Docstoc

Performance Characteristics of Simple Radioimmunoassay for

Document Sample
Performance Characteristics of Simple Radioimmunoassay for Powered By Docstoc
					                     SEMINARS IN THROMBOSIS AND HEMOSTASIS-VOL. 10, NO. 4, 1984




                      Performance Characteristics of a Simple
                      Radioimmunoassay for Fibrinopeptide A

                JEANINE M. WALENGA, B.S., M.T.(ASCP),
                DEBRA HOPPENSTEADT, B.S., M.T.(ASCP),
      R. MARTIN EMANUELE, M.S., and JAWED FAREED, Ph.D., F.A.C.B.


     Although the endogenous generation of fibrino-                 Fibrinopeptide A is a small peptide released
peptide A (FPA) has been known for many years, it              from the N-terminal of the alpha chain of fibrino-
has been primarily used as a research tool rather              gen. Upon the action of thrombin, two peptides
than a diagnostic measure.36,37,39,41,42 Early                 from the alpha chain and two peptides from the
methods for quantitation of FPA did not provide                beta chain are generated as the first products of
specificity, were expensive, complicated, and time-            cleavage. Table 1 illustrates this reaction. FPA
consuming. Today, there are commercially available             consists of 16 amino acids with a molecular weight
FPA kits that provide reliable data by either radio-           of 1535 daltons. The circulating half-life of FPA
immunoassay (RIA) or enzyme-linked immuno-                     is 3 to 4 minutes.1,31 The use of the earliest re-
sorbent assay (ELISA) methods. These assays are                lease products from fibrinogen in a clinical setting
not difficult to perform and are less time-consum-             can provide sensitivity not offered by any previous
ing than the earlier methods. Since RIA and ELISA              marker of thrombosis.3,11,12 Other assays, such as
methodologies are common to clinical laboratories,             the prothrombin time (PT) and partial thrombo-
FPA quantitation can be adapted to any laboratory              plastin time (PTT), the measurement of fibrin(ogen)
to perform this type of testing.                               degradation products, and the protamine paracoag-
     At present, commercial assays are available               ulation or ethanol gelation tests for fibrin mono-
from: Diagnostica Stago (Asnieres, France), As-                mers, are much less sensitive and specific than FPA
serachrom FPA (ELISA); IMCO (Stockholm,                        measurement. Well before any change is observed
Sweden), fibrinopeptide 125I assay kit (RIA); and              in any of these assays, the generated FPA will show
Mallinckrodt (St. Louis, MO), RIA Quant FPA test               an elevation above normal, since determinations are
kit (125I RIA). Our laboratories have evaluated all            made in nanogram per milliliter amounts.
three kits and found excellent correlation among                     Although FPA can be used to aid in the diag-
them; however, the data presented in this article              nosis of the common thrombotic problems, such as
were obtained using the Mallinckrodt 125I kit. This            deep vein thrombosis and pulmonary embolism,17,
                                                               24,26,40,49
is the method routinely used because an iodination                         due to its high sensitivity toward detecting
step is not required before performing the assay, as           thrombotic activity, it is also potentially useful in
must be done with the IMCO kit, and solid phase                the diagnosis and prognosis of such disorders as
support problems are never encountered, as is the              myocardial infarction, malignancies, lupus erythe-
case with the Diagnostica Stago kit. Application of            matosus, and nephritis. 6,7,10,20,34-38,44-46,52 Addi-
high-performance liquid chromatography tech-                   tionally, burns, fractures, and surgery, which can
nology has also been done by several investi-
gators.21,25                                                        TABLE 1. Conversion of Fibrinogen to Fibrin



    From the Department of Pathology, Hemostasis Research
Laboratories, Loyola University Stritch School of Medicine,
Maywood, Illinois.
    Reprint requests: Ms. Walenga, Loyola University Medical
Center, Department of Pathology, 2160 South First Avenue,
Maywood, IL 60153.

                     Publisher: Thieme-Stratton Inc., 381 Park Avenue South, New York, NY 10016                    219
220                                             SEMINARS IN THROMBOSIS AND HEMOSTASIS-VOL. 10, NO. 4, 1984


