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Immunologic survey of the factor IX molecule in hemophilia B+ patients, carriers and cord blood
0049-3848/81/040391-08$02.00/O TRROM8OSIS RESEARCH 21; 391-398, 1981 Printed in the USA. All rights reserved. Copyright (c) 1981 Pergamon Press Ltd
IMMJNOLOGICSURVEY OF THE FACTOR IX MOLECULE IN HEMOPHILIA B+ PATIENTS, CARRIERS AND CORD BLOOD Cheryl Y. Tiarks and Liberto Pechet Departments of Medicine and Pathology University of Massachusetts Medical Center Worcester, MA 01605 (Received 6.5.1980; in revised form 23.11.1980. Accepted by Editor Y. Sultan. Received in final form by Executive Editorial Office 5.2.1981) ABSTRACT Crossed antigen-antibody electrophoresis was performed on 62 hemophilia B (CM) patients and 10 obligatory carriers. The assays were performed in TRIS buffer, or in buffer with the addition of either 2.0 ml4EDTA to bind calciun, or 2.2 r&l calcium lactate, to provide excess calciun. The results revealed migrational differences among normal individuals. To standardize the assay an internal reference (anti prealbumin) was used. A range of normal migrational distance in relation to the reference was established using 30 normal individuals. When comparing atient migration to the normal migrational range (i 2 S.D.P abnormalities were documented in only four patients and in none of the carriers. Even in these cases, mixtures of normal with each patient's from one hemophilia B+ patient revealed a biphasic arc in the presence of EDTA. When mixed with normal plasma, the pattern normalized. Six cord bloods examined were normal. Ue conclude that crossed antigen-antibody electrophoresis requires stringent quality control for valid conclusions, that the method detects very few cases with anomalous migration and that in general, it is not useful for predicting the carriership of hemophilia B. We previously developed a precipitating monospecific antibody to human factor IX (1) which we used to quantitate factor IX antigen (FIX-AS) in a large number of hemophilia B patients and carriers, and factor IX concentrations (2). In addition, crossed antibody electrophoresis utilizing this antiserum revealed migrational differences between factor IX concentrates and normal human plasma. These differences were more pronounced when the electrophoresis was run in the presence of excess KEYWORDS:
Hemophilia B, crossed Antigen-Antibody carriers of Hemophilia B. 391
electrophoresis,
392
CAA!ZIN HEMOPHILIAB
Vo1.21, No.415
calcium (decreased mobility) or in the presence of a Ca++-chelating agent, EDTA (increased mobility). The purpose of the experiments described in this article were two-fold: 1) to discover whether an anomalous migration of FIX-Ag in hemophilia B+ (CRM+) patients could be demonstrated in a crossed antigen-antibody electrophoretic system; 2) to study carriers from these pedigrees by the same methodology to ascertain whether normal and abnormal factor IX molecules could be discerned in their plasma; if abnormal migration were found, this methodology could be used to improve detection of carriers who have high levels of FIX-AS and in whom electroimnunoassays and coagulant assays are not sufficiently discriminant (3). This study presents the results of our studies on 62 hemophilia B+ patients and 10 obligatory carriers of this type; in addition 6 cord bloods from normal newborns, kindly sent to us by Dr. Jack Lazerson of the Medical College of Wisconsin were also studied. METHODS The antiserum to hunan factor IX was prepared as previously described (1). Laurell's electroinmunoassay (4) and crossed antigen-antibody In electrophoresis (5) were performed as previously described (1). order to internally standardize migration, we used hunan prealbumin as a marker; commercial rabbit antiserum to hunan prealbumin (Miles Lab, Elkhart, IN) was added to the gel in the second dimension at a concentration of 80 ~1/6 ml agarose. The migrational distance in reference to the prealbumin was calculated as follows: the arcs for FIX and prealbumfn were bisected and the distance (mn) from the anodal edge of the application well to the middle of each arc was measured. The values described in the results with respect to migration were calculated by dividing the migrational distance of the FIX arc by that of prealbumin. To bind calcium, 2.0&i EDTA was added in the first dimension to the TRIS buffer pH 8.8, used in making the gel. In the same manner, excess calciun was provided by adding 2.2rnMcalciun lactate. The methodology was standardized using normal pooled plasma, prepared as previously described (1). The normal pool was run in parallel with each patient. In addition. 30 normal plasmas ware run individually. These samples were either used fresh, or frozen for a maximum of one week at -7OOC. The hemophilia B+ patients and carriers studied were selected from those previously described (2), all containing lneasureableamounts of factor IX antigen by electrofmmunoassay. All samples had been collected 'as previously described and kept frozen at -7OOC. RESULTS All results refer to crossed antigen-antibody electrophoresis, unless otherwise specified. Normals. Studies on 30 individual normals and repeated assays of normal WInvariably demonstrated a single symmetrical arc in the presence of buffer alone or calcium and a slightly asynnnetricalarc in the presence of EDTA (Fig. 1). The migrational distance of the 30 individual samples
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CAAE IN HEMOPHILIA B
with respect to prealbumin revealed variations under all three conditions studied (Table I). These variations, were less pronounced on 37 repeats of the normal pool (Table I).
