s Guide, Second Edition. 2010

WHO Malaria Microscopy Quality Assurance Manual. Version 1. 2009

7. DOCUMENT HISTORY

Date Version Comments Responsible Person

Oct 2014 1 SOP introduced and formatted NMCP

Feb 2016 2 Major revision; new/separate

SOP; flowchart introduced

NMCP

Page 7 of 7

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39 SOP 7: Recording and reporting of malaria results

National Malaria Control Program

Department of Public Health

Ministry of Health and Sports

Republic of the Union of Myanmar

Document Control No:

NMCP-MM-SOP-7

Version – 2

Giemsa Malaria Microscopy Date of issue: 15 September 2017

Title:Recording and reporting of malaria results

Total Page: 3

1. PURPOSE AND SCOPE

This SOP describes the procedure for proper documentation and correct reporting of microscopy results.

2. PRINCIPLE

The accuracy of reporting of microscopy results has an impact on the way a patient’s illness is managed by the clinician or health worker. Similarly, documentation of cases affects the reliability of epidemiological data that serves as basis for control policies. Therefore, it is imperative to ensure that laboratory reports and malaria registry are accurate and complete.

3. SUPPLIES AND MATERIALS

a. Malaria registry book or results record book b. Patient result form c. Calculator (for estimating parasite density) d. Pens

4. PROCEDURE

a. After identifying all the species present and calculating the parasite density, record results immediately on the malaria registry or results logbook, along with other relevant information about to the patient.

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40 SOP 7: Recording and reporting of malaria results

The most commonly used method of recording of findings after microscopic examination of malaria in NMCP are as follows:

b. Prepare a report for the clinician and/or patient. Report all species and stages seen and the parasite density as in the following examples.

Example 1: Plasmodium falciparum trophozoites counted = 155 White cells counted relative to parasites = 208 Using the following formula:

Parasite count: 155 x 8,000 = 5,962 parasites/µl blood

208 Report as: P. falciparum trophozoites = 5,962 p/µl blood

Example 2: P. vivax trophozoites counted = 88 White cells counted relative to parasites = 505 Actual white cell count of patient = 6500 Parasite count: 88 x 6,500 = 1,133 parasites/µl blood 505 Report as: P. vivax trophozoites = 1,133 parasites/µl blood

c. In mixed or more than one species infections, count all the species together (sexual and asexual stages) and express the results as in the example below:

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Malaria Species Results Plasmodium falciparum

- Ring only - Ring and gametocytes - Gametocyte only

Pf Pf + g Pfg

Plasmodium vivax - All stages at once

Pv

Plasmodium ovale - All stages at once

Po

Plasmodium malariae - All stages at once

Pm

Mixed infection (More than one species)

Seen (Eg. F + V)

No malaria parasite seen (After 200 thick film fields)

NMPS

Parasites per µL blood = Number of parasites counted x 8000 WBCs/µL

Number of WBCs counted

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41 SOP 7: Recording and reporting of malaria results

Example 3: P. falciparum gametocytes + P. vivax trophozoites = a total of 360 parasites (all stages) counted against 202 white cells Report as: P. falciparum gametocytes + P. vivax trophozoites = 14,257 parasites/µl blood

d. Report also the presence of the following: Gametocytes - a separate count is made of the gametocytes of P. falciparum but

when this is done, they are still included in the general parasite count. It is rarely possible to separate the gametocytes of P. vivax or P. malariae from the asexual parasites with sufficient accuracy to justify a gametocyte count.

Schizonts - since they might be an indication of disease severity.

e. If no malaria parasites are observed in the blood film, report as “Negative for malaria” or “no malaria parasites seen”.

The ‘plus system’ is an old method, which is simple but far less accurate for establishing parasite density in thick blood films. Because of its unreliability, it has been replaced by the method described above and is no longer recommended. Its use persists in places where the quantitative method cannot be used. Studies have shown that many workers forget the finer details of the system and mix up the code (the number of plus signs) and the count (the number of parasites per field or per 100 fields), resulting in unreliable information on parasite density. In this system,

+ = 1–10 parasites per 100 oil-immersion thick film fields

++ = 11–100 parasites per 100 oil-immersion thick film fields

+++ = 1–10 parasites per single oil-immersion thick film field

++++ = more than 10 parasites per single oil-immersion thick film field

5. REFERENCES Basic Malaria Microscopy. Part I. Learner’s Guide, Second Edition. 2010 WHO Malaria Microscopy Quality Assurance Manual. Version 1. 2009 6. DOCUMENT HISTORY Date Version Comments Responsible Person

Oct 2014 1 SOP introduced and formatted NMCP

Feb 2016 2 Separate SOP introduced NMCP

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43 SOP 8: Cross-checking of malaria blood films

National Malaria Control Program

Department of Public Health

Ministry of Health and Sports

Republic of the Union of Myanmar

Document Control No:

NMCP-MM-SOP-8

Version – 2

Giemsa Malaria Microscopy Date of issue: 15 September 2017

Title:Cross-checking of malaria blood films

Total Page:8

1. PURPOSE AND SCOPE

This SOP describes the procedure for regular cross-checking of malaria blood films from the provincial hospitals.

2. PRINCIPLE

Cross-checking or validation of malaria blood films plays a vital role in monitoring the accuracy of diagnosis to ensure that high quality microscopy services are provided and maintained in the hospitals.