      TABLE 2. Diagnostic Value of Fibrinopeptide A             nite is required. Recovery studies using FPA show
                                                                no interference of the bentonite treatment on FPA
             1. Hypercoagulable state
                                                                in the sample, and the data are shown in Table 3.
             2. Thrombotic disease
                  Deep vein thrombosis                          This is much simpler and easier than the dialysis
                  Pulmonary embolism                            procedures recommended by earlier investigators,
             3. Malignancy                                      thus reducing turnaround time of the assay to ap-
             4. Burns                                           proximately 2 hours, depending on the number of
             5. Fractures                                       samples analyzed.31
             6. Postsurgical thrombosis                               The test procedure is based on a competitive
             7. Lupus erythematosus                             binding between radiolabeled FPA and unlabeled
             8. Nephritis                                       (test) FPA for a limited number of binding sites on
             9. Procoagulant and anticoagulant therapy          exogenously added FPA antiserum. A polyethylene
                                                                glycol second antibody solution is used to separate
                                                                the free and antibody-bound FPA. After centrifuga-
produce thrombosis, can be monitored for FPA to                 tion, the pellet containing the labeled FPA-antibody-
reduce serious complications.27'33 The most impor-              second antibody complex is counted for radio-
tant use of FPA is in the detection of a hypercoagu-            activity. This procedure is illustrated in Table 4. An
lable state before it leads to thrombus and embolus             increased FPA level in the test sample will allow less
production. Table 2 summarizes the diagnostic                   labeled FPA to bind to the limited antibody, and
value of FPA measurement. Hopefully, such painful               thus less label is precipitated into the pellet (most
invasive procedures as venographics and Doppler                 will be removed in the supernatant). Therefore, an
plethysmography can be replaced by the more sensi-              increased FPA test level results in a lower radio-
tive FPA. The newer antithrombotic and prothrom-                activity count. Standards and controls are provided
botic therapies can be readily assessed by FPA,                 in the kit for a standard curve against which test
because the most commonly used methods are not                  levels can be determined. A typical standard curve
sensitive enough to detect the action of these pro-             as performed in our laboratory is shown in Figure 1.
ducts. 43,48,53 Special products for therapeutic use as
blood components or prothrombin concentrates can
be subjected to quality control prior to human con-                   TABLE 4. Flow Sheet for Fibrinopeptide A
                                                                                Quantitation by RIA
sumption.17,50 Also, certain procedures, such as
leukophoresis, kidney dialysis, and extracorporeal                 Pretreat test plasma with bentonite to remove antigenically
blood circulation, can be monitored for safety to                       cross-reacting fibrinogen and fibrin split products
the patient by assessing the extent of blood acti-
vations.8,9                                                                       Add 125I FPA to test plasma

      METHODOLOGIC CONSIDERATIONS
                                                                                      Add FPA antiserum
    The Mallinckrodt FPA assay is an 125I labeled                                            Incubate

RIA that provides sensitivity from 0 to 40 ng/ml.                   Competitive binding between the antiserum, labeled and
For specificity to the FPA without cross-reactivity                            nonlabeled (patient) FPA occurs
from fibrinogen, a simple pretreatment with bento-
                                                                                      Add second antibody
TABLE 3. Fibrinopeptide A Recovery Studies for the                                     Centrifuge; decant
               Mallinckrodt RIA Kit*
        Plasma Diluent                   Standard Diluent                 Separation of the antibody-bound FPA and
                                                                                  nonbound FPA takes place
  Expected         Recovered         Expected       Recovered
   (ng/ml)         (ng/ml)†           (ng/ml)        (ng/ml)
      30.0            30.0              27.5             27.5
      24.0            24.7              22.0             22.8       Discard unbound-FPA            Radiolabeled and nonlabeled
      18.0            18.6              16.5             16.8           in supernatant            antibody-bound FPA in pellet
      12.0            12.2              11.0             11.3
       6.0             6.3               5.5              6.0
       3.0             2.9               2.8              2.4                                     Count radioactivity of pellet*
       1.5             1.4               1.4              1.3
                                                                *An inverse relationship exists between the level of radioactivity
*This study was performed at the Mallinckrodt laboratories.      measured and the amount of FPA in the test plasma; that is,
 Dilutions were made with plasma or the 0.0 standard supplied    higher concentrations of patient test FPA cause less labeled
 with the kit prior to the bentonite treatment.                  FPA to bind to the antibody, thereby producing lower radio-
†Corrected for plasma background FPA.                            activity counts in the final pellet.
PERFORMANCE CHARACTERISTICS OF A RIA FOR FPA - WALENGA, ET AL                                                                  221



                                                                            Reproducibility of the Mallinckrodt FPA assay
                                                                       depends on carefully following the manufacturer's
                                                                       instructions because of the extreme sensitivity of the
                                                                       assay, which will detect any mishandling of reagents
                                                                       or specimens. However, these precautions involve
                                                                       common sense that should be routinely employed
                                                                       by practicing laboratory personnel. Table 5 shows
                                                                       data for intra-assay variability on three control
                                                                       levels and two plasma levels for which the coeffi-
                                                                       cient of variation ranged from 3.16 to 6.46%. The
                                                                       interassay variability, shown in Table 6, ranges
                                                                       from 3.78 to 11.20%.31

                                                                        SAMPLE COLLECTION AND HANDLING

                                                                            In order to obtain appropriate samples for FPA
                                                                       determinations, it is necessary to use special collec-
                                                                       tion and handling techniques and anticoagulants.
                                                                       Usually, blood plasma is utilized for FPA; how-
                                                                       ever, spinal fluid, urine, ascites, and other body
                                                                       fluids can be used. Due to the fact that FPA is pro-
                                                                       duced as the earliest marker of thrombin's action on
                                                                       fibrinogen, special precautions in sample handling
                                                                       are necessary in order to prevent an increase in the
FIG. 1. Standard curve for the Mallinckrodt fibrinopeptide             values by mechanical or other exogenous activation.
A RIA assay. This is a typical standard curve of bound/                Harsh treatment of a blood sample, poor venipunc-
unbound label (%) versus standard concentrations of fibrinopep-
                                                                       ture, improper centrifugation, time, and tempera-
tide A.
                                                                       ture can cause exogenous release of FPA. The
                                                                       sensitivity of this assay is critical to its diagnostic


