Download Genetic Variants of a1-Antitrypsin and

vitro hemolysis on
24:1966-70.
chemical values for serum. Clin Chem 1978;
5. Laessig RH, Hassemer DJ, Paskey TA, Schwartz TH. The
effects of 0.1 and 1.0 percent erythrocytes and hemolysis on serum
chemistry values. Am J Clin Pathol 1976;66:639-44.
6. Randall UG, Garcia-Webb P, Beilby JP. Interference by haemolysis, icterus and lipaemia in assays on the Beckman Synchron
CX5 and methods for correction. Ann Cliii Biochem 199027:34552.
7. Fairbanks VF, Klee GO. Biochemical aspects of hematology. In:
Tiets NW, ed. Textbook of clinical chemistry. Philadelphia: WB
Saunders, 1986:1495-588.
8. Perlstein MT, Thibert RJ, Zak B. Bilirubun and hemoglobin
alterations
in several hydrogen peroxide generating
procedures.
Microchem J 197823:13-27.
9. Tietz NW, Pruden EL, Siggaard-Anderaen 0. Electrolytes. Op.
cit. (ref. 7):1172-91.
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of lipase activity. Cliii Chim
Acta 1975;60:273-9.
CLIN.CHEM.38/4, 577-580 (1992)
Genetic Variants of a1-Antitrypsin and Hemoglobin Typed by Isoelectric Focusing in
Preselected Narrow pH Gradients with PhastSystemTM
Jan-Olof
Jeppsson1
and Roland Einarsson2
In this method for automatically running and staining
isoelectric focusing (IEF) gels, pre-made dehydrated polyacrylamide gels were rehydrated before assays run with
the PhastSystem
(Pharmacia LKB Biotechnology). The
typing of genetic variants of hemoglobin and a1-antitrypsin in narrow pH gradients (pH 6.7-7.7 and 4.2-4.9,
respectively) was simple, convenient, and reproducible.
The clinically important variants of a1-antitrypsin (ZZ and
SZ) were identified from serum or dried blood on filter
paper. The fast screening of abnormal hemoglobin samples (HbS) for cases in clinical medicine was easily
performed. The total analysis time for the phenotyping
with conventional protein staining was -60 mm.
Additional
Keyphra.es: screening
electroplioresis
polyaciylamide gel
.
sickle cell disease
gycohemogkbin
Isoelectric
focusing
(JEF) of proteins
in polyacrylamide gels has been used in various applications
in
clinical and forensic medicine to reveal detailed protein
microheterogeneity
not obtained with other techniques.
Considerable
time, effort, and skill are required
to
achieve acceptable and reproducible results with current techniques for JEF, which restricts the use of the
technique in routine clinical settings. Many developments have been made in these areas, including the use
of ultrathin
gels to shorten the distance for heat transportation
so that the voltage can be increased (for
greater resolution), introduction of tailor-made
ampholines with narrow pH gradients,
and plastic backings to
simplify the handling of the gels during staining
and
drying.
Most of these new ideas for electrophoresis
have been
‘Department of Clinical Chemistry, General Hospital, Lund
University, 5-21401 MahnO, Sweden.
2Pharmacia Diagnostics AB, S-751 82 Uppeala, Sweden (author
for correspondence).
Received May 21, 1991; accepted February 3, 1992.
integrated in PhastSystemm
(Pharmacia
LKB Biotechnology, Uppsala, Sweden), a dedicated system for horizontal electrophoresis
and JEF in small gels, with automated fixation, staining, and destaining
(1,2). Here we
describe an IEF method performed with PhastSystem
with a new dry polyacrylamide
gel, which is soaked in a
narrow-pH
gradient solvent before use in analysis for
genetic variants
of a1-antitrypsin
and hemoglobin.
MaterIals and Methods
Blood and serum samples for typing genetic variants
of a1-antitrypsin and hemoglobin were obtained from
the Department of Clinical Chemistry,
General Hospital, MalmS, Sweden.
PhastSystemand Gels
The apparatus comprises electrophoresis
and automated staining and destaining
units, which can be
programmed and operated independently (1,2).
