| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
1
The Futhan/EDTA tubes are produced by Terumo Europe and can be purchased from PerSeptive Biosystems, 500 Old Connecticut Path, Framingham, MA 01701. At present, the Futhan/EDTA tubes are approved only for research use in the United States.
a Address correspondence to this author at: Department of Immunology/IMM-18, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Fax 619-784-8307; e-mail hugli{at}scripps.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Methods: BiotrakTM RIAs were used to quantitatively measure C3a and C4a in blood and/or plasma samples from healthy individuals (controls) and from liver transplant patients. Blood samples were routinely drawn into either EDTA (1 g/L) tubes or into tubes containing both EDTA (1 g/L) and Futhan (0.1 g/L) and immediately centrifuged at 2000g for 15 min at 4 °C.
Results: In controls, C4a, but not C3a, in fresh samples (time 0) was higher in EDTA plasma than in Futhan/EDTA plasma (n = 20; P = 0.002). Futhan/EDTA prevented C3a and C4a generation in blood and plasma samples held at room temperature (22–23 °C) for 1 h and in plasma held for 24 h at 4 °C or -70 °C. The mean C3a concentration (1.76 mg/L; n = 19) at time 0 in EDTA plasma samples from liver transplant patients was significantly higher than for controls (0.34 mg/L; n = 11). In these patients, the mean C3a in EDTA samples increased to 13.8 mg/L after 60 min at room temperature, but there was no change in the C3a concentration of an EDTA plasma from a control. In the patients, C3a concentrations were lower in Futhan/EDTA plasma than in EDTA at time 0 and after 60 min at room temperature (1.40 and 2.02 mg/L, respectively). The mean patient C4a was 4.02 mg/L in EDTA plasma at time 0 vs 0.24 mg/L for controls; it increased to 16.9 mg/L after 60 min at room temperature compared with 0.76 mg/L for controls. The mean patient C4a was 0.83 mg/L in Futhan/EDTA plasma at time 0 vs 0.1 mg/L for controls. Neither patient nor control C4a concentrations increased vs time in Futhan/EDTA.
Conclusion: The combination of Futhan (0.1 g/L) and EDTA (1 g/L) eliminates in vitro C activation.© 1999 American Association for Clinical Chemistry
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In designing a routine diagnostic complement assay for the clinical laboratory, sample stabilization becomes a key issue. It is well established that neither of the major C-activation pathways (i.e., the classical and alternative pathways) can function in the presence of the metal chelator EDTA (26). Consequently, understanding the mechanism of in vitro C activation is as important as developing a method to control it.
It has been known for some time that amidine and guanidine derivatives will inhibit C-mediated hemolysis (i.e., C activation) (27). Fujii and Hitomi (28) designed, synthesized, and characterized a potent synthetic inhibitor of complement and coagulation proteinases, based in part on this information. This inhibitor is 6-amidino-2-naphthyl-4-guanidinobenzoate dimethanesulfonate, commonly referred to in the literature by the trade name Futhan or FUT-175, or by the generic name of nafamostat mesilate. The advantage of this particular proteinase inhibitor as a blood additive is that it has an unusually broad specificity for serine proteases, being a potent inhibitor of both coagulation and complement proteinases. Futhan inhibits thrombin, plasmin, and kallikrein (both plasma and pancreatic), all of which can degrade C components C3, C4, and/or C5 (29)(30). Previous studies have focused on identifying the complement proteinases that are targeted by Futhan, which include C1r, C1s, and factors B and D (31). This inhibitor was also effective toward Hageman factor and factor Xa at submicromolar concentrations (32). Several studies have examined the in vivo protective effects of Futhan in various models of immunological reactions including Forssman shock in guinea pigs, passive cutaneous anaphylaxis in rats, and delayed hypersensitivity reactions and endotoxin shock in mice (33)(34). Because Futhan inhibits C-mediated hemolysis by both the alternative and classical pathways (35), it was concluded that the major effect of this compound on in vivo immunological reactions resulted from direct inhibition of enzymes in the C-activation pathways.
