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Clinical Chemistry 48, No. 9, 2002 1601
Correlation of serum immunoglobulin free light chain quantification with
Table 1. Distribution of diagnoses with indication of urinary Bence Jones protein in light chain myeloma. Clin Chem 2002;48:
number of abnormal FLCs. 655–7.
Abnormal
free /free Both FLCs
ratio, n increased,
Clinical diagnosis (%) n (%)
Chronic immune stimulation (n 7) 4 (57%) 5 (71%)
Primer-Extension Preamplified DNA Is a Reliable Tem-
MGUS (n 8) 6 (75%) 3 (37%)
plate for Genotyping, Helena Kuivaniemi,1,2 Sungpil Yoon,1
Primary (AL) amyloidosis (n 1) 1 (100%) 0 (0%)
Hidenori Shibamura,1 Magdalena Skunca,1 Sompong Vong-
B-cell-derived malignant disease (n 34) 24 (71%) 4 (12%)
punsawad,1 and Gerard Tromp1* (1 Center for Molecular
Medicine and Genetics and 2 Department of Surgery,
Wayne State University School of Medicine, Detroit, MI
total and was performed by nephelometry using 48201-1928; * address correspondence to this author at:
Beckman-Coulter reagents on the Immage instrument. Center for Molecular Medicine and Genetics, 3116 Gordon
Quantification of the FLCs revealed an abnormal free H. Scott Hall of Basic Medical Sciences, 540 East Canfield
/free ratio in all samples, whereas quantification of the Ave., Detroit, MI 48201-1928; fax 313-577-5218, e-mail
total light chains revealed an abnormal / ratio in only tromp@sanger.med.wayne.edu)
5 of the 16 samples (data not shown).
Of 12 sera from patients with an intact immunoglobulin A common constraint faced by genetic studies is the
M protein of type (1 IgG, 6 IgA, and 5 IgM), only 4 had limited amount of DNA available from study partici-
an increased free / ratio (between 1.5 and 5.2). Nine of pants. It is often not practical or possible to either collect
21 sera from patients with an intact immunoglobulin M a large amount of tissue such as liver or blood from
protein of type (6 IgG, 8 IgA, 6 IgM, and 1 IgD) had individuals or to ask for a second sample, particularly if
decreased free / ratios (between 0.037 and 0.2). the critical individuals died since the first sample was
The distribution of diagnoses with an indication of the taken. Immortalization of white blood cells by use of
number of abnormal FLC ratios is shown in Table 1. Of Epstein–Barr virus in theory provides an unlimited source
seven patients with chronic stimulation of the immune of material. The immortalization protocol itself, however,
system, the free /free ratio was abnormal in four (57%). must be carried out within 48 h of blood collection and is
In one of these four cases, the ratio was only modestly time-consuming.
increased (1.09). In patients with MGUS and B-cell- DNA linkage studies in which microsatellite or other
derived malignant disease, the free / ratio was abnor- polymorphic DNA markers are used for a genome scan
mal in 75% and 71%, respectively. Concentrations of both require appreciable amounts of DNA. As multiple PCRs
free and free were increased in five of the seven are carried out with each sample, valuable genomic DNA
patients (71%) with chronic immune stimulation. By con- is easily depleted. To prevent loss of irreplaceable sam-
trast, concentrations of both free and free were ples and to maximize the available genetic materials for
increased in only 7 of the 43 patients (16%) with MGUS, DNA linkage and genetic association studies as well as for
B-cell-derived malignancy, or primary amyloidosis. future studies of screening for mutations in candidate
In conclusion, FLC nephelometric measurement in se- genes, we used the primer-extension preamplification
rum seems to be a convenient assay for detection of FLC (PEP) protocol (1 ) to carry out whole-genome amplifica-
M proteins in serum. This technique is more reliable than tion.
