Tissue Antigens (1977), 10, 379-393 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)

MLC (HLA-D) Typing: A Family Study Fritz Jbrgensen, Tom Kristensen, Lars U. Lamm and Flemming Kissmeyer-Nielsen The Tissue Typing Laboratory, University Hospital, Aarhus, Denmark The genetics of five HLA-D specificities (Dwl, Dw2, Dw3, Dw4 and Dw6) have been assessed in 21 normal families with four or more children. The HLA-D traits, as defined by typing response against homozygous typing cells, normally behave as dominant characters. The data support the concept of allelic factors. The logical flaw in the basic algorithm of MLC typing (HLA-D typing), i.e. to draw positive conclusions from negative observations, has been amply reinforced in the following studies. Five assignments could not be verified genetically under the assumption of’ dominant traits. Homozygous lack of specific response genes is among the mechanisms proposed as a cause for the phenomenon which has not yet been fully explained. The estimated magnitude of the frequency of false assignments is approximately 10%. Received for publication 9 May, accepted 17 June 1977

In 1971 Yunis and Amos demonstrated a named following the VIth International sibship where two HLA-identical siblings - Workshop on Histocompatibility Testing judged by serology -stimulated each other, in Aarhus in 1975 (WHO - IUIS Nomenwhereas one of them failed to react with a clature Committee, 1975). Six HLA-D third sibling differing for one haplotype specificities, Dwl through Dw6, were (Yunis & Amos 1971). This was taken as rather well-defined and two more, LD107 evidence for the existence of a separate, and LD108, seemed apparent (Thorsby & controlling MLC locus (or region) within Piazza 1975). the HLA system, which had been suggested Several large population studies of the almost from the very beginning of the MLC HLA-D series have been published ‘era’ (Amos & Bach 1968). (Jbrgensen et al. 1974, Grosse-Wilde e t al. For some years, MLC typing has been 1975, Keuning et al. 1975 Thomsen et al. possible; i.e. to classify determinants or 1975, Thorsby et al. 1975 Dupont et al. antigens genetically controlled by part of 1977). In this way, antigen and gene frethe HLA system - the HLA-D locus, so quencies were estimated, and linkage This work was supported by the Danish Medical disequilibrium established, for most of the Research Council. HLA-D specificities.

3 80

JORGENSEN ET AL.

Studies of the inheritance of HLA-D are rarer, especially studies of the genetic segregation. In 42 informative backcross families, the distribution among positive and negative haplotypes was found to be “balanced” (Grosse-Wilde et al. 1975). This supports the idea of dominant (or codominant) inheritance of the factors, although the authors refrained from drawing firm conclusions from their results. Dominant inheritance, as well as the linkage to the rest of the HLA system, is further amply evidenced via the assignment within families of an HLA-D specificity to a given HLA-A, -B, -C haplotype as defined by serology (e.g. Jbrgensen et al. 1973, Carbonara e t al. 1975). Further, in families with an MLC reaction pattern best explained by a recombination between the MLC controlling part and the rest of the HLA region, it has been possible to demonstrate HLA-B, -D, recombination by HLA-D typing (Keuning e t al. 1975, Netzel et al. 1975, Lamm et al. 1976). The HLA-B to -D map distance has been estimated a t 0.38 - 1.81 centiMorgans (for a review, see Lamm e t al. 1977). The HLA-D antigens or determinants are functional entities, but their exact genetic background is still unclear; pertinent here is whether they are controlled by one o r several loci. The aims of this investigation have been to elucidate the genetics of the trait defined by MLC typing and to test the validity of the HLA-D assignment in a family material primarily selected €or this purpose.

Material and Methods

Twenty-one normal Caucasian families were studied. The only criterion for selection was that both parents and at least

four children should be available for investigation. The following investigations were performed: 1)ABO and Rhesus blood grouping with standard procedures. 2) HLA-A, -B, -C, typing using the local lymphocytotoxicity microtechnique (Kissmeyer-Nielsen & Kjerbye 1967) and the micromodification of the complement fixation test with platelets as antigen (Colombani et al. 1971). 3) HLA-D typing (MLC typing) and combinatorial one-way MLC crosstesting “chess-board test” - within each family by use of our standard procedure (Jbrgensen & Lamm 1974), including three stimulator pools each of three individuals (Osoba & Falk 1974) to define the reference value for the caiculation of the relative response. 4) Finally, typing for PGM3 - phosphoglucomutase 3 - (Lamm 1969), Bf - B factor of the properdin system - (Alper et al. 1972) and Glo - glyoxylase - (Kompf et al. 1975) was carried out.

