KAN-TN-LM 02 - KAN Requirement for Accreditation of Clinical Microbiology

KAN-TN-LM 02

KAN TECHNICAL NOTES ON
MEDICAL LABORATORY IN
THE FIELD OF CLINICAL
MICROBIOLOGY

Issue Number: 3
April 2016

Komite Akreditasi Nasional
National Accreditation Body of Indonesia
Gedung I BPPT Lt. 14
JI. M.H. Thamrin No. 8, Kebon Sirih, Jakarta 10340 - Indonesia
Tel.
: 62 21 3927422
Fax.
: 62 21 3927528
Email
Website

: laboratorium@bsn.go.id ; labmedik kan@yahoo.com
: www.kan .or.id

VKAN
Issue Number: 3

KAN-TN-LM 02

7 April 2016

APPROVAL SHEET

Reviewed by

ｾ

Ｇ＠

Quality Manager of National Accreditation Committee
of Indonesia (KAN)

Approved by :

ｾ Ｇ＠
Director of National Accreditation Committee
of Indonesia (KAN)

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LIST OF AMENDMENT

No.

1

Date

Part Number
Revised

January 2008 All

2

7 April 2016

Identification
document
KAN-R-LM
02

3

7 April 2016

Clause 1

4

7 April 2016

All

5

7 April 2016

Clause 2

6

7 April 2016

Clause 3 a iii

7

7 April 2016

Clause 3

8
9

7 April 2016
7 April 2016

Clause 5 d
Clause 5 k ii

10

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Clause 61

11

7 April 2016

Clause 6 m

Part
Revision
Number

Brief Description of Changes
SR.LM.KAN 002 was revised as a whole
(except introduction)

1

Identification document changed to KANTN-LM 02

2

Insert the term "safety" including "biosafety
and biosecurity"
Replace "Analyst" into "Technician"
- Add " ... certificates of training .."
- Delete " ... health analysis vocation .."
- Add "d v Appropriate numbers of
laboratory
technician (1 laboratory
technician handles for 1
15
specimens)"
Restructure e until j into e by adding
"biosafety and biosecuritv"
Replace " .. . resistant.. " into " ... clean, dry .. "
- Restructure 3 a iv and 3 a v into "3 a iv
Design laboratory is complied with a
regulation on Health Minister Decree

(KMK No. 1244/Xll/1994)."
- Add b
- Restructure 3 c and 3 d into 3 c and
replace the term "chamber'' into
"bioloQical safety cabinet"
Add " ....and calibrated routinely."
Delete " ... .when aooropriate. "
Replace "Clinical microbiology, standards
and reference materials and cultures ... "
into "Reference cultures (stock cultures,
lyophilized) ... "
Delete "Clinical microbiology .." and " .. . of
clean media."
Replace "areas" into "room "

-

1
3

1

1

1

1
1
1

1
2

ii

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KAN TECHNICAL NOTES ON MEDICAL LABO RA TORY IN THE FIELD OF
CLINICAL MICROBIOLOGY

1

INTRODUCTION

a

This technical notes are an interpretation of the general requirements of
SNI ISO 15189:2012.

b

This accreditation technical notes are applicable to clinical microbiology
examination. Clinical microbiology is concerned with the investigation,

diagnosis and, in an advisory capacity, management of infection caused
by microbes (viruses, bacteria, parasites and fungi).

c

These examinations also include procedures for determining, measuring
or otherwise describing the presence or absence of various substances or
micro-organisms. Facilities which only collect or prepare specimens, or
act as a mailing or distribution centre, are not considered to be medical or
clinical laboratories, although they may be part of a larger laboratory
network or system.

d

This accreditation technical notes are applicable to collection , transport of
a clinical specimen for examination or processing, quality control, quality
assurance and requirements for safety (biosafety and biosecurity) in the
laboratory of clinical microbiology.

e

Clinical microbiology are examination of microbes, which are unicellular or
cell-cluster microscopic organisms. That are taken to include detection,
isolation , enumeration , identification of pathogens and antimicrobial drug
assays.

2

PERSONNEL

a

The Laboratory technician has been working in clinical microbiology areas
at least two years with certificates on training of microbiology techniques.

b

Technical manager, laboratory supervisor and laboratory technician shall
possess a basic education in biological analysis, or a related science.

