Acta haemat. 55: 174-180 (1976)
The Action of Seed and Other Reagents on H EM P AS Erythrocytes G. W. G. B ird and J une W ingham Regional Blood Transfusion Service, Birmingham
K ey Words. Blood groups • Congenital dyserythropoietic anaemia • HEMPAS •
Hereditary anaemias • Lectins • Seed agglutinins
Hereditary erythroblastic multinuclearity with a positive acidified ser um test (HEMPAS), or congenital dyserythropoietic anaemia, type II, is an inherited (autosomal recessive) anaemia in which there is a structural abnormality of the red cell membrane [6, 7, 14] which may be responsi ble for defective cell division, failure of the multinucleated erythroblasts to expel their nuclei and for characteristic serological reactions. HEM PAS red cells are agglutinated at 20 °C by about one in three normal (acidified) sera, which contain a naturally occurring IgM alloantibody, anti-HEMPAS, which can be specifically absorbed by HEMPAS cells. In the presence of complement, this antibody lyses HEMPAS cells. The anti body is not present in the sera of HEMPAS patients. The amount of lysis depends on the strength of the antibody, the temperature of incubation and the serum-to-cell ratio [6]. Because HEMPAS red cells are agglutin ated by many human sera, irrespective of blood group, they may be clas sified as polyagglutinable. The i-antigen strength of HEMPAS is greatly enhanced and may exceed the i-antigen strength of newborn infants. The strength of the I antigen may be either within normal limits or increased, depending on the source of anti-I, and HEMPAS cells are readily prone to lysis by anti-I and anti-i in the presence of complement.
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Abstract. Erythrocytes of a patient with hereditary erythroblastic multinuclearity with a positive acidified serum test (HEMPAS) were tested with many seed extracts and various other reagents. Serological evidence of membrane abnormality was con firmed. Various anti-H reagents reacted relatively poorly with HEMPAS cells. HEMPAS cells have both enhanced i and depressed H antigens. A brief note on a ‘new’ anti-H lectin (C ytisus glabrescens) is provided.
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Because seed agglutinins have been usefully applied to the study of various red cell membrane anomalies and to the elucidation of various forms of erythrocyte polyagglutination [1, 4], we have studied the action of seed and other reagents on HEMPAS erythrocytes.
Materials and Methods Seed extracts were prepared and tested by standard methods [2-4]. HEMPAS erythrocytes were obtained through the kindness of Mrs. M arie Crookston of To ronto (4 samples) and Dr. A. G erbal of Paris (3 samples). All tests were carried out on the cells of M. F. (group O), the patient described by Crookston et at. [7]. The other HEMPAS cells were used to confirm apparently significant observations made on the cells of M. F. Appropriate normal control cells were tested throughout. Preliminary tests for avidity (speed of reaction by the tile method) were done first, and titre scores by the tube method were determined whenever there were significant differences in avidity. In our experience, avidity of agglutination on a tile often gives a sharper indication of seed agglutinin specificity than titres or titre scores obtained in tube tests.
Tests on M. F. were carried out in 1973 and the confirmatory tests on other HEMPAS cells in 1974 and 1975. The results are recorded in ta bles I-V. Since one of the blood-group-specific seed agglutinins usefully applied in this study has not been previously reported, we have included a brief description of it at the end of this paper. The important observations on HEMPAS erythrocytes were as follows: (1) no specific seed antiHEMPAS reagent was found; (2) the greatly enhanced reactions of HEM PAS cells with anti-i sera were confirmed; (3) the I-antigen strength of our patient was within normal limits; (4) the H antigen as measured by several anti-H reagents was clearly shown to be depressed. This was par ticularly noticeable with the anti-H agglutinins from Cytisus sessilifolius seeds and from the eel (Anguilla anguilla), which react much less avidly and have lower titer scores with HEMPAS than with normal cells; (5) some ‘non-specific’ seed reagents, e.g. Maclura aurantiaca and Wistaria sinensis reacted more strongly and had higher titre scores with HEMPAS than with control cells, and (6) the agglutinin from the albumin gland of the snail, Helix pomatia, weakly agglutinated group O HEMPAS cells, but not control cells.
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Results
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Table I. Reagents which acted with about equal avidity with HEMPAS and control cells
Aggregators: polybrene, Alcian blue, ruthenium red, lanthanum nitrate, protamine, polylysine, DEAE-dextran, methylcellulose Lectins/protectins: Amaranthus caudatus, Amaranthus hypochondriacus, Cytisus glabrescens1, Datura fastuosa. Datura metelloides, Erythrina indica, H elix ( Cepea) hortensis. Lens esculenta, Sophora japónica, Solanum tuberosum, Tamarix gallica, Viscum album
Human sera: anti-I 1 Had lower titre score however (table V).