Typing Cell Panel D w l : 13-007 and local ARH330; Dw2: 13-009 and ARH216; Dw3: ARH217, ARH220 and ARH225; Dw4: 13-008 and ARH250; Dw6: ARH307 and ARH311. The 13-007, -008 and -009 cells were closely matched with the other cells within the respective specificity group during the sixth workshop (Thorsby & Piazza 1975) while the Dw3 cells were compared locally with the Dw3 cells of the workshop; finally the Dw6 cells were confirmed in the European control laboratory for the Dw6 specificity (Oslo). General MLC Protocol With few exceptions the families were bled late in the afternoon and defibrinated

381

HLA-D TYPING

Table 1 The parents as a population sample. N = 42 No. pos. a)

f b,

present

’)

p(Dw) WS6 ‘)

previousc)

~~~~~~

Dw 1 Dw2 Dw3 Dw4 Dw6 Blank

7 12 4 9 3

0.168 0.286 0.095 0.214 0.071

0.087 0.155 0.049 0.114 0.036 0.559

~

0.102 0.078 0.085 0.082 0.054 0.599

--

0.078 0.146

0.123

-

~~

a) the number of (consistent) assignments b) the antigen frequency “Present” = this c) the gene frequency calculated according to Bernstein’s formula P = 1 study, “WS6” =joint report from the Sixth Workshop (Thorsby & Piazza 1975) and “previous” = our previous population study (Jdrgensen et al. 1974).

blood with the clot removed was stored overnight at room temperature. The lymphocytes were isolated, frozen and stored over liquid nitrogen (cf. standard procedure above). In the subsequent typing experiments all parents were initially typed. The sibships were then grouped in order to avoid too many typing responses of a given specificity within each experiment. The cpm data were converted to relative response, and were further stabilized according to Ryder et al. (1975) (stabilized relative response (SRR)). The typing results were scored as: for SRR < lo%, i.e. determinant is definitely present. f for S R R < 4 0 % , i.e. determinant is probably present. ? for SRR < 60%, i.e. result is equivocal.

f+

- for SRR 2 60% i.e. determinant is not present. Assignment was based on concordant results with the typing cells used to define each specificity. In the chess-board experiments all members of a family and three unrelated individuals were tested. In these experiments the cpm data were only converted to relative response (Jdrgensen et al. 1973). The experiments were only evaluated qualitatively, viz. in positive and negative reactions, and the HLA-A, -B, 4, -D typing was separately compared with the intrafamilial MLC reactions. Unexpected or discrepant results in typing experiments or MLC chess-boards were challenged in repeated experiments to exclude technical errors. The repeated tests were mostly performed on new blood samples.

Table 2 Within each family, the parents are indicated at the top with their respective haplotypes (a/b and c/d for short reference) Below, the children are given by their sex, age (years) and haplotypes, and the interpreted MLC results no stimulation -, autologous control 0) within the sibship are shown (stimulation Uppermost, the different loci are coded: P = PGM, , G = Glo, Bf = glycine-rich betaglyco-protein and A, B, C, D are the HLA loci The sequence indicates the map order, with the centromere to the left The haplotyping, e.g. a/c only refers to the -D-B-C-A stretch indicates a region where recombination may have occurred, / indicates not done and - refers to unknown antigen within the A, B, C series

+,

ad

S

bc

bc

bd

ac

X

/

1 d 17

2 d 15

3 d 14

4 d 10

+

TT

+

lx

-

Bf B C

D

G

P

11

d 41

bc

9

4 9

3 d 13

2

lx

+

3x

4x

P

+

2x

+

3x

o

4x

FAM.no. 3

-

2x

9 40

/

Bf B C A 2 S 8 - 1

G D

9 43

P G D Bf B C A

21ac11

A

D Bf B C A

1 9 20 ac 9 14 ad

P G

d 57

FAM.no. 1

Table 2

1

3 ? 4 d

ac

