- Email: [email protected]
C4 allotyping on plasma or serum: Application to routine laboratories
(:4 AUotyping on Plasma or Serum: Application to Routine Laboratories W. J. Zhang, P. H. Kay, T. J. Cobain, and R. L. Dawkins
ABSTRACT: Allotypes of shefourth componentof complement(C4) can bedetectedby dectrophoresisand immunofixation after treatment of EDTA plasma with neuraminidme (blAse). We bare assessed the value of additional treatment with earbexypeptidmeB (CPseB). Following treatment with CPseB + NAse, each alle:eis resolvedinto a single band, permitting cleardefinition of o~lapping bands seenfollowing treatment with NAse alone, blore importandy, C4 allotypes can be determined using stored heparinized plasma or serum. Most C4 null alle~ can be assigned without requiring family studies. Tke approach describedis suitable for routine use by tissue typing laboratories. ABBREVIATIONS
Bf C4 CPseB EDTA
properdinfactor I~ fourth component of complement carboxypeptidaseB dipotassiumethylenediamine teteaacet~e
MHC NAse SLE
majorhistocompatibilky complex neuraminidase systemiclupus erythematosus
INTRODUCTION The fourth component of complement (C4) is extremely polymorphic, with at least 20 alleles occurring with a frequency of greater than 1%. The detection of these alleles is important in studies of MHC-associated disease susceptibility [1], determination of the significance of C4 concentrations when monitoring the activity of SLE [2], and in renal and bone marrow grafting [3,4]. With the much greater use of paternity testing C4 allotyping may be the simplest and cheapest approach for routine application. For these and other reasons C4 allotyping is a potentially important method for routine immunology laboratories. To date, however, the available method has been difficult to standardize. For example, discordant results were obtained in the 9th International Histocorapatibility Workshop [5] and in the 3rd Asia Oceania Histocompatibility Workshop [6]. In addition, conventional approaches require freshly frozen EDTA plasma, which is often difficult to obtain and store without deterioration.
Fro~ the Departmeatsof ClinicalImmunology,RoyalPerthHospital,The QueeuElizabeth11Medical Centre, and the Universityof WesternAustralia, Perth, WesternAustralia. Addressreprintrequeststo:R. L. Dawkins, Departmentof ClinicalImmunology,RoyalPerthHospital, GPO Box X2213, Perth, WesternAustralia 6001. ReceivedAugust 23, 1987;acceptedDecember4, 1987.
HumanImmunology21,165-171 (1988) ©AmericanSocietyforHistocompatlbilltyand hnmunogenetics,1988
16~
0198-8859188153.50
166
W.J. Zhang et al. We have reevaluated the method of Awdeh and Alper [/], together with the modifications proposed by Sire and Cross [8], and have dv~ecmined the specimen of choice. We conclude that C4 allotyping can be used routinely.
MATERIALS A N D METHODS Samples. The present studies took advantage of EDTA plasma samples that had been (1) tested in other reference laboratories, (2) obtained from fully genotyped subjects, and (3) stored at -70°C or in liquid nitrogen without deterioration.
