Abstracts of the Scientific Posters, 2013 AACC Annual Meeting ...

Cancer/Tumor Markers
Tuesday, July 30, 9:30 am – 5:00 pm
were analysed by Freelite assay (The Binding Site Group Ltd, UK; FLC ratio normal
range 0.26-1.65) and N Latex assay (Siemens, Germany; FLC ratio normal range
0.31-1.56). The sensitivity and specificity of both assays were compared.
Results: Freelite and N Latex provided concordant information in 344/390 (88%)
patients. 308/344 (89%) patients had normal FLC ratios by both assays (Freelite:
median 0.7, range 0.26-1.57; N Latex: median 0.58, range 0.33-1.49). The remaining
36/344 (10%) patients had abnormal FLC ratios by both assays (Freelite: κFLC
median 8.23, range 1.7-1531; λFLC median 0.01, range 0.0001-0.24; N Latex: κFLC
median 3.88, range 1.84-191; λFLC median 0.01, range 0.0002-0.20). The clinical
diagnoses of the 36 patients with abnormal FLC ratio by both assays were: 6 MM, 5
LCMM, 1 cryoglobulinemia, 4 lymphoma, 1 plasmacytoma and 19 MGUS patients.
Freelite was abnormal and N Latex was normal in 19 patients with haematological
disorders (Freelite: κFLC median ratio 2.31, range 1.66-4.82; λFLC median 0.17,
range 0.03-0.25; N Latex: median 1.09, range 0.35-1.55). 14/19 of these patients were
positive by SPEP (3 MM, 1 WM, 2 lymphoma, and 8 MGUS) and the remaining 5/19
patients were negative by both SPEP and N Latex (1 patient subsequently diagnosed
with WM, 4 MGUS). In contrast, N Latex identified 4 MGUS patients with positive
SPEP and normal Freelite ratio (Freelite: median 0.75, range 0.65-0.95; N Latex:
κFLC 1.88, λFLC median 0.15, range 0.12-0.28). Freelite was more sensitive and
specific in identifying patients with hematological disorders and had a better positive
(PPV) and negative (NPV) predictive value compared to N Latex (sensitivity 54%
(95% CI 44-64%) v. 39% (CI 30-49%), specificity 98% (CI 95-99%) v. 94% (CI 91-
96%), PPV 89% (CI 78-95%) v. 70% (CI 56-81%), NPV 86% (CI 81-89%) v 81% (CI
77-85%); respectively).
Discussion: In this study the Freelite assays had a greater sensitivity and specificity
to identify patients with hematological disorders. Furthermore, this data continues to
support the requirement for further clinical evaluation of the N Latex test before it is
used in routine practice and current guidelines for serum FLC measurement using N
latex are not applicable at this time.
A-20
Development of automatic antigen excess detection parameters for
immunoglobulin free light chain (Freelite®) assays on the Roche
cobas® c501
M. D. Coley, H. D. Carr-Smith, D. J. Matters, S. J. Harding, P. J. Showell.
The Binding Site Ltd, Birmingham, United Kingdom
International guidelines, based upon the Freelite® serum free light chain (FLC)
assay recommends its use to aid in the diagnosis of patients with B cell disorders
and to monitor patients with AL amyloidosis, non-secretory and light chain multiple
myeloma. Immunoglobulin light chains are highly variable with over 480 different
genetic combinations for lambda possible prior to antigen exposure. This inherent
variability means there is possibility of antigen excess even in multi-epitope
recognising polyclonal antibody based assays. Here we describe the development of
automatic antigen excess protection for the Freelite assay (The Binding Site group
Ltd) on the Roche cobas® c501. Reaction kinetics of monoclonal patient sera prone to
antigen excess (5 kappa, mean c501 result 5,345.12mg/L, range 502.62-12,672.00mg/
L, and 4 lambda, mean c501 result 4,046.5mg/L, range 1,590.00-6,213.00mg/L) and
4 normal blood donor sera (mean kappa 10.51mg/L, range 9.14-13.18mg/L; mean
lambda 11.41mg/L, range 10.90-12.14; mean ratio 0.93, range 0.79-1.21) were
analysed to set threshold limits. Samples in antigen excess were typified by a high
initial rate of reaction, which rapidly slowed as the detecting antibody became saturated
(early delta OD 125.5, late delta OD 14.0). This is compared to non-antigen excess
samples which showed a slower, more sustained rate of reaction throughout the assay
time (early delta OD 28.0, late delta OD 38.7). Antigen excess capacity was validated
using 67 normal blood donor serum, 68 kappa monoclonal and 33 lambda monoclonal
patient sera which had been collected over a number of years and had previously been
reported as having antigen excess on other analysers or assays. All samples tested
(68/68 kappa, median 265.03mg/L, range 20.06-40,930.00mg/L, and 33/33 lambda,
median 762.00mg/L, range 7.68-56,949.00mg/L) were correctly measured using these
parameters. We conclude that implementation of these parameters will improve assay
throughput and will prevent monoclonal patient samples from being mis-reported.