A new pathway of C activation, called the lectin pathway, was discovered recently (36). This pathway involves a mannan-binding protein or lectin (MBL) that is identical to the bactericidal Ra-reactive factor that binds to the Ra polysaccharides on various strains of bacteria (37). MBL is a multichain, multisubunit protein that functions similarly to the C1q component of the classical pathway. There are two proteinases associated with MBL: MBL-associated serine proteinase-1(MASP-1) and MASP-2 (38). The MBL:MASP-1-MASP-2 complex is activated via MBL binding to neutral sugars, activating the MASP enzymes, which then cleave components C2, C3, and C4 to initiate the classical C pathway (36)(37). These MASP enzymes are likely candidates for the proteases that promote C activation in EDTA plasma.
Although extensive studies have been undertaken to characterize Futhan activity in vivo and to identify the various proteases that it inhibits, its effectiveness as an inhibitor of C activation in drawn blood/plasma samples, particularly for patient samples, is less well characterized. Futhan effectively stabilizes freshly drawn blood samples from healthy individuals, permitting direct measurement of the anaphylatoxins C3a and C4a (39). The effectiveness of Futhan for controlling in vitro C activation in patient blood in which the activating enzyme concentration can be relatively high has not been established. In the present study, we describe parameters for using Futhan, along with EDTA, as effective additives for preventing C activation during routine handling and processing of blood and plasma samples. It was important to show that the combination of Futhan and EDTA is effective when in vitro C activation is relatively high to establish clinical utility of the procedure. Reliable estimates of the circulating concentrations of C3a, C4a, and C5a can be used as an index of in vivo C activation only if in vitro activation is totally eliminated. Because improper handling of blood samples and variable intrinsic proteinase activity can lead to substantial in vitro C activation, even in EDTA plasma, an additional inhibitor must be used. Consequently, we developed a blood-drawing and handling protocol that stabilizes blood or plasma from activation of either component C3 or C4. The ability of Futhan/EDTA to stabilize complement in frozen (-70 °C) plasma samples for months or at 4 °C for >24 h permits research and clinical samples to be stored for later measurement of total circulating C-component concentrations or for determining the concentrations of the C-activation fragments C3a, C4a, and/or C5a.
When applied to patient blood samples, the Futhan/EDTA blood-drawing protocol provided evidence that circulating C3a and C4a in liver (allograft) transplant patients were significantly increased compared with control (i.e., "normal") individuals. None of these patients were experiencing rejection episodes but were considered clinically stable on their respective treatment regimens. The more striking result was that in vitro conversion of C3 and C4 vs time was markedly higher in the EDTA blood or plasma from these patients than observed in EDTA plasma from control individuals.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
blood samples
Blood samples of 5–10 mL were drawn from healthy donors, liver
transplant and SLE patients under an approved Human Subjects protocol
no. 96-293, and after the individuals had signed an informed consent
form agreeing to be voluntary donors. The healthy donors were a random
group of male and female individuals between the ages of 27 and 48
years. Liver transplant patients were selected from a group of patients
being routinely followed at The Scripps Clinic Foundation by Dr. John
Brems, currently at Loyola University Medical Center, Maywood, IL. None
of these organ transplant patients were experiencing acute rejection
episodes or other detectable liver malfunctions at the time that the
blood was drawn.
blood processing protocols
Blood samples were either drawn into EDTA
tubes2 (Venoject; Terumo Corp.) or Futhan/EDTA
tubes (Venoject; Terumo Europe). The plasma was collected immediately
by centrifugation at 2000g for 15 min at 4 °C unless
otherwise indicated. The plasma samples were either processed
immediately for analysis or snap frozen in liquid nitrogen and stored
at -70 °C. Frozen samples were thawed at 4 °C and processed as
described in the text.