3. SUPPLIES AND MATERIALS

a. Giemsa-stained blood films (selected form the provincial hospitals) b. Non- drying Immersion Oil for microscopy (Type A) c. Compound microscope with 100x objective and 10x paired oculars d. Cell counter with multiple tallies e. Calculator, handheld f. Slide tray g. Lens paper h. Forms

- QA Form 1 (Microscopist's Results Form) - QA Form 2 (Validator's Results Form) - QA Form 3 (Validation Report Form)

i. Pens j. Slide boxes (for transporting slides)

4. SAFETY PRECAUTIONS

Slides to be submitted must be packed and shipped according to applicable national and international standards for transporting infectious materials.

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44 SOP 8: Cross-checking of malaria blood films

5. PROCEDURE

5.1. Selection and prequalification of validator(s) a. A validator must be prequalified and must satisfy the following requirements:

Must possess a Level 1 certificate as malaria microscopist obtained from a WHO External Competency Assessment within the last 3 years

Preferably a regular staff of the Ministry of Health (MOH) and willing to accept responsibility as validator

Must have achieved the performance benchmarks for basic malaria microscopy training and/or refresher training within the past 3 years

With at least 3-5 years’ experience working as malaria microscopist b. A validator(s) must always satisfy the standard requirements of training and

assessment listed above, and participate in a WHO ECA every 3 years to maintain the validity of his/her certificate. If he/she fails the assessment, another potential validator should be recruited, assessed, and if qualified, designated.

5.2. Slide validation

a. Slide validation is done on a pre-determined number of slides initially examined by the microscopists, and sent to the validator to be re-examined in a completely independent manner.

b. All malaria blood films examined in the hospitals should NOT be discarded until

validation is done. c. Selection of slides by the hospital microscopists

For the hospitals every month, if the number of slides examined is more than 50 per month, the hospital microscopist must select ALL POSITIVE cases and 20% NEGATIVE cases randomly selected from the hospital malaria register. Alternatively, if the number of slides per month is 50 or less, all slides must be submitted for validation.

For the “elimination” areas, ALL slides examined (positive and negative) within the month must be selected.

d. Submission of slides by the microscopist

All slide submission must be accompanied with QA Form 1 (Microscopist's Results Form).

For the hospitals submission of slides will be every quarter, and must be within 2 weeks after the last month of the preceding quarter

o Example: For the first quarter of 2016 (April-June), submit all selected slides for this quarter on the first 2 weeks of July.

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45 SOP 8: Cross-checking of malaria blood films

For the “elimination” province , submission of slides is every month, and within 1 week after the end of the previous month

o Example: For June 2015, submit ALL slides examined in June on the first week of July 2015.

If the hospital microscopist did not examine any slide for the quarter or month, the microscopist must still submit QA Form 1 (Microscopist Results Form) every quarter or month.

If no blood films were examined during the quarter or month, indicate in the QA Form 1: “No blood films examined”, to differentiate from a situation where the microscopist did not submit his/her slides for validation.

e. Validation of the slides and analysis of results by the validator

The validator must record the validation results on QA Form 2 (Validator's Results Form).

For the hospitals, the validator must examine the slides and analyze the results within 2 to 4 weeks after receiving them.

For the “elimination” area, the validator must examine the slides and analyze the results within 1 to 2 weeks after receiving them.

The validator must analyse the validation results and determine the % agreement between the microscopist and the validator using the analysis tables and tool (Excel?) (Annex). The acceptable agreement must be at least 80%.

The validator must also assess and comment on the quality and staining of the thick and thin blood films submitted.

The results and findings of the validation, including recommendations, must be reported by the validator on QA Form 3 (Validation Report Form). The validator must provide feedback to the microscopy center specifying the level of accuracy, smear and staining quality including recommendations.

f. Sending of validation reports to the province and NMCP-MOHS

For the hospitals, the validator must send out the validation report (QA Form 3) to

the head or supervisor of the hospital laboratory, with a copy provided to the microscopist, within 1-2 months after receiving the slides.

For the “elimination” province, the validator must send out the validation report (QA Form 3) to the head or supervisor of the hospital laboratory, with a copy provided to the microscopist, within 2 weeks after receiving the slides.

All validation reports must be signed by the validator and head of the NMCP and NMRL before they are sent out.

All validation reports must be summarized every quarter and submitted to the NMCP-MOH and NHL.

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46 SOP 8: Cross-checking of malaria blood films

Feedback and resolution of discordant results between the microscopist and validator Discordant validation results must be discussed during supervisory visits, with the

questionable slides available for mentoring purposes. For the microscopist, if the % agreement between the validator and the microscopist is

80% or above, the validator must schedule supervisory visit at least once anytime during the year, to include discussion of discordant results.

If % agreement between the validator and the microscopist is <80% in any quarter, the validator must schedule supervisory visit within 2 months after the report, and institute corrective mentoring to the microscopist.

For the “elimination” area, any discordant validation result must be officially reported immediately to the proper authorities in the hospital and NMCP-MOHS for rapid case investigation. A supervisory visit must be scheduled immediately.

