CLINICAL INTERNSHIP – CLINICAL MICROBIOLOGY

DAY 1 – PRE-ANALYTICAL PROCEDURES;

RECEIVING/RELEASING; LIS
By: Xiao - The Conqueror of Demons, The Vigilant Yaksha, & Alatus, the Golden-Winged King

ORIENTATION o 2 or more: so there is higher chance of isolating

the pathogen
- The best time to isolate the pathogen is during the peak
of infection
- Never extract on the same site
- 4 types:
o Aerobic
o Anaerobic
o Pediatrics
o ARD: Antimicrobial
Removal Device
 For patients under
antibiotic
treatment
- Clinical instructors: - Pedia – 5-10mL
o Zhainal Mark Julaine - Adult – 20-30 mL
o Shiela Mae Peligrino - 2 blood culture bottles – 2 different sites interval of an
hour
PRE-ANALYTICAL, ANALYTICAL, AND POST-
ANALYTICAL PROCEDURES COLLECTION PROCEDURE

1. Locate extraction site

PRE-ANALYTICAL PHASE
2. Disinfect the site with alcohol
- Occurs prior to specimen testing and may include o S. epidermidis is part of the normal microflora of the
variables involving the process of obtaining a specimen skin
3. Disinfect again using povidone-iodine
4. Wipe and disinfect the site using alcohol
Preparation prior Sampling/ Transport/ Preparation prior
5. Then proceed to phlebotomy and collect about 5-10 mL,
to sampling Handling Storage to analysis 20-30mL (adult) of blood from the patient
6. Transfer the extracted blood unto the culture bottles
7. Gently mix the bottle by inversion
8. Label the specimen and make sure to include which site
- Preparation prior to sampling:
o Example: 1 hour fasting for AFB CEREBROSPINAL FLUID (CSF)
- Sampling/Handling:
- To diagnose a disease
o Venipunctures
or condition affecting
o Collection of sputum
the central nervous
- Transport/Storage
system
o Cary-Blair for stool
- Collected thru the
o Amies medium
lower back by a
o Stuart’s transport medium
procedure called
SPECIMEN COLLECTION BLOOD CULTURE lumbar
puncture/spinal tap
- Ordered when the doctor suspects for blood infection
- Performed by a
- 1 or 2 blood culture bottle per patient is usually
physician
requested by the physician; but the doctor can ask for
more
- Bottle collected NASOPHARYNGEAL SWAB
o 1st bottle – Chemistry and Serology
- Tilt patient’s head back 70 degrees
o 2nd bottle – Microbiology
- Gently and slowly insert mini tip swab with flexible shaft
o 3rd bottle – Hematology
(wire or plastic) through the nostril parallel to the palate
o 4th (if there is) – Microbiology
(not upwards) until resistance is encountered
 Why can’t microbiology be the first bottle
- Gently rub and roll the swab
collected? To prevent contamination
- Leave swab in place for several seconds to absorb
THROAT AND NASOPHARYNGEAL SWAB secretions
o If dry: Prone to false negative results
- To check for any signs of infection from the respiratory
- Slowly remove swab while rotating it
secretions
- Place swab, tip first, into the transport tube provided
- Technically any lab processes of each of the swabs (nasal,
NP, throat) are exactly the same

THROAT (OROPHARYNGEAL) SWAB

- Insert swab into the posterior pharynx and tonsillar areas

- Rub swab over both tonsillar pillars and posterior
oropharynx and avoid touching tongue, teeth, gums
- Place swab, tip first, into transport tube

- Thick: Oropharyngeal
- Thin: Nasopharyngeal
SPUTUM

- 30-50 mL capacity (ideal)

- Translucent, clear
o Because macroscopic
assessment of the sample is
performed
o Salivary sample is rejected:
False negative
o Bloody sputum is rejected
in AFB: False positive
- Sterile, single use
- Leak proof, screw cap
- Wide mouth
 - 10x: Low power field
- Mucopurulent, not mucopurelent
- Ideal time of collection of sputum is in the morning and
gargle with water before collection