Method. The standard method has been described previously [7,9]. The addition of CPseB treatment [8] was modified in that 12.5/zl of plasma or serum were treated with 5/tl (1.625 units) ofCPseB (EC 3.4.17.2, Sigma, type I) in 100 mM NaC! and incubated at room temperature (21°--25°(:) for 30 rains. Next 10 ~tl of digest was treated with neuraminidase (NAse, Sigma type VI). lmmunofixation electrophoresis and hemolytic overlay were performed as previously described [7,9]. C4 A : B optic.~3 density ratios were determined and cot apared to those previously provided [10]. To determine the specimen of choice, serum, EDTA plasma (7 raM), serum with 7 mM EDTA, and heparinized plasma (12.5 IU/ml) were incubated at 4 ° or 21°(: for periods of 0, 2, 4, and 8 days prior to testing in parallel with a standard EDTA plasma frozen at -70°C until use. RESULTS Effect of CPseB As shown in Figure 1, CPseB + NAse results in a single band rather than the triplet seen with NAse alone. The simpler pattern facilitates assignment given appropriate reference samples. Rarer C4 Alleles As illustrated in Figure 1, CPseB + NAse is helpful in the recognition of rarer alleles such as C4A1 and C4B3. It is still possible to use hemolytic overlay to distinguish C4B from C4A, as the activity is unaffected by treatment with CPseB. (:4 Null Alleles The visual assessment ~ffrelative density of C4A and C4B bands is simpler after treatment with CPseB + NAse. Densitometric comparisons are still necessary, however, and appear to be unaffected by CPseB (Figure 2). With CPseB + NAse there may be a weak fragment in the A region even with homozygous A Null. We therefore examined C4A/C4B ratios in three samples of C4AQ0 B1 and found that ratios remain low with increasing amounts of CPseB (Figure 3). Further, we were able to show that C4A/C4B ratios were the same in serum as in EDTA plasma (Table 1). (:4 Duplications After conventional NAse treatment it is sometimes difficult to recognize additional products, as, for example, with C4A3 + 2 (Figure 1). With CPseB +
C4 Allotyping on Plasma or Serum
167
• i¸ :
2
3
4
5
§
7
$
FIGURE 1 The addition of CPseB improved C4 allotyping. For the bottom panel, EDTA plasma was treated with NAse alone. For the top panel the same samples were pretreated with CPseB. In both panels the cathode ( - ) is below, and lanes contain samples previously shown to be: 1 = C4A3, 1 ;B1, O0 (std 2 = C4A3, 2 ;B2, 1 3 = C4A3,Q0;B2, 1,96 4 = C 4 A 4 , 4 ;B2, 2 (std 5 = C4AS,3,2;BI, Q0 6 = C4A6, 3 ;B2, 1 7 = C4A3, 3 ;B3, 3 (std 8 = C4A4,Q0;BS, 1 (std
- A3,1) - Azt,B2) - A3,B3) - B5,)
where std indicates that the sample had been tested widely and designated a local standard for the alleles indicated. It can be seen that CPseB simplifies the patterns. For example, in lane 1, AI can be more readily distinguished from the more cathodal B3. The poor definition of B1 w/th NAse alone makes deusitometry less useful in demonstrating that this lane contains BI and BQ0 rather than homozygous BI. In lane 2, the multiple overlapping triplet bands with NAse alone create confusion, but with CPseB it is clear that there are four separate bands. Similarly in lane 3, CPseB makes it clear that there are three C4B alleles plus A3. in lane 4, A4 and B2 are clear in either panel, but CPseB is associated with an extra smudge which could be confusing. In lane 5, A5 and BI are obvious, but CPseB aids in resolving three C4A alleles and in providing an unambiguous A/B deusirometric ratio of 3.5 (and hence assignment of BQ0). In lane 6, A6, A3, and B1 are clear, but A2 and B2 cannot be definitely demonstrated or excluded unless CPseB is used. As shown in lane 7, B3 is readily distinguished from A1, but the trailing band of A3 could create confusion without CPseB. There is no difficulty in identifying B3 and it is obvious that the A/B ratio is close to 1.0, as would be expetxed with A3, 3; B3,3 or A3,Q0; B3, Q0. The assignment of B5 in lane 8 is somewhat difficult without CPseB. Furthermore, the A/B ratio of 0.5 is obvious with CPseB.
168
W.J. Zhang et al. 2.G
2,0'
,.sl
! ~i I.O
O.5
o.;
,.o
,J~
,;
,.~
C4AJBratio(neuraminida~el I~IGURE 2 Comparison of the C4A : 13 O.D. ratios of selected samples following treatment with NAse and CPseB. The correlation (r = 0.99) and slope 0' = x - 0.05) allow previously determined O.D. ratio reference ranges [3] to be used for assignment of null C4A and C4B alleles following CPseB allotyping.