FIG.
1
Cross antigen-antibody electrophoresis (Laurel1 method) of a typical normal plasma showing FIX migration relative to prealbumin, used as an internal reference. During the.lst dimensional electrophoresis the anode was to the right and during the 2nd to the top. Upper frame: buffer alone; Middle frame: buffer plus 2.2 mM calcium lactate in the first dimension; Lower frame: buffer plus 2.flmM EDTA in first dimension. Note, in comparison to buffer alone, the decreased mobility of the FIX molecule in the presence of calcium and the increased mobility in the presence of EDTA.
393
394
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Vo1.21, No.415
In order to evaluate the variability within tests, ten individual normal plasma samples were aliquoted, frozen, then assayed four times. An analysis of variance procedure (Table II) showed no significant difference between the variance of repeated samples on the same persons compared to the variation between persons. However, this group of 10 normals, assayed over a short period of time, also showed significantly less variability among themselves than the previous 30 normals (Table II), except for EDTA experiments (p < .Ol for buffer p < .025 for calcium lactate p > .05 for EDTA). Patients. Plasmas from two patients revealed no arcs on three separate occasions, although by electroimmunoassay they had measurable FIX-Ag (56 and 6196,respectively) thus leaving 60 evaluable patients. In one additional case no arc was obtained only when EDTA was used in the gel (two experiments). Plasma from one patient demonstrated a biphasic arc with EDTA (5 experiments) (Fig. 2). One component migrated 0.63 (decreased migration compared to normal), the other 0.81 (normal migration). A mixture of equal volumes of normal pooled plasma and plasma from this patient, run in the presence of EDTA showed normal migration.
FIG. 2 Cross antigen-antibody electrophoresis in the presence of 2.0 rrt+l EDTA in the 1st dimension, showing a hemophilia 8+ patient who demonstrated a biphasic arc, one component migrating within the normal range and the other component showing a decreased migration.
vo1.21,
CAAE IN HEMOPHILIAB
No.415
395
The vast majority of hemophilia B+ patients, including 3 Bm variants (2), migrated a distance falling within the normal range (Table III), although differences were noticed frequently when compared to the normal pool run concanmitantly. In four hemophilia B+ patients FIX-AS migration fell outside 2 standard deviations of the normal: in one, the migration in the presence of calcium was less than normal (0.31 in two experiments), but normal (0.40) in a third experiment using the same sample; in another patient the migration was greater than normal, with either buffer alone (0.68 and 0.63) or with calcium (0.50 and 0.56); in a third one, migration was greater in buffer in three experiments (0.69, 0.67 and 0.63), but normal in the presence of calcium or EDTA; in the fourth patient migration was greater only with calcium: 0.77 and 0.58 in another experiment. When samples from these four patients were mixed in equal volumes with normal pool plasma, the migration normalized entirely. Obligatory Carriers. The migration of carriers as a group fell within a This may be narrower normal range than that of patients (Table III). related to the smaller number of samples. None of the carriers fell outside the normal range, nor showed anomalous shapes. We did not examine obligatory carriers or family members from the patient with the biphasic arc. The mother of the one patient with decreased migration in calcium, showed normal migration. The mother of the patient with increased migration with buffer and with calcium also had normal migration. Other relatives of patients with abnormal migrations were not available. Cord Bloods. Six cord blood samples were examined (Table IV). showed normal migration.