assay procedure and data analysis
Equal volumes of the plasma sample and the precipitating reagent
supplied in the Biotrak RIA kits were mixed by thorough agitation and
incubated at room temperature for 5 min. The mixture was then
centrifuged at 2500g for 15 min at 4 °C. The supernatant
from each sample tube was collected for analysis in the RIA assay. A
50-µL aliquot of the original or diluted supernatant was mixed with
50 µL of the Biotrak assay buffer in a 12 x 75 mm polypropylene
tube. The 125I-labeled C3a or C4a and the
specific antibody solutions (50 µL of each) were added and incubated
at room temperature for 30 min. A 50-µL aliquot of the second
antibody (goat anti-rabbit) was added to the tube, and the mixture was
incubated for an additional 30 min at room temperature. Isotonic saline
(2 mL) was added, and the tube was centrifuged at 2000g for
10 min at 4 °C to pellet the antigen-antibody complexes. The
supernatants in the tubes were decanted, and the tubes were counted for
1 min in a Cobra Autogamma Model 5002 (Packard Instruments)
scintillation counter. Analysis of each sample was performed in
duplicate. Data were analyzed using RiaSmart software supplied by
Packard Instruments and installed in the Cobra Autogamma counter.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). Although these data were obtained from the plasma of a single
individual, similar data have been obtained from blood and plasma of
other normal donors (see Figs. 2
and
3). When Futhan concentrations were 0.2 g/L, the C3a
concentrations in all of the samples were actually somewhat higher than
at the lower Futhan concentrations. This activation may be a result of
nonspecific effects related to exposing the plasma to increased salt
concentrations.
|
|
These same handling and storage conditions did have variable effects on C4 activation in the EDTA plasma (i.e., in the absence of Futhan). However, the characteristic activation of C4 in the control EDTA plasma was abolished when Futhan was added to these samples. Consequently, the major effect of adding Futhan to the control EDTA plasma was to reduce C4a, but not C3a, generation in vitro. Futhan concentrations as low as 0.01 g/L appeared to be effective in protecting the control plasma from C activation under the conditions described. These data indicated that activation of C in EDTA plasma resulted from either the classical or lectin pathway of activation because primarily C4a was generated. Furthermore, the plasma enzyme(s) clearly prefer cleaving C4 over C3.
Commercial blood-drawing tubes (EDTA/Futhan VenojectTM tubes; Terumo) containing 5 mg of disodium EDTA and 0.5 mg of Futhan per tube were designed for collecting 2.5–5.0 mL of blood. Although Futhan at 0.01 g/L was effective in protecting normal plasma from C4 conversion, it was anticipated that higher concentrations might be required to protect C4 in patient plasma in which the conversion of C4 (i.e., the converting enzyme concentrations) was significantly greater. The EDTA/Futhan vacuum tubes were developed to stabilize C in blood samples being drawn for either research or clinical applications.
Futhan is an inhibitor that exhibits a broad specificity for many of the blood serine proteinases. Therefore, it might be anticipated that Futhan would be superior to many other anticoagulants in preventing complement conversion in whole blood. General serine proteinase inhibitors of the coagulation system such as benzamidine (an inhibitor of Factors VII and Xa, thrombin, plasmin, and C1s) or Trasylol (an inhibitor of thrombin, kallikrein, and plasmin) were compared to Futhan for their effectiveness in controlling C activation. It is well known that certain of the coagulation proteinases can convert C3 and C4 (39); therefore, it is important to inhibit both plasma complement and coagulation enzymes to prevent potential non-complement enzymes from causing C activation.
In the absence of EDTA, neither benzamidine nor Trasylol, either
individually or in combination, could fully stabilize C3 (i.e., prevent
C activation through the alternative pathway) vs time (Fig. 2A
). It
should be noted that in Fig. 1
, the concentrations of C3a generated in
EDTA whole blood at room temperature after 60 min were ~600 µg/L,
whereas in Fig. 2A
, the C3a values were ~200 µg/L under the same
conditions. The differences between these two results reflect
variations in in vitro C activation that are commonly observed between
individual normal donors. The blood donor in Fig. 1
was not the same as
the donor in Fig. 2A
. From these data it is clear that inhibitors of
coagulation proteinases were incapable of preventing C3 conversion.
When EDTA was added to the blood, there was a remarkable increase in C4
conversion, but virtually no C3 was converted. The enzymes responsible
for C4 conversion in the presence of EDTA remain unknown. We
hypothesized the enzyme to be MASP-2 of the lectin pathway
(37)(38). It is not C1s of the classical pathway
because C1 is effectively inactivated by the removal of calcium ions
(40) and any C1s activated would be under the rigorous
control of C1 inhibitor (41).