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c

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The laboratory shall be directed by person who has executive responsible

& competence to assume responsibility for the services of clinical
microbiology test.
d

Laboratory management shall ensure that:
Appropriate numbers of laboratory personnel,
education & training, to meet the

with the required

demands of the service to

customers.
ii

Laboratory personnel have the knowledge, skills, and abilities based
on education, experience, demonstrated skills, and training to perform
their duties.

iii

Laboratory personnel had trained in handling pathogenic agents,
biosafety precautions, procedures for sample collection and handling,
media preparation, sterilization and data handling.

iv

Full educational and professional records of all technical staffs
available to confirm their competence in clinical microbiology testing.

v

Appropriate numbers of laboratory technician (1 laboratory technician
handles for 1 - 15 specimens).

e

The training procedure is applicable to new employees, for the introduction
of new procedures, methods, biosafety and biosecurity, for retraining of
employees, and for re-verification of employee performance.

3

ACCOMMODATION AND ENVIRONMENTAL CONDITIONS

a

The laboratories are designed to:
Provide space, engineering controls, and proper environmental
conditions for optimal sample storage, sample handling, and analyses,
in accordance with microbiology laboratory practices.
ii

Minimize areas with cracks or fibers that could serve to accumulate
debris and serve as an area for growth of microorganisms.

iii

Floors in the laboratories are constructed from a material that is clean ,
dry and easily disinfected.

iv

Design laboratory is complied with a regulation on Health Minister
Decree (KMK No. 1244/Xll/1994).

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b

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A suitable environment is complied with a regulation on Health Minister
Decree

(KMK

No.

1244/MENKES/SK/Xll/1994

and

KMK

No.

1204/MENKES/SK/X/2004) for the tasks carried out clinical microbiology
testing. Laboratory shall have:
Distinct space, in line with the biosafety requirements, for performing
clinical microbiology testing.
ii

Facilities that meet the requ ired environmental conditions, including
any needed separation of work areas to ensure that analyses will not
be adversely affected within resources provided.

iii

Good ventilation is necessary for the protection of the personal from
airborne infectious nuclei. The best way to ensure ventilation is by
use of windows and doors, ensuring that expelled air flow to the
outside environment. To ensure proper ventilation in closed-off rooms
an extraction fan will be required .

iv

Written policies for handling spills of contaminated materials.

v

Laboratory storage areas provide proper storage of samples,
reagents, microbiological media, select agents, standards and
reference materials, and hazardous waste procedures and facilities in
place for collection , storage and disposal of biohazard waste.

vi

Laboratory monitored and documented the air quality of work places
where clean operation is expected (if there are any related
references on air quality of microbiology places, they can be
inserted).

c

Laboratory shall have separate location provided for:
sample receiving
ii

sample collection

iii

sample/specimens preparation

iv

sample examination (manipulation of pathogens relevant to their
hazard level and appropriate biological safety cabinet for handling
bacteria that highly contagious by air-borne routes. Special designs
are required for mycobacteria, mycology, and virology laboratory)

v

media preparation

vi

cleaning of lab-wares, decontamination and sterilization

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vii

molecular

microbiology

(if

laboratory

7 April 2016

has

a

biomolecular

examination).

4

TEST METHOD AND METHOD VALIDATION

a

The test method are based on the valid method or reference method and
adequately documented, including :
title and method number,

b

ii

scope and field of application,

iii

number of revision,

iv

page numbering, total number of pages,

v

references,

vi

principles and definitions,

vii

reagents and materials,

viii

apparatus,

ix

analytical methodology,

x

expression of results,

xi

performance criteria,

xii

storage and shelf-life,

xi ii

procedures for media preparation ,

xiv

procedures for maintenance of stock cultures,

xv

procedures of safety for media handling,

xvi

quality control ,

xvii

colony counting,

xviii

reporting criteria,

xix

issuing authority.

The microbiological test method number and title used in the laboratory
shall the same as the scope of testing whe n carry out assessment for
accreditation.

c

The validation of microbiological test methods should reflect actual test
conditions. This may be achieved by using naturally contaminated products
or products spiked with a predeterm ined level of contaminating organisms.