Table II. Reagents with greater avidity for HEMPAS than with control cells
‘Non-specific’ lectins: Clerodendron trichotomum, Erythrina christa-galli, Lathyrus tingitanus, Maclura aurantiaca, Orobus vernus, Phaseolus caracalla, Phaseolus multiflorus, Pisum sativum, Quercus robur, Ricinis communis, Robinia viscosa, Sambucus nigra, Triticum vulgare, Wistaria sinensis
Human sera: anti-i Specific lectins/protectins: Bauhinia variegata (anti-N) (M.F. is group NN), Cerastium tomentosum (anti-H)1, Evonymus europaeus (anti-B + H)1, H elix pomatia 2 (anti-A: cross-reacts with neuraminidase-treated B or O cells), Moluccella laevis (anti-A+ N) (M. F. is group NN), Petteria ramentacea (incomplete anti-H), Ulex europaeus (anti-H: inhibited by L-fucose)1 1 Had lower titre scores however (table V). 2 Agglutinated M. F. cells weakly; no reaction with control O cells.
Table III. Reagents which reacted less avidly with HEMPAS than with control cells
(anti-H) Anguilla anguilla1, Cytisus sessilifolius1, Laburnum alpinum, Lotus tetragonolobus
1 Appreciably poorer avidity.
The remarkable enhancement of the i antigen in the presence of a nor mal amount of I, which was confirmed in our studies, contradicts the be lief once held that there is a reciprocal relationship between I and i. This contradiction is not confined to HEMPAS; it is also seen in other anae mias associated with bone marrow stress, e.g. thalassaemia major [10]
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Discussion
HEMPAS Erythrocyte Antibodies
111
Table IV. Reagents negative with both HEMPAS and controls, i.e. no HEMPAS-specific
reagent found Lectins: Acacia dealbata, Adonis aestivalis, Agastache mexicana, Akocanthera spectabilis, Anchusa capensis, Angelica archangelica, Anthyllis montana rubra, Asclepias curassavica, Asparagus meyersii, Aucuba japónica, Basella rubra. Beta vulgaris, Bignonia tweediana, Caccalia cochinea, Calpurnia aurea (anti-A + B), Cardiospermum halicacabum, Carica papaya, Catalpa bignonioides, Catananche coerulea, Ceanothus americana, Cladrastis tinctoria, Clarkia elegans, Coleus candida, Cordyline indivisa, Corokia cotoneaster, Coronilla emerus, Cotyledon simplicifolia, Cuphea lanceolata, Cyphomandra betacea, Cytisus maderensis, Cytisus nigricans, Cytisus supinas, Datura suaveolens, Dicentra spectabilis, Didiscus coerulea, Digitalis mertonensis, Dolichos biflorus (anti-Ai), Echium plantaginium, Echium rubrum, Elscholtzia stauntonii, Eruca sativa, Euphorbia lathyrus, Fatsia japónica, Gaura lindlieimerii, Gingko biloba, Gleditschia triacanthus, Glycine soja, Griselinia littoralis, Gypsophila pacifica, Helianthus annuum, Holboiella latifolia, Holmskildia sanguinia, Horminum pyrenaicum, Hovenia dulcís, Hamulus japonicus variegatus, Hyssopus aristatus, Iberis coronaria hyacinthifolia, Iberis jucunda, Iberis sempervirens, Iberis umbellata, Ionopsidium acaule, Isatis tinctoria, Kaulfussia amelloides, Lagenaria leucantha, Lavendula spica, Layia elegans, Leonotis leonuris, Leonto podium alpinum, Leschenaultia biloba, Lespedeza sieboldii, Leucospermum bolusii, Liatris pycnostachya, Limnanthus douglasii, Linum alpinum, Linum utilitissimum, Lonicera caprifoUum, Lotus corniculata, Luffa, Lupinus speciosa, Lychnis coeli-rosa, Martynia louisiana, Medicago echinus, Melia azederach, Monarda didyma, Nepeta macrantha, Nicandro physaloides, Ocimum minimum, Omphalodcs linifolia, Papaver