Quality control in laboratory

1. Quality control
DR. ZAKIR HOSSAIN HABIB
PRINCIPAL SCIENTIFIC OFFICER (PSO)
HEAD, MICROBIOLOGY DEPARTMENT
INSTITUTE OF EPIDEMIOLOGY, DISEASE CONTROL AND RESEARCH (IEDCR),
BANGLADESH
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2. Quality assurance
QA includes (but is not limited to) :
-monitoring
-evaluating
-taking corrective actions
-record keeping
-calibration and maintenance of equipment
-proficiency testing
-training
-competency assessment
-Quality control
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3. Principle of Quality control
 QC programs ensure that information generated by a laboratory is
accurate, reliable, and reproducible.
 This is accomplished by
-assessing the quality of the specimens
-monitoring the performance of test procedures, reagents, media,
instruments, and personnel
- reviewing test results
-documenting the validity of the test methods.
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4. Requiremens
Documents:
 SOPs for all Microbiology laboratory works
 Quality manual
 Record keeping register
 Reference strains for Quality control (QC):
Others:
 Quality ensured Microbiological equipments, media and reagents
 20% Glycerol broth for storage of control strains
 Nutrient agar /Sheep Blood agar plates for working culture growth of control strains
 pH meter
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5. Reference strains for Quality control (QC):
Sl. no Organism ATCC
strain
1 Staph. aureus 25923
2 Salmonella enteric sv
typhimurium
14028
3 Pseudomonas aeruginosa 9027
4 Pseudomonas aeruginosa 27853
5 Enterococcus faecalis 29212
6 Shigella flexneri sv 2b Gp B 12022
7 Escherichia coli 25922
8 Escherichia coli 35218
9 Klebsiella Pneumoniae 700603
10 Streptococcus pneumoniae 6305
11 Streptoccus agalactiae 12386
12 Proteus mirabilis 25933
13 Vibrio parahaemolyticus 17802
14 Acinetobacter lwoffii 17925
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8. IQC of reagents used in Gram’s staining
❑ Prepare all reagents for Gram’s staining following appropriate SOPs.
❑ Check all reagents in every batch soon after preparation and on every month(?)
with Gram-positive and Gram-negative QC strains to make sure that the stains
work properly to provide intended results.
❑ Keep records of the QC results
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9. IQC of bacteriological culture medium
❑ Quality check on dehydrated media: check for-
-Appropriate label of the medium
-Free-flowing granularity
-Date of expiry of the dehydrated medium base.
❑ Check all media plates for-
-Rrecommended pH
-Surface being parallel with base of the plate, and
-Surface free from excess dry, moisture & contamination.
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10. IQC of bacteriological culture medium
❑ Keep always an un inoculated plate of a medium (not containing antimicrobials) in
each batch for overnight incubation at 37ºC for sterility checking prior to inoculation
❑ Inoculate with the recommended control strains, incubate properly and check for
recommended growth characteristics for optimum performance of the media.
❑ Keep records of the quality check for the media.
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11. Media
Commercially prepared media (exempt from QC by the user)
User-prepared or commercially prepared media requiring QC testing-
-Test each new numbered lot of dehydrated medium, reagent, or ingredient in
parallel with an approved lot before it is released for use.
-Maintain a record of the amount of medium prepared, source, lot number,
sterilization method, preparation date, pH, date of initial use, expiration date,
and name of preparer.
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12. Media
❑ Check for contamination by incubating a 5% sample for batches of 100 or less or 10
randomly selected units from larger batches.
❑ Incubate the medium for 48 h at the temperature at which it will be used and then for
an additional 48 h at room temperature.
❑ QC organisms should be derived from well-characterized strains.
❑ The QC method should conform to CLSI document M22-A3 recommendations.
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13. IQC of antibiotic disks for disk diffusion method of ABST
Check antibiotic disks for
 name of the antibiotic
 content of the antibiotic
 date of expiry
 recommended storage conditions
Inoculate over MHA with recommended control strain(s) following SOP for
antibiotic susceptibility testing (ABST) by disk diffusion method,
place antibiotic disk(s) to be tested and incubate properly
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14. IQC of antibiotic disks for disk diffusion method of ABST
 Observe inhibitory zone diameter of the test antibiotic disk with the control
strain and check for acceptable limits as recommended by CLSI
 NOTES:
 Check each batch or new lot of antibiotic disk with reference strains to see
IZDs within acceptable CLSI limits.
