MarkRogers PDF

Biological Indicators (BIs) &
Chemical Indicators (CIs)

Marc Rogers, Ph.D.
Technical Service Specialist
Life Sciences
Formulated Chemistries
St. Louis, MO
USA
AEBioS
Madrid
11 March 2014
Copyright 2014 STERIS Corporation. All Rights Reserved. CONFIDENTIAL and PROPRIETARY to STERIS Corporation.
Agenda
Sterilization Methods
Biological Indicators (56*)
Discs, Strips, Suspensions
SCBI, PCD, Media, Custom BIs
Chemical Indicators (48*)

CI Classification, examples
Air Removal Tests
ISO and EP Guidelines
* Number of items not including VHP

Copyright 2014 STERIS Corporations. All Rights Reserved. CONFIDENTIAL and PROPRIETARY to STERIS Corporation.
Modes of Sterilization
High Temperature
Steam (Moist Heat)

Dry Heat
SOP, SIP, Steam-Air
Superheated water
Gas / Vapor
Ethylene Oxide
VHP, VHP Plasma
Chlorine Dioxide, NO2
Formaldehyde
Peracetic Acid
Ozone
Ionizing Radiation
Gamma Radiation
E-Beam
X-ray
UV light (non-ionizing)
Mechanical (High
Pressure, Filtration)
Liquid Chemical
(oxidizers, aldehydes)
Terminal Sterilization Methods

Medical Device Manufacture
Heat and
Other
E-beam
4%
4%
EO
50%
Gamma
42%
Pharma/ Biopharm Drug

Sterilization
Terminal Sterilization
Steam/Steamair mixture
Superheated
Water
23%
Other
2%
Aseptic Processing
Aseptic
75%
Ethylene Oxide Sterilization

Critical process parameters:
TIME (3-5 hours typical, depends on DEO value)

TEMPERATURE (120 135 F typical)
GAS CONCENTRATION (400-800 mg/L)
HUMIDITY (most 35-80% RH)
Where used:
Terminal sterilize heat / moisture sensitive medical devices (e.g.
catheters, implants, etc.)
Usually done off site at large contract sterilization facilities (i.e.
ISOMEDIX, Griffith Microscience, Ethox, etc.)
Frequency
Accounts for approx. 65% of medical devices sterilized in U.S.
Validated using thermocouples, biological indicators, etc.
Ethylene Oxide Sterilization

Inactivation of Spores by EO:
Reaction of EO with nucleic acids is the primary
cause of sporicidal activity
Alkylation of DNA causes cells to lose ability to
reproduce
VHP
Vaporized Hydrogen Peroxide STERIS VHP
Room Decontamination
Chambers
Vaporized Hydrogen Peroxide

Critical process parameters:
TIME
CONCENTRATION
HUMIDITY
SATURATION
TEMPERATURE
Where used:
Transfer isolator or sterile environment within a barrier
isolator (VHP 1000ED)
Newer application - terminal sterilization of heat

sensitive medical devices (similar to EO) utilizing
deep prevacuum phase (VHP MD and V-Pro)
Dry Heat Sterilization

Critical Process Parameters
Temperature
Time
Commonly used for:

Glass, metal items
Depyrogenation
Generally any dry heat process > 220 C
Rather than utilize BIs, compendia recommend
demonstrating 3-log reduction of endotoxins
Initial Validation with TC and BI

Dry Heat Sterilization
Inactivation of Spores by Dry Heat (B. atrophaeus)

Destruction of microorganisms primarily by oxidation
Damage to DNA to induce mutations
Moist Heat / Steam Sterilization

Equipment
Steam In Place (SIP) equipment
Autoclaves
Pre-Vacuum
Gravity
Steam Sterilization
Critical Process Parameters:
Time
Temperature
Saturated Steam
NOTE: FDA method of choice for the

sterilization of equipment and transfer lines
is saturated clean steam under pressure.
Common uses:
Non heat/moisture-sensitive product components and
supplies
Terminal sterilization of non-heat sensitive drugs
Sterilization of equipment / components used in aseptic
manufacture
Research or test materials, supplies, glassware, culture
media, cages, bedding, etc.
Decontamination of biohazardous or biological wastes
Anatomy of a Sterilization Cycle
Critical parameters of an
Autoclave Cycle
Load Configuration
Temperature Mapping
Sterile Barrier System (Packaging)
Penetration will depend on Density and Penetrability of
materials
Time, Pressure & Temperature parameters
Typical Parameters
121 C for 15 minutes
134 C for 3.5 minutes
Agenda

Air Removal Tests

Sterilization What is Sterility?