Futhan, with or without EDTA added, was effective in stabilizing both
C3 and C4 in normal whole blood for up to 1 h at room temperature
(Fig. 2A
). When blood samples were drawn and immediately placed on ice,
the samples were stabilized for much longer (Fig. 2B
). Note that EDTA
was sufficient to prevent C3a generation in the control blood at
4 °C over a 27-h period. However, even at 4 °C, significant
quantities of C4a can be generated in EDTA blood unless Futhan is
added. These results define a suitable window of opportunity for
collecting stabilized plasma from blood samples drawn into Futhan/EDTA,
and this protocol can be applied to both research and clinical samples.
We examined the C3a and C4a concentrations in EDTA blood obtained from
healthy donors, processed immediately with and without Futhan present.
These data helped to establish a baseline for circulating
concentrations of these factors in healthy individuals. The mean values
for C3a and C4a in EDTA plasma and in EDTA/Futhan plasma were obtained
from a total of 20 individuals. The plasma was recovered immediately
from the blood samples, and analysis gave the data shown in Table 1
. This study involved 11 male and 9 female donors between the
ages of 27 and 48 years. Although on inspection the EDTA/Futhan values
for C4a appeared to be only marginally lower than the EDTA-plasma
values when a careful blood processing procedure was used, there was a
statistically significant protective effect of the Futhan. When the
values in Table 1
(i.e., immediate processing to collect plasma) are
compared with values obtained from EDTA whole blood held on ice for
only a few hours (see Fig. 2B
), it is clear that C4 is not protected in
blood from healthy donors without the addition of Futhan.
|
These data also demonstrated that some donors who present themselves as
normal can have background C4a concentrations >2 SD higher than the
mean value, as shown in row I of Table 1
. The fact that C4a was
significantly lower in Futhan/EDTA samples than in samples with EDTA
alone indicates that low levels of in vitro activation also commonly
occur in normal plasma.
C activation in EDTA plasma was compared to activation in EDTA/Futhan
plasma at room temperature (22–24 °C) over a period of 1 h
(Fig. 3A
). The data are reported as mean plasma concentrations (n
= 11) from a separate group of donors than those reported in Table 1
,
and the only values that changed during this period of time were the
C4a concentrations. C4a was increased approximately three- to fourfold
on average over 60 min in EDTA plasma for this group of 11 normal blood
donors. C4a generation varies significantly even between normal
individuals, and C4a concentrations in normal EDTA plasma usually rose
significantly vs time, even at 4 °C.
|
A dramatically different pattern of C4 activation was observed in the
plasmas of liver transplant patients (42) compared with
normal individuals (Fig. 3B
). These subjects were stable transplant
patients not undergoing a clinically detectable rejection episodes. The
background plasma C3a and C4a concentrations at time 0 were
significantly higher than those in plasma from the normal population.
After 60 min at room temperature, the mean C4a concentration in the
patient EDTA plasma had risen to 25- to 50-fold higher than in normal
EDTA plasma.
We examined activation of C4 in a patient with SLE and found that the
circulating concentrations of C3a and C4a (i.e., in EDTA/Futhan plasma)
were much higher than in normal EDTA/Futhan plasma, which is consistent
with classical pathway activation (Fig. 4
). The C4a concentration in EDTA plasma from the SLE patient
exceeded 10 mg/L after 60 min at 37 °C compared with 8–9 mg C4a/L
in two normal plasmas (data not shown). Although the rate of C4
activation in the SLE patient's EDTA plasma at 37 °C was very high,
this appears to be a typical rate of in vitro C activation at
this temperature. These 37 °C results for C4 conversion illustrate
that if blood is not chilled immediately after it is drawn and/or
Futhan is not added to the stabilized samples, the background
concentration of C4a will rise rapidly even in normal blood.