 Reject a batch of antibiotic disk for which the IZDs is not within acceptable
CLSI limits.
 Keep records of quality check of the antibiotic disks .
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15. IQC of bacteriological reagents
❑ Ensure proper sterilization method recommended for the reagent
❑ Ensure proper labeling with date of preparation and recommended storage
condition
❑ Check for fluidity of the reagent and visible contamination (hazes).
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16. Stains, reagents, and antisera:
Antisera are tested according to the manufacturer's recommendations in parallel with the current lot
before use and every 6 months thereafter.
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17. IQC of bacteriological equipment performance:
 Ensure safe and proper power source for the equipment
 Keep an ID slip with each equipment including following information:
-Name of the instrument
-Stock ledger no
-Manufacturer
-Date of installation
-Date of last PT check
-Next date of PT check
-Person responsible for emergency troubleshoot:
Name:
Cell phone no.
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18. IQC of bacteriological equipment performance:
 Equipments with thermostat for required temperature (e.g. refrigerator, incubator
etc)
-Ensure temperature showcase (meter) fixed at required temperature
-Fix an extra internal thermometer to show internal temperature
-Keep an everyday log of the temperature shown by the equipment, properly
signed by assigned officer
 Equipments for measuring volume of fluids (e.g. pipettes)
 Equipments for measuring weight of reagents (e.g. balance)
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19. A. Instrument and equipment performance
1. Perform preventive maintenance at least as frequently as recommended
by the manufacturer. See manufacturer's instructions.
2. The manufacturer, an independent service company, or the in-house
biomedical engineering service can perform preventive maintenance.
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20. General Quality Control Parameters
A. Specimen collection and transport
1. Include screening tests (e.g., quantitation of WBCs and squamous epithelial cells in
sputum) among the criteria for acceptable specimens.
2. Do not test unacceptable specimens unless it is impossible to re-collect specimens.
When unacceptable specimens must be processed, include a disclaimer in the patient report
indicating that the specimen was not acceptable but was processed at the specific request of
the physician, whose name has been recorded in the test report.
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21. B. Procedure manual
The CAP requires that laboratories maintain a copy of CLSI document GP2-
A4 and that procedures be written in a format largely consistent with
those guidelines.
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22. Patient reports
1. The requester of the test should be notified immediately of any life-threatening
situations indicated by test results (panic values).
2. Options for detection of errors on patient reports
-Review the hard copies of all patient reports.
-Use computer flags for unusual results (e.g., Burkholderia pseudomallei, Vibrio
cholera,vancomycin-resistant staphylococci, or linezolid-resistant Staphylococcus
aureus)
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23. Patient reports
3. When applicable, the laboratory must provide information that is useful for test
interpretation (e.g., normal ranges, sensitivity, specificity, precision, linearity, predictive
value, accuracy, and test interferences)
4. Patient reports and laboratory records must be retained for a minimum of 2 years in an
easily accessible location.
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24. Panic values in microbiology
❑ Organisms seen in CSF
❑ Organisms seen in joint fluids
❑ Positive cryptococcal antigen detection
❑ Positive CSF antigen detection for pneumococci, Streptococcus agalactiae, Neisseria meningitidis,
and Haemophilus infiuenzae type b (now rare)
❑ Positive AFB smear
❑ Positive blood cultures (not contaminated)
❑ Positive CSF cultures
❑ Positive blood films for Plasmodium spp.
❑ Positive eye cultures growing Pseudomonas aeruginosa or Bacillus spp.
❑ Isolation of Mycobacterium tuberculosis
❑ Isolation of Streptococcus agalactiae from pregnant women (cultures taken at 35-37 wks'gestation)
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25. Referral specimens
Reference laboratories must be accredited and/or must meet city,
county, or state standards.
(ISO 15189) FOR MEDICAL LABORATORIES
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26. Proficiency testing program
1. All accredited laboratories must participate in an external proficiency program that reflects
the specialty (e.g., mycology) and level of expertise (e.g., reference laboratory) of the
laboratory.
2. Internal proficiency programs are used to monitor various aspects of laboratory work (e.g.,
uniformity of Gram stain interpretation and identification of parasites or fungi)
Options for an internal proficiency program include the following:
a. Seed a simulated specimen with an unknown organism, and label it as an autopsy
specimen or as from a fictitious patient.
b. Reprocess specimens for analysis by different personnel.
c. Send a portion of the specimen to a reference laboratory for comparison.
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27. QC parameters
QC parameter Guidelines
1.Specimen collection and
transport
Provide instructions for collection and transport.