9

bd

ac 11 a c 2 ? 13

1 P 15

D Bf B

d 40

-

5?12bc/’,i

P G

+

l x

ac 3 9 14 a c 4 9 1 4 ac 2 d 15

1 2 6 F 1 5

0

A

-

2x

-

3x

+

4x

FAM.no. 4

0

+ +

5x

9 37

2 G*S

+ +

4x

2

-

3x

C/d

40

-

2

P G D B f B C A

-

2x

lx

C

c

1-1

Q

9 35

2 I b d I2 2 G F 1 5

P G D B f B C A

1-1

d 44

FAM.no. 2

L

h)

00

W

ad bd

d 43

-

+

-

lx

-

2x

-

-

4x

+

5x

FAM.no. 8

-

3x

9

44

ac

3 d 15 a c 4 9 9 bd

2 d 18 ac

1 ? 20

,I

0

-

+

-

t

+

o + + o

-

2x 3x 4x +

-

lx 0

G D Bf B C A , , P G D Bf 1 2 X S 12 2 1 a c 1 2 Y s 2 2 4 S 1 5 3 2 b d 1 2 2 S

4 3 1 0

ad 5 d 8 b d

2 9 14 3 9 12

:f

d 47

7

9 40 A, , I G D Bf B 1 2 X S 4 0 - 2 8 a c 2 2 Y S 7 3 b d 2 1 X S 40

,l 1 ll I

FAM.no. 5

-

Table 2 (contd).

2:,3

1

2

X s

S

bc

17

4 d 12 bd

bd

ac

21 3 d 14

9

9

1 2 X S 1 8

1 2

46

7 - 3 a c 2 2 X S 1 2 - 2

-

lx

-

3x

P

G

4x

FAM.no. 9

+

2x

+

lx

0

47

Bf

?

+

2x

-

3x

b d

0

4x

1 1 a c 1 2 1 S

m]

d 58

bc

?

P G D B f B C A

8

-

1

B C A

7 - 3 b d 1 1 4 S 1 5 3 9

P G D B f B C A

4 d 10

1 d 21 a c 2 9 17 ad 3 9 15 a c

l

l 2

9

-

-32

d 46

P G D Bf B C A

A,

C

FAM.no. i

co W

W

n

2

2

4

tl

FI

3:

4 d 11 bc

bd

bc

bc

3 9 16

4 d 15

lx

5 8 13 ad t 6 12 bd t

ac

S

1 9 19

3

2 9 18

11 2

8

+ -

2x

+ -

-

3x

+ -

4x

t

5x

- 2 9 l b d l l

o

i

6x

1

Y S121

-

Table 2 (contd).

21

8 - 1

P G D B f B C A

P G D B f B C A

1 1 X S 1 8 - 9 a c 1 1 3 S

9 39

d 41

bc

bc

4 o 11 b d

3 d 16 a d

2 d 20

1 d 22

4 d 10

ad

3 d 14

d 53

bc

3 9 14

FAM.no. 13

bc ac

2 d 16

S

A,

/p

G

FAM.no. 12 D Bf B

9 40

16

2x

3x

b d

4x

1 1 V

+

lx

-

+

+

2x

+

3x

4x

P

FAM.no. 14

-

9 47

S

14

-

E ?

A,

G D Bf B C A

5 7 1

lx

26

5 2 2 a c l l Z S 1 3

S

X

2

2 2 Y

1

Bf B

D

G

,P

bc

4x

Bf B C A

2 d 16

3x

D

1 8 18 ac

2x

G

bc

lx

P

9 37

d 39

1 d 18

d 44

FAM.no. 10 00 P

cy

3 86

J0RCENSEN ET AL.

a 0

m N

cu

d

7 t G

a

a

0

M

N

N

N 0

c

r

4:

N

LL

X 7

c

d z )

4: U

M

d

-+re m ot

X LD

+ + +

+

O1

X

-+ X

m

N :c

+

O

+

X (u

-X 0 X 7

+ + +

387

HLA-D TYPING

FAM.no. 23 d 41

9

P

P G D B f B C A

1 d 15

ac

2 d 14

bc

3 T 12

ac

4 d

ac

9

G

36

D Bf B C A

r/ lx

2x

3x

-

+

-

4x

Table 2 (contd).

Results The distribution of the HLA-D determinants indicates that the parents constitute a representative sample of the population of origin. Table 1 collates the gene frequencies of the HLA-D specificities tested for among the parents, a combined Scandinavian random population obtained in the sixth workshop and population data from Jbrgensen e t al. (1974). In Table 2 the basic data are interpreted in terms of genotypes assuming the minimum number of crossovers. This seems rather safe for the very closely linked markers HLA-A, -B, -C, -D and Bf but it must be taken with caution for the only

closely linked marker Glo and especially for the medium linked PGM3. All the parents are scored as heterozygous at the HLA-D locus, since lymphocytes from none of them behave as typing cells against their children’s cells. In 35 instances an HLA-D specificity has been assigned to at least one member of a family. In the majority of cases - 30 - the assignment is consistent with linkage of HLA-D to HLA-A, -B, -C, and with the intrafamilial MLC results, whereas inconsistency was found in five cases (cf. below). Table 3 shows the segregation of the D specificities. A near 1: 1 ratio appears for each specificity, and the ratio

Table 3 Segregation for five Dw specificities

Dw 3

Dw4

Dw6

10117

719

10117

010

31/57

8/10

010

7/21

3 I4

24/44

18/27

719

17/38

3 14

55/101

Dwl

Dw2

d

4/14

0

619 10123