                      TABLE 5. Intra-Assay Variability of the Mallinckrodt Fibrinopeptide A Kit*

                                                     Lyophilized Control Plasma                   Normal Human Plasma
       Runt                                      I                II              III               I             II
        1 (n = 21)
             ± SD (ng/ml)                   1.37 ± 0.10      7.11 ± 0.25     24.10 ± 0.66      1.12 ± 0.07   8.70 ± 0.23
          CV (%)                                7.44             3.51            2.73              6.07          2.63
       2(n = 21)
            ± SD (ng/ml)                    1.41 ± 0.08      7.13 ± 0.16     24.02 ± 0 . 6 8   1.28 ± 0.06   8.89 ± 0.37
         CV (%)                                 5.45             2.27            2.83              4.90          4.12
       3(n=21)
           ± SD (ng/ml)                     1.35 ± 0.09      7.22 ± 0.27     23.77 ± 1.18      1.12 ± 0.07   8.55 ± 0.30
         CV (%)                                 6.48             3.71            4.95              6.07          3.52
       Composite of 3 runs (n = 63)
           ± SD (ng/ml)                     1.38 ± 0.09      7.15 ± 0.23     23.96 ± 0.84      1.17 ± 0.07   8.71 ± 0.30
         CV (%)                                 6.46             3.16            3.50              5.68          3.42
       *This study was performed in the Mallinckrodt laboratories using three individual kit lots. The compiled data for the
        three separate lots are also given.
       † : mean; CV: coefficient of variability.

                      TABLE 6. Interassay Variability of the Mallinckrodt Fibrinopeptide A Kit*

                                                     Lyophilized Control Plasma                  Normal Human Plasma
       Run†                                      I                II              III               I             II
          ± SD (ng/ml)                      1.29 ± 0.13     7.03 ± 0.31      25.44 ± 1.20      1.16 ± 0.13   8.65 ± 0.33
        CV (%)                                  10.08            4.41              4.72            11.20          3.78
        Number                                 189             189               189              562           562
       *This study was performed at the Mallinckrodt laboratories. Three different lots were assayed.
       † : mean; CV: coefficient of variability.
222                                   SEMINARS IN THROMBOSIS AND HEMOSTASIS-VOL. 10, NO. 4, 1984


value; however, this does present particular con-
siderations in sample handling.
      Therefore, the only blood sample acceptable
for FPA quantitation is one in which there is a clean
venipuncture using a double-syringe technique to
discard the first 3 ml of blood. The collection tube
should be prechilled and the sample should be im-
mediately placed on ice after filling and centrifuged
at 4°C within 30 minutes of collection. If analysis is
not to be performed immediately, the plasma
should be frozen at -70°C. FPA quantitation by
the Mallinckrodt kit after various sample-handling       FIG. 2. Fibrinopeptide levels in a normal male population.
procedures is described by Walenga et al elsewhere       Plasmas were collected in a special anticoagulant and frozen
in this issue of Seminars.                               before assaying.
     The anticoagulant is the most important factor
in obtaining a proper sample. A special anticoagu-
lant developed by Mallinckrodt contains ethylene
diaminetetraacetic acid (EDTA), aprotinin, and a
thrombin inhibitor. This provides inhibition of
coagulation by chelating calcium and by blocking
kallikrein to inhibit the contact system, if any
clotting has occurred, a thrombin inhibitor will stop
any final action on fibrinogen. Data from Loyola
Medical Center clearly illustrate the efficacy of this
anticoagulant. These data have been published else-
where in this issue of Seminars.
                                                         FIG. 3. Fibrinopeptide A levels in a normal female popula-
           CLINICAL APPLICATION                          tion. Plasmas were collected in a special anticoagulant and
                                                         frozen before assaying.
     To assess the Mallinckrodt FPA assay kit and
to define normal range values in our laboratory,         showed higher values than males, 32.0 ± 6.6 µg/ml
specimen from normal human volunteers were as-           as opposed to 27.1 ± 8.4 µg/ml. In addition, the
sayed. Data were categorized by male and female          distribution of FPA levels seemed more concentrated
and plasma and serum. Plasma samples were col-           at higher levels in females, whereas males were
lected in the previously described manner and            evenly distributed. These data, as shown in Figures
frozen at -70°C for 1 week to 4 months. Figure 2         4 and 5, reflect the differences in clotting rates be-
shows a histogram for normal male FPA levels. The        tween males and females.
mean is 1.5 ± 1.1 ng/ml with a fairly normal dis-             Normal human plasma or serum pools, as pre-
tribution. Similarly for females (Fig. 3), the mean is   pared for daily control in our laboratory using a
1.9 ± 1.2 ng/ml, slightly higher than that for           double-syringe technique and sodium citrate as an
males, with a fairly normal distribution. There ap-      anticoagulant, were used to evaluate the perform-
pears to be a higher frequency and narrower distri-      ance characteristics of this FPA kit. Twenty aliquots
bution of the population at lower than mean values       of one lot of normal plasma pool were run simul-
for both male and female groups. Above the mean          taneously to assess the intra-assay variation. Simi-
there is lower frequency and wider distribution of       larly, 20 aliquots of normal serum pool were simul-
population for the higher values of FPA. In other        taneously run. At slightly elevated levels, due to the
words, most normal values will be from 0.1 to 1.5        anticoagulant used, the plasma gave 7.9 ± 1.8 ng/
ng/ml and less frequently from 1.5 to 5.25 ng/ml.        ml and the serum gave 12.5 ± 0.7 µg/ml FPA
Values greater than 2.0 ng/ml should be evaluated        levels. At very high concentrations, the variation was
for the normality of the volunteer and reanalyzed to     not statistically significant; however, at low ranges,
be sure that the true normal value is that high. It is   there was a higher variation. Due to the sensitivity
generally accepted that normal values range from 0       of this assay, that is, measurements in the nanogram
to 2.0 ng/ml.                                            range, these variations are to be expected. However,
     In an identical fashion, serum from males and       from the data to follow, it will be clear that normal
females were analyzed. As expected, FPA levels           and abnormal values are easily distinguished by a
were extremely high; however, the female group           range of values generally accepted for each group,
PERFORMANCE CHARACTERISTICS OF A RIA FOR FPA - WALENGA, ET AL                                                           223