Establish criteria for acceptable specimens.
Establish rejection criteria for unacceptable specimens.
2. Procedure manual Write in CLSI format. Group all procedures, when applicable, as
preanalytical, analytical, and postanalytical
Define test performance, tolerance limits, specimen acceptability, reagent
preparation, QC, calculations, and reporting.
Review and initial annually.
Approve and date all changes.
Make available in work area.
Retain obsolete procedure for 2 years.
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28. QC parameters
QC parameter Guidelines
3. Personnel Employ sufficient numbers of qualified personnel for volume and complexity
of work.
Document continuing-education activities.
Provide employees with written performance standards.
Evaluate employees annually.
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29. QC parameter Guidelines
4. QC records Record all QC results on QC forms or in computer.
Report all out-of-control results to supervisor, and note corrective action(s) taken
on QC forms.
Hold monthly review of QC records with supervisor.
Retain QC records for a minimum of 2 years.
5.Patient reports Report results only to authorized personnel.
Notify test requester of "panic" values immediately.
When rendering verbal reports, record name of individual notified, date, and
time.
Provide normal ranges, when appropriate.
Correct errors in patient reports in a timely fashion
Retain records for a minimum of 2 years. Note: Time may vary in different states
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30. QC parameter Guidelines
6. Referral
specimens
Use only accredited or licensed reference laboratories.
Include the name of the reference laboratory actually performing the testing on
the patients' reports.
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31. QC parameter Guidelines
7. Proficiency testing Participate in an appropriate-level external proficiency-testing program.
Consider internal proficiency-testing program.
8. Instrument or equipment
performance
Document function checks of equipment.
Perform as frequently as necessary to ensure proper function or as
specified by manufacturers.
Document routine preventive maintenance.
Retain maintenance records for life of instrument.
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32. QC parameter Guidelines
9.Commercially prepared media
exempt from QC
Certain primary plating media are exempt from QC testing by
user.
Retain manufacturer's QC protocol.
Obtain written assurance that manufacturer follows CLSI standards
(e.g., package insert, label, protocol)
Inspect each shipment for cracked media or petri dishes,
hemolysis, freezing, unequal filling, excessive bubbles, and
contamination.
Document medium deficiencies and corrective action, and inform
manufacturer.
Perform in-house QC testing until deficiency is corrected.
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33. QC parameter Guidelines
10.User prepared and nonexempt
media
Record amount prepared, source, lot number, sterilization method,
preparation date, pH, expiration date, and name of preparer.
Check medium for proper color, consistency, depth or slant,
smoothness, hemolysis, excessive bubbles, and contamination.
Test medium with QC microorganisms of known physiological and
biochemical properties.
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34. QC parameter Guidelines
11. Stains, reagents, and antisera -Label containers as to contents; concentration; storage
requirements; dates prepared, received, and/or placed in service;
and expiration date or shelf life.
If user prepared, record volume, source, and lot number
Store according to manufacturer's recommendations
Test reagents with positive and negative controls prior to use (with
each batch, lot number, and shipment).
Test Gram stain reagents with control organisms with each batch,
lot number, and shipment and weekly thereafter
Check each batch, lot number, and shipment of antiserum when
prepared or opened and once every 6 months thereafter with
positive and negative controls.
Discard outdated material and reagents that fail performance
standards.
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35. QC parameter Guidelines
12. Commercial kits Test each new batch, lot, and/or shipment. Follow manufacturers'
recommendations for QC testing.
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36. Rejection criteria for microbiological specimens
Clerical errors
 Discrepancy between patient identification on requisition and specimen container label
 No identification on container
 Specimen source or type not noted
 Test not indicated on requisition
General microbiology
 Specimen received in fixative (formalin); exception, stool for parasites and ova
 Foley catheter tip
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37. Rejection criteria for microbiological specimens
Containers
 Unpreserved urine held in refrigerator for >24 h
 Improper or non sterile container
 Leaking container
 Dry swab
 More than one specimen of urine, stool, sputum, wound, or routine throat
specimens submitted on the same day from the same source
 Only one swab submitted with multiple requests for various organisms (bacteria,
AFB, fungi, virus, ureoplasmas, etc.)