somniferens, Paronychia capitata, Perilla frutescens, Pitacelia campanularia, Philodendron bipimatifidum, Phiomis samia (anti-A+ B), Phiomis viscosa (anti-A+ B), Phytolacca americana, Picnostachys urticifolia, Pueraria lobata, Quamoclit lobata, Rumex scutatus, Ruta graveolens, Sal via argentea(iinü-Tn), Salvia farinácea (anli-Tn and anú-Cad), Salvia haematodes (anti-Tn), Salvia horminum (anti-Tn and anti-Cad), Salvia officinalis, Salvia patens, Salvia pratensis (anti-Tn), Salvia sclarea (anti-Tn), Salvia sclarea turkestanica (anti-Tn), Salvia splendens, Sanguinia canadensis, Saponaria calabrica, Sarcococca con fusa, Schefflera digitata, Scutellaria alpina, Scutellaria baicalensis coelestina, Shepherdea argéntea, Silybum marianum, Sorbus acuparia, Stachys lanata, Swainsonia galeg ¡folia, Tcucrium chamaedris, Thermopsis caroliniana (anti-T), Thermopsis montana (anti-T), Thymus serpyllum, Torenia fournieri grandiflora, Verbascum bombyclferum, Visearía candida, Zea mays
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Human sera: ‘incomplete’ Rhesus antibodies e.g. anti-D with HEMPAS D + ve cells in saline
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Bird /W ingham Table V. Titre scores with various reagents
HEMPAS Control Anti-H reagents Anguilla anguilla (anti-H) Cerastium tomentosum (weak extract) Cytisus glabrescens Cytisus sessilifolius Evonymus europaeus (anti-B + H)
Human anti-H (Bombay plasma) Laburnum alpinum Lotus tetragonolobus (weak extract) Ulex europaeus
16 38 5 10 23 44 25 47 44 47 13 21 not tested, extract too weak 7 13 32 51
Miscellaneous 92 46
77 16
and malignant disorders of lymphoreticular tissues [11]. An explanation must await the full chemical characterization of I and i and elucidation of their biosynthesis. A relationship between I, i and H is already known [2]. Our observa tions show that in HEMPAS cells there is a reciprocal relationship be tween H and i. R ochant et al. [12], independently tested the red cells of a HEMPAS patient with an extract of Ulex europaeus seeds, with nega tive results, but observed that anti-H was not present in the patient’s ser um. We do not know whether the H antigen of their patient’s red cells was very much more depressed than those of our patients, or whether their Ulex extract was very weak. We confirmed the weak agglutination of HEMPAS cells by the Helix pomatia protectin reported by B izot and M onis [5]. The Helix hortensis protectin, however, reacted about equally with HEMPAS and control cells. Early in our investigation, we confirmed that HEMPAS cells were dif ferent from other known forms of polyagglutinable erythrocytes: T, Tk, Tn, which are acquired characters, and Cad (Cad 1) which is an inherited form of polyagglutination. The non-identity of HEMPAS with any of these forms of polyagglutination was demonstrated by cross-absorption studies and by the reactions of various seed reagents. For example, HEM PAS cells are not agglutinated by the lectin of A rachis hypogaea (anti-T)
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Maclura aurantiaca (syn. pomífera) Wistaria sinensis
HEMPAS Erythrocyte Antibodies
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A cknow ledgem ents. We are grateful to Mrs. M arie C rookston for sending us HEMPAS bloods, for expert advice and much valuable information; and to Dr. A. G erbal for providing blood samples from HEMPAS patients.