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d

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The validation of microbiological test methods needs for non-standard
methods and laboratory-developed methods. This activity is planned and
assigned to qualified personnel.

e

The method's performance characteristics are based on the intended use
of the method.

Method that used for qualitative microbiological test

analysis there does no need validate the method's linearity over the full
dynamic range of the equipment.
f

Qualitative microbiological test methods, when the result is expressed in
terms of detected I not detected and confirmation and identification
procedures, should be validated by determining, if appropriate, the
specificity, relative trueness, positive deviation , negative deviation , limit of
detection , matrix effect, repeatability and reproducibility

g

Quantitative microbiological test methods, the specificity, sensitivity,
relative trueness, positive deviation, negative deviation , repeatability,
reproducibility and the limit of determination within a defined variability
should be considered and, if necessary, quantitatively determined in
assays.

h

Determination of microbiological test methods performance characteristics
for the validation of non-standard and laboratory developed methods
recommended following:
Meet method system suitability requirements, if applicable. The

suitability of the method is checked and confirmed by comparing with
requirements typical for the intended use of the method. Such as, a
filtration method for a non-filterable food, surface tests for Colony
Forming Units /square area where CFU/gram is needed.
ii

Include un-inoculated medium control to assess contamination from

the laboratory. This control is considered a blank and is to exhibit no
growth .
iii

Prepare

and analyze positive

and negative culture

controls.

Negatives control is a typically negative or no growth and the positive
control is positive or shows microbial growth.
Spike positive culture control is prepared and analyzed. This assesses the

matrix effects as well as the sensitivity of the method. It is recommended
that a 25 gram sample be spiked with inoculums of 30 cells or less.
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Evaluate interferences. This assesses the selectivity and specificity of the

method.
k

Laboratory shall have:
ii

Written procedures for the proper collection and handling of sample
and organism including personal safety and spill handling.

iii

Preparation procedures, protocols and quality control programs for
in-house media.

iiii

Procedures and records of media preparation.

Method is validated or verified in laboratory thorough :
Qualitative tests are based upon performance history and media QC.
ii

Repeatability - estimated using a minimum of 10 replicates of a
known positive sample, or duplicate data collected over a period of
time.

m

iii

Estimation of measurement uncertainty.

iv

Successfully participate in proficiency testing.

Quality control programs for in-house media
Physical appearance.

n

ii

pH checking.

iii

Sterility results after incubation.

iv

Performance checks using positive and negative control organisms.

Supporting work instructions shall available to:
work instructions for sample disposal,
ii

work instructions

for

disinfection I sterilization and disposal of

biohazardous material,
iii

work instructions for glassware cleaning and sterilization ,

iv

work instructions for equipment instruction manuals ,

v

work instructions for computer software related data entry and
approval.

5

EQUIPMENTS AND MEASUREMENT TRACEABILITY

a

Laboratory shall have:
List of the major equipment including specification, date received ,
manufacturer, price, and maintenance service.
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ii

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Procedure specifies the schedule and requirements for maintenance,
performance,

calibration,

and verification of laboratory testing

equipment that meet the criteria of the maintenance and calibration
parameters needed to achieve the accuracy of instruments used for
analytical testing.
iii

Equipment records containing description of the instrument, critical
accessories and software, manufacturer's name, type identification
and serial number; laboratory number; installation qualification (IQ)
and operational qualification (OQ) records obtained from the installer
or manufacturer; and other related material such as instrument
service and repair, warranty information , service contract conditions
and specifications.

iv

Laboratory shall have operating instructions for each instrument,
including starting and shutting down the instrument.

v

pH meter at least available two buffer to cover the range of pH
needed in the tests methods for determination of pH standards,
buffers available within their expire dates.

vi

Probes of pH meter maintained with cap on and/or in buffer solution
or distilled water.

vii

Biosafety cabinet shall have documented protocol and record of
decontamination and calibration (complied with ISO 15190 page 16).

viii

Program established to check regularly the rate of airflow and particle
count in the Biohazard hoods or lam inar flow cabinets (complied with
ISO 15190 page 16).

b

The effectiveness of the autoclave operation shall checked monthly with a
biological indicator and thermal indicator.