Dehydrated polyacrylamide gels were rehydrated before use by applying a droplet of ampholyte solution on
a plastic surface and either placing the dry gel upsidedown on the droplet for 30 mm or using a reswelling
casette
(Pharmacia
LKB Biotechnology).
For typing
of
a1-antitrypsin, we applied 1.3 mL of Pharmalyte,
4.24.9, diluted 16-fold with water onto the plastic surface;
for typing of hemoglobin, we applied 1.3 mL of Pharmalyte, 6.7-7.7, also diluted 16-fold with water. A spacer
molecule (-alanine,
17 mmol/L) was added in some
experiments
to better
separate
the glycohemoglobin
fraction from nonglycated hemoglobin (3).
IEF of a1 -Antitrypsin
Samples
were prepared either from dried blood on
filter paper or from serum, essentially
as previously
described
(4, 5). Briefly, dried blood on filter paper
(4-mm-diameter
disc) was eluted in 15 pL of 1 mol/L
glycine,
pH 7.4, containing
cysteine
hydrochloride,
0.5
CLINICAL CHEMISTRY, Vol. 38, No. 4, 1992 577
For serum samples, we mixed 90 L of serum
with 10 pL of cysteine hydrochloride
(0.3 mol/L, pH 7.4).
The blood and serum samples were incubated overnight at 4#{176}C.
Cysteine eliminates
the adducts bound to
the active thiol group in a1-antitrypsin
and thus increases the sharpness
of the IEF patterns (4). This effect
is especially
pronounced
for dried blood and old serum
samples. Fresh serum can be run without pretreatment.
Assay:
Aliquots (3.6 L) of cysteine-reduced
serum or
eluted whole blood were applied to PhastSystem
rehydrated polyacrylamide IEF gels according to the manufacturer’s instruction.
IEF was carried out according to a
modified
electrophoresis
program
(Table
1) with
PhastSystem.
The time for separation was 30 mm. After
electrophoresis
the gels were fixed for 5 mm in trichloroacetic acid (200 g/L) and stained with either Coomassie
Brilliant
Blue (PhaatGel Blue R, 0.5 g/L) or automated
silver staining (PhastGel
Silver Kit), according to the
methods given in the PhastSystem
Owner’s Manual
(1986). Gels were destained in methanol/acetic acid/water (3/1/6, by vol). The processed gel was treated with
glycerol, 50 mi/L in 50 mLfL acetic acid solution, to
prevent deforming
of the gel. The stained gels were
evaluated
by visual comparison with a reference pattern.
mol/L.
IEF
Samples
were prepared
according
to a previously
procedure
(6). Briefly, blood samples were
collected into EDTA-containing
tubes or as capillary
blood into heparinized
microtubes.
The blood cells were
washed in isotonic saline, hemolyzed by shaking
the
cells in a mixture of water and Cd4 for 30 s, and then
centrifuged.
The clear supernate was diluted with water
described
Assay:
concentration
We applied
of -5 g/L.
1 p.L of hemolysate
to the rehy-
drated polyacrylamide
gels for IEF. The electrophoresis
was performed according to the method given in Table 1.
The protein precipitation and staining procedures
were
as described for a1-antitrypsin.
Resufts and DiscussIon
a1-Antitrypsin
Pi-typing (protease
inhibitor typing) of a1-antitrypsin
was performed by IEF in a narrow pH gradient, 4.2-4.9.
Figure 1 demonstrates
a gel pattern with the typical
microheterogeneity
of some common
a1-antitrypsin
variants of clinical importance after Coomassie
BrilTable 1. isoeiectrlc Focusing Analysis for
a1-Antitrypsln (AT) and Hemoglobin (Hb) with
Rehydrated Polyacrylamide Gels
current,
Step
Prefocusing
Sample
application
Separation
AT
Hb
578
Potential,
2
V
2000
200
Powei,
w
limp,
mA
2.0
2.0
3.5
3.5
15
15
4
-
-
4.48
6
-
-
4.55
7
4.59
8
4.67
zz
SZ
FZ
M1
Fig.1. Isoelectric focusing of variousa1-antltnjpslnPiphenotypes in
the pH range4.2-4.9 after Coomassle Brilliant Bluestaining(anode
CLINICAL CHEMISTRY,
5.0
7.5
3.5
5.0
y.axls
Figure 1 also shows a schematic
the position of the bands. The variants of particular
clinical interest are the ZZ and SZ
phenotypes, which are associated
with a1-antitrypsin
deficiency. These variants were easily detected with this
reproducible narrow pH gradient, as were the heterozyliant Blue
staining.
drawing indicating
gotes FZ and MZ.