|
The plasmas from the organ transplant patients, whose mean C3a and C4a
concentrations are shown in Fig. 3B
, gave even more interesting
patterns when each individual sample was examined. The patterns
observed in Fig. 3B
for EDTA vs Futhan/EDTA plasma C activation
indicated that these patients had high concentrations of active plasma
enzyme. When Futhan and EDTA were used to stabilize the plasma samples,
there was no significant C3a or C4a generation in any individual
patient's plasma vs time at room temperature (Fig. 5
). However, the concentrations of both C3a and C4a in
Futhan-stabilized plasma, which should represent the true circulating
concentrations, were increased in all patients compared with healthy,
nontransplant individuals. One sample (patient 15) gave a markedly
increased C3a value after 1 h of incubation, and we concluded that
mishandling during the assay procedure was responsible for this
artifactual result. The incremental increases in Futhan/EDTA plasma C3a
or C4a after 1 h at room temperature were minimal in all patients
except patient 15.
|
A more dramatic result was observed when the EDTA plasmas from these
patients were evaluated. The scale in Fig. 6
is more than 10-fold higher to accommodate the large increases
in C3a and C4a occurring after 1 h at room temperature. Although
the mean values for C3a and C4a were markedly increased, there were
extreme differences between individual patients. Patients 4, 6, 8, 9,
11, 12, 15, and 16 showed extensive in vitro C activation vs time,
which we speculate to be caused by the MASP enzymes of the lectin
pathway. The fact that only some of these patients demonstrated marked
increases in C3a and C4a generation in vitro indicates that a
discriminating method exists to detect or evaluate these patients once
the underlying cause for activating the converting enzyme(s) is
understood. Because these transplant outpatients demonstrated no
clinical signs of organ rejection or infection when these plasma
samples were taken, no biopsies were obtained to further evaluate the
status of the organ.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The primary advantages of measuring these two activation fragments of components C3 and C4 are (a) C3a and C4a concentrations are direct indicators of the type and extent of activation because C4a is generated only during classical or lectin pathway activation and C3a generation in the absence of C4a confirms alternative pathway activation; (b) The desArg forms of C3a or C4a no longer bind to their specific cellular receptors and thus circulate freely without further degradation, and once C3a and C4a are released into the blood, they are rapidly converted to the desArg forms (C3a desArg and C4a desArg) by serum carboxypeptidase N (43); (c) C3 and C4 are abundant blood proteins, and even minor C activation can be detected by sensitive assays for these breakdown products; (d) the factors C3a desArg and C4a desArg are extremely stable and soluble proteins and are not denatured by handling; and (e) generation of C3a or C4a signals the production of numerous other physiologically important breakdown products of the C cascade, namely C3b, C4b, C3bi, C4bi, C3d, C4d, and C5b-9, each having known biologic functions and activities.
Complement is not stable in EDTA plasma vs time, and numerous earlier
attempts to stabilize complement in blood or plasma have been reported.
The most successful procedure to date involved the use of a
broad-spectrum serine proteinase inhibitor called Futhan
(31)(32)(33)(34)(35). A variety of inhibitor and stability studies using
Futhan have been reported in the literature; unfortunately, many of
these reports are published in Japanese journals, and these results are
not widely known. Our goal was to design a protocol for blood or plasma
collection and handling that can effectively stabilize C, even in
patient blood samples in which in vitro C activation is extensive.
Previous studies using Futhan to prevent C activation were focused
mainly on normal blood samples. In normal EDTA blood or plasma held at
4 °C, we observed significant activation of C4 vs time. There was
little conversion of C3 in normal EDTA blood or plasma at 4 °C up to
24 h, indicating that the proteinase responsible for C4a
generation selectively cleaves C4 over C3. The addition of 0.01 g/L
Futhan to the EDTA plasma produced a measurable reduction in C4
conversion vs time, and 0.10 g/L Futhan virtually eliminated C4
cleavage over a 24-h period. Because 0.10 g/L Futhan is the
concentration of the inhibitor used in commercial Futhan/EDTA
blood-drawing tubes, many of our experiments were performed using this
concentrations of the inhibitor. It was important to test whether the
combination of Futhan and EDTA in blood or plasma would be adequate to
stabilize the C4 over a 24-h period. This would indicate that blood or
plasma samples could be drawn at one site and shipped for analysis,
either on ice or frozen, to another site. It appears that Futhan/EDTA
plasma, but not whole blood, can be collected and safely stored at
4 °C for at least 24 h, based on the data shown in Fig. 2B
. The
EDTA/Futhan plasma appears to provide an appropriate sample for
obtaining reliable circulating concentrations of C3a and C4a (or C3 and
C4) while avoiding in vitro activation that could render the data
invalid.