- AFB: An hour prior to collection, the patient should fast

o Because the sample might be contaminated with
food particles
o Required that 2 sample be submitted: 1st morning
specimen, and 1 hour after 1st morning specimen
- PMNs > Epithelia cells: Accept
- Use of mouthwash should be avoided
- Epithelial cells > PMNs: Reject
- If the patient cannot provide a sample, the physician
must be informed Bartlett’s classification:
- May often be contaminated with normal flora so it is
important to evaluate the quality - Scores of 0 or less: Lack of inflammation; Presence of
- Note the number of squamous epithelial/LPF and PMNs saliva = Reject the sample
to evaluate acceptability of specimen - Scores greater than 0 = Accept the sample
- Perform Gram stain
URINE

- 30-50 mL capacity
- Translucent/clear
- Sterile, single use
- Leak proof, screw cap
- Wide mouth
- Specimen of choice:
o Midstream clean catch
o Catheterized for those
unable to produce urine
o Suprapubic if anaerobic culture
BARTLETT’S CLASSIFICATION - Must be preserved or refrigerated if not processed
immediately
- Boric Acid – suitable preservative
- If patient’s request is coupled with urinalysis, process first
in microbiology
o Which is performed first, AFB or culture? Culture

STOOL

- Stool specimen/Rectal swabs must be placed in sterile

container or appropriate size with tight-fitting, leak-
proof lid
- Processed within 2 hours of collection; if not, place in
transport media (CARY-BLAIR)
GENITAL TRACT SPECIMEN
- Collected by the physician (OB-GYNE)
- All specimens should be collected during a pelvic exam
using speculum
- Speculum may be moistened with warm water before use
- Antiseptics or gynaecological exploration cream should
not be used (lethal to gonococci)
- Vaginal swab/discharge – posterior fornix of vagina
- Gonococcal/Chlamydial culture – endocervix
- After inserting speculum, cervical mucus should be
wiped off with cotton
- Sampling swab should then be introduced into the
cervical canal and rotated for at least 10 seconds before
withdrawal
- For collection of urethral specimens, a swab with narrow
diameter or sterile bacteriological loop should be
inserted 3-4 cm into the urethra and gently rotated
before withdrawal.
- Purulent discharge can be collected directly on a swab or
on the inoculating loop. The composition of both the tip
and the shaft of the swab is important.
- For the culture of Neisseria gonorrhoeae, charcoal-
treated cotton tips or calcium alginate or Dacron tips are
preferred
- Collected by the physician
- If immediate plating and incubation are not possible, a
transport medium such as Amies or Stuart transport
medium should be used
GUIDELINES IN RECEIVING OF REQUEST AND SPECIMEN
FOR MICROBIOLOGY EXAMINATION
- Check laboratory request for completeness of data
o Name, age, physician, room number, ward, etc.
- Check receipt for correct billing of laboratory tests
o Request form and receipt form should match
- Evaluate suitability of specimen for requested lab test
o Blood stained sputum sample for AFB should be
rejected; it will lead to false positive results
- Evaluate specimen if fit for processing (enough amount)
- Check if there’s any missing data in LIS
- Inform ward personnel/watcher for any discrepancy
regarding specimen
- Claim stub with requested test will be issued to watcher
and informed of the date to be claimed
- Endorse request and specimen to the processing area
o Doctors can request for STAT culture: this means the
sample must be collected immediately
o Blood culture samples usually require 5 days of
reading before reporting is performed or no growth
is concluded
 Urine and sputum = 48 hours
 Other body fluids = 72 hours
PRE-ANALYTICAL ERRORS
- Inappropriate quality of specimen
- Wrong identification of the patient
o Label is according to the serial number, not the
patient’s name
- Missing physician’s order
o Doctor’s request is required
- Use of inappropriate container
o Urine placed in vials which were not autoclaved are
rejected
- Patient not appropriately prepared for the test
- Wrong procedure of sample collection
- Errors in specimen transport to the laboratory
- Blood collection vials not properly labelled
- Requisition form not properly filled (wrong patient’s
name, wrong tests entered)
- Inadequate sample volume
o Especially for automated blood culture machines;
requires larger volume/amount of specimen
- Referral of specimen
- Concerning the pre-analytical phase, CLIA (Clinical
Laboratory Improvement Amendments) requires that
laboratories:
o Have written procedures for the pre-analytical phase
o Provide documentation of personnel qualifications
and training
 There are procedures that must be performed by
Medtechs who has undergone special training
o Monitor sample quality indicators
ANALYTICAL PHASE BENCH C1