A summary of the proposed slide validation scheme is presented in Table 1. Table 1. Slide Validation Scheme

Process/ Activity

Areas reporting cases to or more than 30 slides/ month

(Control Areas)

Areas reporting of less than 30 slides/ month

( Elimination Areas)

Related Forms/

Documents

Selection of slides by the hospital microscopist

Every month, If total slides examined is >50, select

ALL POSITIVE cases 20% NEGATIVE cases

randomly selected

If total slides examined is <50, select ALL slides. Note: All malaria blood films examined should NOT be discarded until validated.

Every month, Collect ALL slides examined Note: All malaria blood films examined should NOT be discarded until validated.

Hospital microscopist will randomly select negative slides based on the hospital Malaria Register.

Submission of slides by the hospital microscopist to the NMCP or NMRL

Quarterly Submit within 2 weeks after

the last month of the preceding quarter

Example: For the 2nd quarter of 2015 (April-June), submit all selected slides for this quarter (based on above) on the first 2 weeks of July.

If no blood films were

examined during the quarter, indicate in QA Form 1: “No blood films examined”

Monthly Submit within 1 week

after the previous month

Example: For June 2015, submit ALL slides examined within the month on the first week of July.

If no blood films were

examined during the month, indicate in QA Form 1: “No blood films examined”

Hospital microscopist will accomplish and submit to the NMRL the QA Form 1 (Microscopist's Results Form), with or without slides examined/ submitted

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47 SOP 8: Cross-checking of malaria blood films

Validation/ Cross-checking of the slides by the NMCP or NMRL validator

2 to 4 weeks after receiving the slides from the hospitals

1 to 2 weeks after receiving the slides

Validator will accomplish QA Form 2 (Validator’s Results Form) and QA Form 3 (Validation Report Form)

Analysis results and preparation of reports by the NMCP or NMRL validator

Analysis:(develop a simple Excel tool for this)

% agreement between microscopist and validator

Acceptable agreement is 80%.

Analysis:(develop a simple Excel tool for this)

% agreement between microscopist and validator

Acceptable agreement is 80%.

% agreement w/o species ID = (A+D) x 100 A + B+ C + D

% agreement w/ species ID = (A+D) x 100 A + B+ C + D

Microscopist results

Validator’s resultPositive Negative

Positive A: number of slides reported as positive by both readers

B: (False Positives) number of slides reported as positive by microscopist but reported as negative by validator

Negative C: (False Negative) number of slides reported as negative by microscopist but reported as positive by validator

D: number slides reported as negatives by both readers

Microscopist results

Validator’s resultPositive Negative

Positive A: number of slides reported as Pf (single/ mono or mixed infection) by both reader

B: number of slides reported as Pf by microscopist but Pf was not reported by validator (incorrect species ID)

Negative C: number of slides reported as Pf negative by microscopist but found to be positive by validator (single/mono or mixed infection)

D: number slides reported as Pf negatives by both readers

Sending of validation reports to the S/R and NMCP-MOHS

1 to 2 months after receiving the slides

2 weeks after receiving the slides

Validator will send out QA Form 3 (Validation Report Form)

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48 SOP 8: Cross-checking of malaria blood films

Feedback and resolution of discordant results between the microscopist and validator

If % agreement is 80% or above, discuss results during scheduled supervisory visits (1-2 times per year)

If % agreement is <80% in any quarter, schedule a supervisory visit within 1-2 months after the report to discuss results and do corrective action

For every discordant result in any quarter, 2nd validator should be checked for correct result. If the result is discordant, officially notify authorities immediately and schedule a supervisory visit within 1 month of the report and do corrective action.

Validator will schedule visit and accomplish (Supervisory Visit Checklist)

For the “elimination” province, any discordant validation result must be officially reported immediately to the proper authorities in the hospital and NMCP-MOHS for rapid case investigation. A supervisory visit must be scheduled immediately or within 1 month after the report.

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49 SOP 8: Cross-checking of malaria blood films

Form 1: Malaria Blood Films for Validation

(1) Month: …………………………………. (2) Year ……………………….. (3) State/Region ………………..…...…….……

(4) Township …………………………………………… (5) Station ………………………………………………..

(6) Health Facility Name: ……………………………………………………………………………………………..

(7) Total No. of slides submitted: / / / /

(8) Total No. of Positive for the month: …………………………

(9) Total No. of slides examined for this month: ………………………

(10) Slide ID

No.

(11) Date Examined (mm/dd/yyyy)

Malaria Smear Result Remarks

(12) Species

(13) Parasites/μl blood (t,s)

(14) Parasites/μl blood (gametocytes

1 2 3 4 5 6 7 8 9

10

Examined by: ………………………………………………………. Date of Submission: ………………… Name of Examined microscopist: ……………………………………….. Noted by: ………………………………………………………………………….… Township Medical Officer: …………………………………………………..

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50 SOP 8: Cross-checking of malaria blood films

Form 2: Malaria Blood Films Report By Validator (1) Month …………………………………….. (2) Year …………………… (3) State/Region………………………….....(4) Township …………………………………………… (5) Station ………………………………………………..(6) Health Facility Name: …………………………………………………………………………………………….. (7) Total No. of blood films received: / / / (8) Date blood films examined: …………………

(9) Slide

ID No.

