Celine - Automation Flashcards
Why is micro slow to change over to automation?
(5)
Micro too complex to automate -> there is a large variety of specimens, specimen collection systems, specimen processing and culture requirements
Automation can’t replace subjective decision, making skills and interpretative expertise required
High cost - complexity of specimens poor return on instrument investment
Scale of economy lost in micro - large scale tracking automation systems under-utilised
Microbiologists don’t like change
What are the four main drivers for automation in micro
Increased demands on service increase by 10% per year
More challenges - mutli-resistant organisms, new pathogens
Reduced funding - reduced budget and staff number
Accreditation - need for standardisation and traceability
Explain why automation is being introduced to the micro lab
(3)
Lab under immense pressure to produce faster, better, standardised results in face of increasing work volume
Microbiologist recognise that automation need not replace cognitive decision-making but rather replace tedious, repetitive steps
Automation offers the opportunity to reduce TAT, optimise workflow and reduce costs
What happens on Day 1
(4)
Specimen in
- Microscopy
- Processing
- Culture
- Isolate
What happens on day 2?
Identify
Susceptibility
Comment on automation for day 1 specimen processing
(2)
Automated systems for specimen preparation and culture are complex
Front end processing only recently developed and only evident in large diagnostic micro labs
Comment on automation for day 2, pathogen
Austomated systems for ID and AST first developed widespread use in all diagnostic micro labs
Where is the most manual work done in micro
Plate and broth medium inoculation
Medium and plate reading
Write about the role of automation in Day 1: Microscopic investigation of clinical specimen
(6)
Microscopic investigation consists of direct gram stain and/or white cell count (CSF and Urine)
Direct gram stain can be automated but microscopy is still manual
Microscopic cell count increasingly automated
Urine is the most common sample in the lab
Automation of urine microscopy using fluorescence flow cytometry
Negative screening method for urine -> 60-80% negative, if WCC count normal no need for culture
What machine can be used for urine microscopy?
Sysmex UF-5000 - urine microscopy
How does the Sysmex UF-5000 work?
(3)
Urine flow cytometer
Enumerates WBC, RBC, bacteria and epithelial cells by cell size and staining with fluorescent dyes
105 samples/hour
Comment on the role of automation in Day 1 processing and culture
(6)
Remains largely manual
Front end processing evolving at a rapid pace
Liquid samples readily compatible with automation but swabs required manual handling
Liquid-based ESwab facilitated change
The Eswab sits in liquid media - vortex so flocked head releases organism into liquid media -> this allows for automated smear preparation for gram stain and for plate inoculation
New generation of liquid-based specimen processors now in large diagnostic labs with high specimen throughput
Explain the role of Eswabs in automation
The Eswab sits in liquid media - vortex so flocked head releases organism into liquid media -> this allows for automated smear preparation for gram stain and for plate inoculation
Give an example of a specimen processor used in the labs, capable of plate culture
WASP Processor (Copan Diagnostics)
What is the WASP processor and how does it work
(8)
2 robot arms which move specimens and plates
Can work with all specimen types
Has 9 media silos and a capacity of 370 plates
Uses reusable chrome loops
Can inoculate 180 plates per hour
Can gram stain prep
Capable of auto decapping and recapping
Only one installed in Ireland
Write about the role of automation in blood culture processing
(4)
Automation is important for the early detection of BSIs -> rapid diagnosis, prognosis and appropriate therapy
Automation for Blood cultures is well established
BacT/Alert SD is the most common BC system available
BacT/Alert SD allows for the continuous monitoring for detection of bacteria, fungi and mycobacteria
What is a BC bottle
(3)
A blood culture bottle is used to investigate BSI
Blood is injected into bottles with culture media at bedside
The BC is then sent to the lab
What is the most common BC automation system
BacT/Alert 3D
What is the principle behind the BacT detecion system
(5)
If organisms are present in blood they will multiply in nutritious liquid media in BC bottle to generate CO2
CO2 production lowers the pH of the medium and produces a colour change in a sensor at the base of each bottle
Resins in the media neutralise antimicrobials in patient blood enhancing early recovery of pathogens
The bottles are constantly agitated and read every 10 minutes
When the bottle flags positive the bottle is removed from the BacTAlert -> withdraw a sample and perform a gram stain and culture on appropriate culture plates
What is present in BC bottles that neutralise antimicrobials?
Resins
What two aspects of confirming identification on Day 2 is compatible with automation
Detection of biochemical properties via Vitek/Manual
Determine protein profile via MALDI
In general what is the Vitek 2 capable of doing
Identifying biochemical properties
Antimicrobial Susceptibility Testing
In general what is the MALDI TOFF capable of doing
Id only based on protein profile
In general what is the pheonix capable of?
Biochemical properties and AST
Why is automation so important in pathogen identification
(2)
These systems (Vitek and MALDI) are reproducible, reduce hands on time and quicker turn around time
Systems are also interfaced with hospital computer systems - automate result entry
What is the Vitek comprised of and how does it work?
Filler-sealer module -> draw bacterial suspension in 64 microwell card
Incubator and photometer to detect optical density for bacterial growth and biochemical reaction colour
What are the five ID cards for the Vitek 2?
(5)
Gram negative
Gram positive
YST(Yeast)
Neisseria/Haemophilus
ANC (anaerobe)
Explain in your own words how the vitek is used
(6)
Select the correct ID card
The Vitek will draw the bacterial suspension into the 64 microwells of the ID cardd
These wells contain test substrates which are used to measure a variety of metabolic activities, enzyme hydrolysis and growth in presence of inhibitory substrate
The growth/colour change is determined every 15 minutes
The series of 64 biochemical reactions is then compared to a database of known organisms
Gives results in 8 to 16 hours
What is measured by the Vitek?