A-21
Validation of an automated immunoassay for the quantitative
measurement of hemoglobin in stool
J. Lu 1 , S. Clinton 2 , D. G. Grenache 2 . 1 ARUP Laboratories, Salt Lake City,
UT, 2 University of Utah, Salt Lake City, UT
Background: Fecal occult blood (FOB) has clinical utility as a colorectal cancer
(CRC) screening test and has been shown to significantly reduce CRC mortality.
Immunochemical detection of FOB (iFOB) is considered to be superior to guaiac
tests, the latter of which are prone to false-positive results. iFOB requires no patient
preparation or dietary restrictions before collection and is specific for human
hemoglobin A (HbA).
Objective: To validate the OC-Auto Micro 80 (Polymedco Inc. Cortlandt Manor, NY)
immunoassay for the detection and measurement of HbA in stool.
Methods: In accordance with manufacturer instructions, residual patient stool samples
were extracted with a stabilizing buffer and the sample added to latex particles coated
with anti-human HbA antibodies. Any HbA present in the sample binds to the beads
resulting in an agglutination reaction and the change in optical density is directly
proportional the HbA concentration. Analytical characteristics including linearity,
precision, analytical sensitivity, analyte stability, and accuracy were determined.
Results: Linearity was determined by adding diluted, lysed whole blood (HbA 16-
909 ng/mL) to a set of five HbA-negative stool samples and testing each sample in 3
replicates. Linear regression analysis produced a slope of 0.911 and a y-intercept of
-3.84. Precision was assessed by adding diluted, lysed whole blood to HbA-negative
stool samples to prepare a set of 3 samples with different HbA concentrations. Each
sample was tested in 3 replicates once per day for 10 days. Within-laboratory CVs
were 14.1, 13.5 and 25.4% at HbA concentrations of 549.1, 93.9 and 33.6 ng/mL,
respectively. The limit of blank was determined to be 1.0 ng/mL by measuring sample
buffer in 10 replicates. Stability of stool samples in stabilizing buffer was evaluated
by determining HbA in two sample pools with mean HbA concentrations of 559 and
98 ng/ml at time 0, stored at ambient temperature and at 4 °C for 14 and 29 days,
respectively, and then tested in two replicates. In both pools, the change in HbA
concentration was within 15% compared to time 0. 20 samples were tested for iFOB
and by guaiac testing. 90% (9/10) of guaiac-negative samples had no detectable HbA
by iFOB and 10% (1/10) had an HbA concentration of 517 ng/mL. 100% (10/10)
of guaiac-positive samples had HbA detected by iFOB (range, 420-2,078 ng/mL).
Recovery was evaluated by adding diluted, lysed whole blood to HbA-negative stool
samples and the recoveries were 93 and 94% at the mean HbA concentrations of 465
(n=4) and 94 (n=4) ng/mL.
Conclusion: We have validated an automated immunoassay for the measurement of
fecal occult blood that provides high accuracy and specificity for HbA and does not
require special dietary restrictions prior to sample collection.
A-22
Comparison of the analytical performance of polyclonal and
monoclonal antibody based FLC assays in refractory multiple
myeloma patients
S. J. Harding 1 , R. Popat 2 , O. Berlanga 1 , H. Sharrod 1 , J. Cavenagh 2 , H.
Oakervee 2 . 1 The Binding Site Ltd, Birmingham, United Kingdom, 2 St
Bartholomew’s Hospital, London, United Kingdom
Background: Current international guidelines for the identification of B cell disorders
recommend a screening algorithm of serum protein electrophoresis and serum free
light chain (FLC) testing based upon the polyclonal, multi-epitope Freelite® assay.
A new monoclonal antibody (single epitope) based assay, N Latex FLC (Siemens,
Germany) has been developed for measuring serum FLC levels. Here, we compare the
analytical performance of the two assays in a population of MM patients.
Methods: Baseline sera from 91 refractory MM patients (26 IgGκ, 14 IgGλ, 5 IgAκ, 4
IgAλ, 3 biclonal, 2 LC only; 13 IFE negative; 24 IFE not available; 52 males; median
age 62 years (30-85)) were analysed for FLC levels by Freelite and N Latex FLC on
the BN TM II nephelometer (Siemens, Germany). Reported values were compared using
Passing-Bablok (PB) and linear regression (R 2 ) using Analyze-It software; an R 2 ≥0.95
was considered to be identical analyte measurement in keeping with CLSI guidelines.
FLC ratio normal range by Freelite: 0.26-1.65; by N Latex FLC: 0.31-1.56.
Results: In 21 patients with normal kappa/lambda FLC ratios by both assays showed
moderate correlation for kappa FLC (PB: 3.18+0.65x; R 2 =0.92), with poor correlation
for lambda FLC (PB: 0.20+1.04x; R 2 =0.47). Similarly, in 47 patients with an abnormal
CLINICAL CHEMISTRY, Vol. 59, No. 10, Supplement, 2013
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