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or Salvia sclarea (anti-Tn), Salvia horminum (anti-Tn + anti-Cad) or Dolichos biflorus (anti-A1; anti-Tn and anti-Cad) [1, 4], C rookston and C rookston [8] have shown that HEMPAS is different to T, B izot and M onis [5] that it is different to Cad, and R ochant et al. [12] that it is not T, Tn or Cad. The En(a-) character, in which there is a red cell membrane defect, was also distinguished from HEMPAS. En(a-) cells are not polyagglutinable. They are agglutinated in saline by ‘incomplete’ Rhesus antibodies and by the lectin of Sophora japonica [9]. We found that HEMPAS cells (M. F.) were not agglutinated either by ‘incomplete’ Rhesus antibodies in saline or by the Sophora japonica lectin which reacts strongly with En(a-) cells or those of En(a+) heterozygotes; this was first observed by Sanger [13] who also observed that these cells in saline do not react with incom plete anti-D, unlike En(a-) or En(a+) heterozygous cells, that they react ed normally with rabbit or human anti-Ena and reacted more strongly than a normal NN control with Vicia graminea and Bauhinia purpurea lectins. Furthermore, the Maclura aurantiaca lectin, which reacts relative ly poorly with En(a-) cells [3], reacts more strongly with HEMPAS cells (table V). Brief note on a ‘new’ anti-H lectin. Because of the difficulty sometimes experienced in obtaining seeds of various plants for the preparation of blood-grouping reagents, it is always useful to learn of new ones. In March 1973, during one of our periodic searches for ‘new’ lectins, we found an anti-H agglutinin in the seeds of Cytisus glabrescens. Extracts of the seeds reacted on red cells in the following descending order of avidity: O, A2, B, A,. Oh cells were not agglutinated. Titre scores were as under: O = 56, A2 = 52, B = 25, A, = 10. The avidity and titration results are typical of anti-H [2]. The agglutinin was inhibited by secretor, but not non-secretor, saliva. L-fucose did not inhibit, IV-acetylglucosamine inhibit ed only slightly, but salicin neutralized the lectin (titre: 32). Thus the Cy tisus glabrescens anti-H agglutinin is of the Cytisus sessilifolius type, not the Lotus tetragonolobus eel serum type of anti-H, which is neutralized by L-fucose. It is quite a good anti-H and would be suitable for routine laboratory use. The Cytisus glabrescens agglutinin, as did other anti-H re agents, reacted relatively poorly with HEMPAS cells (table V).
180
Bird /W ingham
References 1 Bird , G. W. G.: Erythrocyte polyagglutination. Nouv. Revue fr. Hémat. 11: 885-896 (1971). 2 Bird , G. W. G. and W ingham , J.: Anti-H from Cerastium tom entosum seeds. A comparison with other seed anti-H agglutinins. Vox Sang. 19: 132-139 (1970). 3 Bird , G. W. G. and W ingham , J.: The action of seed and other reagents on En(a-) erythrocytes. Vox Sang. 24: 48-57 (1973). 4 Bird , G. W. G. and W ingham , J.: Haemagglutinins from Salvia. Vox Sang. 26: 163-166 (1974). 5 Bizot , M. et M onis, M.: Un cas de dysérythropoïèse congénitale de type II. Re vue fr. Trans. 17: 147-150 (1974). 6 C rooskton, J. H.; Crookston , M. C.; Burnie, K. L.; F rancombe, W. H.; D acie, J. V.; D avis, J. A., and L ew is , S. M.: Hereditary erythroblastic multinuclearity with a positive acidified serum test: a type of congenital dyserythropoietic anae mia. Br. J. Haemat. 17: 11-26 (1969). 7 C rookston, J. H.; C rookston , M. C., and R osse, W. F.: Red cell abnormalities in HEMPAS (hereditary erythroblastic multinuclearity with a positive acidified serum test). Br. J. Haemat. 23: 83-91 (1972). 8 C rookston, J. H. and C rookston , M. C.: Hereditary anemia with multinuclear erythroblasts (HEMPAS). Birth Defects 8: 15-19 (1972). 9 F uruhjelm , U.; M yllyla, G.; N evanlinna, H. R.; N'ordling , S.; P irkola, A.; G avin, J.; G ooch , A.; Sanger, R., and T ippett , P.: The red cell phenotype En(a-) and anti-Ena; serological and physicochemical aspects. Vox Sang. 17: 256-278 (1969). 10 G iblett , E. R. and C rookston , M. C.: Agglutinability of red cells by anti-i in patients with thalassaemia major and other haematological disorders. Nature, Lond. 201: 1138-1139 (1966). 11 R ochant, H.; T on T hat , H.; N’G o M inh , M.; L efaou, J.; H enri, A. et D rey fus , B.: Etude quantitative des antigènes érythrocytaires I et i en pathologie. Nouv. Revue fr. Hémat. 13: 307-318 (1973). 12 R ochant, H.; N’Go M inh , M.; T on T hat, H.; H enri, A.; Basch, A.; Sultan, C. et D reyfus, B.: Un nouveau cas de dysérythropoïèse congénitale de type II. Caractères immunologiques et transmission héréditaire de l’antigène de polyagglutinabilité distinct des antigènes T, Tn et Cad. Nouv. Revue fr. Hémat. 13:
Dr. G. W. G. Bird , Regional Blood Transfusion Service, Birm ingham , B15 2SG (England)
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649-660 (1973). 13 Sanger, R.: Personal communication to M. C. C rookston (1969). 14 V ervilghen , R. L.; L ew is , S. M.; D acie, J. V.; Crookston , J. H. and Crook ston , M. C.: HEMPAS: congenital dyserythropoietic anaemia (type II). Q. J1 Med. 42: 257-278 (1973).