FIGURE 3 The C4A : C4B ratios in three examples of C4AQ0 B1 following treatment of EDTA plasma with NAse and CPseB. The C4 phenotypes were previously determined using NAse treatment alone. Increasing amounts of CPseB do not alter the O.D. ratios, suggesting the weak hand in the C4A3 position is not due to incomplete digestion with CPseB. A C4A/B O.D. ratio of less than 0.2 allows assignment of complete C4A deficiency.
C4A:C4B 0.20
0.15
o.____._______.._.-o
o °
0.10
I
1.6
m
:
2.4
3.2
Amount of c~boxyl~l)tidase B {unit)
C4 Allotyping on Plasma or Serum TABLE 1
169
Comparison o f C 4 A / C 4 B optical density ratios in serum and E D T A plasma samples following CPseB + NAse*
C4 pheno~pe
C4A:C4B~fio
A
B
phsm.a
serum 1.1 1.1 0.9 0.8 2.5
3 3 3 3
1 1 1 1
1.0 l.t 0.9 0.8
3 3,QO
1,Q0 1
2.7 0.3
0.4
1.0
*r=
N A s e , duplications are generally m o r e obvious and optical density remains useful (Figures 2 and 3). Specimens for Routine Use A comparison o f serum, E D T A , and heparin plasma is shown in Figure 4. It can be seen that allotyping is quite possible with serum; this is true after a few days at FIGURE 4 Degradation of C4A (closed triangles) and C4B (open triangles) is complete after 8 days at 21°(2 (line) but is reduced by storage at 4°(2 (dottedline). With serum, typing is still possible after several days at room temperature, but this is not the case with EDTA plasma and serum + EDTA. Storage for I week at 4°(2 is not detrimental to hepar~nized plasma. In all instances C4A and C4B are affected similarly. The band concentrations of C4A and C4B are given relative to a standard EDTA plasma sample that was frozen to - 7 0 " C immediately after collection, separation, and aliquoting. The relative band concentration was calculated as O.D. C4A or C4B for test specimen O.D. C4A or C4B for standard x 100% and shown along the Y axis. 140
ttO
./
•
C4A
~
6
C4B
---
Z~:
4°C
\
P+
~u
.l
0
2
tiepin'in ~
4
8
0
=
Bedim
4
B
8
I~DTAt~amm 114CUBAYIOH (DAYS)
2
4
Serum + I~TA
8
170
W.J. Zhang et al. 4°C and even at room temperature. Pragmatically we have found it possible to use serum samples collected and stored without any special precautions. Heparin plasma is somewhat better, especially after storage at 4°C for periods of I week. Indeed heparin plasma appears to be the specimen of choice for typing of samples with a low serum C4 concentration. EDTA plasma is also useful but requires careful storage because there is relatively rapid deterioration at room temperature. The addition of EDTA to serum resulted in a very similar profile to that seen with EDTA plasma (Figure 4).
Disadvantages of CPseB To date we are aware of only two disadvantages of CPseB treatment. The faint "ghosting" bands at a1~proximately the C4A3 position with all C4A null can create some confusion. However, these bands are clearly of lower concentration than the corresponding C4B alleles and a C4A/C4B ratio of less than 0.2 therefore indicates homozygous C4AQ0 (Figure 3). Occasionally there are additional bands visible (e.g., lane 4 in Figure 1).