                                                                 FIG. 6. A low level FPA control supplied in the Mallinckrodt
                                                                 FPA kit.
FIG. 4. Fibrinopeptide A levels in normal serum of males.
Serum was collected in glass and frozen before use.




                                                                 FIG. 7. A high level FPA control supplied in the Mal-
                                                                 linckrodt kit.
FIG. 5. Fibrinopeptide A levels in normal serum of females.
Serum was collected In glass and frozen before use.              marker. For thrombotic diseases, such as deep vein
                                                                 thrombosis in which there is ongoing coagulation
                                                                 activity, FPA is an obvious choice to monitor the
if proper collection technique is followed. Table 7              disease. Evaluations were also done for cases of
illustrates these data.                                          malignancy in which thrombotic activity is second-
      To assess interassay variability, both high and            ary to the initial disease. Table 8 lists data for 18
low controls supplied with the kit were included in              various neoplasias and their accompanying FPA
daily runs. The low control (Fig. 6) gave a mean of              levels. These plasmas were obtained from San
1.1 ± 0.3 ng/ml with a normal distribution and the               Joaquin Hematology/Oncology Group in Bakers-
high control gave a mean of 7.6 ± 0.6 ng/ml with a               field, California. Samples were collected as recom-
normal distribution (Fig. 7). Again at lower concen-             mended and frozen prior to analysis. Half of the
trations the variation was higher than that for above            samples show elevated levels. A more complete
normal concentrations, as seen in the intra-assay                study should be undertaken in which a larger
data. However, this does not interfere in clinical               number of samples per group at various stages of
diagnoses. The Mallinckrodt FPA kit was used in                  each type of malignancy are analyzed. However, in
investigations of numerous disease states to deter-              this preliminary study, FPA appears to be an
mine whether FPA could be included as a diagnostic               appropriate indicator of ongoing malignancy. This
                                                                 analysis will also be beneficial not only in proper
 TABLE 7. Comparison of Fibrinopeptide A Levels in               diagnosis but also in selecting the proper therapy
       Plasma and Serum in an Intra-Assay                        for all secondary clinical involvements. Previously
              Performance Study*                                 published articles 2,4,5,13-16,18,19,22,23,28-30,32,47,51 as
                         Human Plasma        Human Serum         well as other articles in this issue of Seminars report
                                                                 on further clinical uses of FPA quantitation.
     ± SD (ng/ml)t         7.9 ± 1.8         12,500 ± 700
   CV (%)†                    22.8                5.6                  Table 9 shows the results of FPA levels in 14
                                                                  patients with suspected hypercoagulable state due
*Twenty individual aliquots of each sample were assayed to as-
 sess the performance characteristics of the Mallinckrodt FPA
                                                                  to deep vein thrombosis (OVT), low antithrombin
 kit. Plasma was collected in sodium citrate.                     III (AT III) levels, and shortened PT and PTT.
† : mean; CV: coefficient of variation.                           In all of these patients the FPA levels are signifi-
224                                            SEMINARS IN THROMBOSIS AND HEMOSTASIS-VOL. 10, NO. 4, 1984