CZE and nephelometric measurement of total and . For The advantages of using the PEP protocol are that only
intact immunoglobulin M proteins, the free / ratio was a small amount of genomic DNA is needed for the entire
within reference values in 60% of the cases in which CZE
genome scan and the remaining genomic DNA can be
failed to detect an M protein. Future studies should
stored for future analyses. One possible caveat is that PEP
address the question whether the presence of increased
may not amplify both parental copies of an individual’s
FLCs together with a low concentration intact immuno-
DNA or may not amplify them equally at all loci. In turn,
globulin M protein has a prognostic clinical value.
genotyping inaccuracies may potentially obscure allele
sharing. To investigate the reliability of genotyping re-
References
1. Katzman JA, Clark R, Sanders E, Landers JP, Kyle RA. Prospective study of sults when PEP was used to produce PCR templates, we
serum protein capillary electrophoresis immunotyping of monoclonal pro- compared the results for PCR products from PEP with
teins by immuno-subtraction. Am J Clin Pathol 1998;110:503–9. those obtained with genomic DNA templates. All exper-
¨
2. Bossuyt X, Marien G. False-negative results in detection of monoclonal
proteins by capillary zone electrophoresis: a prospective study. Clin Chem iments were carried out according to protocols approved
2001;47:1477–9. by the Institutional Review Board of Wayne State Univer-
3. Bradwell AR, Carr-Smith HD, Mead GP, Tang LX, Showell PJ, Drayson MT, et
al. Highly sensitive automated immunoassay for immunoglobulin free light
sity School of Medicine.
chains in serum and urine. Clin Chem 2001;47:637– 80. Before PEP, 30 –250 ng of the genomic DNA was
4. Drayson M, Tang LX, Drew R, Mead G, Carr-Smith H, Bradwell AR. Serum free denatured at 95 °C for 5 min and cooled on ice. The PEP
light-chain measurement for identifying and monitoring patients with nonse-
cretory multiple myeloma. Blood 2001;97:2900 –2. was 45 cycles of denaturation at 92 °C for 1 min, annealing
5. Abraham RS, Clark RJ, Bryant SC, Lymp JF, Larson T, Kyle RA, et al. at 37 °C for 2 min (programmed increase in time of 4
1602 Technical Briefs
s/cycle and increase in temperature of 0.4 °C/cycle so The reliability of the PEP protocol (1 ) was tested by (a)
that in the final, 45th, cycle the annealing was carried out sequencing the PCR products (ThermoSequenase Cycle
at 55 °C), and a polymerase extension step of 4 min at Sequencing Kit; USB) generated with and without PEP
55 °C (PEC9600; PE Biosystems). The PEP products were from 16 individuals for a 306-bp PCR product from the
diluted 100-fold in two steps. In the first step, the 50- L MMP-8 promoter and (b) by comparing the genotyping
PCR was added to 750 L of 10 mmol/L Tris–1 mmol/L results obtained from PEP DNA templates with those
EDTA (pH 8.0). In the second step, 16 L of the first obtained with genomic DNA templates for a total of 881
dilution was added to 84 L of 10 mmol/L Tris–1 genotypes from five different polymorphic markers typed
mmol/L EDTA (pH 8.0). The second diluation was used with 332 different templates (Table 1). DNA sequencing
for genotyping. did not reveal any differences in the PCR products with or
For genotyping, we used previously published primer without PEP (not shown). Furthermore, there was 100%
sets and PCR conditions for the matrix metalloprotein- concordance between the genotypes from PEP DNA and
ase-1 (MMP1) (2 ), matrix metalloproteinase-3 (MMP3) genotypes from the corresponding genomic DNA sam-
(3 ), and plasminogen activator inhibitor-1 (PAI-1) (3 ) ples where both produced a result (Table 1). The PEP-
genes. A primer set was designed for MMP13 (GenBank amplified DNA successfully yielded PCR products for
accession no. X75308) PCR (sense, 5 -GATACGTTCTTA- polymorphic regions scattered throughout the chromo-
CAGAAGGC-3 ; antisense, 5 -ACCCATCTGGCAAAA- somes. Additionally, 6000 genotypes generated with 54
TAAAC-3 ) and purchased from Integrated DNA Tech- microsatellite markers and PEP DNA templates yielded
nologies. Primers for dinucleotide repeat markers no inconsistent inheritance in pedigrees (not shown). For
(MapPairs) were purchased from Research Genetics. One highly variable microsatellite markers, the number of
of the genotyping primers was radioactively labeled using heterozygotes observed was consistent with expectation.