Sterility Assurance Level
In microbiology, it is impossible to prove that all
organisms have been destroyed/inactivated
SAL: Probability of a single viable microorganism
occurring on an item after sterilization (e.g., 10-6)
PNSU: Probability of a Non-Sterile Unit
From PDA Technical Report #1 (Revised 2007), Validation of Moist Heat
Sterilization Processes: Cycle Design, Development, Qualification, and
Ongoing Control
EN556-1:2001 Requirements for medical devices to be designated
STERILE defines sterilization as an SAL = 10-6
17
Sterility Assurance
European approach vs. U.S. approach
EU based on parametric release (declares
product is sterile based on records that process
parameters were delivered within specified
tolerances (ISO 11139)
U.S. - based on BI monitoring of
each load (CIs support BIs)
CIs are an intrinsic part of each
process indicators, in-pack indicators,
Bowie Dick tests
What is Sterility Assurance?

A way of evaluating or monitoring a sterilization process/ products
to ensure that all pathogens (disease causing microbes) have
been inactivated/ destroyed and to provide protection from
recontamination; used as a supplement to physical monitoring.
Higher more
quantifiable level of
sterility assurance
3 Levels of Sterility Assurance
Biological Indicators
Chemical Integrators
Chemical Indicators
19
What are they?
Living microorganisms intended to present a worstcase for a sterilization process (i.e. steam)
Working principle
If a high number (population) of the most resistant
organisms to a particular sterilant can be inactivated
by sterilization , there is a high PROBABILITY that all
other organisms at that location in the sterilizer will
also be inactivated
20
Common BI Uses:
Qualification testing of sterilization equipment.
Validation of a sterilization process or cycle, or for
periodic revalidation, in conjunction with type T
thermocouples, Kaye Validators, data loggers,
recorders, etc.
Routine monitoring of a sterilization cycle.
21
Biological Indicator Definitions
Spores
The dormant stage of some microorganisms most resistant to

environmental stress
Media
The perfect food to grow spores (i.e., convert them into bacteria
that can reproduce)
Incubation
Spores usually grow fastest within fairly narrow temperature

ranges. The process of heating the media and spore to grow is
incubation
Population The number of spores on a BI usually measured as 106 (i.e.,

1,000,000)
Controls
Positive control (not sterilized) & negative control (sterile, so

should have no growth) used in validation
22
What spores are used?

G. stearothermophilus
STEAM (& VHP)

Geobacillus stearothermophilus
(GST, Gram positive)
Formerly Bacillus stearothermophilus
Thermophile (heat loving)
Growth at 55 to 60C (131 to
140F)
No growth at 37C (98.6F)
Not pathogenic to humans
Singh MK et al. 2009. New

J Phys 11:115027
EO & Dry Heat

Bacillus atrophaeus (formerly
B. subtilis var. niger)
Plomp M et al. 2005. Langmuir 21(23):10710-6.
23
BI Survivor Curve
Log Number of Survivors
Number of Survivors
10,000
1,000
100
10
1
D-Value
D-Value is the time to reduce

the microbial population by
90% or 1 log
0.1
-1
-2
0.01
0
Exposure Time (Minutes)
10
11
12
24
BI Formats Pros/Cons
SCBI
Strips
Discs
Suspensions
Advantages
Complete
system
No transfer
required
No micro lab
required
Broad
applicability
Cost
Size (6.3 mm
diameter)
Cost
Flexibility in
applications
Allows for
direct
inoculation
Disadvantages
Not
submersible
Size
Requires
aseptic
transfer
Not
individually
packaged
Requires
aseptic
transfer
D-value may
need to be
determined for
different
substrates
25
Suspensions
10 mL vial of bacterial spores in water
Comes with syringe for use in inoculation of product
One vial is good for approximately 100 product inoculations
Spores GST & BA

Population 103 to 109/0.1 mL
3,4,5,8,9 logs are Special Order
Resistance Available for:

Steam Sterilization 1.5 to 2.5
Dry Heat Sterilization 1.0 to 3.0
EO Sterilization 2.6 to 4.5
Expiration 18 mo. from DOM

Storage at 2-8C (No RH req.)
26
Suspensions
Applications
Steam, Dry Heat and EO
To directly inoculate a medical device to determine
the worst-case location where a spore strip or disc
cannot be used
To directly inoculate a product in the places least
accessible to the sterilization agent
To manufacture biological indicators using a substrate
similar or the same as the product being sterilized
To monitor sterilization of liquids
27
Strips
1.5" X .25" strip of filter paper packaged in a
glassine envelope
Spores - GST & BA
Spordex single species
Spordi dual species
Population - 1.0 X 104 to 5 X 106

Resistance D value 1.0 to 4.5 min
Expiration -18 mo. from DOM
Storage at 2-24C and 30-80% RH
No Direct Sunlight or oxidative chemistry exposure
28
Strips
Applications
Most widely used format
Validation and routine monitoring of steam, EO, and
Dry Heat sterilization cycles
Used in PCDs
EO and Dry Heat standards
USP, ANSI/AAMI/ISO/EP
Steam standards
USP and ISO
29
Discs
.25 round unpackaged filter paper
disc inoculated with spores
Spores GST & BA
Population - 1.0 X 106 to 5 X 106
Resistance D value 1.0 to 4.5
minutes
Storage at 2-24C and 30-80% RH
No direct sunlight or oxidative
chemistry
30
Discs
Applications
Dry heat, steam and EO sterilization
Commonly used in development of PCDs
Because of their size they may be used in areas
where strips will not physically fit
Tool in determining worst case location within a
product
USP
31
BI Media (Spordex)
Sterile modified soybean

casein broth
Each lot validated for use with
Spordi and Spordex strips
Meet current USP
requirements for growth
promotion
All D-values for the BIs are
based on testing using
Spordex Media
To duplicate our D-value, our
media should be used
SCBIs
BI system designed to eliminate
the challenges and time needed
for aseptic handling
Spores GST & BA
Population 1 X 105 to 5 x 106
Resistance D value 1.5 to
5.8 minutes
Expiration 12 mo from DOM
Storage 20-25C and 30-80%
RH
33
SCBI Configuration
Plastic vial and cap
Paper disc inoculated with bacterial spores
Crushable glass ampoule containing culture
media and pH indicators (bromthymol blue,
bromocresol purple)
Process chemical indicator for steam or EO
sterilization
Dual species(S3060, S3061) and single
species (S3111, S3112)
Colors are an approximation
S3111 and S3112 are tested in accordance

with International Standard ISO 11138 and
EU Standard EN866; Facilitates
harmonization of BIs between global
customer sites
Positive
(Failure)
Caramelized
SCBI Processing
1) Place the SCBI in the special

tool (Activator Set S3075)
3) Push or pull the Verify SCBI completely

through the gap on the side of the activator
to crush the media ampoule.
2) Press down
the blue cap to
close vial to
protect SCBI
from outside
contaminates
4) Place the
Verify SCBI in
an incubator
at the
appropriate
temperature.
35
Incubating the SCBI
Available in 120 VAC

and 220 VAC
Incubation Temperature:
37C (35C - 39C) EO

57C (55C - 60C) Steam
Media does not evaporate if incubated for 7 days

- 24 hour reduced incubation time for steam (S3060,S3061)
- 48 hour reduced incubation time for EO (S3060, S3061)
- Customers can validate a reduced incubation time (less than 7 days) for S3111, S3112
PCD
Spores - GST & BA
Population 1 X 105 to 5 x 106
Resistance - 1.4 to 5.8
minutes
Storage at 20-25C and 3080% RH
Accessories
Incubator
Activator tool S3075
S3065 Steam
EO PCD
Custom BI Products
Pinecone Biologics can offer
a variety of custom products
Pricing and timing are given
once the form in submitted
Form available on the
intranet
Life Sciences
Sales and Marketing
FC Pricing
Agenda

Air Removal Tests

What are chemical indicators

(CIs)?
A chemical indicator is any substance that gives
a visible sign, usually by a color change, of the
presence or absence of a threshold
concentration of a chemical species or physical
parameter
In terms of steam sterilization, chemical
indicators may indicate:
Time
Temperature
Steam/Water
40
What are chemical indicators