~~~

d+?

Total

~

Number of children having a given specificity out of the total number of children of parents with the particular specificity Children whose parents share a specificity are excluded, when this specificity is counted ( D w l , fam. 10; Dw2, fam. 17 & 21; Dw3, fam. 13 and Dw6 fam. 2 )

3 88

J0RGENSEN ET AL.

in the combined data is 5 5 :46,indicating a dominant (or codominant) inheritance. There are only three parents with two D specificities (none of which is shared with the spouse) vzz, the mothers of families 3 and 9 and the father of family 20. They have altogether 13 children, and they always transmit either one or the other of their D specificities, never none or both. This favors allelism (or pseudo-allelism) of the genes controlling the D determinants, but the material is too small to provide critical evidence. In three families a recombination is evident between the B and D locus, i.e. families 2, 18 and 20; all three are maternal crossovers. In two cases (families 2 and 18) the crossing over is directly demonstrable by the D typing results, whereas in family 2 0 the recombination can only be asserted indirectly (child 3 reacts with child 2, but not with child 1 or child 4). This material allows for a combined direct and indirect estimate of the B, D map distance, 1.8

centiMorgans (Lamm et al. 1977 (this issue)). The findings in family 2 have been published separately as a contribution to the mapping of the Bf locus between the HLA-B and -D loci ( L a m et al. 1976). Children 1 and 5 in this family are donors of our Dw6 typing cells. A surprising finding is the high number of “inconsistent assignments”, viz; the five above-mentioned cases of inconsistency between HLA-A, -B, -C typing and MLC. A survey of these findings is given in Table

4. One family (15) shows “positive child, negative parents”. The extensive studies of the other genetic markers: the HLA-A, -B, -C antigens, serum and enzyme types, blood groups and heteromorphic fluorescence characteristics of the chromosomes d o not give evidence as to illegitimacy. Thus, this case represents inconsistent “false” HLA-D assignments, achieved on the basis of reactivity against typing cells. In four families (4, 15, 21, and 23) a

Table 4 Survey of data on genetically inconsistent assignments

Position in family no.