T4 polynucleotide kinase (New England BioLabs) and Thus, the PEP protocol did not cause selective amplifica-
[ -32P]ATP (Amersham). PCRs were carried out in 15- L tion or loss of one allele as determined by concordance,
reaction volumes that included 5 L of 100-fold diluted heterozygosity, and consistent inheritance in pedigrees
template from PEP (1.7–7 ng of DNA based on absorbance (loss of one allele could also yield inconsistent inheritance
reading; see below) or 30 –100 ng of genomic DNA as in pedigree). Not only did the PEP protocol yield high-
template, 5 pmol of unlabeled primer, 5 pmol of radiola- fidelity copies, the PEP DNA appeared to be a more
beled primer, 200 M deoxynucleotide triphosphates suitable template for the PCR because a greater propor-
(dNTPs; final concentration), 0.3 U of Taq DNA polymer- tion of the PCRs were successful (Table 1). In addition, the
ase [AmpliTaq Gold (PE Biosystems) for MMP1, MMP13, intensity of genotyping products was more uniform from
PAI-1, and all microsatellite markers, and Taq DNA poly- sample to sample when PEP DNA templates were used
merase (Qiagen) for MMP3]. Several reactions containing than when genomic DNA templates were used, making
all the reagents except template DNA, which was re- interpretation of results easier.
placed by water, were carried out as negative controls in The amount of DNA generated in the PEP was deter-
the PCRs along with the experimental samples, and they mined after the PEP products were purified from the
were consistently negative. PCR products were analyzed remaining primers and nucleotides (Microcon YM-30
on 7% polyacrylamide gels containing formamide or 6% Centifugal Filter Devices; Millipore Corporation) and
sequencing gels (Sequagel-6; National Diagnostics) to quantified by spectrophotometry at 260 nm. The PEP
resolve the 1- to 4-base differences between different produced up to a 110-fold increase in the amount of DNA
alleles. After fixing and drying, the gels were exposed to (Fig. 1), a nearly linear increase in DNA. In our hands, the
x-ray film (Eastman Kodak Company) at 80 °C for PEP protocol yielded sufficient amounts of product to
autoradiography. perform 400 genotyping PCRs when as little as 30 ng of
Table 1. Genotyping success rates when PEP and genomic DNA templates were used in PCR.
Successful PCRs, n (%)
Gene Product Genomic Heterozygotes,c n
(chromosome) Polymorphism size,a bp nb PEP DNA (%)
MMP1 (11) G1/G2 148 237d 235 (99) 194 (82) 117 (50)
MMP3 (11) A5/A6 124 195d 189 (97) 160 (82) 37 (20)
PAI-1 (7) G4/G5 104 128d 127 (99) 106 (83) 40 (19)
MMP13 (11) A11/A12 123 226d 213 (94) 184 (81) 51 (40)
D17S928 (17) (CA)n 135 95e 94 (99) 76 (80) 76 (81)
Total 881 858 (97) 720 (82)
a
If sizes of different alleles vary, the size of the smallest allele is shown.
b
Number of samples used to study the specific polymorphism.
c
Number and percentage of those individuals whose DNA worked in genotyping after PEP and who were heterozygotes.
d
DNA was isolated from 237 liver samples taken at autopsy; the indicated number of samples were used to study the polymorphism.
e
DNA was isolated from 95 whole-blood samples.
Clinical Chemistry 48, No. 9, 2002 1603
spontaneous lethal outcomes, where samples are difficult
to obtain and frequently the only available samples are
paraffin-embedded tissue blocks or microscope slides
prepared from tissue sections.