(CIs)?
Chemical indicators do not:
Indicate sterility: CIs provide a visual indication that
the indicator has been exposed to the physical
parameters (time, temperature, steam) required to
achieve sterility
React based on lethality: Although microorganisms
may be killed at sub lethal temperatures (<121C), a
CI will not indicate a pass unless ALL parameters
have been met
41
Classifications of CIs
The ANSI/AAMI/ISO 11140-1:2005 document
defines six classes of chemical indicators
The indicators are subdivided within these 6
classes based upon the information they relay to
the user
The classification structure is used solely to
denote the characteristics and intended use of
each type of indicator as defined by the
manufacturer. The classification has no
hierarchical significance.
42
Class
ANSI/AAMI/ISO 11140-1: Practical Application

2005 Definition
Class 1:
Process Indicators
Process indicators are

intended for use with
individual units, (e.g.,
packs, containers) to
indicate that the unit has
been directly exposed to
the sterilization process
and to distinguish
between processed and
unprocessed units. They
shall be designed to react
to one or more of the
critical process
variables.
(PCC003, PCC004)
Verify EO
(PCC009, PCC010)
Verify Steam
(260010) steam tape
(410100) EO tape
Indicator tapes, indicator

labels, and load cards
are
examples of externally
visible Chemical
Indicators that
are Process Indicators
used for exposure control
VHP and Dry Heat
chemical indicators
commonly fall into this
category
43
Class
ANSI/AAMI/ISO 11140-1:
2005 Definition
Practical Application
Class 2:
Indicators
for use
in Specific
Tests
Class 2 indicators are

intended for use in specific
test procedures as defined
in relevant
sterilizer/sterilization
standards.
Bowie Dick type tests are specific

tests used for equipment control to
evaluate the sterilizer
performance.
e.g. EQC003 Steraffirm Bowie Dick Test Pack; EQC009, EQC010 Verify
Bowie Dick Test Packs; NB113, NB125 DART Daily Air Removal Test; S3095
Verify Bowie Dick Test Card
44
Class
ANSI/AAMI/ISO 11140-1:
2005 Definition
Class 3:
Single
Variable
Indicators
A single variable indicator

shall be designed to react to
one of the critical variables
and is intended to indicate
exposure to a sterilization
process at a stated value
(SV) of the chosen
variable.
An example of a Single Variable

Indicator is a
temperature tube that contains a
chemical pellet that melts at a
specific temperature. Single
variable indicators may be used for
pack control monitoring but would
not provide as much information
as a Class 4 or Class 5 Chemical
Indicator. Single Variable
Indicators may also be used for
exposure control monitoring.
45
Class
ANSI/AAMI/ISO
11140-1: 2005
Definition
Class 4:
Multivariable
Indicators
A multi-variable
indicator shall be
designed to react
to two or more of
the critical
variables and is
intended to indicate
exposure to a
sterilization cycle at
SVs of the chosen
variable.
Multi-variable Chemical Indicators are used

for pack control. These internal Chemical
Indicators are usually paper strips printed
with a Chemical Indicator.
e.g. Steraffirm Sterilizer Control Tube 121C 15 min (PCC024), and 12 min
(PCC025); at 124C for 3 min (PCC026)
46
Class
ANSI/AAMI/ISO
11140-1: 2005
Definition
Class 5:
Integrating
Indicators
Integrating
indicators shall be
designed to react
to all critical
variables. The SVs
are generated to
be equivalent to, or
exceed the
performance
requirements given
in the ISO 11138
series for BIs.
Integrating Indicators are the most accurate