More than 75 different variants
have been identified
by IEF of serum (7). The protein separation can be
further improved by hybrid IEF in Immobiline, with use
of an ultra-narrow
pH gradient
(8). More than six
subtypes of the M variant have been identified, three of
which (Ml, M2, and M3) are clearly detected with the
PhastSystem dry-gel technique.
Furthermore,
the typical homozygous
patterns-two
major bands and three minor ones-are
easily detected
(numbered
2-8 on Figure 1). These patterns
are due to
the variation of the content of sialic acid and the length
of the polypeptide chain (9). We included in each exper-
75
iment a sample for heterozygote MZ (Figure 1) as a
marker for phenotyping and for quality control.
This
heterozygote
constitutes
one of the most heterogenous
15
patterns,
Duration,
#{149}c V’h
15
15
Vol.38, No. 4, 1992
M1Z
at top)
Inthe schematic drawing, the open bass represent the Z-aIIele products. The
anodal major Z-isoform is superimposed on the majorcathodal S-isoform.The
isoelectric points and isoform number for the M Isoforms are given on the
being
a combination
of the normal M-allele
Z-allele products of the human
a1 -Pi
gene. The resolution on this ultrathin polyacrylamide
gel is good, despite the very short focusing time (30
mm). Using dried blood on filter paper (Guthrie spots) in
and the mutant
2000
2000
p1
4.42
M1
of Hemoglobin
to a hemoglobin
0
N:o
450
550
combination
with the sensitive silver-staining
method
can help identify low-a1-antitrypsin-concentration
phenotypes (Pi ZZ or SZ) in future screening programs. The
PhastSystem
typing procedure
is very simple and rapid
compared with the hybrid IEF method based on miniaturized immobilized
pH-gradient gel (8); the latter gives
excellent resolution but is too complex and time consuming for screening purposes. An extensive review of
the genetic, functional, and medical aspects of a1-antitrypsin was published recently (7), demonstrating
the
need for phenotyping.
Hemoglobin
Screening
requested
for abnormal hemoglobins is a frequently
in the clinical laboratories (10). For
procedure
optimal separation, high-resolution
IEF polyacrylamide
gels in a narrow pH gradient have been used. More than
500 hemoglobin
variants
have been characterized
with
defined amino acid substitutions (11). The most common
of these endemic variants
is HbS, but HbC and HbE are
also common. Many hemoglobin variants
are identified
in single families or only a few families.
Figure 2 demonstrates
the hemoglobin
patterns
obtained with PhastSystem
high-resolution
IEF in a nar-
row pH gradient,
6.7-7.7;
a schematic
drawing
is in-
cluded for comparison. The lane to the left represents a
normal hemolysate
gel pattern
with the major HbA1
fraction (pI 7.12) and the normal amounts
of HbF and
HbA2 (p1 7.42) fractions (-1% and -2-3%, respectively). Anodal to HbA1 is the glycated form of hemoglobin,
HbA1C (normal concentration
4-5.3%). The most anodal
protein fraction is HbA3, a gluthathione
adduct, which
increases
with time and storage of the samples. The
second lane (Thal) shows increased
HbA2, which is
typical for /3-thalassemia minor. The third lane demonstrates a sample from a diabetic patient with increased
HbA1C and HbF, accidentally
found in ion-exchange
chromatography
for quantification of glycohemoglobin
(12). The last three lanes show the most common hemoglobin variants AE, AS, and AC. HbE and HbC are
superimposed
on the HbA2 fraction, E being slightly
more anodal and C more cathodal than A2.
The commonly encountered
abnormal
hemoglobins
were well resolved in the actual pH gradient 6.7-7.7, and
further
gel electrophoresis in alkaline or acid buffers was
not necessary (13). The small mobility differences, which
depend on the amino acid substitutions,
with the narrow
can be detected
pH gradient
used. Electrofocusing
in
gels rehydrated
in diluted and
pre-made polyacrylamide
defined Pharmalyte
solutions gives reproducible and reliable results. Furthermore,
the high field strength eliminates problems of diffusion and allows the extremely
high resolution necessary for the typing procedure.
