Lab stuff!

Question 1

Describe library prep in sequencing

Answer 1

EFAL B (souds like awful- which this exam is)
after Extraction, there if Fragmentation of genetic material in to smaller sequences, Adapter Ligated to fragments, Barcoded

Question 2

Define read length and through put

which methods have high and low of each of these

Answer 2

Read length: total amount of nucleotides sequenced (measured in base pairs or kilobases)

Throughput: total amount of sequences data generated, and overall capacity of the sequencing platform

Sanger (chain terminating) sequencing: High read length, low through put

NGS: Low read length, high through put (this means a higher error rate however)

Question 3

In NGS what is index hopping?

Answer 3

AKA swapping

Misalignment. When DNA fragments from one sample are mistakenly assigned to the index of another sample. Can lead to to misinterpretation of results

Improvements in next-generation sequencing (NGS) technology have greatly increased sequencing speed and data output, resulting in the massive sample throughput of current sequencing platforms. A key to utilizing this increased capacity is multiplexing, which adds unique sequences, called indexes, to each DNA fragment during library preparation. This allows large numbers of libraries to be pooled and sequenced simultaneously during a single sequencing run.

Gains in throughput from multiplexing come with an added layer of complexity, as sequencing reads from pooled libraries need to be identified and sorted computationally in a process called demultiplexing before final data analysis. However, with multiplexing, the potential for index hopping is present regardless of the library prep method or sequencing system used. Index hopping may result in assignment of sequencing reads to the wrong index during demultiplexing, leading to misalignment.

Question 4

What are the features of an infection that deem it category 4 HG?

What distinguished this from HG 3?

Answer 4

-causes severe human disease and is a serious hazard to
employees
-it is likely to spread to the community and there is usually
- no
effective prophylaxis or treatment available

In cat three there is a prophylaxis or treatment available

Question 5

Define Limit of detection

Answer 5

Lowest amount of analyte in a sample which can be detected but not necessarily quantified. Lowest concentration level that can be determined as statistically different from a blank at a certain level of confidence (95% of the time).

measure of analytic sensitivity

Question 6

Define limit of quantification

Answer 6

lowest analyte concentration that can be quantitatively detected with a stated accuracy and precision

Question 7

What is a compliment fixation test

Answer 7

Antibody present=no hemolysis (pellet of blood in the bottom). Antibody absent=hemolysis (looks cloudy).

https://microbenotes.com/complement-fixation-test/

Patients serum mixed with a known antigen and compliment proteins
If patients serum contains specific antibodies they will form antigen-antibody complexes
Activated complement proteins then lyse cells that have been sesnitised

Positive result: lack of complement activity (as it is bound to antibody)
= no lysis
Negative result (i.e no antibodies)

Compliment haemolysis red cells. Antibodies stop this process

i.e. the antibodies “fix” the complement and stop them from going nuts

Question 8

What are the legends (i.e x and y axis and dotted lines) on a bland altman analysis

Answer 8

X axis is mean or average
y axis is the difference between the to assays (0 is in the middles, then goes - or +)
Needs limits of agreement

Question 9

What is a bland altman used for

Legends: X is mean, y is difference

Needs limits of agreement

Answer 9

Compares measurements by two different methods

May be used in virology for:

Introducing new methods or instruments
Check consistency between parallel instruments
To check performance of new reagents

Caveats:
Measurements recorded on
the same patient could be expected to vary less than measurements recorded from separate patients

Question 10

What is accreditation

Answer 10

External audit of quality assurance programe

Accreditation is an external audit of an applicant department’s organization and quality assurance program.

UKAS is the accreditation authority

ISO 15189 is used as the standard for all accreditation bodies in order to provide uniformity of standards and cross-recognition.