All
TABLE I Migration Data on 30 Normal Individuals and 37 Repeats of the Normal Pool. Normal
Normal Pool
Range
Mean
S.D.
Range
Mean
S.D.
Buffer
0.46-0.69
0.544
0.057
0.43-0.69
0.515
0.038
Ca Lactate
0.31-0.50
0.415
0.048
0.31-0.55
0.408
0.034
EDTA
0.72-0.89
0.82
0.033
0.73-0.89
0.816
0.027
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CAAX IN HEMOPHILIAB
396
TABLE II Normals Repeated 4 Times Each Compared to 30 Normals
Migration Data on.10
Done Previously S.D. of Replications on the 10 Individuals
Mean of 10 Individuals Means (4 experiments)
S.D. Between 10 Individuals
S.D. Retween 30 Individuals
Buffer
0.4796
0.020
0.026
0.057
Ca Lactate
0.3773
0.025
0.025
0.048
EDTA
0.7847
0.026
0.036
0.933
TABLE III Migration of 60 Patients and 10 Carriers Carriers
Patients Range
Mean
S.D.
Range
Mean
S.D.
Buffer
0.43-0.69
0.53
0.06
0.47-0.57
0.517
0.034
Ca Lactate
0.31-8.55
0.415
0.057
0.34-0.43
0.393
0.027
EOTA*
0.73-0.89
0.803
0.04
0.77-0.85
n.801
0.028
*exclusive of cases
55 (biphasic arc) and 387 (no visible arc) TABLE IV Migration Data on 6 Cord Bloods Range
Mean
S.D.
Buffer
0.43-0.56
0.47
0.043
Ca Lactate
0.38-0.46
0.40
0.028
EDTA
0.80-0.85
0.82
0.017
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C&G IN HEMOPHILIAB
DISCUSSION The results of this study suggest that crossed antigen-antibody electrophoresis has intrinsic variability (Table II) as well as variation among normal individuals (Table 1). Since the original description of the method by Laurel1 it has been custanary to compare unknown plasmas with normal controls run simultaneously. Based on experiments described above, we conclude that anomalies in the shape of the Factor IX arc may be significant, if reproducible, but that, in general, anomalous migration in comparison with a simultaneously run control may not represent an abnormality. A statistical comparison between patients' migrational distance with that of the normal range of migration must be made in order to draw valid conclusions. Our normal range was established by surveying 30 normal subjects. It is unlikely that the variability we observed is related to technique alone, since we performed our experiments under identical conditions with respect to buffers, molarity, pH, temperature, equipment, and length of electrophoresis time. In addition, repeated assays of 10 normal plasmas revealed less variability (Table II), with a coefficient of variation of 5%. Interestingly, the variation between this group of 10 normals was significantly less than previously observed in assays of 31)normal controls with the exception of EDTA experiments (Table I). We have no explanation for this observation, which should not invalidate the basic observation of inter-individuals variation and the need for stringent standardization of the assay. Repeated assays of the normal pool, as expected, also showed less variability than that observed in comparisons of the 30 individual normals. We observed in our studies with factor IX concentrates (1) that in the crossed antigen-antibody electrophoretic assay the factor IX molecule has a greater migrational mobility in a calcium-free medium than in the presence of calcium. We therefore performed our present experiments in the presence of excess Ca++ or in its absence through chelation by EDTA. This study presents the results in 62 hemophilia B+ (CRl@) patients and 10 obligatory carriers. If we had compared the migration of each factor IX deficient plasma to the concomnritantlyrun control, we would have erroneously concluded that a large number of samples had anomalous migration. However, by internally standardizing the measurement of migration relative to prealbumin, 56 of the 60 patients' plasmas fell within 2 standard deviations of the calculated mean for 30 normals under all conditions studied. JJsingthese criteria, all the carriers and the cord bloods studied showed normal relative migration (Tables III, IV). Of the 60 cases which formed visible arcs we found only one case in which the capacity to bind calcium seemed to be diminished (greater mobility in the presence of excess calcium) a rare occurrence, confirming the findings of Hemmendorf et al. (6); three other cases had other abnormalities in mobility, and a fifth had a biphasic arc. Even in these cases, doubt exists concerning the significance of the findings, since the patterns normalized when mixtures of patient and normal plasma were assayed. We must therefore conclude that the technique of crossed antigen-antibody electrophoresis as used in these experiments detects very few cases with anomalous migration in hemophilia B+. Moreover, the method
397
CAAE IN HFZOPHILIAB
398
Vo1.21, No.415
is not helpful in predicting the carriership of hemophilia B. It may, however, be useful in detecting occassional abnormal factor IX molecular forms on which further biochemical characterization can be performd. ACKNOWLEDGEMENTS
The authors wish to thank Dr. Leslie Lipworth for his invaluable help in data analysis.