DISCUSSION In this study we have evaluated the utility of the recently described method of CZi allot/ping using CPseB + NAse [8] and show that it has major advantages. In particular it is possible to use serum or heparin plasma rather than freshly frozen EDTA plasma, as is recommended when using NAse alone. This advantage results in greater convenience and improved cost-effectiveness. Digestion of plasma with CPseB does not affect the functional properties of C4A and C4B locus products. Therefore a hemolytic overlay may still be used to distinguish locus of origin. Because C4A:C4B densitometric ratios are not affected by CPseB + NAse treatment, the same reference ranges can be used to assign null aUeles [10]. Assignments are easier with CPseB + NAse because of the removal of overlapping triplets. The CPseB + NAse method will be helpful in assigning null, rare, and duplicated C4 alleles and therefore disease-associated supratypes [I]. Similarly it should be possible for more laboratories to use C4 allot/ping in routine applications such as paternity testing. Previously we have shown that serum C4 concentrations are largely dependent on the number of expressed C4 genes [11]. Better recognition of null alleles allows more appropriate use of serum C4 concentration reference ranges in monitoring disease activity such as SLE [2]. Further, CPseB + NAse is useful when serum C4 concentrations are relatively low, and heparin plasma may be especially valuable in this respect. The improved typing method has important implications in transplantation because it is now clear that matching for complete MHC supratypes improves graft survival [4]. It may be possible to develop a more rapid C4 allot/ping method for use in cadaver donor-recipient matching. In oar experience CPseB + NAse C4 allotyping is capable of distinguishing between alleles with small differences in electrophoretic mobilit/such as C4A1 and C4B5. The correct assignment of these alleles should be less difficult than previously [ 12]. In conclusion, it is recommended that the use of CPseB + NAse be adopted as the method of choice for routine C4 allot/ping.
C4 Allotyping on Plasma or Serum
171
ACKNOWLEDGMENTS This work was assisted by financial support from the Western Australian Arthritis and Rheumatism Foundation and the National Health and Medical Research Council of Ausrtalia. Publication 8718 of the Departments of Clinical Immunology, Royal Perth Hospital and The Queen Elizabeth II Medical Centre, Perth, Western Australia. REFERENCES 1. Dawkins RL, Christiansen FT, Kay PH, Garlepp M, McCluskeyJ, Hollingsworth PN, Zilko pJ: Disease associations with complotypes, supratypes and haplotypes, lmmunol Rev 70:5, 1983. 2. Uko G, Christiansen FT, Dawkius ILL:Serum C4 concentration in the moulmring of systemic lupus erytbematosus: Requirement for C4 allotyping. Rheumatol Int 6:111, 1986. 3. Awdeh ZL, Alper CA, Eynon E, Alosco SM, Stein R, Yunis EJ: Unrelated individuals matched for MHC extended haplotypes and HLA-identical siblingsshow comparable responses in mixed lymphocyte culture. Lancet ii:853, 1985. 4. Wilton AN, Christiansen FT, Dawkius RL: Supratype matching improves renal transplant survival. Transplant Proc 17:2211, 1985. 5. Ritmer C, Mauff G: C4 polymorphism. In E Albert, M Banr, W Mayr (eds): Histocompatibility Testing 1984. Berlin, Springer, 1984, p 318. 6. Proceedings of ~he 3rd Asia Oceania HistocompatibilityWorkshop and Conference. In Aizawa M (ed): HI.A in Asia-Oceania. Sapporo, Japan, Hokkaido University Press, 1986. 7. Awdeh AL, Alper CA: Inherited structural polymorphism of the fourth component of human complement. Proc Natl Acad Sci USA 77:3576, !980. 8. Sire E, Cross SJ: Pheaotyping of human complement component C4, a Class 111HLA antigen. Biochem J 239:763, 1986. 9. Mauff G, AIPOr CA, Awdeh Z, Batchelor JR, Bertrams T, Braun-Petterseo G, Dawkins RL, Demant P, Edwards J, Gross-Wilde H, Hauptmann G, Klonda P, |.~mn3 L, Mullenhaner E, Nerl C, Olaisen B, O'Neill GJ, Ritmer C, Roos MH, Skanes V, Teisberg P, Wells L: Statement on the nomenclature of human (24 allotypes. Immunobiology 164:184, 1983. 10. Christiansen FF, Dawkins RL, Uko G, McCluskeyJ, Kay PH, Zilko PJ: Complement allotyping in SLE: Association with C4A null. Aust N Z J Med 13:483, 1983. 11. Uko G, Christiansen FF, Dawkins RL, McCann VJ: Reference ranges for serum C4 concentration in subjects with and without (24 null alleles. J Clin Pathol 39:513, 1986. 12. Kay PH, Dawkins RL: Complement genetics and disease: Genetically determined polymorphism of complement components. In K Whaley (ed): Complement in Health and Disease. UK, MTP Press, 1987, p 79.