Question 11

What is primary and secondary containment in lab

Answer 11

1ry: Protection of worker and immediate environment
2ry: protection of people and environment outside of lab

Question 12

Define sterilisation

Answer 12

Sterile is an abscence of living organisms
Sterilisation removes organsims and their spores

Question 13

What is SGSS

Answer 13

Second generation surveillance system

Question 14

Design features of CL3 labs

Answer 14

In addition to CL2 +
Sealable for fumigation
Negative pressure
HEPA (High efficiency Particulate Air) filitered
Single-pass air- typically 10-12 air changes/hour
Alarm systems for air leaks
Ventilation should be dedicated to lab

Question 15

Difference between affinity and avidity

Answer 15

Affinity is strength of a single interaction and a single antigen binding site i.e. monovalent

Avidity is overall binding strength polyvalent

Question 16

What is the VIDAS assay

Answer 16

Enzyme linked fluorescence assay (ELFA)

Quick and easy to set up

Question 17

Compliment fixation test

Answer 17

Change in acute and chronic phase

Antigen or antibody detection

Sheep red blood cells + compliment

Question 18

What is the DS2

Answer 18

plate washer

Question 19

ECLIA

Answer 19

Change is in signal before and after reaction

Measure luminesance

Sensitive

Large dynamic range

https://www.labtestsguide.com/clia-vs-eclia

Question 20

Netralisation

Answer 20

Crystal violet will only stain live cells

(incomplete)

Question 21

Prozone

Answer 21

Antibody excess, leads to false negative result

Question 22

Problems with using Ct values as a quantitave value

Answer 22

Ct values are assay specific, highly variable
Specimen specific variability

https://www.idsociety.org/globalassets/idsa/public-health/covid-19/idsa-amp-statement.pdf

Question 23

Standard curves quality measures

Answer 23

Appropriate dynamic range
Efficiency between 90-110%
Reproducible
R2 close to 100

Question 24

Digital PCR pros and cons

TBC

Answer 24

Pros:Accurate at low levels of detection
Gives results as copies per ml
Absolute quantification

Cons: limited dynamic range

Question 25

Hypochlorite when do you use, examples
1) 10,000ppm
2) 20,000 ppm
3) 1,000 ppm

Answer 25

1) Blood spills ?BBV
2) Prion cleaning
3) general cleaning including norovirus

Don’t use on metals- corrosive

Question 26

Targets for an Mpox Assay

Answer 26

G2R_WA, E9L-NVAR

Question 27

How to calculate specificity

Answer 27

ability of a screening test to detect a true negative, being based on the true negative rate, correctly identifying people who do not have a condition

specificity is the proportion of people without a condition who are correctly identified by a screening test as indeed not having the condition.

True negatives/ (true negatives + false positives) x100

Spin:When specificity is high, can be ruled in

Snout: When sensitivy is high can rule out when a test is negative

Question 28

Calculate specificity

Answer 28

Of those that don’t have the condition (ie have something else), what percentage tested negative

True negatives/ (true negatives + false positives)

Question 29

Define and calculate PPV (screening usually)

Answer 29

Whether people who test positive actually have the condition

True positives/ (true positives + false positives)

All the positives

Question 30

Negative predictive valcue

Answer 30

Whether people who tested negative, do not have the condition
True negative/ (true negative + false negatives)

All the negatives

Question 31

List 3 major Westguard rules

Answer 31

1 3SD- random error
R 4 SD - random error (but may be systematic)
10x (when 10 fall on one side of mean)- systematic error

violations of the Westgard QC 4 1SD or 10x rules may be investigated through equipment monitoring. Reduced incubator temperature would indicate equipment failure, whereas correct temperatures may suggest reagent
deterioration

Question 32

Westguard Rule 4 1SD is violated what should you do

Answer 32

This rule detects systematic error. The rule is violated when 4 consecutive values
exceed the same mean ±1SD.

The run does not need to be rejected if this rule is
violated but should trigger recalibration or equipment maintenance.