---
In conclusion, the total time for analysis is very short
because of the short separation time and because of the
rapid and efficient staining and destaining
in the development unit, which makes the typing on PhastSystem
-
suitable
A
clinical diagnosis.
The techniques
intended for screening
purposes.
Characterization
of unusual or new variants, of course,
requires specialized laboratories with knowledge
of peptide mapping, polymerase
chain reaction,
and DNA
sequence techniques.
A1
The skillful technical assistance
appreciated.
-
-
for routine
presented
0
p1
A1
7.12
F
S
-
7.42
A2
N
Thai Diab AE
AS
AC
Fig. 2. Isoelectnc focusing patterns of hemolysates of a control and
patients’ samples in the pH range 6.7-7.7 after Coomassie
five
Brilliant Blue staining (anode at top)
In the schematic drawing of the six different sample types (normal,
p-thalassemia, diabetic, and three hemoglobin vanants),the isoelectricpoints
and typeof hemoglobinproteinbandare shown on the y-axis
are
mainly
of Anna Arnetorp is highly
References
1. Olsaon I, Axio-Fredriksson U-B, Degerman M, Olsson B. Fast
horizontal electrophoresis. I. Isoelectric focusing and polyacryl-.
amide gel electrophoresis using PhastSystem’.
Electrophoresis
1988;9:16-22.
2. Olsson I, Wheeler R, Johansson
C, et al. Fast horizontal
electrophoresis.
II. Development
of fast automated
staining
procedures using PhastSystem”.
Electrophoresis 1988;9:22-7.
3. Jeppsson J-O, Franz#{233}n
B, Nilsson K-O. Determination of the
glycosylated hemoglobin fraction HbAIC in diabetes mellitus by
thin-layer electrofocusung. Sci Tools 1978;25:69-72.
4. Jeppeson J-O, Franzen B. Typing of genetic variants of a1antitrypsun by electrofocusing. Clin Chem 1982;28:219-25.
5. Jeppason ,J-0, Sveger T. Typing of genetic variants of a1antitrypsin using dried blood from the Guthrie test. Scand J Cliii
Lab Invest 1984;44:413-6.
6. Jeppsson J-O, KAilman I, Lindgren G, Fagerstam G. HbLinkoping (-36 Pro-Thr): a new hemoglobin mutant characterized
by reversed-phase
high-performance liquid chromatography.
J
Chromatogr 19&4;297:31-6.
7. Cox-Wilson D. a1-Antitrypsin
deficiency. In: Scriver CHR,
CLINICALCHEMISTRY,Vol.38, No.4, 1992
579
Beaudet AL, Sly XS, Valle D, eds. The metabolic basis of inherited
disease, 6th ed. New York: McGraw Hill, 1989:2409-38.
8. Alonso A. Human alpha1-antitrypsin
subtyping by hybrid isoelectric focusing in miniaturized polyacrylamide gel. Electrophoresis 1989;10:513-9.
9. Jeppsson J-O, LiIja H, Johansaon M. Isolation and characterization of two minor fractions of a1-antitrypsun by high performance liquid chromatofocusing.
J Chromatogr 1985;327:173-7.
10. Basset P, Benzard Y, Garel MC, Rosa J. Isoelectric focusing of
human hemoglobin: its application to screening, to the character-
ization of 70 variants, and to the study of modified fractions of
normal hemoglobuns. Blood 1978;51:971-82.
11. H’iiemin THJ. Policies of the International Hemoglobin Information Center. Hemoglobin 1991;15:139-245.
12. Jerntorp P, Sundkvist G, Fex G, Jeppsson J-O. Clinical utility
of serum fructosamune in diabetes mellitus compared with hemoglobin A1. Clin Chim Acta 1988;175:135-42.