The Action of Seed and Other Reagents on H EM P AS Erythrocytes G. W. G. B ird and J une W ingham Regional Blood Transfusion Service, Birmingham
K ey Words. Blood groups • Congenital dyserythropoietic anaemia • HEMPAS •
Hereditary anaemias • Lectins • Seed agglutinins
Hereditary erythroblastic multinuclearity with a positive acidified ser um test (HEMPAS), or congenital dyserythropoietic anaemia, type II, is an inherited (autosomal recessive) anaemia in which there is a structural abnormality of the red cell membrane [6, 7, 14] which may be responsi ble for defective cell division, failure of the multinucleated erythroblasts to expel their nuclei and for characteristic serological reactions. HEM PAS red cells are agglutinated at 20 °C by about one in three normal (acidified) sera, which contain a naturally occurring IgM alloantibody, anti-HEMPAS, which can be specifically absorbed by HEMPAS cells. In the presence of complement, this antibody lyses HEMPAS cells. The anti body is not present in the sera of HEMPAS patients. The amount of lysis depends on the strength of the antibody, the temperature of incubation and the serum-to-cell ratio [6]. Because HEMPAS red cells are agglutin ated by many human sera, irrespective of blood group, they may be clas sified as polyagglutinable. The i-antigen strength of HEMPAS is greatly enhanced and may exceed the i-antigen strength of newborn infants. The strength of the I antigen may be either within normal limits or increased, depending on the source of anti-I, and HEMPAS cells are readily prone to lysis by anti-I and anti-i in the presence of complement.
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Abstract. Erythrocytes of a patient with hereditary erythroblastic multinuclearity with a positive acidified serum test (HEMPAS) were tested with many seed extracts and various other reagents. Serological evidence of membrane abnormality was con firmed. Various anti-H reagents reacted relatively poorly with HEMPAS cells. HEMPAS cells have both enhanced i and depressed H antigens. A brief note on a ‘new’ anti-H lectin (C ytisus glabrescens) is provided.
BlRDA V INGHAM
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Because seed agglutinins have been usefully applied to the study of various red cell membrane anomalies and to the elucidation of various forms of erythrocyte polyagglutination [1, 4], we have studied the action of seed and other reagents on HEMPAS erythrocytes.
Materials and Methods Seed extracts were prepared and tested by standard methods [2-4]. HEMPAS erythrocytes were obtained through the kindness of Mrs. M arie Crookston of To ronto (4 samples) and Dr. A. G erbal of Paris (3 samples). All tests were carried out on the cells of M. F. (group O), the patient described by Crookston et at. [7]. The other HEMPAS cells were used to confirm apparently significant observations made on the cells of M. F. Appropriate normal control cells were tested throughout. Preliminary tests for avidity (speed of reaction by the tile method) were done first, and titre scores by the tube method were determined whenever there were significant differences in avidity. In our experience, avidity of agglutination on a tile often gives a sharper indication of seed agglutinin specificity than titres or titre scores obtained in tube tests.
Tests on M. F. were carried out in 1973 and the confirmatory tests on other HEMPAS cells in 1974 and 1975. The results are recorded in ta bles I-V. Since one of the blood-group-specific seed agglutinins usefully applied in this study has not been previously reported, we have included a brief description of it at the end of this paper. The important observations on HEMPAS erythrocytes were as follows: (1) no specific seed antiHEMPAS reagent was found; (2) the greatly enhanced reactions of HEM PAS cells with anti-i sera were confirmed; (3) the I-antigen strength of our patient was within normal limits; (4) the H antigen as measured by several anti-H reagents was clearly shown to be depressed. This was par ticularly noticeable with the anti-H agglutinins from Cytisus sessilifolius seeds and from the eel (Anguilla anguilla), which react much less avidly and have lower titer scores with HEMPAS than with normal cells; (5) some ‘non-specific’ seed reagents, e.g. Maclura aurantiaca and Wistaria sinensis reacted more strongly and had higher titre scores with HEMPAS than with control cells, and (6) the agglutinin from the albumin gland of the snail, Helix pomatia, weakly agglutinated group O HEMPAS cells, but not control cells.