ABSTRACT: Allotypes of shefourth componentof complement(C4) can bedetectedby dectrophoresisand immunofixation after treatment of EDTA plasma with neuraminidme (blAse). We bare assessed the value of additional treatment with earbexypeptidmeB (CPseB). Following treatment with CPseB + NAse, each alle:eis resolvedinto a single band, permitting cleardefinition of o~lapping bands seenfollowing treatment with NAse alone, blore importandy, C4 allotypes can be determined using stored heparinized plasma or serum. Most C4 null alle~ can be assigned without requiring family studies. Tke approach describedis suitable for routine use by tissue typing laboratories. ABBREVIATIONS
Bf C4 CPseB EDTA
properdinfactor I~ fourth component of complement carboxypeptidaseB dipotassiumethylenediamine teteaacet~e
MHC NAse SLE
majorhistocompatibilky complex neuraminidase systemiclupus erythematosus
INTRODUCTION The fourth component of complement (C4) is extremely polymorphic, with at least 20 alleles occurring with a frequency of greater than 1%. The detection of these alleles is important in studies of MHC-associated disease susceptibility [1], determination of the significance of C4 concentrations when monitoring the activity of SLE [2], and in renal and bone marrow grafting [3,4]. With the much greater use of paternity testing C4 allotyping may be the simplest and cheapest approach for routine application. For these and other reasons C4 allotyping is a potentially important method for routine immunology laboratories. To date, however, the available method has been difficult to standardize. For example, discordant results were obtained in the 9th International Histocorapatibility Workshop [5] and in the 3rd Asia Oceania Histocompatibility Workshop [6]. In addition, conventional approaches require freshly frozen EDTA plasma, which is often difficult to obtain and store without deterioration.
Fro~ the Departmeatsof ClinicalImmunology,RoyalPerthHospital,The QueeuElizabeth11Medical Centre, and the Universityof WesternAustralia, Perth, WesternAustralia. Addressreprintrequeststo:R. L. Dawkins, Departmentof ClinicalImmunology,RoyalPerthHospital, GPO Box X2213, Perth, WesternAustralia 6001. ReceivedAugust 23, 1987;acceptedDecember4, 1987.
HumanImmunology21,165-171 (1988) ©AmericanSocietyforHistocompatlbilltyand hnmunogenetics,1988
16~
0198-8859188153.50
166
W.J. Zhang et al. We have reevaluated the method of Awdeh and Alper [/], together with the modifications proposed by Sire and Cross [8], and have dv~ecmined the specimen of choice. We conclude that C4 allotyping can be used routinely.
MATERIALS A N D METHODS Samples. The present studies took advantage of EDTA plasma samples that had been (1) tested in other reference laboratories, (2) obtained from fully genotyped subjects, and (3) stored at -70°C or in liquid nitrogen without deterioration.