- Includes what is usually considered the “actual” - Reading of Biochemical, Preliminary and Confirmatory
laboratory testing or the diagnostic procedures, tests
processes, and products that ultimately provide results o TSI
o LIA
o Citrate
PROCESSING ANALYSIS REVIEW o Urease
o SIM
- Evaluation of isolate
GUIDELINES IN PROCESSING OF REQUEST AND SPECIMEN - Identification of isolate
- Evaluation of Identified organism according to
PROCESSING AREA (BENCH A) antibiogram requirement
- Performance of Susceptibility Test
1. Receiving of request and specimen in the Microbiology
Receiving Area BENCH C2:
2. Classification of Request/Specimen AFTER 24 HOURS OF INCUBATION
A. Request are classified according to the type of
Microbiology examination: - Arrangement of incubated susceptibility plate according
o Culture and Sensitivity to their respective specimen groupings
o Direct Smears - Evaluation of Zone of Inhibition
o Gram’s Stain - Measurement of Zone of Inhibition
o Acid-Fast Stain - Interpretation of Zone of Inhibition
o KOH Preparation o Resistant
o India Ink o Intermediate
o Wet Mount o Sensitive
B. Specimen are classified according to type: - Documentation of results on corresponding worksheet
o Urine folders
o Exudates
BENCH D
o Body Fluids
o Blood - Disinfection
o Stool - Decontamination
o Other Respiratory Specimen - Media Preparation
o Cervico-Vaginal/Urethral Discharge
3. Entry of data to corresponding Receiving and Processing ANALYTICAL ERRORS
Worksheet - Sample lost
o Name of patient is listed in receiving worksheet - Sample mix up
o Individual worksheet is prepared to each - Equipment failure
request/specimen and filed to corresponding - Undetected failure in quality control
specimen folder - Procedure not followed correctly
4. Processing of Specimen
o Preparation of smears - Concerning the analytical phase, CLIA requires that
o Inoculation of Specimen to Appropriate Culture laboratories:
Plate and Broths o Establish and maintain written policy, process, and
ISOLATION AREA: procedure manuals
AFTER 24 HOURS OF INCUBATION (BENCH B) o Provide training and perform competency
evaluations of personnel
- Arrangement of incubated Culture Plates/Broths o Participate in a proficiency testing program
- Reading of Culture Plates/Broths (annually) appropriate for their test menu and
- Performance of Work-up procedures on culture plates specialties
- Incubation of Work-up tubes and plates at appropriate o Verify instrument performance and track instrument
temperature (35-37oC) for 18-24 hours calibration and maintenance
- Reading of growth in culture plates/broths o Maintain records of the laboratory environment (e.g.,
temperature, humidity, refrigeration)
POST-ANALYTICAL PHASE LABORATORY INFORMATION SYSTEM (LIS)
- Refers to recording, releasing, and interpretation of test What is a laboratory information management system?
results by physicians to formulate diagnosis to guide
patient management - A computer software that processes, stores, and
manages data from all stages of medical processes and
tests
- Involved in storing, recording, recovering, and
RECORDING OF RELEASING OF INTERPRETATION
TEST RESULTS TEST RESULTS OF TEST RESULTS
consolidating lab information from the clinical and
anatomic sections of the laboratory
- It standardizes workflows, tests, and procedures, while
providing accurate controls of the process
- Documentation of Results on corresponding worksheet - Hospital: Tertiary Lab
folders - Free standing: Secondary
- Recording of results on corresponding logbook
- Preparation of result CLASSIFICATION BY FUNCTION
- Preparation of second copy of the official result - Clinical Pathology
- Presentation of claim stub at receiving/releasing area o Clinical Chemistry
(OPD only) o Hematology
- Endorsement of claim stub at microbiology receiving o ImmunoSerology
area (OPD only) o Blood Banking
- Release of official result signed by the microbiology staff o Clinical Microscopy
NOTE o Microbiology
o Molecular Biology
o Documentation should include the person who o Toxicology
performed the biochemical tests, the culture, etc. o Cytogenetics
o Therapeutic Drug Monitoring
POST-ANALYTICAL ERRORS
- Anatomic Pathology
- Transcription error o Medtechs assist the pathologist here
- Delayed report of result
- Failure to report the results o Surgical pathology
- Loss of test results o ImmunoHistopathology
- Incorrect interpretation of results o Cytology
o Autopsy
- Concerning the post-analytical phase, CLIA requires that o Forensic pathology
laboratories: o Molecular pathology
o Report STAT results and critical values promptly have
GOALS OF LIS
a written quality assurance program
o Generate Levy-Jennings or trend plots of QC results - Conform to standard classifications and validity of data
to identify changes in test performance set by the DOH
o Reduce human error through root-cause analysis, o HFSRB – Health Facilities and Services Regulatory
process control, and education/communication Bureau
 Sets laws and requirements for lab accreditation;
Under the DOH
- Ensure the integrity and internal consistency
- Maintain patient confidentiality and security of the
information
- Allow easy access by the laboratory managers up to the
health professionals
- Allows various data to be integrated to the laboratory
data
WHAT IS THE PURPOSE OF THE LIS? SPECIMEN REJECTION