(10) Microscopist's results (11)Blood Films results by Validator (12) Percent agreement (13) Smear and staining Quality Assessment

(10a) Type of Species

(10b) Parasite

count (asexual)

(10c) Parasite

count (Sexual)

(11a) Species

(11b) Parasites/μl blood (t,s)

(11c) Parasites/μl

blood (gametocytes)

(12a) Accuracy of

crosschecking without species

differentiation

(12b) Accuracy of

crosschecking with species

differentiation

(13a) Smear G-good P-

poor smear

(13b) Staining G-good , U-

under , O-over stained

(13c) Remarks (Describe

smear assessment)

1 2 3 4 5 6 7 8 9

10

Total

Examined by: ……………………………………………………….

Name of Examined microscopist: ……………………………………….. Signature

Noted by: ………………………………………………………………………..…… Name of Validator ……………………………..

Township Medical Officer: ………………………………………………….. Date …………...………….…………

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51 SOP 8: Cross-checking of malaria blood films

Form 3: Summary validation report

VALIDATION SUMMARY REPORT Report prepared by: Date of report prepared: For TMO: For the attention of: SUMMARY OF RESULTS: No. of blood films received: …………… malaria blood smears Date received: Date of validation: % Agreement in parasite detection = (A+D) x 100 A+B+C+D Routine laboratory result Cross-check Positive Negative Positive A B Negative C D % Agreement in falciparum detection = (A+D)x100 A+B +C+D Routine laboratory result Cross-check Positive Negative Positive A B Negative C D Interpretation of results: Microscopist had a false positive result, with a % agreement of ……% on species detection and ……% on species identification, …% of smear assessment are in good quality. Validator’s comments and recommendation: Schedule for next validation: Validator: 6. REFERENCES Basic Malaria Microscopy. Part I. Learner’s Guide, Second Edition. 2010 WHO Malaria Microscopy Quality Assurance Manual, Version 1. 2009. WHO Malaria Microscopy Quality Assurance Manual, Version 2. 2015. 7. DOCUMENT HISTORY

Date Version Comments Responsible Person

August 2017 1 New SOP developed and

introduced

NMCP

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53 SOP 9: Instruction for laboratory aspect of infection prevention and control (IPC)

National Malaria Control Program

Department of Public Health

Ministry of Health and Sports

Republic of the Union of Myanmar

Document Control No:

NMCP-MM-SOP-9

Version – 2

Giemsa Malaria Microscopy Date of issue: 15 September 2017

Title:Instruction for Laboratory aspect of Infection

Prevention and Control

Total Page: 7

1. PURPOSE AND SCOPE

This SOP describes the instruction for laboratory aspect of infection prevention and control for routine staining of malaria blood films.

2. STANDARD PRECAUTIONS IN MEDICAL LABORATORIES

1. Hand washing 2. Housekeeping practices and general sanitation 3. Cough etiquettes 4. Use of PPE 5. Laboratory water quality monitoring 6. Biomedical waste management 7. Health checkup and screening of all laboratory staff and vaccination for Hepatitis B,

etc. 8. Use of Post-exposure prophylaxis (PEP) by health staff in case of potential exposure to

infected blood and other body fluids 9. Collaboration and communication with hospital infection control committee

3. COMPONENTS OF UNIVERSAL PRECAUTION

1. Use of protective barriers, PPE (gloves, gowns, aprons, face-mask, goggles, etc.) 2. Prevention of accidents particularly injuries by sharps 3. Proper use of disinfection and sterilization techniques to render contaminated

material, instruments and surfaces safe 4. Safe discard and disposal of contaminated waste

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54 SOP 9: Instruction for laboratory aspect of infection prevention and control (IPC)

4. BIOSAFETY

Biosafety aims to protect all those who are exposed, directly or indirectly to infectious agents while handling laboratory specimens Biosafety level of risks with organisms It depends on the basis of risks to laboratory staff, spread in the community, pathogenicity and availability of effective prophylaxis and treatment. Risk group (1) organisms Risk group (1) organisms are harmless or pose a minimal hazard to laboratory staff and community. Risk group (2) organisms Risk group (2) organisms pose moderate potential hazard for laboratory staff but limited risk for community. Effective preventive measures and treatment are available. Risk group (3) organisms Risk group (3) organisms cause serious human disease and pose serious hazards to laboratory staff. These organisms are transmitted through aerosol but do not readily spread from one infective individual to another. They are low risk for the community. Effective prophylaxis and treatment are normally available. Risk group (4) organisms Risk group (4) organisms pose severe human disease and are high risk for laboratory personnel. These organisms readily spread from one infected individual to another in the community. There is no effective treatment or prophylaxis, and maximum containment facilities during handling are required.