REFERENCES 1.
PECHET L, TIARKS CY, CHANG CN, SUDHINDRA R, LIPUORTH L: Characterization and experimental use of a monospecific antiserum to factor IX. Thromb. and Ham. j&454-464, 1978.
2.
PECHET L, TIARKS CY, STEVENS J, SUDHINDRA R, LIPWORTH L: Relationship of factor IX antigen and coagulant in hemophilia 6 patients and Thranb. and Hwn. %:465-477, 1978. carrfers.
3.
LIPWORTH L, TURKS CY, RICE C, PECHETL: The laboratory prediction of the carrier state in hemophilia B: Role of replication of assays. Am. J. of Clin. Path. -72:1000-1004, 1979.
4.
LAURELL CB: Quantitative estimation of proteins by electrophoresfs in agarose gel containing antibodies. Ann of Biochem. s:45-2, 1966.
5.
LAURELL CB: Antigen-antibody crossed electrophoresis. Ann. of Biothem. g:358-361, 1965.
6.
HEMnENDORFF B, BORG H, LINDER A and ANDERSSON LO: Studies on hunan factor IX by imnunoelectrophoresis and imunodiffusion methods. Thromb. Res. g:131-139, 1977.
IMMJNOLOGICSURVEY OF THE FACTOR IX MOLECULE IN HEMOPHILIA B+ PATIENTS, CARRIERS AND CORD BLOOD Cheryl Y. Tiarks and Liberto Pechet Departments of Medicine and Pathology University of Massachusetts Medical Center Worcester, MA 01605 (Received 6.5.1980; in revised form 23.11.1980. Accepted by Editor Y. Sultan. Received in final form by Executive Editorial Office 5.2.1981) ABSTRACT Crossed antigen-antibody electrophoresis was performed on 62 hemophilia B (CM) patients and 10 obligatory carriers. The assays were performed in TRIS buffer, or in buffer with the addition of either 2.0 ml4EDTA to bind calciun, or 2.2 r&l calcium lactate, to provide excess calciun. The results revealed migrational differences among normal individuals. To standardize the assay an internal reference (anti prealbumin) was used. A range of normal migrational distance in relation to the reference was established using 30 normal individuals. When comparing atient migration to the normal migrational range (i 2 S.D.P abnormalities were documented in only four patients and in none of the carriers. Even in these cases, mixtures of normal with each patient's from one hemophilia B+ patient revealed a biphasic arc in the presence of EDTA. When mixed with normal plasma, the pattern normalized. Six cord bloods examined were normal. Ue conclude that crossed antigen-antibody electrophoresis requires stringent quality control for valid conclusions, that the method detects very few cases with anomalous migration and that in general, it is not useful for predicting the carriership of hemophilia B. We previously developed a precipitating monospecific antibody to human factor IX (1) which we used to quantitate factor IX antigen (FIX-AS) in a large number of hemophilia B patients and carriers, and factor IX concentrations (2). In addition, crossed antibody electrophoresis utilizing this antiserum revealed migrational differences between factor IX concentrates and normal human plasma. These differences were more pronounced when the electrophoresis was run in the presence of excess KEYWORDS:
Hemophilia B, crossed Antigen-Antibody carriers of Hemophilia B. 391
electrophoresis,
392
CAA!ZIN HEMOPHILIAB
Vo1.21, No.415
calcium (decreased mobility) or in the presence of a Ca++-chelating agent, EDTA (increased mobility). The purpose of the experiments described in this article were two-fold: 1) to discover whether an anomalous migration of FIX-Ag in hemophilia B+ (CRM+) patients could be demonstrated in a crossed antigen-antibody electrophoretic system; 2) to study carriers from these pedigrees by the same methodology to ascertain whether normal and abnormal factor IX molecules could be discerned in their plasma; if abnormal migration were found, this methodology could be used to improve detection of carriers who have high levels of FIX-AS and in whom electroimnunoassays and coagulant assays are not sufficiently discriminant (3). This study presents the results of our studies on 62 hemophilia B+ patients and 10 obligatory carriers of this type; in addition 6 cord bloods from normal newborns, kindly sent to us by Dr. Jack Lazerson of the