Microsatellite markers have been used to detect aneu-
ploidy in small amounts of fetal material amplified by
quantitative PCR (5, 6 ) or after whole-genome amplifica-
tion using comparative genome hybridization (7 ). Al-
though our genotyping experiments were not quantita-
tive, they were encouraging in the sense that both alleles
seemed to be amplified. Detailed quantitative analyses are
necessary to establish the usefulness of PEP-amplified
templates in quantitative PCRs. Several investigators have
noted the problem of poor fidelity when PEP or other
whole-genome amplification methods, such as the degen-
erate oligonucleotide-primed PCR, tagged PCR, or Alu-
PCR, were used with single cells (8 –10 ). We would,
Fig. 1. Fold amplification as a function of amount of template DNA.
therefore, not recommend using these methods with such
The amount of DNA synthesized during PEP amplification was divided by the
limited amounts of genetic material. Rather, our study
amount of DNA used as template. Data were generated for eight different was meant to show that PEP would reduce the amount of
genomic DNA templates at the amounts indicated. The curves with dashed lines template DNA required by 300-fold from the amounts
indicate the fold amplification that would have been obtained if all tubes had DNA
yields equal to the minimum ( ), average (f), and maximum (Œ) amounts. The currently requested by large genotyping laboratories and
thick solid line is the observed data ( ). The experimental data agree well with would improve the uniformity of intensity and reproduc-
the prediction that amplification reaches a limit either because of the depletion ibility of the genotypes.
of nucleotides or because the concentration of DNA generated by amplification
becomes limiting (12 ). The observed maximum amount of DNA corresponds to In conclusion, we have found that the PEP protocol is
52– 64% of the amount of DNA that can be synthesized from the available simple, robust, and reliable. With PEP, the amount of
nucleotides, i.e., when the reaction reaches its limit, the concentration of dNTPs
is between 36 and 48 mol/L. This is well above the Km for the enzyme,
genomic DNA used for one conventional genotyping PCR
suggesting that the limit is attributable to the concentration of DNA (12 ). (30 ng) is more than sufficient for an entire genome scan
at 10-centimorgan spacing [ 400 markers (11 )].
was used as the genomic DNA template. Typically, we
carried out the PEP with 100 ng of genomic DNA and
aliquoted the PEP for future use for thousands of geno- This work was supported in part by grants from the NIH
typing reactions. In negative-control experiments, (Grants NS34395 and HL64310), and a grant from the
genomic DNA that was processed through the PEP pro- American Heart Association-Michigan Affiliate.
cedure without the enzyme, primer, and dNTPs yielded
PCR products only for exceedingly robust PCRs because References
1. Zhang L, Cui X, Schmitt K, Hubert R, Navidi W. Whole genome amplification
30 ng of genomic DNA was diluted to 11 haploid from a single cell: implications for genetic analysis. Proc Natl Acad Sci U S
genome templates in the final PCR. A 1992;89:5847–51.
The majority of our data were for products of 250 bp 2. Rutter JL, Mitchell TI, Buttice G, Meyers J, Gusella JF, Ozelius LJ, et al. A
single nucleotide polymorphism in the matrix metalloproteinase-1 promoter
or fewer. We have, however, successfully amplified a creates an Ets binding site and augments transcription. Cancer Res
665-bp polymorphic region in the elastin gene (4 ) from 1998;58:5321–5.
PEP products, but we failed to amplify a 1200-bp region 3. Yoon S, Tromp G, Vongpunsawad S, Ronkainen A, Juvonen T, Kuivaniemi H.
Genetic analysis of MMP3, MMP9, and PAI-1 in Finnish patients with
when we used a PEP template from the same gene (not abdominal aortic or intracranial aneurysms. Biochem Biophys Res Commun
shown). Because the PEP generates fragments that are 1999;265:563– 8.
shorter than the template, the proportion of intact copies 4. Tromp G, Christiano A, Goldstein N, Indik Z, Boyd C, Rosenbloom J, et al. A
of template for long PCRs is expected to decrease expo- to G polymorphism in ELN gene. Nucleic Acids Res 1991;19:4314.