Because Futhan is a serine proteinase inhibitor that inhibits
coagulation proteinases as well as complement proteinases, we examined
several other proteinase inhibitors that are known to inhibit
proteinases in both blood systems (Fig. 2A
). We were surprised to find
that both benzamidine and Trasylol (Kunitz inhibitor) were relatively
ineffective in preventing C3 conversion in whole blood over a 60-min
period. This is presumably because these inhibitors more effectively
block the complement serine proteinase factor I than factor B. Factor I
is a control proteinase that inactivates C3 convertase (C3b,Bb) by
cleaving the essential cofactor C3b to C3bi, destroying the active
enzyme complex. Therefore, if factor I is inhibited, small quantities
of the C3b,Bb complex will form in the blood and permit the alternative
pathway to progress unimpeded. When EDTA was added to these same blood
samples, C3 conversion by the Ca2+-dependent
classical and alternative pathways was prevented.
C4 conversion in the presence of EDTA indicated activation of a plasma
proteinase in the absence of calcium. Neither of the coagulation
proteinase inhibitors used here were effective in blocking C4
conversion; however, Futhan appears to be fully effective in inhibiting
this proteinase. We hypothesize that the proteinase responsible for the
in vitro activation of C4 may be the MASP-2 enzyme of the lectin
pathway (38). The activation or induction of MASP-2 activity
in plasma by EDTA has not yet been demonstrated, and this mechanism is
currently being explored. Consequently, we suggest that the MASP-2
enzyme of the lectin pathway may be primarily responsible for in
vitro C activation; however, the proteinase(s) in EDTA plasma have
not been identified (see Table 2
).
|
In Fig. 3A
, the C4a values in EDTA plasma were the only ones to
increase vs time, and these concentrations remained quite low. These
data matched published data for best estimates of the minimal
background or baseline circulating concentrations of C3a or C4a in the
general population (35). The C4 conversion values in a group
of liver transplant patients shown in Fig. 3B
were remarkably high
(20-fold higher than the control group) even at time 0 (i.e., the
actual time it takes to spin down the blood, recover the plasma, and
add precipitating agent to stop the activation). After 60 min, the
conversion of both C3 and C4 was extensive in EDTA plasmas from some of
the organ transplant patients. These data indicated that, although the
plasma proteinases appeared to prefer C4 over C3, at high
concentrations, these enzymes were capable of cleaving both C3
and C4. In fact, there are reports that MASP-1 preferentially cleaves
C3 (44). The key information provided by these studies, in
terms of potential clinical applications for the C3a/C4a assay system,
was that Futhan remains protective even when the proteinase
concentrations are markedly higher than in normal plasma.
A recent report describing the C-activation profiles of C vs time in
liver transplant patients suggested that pathologic events such as
acute rejection episodes, hepatitis C, or cytomegalovirus infections as
well as bacterial infections can be detected by monitoring the
circulating concentrations of C3a and C4a (42). Many of the
events monitored in these liver transplant patients appeared to involve
primarily the classical pathway (i.e., only C4a concentrations were
increased). Considering the results shown in Figs. 5
and 6
in this
study, we hypothesize that components C3 and C4 are being activated in
vitro either by a non-complement proteinase or by MASP enzymes of the
lectin pathway. It is clear that the pathology leading to expression of
the enzyme activity in EDTA plasma has some specificity because only 8
of 19 patients showed marked increases in C activation. The condition
or process responsible for this high enzyme activity remains unknown in
this patient population that exhibited no overt clinical signs of organ
malfunction or rejection. Consequently, many such complications in
organ transplant patients that lead to increased enzyme activity in
EDTA plasma have remained undetected and undiagnosed. Many other immune
diseases, particularly autoimmune diseases such as SLE (Fig. 4
) and
rheumatoid arthritis, have been associated with C4 conversion solely by
the classical pathway and not the lectin pathway. Futhan/EDTA plasma
samples are clearly recommended as a means to monitor these diseases.