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55 SOP 9: Instruction for laboratory aspect of infection prevention and control (IPC)

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56 SOP 9: Instruction for laboratory aspect of infection prevention and control (IPC)

Biosafety level Biosafety level is divided into four levels depending on risk of organisms. Biosafety level (1)

- Organisms which are not known to cause disease in healthy adult humans - Conducted on open benches with no special containment equipment

Biosafety level (2) - Work involving agents of moderate potential hazard to staff and environment - Staff take universal precaution and follow good microbiological techniques - Procedure which create infectious aerosols are performed in biological safety cabinets

Biosafety level (3) - Work with agents that may cause serious or potentially lethal disease as a result

of exposure by inhalation - All procedures are conducted within biological safety cabinets by wearing protective

clothing Biosafety level (4)

- Organisms that cause life-threatening disease - Apart from level (3), include airlock entry, shower exit and special waste disposal facilities, class III biosafety safety cabinets, positive pressure suits, double-ended autoclaves and filtered air are essential safety requirement

5. LABORATORY PROCEDURES AND MODE OF TRANSMISSION OF HIV, HBV, HCV AND

OTHER BLOOD BORN AGENTS

Procedure Mode of transmission Collection of blood sample - Skin puncture by needle or broken specimen

container - Contamination of hand by blood

Transfer of specimen (within laboratory)

- Contaminated exterior of specimens container - Broken specimen container - Spill or splash of specimen

HIV serology - Skin puncture or contamination of mucous membrane

- Contaminated exterior of specimens container - Contaminated work surface - Broken specimen container - Perforated gloves

Cleaning and maintenance - Skin puncture or skin contamination - Spill or splash - Contaminated work surface

Waste disposal - Contact with contaminated waste - Puncture wounds and cuts

Shipment of specimens (to other centres)

- Broken or leaking specimen containers

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57 SOP 9: Instruction for laboratory aspect of infection prevention and control (IPC)

2. GUIDELINES FOR COLLECTION OF BLOOD SAMPLE

- Gloves should always be worn while taking blood - If blood spills on the gloves, they should be discarded - Care should be taken to avoid contamination of hands while taking blood - Hands should be washed with soaps and water immediately after any contamination with

blood and after work is completed - Laboratory gowns should be worn - Used needle and syringes should be placed in puncture resistant container, used

needles should not be recapped, nor should they be removed from syringes - Specimen containers should be sealed securely, and outside of the container should be

wiped clean of blood contamination with disinfectant. Dos and Don’ts Dos - Do keep your hair tied - Do wash your hands after completing your work - Do wear protective clothing including gloves - Do disinfect the workplace after completion of every activity as well as at the end of every

working day - Do keep your nails trimmed all the times Don’ts - Do not eat or smoke or apply cosmetics in the laboratory - Do not lick labels, but instead use gum or adhesive - Do not touch your eyes, nose or other exposed parts while working with infected material - Do not pipette by mouth - Do not wear loose clothes while in the laboratory or while engaged in handling

infectious material anywhere

3. BASIC REQUIREMENTS FOR LABORATORIES - For handling of infectious material, clearly identified working area should be provided in

the laboratory - Walls, ceilings and floor of the laboratory should be smooth, easy to clean, impermeable

and resistant to the chemicals and disinfectants normally used in the laboratory - Floors should be non-slippery - The bench tops should be impervious and resistant to disinfectants, acids, alkalis, organic

solvents and moderate heat and laboratory furniture should be sturdy and easy to clean - Wash basins should be provided in each laboratory, preferably near the exit - Doors to the laboratory should be self-closing and have vision panels and windows should

be fitted with fly screens

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58 SOP 9: Instruction for laboratory aspect of infection prevention and control (IPC)

- Autoclave for decontamination of infectious laboratory material and waste should be available in the same building

- Facilities for storing outer clothing and personnel items and space for eating and drinking should be provided outside the workroom

How to do when exposed by HIV specimens

1. Management system

- Post-exposure prophylaxis manual

2. Procedure for exposure

- Use soap and water to wash any wound or skin contact with infected blood or fluid - Flush exposed mucous membrane with water - Irrigate an open wound with sterile saline or disinfectants - Eye should be irrigated with water, saline or sterile eye irritants - Counseling and HIV antibody testing to rule out pre-existing seropositive - Use anti-retroviral therapy

3. Follow up

- HIV antibody testing is repeated 3 month, 6 month, 1 year after exposure

4. Information flow

- Responsible person / biosafety officer - Laboratory supervisor - Laboratory In-charge - Hospital infection control committee

5. Preventive measures for exposure to HIV specimens

- Use of universal precaution - Use of gloves, face mask, gowns, shoe cover and goggle (PPE) - Avoiding recapping of needle - Use of impervious needle-disposal container - Transport of samples in triple container - Availability of ART on side or easily - Training personnel for post-exposure

Decontamination of spills - Wear PPE - The affected area is covered with absorbent paper towels and flooded with an appropriate disinfectant (5-10% sodium hydrochloride or available chlorine) and leave it for 30 minutes - Other workers are warned to avoid this area - Autoclavable dustpan and forceps are used to pick up absorbent paper towels and any solid materials with gloved hands and put in a bag. This area is swabbed with fresh disinfectant - Absorbent Paper towels, dustpan and forceps are decontaminated by autoclaving

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59 SOP 9: Instruction for laboratory aspect of infection prevention and control (IPC)

4. DECONTAMINATION - Autoclave - If not available, pressure cooker should be used the highest possible pressure - Use of boiling water for 30 minutes is also effective for decontamination - Appropriate concentration of sodium hypochlorite, formaldehyde or glutaraldehyde is

desirable for disinfection - Sodium hypochlorite is used universally as a disinfectant for disinfection of blood

spillages and organic matter

5. DISPOSAL OF WASTE AND CONTAMINATED MATERIAL - Specimens, cultures, gloves, gowns, needles, syringes, used pipettes, slides, tissues,

paper towels, tissue used to wipe benches, equipment and surfaces - Different for disposables and reusable

6. DOCUMENT HISTORY

Date Version Comments Responsible Person

January 2017 1 Use instruction of infection

control from NHL

National Health Laboratory

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61 SOP 10: Medical laboratory waste management instruction

National Malaria Control Program

Department of Public Health

Ministry of Health and Sports

Republic of the Union of Myanmar

Document Control No:

NMCP-MM-SOP-10

Version - 2

Giemsa Malaria Microscopy Date of issue: 15 September 2017

Title: Medical Laboratory Waste Management

Instruction

Total Page: 3

1. PURPOSE AND SCOPE

This SOP describes the instruction for medical laboratory waste management for routine examination of malaria blood films.

