Genitourin Med 1985;61:88-94

Internal quality control in serological tests for

syphilis
G D WASLEY
From the Department of Immunology, St Thomas's Hospital Medical School, London SE] 7EH

SUMMARY The importance of syphilis serological tests demands that laboratory reports are
reliable. Internal quality control applied to the organisation of a syphilis serology service improves
laboratory bench performance and reporting. Described here are internal quality control
procedures of a department that serves a genitourinary medicine clinic and conducts 70 000 tests a
year to investigate for syphilis.

Introduction IMPLICATION OF INTERNAL QUALITY CONTROL

Table I lists the procedures undertaken in the
The history of testing serum for lipoidal and organisation of a syphilis serology service. At any
treponemal antibodies is well documented. stage from (a) to (i) an error can occur, and these
Wassermann et al applied the complement fixation errors may be multiplied. To prevent mistakes it is
reaction to investigating syphilis.' Thereafter
numerous serological tests have been described with TABLE I Organisation of syphilis serology service
the objectives of improved specificity and sensitivity.
Diagnostic confirmation of early clinical syphilis is (a) Sources and types of requests
by the recognition of viable Treponema pallidum (b) Reception of specimen
Sorting; clerking in; preparation; storage; selection of
from suitable lesions using dark field microscopy. In tests
recent years several workers including Daniels and (c) Daily organisation
Work sheets; timing and sequence of operations
Ferneyhough have proposed using a direct (d) VDRL method (i) Reagents
fluorescent antibody staining technique as an (ii) Dilutions of serum
alternative method.2 The clinician will request (iii) Conduct of tests
(e) TPHA method (i) Reading individual results
serological tests to support positive microscopical (ii) Batch testing
results. Microscopy sometimes gives negative results (f) FTA-ABS
(g) Recording of results
when the clinical history of the patient indicates early Individual tests
syphilis: it is then that serological tests are essential to (h)
Batch testing
Reporting and surveillance of results
confirm diagnoses based on the clinician' s Individual test forms
experience. A battery of serological tests provides an Procedures for scrutiny of results
antibody profile suggesting active or past treponemal Reporting results to clinics
Monthly and quarterly returns
infection. Problem results can be checked by using (i) Retrieval of results
the facilities of a reference laboratory. Untreated and Individual tests
Batches and categories
treated disease usually follows predictable serological (j) Quality control
patterns with individual variations. The choice of Use of reference reagents
tests depends on local policy. I consider three such Quality control procedures
Participation in external quality assurance
tests: the Venereal Disease Research Laboratory (k) Repeats and referrals
(VDRL) test, the Treponema pallidum Indications for repeating tests on single specimens
Indications for requesting repeat specimens
haemagglutination assay (TPHA), and the Indications for referring patients
fluorescent treponemal antibody absorbed (FTA- Indications for referring serum sample to reference
ABS) test. laboratory
(1) Organisational and financial
Handling, storage, clerking in
Performance of tests
Reportinig, sorting, and retrieval of results
Address for reprints: Mr G D Wasley, Department of Immunology, Weighting of individual tests
St Thomas's Hospital Medical School, London SEI 7EH Anticipated workload
Anticipated costs
Accepted for publication 25 May 1984
88
Internal quality control in serological tests for syphilis 89