(2)
Metabolic activities
Enzyme hydrolysis and growth in presence of inhibitory substrate
What does MALDI TOF stand for
Matrix-Assisted Laser Desorption/Ionisation - Time of Flight
What is the MALDI-TOF MS used for
MALDI-TOF mass spectrometry used for species ID
What are some of the benefits of MALDI?
(6)
Widely used for ID of bacteria and fungi
Its reproducible and sensitive - powerful tool
Analysis is very short -> TAT only takes 6 minutes/bacteria
Simple sample preparation and result acquisition
Cost is 5 times cheaper than conventional ID
MALDI analyses the protein composition of the organism -> there is no need to wait for growth -> can test directly on colony
How is the MALDI used/How does it work?
(8)
Prepared single colonies from Day 2 plate used (directly or inactivated)
Spot colony on biotarget teflon plate
Add matrix (crystallized molecules), air dry, place on MALDI
Sample ionised with laser burst, releasing ‘cloud’ of proteins
Proteins accelerated with an electric charge through a flight tube to mass spectrometer
Time of flight recorder, lighter proteins travel faster than slower heavier ones
Protein profile determined by mass/charge ratio
Protein profile of test organism compared with profile database of well-characterized strains
How do you prepare your colonies for MALDI
Single colonies from Day 2 plate used directly or inactivated with alcohol/formic acid which allows for cell wall disruption and protein extraction
Where do you add your colony for the MALDI?
On biotarget teflon plate
What do you add to your colony on the teflon plate for MALDI
Add matrix (crystallized molecules), air dry then place on MALDI
What is the point of the matrix for MALDI
Acts as a buffer between bacteria and laser and helps ionise the sample, carrying it along the flight tube
What is the most common MALDI-TOF instrument
Bruker Biotyper system
Write about the Burker Biotyper system, how accurate is it?
98% ID at genus level
87% ID at species level
Misidentification most common in S. pneumonia, coagulase negative staph, and shigella Vs E. Coli
The database will expand to resolve many of the current identification problems
Comment on the role of automation in AST
(5)
Many automated ID systems are adapted to accommodate automated susceptibility testing
Most common automated AST system is the Vitek 2
The AST cards contain 64 micro wells with a range of 8 to 9 antibiotics suitable for a wide range of organisms
Each antibiotic present at concentrations that convers the threshold/breakpoint for susceptible, intermediate and resistant
Employ short incubate with results in 8 to 16 hours
AST can be measured using the VITEK for which organisms
(4)
Staphylococci
Streptococci
Enterobacterales
Pseudomonas
Comment on future total lab automation
(2)
There are 2 Total lab automation microbiology systems in development
Th most common TLA system in Micro is the Kiestra
What are some common features of the TLA systems
(4)
Robotic plate management to automate specimen processing and workup
Conveyor/track systems to move plates to and from incubators
Automated incubators with digital reading stations
Digital cameras capture plate images at timed intervals
Is TLA a reality?
(7)
Total processing time takes 26 hours and AST appears feasible
They have been said to improve turn around time which could have a positive impact on clinical decisions and patient outcome
But there are few peer-reviewed publications
Benefits are inferred not proven
Need large studies to accurately assess the financial operation and clinical impacts of TLA
There have been 68 installations to date
One study found 2 fold in lab productivity despite 27% increase in workload but there was a decrease in 2.4 staff members
What is molecular diagnostics considered the gold standard for?
(5)
Non-culturable organisms - viruses, chylamdiae
Fastidious organisms - N. gonorrhoeae
Slow growing orgnaisms - Mycobacteria
Important HCAI pathogens - MRSA, VRE
Multiple potential pathogens implicated
Give an example of a piece of automation used for molecular diagnostics
COBAS 4800 Ct/Ng assay
What is the COBAS 4800 Ct/Ng assay
(5)
Multi-plex Realtime PCR-C trachomatis and N. gonorrhoeae
System combines fully automated DNA extraction followed by PCR preparation + Real time PCR detection
97% sensitivity
Specimens include urine samples and genital samples
Processes 94 specimens in 5 hours with only 40 minutes hands-on-time
Give the most commonly used automation in molecular diagnostics
(4)
GeneXpert Real Time PCR
DNA extraction, amplification and detection in a single cartridge
60 minutes turn around time
Swab samples transferred to cartridges and then inserted into machine
Write about the Real-Time Enteric Bio Assay
(6)
EntericBio Gastro Panel 1
Real time PCR kit for simultaneous detection Campylobacter spp, Salmonella enterica, Shigella spp and Vero Cytotoxigenic E. Coli (VTEC)
Panel 2 plus Cryptosopidium + Giardia
Multiplex detection of pathogens directly from faeces without pre-enrichment or DNA extraction
PCR performed on Roche 480 Light Cycler Platform
3 hour turn around time
Comment on automation in serology
(5)
Manual methods utilise ELISA-based micro-titre plate which is very labour intensive
Fully automated systems are widely used for Serology
Main application diagnosis of infection caused by non-culturable/slow growing/fastidious pathogens
Main application in virology for HBV, HCV, HIV, CMV
Main application in syphilis serology - STI agent
Give three applications of automation in serology
Main application diagnosis of infection caused by non-culturable/slow growing/fastidious pathogens
Main application in virology for HBV, HCV, HIV, CMV
Main application in syphilis serology - STI agent
Give an example of an analyser used in the automation of serology
(6)
Immunoassay analyser
Assays in Clinical Chemistry and Microbiology
Primary sampling - no sample separation required
Random access - multiple tests performed at the same time
65 sample load
Throughput 50-100 tests/hr