D -B genotype of proband

Child no. 1

x -7 y-17

Dw4

X-14 y--13

Dw3

4

*45 (4) 25-65

48(4) 42-58

5 5 (4) 51-64

51 (2) 50-51

72(4) 39-87

Mother

y__ -17

Dw3

*29(4) 16-40

130(4) 90-175

72(4) 55-90

107 (4) 54-143

73 (4) 61-87

Dw4

‘20 (3) 14-29

58 (8) 30-85

65 (8) 54-86

89 (4) 88-90

67(4) 57-74

Dw4

*18 (4) 6-38

75 (4) 71-94

89(4) 64-109

76(4) 68-81

84(4) 78-94

15

Father

1s

2-12

Father

2-7 -

21

Father 23

Specificty involved

2-7 12

*44 (4)’)

9 0 (1)

110 (3) 81-145

31-71

x-7

x-

Mean stabilized relative response, (no. of observations) and range.

* “Inconsistent” typing result as SRR Each line gives the result obtain with the proband, followed by the results from the relevant members of the rest of the family, for child 1 the father and mother, for the remaining four parents their respective four children in descending birth order

HLA-D TYPING

parent is assigned a specificity which cannot be found among the children although both HLA haplotypes have been transmitted judged from the A, B, C serology. The father in family 4 has an average response of 45 against the two Dw3 typing cells; however, we d o not violate our assignment criteria as the high average is the result of a 29% and 25% response against one of the cells and 60% and 65% against the other. None of the children shows this pattern, they are all negative. It thus seems that the father possesses a Dw3-like determinant not transmitted to the children. Child 4 is the only one who has inherited the b haplotype. If this is a B, D recombination the Dw determinant will escape detection in the child. However, a critical combination to test this possibility in MLC is not available. In the remaining three families recombination can be ruled out as the reason a parental determinant unexpectedly is not found among the children, because of: (a) intra-familial chess-board testing, (b) the rest of the D typing or (c) the need for the assumption of repeated crossings over in the same family. So also in these “positive parents, negative children” families we can conclude that inconsistent assignments are present. No triplets of D-assignment occur by the inconsistent assignments. In contrast, positive assignment could only be performed in two out of 10 theoretically possible instances (two times Dw2). This means that the persons involved are characterized by not having the more common specificities. Finally, the chess-boards have disclosed several typing cells or putative typing cells (families 2, 5, 7, 10, 13 and 21). Some (families 5 and 7) are of unknown specificity to us.

3 89

Discussion The present material confirms that dominant inheritance is the genera1 feature of the trait “typing responses against homozygous typing cells”. As to the detailed mapping of the genetic region determining the trait (alleles or pseudoalleles, one or more loci) the overall data are, however, still meagre. The segregation seen in parents with two recognized HLA-D specificities favors allelism, but our data provide barely conclusive evidence. Population data, e.g. the joint report from the sixth workshop (Thorsby & Piazza 1975) and the report from the post workshop cell exchange (Lamm et al. 1977) have shown that the HLA-D specificities are negatively correlated to each other and in Hardy-Weinberg equilibrium. The assumption of allelism agrees well with these findings which, however, d o not exclude pseudoallelism. Our experience over a time shows that MLC typing is difficult and tedious; it demands frequently repeated testing; preferentially more than one typing cell per specificity should be used; and one has to be very cautious in the interpretation. Pertinent to the present study is the high frequency of assignments which cannot be verified in the family. Since they are reproducible and thus not due to technical factors, they should be considered as biological phenomena. The typing responses in these cases behave as recessive traits or dominant traits of varying penetrance, viz. positive parents with negative children and positive children from negative parents. With no attention paid to the Achilles heel of MLC typing (its questionable basic logic), to draw positive conclusions on negative observations, the typing response

3 90

J0RGENSEN ET AL.