      TABLE 8. Fibrinopeptide A Levels in Various                          TABLE 10. Fibrinopeptide A Level in Various
                     Malignancies*                                                 Disorders of Fibrinolysis*
                                                Fibrinopeptide A                Disorder                      FPA    (ng/ml)
Malignancy                         No.          (ng/ml ± 1 S.D.)
                                                                                Primary fibrinolysis             6.1 ± 3.2
Gastric                              1                10                          Malignancy
Neck                                 1                12.5                        Pregnancy
Breast                              15                 8.5   ± 10.7
                                                                                Secondary fibrinolysis          14.1 ± 6.4
Squamous cell                        2                 5.2   ± 5.1
                                                                                  DIC
Prostatic                            3                 5.3   ± 3.6
Gallbladder                          1                 5.0                      Normal                          2.8 ± 0.9
Renal cell                           1                 4.8            *All results are expressed as means of ten individual determina-
Esthesioneuroblastoma                1                 3.7             tions ± 1 SD.
Colon                                6                 3.2   ± 2.8
Pancreatic                           3                 3.1   ± 1.6
Hairy cell leukemia                  1                 2.6
Chronic myelocytic leukemia          1                 2.4                  Many investigators realize the importance and
Multiple myeloma                     1                 2.4            direct application of FPA in the clinical laboratory,
Chronic lymphocytic leukemia         2                 2.4   ± 0.07   but they have not been able to appreciate it fully,
Liposarcoma                          1                 2.1            since earlier methods were time-consuming and
Fibrocystocytoma                     1                 2.0
Endometrial                          2                 1.3   ± 0.14
                                                                      tedious. With the current availability of the Mal-
Ovarian (with liver metastases)      1                 1.2            linckrodt RIA kit, one can now easily incorporate
                                                                      the FPA evaluation in a hemostatic workup. Cur-
*Plasma samples obtained from the San Joaquin Hematology/
 Oncology Group in Bakersfield, California, were collected in         rent research is also using FPA levels to develop
 special anticoagulant and frozen before assaying.                    new assays, such as an FPA generating ability of
                                                                      plasma to assess the capacity of an individual's
                                                                      whole blood or plasma system to activate thrombin.
  TABLE 9. Fibrinopeptide A Levels in Patients w i t h                This can be used as a measure of tendency to
       Suspected Hypercoagulable States*
                                                                      develop a hypercoagulable state or in evaluating
             Patient                     FPA    (ng/ml)               antithrombotic therapy. Figure 8 shows circulating
                1                               4.2                   FPA levels in 23 cases of total hip replacement. As
                2                               4.6                   can be seen, the activation of coagulation reaches a
                3                               3.4
                                                                      peak on day 1 and progressively returns to the base-
                4                              10.0
                5                               8.2                   line level within a week. This method is very useful
                6                               9.2                   in monitoring drugs that are not measurable using
                7                              23.0                   global tests.
                8                               6.6
                                                                           FPA can be used as a quality control device for
                9                              10.0
               10                               4.2                   plasma-based laboratory controls, reagents, and
               11                               9.0                   therapeutic agents. By making use of the extreme
               12                               6.0                   sensitivity of this marker, these products can be
               13                              11.5
               14                               4.6
*Patients with deep vein thrombosis, low AT III, shortened PT
 and PTT were considered to be hypercoagulable.



cantly higher than the general baseline levels. This
result suggests that all of these patients have some
form of activation of the coagulation system that
results in the generation of FPA.
     Table 10 shows the results of FPA levels in
various disorders of fibrinolysis. In ten patients
with malignancy and fibrinolysis a significant in-
crease in the FPA levels was obvious. However, in
secondary fibrinolysis (DIC) a marked increase in
the FPA levels was observed. This suggests that al-
though a nonspecific increase of FPA in the so-
called primary fibrinolysis is observed, the genera-                  FIG. 8. Circulating fibrinopeptide A levels following total
tion of this marker is much higher in DIC.                            hip replacement in 23 cases. Average values ± 1 SD.
PERFORMANCE CHARACTERISTICS OF A RIA FOR FPA - WALENGA, ET AL                                                                 225


assessed for batch to batch variability in a manu-                     TABLE 12. Fibrinopeptide A Levels in Various
facturing procedure if activation of the plasma                         Activated and Nonactivated Prothrombin
would be deleterious to the performance of the pro-                              Complex Concentrates*
duct. This is an important consideration for plasma                          Concentrate                     FPA (ng/ml)