Major rules: 1 3SD
10 x (10 consequtive fall one side)
R4sd

Question 33

What dose 10 x rule indicate

Answer 33

Sytematic error-

gradual deterioration of control materials, issues with storage
conditions and change in the reagent batch or modifications in testing method

Question 34

What is a horizontal audit

Answer 34

Assess one element of a quality system. Does not assess whole system

Question 35

What is a vertical audit

Answer 35

Process of following whole sample through process from receipt to report- include all activities which
relate to the testing of the sample

(remember following something through, something falling down a drain vertically? My kids would say followng a poo from formation down the toilet to a sewer- Claire is obviously more mature than this…)

Question 36

Define limit of detection

Answer 36

Lowest amount of analyte that can be detected in a sample (not quantifed)

Question 37

Problems with unique detections from metagenomics

Answer 37

May be part of normal flora
Contamination
Artefact (problem with sequencing or pipeline)

Question 38

Define lower limit of quantification

Answer 38

LoQ is the lowest concentration at which the analyte can not only be reliably detected with an acceptable level of repeatability and trueness

Question 39

Define prevelance

Answer 39

Number of cases present at a particular moment in time

(can alter the perceived performance of a test)

Vs incidence: measure of new cases over a time period

Question 40

Nanopore is high throughput T/F

Answer 40

T
Advantages: long read length
Small machine- saves space in lab

Question 41

Benefits drawbacks of illumina squencing

Answer 41

High throughput
Cost effective per base per is low
Lots of experience with using the technology
Low error rate (0.5%- compared to Pacbio and nanopore)

Cons:
Short read lengths (makes WGS more complicated)
Needs bioinformatics and pipeline

Question 42

Indications for doing an external quality control

Answer 42

Every 6 months
Change of reagent (either batch or shipment)
Any change in major components of assay (including repairs)
Following maintenance

Question 43

Your supplier has had a fire in their warehouse and there are no reagents. Identify FOUR distinct individuals or groups who you think should be notified about this situation

(question modified from micro part 2) 2 marks

Answer 43

Clinical lead
Medical Director
IPC
Lab manager
User Groups
Public health team
Local Medical Committee - (GP Liaison group)

Question 44

Your supplier has had a fire in there warehouse and there are limited reagents.

List FIVE factors (both clinical and laboratory)
that you would consider when deciding how to prioritise use of testing?

5 Marks

Answer 44

Model answer from micro exam

Prioritise
- unwell patients (ICU)
- immunosupressed patients
- sterile site specimens (eg blood,CSF)
- specimens that are difficult to repeat (eg BAL)
- Prioritise specimens/organisms of medico-legal or public health importance

Expected duration of shortage

Organisms likely to survive storage pending reinstatement of supply
Access to alternative sens testing/inference methods (eg VITEK, PCR, latex tests)
Other sensible options accepted

For viro other things:
- access to alternative labs/send away
- look to business continuity plan
- consider storage of samples

-? call a meeting- who would you invite

Question 45

Error in serology what do you do

(answer from GSTT SOP- needs editing)

Pre- analyticl
Analytical
Post-analytic
Report (IR1, and ammend reports, check SOP uptodat)

Answer 45

1.4.1 Actions
Record all of these actions in an IR1 incident notification

∙Inform Duty Consultant and Lead serology BMS
∙Ensure Primary tubes and aliquots -retain them, check labels, check whether done from primary or alliquot

Notify clinical team responsible for the patient;

IR1
Explain discrepant results received, an investigation and IR1 will be started, obtain names from clinical team to include on IR1, request a repeat HIV test with confirmation of patient identity

Change report on LMS, and state as ammended report

∙Initiate retesting of the original sample and record the original individual assay testing values in the IR1

∙If required genetic testing of the serum can be performed by virology at KCH to determine if one of
the samples is from a separate individual

Question 46

Steps of sanger sequencing

6 of them- following extraction and purification

DADDY G

Answer 46

1) denature
2) primer attached
3) add dNTPs and ddNTP
4) DNA synthesis reaction initiates and the chain extends until a termination nucleotide is randomly incorporated
5) Have fragments which are denatured
6) denatured fragments are separated by gel electrophoresis and the sequence is determined

DADDY G
Denature, Attach primer (similar to annealing), ddNTPs (for chain terminating extension using polymerase), Denature fragments, Gel electrophoresis