is interpreted as the expression of the presence of an HLA-D determinant on the responder cells similar to that on the typing cell. This concept almost inevitably calls for a dominant inheritance and an HLA-haplotypewise transmittance. The concept of heterogeneity of the determinants or the presence of multiple determinants on one haplotype (Dupont et al. 1973, 1976) cannot generally or p e r se account for the apparent discrepancies, as, in this material, we are dealing with determinants which are identical by descent. At the determinant level it may be possible that the “opposite”, i.e. the homologous, haplotype in some way influences the MLC typing. Can factors, not necessarily the Ddeterminants p e r se, but governed by the two haplotypes, act in concert in such a way that a typing response is obtained although the relevant D-determinant is absent from the responder cells, or can they mask a typing response so it evades detection? In both cases the effect will probably be lost by segregation. Recently, Jensen e t al. (1977) attempted to answer this question by an analysis of variance in selected families from the present material. The study gave only weak evidence that the level of typing response to selected homozygous typing cells may be influenced by factors coded for by the homologous HLA-D region. Non-response, not due to the presence of a given determinant on the responder cells, but due to lack of specific response capability may be caused by the absence of specific immune response genes, or it may be the result of specifically altered immunoresponsiveness, i.e. either specific tolerance or cytotoxicity due to alloimmunization or microbial immunization. Recessive immunogenetic traits are wellknown in red cell serology, e.g. group 0

and Rhesus D-negative. Apart from these, probably the most well-known recessive immunogenetic characteristics are the specific immune non-responsiveness controlled by immune response genes probably located all ovcr the genome. Several are located within the major histocompatibility region of a species, e.g. H-2 of the mouse (for a review, see Klein 1975) such genes may also exist in the HLA system of man (Levine 1972). The homozygous absence of a given immune response gene is disclosed phenotypically as the lack of response against a particular immunodeterminant. Such a mechanism may be the cause of the observed inconsistent assignments. We observed a Dw2 homozygous typing cell giving typing response against several non-Dw2 typing cells of different specificities. Thest: induced normal response in heterozygotes ascertained by the above Dw2 typing cell (Jdrgensen e t al. 1975). The fact that the typing cell donor is a child of first cousins tempts one to suggest a homozygous lack of certain immune response genes. Recently, Thomsen et al. (1976) convincingly demonstrated an individual who gave typing response against cells of four different specificities, viz. HLA-Dwl, 2, 3 and LD107, but only two, HLA-Dw2 and LD107, could be verified by family typing and primed lymphocyte typing, PLT, as described by Sheehy et al. (1975). The genetically inconsistent assignments will primarily be of unportance in population genetic studies, disease association studies, and other situations where family investigations are not normally performed in parallel. The errors will remain quiescent except when triplets emerge. In the sixth workshop there were approximately 4% trip-

391

HLA-D TYPING

lets, i.e. seven out of a panel of 178 Caucasians. Among the 171 non-triplets 39 had two assignments. Thus the frequency of false positive assignment is approximately seven out of 46, i.e. 15%. The equivalent in the present material is 1O%, i.e. four inconsistent positive assignments among the 42 parents. In addition, our material contains one example of a possible false negative assignment, i.e. the father or the mother in family 15. Approximately one out of 10 individuals will be incorrectly typed in a population study. This will have consequences for the validity of the estimates of certain population parameters, e.g. the gene frequencies, the delta values or specific disease incidence ratios. Alternative methods of detecting the HLA-D antigens seem available, i.e. the PLT test and serological tests on B-cell enriched suspensions by use of lymphocytotoxicity techniques, e.g. van Rood e t al. (1975). Several small workshops have already been held on this new technique in preparation for the Seventh International Workshop on Histocompatibility Testing (e.g. see Tissue Antigens 8: issue 5, pp. 327 -384). Critical evidence that typing cells and antibodies detect the same structures in the celI membrane remains to be given. In this context a collation of the above inconsistent HLA-D assignments and serological results obtained with anti-Ia sera (= anti-HLA-D?) may be very informative. There will, however, still be room for HLA-D typing with homozygous typing ceUs and for MLC as such, since the culture system is also a functional test with options unattainable in serology.