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38. Rejection criteria for microbiological specimens
Aerobic bacteriology
 Gram stain for Neisseria gonorrhoeae on specimens from cervix, vagina, and anal
crypts
 Sputum specimen with <25 WBC, >10 epithelial cellsl/lpf
 Stool specimens submitted for culture for inpatients after third hospital day
Mycobacteriology/mycology
 24-h collection of urine or sputum for APB or fungus culture
 Inform physician or nurse that according to laboratory manual, three separate first morning specimens of sputum
or of urine are the best samples for analysis; reject 24-h specimens.
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39. Rejection criteria of urine
1. Request a repeat urine specimen when there is no evidence of refrigeration or preservation
and the specimen is >2 h old.
2. Request a repeat specimen or obtain the information when the collection time and method
of collection have not been provided.
3. Reject 24-h urine collections.
4. Reject urine specimens obtained with the same collection method within 48 h of receipt of
first specimen as a duplicate specimen.
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40. Rejection criteria of urine
6. Neonates, infants, and young toddlers cannot cooperate to provide a voided sample.
Although collection of urine by in-and-out straight catheterization is the recommended
method to obtain an accurate urine culture sample, catheterization is not always possible,
particularly in neonates.
7. Reject Foley catheter tips as unacceptable for culture; they are unsuitable for the diagnosis
of urinary infection
8. Reject urine from the bag of a catheterized patient.
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41. Rejection criteria of urine
9. Reject specimens that arrive in leaky containers.
10. Except for suprapubic bladder aspirates, reject specimen requests for anaerobic
culture.
11. If an improperly collected, transported, or handled specimen cannot be replaced,
document in the final report that specimen quality may have been compromised and
who was notified. Generally, voided urine from inpatients is easily recollected.
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42. Rejection criteria of stool
1. Reject stools not in transport medium received >2 h after collection, as changes occur that
are detrimental to most Shigella spp.
2. If specimen in transport medium is delayed for more than 48 h at 4°C or is delayed more
than 24 h at 25°C, request immediate re-collection since the specimen may be compromised
3. Multiple stool samples from the same patient that were submitted on the same day should
not be cultured.
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43. Rejection criteria of stool
❑ Reject fecal cultures received from adults and pediatric patients hospitalized for >3 to 4
days, unless the patient is known to be human immunodeficiency virus positive or
there is a cluster epidemic within the institution.
❑ Do not reject stool samples from infants and toddlers until after the fourth day of
hospitalization, since studies have shown that it may take longer to Collect a stool
sample from pediatric patients admitted with gastroenteritis that are placed on bowel
rest and are not eating a normal diet.
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44. Rejection criteria of stool
5.Notify caregiver if transport tube is filled above the line, indicating that too much
specimen was submitted in the vial.
7. Do not process hard, solid stools that cannot be sampled for inoculation.
8. Do not process dry swabs.
9. Do not process stools with barium.
10. Reject more than three stools from the same patient in a 3-week period or multiple
specimens received on the same day.
11. Do not use specimens submitted in bacteriology transport tubes for parasitology
examination.
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45. Rejection criteria for sputum and endotracheal aspirate specimens
1. Reject duplicate specimens received on the same day unless the initial sample was
inadequate for culture according to microscopic evaluation.
2. Do not accept repeat cultures at intervals of less than every 48 h.
3. Reject the following specimens for diagnosis of lower respiratory tract disease.
-24-h sputum collection
-Contaminated sputum and endotracheal specimens per Gram stain rejection criteria
- Specimens that are visually saliva only
-Specimens that are visibly contaminated by toothpaste or other substances
- Throat specimens, since they are not diagnostic of lower respiratory tract infection
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46. Rejection criteria of wound swab/pus
1. Do not accept specimens for microbiological analysis in container with formalin.
2. If numerous squamous epithelial cells are present on the Gram stain, especially from
swab specimens, request a recollection if there is evidence of infection.
3. Discourage submission of specimens to determine if an infection is present.
4. Since commercial swab transport systems have been documented to preserve most
pathogens for up to 24 h, swabs are generally not STAT transported either in the hospital
or in the ambulatory setting.
5. For multiple requests (acid-fast bacilli, fungal, bacterial, and viral) but little specimen,
contact the physician to determine which assays are most important and reject the others
as "Quantity not sufficient."
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47. QC-testing schedule for identification tests used in the laboratory
QC SCHEDULE DESCRIPTION
Daily - Routine QC is performed once when a new vial or batch is introduced and on
each subsequent day of laboratory operation.
Daily - Ad Hoc QC is performed once when a new vial or batch is introduced and on
each subsequent day that the test is performed; i.e., QC testing is only
done when the test is run for a patient isolate).