2. MEDICAL LABORATORY WASTE

- Waste generated within health-care services related to medical laboratory procedures. (including those contaminated with biological agents and all sharps, whether contaminated or not)

3. CATEGORIES OF MEDICAL LABORATORY WASTE

1. Non-hazardous waste - General waste - Communal waste - Domestic waste

2. Hazardous waste - Infectious waste - Sharp waste - Chemical waste - Pathological waste

1. Non-hazardous waste Two types

- Dry- paper, leaves, plastic, can, wood - Wet- chemicals, washing solutions Sources - laboratory room, staff room, admin room

2. Hazardous waste Infectious waste

- Gloves, blood and blood products, used alcohol pads Sources - laboratory, sample collection room Sharp waste - Needles and syringes, vacutainer, blades, contaminated broken glass, blood collection tubes / bottles Sources - laboratory, sample collection room

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62 SOP 10: Medical laboratory waste management instruction

Chemical waste - Solvents, reagents, corrosive or explosive chemicals and acids Sources - laboratory room Pathological waste - Tissues, organs, body parts, cytology fluids Sources - Histopathology section, Cytopathology section

4. FIVE ELEMENTS OF MEDICAL LABORATORY WASTE MANAGEMENT

(1) Separation (2) Identification (3) Handling: - Collection

Measurement Storage Transport

(4) Treatment (5) Disposal Separation

- Laboratory waste shall not be mixed with other wastes. - They must be separated at the point of waste generation.

Identification - WHO color coding

Yellow - infectious wastes and sharps Red - highly infectious waste Black or Blue - non-hazardous (communal waste) Green - anatomical waste

Handling Handling includes collection, measurement, storage and transport.

Collection of Waste - Start immediately after laboratory procedure. Measurement - Daily output of waste should be measured to estimate future waste load.

Storage and Transport

Transport of waste from sources to interim storage. Should be completely closed system. - Interim storage area • Storing waste within the facility for final treatment of disposal. • Should not be stored beyond 48 hours.

Treatment

There are four principal medical laboratory waste treatment technologies. 1. Incineration 2. Autoclave 3. Boiling

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63 SOP 10: Medical laboratory waste management instruction

4. Chemical disinfection a. 5% Lysol solution (lycresol) b. Hypochlorite solution (0.5%, 1%, 10%).

Disposal Final disposal is done by following methods 1. Incineration 2. Digging – must be 3 meter deep and 50 meter away from water source 3. City Development Committee

5. SUMMARY OF GUIDELINES FOR MEDICAL LABORATORY WASTE

I. Sharps (needles, syringes, scalpel blades, etc.) 1. Place intact "sharps," whether contaminated or not, in a puncture resistant "sharps"

container. The containers can be purchased from the Chemistry Stores or laboratory safety equipment vendors.

2. Do not recap needles. 3. Fill 3/4 full, snap the lid closed and secure with tape. Overfilling or forced filling may

result in puncture wounds. Do Not Overfill. II. Non-Sharp Solid Waste for Autoclave

1. Collect non-sharp solid biological waste in autoclavable bags. It is preferable that autoclave bags be white or clear and without the word "biohazardous" or the universal symbol for biohazardous material.

2. Place filled bags into the autoclave pan for transport from the laboratory to the autoclave.

3. Add 250 mL of water to the bag and close loosely to allow the steam to escape and air to enter.

4. Autoclave the pan and bag at 121°C, 15 lb/cm3 for 15 minutes. 5. Allow the pan and autoclaved material to cool. 6. Put autoclaved bags into trash cans lined with heavyweight, opaque plastic bags, and

then transport them to the building dumpster. III. Non-Sharp Solid Waste for Incineration

1. Twist the plastic bag(s) at the top; bend the twisted portion to form a loop and seal with tape.

2. Seal bags individually when double bagging. 3. Place the waste material in a plastic liner inside a biohazardous waste box ("burn

box"). Double bag wet material with absorbent material in the inner bag. 4. Secure the waste box with tape.

6. DOCUMENT HISTORY

Date Version Comments Responsible Person

January

2017

1 Use instruction of waste

management system from

NHL

National Health Laboratory

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65 SOP 11: Supervisory visits to laboratories providing malaria microscopy services

National Malaria Control ProgramDepartment of Public Health Ministry of Health and Sports

Republic of the Union of Myanmar

Document Control No:NMCP-MM-SOP-11

Version – 2

Giemsa Malaria Microscopy Date of issue: 15 September 2017

Title: Supervisory Visits to Laboratories Providing

Malaria Microscopy Services

Total Page: 8

1. PURPOSE AND SCOPE

This SOP describes the procedure for conducting regular supervisory visits to provincial hospital laboratories providing malaria microscopy services. This procedure is to be modified only with approval of the National Malaria Programme Manager or supervisor/head of the National Malaria Reference Laboratory (NRL) All procedures specified herein are mandatory for all malaria microscopists working in the laboratories.

