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Evaluation of cell sensitivity

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Preparation and distribution of standards, reference reagents and training materials ... Titration of fresh aliquot of the same batch. Review of inoculation procedure ... – PowerPoint PPT presentation

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Title: Evaluation of cell sensitivity


1
Evaluation of cell sensitivity
  • Javier Martin

2
National Institute for Biological Standards and
Control (NIBSC)Potters Bar, HertfordshireUnited
Kingdom
3
Global Specialized Laboratories
  • Definitive identification of poliovirus isolates
  • Preparation and distribution of standards,
    reference reagents and training materials
  • Preparation and distribution of proficiency
    panels
  • Evaluation and training
  • Participation in collaborative studies
  • Research
  • Report results in a timely manner
  • Coordination and implementation containment
    activities
  • Coordination with EPI case investigators

4
3.1 The basis of Laboratory Quality
Assurance Laboratory Quality Assurance (LQA) is
concerned with the organisational processes and
the conditions under which laboratory activities
are planned, performed, monitored, recorded and
reported. Setting up a LQA system in a
laboratory means defining the organisational
structure, responsibilities, procedures,
processes and resources necessary to achieve the
following objectives To prevent risks. To
detect deviations. To correct errors. To
improve efficiency. To ensure data quality and
integrity
5
Monitoring sensitivity of cells
  • Routine monitoring of the sensitivity of cell
    lines for virus isolation is an important
    component of the laboratorys quality assurance
    programme.
  • Provides reassurance of cell lines ability to
    detect poliovirus infection
  • Impact factors include Mycoplasma contamination,
    quality of media, growth conditions, etc.
  • Morphological and other physical characteristics
    are not good markers for cell sensitivity

6
Evaluation of cell sensitivity
  • Using well characterised reference virus
    preparations of known and reproducible titre
  • Periodic titration of laboratory standards will
    provide quantitative data on the sensitivity of
    cells for polioviruses

7
Advantages of standard Sabin strains
  • Are authenticated as Sabin strains
  • Have an assigned titre so they can be used to
    monitor the sensitivity of cell cultures within a
    laboratory
  • Use of the same standard preparation will enable
    direct comparison of results between laboratories

8
Standard Sabin strains for titration
  • Standards for each serotype were prepared at
    NIBSC and are being supplied to lab network
    (2,500 vials per type)
  • Laboratories are requested to discard old stocks
    labelled as Sabin and replace with authenticated
    Sabin strains
  • (OPV vials are not recommended, since there may
    be small variations in titre between OPVs from
    different sources)

9
Characteristics of materials used to prepare
standard strains
10
Preparation of Sabin standards
  • Bulk materials diluted to a target titre of 5
    log10/0.1ml
  • Distributed into vials and labelled with unique
    code number
  • Titre, serotype and nucleotide sequence (5NCR,
    VP1 and 3D) determined at NIBSC
  • Stored at -70 C

11
SOP for virus titration
  • The WHO standard procedure used for vaccine
    potency assays will be used
  • Key parameters are dilutions intervals and number
    of replicate wells inoculated (recommend 10 wells
    per dilution)
  • The time of incubation after inoculation of virus
    and before reading is also defined

12
Virus titre determinations
  • The dilutions used are chosen to cover the 100
    to 0 dose response range
  • The precision of the assay depends on the
    intervals between dilutions and the number of
    replicates inoculated

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Calculation of the virus titre by the Kärber
formula
log CCID50 L d (S 0.5), where L log of
lowest dilution used in the test d difference
between log dilution steps S sum of proportion
of positive tests (i.e. cultures showing
CPE). In this example L -3.0 d 1.0 S 1
0.9 0.8 0.7 0.4 0.0 then log CCID50 -
6.30 Virus titre 106.30 CCID50 / 0.1 ml
15
Validation of Sabin standards
  • Titration in both L20B and RD cells in a
    collaborative study to assign a titre

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Mean titres and lower 1 1-tail limits
were (based on the between lab standard
deviation) Cell Type Mean Lower
Limit RD 1 5.10 4.53 RD 2 5.10 4.53 RD 3 5
.31 4.75 L20B 1 4.93 4.37 L20B 2 4.79 4.22
L20B 3 4.92 4.35
23
Supplies to laboratories
  • 5 ampoules per Sabin serotype are being supplied
    to each laboratory
  • SOP for the preparation of laboratory quality
    control standards to be included in the Polio
    Mnual
  • SOP for virus titration to be added to the Polio
    Manual

24
Cell evaluation schedule
  • Frequency of testing should be at least once soon
    after cells are initiated if possible at
    mid-point and end of passage
  • The sensitivity of the cell culture for
    poliovirus detection should be evaluated after a
    new lot of serum, new incubator, new technician
    or any other major change in the procedure has
    been introduced.

25
Documentation
  • Records to be kept on laboratory control charts
  • Regular reporting of results on standard forms to
    Laboratory Co-ordinators, HQ and NIBSC

26
Interpretation of results
  • If the observed titre is above the lower limits
    of the assigned titre for the reference
    preparation, then the cells are of adequate
    sensitivity
  • The observed titre should also be compared to a
    control chart of previous results from the
    laboratory to look for trends in virus titres

27
Actions to be taken
  • If cells found to be of low sensitivity
  • Titration of fresh aliquot of the same batch
  • Review of inoculation procedure
  • Replacement with new batch of documented
    sensitivity
  • Order new cells from WHO RRL
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