controls used in the newer, more sensitive assays                            Proplex (Hyland)                  7.5 ±    2.0
other than PT and PTT when a defined normal                                  Autoplex (Hyland)                 3.5 ±    1.2
plasma is required. An elevated FPA level will indi-                         Konyne (Cutter)                  15.0 ± 19.0
cate that some extent of a thrombotic event has                              AICC (Cutter)                    17.5 ±    4.0
occurred and the plasma is no longer normal. In                              FEIBA (Immuno)                  187.0 ± 19.0
addition, FPA on other activation products may                               Preconativ (Kabi)               210.6 ± 23.0
exhibit feedback mechanisms on serine proteases in
                                                                   *All FPA concentrations are expressed as ng/ml/U of the con-
the coagulation network and thus interfere in a                     centrate. Lyophilized concentrates were diluted with sterile
normal control response. Quality control by FPA                     water.
will allow a plasma pool to be used as a standard
preparation in assays in which the commercially                        TABLE 13. Generation of Fibrinopeptide A by
available controls may not provide a normal human                         Prothrombin Complex Concentrates*
value. Such controls either contain animal products                   Complex              Manufacturer   FPA Generated (ng/ml)
or substances from processing that may interfere in
                                                                      AICC                   Cutter            1825 ± 247
the newer assays, such as heparin. Table 11 shows
                                                                      Konyne                 Cutter             250 ± 1
data from ten individual lots of in-house normal
                                                                      Proplex                Hyland             210 ±    56
human plasma pool preparations. Although these
                                                                      Autoplex               Hyland             612 ±    53
are samples collected in citrate, one can determine a
                                                                      FEIBA                  Immuno             412 ±    18
control level for each pool. Additionally, batch to
                                                                      Preconativ             KABI               290 ±    21
batch variations can be evaluated. It is also ap-
parent that if proper venipuncture is carried out, a               *A 0.9 ml of normal human plasma (diluted 1:10) was incubated
                                                                    with 0.1 ml of each prothrombin complex concentrate (50 U/ml
clean, nonactivated pooled control can be obtained                  for 30 minutes at 37°C. Fibrinopeptide A measurements were
without the proper anticoagulant. However, this is                  then determined.
not recommended for routine use.
      Table 12 shows FPA levels in various commer-                 the concentrates. The prothrombotic activity of
cially available prothrombin complex concentrates.                 each concentrate was assessed by its ability to
All concentrates were made to 1 U/ml sterile water                 generate FPA from the plasma. There is a tremen-
(manufacturer's units) and assayed for FPA. The                    dous amount of variability between each prepara-
individual manufacturing procedures can be evalu-                  tion, even after background concentrations (Table
ated for consistency of plasma activation by this                  12) are taken into consideration. In this manner,
method. This may or may not be desirable, depend-                  potency evaluations of each concentrate can be
ing on the use of the product.                                     made.
     On the other hand, Table 13 shows data of the
same concentrates but for a modified application of                                         SUMMARY
the FPA quantitation. In this method normal
human plasma was incubated at 37°C with each of                          FPA, although identified 15 years ago, is now
                                                                   becoming an increasingly important diagnostic tool
TABLE 11. Fibrinopeptide A Determination in Normal
                                                                   in the evaluation of the hemostatic process. Since
        Human Pool Plasma Preparations*                            this peptide is generated in very small amounts,
                                                                   only very sensitive methods, such as RIA, are use-
         Preparation              Fibrinopeptide A (ng/ml)
                                                                   ful for its quantitation. Measurement of this
               1                             13.0
                                                                   peptide allows for a most precise and reliable
               2                              1.8
               3                              2.3                  monitor of any ongoing thrombotic event in which
               4                              1.9                  thrombin is generated.
               5                              3.8                       Commercial kits have become available for fast
               6                             16.1                  and simple clinical evaluations of FPA. The Mai-
               7                             11.4
                                                                   linckrodt RIA Quanti FPA kit has proved its reli-
               8                             12.1
               9                              3.6                  ability in precision, accuracy, fast turnaround time,
              10                             11.0                  and applicability to a routine laboratory setting.
*Plasmas were collected in sodium citrate using a double syringe         This assay kit was evaluated in our laboratory
 technique. No special precautions were taken in specimen          in various aspects. The following points summarize
 handling.                                                         our finding:
226                                           SEMINARS IN THROMBOSIS AND HEMOSTASIS-VOL. 10, NO. 4, 1984