2. PRINCIPLE

A key element of any quality assurance scheme is effective supervision and monitoring to ensure that high quality services are sustained. During these visits, actual working conditions are observed and necessary corrective actions are implemented immediately. It provides an environment for dialogue between the microscopist and validator/supervisor where they can identify and discuss problems or difficulties that are encountered in their work, so that technical advice or supplementary coaching may be provided by the supervisor, where necessary. One major disadvantage of these visits is the considerable resources they entail, i.e. travel costs and time. These activities should therefore be organized and integrated into an overall plan, with funds provided for and approved by the authorities in the NMP-MOH and /or NRL. The person designated to conduct these visits must be adequately trained and have considerable expertise on malaria microscopy.

3. SUPPLIES AND MATERIALS

a. Form 1-3 (Supervisory Visit Checklist) b. Blood films with discordant results (from recent validation) c. Blood films for on-site mentoring d. Pen e. Camera (if available) f. Laboratory equipment and reagent (necessary supplies)

4. PROCEDURE a. Prepare a schedule of the supervisory visits depending on the extent of errors (%

disagreement) detected between the validator’s and microscopist’s results. Page 1 of 8

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66 SOP 11: Supervisory visits to laboratories providing malaria microscopy services

For the hospitals, if % disagreement is>20%, on-site supervision must be done within 1-2 months after the report.

If % disagreement is<20%, on-site supervision must still be done at least once a year. For the “elimination” area, any discordant result in a supervisory visit as soon as

possible to implement rapid case investigation and corrective action. Any microscopist who consistently report and submit negative cases every 6 months

and confirmed correct after validation will undergo panel testing consisting of 15 panel slides (any species, preferably of low parasite density). This is to maintain the skill of the microscopist in detecting and identifying malaria parasites.

For laboratories that did not submit slides for validation for 2 consecutive quarters, a supervisory visit must be done during the next quarter to assess the situation.

b. Communicate the schedule of the visits in advance to the microscopist and his/her supervisor to ensure their presence during the activity.

c. During the visit, observe the working conditions of the laboratory and practices of the microscopist using the standard checklist for conducting supervisory visits (Form 1: Supervisory Visit Checklist).

d. Review the malaria records (registry for logbook) for completeness and accuracy. e. Randomly select 10 at least blood films from the slide box and assess their quality and

staining. (Form 2: Accuracy of microscopic examination of blood slides) f. Do a rapid on-site cross-checking of these slides and comment on the results. g. Before leaving the laboratory, summarize and discuss important findings to the

microscopist and with his/her supervisor in a positive and non-confrontational manner. Provide a feasible solution or recommendation to observed problems, as necessary.

h. Comment also on the observed positive aspects of the workplace and the staff. i. An official report signed (Form 3: Supervisory Visit Feedback), by the

validator/supervisor, must be forwarded to the laboratory, addressed to the head/supervisor of laboratory or Township Medical Officer and copied to the microscopist, within 2 weeks after the visit.

j. Key findings from all supervisory visit reports must be summarized every 6 months and reported to the NMP-MOH.

5. REFERENCES

Basic Malaria Microscopy.Part I. Learner’s Guide, Second Edition. 2010 WHO Malaria Microscopy Quality Assurance Manual, Version 1. 2009. WHO Malaria Microscopy Quality Assurance Manual, Version 2. 2015

6. DOCUMENT HISTORY

Date Version Comments Responsible Person February 2017 1 New SOP developed and

introduced NMCP

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67 SOP 11: Supervisory visits to laboratories providing malaria microscopy services

Form – 1 CHECKLIST FOR SUPERVISION (OTSS) OF MALARIA MICROSCOPY LABORATORY STATUS

Region/ State ………………………………Township …………………………………Date:……………………………

General Hospital/Station Hospital/RHC ……………….……………………………………..……………………….

Name of staff…...Designation…………………….……………….….………

Name of Technicians

Years of experience

Training Frequency Days Last date Topic

Slides examined/day – ( ) No. Slides examined/year – ( ) Y = 1, N = 0 GENERAL CONDITION OF LABORATORY

Checklist Question Y/N Remark

1 Is the room or work place kept neat and tidy?

2 Are the equipment, chemical and utensil kept properly?

3 Is the room well ventilated with enough light?

4 Electricity

5 Water supply

6 Waste management system - properly set up?

Total

CHEMICALS FOR STAINING

Checklist Question Y/N Remark 1 Is the staining solution (working solution) used within one

hour?

2 Are the solution and chemical bottles kept in cool, dry place and away from sunlight?

3 Are the staining solution and chemicals enough in stock? 4 Is the stock Giemsa stain bottle properly closed when it is

not in use (Screw tight)?

5 Do you use (10%) Giemsa staining solution? 6 Is working solution prepared properly?

Total

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68 SOP 11: Supervisory visits to laboratories providing malaria microscopy services

MICROSCOPIC SLIDES

Checklist Question Y/N Remark 1 Are the glass slides cleaned before use? 2 Are the glass slides well packed and kept properly before

use?