Method. The standard method has been described previously [7,9]. The addition of CPseB treatment [8] was modified in that 12.5/zl of plasma or serum were treated with 5/tl (1.625 units) ofCPseB (EC 3.4.17.2, Sigma, type I) in 100 mM NaC! and incubated at room temperature (21°--25°(:) for 30 rains. Next 10 ~tl of digest was treated with neuraminidase (NAse, Sigma type VI). lmmunofixation electrophoresis and hemolytic overlay were performed as previously described [7,9]. C4 A : B optic.~3 density ratios were determined and cot apared to those previously provided [10]. To determine the specimen of choice, serum, EDTA plasma (7 raM), serum with 7 mM EDTA, and heparinized plasma (12.5 IU/ml) were incubated at 4 ° or 21°(: for periods of 0, 2, 4, and 8 days prior to testing in parallel with a standard EDTA plasma frozen at -70°C until use. RESULTS Effect of CPseB As shown in Figure 1, CPseB + NAse results in a single band rather than the triplet seen with NAse alone. The simpler pattern facilitates assignment given appropriate reference samples. Rarer C4 Alleles As illustrated in Figure 1, CPseB + NAse is helpful in the recognition of rarer alleles such as C4A1 and C4B3. It is still possible to use hemolytic overlay to distinguish C4B from C4A, as the activity is unaffected by treatment with CPseB. (:4 Null Alleles The visual assessment ~ffrelative density of C4A and C4B bands is simpler after treatment with CPseB + NAse. Densitometric comparisons are still necessary, however, and appear to be unaffected by CPseB (Figure 2). With CPseB + NAse there may be a weak fragment in the A region even with homozygous A Null. We therefore examined C4A/C4B ratios in three samples of C4AQ0 B1 and found that ratios remain low with increasing amounts of CPseB (Figure 3). Further, we were able to show that C4A/C4B ratios were the same in serum as in EDTA plasma (Table 1). (:4 Duplications After conventional NAse treatment it is sometimes difficult to recognize additional products, as, for example, with C4A3 + 2 (Figure 1). With CPseB +
C4 Allotyping on Plasma or Serum
167
• i¸ :
2
3
4
5
§
7
$
FIGURE 1 The addition of CPseB improved C4 allotyping. For the bottom panel, EDTA plasma was treated with NAse alone. For the top panel the same samples were pretreated with CPseB. In both panels the cathode ( - ) is below, and lanes contain samples previously shown to be: 1 = C4A3, 1 ;B1, O0 (std 2 = C4A3, 2 ;B2, 1 3 = C4A3,Q0;B2, 1,96 4 = C 4 A 4 , 4 ;B2, 2 (std 5 = C4AS,3,2;BI, Q0 6 = C4A6, 3 ;B2, 1 7 = C4A3, 3 ;B3, 3 (std 8 = C4A4,Q0;BS, 1 (std
- A3,1) - Azt,B2) - A3,B3) - B5,)
where std indicates that the sample had been tested widely and designated a local standard for the alleles indicated. It can be seen that CPseB simplifies the patterns. For example, in lane 1, AI can be more readily distinguished from the more cathodal B3. The poor definition of B1 w/th NAse alone makes deusitometry less useful in demonstrating that this lane contains BI and BQ0 rather than homozygous BI. In lane 2, the multiple overlapping triplet bands with NAse alone create confusion, but with CPseB it is clear that there are four separate bands. Similarly in lane 3, CPseB makes it clear that there are three C4B alleles plus A3. in lane 4, A4 and B2 are clear in either panel, but CPseB is associated with an extra smudge which could be confusing. In lane 5, A5 and BI are obvious, but CPseB aids in resolving three C4A alleles and in providing an unambiguous A/B deusirometric ratio of 3.5 (and hence assignment of BQ0). In lane 6, A6, A3, and B1 are clear, but A2 and B2 cannot be definitely demonstrated or excluded unless CPseB is used. As shown in lane 7, B3 is readily distinguished from A1, but the trailing band of A3 could create confusion without CPseB. There is no difficulty in identifying B3 and it is obvious that the A/B ratio is close to 1.0, as would be expetxed with A3, 3; B3,3 or A3,Q0; B3, Q0. The assignment of B5 in lane 8 is somewhat difficult without CPseB. Furthermore, the A/B ratio of 0.5 is obvious with CPseB.
168
W.J. Zhang et al. 2.G
2,0'
,.sl
! ~i I.O
O.5
o.;
,.o
,J~
,;
,.~
C4AJBratio(neuraminida~el I~IGURE 2 Comparison of the C4A : 13 O.D. ratios of selected samples following treatment with NAse and CPseB. The correlation (r = 0.99) and slope 0' = x - 0.05) allow previously determined O.D. ratio reference ranges [3] to be used for assignment of null C4A and C4B alleles following CPseB allotyping.
FIGURE 3 The C4A : C4B ratios in three examples of C4AQ0 B1 following treatment of EDTA plasma with NAse and CPseB. The C4 phenotypes were previously determined using NAse treatment alone. Increasing amounts of CPseB do not alter the O.D. ratios, suggesting the weak hand in the C4A3 position is not due to incomplete digestion with CPseB. A C4A/B O.D. ratio of less than 0.2 allows assignment of complete C4A deficiency.