Medical College of Wisconsin were also studied. METHODS The antiserum to hunan factor IX was prepared as previously described (1). Laurell's electroinmunoassay (4) and crossed antigen-antibody In electrophoresis (5) were performed as previously described (1). order to internally standardize migration, we used hunan prealbumin as a marker; commercial rabbit antiserum to hunan prealbumin (Miles Lab, Elkhart, IN) was added to the gel in the second dimension at a concentration of 80 ~1/6 ml agarose. The migrational distance in reference to the prealbumin was calculated as follows: the arcs for FIX and prealbumfn were bisected and the distance (mn) from the anodal edge of the application well to the middle of each arc was measured. The values described in the results with respect to migration were calculated by dividing the migrational distance of the FIX arc by that of prealbumin. To bind calcium, 2.0&i EDTA was added in the first dimension to the TRIS buffer pH 8.8, used in making the gel. In the same manner, excess calciun was provided by adding 2.2rnMcalciun lactate. The methodology was standardized using normal pooled plasma, prepared as previously described (1). The normal pool was run in parallel with each patient. In addition. 30 normal plasmas ware run individually. These samples were either used fresh, or frozen for a maximum of one week at -7OOC. The hemophilia B+ patients and carriers studied were selected from those previously described (2), all containing lneasureableamounts of factor IX antigen by electrofmmunoassay. All samples had been collected 'as previously described and kept frozen at -7OOC. RESULTS All results refer to crossed antigen-antibody electrophoresis, unless otherwise specified. Normals. Studies on 30 individual normals and repeated assays of normal WInvariably demonstrated a single symmetrical arc in the presence of buffer alone or calcium and a slightly asynnnetricalarc in the presence of EDTA (Fig. 1). The migrational distance of the 30 individual samples
Vo1.21, No.415
CAAE IN HEMOPHILIA B
with respect to prealbumin revealed variations under all three conditions studied (Table I). These variations, were less pronounced on 37 repeats of the normal pool (Table I).
FIG.
1
Cross antigen-antibody electrophoresis (Laurel1 method) of a typical normal plasma showing FIX migration relative to prealbumin, used as an internal reference. During the.lst dimensional electrophoresis the anode was to the right and during the 2nd to the top. Upper frame: buffer alone; Middle frame: buffer plus 2.2 mM calcium lactate in the first dimension; Lower frame: buffer plus 2.flmM EDTA in first dimension. Note, in comparison to buffer alone, the decreased mobility of the FIX molecule in the presence of calcium and the increased mobility in the presence of EDTA.
393
394
CAAE IN HEMOPHILIAB
Vo1.21, No.415
In order to evaluate the variability within tests, ten individual normal plasma samples were aliquoted, frozen, then assayed four times. An analysis of variance procedure (Table II) showed no significant difference between the variance of repeated samples on the same persons compared to the variation between persons. However, this group of 10 normals, assayed over a short period of time, also showed significantly less variability among themselves than the previous 30 normals (Table II), except for EDTA experiments (p < .Ol for buffer p < .025 for calcium lactate p > .05 for EDTA). Patients. Plasmas from two patients revealed no arcs on three separate occasions, although by electroimmunoassay they had measurable FIX-Ag (56 and 6196,respectively) thus leaving 60 evaluable patients. In one additional case no arc was obtained only when EDTA was used in the gel (two experiments). Plasma from one patient demonstrated a biphasic arc with EDTA (5 experiments) (Fig. 2). One component migrated 0.63 (decreased migration compared to normal), the other 0.81 (normal migration). A mixture of equal volumes of normal pooled plasma and plasma from this patient, run in the presence of EDTA showed normal migration.