of the internal Chemical Indicators.
Integrating Indicators are used for pack
control monitoring. They can also be used as
an additional monitoring tool to release loads
that do not contain implants. For this
additional monitoring the Class 5 Integrating
Indicator must be used in the appropriate
challenge test pack or Process Challenge
Device (PCD). These indicators must now
have SVs at 121C/250F, 135C/276F, and
at least one more temperature in between.
Also, the SV at 121C MUST be greater than
16.5 minutes to ensure performance is
comparable to BIs in saturated steam.
PCC039
VERIFY
Integrating
Indicator
for Steam
47
Class
ANSI/AAMI/ISO
11140-1: 2005
Definition
Class 6:
Emulating
Emulating indicators are cycle
Indicators verification
indicators which
Steraffirm
shall be designed to
Sterilizer
react to all critical
Control
variables for
Tubes
PCC027,
specified
PCC028
sterilization cycles.
The SVs are
Steraffirm
Steam Class generated from the
6 Emulating
critical variables of
Indicators
the specified
PCC012
sterilization
PCC013
PCC014
process.
Emulating Indicators can be used as

internal Chemical Indicators for pack
control. These indicators are indentified for
specific sterilization cycles which means an
end user will need to inventory a different
Class 6 Emulating Indicator for each
sterilization cycle time and temperature
(i.e., 3 min, 5 min, 10 min, 18 min, 40
min,etc.) performed in the facility. The
response of a Class 6 Emulating Indicator
does not necessarily correlate to a BI so the
indicator cannot be used as an additional
monitoring tool to release loads that do not
contain implants. (See Class 5 definition)
48
Chemical Indicators
May consist of sensitive
chemical ink/dye applied to
paper or in liquid
Immediate visual indication of
exposure to a specific sterilant
(e.g. steam, EO, dry heat)
Assist detection of sterilizer
malfunctions and personnel
errors
Many different types
available
Steraffirm Class 6 Emulating

Indicators
Cycle Specific
Steam sterilization
Measures temperature, saturated steam and time
121C/250F for 15 minutes

Use inside individual packs, trays or pouches
Other indicators available for different cycles
50
Verify Integrating Class 5

Indicator (PCC039)
Steam sterilization
Measures temperature, saturated steam and time
Stated Values are 121C (18 min), 132C (3 min), and

134C (2.5 min)
Placed into each pack to be steam sterilized
No ink color interpretation
Clear and unambiguous
Correlated to bacterial spore death
51
Difference between Class 5 & 6

Class 6 emulating indicators
Cycle specific
React to all critical variables within a specific cycle
Class 5 integrating indicators

Not cycle specific
React to all critical variables, designed to meet or
exceed the BI performance requirements
52
Steraffirm Sterilization Control

Tubes
Glass tube with a heat sensitive liquid

which will change color from red to green
Used routine monitoring of gravity,

prevacuum or liquid cycles
Product Range:
Steam (ISO Class 4) - dry goods or liquids
134C 3 mins
121C 15 mins
121C 12 mins
Dry Heat (ISO Class 6)
160C 120 mins
160C 60 mins
(Note: can vary times and temperatures
based on Tech Data e.g. low temp, raise

time)
VHP BIs and CIs
Spordex S24 SCBI (NA340), 24-h readout

Verify S24 SCBI Activator (LCB004)
Steraffirm
Process Indicator (PCC049, PCC050)
Steraffirm
Process Indicator PCC051
PCC049
VHP BIs and CIs
VHP BIs and CIs
VHP BIs and CIs

NA117, culture
Media for use with
NA333 BIs, 24-h
RIT
NA333, E6 GST 12980 on SS in

Tyvek
Steraffirm PCC060 for

Intermediate to high
H2O2 vapor concentrations
Agenda

Air Removal Tests

Testing for Air Removal

Bowie and Dick Type tests for air removal
Utilize a steam sensitive chemical indicator and
porous barrier or challenge material (e.g., paper,
cloth)
Test pack serves as a heat sink in otherwise empty
chamber
Any air not removed during prevac cycles will be
concentrated in test pack
Residual air prevents steam contact with chemical
indicator, preventing color change
59
How the Bowie Dick Test Detects

Air (Illustrated)
AIR
AIR
STEAM
AIR
BD TEST
STERILIZER DRAIN
AIR
AIR
CHAMBER TRAP
60
Air Removal Indicators

Steraffirm Bowie Dick
Test Pack
Tests air removal of prevacuum sterilizer cycles
with cycles of 121-124 C
Most popular
Easy to use - single use
Diagnostic capability
pattern of failure can help
isolate problem
Lead free ink
Available Bowie Dick Test Packs