5. Pert B, Yau SC, Sherlock J, Davies AF, Mathew CG, Adinolfi M. Rapid
nentially. PEP DNA may, therefore, be unsuitable for molecular method for prenatal detection of Down’s syndrome. Lancet
amplifying long products. When the PEP products were 1994;343:1197– 8.
diluted 1:100 in molecular biology-grade water (Eppen- 6. Sherlock J, Cirigliano V, Petrou M, Tutschek B, Adinolfi M. Assessment of
diagnostic quantitative fluorescent multiplex polymerase chain reaction
dorf), degradation occurred in products stored at 4 °C assays performed on single cells. Ann Hum Genet 1998;62:9 –23.
after 2 months. When the diluted products were stored 7. Wells D, Delhanty JD. Comprehensive chromosomal analysis of human
at 20 °C or Tris-EDTA buffer was used as the diluent, preimplantation embryos using whole genome amplification and single cell
degradation was prevented, suggesting that the low pH comparative genomic hybridization. Mol Hum Reprod 2000;6:1055– 62.
8. Wells D, Sherlock JK, Handyside AH, Delhanty JDA. Detailed chromosomal
(5.5) of the water led to slow chemical degradation of the and molecular genetic analysis of single cells by whole genome amplification
PEP products. and comparative genomic hybridization. Nucleic Acids Res
Modifications of the PEP, including fewer cycles and 1999;27:1214 – 8.
9. Foucault F, Praz F, Jaulin C, Amor-Gueret M. Experimental limits of PCR
lower primer concentrations, may be suitable for near- analysis of (CA)n repeat alterations. Trends Genet 1996;12:450 –2.
linear amplification of DNA from paraffin-embedded 10. Cheung VG, Nelson SF. Whole genome amplification using a degenerate
tissues, an important consideration for diseases with oligonucleotide primer allows hundreds of genotypes to be performed on
1604 Technical Briefs
less than one nanogram of genomic DNA. Proc Natl Acad Sci U S A dilution and the final reagent concentrations were 123
1996;93:14676 –9.
11. Yuan B, Vaske D, Weber JL, Beck J, Sheffield VC. Improved set of short mmol/L citrate buffer (original pH 4.5) and 4.6 mmol/L
tandem repeat polymorphisms for screening the human genome. Am J Hum substrate. The increase in liberated 6-methyl-2-pyridine-
Genet 1997;60:459 – 60. thiol was measured at 340 nm over forty-five 10-s inter-
12. Kainz P. The PCR plateau phase—towards an understanding of its limita-
tions. Biochim Biophys Acta 2000;1494:23–7. vals, and the rate of reaction was converted to NAG
activity. The procedure was calibrated at 36 U/L (K-Assay
NAG Calibrator KAE-003C) and monitored using a 13
U/L control material (K-Assay NAG Control KAE-004C),
all prepared by reconstituting lyophilized bovine normal
Two Assays for Urinary N-Acetyl- -D-glucosaminidase kidney preparations with distilled water. The mean re-
Compared, Pamela L. Drake,1* Edward Krieg,2 Alexander W. sults for the two control samples were 104% of the
Teass,2 and Val Vallyathan3 (1 National Institute for Occu- nominal concentrations.
pational Safety & Health, Spokane Research Laboratory, The 3-cresol purple assay (6 ) was performed with the