13. Huisman THJ, Jouxis JHP. The haemoglobinopathies, techniques of identification. New York: Marcel Dekker, 1977.
CLIN. CHEM. 38/4, 580-584 (1992)
Rapid Immunometric Measurement of C-Reactive Protein in Whole Blood
Petter Urdal,’
Stig M. Borch,2
Sverre Landaas,’
May B. Krutnes,2
We examined an instrument-free test for C-reactive pro-
tein (CAP) in whole blood. The NycoCard CRP Whole
Blood test uses a ceil-solubilizing dilution liquid, a membrane-bound antibodythat binds CAP, and a gold-conjugated antibody for making visible the bound CRP. We
obtained essentially
identical dose-response curves in
citrate-, hepann-, and EDTA-treated blood. CVs were
6.7-12.5% within series and 10.1-14.7% between series.
The detection limit was 12 mg/L. lntralipidadded to blood
increased measured CRP by 10-20%, whereas no
change was seen with added bilirubin, added serum
amyloid P component,
or the presence of rheumatoid
factor. In 234 patients’ blood samples the results of the
NycoCard Whole Blood test correlated well (r = 0.96) with
those of a turbidimetric
serum method. The test allows
reliable measurement of CAP from a small volume of
whole blood (25 L) without using expensive equipment;
it should be useful for decentralized testing in hospital
departments, emergency units, and primary health care
centers.
AddItional Keyphraeee:
gated antibody
intermethod comparison
-
gold-conju-
C-reactive protein (CRP) is one of the more characteristic acute-phase
proteins and is considered a reliable
indicator of disease activity in various clinical conditions (1). Its concentration
in blood increases rapidly by
as much as 1000-fold upon exposure to various inflammatory stimuli, decreasing
rapidly when the stimulus
declines, e.g., after effective antibacterial
treatment (2).
CRP has been used successfully for clinical diagnosis
and monitoring of a variety of infections and diseases,
including infections
caused by bacteria, fungi, and viruses (3-7); intercurrent infections in leukemia (8,9) and
‘Department of Clinical Chemistry, Ullevtl Hospital, N-407
Oslo 4, Norway; 2Reaearch and Development Unit, Diagnostics
Division, Nycomed Pharma AS; and 3Department of General
Practice, University of Oslo, Norway.
Received July 22, 1991;accepted February 6, 1992.
580 CLINICALCHEMISTRY,Vol.38, No.4, 1992
Geir O Gogstad,2
and Per Hjortdahl3
systemic lupus erythematosus
(10); noninfectious
inflammatory diseases such as rheumatoid arthritis
(11); and
diseases with cellular necrosis such as myocardial
infarction (12,13). Measurements of CRP are especially useful
in distinguishing
viral from bacterial infections (3,5,14).
Among the methods used to measure
CRP in serum
are radioimmunoassay
(15); radial
immunodiffusion
(16); latex agglutination
(17), which also is available in
a quantitative microtiter
version (18); lipid agglutination (19); turbidimetry
(16, 20, 21); nephelometry
(16);
particle-enhanced
turbidimetry
(22); enzyme immunoassays
(23, 24); and fluorescence polarimetry
(25).
These methods assay CRP in serum, either as instrument-based quantifications
or as instrument-free,
qualitative
to semiquantitative
agglutination
tests. The
acute nature of many diseases in which CRP is relevant
for diagnosis and monitoring
requires a rapid, easily
interpreted,
instrument-free,
quantitative
test that may
be applied directly to whole blood. We present here a
test with such characteristics.
Materials and Methods
Materials
Bilirubin was obtained
from Sigma Chemical Co. (St.
Louis, MO), serum amyloid P component from Calbiochem Corp. (San Diego, CA), and Intralipid from KabiVitrum (Stockholm,
Sweden).
Reference Preparations
We used the 1st International
Standard
(World
for human CRP (National
Institute for Biological Standards
and Controls,
London,
U.K.) to establish the concentration
of CRP in the
reference preparations.
These preparations
contained
CRP at <5 to 1000 mg/L of plasma: purified CRP (Scipac
Ltd., Kent, U.K.) added to whole blood (anticoagulated
with citrate, heparin, or EDTA) obtained from healthy
donors. We similarly prepared controls containing CRP
concentrations
of <5,30, and 125 mg/L in plasma, using
blood from one donor, and stored these at 4#{176}C.
The
controls were used for estimating
within- and betweenHealth
Organization)