obviously necessary that an internal quality control Materials and methods

scheme should be implemented. Examples of factors
affecting laboratory results are: mislabelled blood REFERENCE SERUM
specimens; contaminated serum; clerical errors; International biological standards are used to
errors of transfer of liquid; errors of technique; compare similar materials under the same test
inadequate evaluation of reagents; insufficient conditions. In referring to their use it is necessary to
reagent controls; unofficial modification of identify the status of the material in the categories of
protocols; inadequate performance of equipment; the World Health Organisation (WHO)
lack of understanding of laboratory routine; nomenclature of standards.3 The WHO reference
insufficient supervision; and poor scrutiny of results. serum (ref 3-1980) for syphilis serological tests is a
Although the onus is on individual laboratories to WHO international reference reagent. The serum is a
introduce their own internal quality control lyophilised pooled preparation; it is used to calibrate
procedures, no active encouragement in this field has in house standards used in routine serological tests
been given to laboratories undertaking syphilis for treponemal infections.
serological tests through the agency of the external
quality assurance (EQA) scheme of the United CALIBRATING IN HOUSE STANDARDS FOR
Kingdom. Participation in an external quality THE TPHA AND VDRL TESTS
assurance scheme provides retrospective comparison The WHO reference serum is not a consumable
of laboratory performance against a target value or a product to be used in every batch of tests. It is
consensus of results. A laboratory participating in therefore necessary to establish in house standards
external quality assurance without its own internal whose titre relative to that of the WHO reference is
quality control, however, may be under an illusion measured using precisely the conditions and reagents
regarding its own routine performance because it used for routine tests. The remainder of the reference
may set up special procedures for external quality material is stored as a master serum and used
assurance samples. Internal quality control gives periodically to check the in house standard for
information on routine laboratory performance at evidence of drift, to establish a new batch of local
the time of testing. It acts as an early warning system standard material, or for recalibration when
to enable errors, malfunctions of equipment, or modifications or new reagents have been introduced
substandard reagents to be identified and corrected. into the existing method. Preparations are mnade in
Scrutinising data on patients can detect mistakes of advance of the proposed date of calibration to ensure
sampling, collecting, handling, and clerking that that sufficient volumes of serum are available. This
external quality assurance schemes do not disclose. entails using a serum bank ( 700C) with the relevant
-

Improved performance derived from internal quality records of the source, volume, and serological profile
control will benefit the management of patients. of serum specimens. All banked serum samples are
Implementing internal quality control is not HBsAg negative.
difficult but requires discipline from the laboratory In house standards for the TPHA and VDRL tests
staff in respect of monitoring and recording specific are calibrated against the WHO reference serum. An
data. It also costs time; an estimated 12/o of a in house standard titre does not have to equal the
specific work load is taken up by internal quality reference titre. Whenever further comparisons are
control, and laboratories should include this as a made between the two serum samples, however, the
separate item of expenditure in their financial in house standard is expected to maintain its titre in
budgets. relation to the WHO reference serum. Serum samples

TABLE 11 Interpretation and designation of categories of test results

Appearances and titres of test reactions designated:
Positive or reactive
Negative or
non-reactive Borderline Minimal Moderate High Very high
VDRL flocculation No clumps Small clumps Small clumps Medium or large. clumps
VDRL titre 1/0 1/1-1/2 1/4-1/8 1/16-1/64 >1/128
TPHA titre 1/0-1/40 1/80-1/160 1/320-1/640 1/12801/5120 >1/5120
FTA-ABS fluorescence 0 + ++ + + + , +++
(shades of apple green) (faintly visible) (very bright) (brilliant)
VDRL = Venereal Disease Research Laboratory test. TPHA = Treponema pallidum haemagglutination assay. FTA-ABS = fluorescent
treponemal antibody absorbed test.
909 D Wasley
with an antibody titre designated "moderate" in cannot be met by the manufacturer as the ultimate
TPHA and VDRL tests are chosen for in house value depends on the microscope, substrate, and
standards (see table II). reagents used in any individual laboratory. To
Titrations of the reference serum and the proposed measure the working dilution a two dimensional
in house standard are performed in parallel at least (chessboard) titration is performed using a known
30 times, from which the geometric mean titre and middle layer reactive control (see fig 1). A pooled
geometric standard deviation are calculated. Each in normal serum control is included to detect "serum
house standard is further tested to establish its induced" non-specific staining, and a buffer
suitability under routine bench conditions. A blind (phosphate buffered saline (PBS)) control to detect
series of tests is carried out to eliminate bias. non-specific staining by the conjugate. A satisfactory
Satisfactory standards are sterilised by membrane chessboard titration would require a titre of in house
filtrations. Aliquots of 25 MI serum are distributed serum (the middle layer) at which minimal
into vials and stored at - 70°C. The unused WHO fluorescence reactivity (see table II) occurs over a
reference serum is also distributed in similar volumes range of at least three conjugate dilutions.4 This
and stored in the serum bank. In house standards are critical serum titre is the "plateau level". The end of
included in routine test batches at random so they are the range of conjugate dilutions over which
not identifiable by the laboratory worker performing fluorescence occurs at the plateau level is reported as
the tests. The reactivity of each batch of freshly the plateau end poiont. The working titre of the
prepared VDRL antigen is checked using an aliquot conjugate is calculated as being twice the
of in house standard before being used in routine concentration, or half the titre, of the plateau end
tests. point when using doubling dilutions. The length of
THE FLUORESCENT TREPONEMAL ANTIBODY
the plateau level under the conditions described
ABSORBED TEST
above is important. The higher the concentration of
The FTA-ABS test is a confirmatory test for antibody in a conjugate the more it can be diluted but
still retain its plateau level (titre). Conjugates with
detecting antitreponemal IgG. It is extremely long plateau levels, and hence high working
sensitive, and unofficial alterations of laboratory
protocols will lead to unreliable results. Evaluation
of all reagents is necessary. Laboratory bench
records for FTA-ABS tests must include a record of Plateau end point
the working titre and batch number of the reagents 1/10240
used on each occasion and the reading score for each Plateau level - -