315 East Montgomery Ave., Spokane, WA 99207; 2 Na- NAG assay from Boehringer Mannheim Biochemica (cat.
tional Institute for Occupational Safety & Health, Division no. 875406) adapted to the Cobas Fara II analyzer as an
of Applied Research & Technology, 4676 Columbia end-point assay. Sample was added to the reaction me-
Pkwy., Cincinnati, OH 45226; 3 National Institute for dium (original pH 5.14) at 37 °C, to obtain a 1:7 dilution (1
Occupational Safety & Health, Health Effects Research part sample to 6 parts reagent) of the sample and a final
Laboratory, 1095 Willowdale Rd., Morgantown, WV concentration of 36 mmol/L citrate buffer, 2.0 mmol/L
26505; * author for correspondence: fax 509-354-8099, e- substrate, and 2.2 mmol/L borax. After incubation for 15
mail pdrake@cdc.gov) min, the reaction was stopped by the addition of aqueous
sodium carbonate (pH 10.86), and the absorbance of the
In the course of investigating the exposure of gold miners solution at 580 nm was measured over 10 min. The
and other workers to mercury, investigators at the Na- procedure was calibrated at 27 U/L (Boehringer Mann-
tional Institute for Occupational Safety & Health (NIOSH) heim NAG Standard 982962) and monitored with a 9 U/L
measured urine N-acetyl- -d-glucosaminidase (NAG; EC control material (Boehringer Mannheim NAG Control
3.2.1.52) activity to monitor for renal injury (1–3 ). Numer- 1164368), all prepared by reconstituting lyophilized bo-
ous methods are available for the urinalysis of NAG vine normal kidney preparations with distilled water. The
activity (4 ). Most of the analyses for the NIOSH studies mean results for the two control samples were 104% of the
were performed by Pacific Toxicology Laboratories, using nominal concentrations.
the Kamiya Biomedical Company reagent set, which was In addition to analyzing standards and control samples
based on NAG-catalyzed hydrolysis of 6-methyl-2-pyri- as described above, we checked the operation of the
dyl-N-acetyl-1-thio- -d-glucosaminide to 6-methyl-2-pyr- Cobas Fara II analyzer routinely and also on changing
idinethiol. However, while planning our most recent reagents or adjusting the operation of the instrument.
investigational visit to a gold mine, we learned that the These checks involved replicate analyses of the Roche
Kamiya Biomedical reagent set was temporarily out of Precision Testing Set (cat. no. 44302); the instrument was
stock. Consequently, we decided to perform the analyses deemed in control if the CV was 2%.
in-house using the reagent set manufactured by Boehr- The performance of the two assays was tested by 20
inger Mannheim Biochemica, which we had used previ- analyses each of control samples at three concentrations.
ously for the determination of NAG in rats. This reagent The mean measured values (CVs) at each concentration
set is based on NAG-catalyzed hydrolysis of the sodium for the two assays were as follows: 6-methyl-2-pyridine-
salt of 3-cresolsulfonphthaleinyl-N-acetyl- -d-glucosami- thiol assay, 3U/L (4%), 14 U/L (2%), 49 U/L (1%); 3-cresol
nide to give 3-cresol purple (3-cresolsulfonphthalein, so- purple assay, 2U/L (3%), 10 U/L (1%), 26 U/L (1%).
dium salt). Because the two assays are based on the Using the data for the lowest concentrations, we esti-
hydrolysis of different substrates, and in light of the mated the detection limits to be 0.35 U/L for the 6-methyl-
differences reported among four NAG assays (including 2-pyridinethiol assay and 0.16 U/L for the 3-cresol purple
the 3-cresol purple) (5 ), we suspected that the 3-cresol assay.
purple assay might not give NAG activity results equiv- Urine specimens were obtained from 42 gold miners, 12
alent to those obtained by the 6-methyl-2-pyridinethiol nonoccupationally exposed persons living in the vicinity
assay used in previous investigations. To obtain data with of the mine, and 18 other nonoccupationally exposed
which to compare the two assays, we applied both assays persons. The protocol for the investigation was approved
to 72 urine specimens. by the NIOSH Human Subjects Review Board, and the
The 6-methyl-2-pyridinethiol assay was performed participants gave their informed consent. Nine of the
with the K-Assay for NAG activity (cat. no. KAE-002) specimens, seven from miners and two from nonoccupa-
from Kamiya Biomedical Company adapted to the Cobas tionally exposed persons living in the vicinity of the mine,
Fara II analyzer (Roche Diagnostic Systems) as a kinetic were split and submitted as separate samples. Statistical
assay. The instructions supplied with the reagent set were analyses were performed using SAS® (SAS Institute).
followed, yielding a final solution at 37 °C in which the Samples from three workers gave “0” NAG activity by the
sample was at a 1:13 (1 part sample to 12 parts reagent) 3-cresol purple assay and were excluded (the creatinine