The circulating C3a and C4a concentrations can be accurately determined
if Futhan/EDTA plasma samples are used as described in this report.
Independent evaluations of C-activation products either in vivo or in
vitro promise to provide a means to separate, characterize, and
identify involvement of the various C-activation pathways in human
disease.
|
|
Acknowledgments |
|---|
|
Footnotes |
|---|
1 Present address: BMA Biomedicals AG, Rheinstrasse 28-32, CH-4302 Augst, Switzerland. 
A preliminary report of this work, entitled "Plasma C3a and C4a levels in liver transplant recipients: a longitudinal study" (Mol Immunol 1998;35:357), was presented as Abstract 107 at the XVII International Complement Workshop, Rhodes, Greece, October 11–16, 1998.
This is publication 11902-IMM from The Scripps Research Institute.
2 Nonstandard abbreviations: C, complement; C3a and C4a, activation fragments from complement components C3 and C4; SLE, systemic lupus erythematosus; Futhan, 6-amidino-2-naphthyl-p-guanidinobenzoate dimethanesulfonate (FUT-175 or nafamostat mesilate); MBL, mannan-binding lectin; and MASP, MBL-associated serine protease.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  V. E. Burns, K. M. Edwards, C. Ring, M. Drayson, and D. Carroll Complement Cascade Activation After an Acute Psychological Stress Task Psychosom Med, May 1, 2008; 70(4): 387 - 396. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. R. La Bonte, G. Davis-Gorman, G. L. Stahl, and P. F. McDonagh Complement inhibition reduces injury in the type 2 diabetic heart following ischemia and reperfusion Am J Physiol Heart Circ Physiol, March 1, 2008; 294(3): H1282 - H1290. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Honczarenko, M. Z. Ratajczak, A. Nicholson-Weller, and L. E. Silberstein Complement C3a Enhances CXCL12 (SDF-1)-Mediated Chemotaxis of Bone Marrow Hematopoietic Cells Independently of C3a Receptor J. Immunol., September 15, 2005; 175(6): 3698 - 3706. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. E. Banks, A. J. Stanley, D. A. Cairns, J. H. Barrett, P. Clarke, D. Thompson, and P. J. Selby Influences of Blood Sample Processing on Low-Molecular-Weight Proteome Identified by Surface-Enhanced Laser Desorption/Ionization Mass Spectrometry Clin. Chem., September 1, 2005; 51(9): 1637 - 1649. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kirschfink and T. E. Mollnes Modern Complement Analysis Clin. Vaccine Immunol., November 1, 2003; 10(6): 982 - 989. [Full Text] [PDF]
|
|
|
|
 |
|
 H. Tani, C. K. Limn, C. C. Yap, M. Onishi, M. Nozaki, Y. Nishimune, N. Okahashi, Y. Kitagawa, R. Watanabe, R. Mochizuki, et al. In Vitro and In Vivo Gene Delivery by Recombinant Baculoviruses J. Virol., September 15, 2003; 77(18): 9799 - 9808. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Furuya, H. Takeda, S. Azhar, R. M. McCarron, Y. Chen, C. A. Ruetzler, K. M. Wolcott, T. J. DeGraba, R. Rothlein, T. E. Hugli, et al. Examination of Several Potential Mechanisms for the Negative Outcome in a Clinical Stroke Trial of Enlimomab, a Murine Anti-Human Intercellular Adhesion Molecule-1 Antibody: A Bedside-to-Bench Study Stroke, November 1, 2001; 32(11): 2665 - 2674. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||
) were only slightly higher than C3a concentrations in Futhan/EDTA
blood (
) vs time. C4a concentrations in EDTA blood (
) continued
to rise throughout the entire 23-h period, from 0.3 mg/L to >1.70
mg/L. When the blood was drawn into Futhan/EDTA (•), no C4a was
generated over the entire 23-h period.
) or without (