test serum. 1/5120 + + + + + _ -

The FTA-ABS antigen c 1/2560 + 4 + +

4. 4 --I
I -
L
Tpallidum (Nichols strain) maintained by passage in 0 - - -
I

rabbits is the source of FTA-ABS antigen. Antigen 1 1/1280

._
++ + ++ + + + I _
suspensions develop hyperreactivity after storage for
six weeks at 4°C. Slide preparations of antigen have a E 1/640 40 44. 44 4. 4. 4. 4. _

limited shelf life; reduced reactivity occurs after a 2

period of time. Antigen is assessed by comparing it * 1/320 44. 44 44. 4 4. 4. 4. _

with known material using in house standards. At

least 30 treponemal organisms per microscope field 11/160 44-6 +4+4 4.44 444 444. 4. _.

(x 40 objective) are used for each FTA test.

Inadequate numbers of treponemes make it difficult 1l/80 444.4+4. 4+ +_ 4++ 4+ 4. _

for the microscopist to distinguish shades of apple 1/40 ++ 4+ 4. _

green. The preparation of antigen smears is a tedious
but important job, as they need to be spread evenly 1/20 444. III +4- 444. 4+ 4+ _.
before being fixed with 10% methanol. Each smear is
checked under the dark field microscope to ensure
that sufficient treponemes are present. Fixed
preparations are stored in airtight plastic bags at .,( AS eP Ne s\e I\V\
sN
-200C.
Conjugate dilutions
Fluorescein isothiocyanate (FITC) conjugate (substrate Treponemna pallidum (Nichols))
The working dilution of an FITC conjugate must be FIG I Chessboard titration indicating plateau level and
ascertained to evaluate its suitability. This criterion plateau end point.
Internal quality control in serological tests for syphilis 91
dilutions, are more efficient, and also more VENEREAL DISEASE RESEARCH LABORATORY
economical, because they reduce background TEST (VDRL)
staining. Acceptable FITC conjugates are aliquoted The VDRL test was first reported by Harris et al in
into working volumes and stored at - 20°C. 19466 and has since undergone various
modifications. It is a flocculation test, which detects
Middle layer reactive controls mainly IgM class lipoidal antibody.7 Reactions are
Middle layer reactive controls are included with each classified according to the appearance of flocculation
batch of FTA-ABS tests. These reactive controls are indicating the concentration of lipoidal antibody
used in a different way from the middle layer control (table II). Vigilance is required with this simple test to
referred to in the evaluation of the FITC conjugate. ensure that no unauthorised modifications, however
They are calibrated by comparison with the WHO well meant, are introduced. Speeds of rotation,
reference serum using the same batch of antigen. volumes of liquid, storage of reagents, and methods
Dilutions of the serum are selected that will give the of reading reactions are factors that differ when
required reactions. Middle layer controls are comparing manufacturers' protocols.
sterilised by membrane filtration, distributed in A VDRL carbon added reagent, a modification
working volumes, and stored at - 20°C. used in this laboratory, enhances the end point
The following reactive controls are used in each reading of the test. The shelf life of VDRL reagents is
test batch: standard minimum reactive control important, as false reactions will occur if they are
(designated +); reactive (+ + + +) control in PBS; used after the stated expiry date. Serum samples
reactive (+ + + +) control in sorbent. The minimal exhibiting prozones are uncommon. To exclude this
reactive control serum is used as an indicator of the phenomenon, however, samples from patients with
minimum fluorescence that is regarded as showing a primary and secondary syphilis that give negative
positive result (the reading standard). results to the VDRL test require retesting in dilution
to ascertain whether they have any lipoidal antibody
concentrations.
Batch testing of sorbent
New batches of sorbent are quality controlled using a WALL CONTROL CHARTS
non-syphilitic serum that is reactive when diluted in Quality control wall charts indicating limits of
PBS but non-reactive when diluted in sorbent. acceptance are used to plot in house standard values
for each batch of tests performed. Recording the
Non-reactive control distribution of values monitors trends in precision
A non-reactive control serum is included in each and accuracy. Precision is used to describe the ability
batch of tests. Serum samples are confirmed as being to obtain closely related values for duplicated
non-reactive by testing dilutions of them in PBS and specimens (such as in house standards). Accuracy is
sorbent. an expression of the nearness of one of a series of