C4A:C4B 0.20
0.15
o.____._______.._.-o
o °
0.10
I
1.6
m
:
2.4
3.2
Amount of c~boxyl~l)tidase B {unit)
C4 Allotyping on Plasma or Serum TABLE 1
169
Comparison o f C 4 A / C 4 B optical density ratios in serum and E D T A plasma samples following CPseB + NAse*
C4 pheno~pe
C4A:C4B~fio
A
B
phsm.a
serum 1.1 1.1 0.9 0.8 2.5
3 3 3 3
1 1 1 1
1.0 l.t 0.9 0.8
3 3,QO
1,Q0 1
2.7 0.3
0.4
1.0
*r=
N A s e , duplications are generally m o r e obvious and optical density remains useful (Figures 2 and 3). Specimens for Routine Use A comparison o f serum, E D T A , and heparin plasma is shown in Figure 4. It can be seen that allotyping is quite possible with serum; this is true after a few days at FIGURE 4 Degradation of C4A (closed triangles) and C4B (open triangles) is complete after 8 days at 21°(2 (line) but is reduced by storage at 4°(2 (dottedline). With serum, typing is still possible after several days at room temperature, but this is not the case with EDTA plasma and serum + EDTA. Storage for I week at 4°(2 is not detrimental to hepar~nized plasma. In all instances C4A and C4B are affected similarly. The band concentrations of C4A and C4B are given relative to a standard EDTA plasma sample that was frozen to - 7 0 " C immediately after collection, separation, and aliquoting. The relative band concentration was calculated as O.D. C4A or C4B for test specimen O.D. C4A or C4B for standard x 100% and shown along the Y axis. 140
ttO
./
•
C4A
~
6
C4B
---
Z~:
4°C
\
P+
~u
.l
0
2
tiepin'in ~
4
8
0
=
Bedim
4
B
8
I~DTAt~amm 114CUBAYIOH (DAYS)
2
4
Serum + I~TA
8
170
W.J. Zhang et al. 4°C and even at room temperature. Pragmatically we have found it possible to use serum samples collected and stored without any special precautions. Heparin plasma is somewhat better, especially after storage at 4°C for periods of I week. Indeed heparin plasma appears to be the specimen of choice for typing of samples with a low serum C4 concentration. EDTA plasma is also useful but requires careful storage because there is relatively rapid deterioration at room temperature. The addition of EDTA to serum resulted in a very similar profile to that seen with EDTA plasma (Figure 4).
Disadvantages of CPseB To date we are aware of only two disadvantages of CPseB treatment. The faint "ghosting" bands at a1~proximately the C4A3 position with all C4A null can create some confusion. However, these bands are clearly of lower concentration than the corresponding C4B alleles and a C4A/C4B ratio of less than 0.2 therefore indicates homozygous C4AQ0 (Figure 3). Occasionally there are additional bands visible (e.g., lane 4 in Figure 1).
DISCUSSION In this study we have evaluated the utility of the recently described method of CZi allot/ping using CPseB + NAse [8] and show that it has major advantages. In particular it is possible to use serum or heparin plasma rather than freshly frozen EDTA plasma, as is recommended when using NAse alone. This advantage results in greater convenience and improved cost-effectiveness. Digestion of plasma with CPseB does not affect the functional properties of C4A and C4B locus products. Therefore a hemolytic overlay may still be used to distinguish locus of origin. Because C4A:C4B densitometric ratios are not affected by CPseB + NAse treatment, the same reference ranges can be used to assign null aUeles [10]. Assignments are easier with CPseB + NAse because of the removal of overlapping triplets. The CPseB + NAse method will be helpful in assigning null, rare, and duplicated C4 alleles and therefore disease-associated supratypes [I]. Similarly it should be possible for more laboratories to use C4 allot/ping in routine applications such as paternity testing. Previously we have shown that serum C4 concentrations are largely dependent on the number of expressed C4 genes [11]. Better recognition of null alleles allows more appropriate use of serum C4 concentration reference ranges in monitoring disease activity such as SLE [2]. Further, CPseB + NAse is useful when serum C4 concentrations are relatively low, and heparin plasma may be especially valuable in this respect. The improved typing method has important implications in transplantation because it is now clear that matching for complete MHC supratypes improves graft survival [4]. It may be possible to develop a more rapid C4 allot/ping method for use in cadaver donor-recipient matching. In oar experience CPseB + NAse C4 allotyping is capable of distinguishing between alleles with small differences in electrophoretic mobilit/such as C4A1 and C4B5. The correct assignment of these alleles should be less difficult than previously [ 12]. In conclusion, it is recommended that the use of CPseB + NAse be adopted as the method of choice for routine C4 allot/ping.