A range of preassembled, single use Bowie Dick Test Packs:
134C-137C (273F-279F) for 3.5 minutes
132C (270F) for 3 to 4 minutes
121C- 124C (250F-255F) for 8 8.3 minutes*
*For use by any LS customer using a prevacuum steam sterilization cycle

Steraffirm Bowie Dick Test

Packs
AIR REMOVAL
NON-CONDENSABLES
WET STEAM
SUPERHEAT
DART TEST
Preassembled, Single Use Test
Dart Temperature Range:

For prevacuum cycles at 132-134C for 3.5-4 minutes only
Agenda

Air Removal Tests

Regulatory Guidance
Standards Hierarchy
EN Standards
EN ISO 11138 Series

EN ISO 11138-1* was first published 1994,
revised 2006
It specifies requirements for the labeling and
performance of BIs
Subsequent parts give performance
requirements of BIs for specific sterilization
methods, e.g. ethylene oxide (Part 2), moist heat
(Part 3), dry heat (Part 4), formaldehyde (Part 5)
EN ISO 11138-6 hydrogen peroxide vapor
processes in development
*Sterilization of health care products Biological Indicators Part 1: General requirements
EN ISO 11140 Series

EN ISO 11140-1* first published 1995, revised 2005
Specifies requirements for labeling and performance of
CIs
Subsequent parts give performance requirements for
Bowie Dick type indicators
Class 2 indicator systems for use in the Bowie and Dicktype steam penetration test (Part 3)
Alternatives to B-D, European (Part 4)
Alternatives to B-D, USA (Part 5)
Part 6 in development Class 2 indicators and process
challenge devices for use in performance testing of
steam sterilizers
*Sterilization of health care products Chemical indicators Part 1: General requirements
Requirements for BI Spore

Population (per unit)
Requirements for BI D-Value
Sterilization Guidance
EN ISO 14161
First published in 2000
Guidance on selection, use, and interpretation of
results of BIs when used to develop, validate
and monitor sterilization processes
EN ISO 15882
First published 2003
Gives guidance for users on the selection,
use, and interpretation of results of CIs
Process Validation Guidance
Agenda

Air Removal Tests

QUESTIONS?
marc_rogers@steris.com
75

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Biological Indicators (BIs) &

Chemical Indicators (CIs)

Chemical Indicators (48*)

ISO and EP Guidelines

* Number of items not including VHP

Steam (Moist Heat)

Terminal Sterilization Methods

Pharma/ Biopharm Drug

Ethylene Oxide Sterilization

TIME (3-5 hours typical, depends on DEO value)

Ethylene Oxide Sterilization

Vaporized Hydrogen Peroxide

Newer application - terminal sterilization of heat

Dry Heat Sterilization

Commonly used for:

Initial Validation with TC and BI

Dry Heat Sterilization

Inactivation of Spores by Dry Heat (B. atrophaeus)

Moist Heat / Steam Sterilization

NOTE: FDA method of choice for the

Anatomy of a Sterilization Cycle

Chemical Indicators (48*)

ISO and EP Guidelines

* Number of items not including VHP

Sterilization What is Sterility?

What is Sterility Assurance?

3 Levels of Sterility Assurance

Biological Indicator Definitions

The dormant stage of some microorganisms most resistant to

Spores usually grow fastest within fairly narrow temperature

Population The number of spores on a BI usually measured as 106 (i.e.,

Positive control (not sterilized) & negative control (sterile, so

What spores are used?

STEAM (& VHP)

Singh MK et al. 2009. New

EO & Dry Heat

D-Value is the time to reduce

Exposure Time (Minutes)

Spores GST & BA

Resistance Available for:

Expiration 18 mo. from DOM

Population - 1.0 X 104 to 5 X 106

Sterile modified soybean

Colors are an approximation

S3111 and S3112 are tested in accordance

1) Place the SCBI in the special

3) Push or pull the Verify SCBI completely

Incubating the SCBI

Available in 120 VAC

37C (35C - 39C) EO

Media does not evaporate if incubated for 7 days

Chemical Indicators (48*)

ISO and EP Guidelines

* Number of items not including VHP

What are chemical indicators

What are chemical indicators

ANSI/AAMI/ISO 11140-1: Practical Application

Process indicators are

Indicator tapes, indicator

Class 2 indicators are

Bowie Dick type tests are specific

A single variable indicator

An example of a Single Variable