      1. FPA is a useful diagnostic parameter to eval­                    CRM Prentice: Plasma fibrinopeptide A and beta-thrombo-
         uate the activation of coagulation pathways                      globulin in pre-eclampsia and pregnancy hypertension.
                                                                          Thromb Haemost 47:54-55, 1982.
         in various pathologic states.
                                                                   11.    Fareed J, RL Bick, G Squillaci, JM Walenga, EW Bermes:
      2. A study of 170 normal plasma samples re­                         Molecular markers of hemostatic disorders: Implications in
         sulted in 1.7 ± 0.5 ng/ml. No significant                        the diagnosis and therapeutic management of thrombotic
         difference between males and females was                        and bleeding disorders. Clin Chem 29:1641-1658, 1983.
         noted.                                                    12.   Fareed J, HL Messmore, EW Bermes: New perspectives in
                                                                         coagulation testing. Clin Chem 26:1380-1391, 1980.
      3. FPA levels are evaluated in patients with
                                                                   13.   Gaffney PJ, F Joe, M Mahmoud, CA Fossati, M Spitz: A
         hypercoagulable states, DIC, and related                        novel radiometric approach to the assay of components of
         thrombotic states.                                              human haemostasis: 1. Assay of plasma fibrinopeptide A
      4. Our studies have shown that FPA levels are                      levels. Thromb Res 19:815-822, 1980.
         also elevated in certain cancers, postsurgical            14.   Harenberg J, G Stehle, H Schmidt-Gayk, R Zimmermann:
                                                                         Fibrinopeptide A (FPA) radioimmunoassay influence of the
         states, and certain other conditions in which
                                                                         affinity of the second antibody for FPA antiserum. Thromb
         the coagulation process is activated.                           Res 21:531-532, 1981.
      5. During therapeutic heparinization, FPA                    15.   Harenberg J, G Hepp, H Schmidt-Gayk: Fibrinopeptide A
         levels are reduced; thus this form of therapy                   in human plasma. Evaluation of a new radio immunoassay
         can be monitored using FPA levels.                              technique on microtiter plates. Thromb Res 15:513-522,
                                                                         1979.
      6. High-risk population (thrombotic) can be                  16.   Higgins DL, JA Penner, JW Shafer: Fibrinogen Petoskey:
         easily screened using FPA measurement. We                       Identification of a new dysfibrinogenemia characterized by
         propose that a multicenter study on FPA                         altered release of fibrinopeptide A. Thromb Res 23:491-504,
         levels be conducted to prove its clinical rele­                 1981.
         vance to other diseases.                                  17.   Hirsh J: Blood tests for the diagnosis of venous and arterial
                                                                         thrombosis. Blood 57:1-8, 1980.
     The current technologic developments that are
                                                                   18.   Hofmann V, PW Straub: Fibrinopeptide A release by Factor
able to provide clinically applicable kits, such as                      IX concentrates and FEIBA fraction. Thromb Res 20:623-
that from Mallinckrodt, will greatly enhance the                         631, 1980.
diagnostic and prognostic abilities of clinicians as               19.   Hofmann V, PW Straub: A radio immunoassay technique
well as increase understanding of the total hemo­                        for the rapid measurement of human fibrinopeptide A.
static process.                                                          Thromb Res 11:171-181, 1977.
                                                                   20.   Johnsson H, E Orinius, C Paul: Fibrinopeptide A (FPA) in
                                                                         patients with acute myocardial infarction. Thromb Res 16:
                                                                         255-260, 1979.
                     REFERENCES                                    21.   Kehl M, F Lottspeich, A Henschen: Analysis of human
                                                                         fibrinopeptides by high-performance liquid chromatography.
 1. Alkjaersig N, AP Fletcher: Catabolism and excretion of               Hoppe Seyleret's Z Physiol Chem 362:1661-1664, 1981.
    fibrinopeptide A. Blood 60:148-156, 1982.                      22.   Kockum C: Radio immunoassay of fibrinopeptide A. Im-
 2. Andes WA, SI Chavin, G Beltran, WJ Stuckey: Fibrinogen               muno reactivity of fibrinogen and fibrinogen fragments.
    New Orleans: Hereditary dysfibrinogenemia with an A chain            Thromb Res 19:639-646, 1980.
    abnormality. Thromb Res 25:41-50, 1982.                        23.   Kockum C, S Frebelius: Rapid radio immunoassay of human
 3. Bick RL: The clinical significance of fibrinogen degradation         fibrinopeptide A. Removal of cross-reacting fibrinogen with
    products. Semin Thromb Hemost 8:302-330, 1982.                       bentonite. Thromb Res 19:589-598, 1980.
 4. Budzynski AZ, VJ Marder: Determination of human fibrino­       24.   Kockum C: Radio immunoassay of fibrinopeptide A. Clini­
    peptide A by radio immunoassay in purified systems and in            cal applications. Thromb Res 8:225-236, 1976.
    blood. Thromb Diath Haemorrh 34:709-717, 1975.                 25.   Koehn JA, RE Canfield: Purification of human fibrinopep­
 5. Budzynski AZ, VJ Marder, S Sherry: Reaction of plasmic               tides by high-performance liquid chromatography. Anal
    degradation products of fibrinogen in the raido immuno­              Biochem 116:349-356, 1981.
    assay of human fibrinopeptide A. Blood 45:757-768, 1975.       26.   Lane DA, S Wolff, H Ireland, M Gawel, M Foadi: Activa­
 6. Coccheri S, PM Mannucci, G Palareti, W Gervasoni, M                  tion of coagulation and fibrinolytic systems following stroke.
    Poggi, S Vigano: Significance of plasma fibrinopeptide A             Br J Haematol 53:655-658, 1983.
    and high molecular weight fibrinogen in patients with liver    27.   Lane DA, H Ireland, S Wolff, R Grant, S Jennings, T
    cirrhosis. Br J Haematol 52:503-509, 1982.                           Allenmersh: Plasma concentrations of "fibrinopeptide A,
 7. Cronlund M, J Hardin, J Burton, L Lee, E Haber, KJ                   fibrinogen fragment B-β-1-42 and β-thromboglobulin follow­
    Bloch: Fibrinopeptide A in plasma of normal subjects and             ing total hip replacement. Thromb Res 26:111-118, 1982.
    patients with disseminated intravascular coagulation and       28.   Lane DA, H Ireland, E Thompson, TJ Frost, B Luddigan:
    systemic lupus erythematosus. J Clin Invest 58:142, 1976.            Two more abnormal fibrinogens (London III and Sydney)
 8. Davies GC, M Sobel, EW Salzman: Elevated fibrinopeptide              with impaired fibrinopeptide release. Thromb Res 28:821-
    A and thromboxane B2 levels during cardiopulmonary by­               824, 1982.
    pass. Circulation 61:808-814, 1980.                            29.   Lane DA, B Cuddigan, M Van Ross, VV Kakkar: Dys-
 9. Dudczak R, H Niessner, E Thaler, K Lechner, K Kletter, H             fibrinogenaemia characterized by abnormal fibrin monomer
    Frischauf, E Domanig, H Aicher: Plasma concentration of              polymerization and normal fibrinopeptide A release. Br J
    platelet specific proteins and fibrinopeptide A in patients          Haematol 44:483-494, 1980.
    with artificial heart valves. Haemostasis 10:186-194, 1981.    30.   Lane DA, M Van Ross, VV Kakkar, J Bottomley, K Ohir,
10. Duglas JT, M Shah, GDO Lowe, JJF Belch, CD Forbes,                   LPJ Holt, JE Mac Iver: An abnormal fibrinogen with
P E R F O R M A N C E C H A R A C T E R I S T I C S O F A RIA F O R F P A - W A L E N G A , ET AL                                    227