FIG. 2 Cross antigen-antibody electrophoresis in the presence of 2.0 rrt+l EDTA in the 1st dimension, showing a hemophilia 8+ patient who demonstrated a biphasic arc, one component migrating within the normal range and the other component showing a decreased migration.
vo1.21,
CAAE IN HEMOPHILIAB
No.415
395
The vast majority of hemophilia B+ patients, including 3 Bm variants (2), migrated a distance falling within the normal range (Table III), although differences were noticed frequently when compared to the normal pool run concanmitantly. In four hemophilia B+ patients FIX-AS migration fell outside 2 standard deviations of the normal: in one, the migration in the presence of calcium was less than normal (0.31 in two experiments), but normal (0.40) in a third experiment using the same sample; in another patient the migration was greater than normal, with either buffer alone (0.68 and 0.63) or with calcium (0.50 and 0.56); in a third one, migration was greater in buffer in three experiments (0.69, 0.67 and 0.63), but normal in the presence of calcium or EDTA; in the fourth patient migration was greater only with calcium: 0.77 and 0.58 in another experiment. When samples from these four patients were mixed in equal volumes with normal pool plasma, the migration normalized entirely. Obligatory Carriers. The migration of carriers as a group fell within a This may be narrower normal range than that of patients (Table III). related to the smaller number of samples. None of the carriers fell outside the normal range, nor showed anomalous shapes. We did not examine obligatory carriers or family members from the patient with the biphasic arc. The mother of the one patient with decreased migration in calcium, showed normal migration. The mother of the patient with increased migration with buffer and with calcium also had normal migration. Other relatives of patients with abnormal migrations were not available. Cord Bloods. Six cord blood samples were examined (Table IV). showed normal migration.
All
TABLE I Migration Data on 30 Normal Individuals and 37 Repeats of the Normal Pool. Normal
Normal Pool
Range
Mean
S.D.
Range
Mean
S.D.
Buffer
0.46-0.69
0.544
0.057
0.43-0.69
0.515
0.038
Ca Lactate
0.31-0.50
0.415
0.048
0.31-0.55
0.408
0.034
EDTA
0.72-0.89
0.82
0.033
0.73-0.89
0.816
0.027
Vo1.21, No.415
CAAX IN HEMOPHILIAB
396
TABLE II Normals Repeated 4 Times Each Compared to 30 Normals
Migration Data on.10
Done Previously S.D. of Replications on the 10 Individuals
Mean of 10 Individuals Means (4 experiments)
S.D. Between 10 Individuals
S.D. Retween 30 Individuals
Buffer
0.4796
0.020
0.026
0.057
Ca Lactate
0.3773
0.025
0.025
0.048
EDTA
0.7847
0.026
0.036
0.933
TABLE III Migration of 60 Patients and 10 Carriers Carriers
Patients Range
Mean
S.D.
Range
Mean
S.D.
Buffer
0.43-0.69
0.53
0.06
0.47-0.57
0.517
0.034
Ca Lactate
0.31-8.55
0.415
0.057
0.34-0.43
0.393
0.027
EOTA*
0.73-0.89
0.803
0.04
0.77-0.85
n.801
0.028
*exclusive of cases
55 (biphasic arc) and 387 (no visible arc) TABLE IV Migration Data on 6 Cord Bloods Range
Mean
S.D.