Question 47

Describe isothermal NAAT method types

Answer 47

LAMP
NASBA (RNA amplification)
TMA

(expand on this)
See paper by Sherry Denver- chemistry being used in clinical virology

Question 48

Haemagglutination assay and haemaglutinatiojn inhibition

Answer 48

Haemaglutination looks cloudy

For influenza viruses, hemagglutination is the process whereby the viral surface protein hemagglutinin binds to sialic acid sites on the surface of red blood cells (RBCs) thereby creating a sustained suspension of RBCs in solution. Typically RBCs of avian origin (chicken or turkey) or mammalian origin (horse or guinea pig) are used for analysis of influenza viruses. Hemagglutination inhibition titers are determined from the endpoint titration of antibodies in serum that bind to the virus, thereby inhibiting hemagglutination. HAI assays are conducted in 96-well U or V bottom plates, with each row or column consisting of a serial dilution of serum from a single sample. A fixed concentration of influenza virus antigen (4 HA units/25 μl) is added in each well to allow binding between hemagglutinin and anti-HA antibodies. Following an incubation period, a fixed concentration of RBCs is added to each well. If a sufficient number of anti-HA antibodies are present, hemagglutination will be inhibited or blocked and the RBCs will “precipitate” resulting in a solid red “button” (for avian-origin RBCs) or a hollow red “ring” (for mammalian-origin RBCs) at the bottom of the well that represents the non-agglutinated state. As the antibody concentration is decreased, hemagglutination will result and is visually observed as an opaque reddish haze within the well. The transition or endpoint between the non-agglutinated state and the agglutinated state is the titer (1/dilution factor).

Question 49

What is bridge amplication

Answer 49

Method used by illumina
takes place in a flow cell
generates clusters of DNA
Adaptor ligands on DNA bind to oligonucloetides formng a bridge-> replication (polymerase) -> denatured, forming two separate strands of DNA

https://frontlinegenomics.com/dna-amplification-techniques/

Question 50

Steps in illumina sequencing
ELBS

Answer 50

Extraction
Library prep
Bridge amplification
Sequencing by synthesis

Question 51

Where can you get external QA material

Answer 51

From SMI

Practice should, where possible be supported by reference
materials such as World Health Organization (WHO) or National Institute for Biological
Standards and Control (NIBSC) International Standards reference materials.
Standardisation of methods and results is key to the use of pathology data between
laboratories and for wider purposes such as surveillance

Question 52

Define validation

Answer 52

Confirmation through objective evidence that the requirements for a specific intended use or application have been fulfilled

Question 53

Define verification

Answer 53

Confirmation through objective evidence that the specified requirements have been fulfilled

Verify ability to achieve acceptable results with the method in question that has already been through validation

Question 54

Define avidity

Answer 54

Measures antibody maturity

Avidity
Avidity measures antibody maturity by determining the binding strength of antibody-antigen interactions. IgG avidity tests may be used as an additional diagnostic tool especially in patients with IgM test reactivity. Avidity is initially low after primary infection and increases over time, usually about 3 months. High IgG avidity suggests that infection occurred over 3 months ago. Low or moderate IgG avidity results should not be interpreted as diagnostic of recently acquired infection, as low or moderate avidity antibodies may persist for many months following infection in some individuals. Health care providers and clinical laboratories involved in pregnancy care should be aware that avidity testing is an adjunct to the other tests and should be interpreted with consideration of the other serological tests and ideally earlier results.

Question 55

Interpresting Western Blot

Answer 55

https://asm.org/Lesson-Plans/Interpretation-of-ELISA-and-Western-Blot-Assays-fo

Positive result At least two of the following HIV bands are recognized: p24, gp41, &
gp120/160. The presence of both gp160 and 120 counts as one band.
Note: Any number of additional bands may be present.
Negative result No bands whatsoever are recognized.
Indeterminate One or more bands of any size are recognized, but the criteria for a
“positive” result are not met.