      delayed fibrinopeptide A release. Br J Haematol 46:89-98,               anticoagulant treatment measured by fibrinopeptide A (FPA)
      1980.                                                                   in patients with venous thromboembolism. Thromb Res 18:
31.   Mallinckrodt FPA: Quant RIA test kit. St. Louis, MO, 1983.              33-43, 1980.
32.   Marciniak E, MF Greenwood: Acquired coagulation inhibi­           44.   Peuscher RW, FJ Cleton, L Armstrong, EA Stoepman-
      tor delaying fibrinopeptide release. Blood 53:81-92, 1979.              van Dalen, JA Van Mourik, WG van Aken: Significance of
33.   Medden-Britth G, K Radegran: Fibrinopeptide A as a mea­                 plasma fibrinopeptide A (FPA) in patients with malignancy.
      sure of coagulation activity induced by surgery. An experi­             J Lab Clin Med 96:5, 1980.
      mental study in dogs. Thromb Res 18:333-341, 1980.                45.   Rickles FR, RL Edwards, G Bard, M Cronlunc: Abnormal­
34.   Mettinger KL: A study of hemostasis in ischemic cerebro­                ities of blood coagulation in patients with cancer. Fibrino­
      vascular disease. IV. A five year follow-up of some blood               peptide A generation and tumor growth. Cancer 51:301-307,
      coagulation parameters also including fibrinopeptide A,                 1983.
      factor XII and prekallikrein. Thromb Res 27:155-160, 1982.        46.   Serneri GG, GF Gensini, R Abbate, C Nugnaini, S Favilla,
35.   Mombelle G, A Roux, A Haeberli, PW Straub: Comparison                   C Brunelli, S Chierchia, O Parodi: Increased fibrinopeptide
      of 125 I fibrinogen kinetics and fibrinopeptide A in patients           A formation and thromboxane A2 production in patients
      with disseminated neoplasia. Blood 60:381-388, 1982.                    with ischemic heart disease: Relationship to coronary patho-
36.   Myers TJ, FR Rickles, C Brab, M Cronlund: Fibrinopeptide                anatomy, risk factor, and clinical manifestations. Am Heart
      A in acute leukemia: Relationship of activation of blood                J 101:185-194, 1981.
      coagulation to disease activity. Blood 57:518-525, 1981.          47.   Stehle G, J Harenberg, H Schmidt-Gayk, R Zimmermann:
37.   Nichols AB, J Owen, KL Kaplan, RR Sciacca, PJ Cannon,                   Fibrinopeptide A radioimmunoassay; a rapid and sensitive
      HL Nossel: Fibrinopeptide A, platelet factor 4, and β-                  measurement of fibrin generation in vivo. J Clin Chem Clin
      thromboglobulin levels in coronary heart disease. Blood 60:             Biochem 21:91-95, 1983.
      650-654, 1982.                                                    48.   Torngren S, I Noren, G Savidge: The effect of low-dose
38.   Nossel HL, FR Smith, AH Seplowitz, RB Dell, RR Sciacca,                 heparin on fibrinopeptide A, platelets, fibrinogen degrada­
      C Merskey, DS Goodman: Normal levels of fibrinopeptide                  tion products and other haemostatic parameters measured
      A in patients with primary hyperlipidemia. Circ Res 45:347-             in connection with intestinal surgery. Thromb Res 14:871-
      350, 1979.                                                              879, 1979.
39.   Nossel HL, VP Butler Jr, GD Wilner, RE Canfield, EJ               49.   van Hulsteijn H, R Bertina, E Briet: A one year follow up
      Harfenist: Specificity of antisera to human fibrinopeptide A            study of plasma fibrinopeptide A and beta-thromboglobulin
      used in clinical fibrinopeptide A assays. Thromb Haemost                after deep vein thrombosis and pulmonary embolism.
      35:101, 1976.                                                           Thromb Res 27:225-229, 1982.
40.   Nossel HL, VP Butler Jr, RE Canfield, I Yudelman, M Ti,           50.   Vigano S, M Cattaneo, W Gervasoni, PM Mannucci: In­
      K Spanondis, T Soland: Potential use of fibrinopeptide A                creased fibrinopeptide A after prothrombin complex con­
      measurements in the diagnosis and management of throm­                  centrates. Thromb Haemost 44:72-75, 1980.
      bosis. Thromb Diath Haemorrh 33:426, 1975.                        51.   Woodhams BJ, PB Kernoff: Rapid radioimmunoassay for
41.   Nossel HL, I Yudelman, RE Canfield, VP Butler, K                        fibrinopeptide A in human plasma. Thromb Res 22:407-416,
      Spanondis, GD Wilner, GD Quereshi: Measurement of                        1981.
      fibrinopeptide A in human blood. J Clin Invest 54:43-53,          52.   Yoda Y, A Tsukasa: Fibrinopeptide A (FPA) level and
      1974.                                                                   fibrinogen kinetics in patients with malignant disease.
42.   Nossel HL, LR Younger, GD Wilner, T Procupez, RE Can-                   Thromb Haemost 46:706-709, 1981.
      field, VP Butler: Radio immunoassay of human fibrinopep­          53.   Yudelman I, J Greenberg: Factors affecting fibrinopeptide
      tide A. Proc Natl Acad Sci USA 68:2350-2353, 1971.                      A levels in patients with venous thromboembolism during
43.   Peuscher FW, WG van Aken, OTN Flier, EA Stoepman-                       anticoagulant therapy. Blood 59:787-792, 1982.
      van Dalen, TA Cremer-Goote, JA van Mourik: Effect of

				
DOCUMENT INFO
Shared By:
Categories:
Tags:
Stats:
views:10
posted:12/17/2011
language:English
pages:9