3 Are the steps performed in taking blood of patients correct? 4 Are the steps performed in making blood films on the slides

correct?

Total

SAMPLE PREPARATION AND EXAMINATION

Checklist Question Y/N Remark 1 Do you prepare both thick and thin smears from one

patient?

2 Do you prepare both thick and thin smears on one glass slide?

3 Do you observe (200) fields in thick smear before giving the result?

4 Do you report result from one patient within (45) minutes? 5 Do you report the MP result in species? 6 Do you report MP result in parasite’s stage? 7 Do you report the parasites count in the result? 8 Do you send slides to Region/State VBDC or Central VBDC

(or any other facilities) for CROSS-CHECKING?

Total

RDT

Checklist Question Y/N Remark 1 Do you examine RDT along with Microscopic examination? 2 If YES, who request for RDT examination? (Medical Doctor-

1, Nurse-2, Lab tech-3, Microscopist –4,Patient -5, Other -6)(Tick according to the answer)

1 2 3 4 5 6

3 Brand of RDT

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69 SOP 11: Supervisory visits to laboratories providing malaria microscopy services

STORAGE AND MAINTENANCE OF EQUIPMENT AND REAGENTS

Checklist Question Y/N Remark1 What do you use to wipe out immersion oil on objective

lens? (Cloth-1; No need to wipe because we use Anisole-2; Others -3)

1 2 3

2 Do you cover the microscope with Plastic Dust Cover when the lab is closed (especially at night, weekend, national holidays)

3 Do you put the microscope back in the box or cabinet when the lab is closed (especially at night, weekend, national holidays)

4 Do you use special method /items to avoid mold? 5 Do you have special container, box or shelf to store

glassware?

6 Do you have special container, box or shelf to store reagents?

7 Who manages the Key of the lab? Total

SUPPLY SYSTEM

Checklist Question Y/N Remark 1 Do you have a file for stock in and out? 2 Do you have stock request form/ indent form?

TotalITEMS Source & Frequency of SUPPLIES

VBDC CMSD NHL OTHERS HOW OFTEN Remark Lancets Glass Slides Giemsa stain Immersion Oil Methanol RDT

REPORTING SYSTEM

Checklist Question Y/N Remark 1 Do you use malaria microscopy? How many blood smears

did you examine in last year? ……………….

slides 2 Do you have a format of reporting form to fill the result

(on Malaria Microscopy)?

3 Do you report malaria microscopy data? Where and when do you report your routine malaria slides examination?

……………………. ……………........

4 Do you report parasite species with staging and parasite count?

Total

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70 SOP 11: Supervisory visits to laboratories providing malaria microscopy services

General findings and recommendations

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Supervisor / Validator’s comments:

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71 SOP 11: Supervisory visits to laboratories providing malaria microscopy services

FORM -2 ACCURACY OF MICROSCOPIC EXAMINATION OF BLOOD SLIDES (Checking with Standard Slide Set during Supervision (OTSS) Visit)

Region/State …………………….……Township ……………….…………………Date………………..………………… VBDC/General Hospital/Township Hospital/ Station Hospital/RHC …………………………………………………………………………………………………….………………………………………… Name of staff…………………………………..… Designation……………………………………………………………… Sr Slides

No Parasite Species

No of asexual

stages Count

Correct Species

Correct Count

Scoring Remark

1 2 3 4 5 6 7 8 9 10

MICROSCOPE

Sr Microscope Brand Model Bi/Monocular Light/Electric Function +/- 1 2 3 4

PARTS OF MICROSCOPE

Parts EXD: Microscope Brand/Model Any defect identified Repaired during visitEye Pieces Objectives Condenser Mirror Light Source Signature of Supervisor……………………………….. Name of Supervisor......................................... Designation …………………..................................

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72 SOP 11: Supervisory visits to laboratories providing malaria microscopy services

FORM – 3 FEEDBACK FOR SUPERVISION ASSESSMENT (OTSS) OF LABORATORY (For Feedback to the TMO or concerned authority immediately after supervision)

Region/State…………………………Township………………………………Date of Supervision…………………

Place of supervision - VBDC/ General Hospital/Township Hospital/ Station Hospital/RHC

..............…………………………………………………………………………………………………………………………………

Name of laboratory in-charge ……………………………………..Designation………………………………………

CHECKLIST MAX SCORE

RESULT SCORE

REMARKS

GENERAL CONDITION OF LABORATORY 6 CHEMICALS FOR STAINING 6 MICROSCOPIC SLIDES 4 SAMPLE PREPARATION AND EXAMINATION 8 MICROSCOPE FUNCTIONING 1 STORAGE AND MAINTENANCE OF EQUIPMENT AND REAGENTS

5

SUPPLY SYSTEM 2 REPORTING 4

TOTAL 36 A Score on observation of laboratory = ……A…………

% of scoring result of laboratory = ……A…/36 = …………….

No

Name

Designation

No of Slide

True(+) ve

False(+) ve

False (-)ve

True(-)ve

Malaria

knowledge (Theory)

% of

Agreement result

Result of Proficiency testing = ……………….. (% of agreement result formula= TP+TN/Total slides) General findings and recommendations

--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Signature of Supervisor………………………………….. Name of Supervisor………………………………..……… Designation.......................................................

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