- Improve – quality of data because it plays a central role
on laboratory decision making
- Optimize – efficiency in laboratory operations for
healthcare delivery
- Connects – with patient registration, billing systems, and
Electronic Hospital Records
- Provide – accurate and timely results
- Assist – in the decision-making of health professionals
for the promotion, diagnosis, treatment and
management of health
- The information on the label does not match the
information on the request form
- The specimen has been transported at the improper
temperature
- The specimen has not been transported in the proper
medium
- The quantity of the specimen is insufficient for testing
- The specimen is leaking
- The specimen has received for anaerobic culture from a
site known to have anaerobes as part of the normal flora
- The specimen is dried up
- The specimen is unpreserved
- The specimen was received in a fixative (formalin)
- The specimen transport time exceeds 2 hours post
collection

SPECIMEN STORAGE

- Anaerobic bacteria should not be refrigerated

- CSF should be kept at 37 degrees Celsius
- Urine, stool, viral specimens, sputum, swabs, and foreign
devices should be stored at 4 degrees Celsius
- Serum for serologic studies may be frozen at -20 degrees
Celsius
- Tissues or specimens for long term storage should be
frozen at -70 degrees Celsius
COMMON ERRORS IN THE LABORATORY WORKFLOW
- 20 degrees Celsius = ref temp
PRE-ANALYSIS
TRANSPORT MEDIA
- Obtaining inadequate or insufficient samples
- Stuarts system
- Samples taken from the wrong patients
o Consists of swabs in a test tube with transport media
- Specimen replaced in the wrong chemical/container or
o H. influenza, S. pneumoniae, S. pyogenes, C.
tube
diptheriae
- Ordering wrong tests
- Amies
- Identification problems related to the patient and the
o A modified stuart’s medium
source of the specimen
o H. ducreyi, N. gonnorhoeae
- Lack of clinical information when required
- Carry-Blair
ANALYSIS o Designed for transport of stool specimen and for
enteric pathogens
- Bad reagent lot o Vibrio, Shigella, Salmonella, E. coli O157:H7
- Instrumentation failures
- Wrong data entry
- Erroneous transcript

POST-ANALYSIS

- Delay of the delivery of results

- Failure to report or reporting to the wrong healthcare
provider
- Transcription errors
- Incorrect results
- Misunderstanding of the results by the clinicians
- Failure of the clinicians to see the reports