References Alper, C. A., Boenish, T. & Watson, L. (1972) Genetic polymorphism in human glycinerich betaglyco-protein. J . exp. Med. 135, 68-80. Amos, D. B. & Bach, F. H. (1968)Phenotypic expressions of the major histocompatibility locus in man (HL-A), leucocyte antigens and mixed leucocyte culture reactivity. J. exp. Med. 128,623-637. Carbonara, A. 0..Miggiano, V. C., De Marchi, M., Trucco, M., Galfrd, G., Baricordi, R., Belvedere, M. C., Curtoni, E. S., Savina, C., Varello, 0. & Ceppelini, R. (1975)Collaborative participation to the Sixth International Workshop. 11: The LD data. Histocompatibility Testing 1975, pp. 494-500. Munksgaard, Copenhagen. Colombani. J., D'Amaro, J ., Graab, B., Smith, G. & Svejgaard, A. (1971) International agreement on a microtechnique of platelet complement fixation (P1.C Fix). Transplant. Proc. 3,121-126. Dupong, B., Hansen, J. A,, Whitsett, C., Slater, L. & Tsung, D. L. (1977)Population gentics for five HLA-D determinants in Caucasians P ~ o c 3,121-126. . Dupont, B., Hansen, J. A. Whitsett, C., Slater, Human mixed lymphocyte culture reaction: Genetics, specificity and biological implications. Advances in Immunology 23, 107202. Dupont, B., Jenlid, C., Hansen, G. S., Nielsen, L. S., Thomsen, M. & Svejgaard, A*. (1973) Multiple MLC (LD) determinants on the same HL-A haplotype. Transplant Proc. 5 , 14811487. Grosse-Wilde, H., Netzel, B., Kempel, W., Ruppert, W., Brehm, G., Bertrams, J., Ewals, R., Lenhard, V., Rittner, Ch., Scholz, G. & Albert, E. D. (1975) Immunogenetics of LD determinants in man. Histocompatibility Testing 1975, pp. 526-532. Munksgaard, Copenhagen. Jensen, E. B., Kristensen, T., Jdrgensen, F. & Lamm, L.U. (1977)HLA-D typing by homozygous typing cells. A statistical analysis of experimental and biological variation. Tissue Antigens 10,83-98.

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JQrgensen, F., Lamm, L. U. & Kissmeyer-Nielsen, F. (1973) Mixed lymphocyte cultures with inbred individuals: An approach t o MLC typing. Tissue Antigens 3, 323-339. Jdrgensen, F. & Lamm, L. U. (1974)MLC - A micro-modification of the mixed leucocyte culture technique. Tissue Antigens 4, 482494. JBrgensen, F., Lamm, L. U. & Kissmeyer-Nielsen, F. (1974) Three LD (MLC) determinants. A Danish population study. Tissue Antigens 4,419-428. JQrgensen,F., Lamm, L. U., Ferrara, G. B., Ipsen, S., JQrgensen, H. & Kissmeyer-Nielsen, F. (1975) LD report from Aarhw, Denmark. Histocompatibility Testing 1975, pp. 519525.Munksgaard, Copenhagen. Keuning, J. J., Termijtelen, A., Blussd van Oud Alblas, A., Gabb, B. W., D’Amaro, J. & van Rood, J . J. (1975)LD (MLC) population and family studies in a Dutch population. Histocompatibility Testing 1975, pp. 533-543. Munksgaard, Copenhagen. Kissmeyer-Nielsen, F. & Kjerbye, K. E. (1967) Lymphocytotoxic microtechnique. Purification of lymphocytes by flotation. Histocompatibility Testing 1967, pp. 381-383, Munksgaard, Copenhagen. Klein, J. (1975) Biology of the mouse. Histocompatibility-2 Complex. Springer-Verlag, Berlin Heidelberg, New York. Kompf, J., Bissbort, S. & Ritner, H. (1975)Red Cell Glyoxalase I (E.C.:4.4.1.5): Formal genetics and linkage relations. Humangenetik 28,249-251. Lamm, L. U. (1969)Detection of the third phosphoglucomutase locus (PGM, ) in human leucocytes. Hereditas 61,282-285. Lamm, L. U., JBrgensen, F. & Kissmeyer-Nielsen, F. (1976)Bf maps between the HLA-A and D loci. Tissue Antigens 7,122-124. Lamm, L. U., JBrgensen, F. & Thorsby, E. (1977) Report from a post-workshop exchange of HLA-D typing cells. Tissue Antigens 9, 45-53. Lamm, L. U., Kristensen, T., Kissmeyer-Nielsen, F. & JBrgensen, F. (1977) On the HLA-B, D map distance. (This issue) Levine, B. B., Stender, R. H. & Fotino, M. (1972) Ragweed hay fever: Genetic control and linkage to HL-A haplotypes. Science 178,12011203.