estimates to what is accepted to be the true value of
(TREPONEMA PALLIDUM HAEMAGGLUTINATION the substance being measured. A wall chart is
ASSAY (TPHA) prepared by taking the geometric mean and
This passive haemagglutination test is used to detect geometric standard deviation values. Fig 2 shows
antitreponemal IgG, and its sensitivity was reviewed
by Notowicz and Menke in 1981.5 Comparative tests (a) Poor precision (out of control)
are made on all new batches of reagents, as variation GSD 22- ---
occurs between batches. The recognition of the end
point is made absolutely clear to staff, as the subtle Mean - ----
changes in the haemagglutination pattern can result
in variable reading of the end point. TPHA results GSD -2---
are influenced by chemical contamination, and all
glass and plastics used must therefore be (b) Maintained accuracy (decreased precision)
scrupulously cleaned. During the preparation of GSD .2-----
reagents and in the actual performance of the test
close attention must be given to accuracy in Mean - . - - -
measuring fluid volumes, and in the precise way
volumes of liquid are transferred. Manufacturers' GSD -2
instructions to prepare TPHA reagents daily are GSD = geometric standard deviation
followed to retain the calibrated sensitivity of the FIG 2 Example of wall charts used for plotting in house
test. standard values.
92 G D Wasley
examples of results with differing precision. The Scrutiny of laboratory results
distribution should indicate that: Syphilis serology results are scrutinised by an
95 501 of reactive control results are within two authorised member of staff. A problem area is
geometric standard deviations from the mean. commenting on an individual result from a large
batch of specimens received from a department of
Mean and standard deviation values may not genitourinary medicine, antenatal clinic, or blood
coincide with actual titres, but that in no way donor unit. These serum samples will often arrive in
invalidates the result. batches accompanied by lists of names as opposed to
individual request forms with relevant information.
GENERAL ASPECTS OF INTERNAL QUALITY Fig 3 shows a record card, which is used to scrutinise
CONTROL results and detect errors in transcription or transfer
Scnrtiny of incoming blood specimens of liquid. Suspect test results are repeated from the
Identification of a blood specimen with the original clot specimen. All serum samples giving
corresponding request form appears to be simple positive test results are filed in the serum bank.
enough, but for a variety of reasons that include
misreading of names or numbers, inadequate Skilled performance
information, and sorting of specimens, mistakes do Internal quality control includes monitoring
occur. Ideally, permanent staff should be used to individual skills, the levels of which appreciably
prepare specimens. If temporary help is given influence results. Adequate skilled performance
because of increased workloads or staffing problems, should not be assumed to be maintained all the time.
intensive supervision must be used to control the If the performance of a skilled worker is compared
clerking and preparation of the blood. Laboratory with those less skilful, the difference lies not so much
meetings should be held periodically to discuss this in the movements as in the way the senses are
area of control with the objective of improving the channelled and organised and in the precise and
system. timely way decisions are made. Fig 4 shows how a
VDRL test can be analysed, with each component
part further divided to obtain a critical analysis.
Preparation of serum
Clear serum samples are used from centrifuged Discussion
clotted blood specimens. House rules require
identification of specimens from people known to be Numerous factors can affect syphilis serological
in "danger of infection". Blood is separated under a tests. Many of these are identified using internal
cabinet, and serum is transferred using disposable quality control procedures. Other factors that
drinking straws. It is stored at 40C and is not influence results are less easy to define. The style of
subjected to heat treatment before being tested. laboratory management can influence performance.
Original clot specimens are kept for seven days. Stress is of particular concern as this is linked with

572136
Date VDRL TPHA FTA (G) (M) Latex RW Lab
Ref.
2.6.81 16 1280 Pos 3241
Age 32 4.6.81 16 1280 Pos 4018
D.OB. 1. 3.49 15.6. 81 8 1280 Pos 6301
26.6.81 Neg Neg Neg 8416

1st Presented 2. 6.81

A Primary

Rx Pen: 2.6.81 Laboratory specimen 8416 discordant result repeat tests

from original clot.