Weekly - Routine QC is performed once when a new vial or batch is introduced and on
each subsequent week of laboratory operation.
Monthly - Routine QC performed once when a new vial or batch is introduced and on
each subsequent month of laboratory operation.
One-Time QC performed only once when a new vial or batch is introduced.
Note: no further QC is required.
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48. ABERRANT RESULT PROBABLE CAUSE CORRECTIVE ACTION REPORTING
Single disc result above or below
control limit
1. Error in reading
2. “Fuzzy” zone edge
3. Clerical error
4. Bad disc
Recheck original reading and/or
Ask for second opinion
Report other in-control agents.
Repeat QC on out-of-control disc
before reporting clinical results for
that agent.
Zones universally too large on
control plates
1. Inoculum too light
2. Slow-growing strain
3. MH too thin
1. Ensure 0.5 McFarland density
2. Retest with MH 4-6 mm in depth
3. Poor nutritional value of MH
1. Hold results until repeat QC
passes
2. Do not report KB results for slow
growers
3. Do not use this batch of MH for
testing
Zones universally too small on
control plates
1. Inoculum too heavy
2. MH too thick
1. Ensure 0.5 McFarland density
2. Retest with MH 4-6 mm in depth
1. Hold results until repeat QC
passes
2. Do not use this batch of MH for
testing
Colonies within zone of inhibition for
clinical isolates
1. Mixed culture
2. Resistant mutants within zone
Check purity plate for mixed growth.
Gram stain or do other test to rule out
contaminant
Isolate, identify and retest pure
growth.
1. Do not attempt to report KB
results
2. If repeat test of pure growth
shows resistant mutants in zone;
interpret innermost zone
without any growth
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49. ABERRANT RESULT PROBABLE CAUSE CORRECTIVE ACTION REPORTING
Zone-sizes decrease over
days or weeks with QC
strains
Antibiotic discs lose potency
towards expiration.
Improper storage
contributes to this
phenomenon
Ensure that freezer is held at
constant temperature
Check expiration date of
discs
Repeat testing with newest
lot number of discs.
Report clinical isolate results
only after QC results are
within expected limits.
Poor growth of test
organism
1. Organism may not be
set-up on the correct
medium
2. Strain may have
atypical growth
characteristics
3. Organism may have
been in saline too long
before inoculation
1. Check Gram stain
and/or Identification of
organism; it may not be
set-up on the correct
medium
2. If medium is
appropriate for
organism, do not try
alternate, un-approved
medium – perform
dilution susceptibility
testing (if available)
May have to send to
reference lab for testing if the
organism is an atypical
grower.
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50. ABERRANT RESULT PROBABLE CAUSE CORRECTIVE
ACTION
REPORTING
Gentamicin or
Tobramycin zones too
small with P. aeruginosa
QC strain; rest of
antibiotics are OK
Ca2+ and/or Mg2+ content
too high in medium -
neutralizes
aminoglycosides
Repeat KB testing with
newest or different lot
number of discs. Repeat
testing with a different lot
number of Mueller-Hinton
agar. If zones still too
small look into details
recorded for manufacture
of batch.
Do not report
aminoglycoside results for
P. aeruginosa or
Acinetobacter spp until QC
limits are met.
Gentamicin or
Tobramycin zones too
large with P. aeruginosa
QC strain; rest of
antibiotics are OK
Ca2+ and/or Mg2+ content
too low in medium
Repeat KB testing with
newest or different lot
number of discs. Repeat
testing with a different lot
number of Mueller-Hinton
agar. Ensure MH is 4-6
mm in depth. If zones still
too small look into details
recorded for manufacture
of batch.
Do not report
aminoglycoside results for
P. aeruginosa or
Acinetobacter spp until QC
limits are met.
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51. ABERRANT RESULT PROBABLE CAUSE CORRECTIVE
ACTION
REPORTING
Indistinct zones with
TMP/SMX or
sulfamethoxazole with
QC strains
1. Inoculum too heavy
2. Thymidine, which
inhibits action of these
agents, is at high
levels in MH
Test MH as follows:
1. Test E. faecalisATCC
29212 versus
TMP/SMX according to
KB method.
2. Medium is satisfactory
if zone of inhibition is
clear and  20 mm in
diameter.
3. Look into details
recorded for
manufacture of batch or
contact media supplier
if medium is
unsatisfactory by above
criteria.
Do not report TMP/SMX
or sulfonamides for clinical
isolate if the E. faecalis
ATCC 29212 result fails.
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