Buffer
0.43-0.56
0.47
0.043
Ca Lactate
0.38-0.46
0.40
0.028
EDTA
0.80-0.85
0.82
0.017
vo1.21, No.415
C&G IN HEMOPHILIAB
DISCUSSION The results of this study suggest that crossed antigen-antibody electrophoresis has intrinsic variability (Table II) as well as variation among normal individuals (Table 1). Since the original description of the method by Laurel1 it has been custanary to compare unknown plasmas with normal controls run simultaneously. Based on experiments described above, we conclude that anomalies in the shape of the Factor IX arc may be significant, if reproducible, but that, in general, anomalous migration in comparison with a simultaneously run control may not represent an abnormality. A statistical comparison between patients' migrational distance with that of the normal range of migration must be made in order to draw valid conclusions. Our normal range was established by surveying 30 normal subjects. It is unlikely that the variability we observed is related to technique alone, since we performed our experiments under identical conditions with respect to buffers, molarity, pH, temperature, equipment, and length of electrophoresis time. In addition, repeated assays of 10 normal plasmas revealed less variability (Table II), with a coefficient of variation of 5%. Interestingly, the variation between this group of 10 normals was significantly less than previously observed in assays of 31)normal controls with the exception of EDTA experiments (Table I). We have no explanation for this observation, which should not invalidate the basic observation of inter-individuals variation and the need for stringent standardization of the assay. Repeated assays of the normal pool, as expected, also showed less variability than that observed in comparisons of the 30 individual normals. We observed in our studies with factor IX concentrates (1) that in the crossed antigen-antibody electrophoretic assay the factor IX molecule has a greater migrational mobility in a calcium-free medium than in the presence of calcium. We therefore performed our present experiments in the presence of excess Ca++ or in its absence through chelation by EDTA. This study presents the results in 62 hemophilia B+ (CRl@) patients and 10 obligatory carriers. If we had compared the migration of each factor IX deficient plasma to the concomnritantlyrun control, we would have erroneously concluded that a large number of samples had anomalous migration. However, by internally standardizing the measurement of migration relative to prealbumin, 56 of the 60 patients' plasmas fell within 2 standard deviations of the calculated mean for 30 normals under all conditions studied. JJsingthese criteria, all the carriers and the cord bloods studied showed normal relative migration (Tables III, IV). Of the 60 cases which formed visible arcs we found only one case in which the capacity to bind calcium seemed to be diminished (greater mobility in the presence of excess calcium) a rare occurrence, confirming the findings of Hemmendorf et al. (6); three other cases had other abnormalities in mobility, and a fifth had a biphasic arc. Even in these cases, doubt exists concerning the significance of the findings, since the patterns normalized when mixtures of patient and normal plasma were assayed. We must therefore conclude that the technique of crossed antigen-antibody electrophoresis as used in these experiments detects very few cases with anomalous migration in hemophilia B+. Moreover, the method
397
CAAE IN HFZOPHILIAB
398
Vo1.21, No.415
is not helpful in predicting the carriership of hemophilia B. It may, however, be useful in detecting occassional abnormal factor IX molecular forms on which further biochemical characterization can be performd. ACKNOWLEDGEMENTS
The authors wish to thank Dr. Leslie Lipworth for his invaluable help in data analysis.
REFERENCES 1.
PECHET L, TIARKS CY, CHANG CN, SUDHINDRA R, LIPUORTH L: Characterization and experimental use of a monospecific antiserum to factor IX. Thromb. and Ham. j&454-464, 1978.
2.
PECHET L, TIARKS CY, STEVENS J, SUDHINDRA R, LIPWORTH L: Relationship of factor IX antigen and coagulant in hemophilia 6 patients and Thranb. and Hwn. %:465-477, 1978. carrfers.
3.
LIPWORTH L, TURKS CY, RICE C, PECHETL: The laboratory prediction of the carrier state in hemophilia B: Role of replication of assays. Am. J. of Clin. Path. -72:1000-1004, 1979.
4.
LAURELL CB: Quantitative estimation of proteins by electrophoresfs in agarose gel containing antibodies. Ann of Biochem. s:45-2, 1966.
5.
LAURELL CB: Antigen-antibody crossed electrophoresis. Ann. of Biothem. g:358-361, 1965.
6.
HEMnENDORFF B, BORG H, LINDER A and ANDERSSON LO: Studies on hunan factor IX by imnunoelectrophoresis and imunodiffusion methods. Thromb. Res. g:131-139, 1977.