Netzel, B., Grosse-Wilde, H., Ritmer, Ch., Pretorius, A. M. G., Scholz, S. & Albert, E. D. (1 975) HL-AIMLC recombination frequency and LD typing in HL-AIMLCIBfIPGM, recombinant families. Histocompatibility Testing 1975, pp. !?55-959. Munksgaard, Copenhagen. Osoba, D. & Falk, J . (1974)The mixed leucocyte reaction in man. Effect of pools of stimulating cells selected on the basis of crossreacting HL-A specificities. Cdl. Immunol. 10,117135. Ryder, L. R., Thomsen, NI., Platz, P. & Svejgaard, A. (1975) Data reduction in LD-typing. Histocompatibility Testing 1975, pp. 557559.Munksgaard, Copenhagen. Sheehy, M. J., Sondel, P. M., Bach, F. H., Sapori, M. G. & Bach, M. L. (1975)Rapid detection of LD determinants: the PLT assay. Histocompatibility Testing 1975, pp. 569-575. Munksgaard, Copenhagen. Thomsen, M., Jacobsen, 13., Platz, P., Ryder, L. P., Staub Nielsen, L. 1% Svejgaard, A. (1975) LD typing, polymorphism of MLC determinants. Histocompatibility Testing 1975, pp. 509-5 18.Munksgaard, Copenhagen. Thomsen, M., Morling, N., Platz, P., Ryder, L. P., Nielsen, L. S. & Svejgaard, A. (1976)Specific lack of responsivenesr to certain HLA-D (MLC) determinants with notes on primed lymphocyte typing (PLT). Transplant. Proc. 8,455-459. Thorsby, E., DuBok, R., Bondevik, H., Dupont, B., Eijsvogel, V. P., Hansen, J. A., Jersild, C., JQrgensen. F., Kissmeyer-Nielsen, F., Larnm, L. U., Schellekens, P. 111. A., Svejgaard, A. & Thomsen, M. (1974) Joint report from a mixed lymphocyte cu1t:ure workshop. Tissue Antigens 4,507-525. Thorsby, E., Helgesen, A., Rankin, B., Moller, E. & Kaakinen, A. (1975) Identification of five lymphocyte activating determinants in man. Tissue Antigens 6 , 147-160. Thorsby, E. & Piazza, A. (1975) Joint report from the sixth international histocompatibility workshop conference. 11. Typing for HLA-D (LD-1 or MLC) determinants. Histocompatibility Testing, 1975, pp. 414422.Munksgaard, copenhagen. van Rood, J. J., van Leeuiwen, A., Keuning, J . J . & Blussd van Oud Alblas, A. (1975)The serological recognition of the human MLC

HLA-D TYPING determinants using a modifed cytotixicity technique. Tissue Antigens 5,73-79. WHO-IUIS Terminology Committee (1975) Histocompatibility Testing 1975, pp. 5-11 Munksgaard, Copenhagen. Yunk, E. J. & Amos, D. B. (1971)Three closely linked genetic systems relevant to transplantation. Proc. nut. Acad. Sn'. (Wash). 6 8 , 3031 -3035.

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Address: Fritz Jbrgensen

The Tissue Typing Laboratory University Hospital Aarhus Den mark

Note:

During the VIIth International Workshop on Histocompatibility (Oxford, Sept. 1977) all the families were tested with a subset of the workshop Ia antisera. Reactivity with the Ia sera was associated with Dwspecificities, and all assigned Dwwssociated specificities (Das) segregated with HLA and followed the D-marker in HLA-B, D recombinants. All Dwassignments had equivalent Das-assignments, whereas several Das-assignments

could be made without corresponding Dw-assignments. In the four families with genetically inconsistent HLA-D inheritance, B-lymphocyte typing confirmed the positively assigned Dwspecificities, and inconsistent Dw-negative family members were found to be Daspositive. We are not able to explain these falsely negative Dwassignments for the present.

MLC (HLA-D) typing: a family study.

Tissue Antigens (1977), 10, 379-393 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission f...
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