c

Thermometers and or thermocouples used for checking temperature of
incubators, water baths

refrigerators, and freezers calibrated against a

reference thermometer by calibration laboratory and traceable to a national
standard.
d

Automatic pipettes is there an initial verification of volume delivered
checked and calibrated routinely.

e

There are regular checks to ensure that the equipments is performing
within the specifications.
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f

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Hygrometer shall calibrated by calibration laboratory and traceable to a
national standard, when humidity is influence to the outcome of the test.

g

Equipment that is not operating properly is clearly marked to show that it is
out of service.

h

When an instrument is discovered to be improperly operating, it is tagged
and taken out of service. Equipment is not returned to service until
performance

checks

and

verification

have

been

performed

and

documented.
Each instrument has an established schedule specifying performance
checks, including the testing frequency and acceptable performance
specifications. These performance checks ensure the instrument is
operating properly and consistently prior to analysis.
All instruments required for the routine test in clinical microbiology field are
available:
Bunsen burners or other flame source functioning properly.
ii Colony counter for pour plate and spread plate method functioning
properly.
iii Incubators functioning

properly, maintained within

the

specified

temperature range, temperatures monitored and recorded and spatial
variability in incubators checked periodically.
iv Water baths functioning properly, maintained within the specified
temperature range; temperatures monitored and recorded periodically.
v Working thermometers are traceable to national standards.
vi Microscopes covered with dust cover when not in use.
vii Optical surfaces of microscopes cleaned after each use.
viii Autoclave

is

functioning

properly,

monthly

test

of

autoclave

performance using a spore strip or spore suspension of Bacillus
stearothermophilus, (that are capable of demonstrating a 6 log kill, of
Bacillus stearothermophilus).

ix Regularly

scheduled

maintenance

program

for

each

piece

of

equipment, where appropriate and records of service where service
was required.

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x Distilled water that producing in-house and or purchasing from external
supplier and used to make media or reagents in clinical microbiology
tests shall check the conductivity periodically.
xi If total heavy metals may influenced the results of tests method for
specific clinical microbiology test, distilled water that producing inhouse and or purchasing from external supplier and used to make
media or reagents in clinical microbiology tests shall be check the total
heavy metal periodically.
k

Laboratory shall have arrangement for avoidance of cross-contamination
arising from equipment, such as:
Disposable equipment shall be clean and sterile.
ii

Re-used glassware should be properly cleaned and sterilized.

iii

Laboratories should have a separate autoclave for sterilization.
However, one autoclave is acceptable provided that adequate
precautions are taken to separate decontamination and sterilization
loads, and a documented cleaning programmed is in place to
address both the internal and external environment of the autoclave.

Laboratories should carry out initial verification of volumetric equipment
such as automatic dispensers,

dispenser/diluters, mechanical hand

pipettes and disposable pipettes and then make regular checks to ensure
that the equipment is performing within the required specification.
m

Verification should not be necessary for class A glassware which has been
certified to a specific tolerance.

n

Equipment should be calibrated at least once a year for the accuracy of
the delivered volume against the set volume (for several different settings
in the case of variable volume instruments) and the precision of the repeat
deliveries should be measured.

o

Where centrifuges are used in test procedures, an assessment should be
made of the criticality of the centrifugal force. Where it is critical, the
centrifuge will require calibration.

p

Conductivity meters, pH meters and other similar instruments should be
verified regularly or before each use.

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REAGENTS AND REFERENCE CUL TURES

a

Laboratory shall have sufficient & appropriate quantities of reagents /media
I commercial kits available to carry out the volume of work following the

required test methods.
b

Raw materials (dehydrated formulations and individual constituents) shall
be stored under appropriate conditions, e.g. cool, dry and dark.

c

All containers, especially those for dehydrated media, should be sealed
tightly and suitable for each material.

d

Dehydrated media that are caked or cracked or show a color change
should not be used.

e

Shelf life of prepared media under defined storage conditions shall be
determined and verified.

f

Stored reagents, reference materials and supplies shall be under the
appropriate conditions and in a secure manner to ensure the separation of
incompatible materials.

g

Solutions of reagent, standard and any other such as mobile phase
properly labeled with solution name, concentration , date of preparation ,
expiration date, and identity of person who prepared.