C4 Allotyping on Plasma or Serum
171
ACKNOWLEDGMENTS This work was assisted by financial support from the Western Australian Arthritis and Rheumatism Foundation and the National Health and Medical Research Council of Ausrtalia. Publication 8718 of the Departments of Clinical Immunology, Royal Perth Hospital and The Queen Elizabeth II Medical Centre, Perth, Western Australia. REFERENCES 1. Dawkins RL, Christiansen FT, Kay PH, Garlepp M, McCluskeyJ, Hollingsworth PN, Zilko pJ: Disease associations with complotypes, supratypes and haplotypes, lmmunol Rev 70:5, 1983. 2. Uko G, Christiansen FT, Dawkius ILL:Serum C4 concentration in the moulmring of systemic lupus erytbematosus: Requirement for C4 allotyping. Rheumatol Int 6:111, 1986. 3. Awdeh ZL, Alper CA, Eynon E, Alosco SM, Stein R, Yunis EJ: Unrelated individuals matched for MHC extended haplotypes and HLA-identical siblingsshow comparable responses in mixed lymphocyte culture. Lancet ii:853, 1985. 4. Wilton AN, Christiansen FT, Dawkius RL: Supratype matching improves renal transplant survival. Transplant Proc 17:2211, 1985. 5. Ritmer C, Mauff G: C4 polymorphism. In E Albert, M Banr, W Mayr (eds): Histocompatibility Testing 1984. Berlin, Springer, 1984, p 318. 6. Proceedings of ~he 3rd Asia Oceania HistocompatibilityWorkshop and Conference. In Aizawa M (ed): HI.A in Asia-Oceania. Sapporo, Japan, Hokkaido University Press, 1986. 7. Awdeh AL, Alper CA: Inherited structural polymorphism of the fourth component of human complement. Proc Natl Acad Sci USA 77:3576, !980. 8. Sire E, Cross SJ: Pheaotyping of human complement component C4, a Class 111HLA antigen. Biochem J 239:763, 1986. 9. Mauff G, AIPOr CA, Awdeh Z, Batchelor JR, Bertrams T, Braun-Petterseo G, Dawkins RL, Demant P, Edwards J, Gross-Wilde H, Hauptmann G, Klonda P, |.~mn3 L, Mullenhaner E, Nerl C, Olaisen B, O'Neill GJ, Ritmer C, Roos MH, Skanes V, Teisberg P, Wells L: Statement on the nomenclature of human (24 allotypes. Immunobiology 164:184, 1983. 10. Christiansen FF, Dawkins RL, Uko G, McCluskeyJ, Kay PH, Zilko PJ: Complement allotyping in SLE: Association with C4A null. Aust N Z J Med 13:483, 1983. 11. Uko G, Christiansen FF, Dawkins RL, McCann VJ: Reference ranges for serum C4 concentration in subjects with and without (24 null alleles. J Clin Pathol 39:513, 1986. 12. Kay PH, Dawkins RL: Complement genetics and disease: Genetically determined polymorphism of complement components. In K Whaley (ed): Complement in Health and Disease. UK, MTP Press, 1987, p 79.