SEROLOGY RECORD
FIG 3 Sample serology record card.
Internal quality control in serological tests for syphilis 93

1st Analysis 2nd Anolysis 3rd Analysis

Collection of blood,
request form, transnort
Receipt of specimen,
Collection and clerking in
1st Component
transport of specimen Liquid transfer,
Collection and prepar- separation by centrifuge
ation of blood specime Receipt and
preparation Separation of serum,
transfer to working
tube

Check working temp

Identify reagents of reagent
2nd Component
from storage Calculate volume,

I Preparation of reagents Prepare working

dilutions of reagent
Prepare reagent
Identify and check
antigen dropper
Prepare glass slides
for test

Identify serum transfer

pipette
Transfer test and Transfer serum volume
3rd Component control serum to slide on slide, place on
The test proper Add volume of antigen orbital shaker
to serum mix Mix, check speed of
rotation and time

Examine results
record
4th Component Record results
Report and scrutiny
Laboratory reporting
Prepare lab report Locate report venue,
dispatch report
File lab report copy

FIG 4 Analysis of VDRL test (each component part may be analysed further if necessary.)

the performance of staff.8 Whether the cost incurred advance towards achieving standardisation in syphilis
in reducing errors is more than current budgets can serology, and its use as a biological standard should
afford to pay is a matter of policy. If the current be exploited. Technical excellence is fundamental in
economic climate denies laboratories additional achieving consistency; this embraces technique,
financial resources to implement internal quality equipment, tools, reagents, and skills, all of which
control, it may be that this should still be undertaken need periodical review. Serological testing for
by reappraising workloads. The availability of the syphilis, particularly the mass screening of blood, is
WHO reference serum (ref 3-1980) is an important tedious. This does not alter its importance as a
94 0 D Wasley
routine serological test. Confidence in reagents will References
improve if manufacturers standardise their products 1. Wassermann A, Neisser A, Bruck C. Eine Serodiagnastiche
based on the WHO reference serum. Reaktion bei Syphilis. Dtsch Med Wochenschr 1906;32:745.
I recommend that departments responsible for 2. Daniels KC, Ferneyhough HS. Specific direct fluorescent
antibody detection of Treponema pallidum. Health Lab Sci
serological tests for syphilis should implement 1977; 14:164-7 1.
internal quality control based on the WHO reference 3. Guidelines for the preparation and establishment of reference
materials and reference reagents for biological substances.
serum, and should monitor their daily performance WHO Tech Rep Ser 1978; No 26:102-5.
by using wall charts. Internal quality control includes 4. Johnson GD, Dorling J. Immunofluorescence and immuno-
surveillance of all activities that can affect laboratory peroxidase techniques. In: Thompson RA, ed. Techniques in
clinical immunology. London: Blackwell Scientific
results. A senior member of the laboratory should be Publications, 1951:106-37.
appointed to administer these procedures. The 5. Notowicz A, Menke HE. Routine diagnostic procedures in
treponemal disease. In: Harris JRW, ed. Recent advances in
importance of establishing a reliable syphilis serology sexually transmitted diseases. Edinburgh: Churchill
service in a laboratory must, however, be seen as a Livingstone, 1981:93-100.
6. Harris A, Rosenberg AA, Reidel LM. Microflocculation test
basis for obtaining national concordance between for syphilis using cardiolipin antigen. Journal of Venereal
laboratories. It is hoped that regional or national Disease Information 1946; 27:167-74.
7. O'Neill P. A new look at the serology of treponemal disease.
reference serum samples will eventually become British Journal of Venereal Diseases 1976; 52:296-9.
available to meet this need. 8. Griffin P, Klun C. Laboratory stress. Am J Med Technol
1980;46:490-4.
I thank Dr James Taylor and Dr Rt N Thin for their
advice on the preparation of this paper.