h

Disposed of reagents and standards solutions appropriately followed to
National and or local regulation.
Laboratory shall have documented verification or comparison of standards
and/or reference materials before use and the protocol when nonconforming results are encountered
Biochemical kit and rapid test manufacturers may specify control cultures
for use with their products. If not specified , quality control procedures for
biochemical tests and test media should include cultures that will
demonstrate pertinent characteristics of the product.

k

Distilled or de-ionized water systems are monitored for conductivity,
bacterial content, and total chlorine periodically. Heavy metals analysis,
water quality test and use test are performed annually on the water
systems.
Reference cultures (stock cultures, lyophilized) are stored separately from
samples.
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m

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Media preparation and sterilization room are separated from work room to
prevent contamination.

n

Laboratories shall ensure that all reagents including stock solutions, media,
diluents, and other suspending flu ids are adequately labeled to indicate, as
appropriate, identity, concentration , storage conditions, preparation date,
and validated expiry date and I or recommended storage periods.

o

Reference cultures and reference material are requ ired for establishing
acceptable performance of media including test kits, for validating methods
and for assessing I evaluating on-going performance.
Traceability is necessary, when establishing media performance for
test kit and method validations.
ii

To demonstrate traceability, laboratories must use reference strains
of microorganisms obtained directly from a recognized national or
international collection , where these exist.

iii

Commercial derivatives of reference strains for which all relevant
properties have been shown by the laboratory to be equivalent at the
point of use may be used as alternatively.

iv

Reference strains may be sub-cultured once to provide reference
stocks. Purity and biochemical checks should be made in parallel as
appropriate.

v

It is recommended to store reference stocks in aliquots either deepfrozen or lyoph ilized.

vi

Working cultures for routine use should be primary subcultures from
the reference stock. If reference stocks have been thawed, they must
not be re-frozen and re-used.

vii

Working stocks should not be sub-cultured unless it is required and
defined

by a standard

method

or laboratories

can

provide

documentary evidence that there has been no change in any relevant
property.
viii

Working stocks shall not be sub-cultu red to replace reference stocks.
Commercial derivatives of reference strains may only be used as
working cu ltures.

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ASSURING THE QUALITY OF TEST RESULTS

a

Laboratory quality control is an essential aspect of ensuring that data
released is fit for the purpose determined by the quality objectives.

b

Approach of quality control is the principal recourse available for ensuring
that only qualified data is released.

c

The principle of the laboratory quality control program is its internal quality
control to monitoring of analytical performance, and its external quality
control based on the laboratory's performance in proficiency testing
programs.

d

Laboratory management is responsible for establishing a laboratory quality
control program and ensures that quality control is performed and reviewed
of quality control data for acceptability.

e

Technicians are responsible for conducting quality control analyses in
accordance with the laboratory quality control program.

f

Internal quality control is used to measure accuracy, contamination , and
matrix effects. The laboratory determines, where feasible, the accuracy of
all analyses performed.

g

Accuracy and precision control charts are used to determine if the
measurement system process is in control and whether the results
generated by the measurement system are acceptable.

h

Quality control program for media shall including:
pH of medium checked before autoclaving.
ii

Positive control culture to reference culture performed per batch of
media.

iii

Negative control culture to reference culture performed per batch of
media.

iv
v

Checked of sterility per batch of media.
Media that used for quantitative methods, quality control program
including compare positive control cultures on selective media and
non-selective media once per batch of media.

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BIBLIOGRAPHY

Centers for Disease Control and Prevention and National Institutes of
Health). BioSafety in Microbiological and Biomedical Laboratories4

th

ed.

U.S. Government Printing Office, Wash ington , D.C. 1999.
2

Usefulness of EC4 Essential Criteria for Quality Systems of Medical
Laboratories as Guideline to the ISO 15189 and ISO 17025 Documents,
Clin Chem Lab Med 2000; 38(10):1057-1064 © 2000 by Walter de
Gruyter.

3

Lennette EH , Balows A, Hausler WJ, Shadomy HJ. Manual of clinical
microbiology. Washington : Am. Soc. Microbial, 1985.

4

Eurochem/EA Guide 04/10. Accreditation for Microbiological Laboratories.

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