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Results
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BIRD/WINGHAM
Table I. Reagents which acted with about equal avidity with HEMPAS and control cells
Aggregators: polybrene, Alcian blue, ruthenium red, lanthanum nitrate, protamine, polylysine, DEAE-dextran, methylcellulose Lectins/protectins: Amaranthus caudatus, Amaranthus hypochondriacus, Cytisus glabrescens1, Datura fastuosa. Datura metelloides, Erythrina indica, H elix ( Cepea) hortensis. Lens esculenta, Sophora japónica, Solanum tuberosum, Tamarix gallica, Viscum album
Human sera: anti-I 1 Had lower titre score however (table V).
Table II. Reagents with greater avidity for HEMPAS than with control cells
‘Non-specific’ lectins: Clerodendron trichotomum, Erythrina christa-galli, Lathyrus tingitanus, Maclura aurantiaca, Orobus vernus, Phaseolus caracalla, Phaseolus multiflorus, Pisum sativum, Quercus robur, Ricinis communis, Robinia viscosa, Sambucus nigra, Triticum vulgare, Wistaria sinensis
Human sera: anti-i Specific lectins/protectins: Bauhinia variegata (anti-N) (M.F. is group NN), Cerastium tomentosum (anti-H)1, Evonymus europaeus (anti-B + H)1, H elix pomatia 2 (anti-A: cross-reacts with neuraminidase-treated B or O cells), Moluccella laevis (anti-A+ N) (M. F. is group NN), Petteria ramentacea (incomplete anti-H), Ulex europaeus (anti-H: inhibited by L-fucose)1 1 Had lower titre scores however (table V). 2 Agglutinated M. F. cells weakly; no reaction with control O cells.
Table III. Reagents which reacted less avidly with HEMPAS than with control cells
(anti-H) Anguilla anguilla1, Cytisus sessilifolius1, Laburnum alpinum, Lotus tetragonolobus
1 Appreciably poorer avidity.
The remarkable enhancement of the i antigen in the presence of a nor mal amount of I, which was confirmed in our studies, contradicts the be lief once held that there is a reciprocal relationship between I and i. This contradiction is not confined to HEMPAS; it is also seen in other anae mias associated with bone marrow stress, e.g. thalassaemia major [10]
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Discussion
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Table IV. Reagents negative with both HEMPAS and controls, i.e. no HEMPAS-specific
reagent found Lectins: Acacia dealbata, Adonis aestivalis, Agastache mexicana, Akocanthera spectabilis, Anchusa capensis, Angelica archangelica, Anthyllis montana rubra, Asclepias curassavica, Asparagus meyersii, Aucuba japónica, Basella rubra. Beta vulgaris, Bignonia tweediana, Caccalia cochinea, Calpurnia aurea (anti-A + B), Cardiospermum halicacabum, Carica papaya, Catalpa bignonioides, Catananche coerulea, Ceanothus americana, Cladrastis tinctoria, Clarkia elegans, Coleus candida, Cordyline indivisa, Corokia cotoneaster, Coronilla emerus, Cotyledon simplicifolia, Cuphea lanceolata, Cyphomandra betacea, Cytisus maderensis, Cytisus nigricans, Cytisus supinas, Datura suaveolens, Dicentra spectabilis, Didiscus coerulea, Digitalis mertonensis, Dolichos biflorus (anti-Ai), Echium plantaginium, Echium rubrum, Elscholtzia stauntonii, Eruca sativa, Euphorbia lathyrus, Fatsia japónica, Gaura lindlieimerii, Gingko biloba, Gleditschia triacanthus, Glycine soja, Griselinia littoralis, Gypsophila pacifica, Helianthus annuum, Holboiella latifolia, Holmskildia sanguinia, Horminum pyrenaicum, Hovenia dulcís, Hamulus japonicus variegatus, Hyssopus aristatus, Iberis coronaria hyacinthifolia, Iberis jucunda, Iberis sempervirens, Iberis umbellata, Ionopsidium acaule, Isatis tinctoria, Kaulfussia amelloides, Lagenaria leucantha, Lavendula spica, Layia elegans, Leonotis leonuris, Leonto podium alpinum, Leschenaultia biloba, Lespedeza sieboldii, Leucospermum bolusii, Liatris pycnostachya, Limnanthus douglasii, Linum alpinum, Linum utilitissimum, Lonicera caprifoUum, Lotus corniculata, Luffa, Lupinus speciosa, Lychnis coeli-rosa, Martynia louisiana, Medicago echinus, Melia azederach, Monarda didyma, Nepeta macrantha, Nicandro physaloides, Ocimum minimum, Omphalodcs linifolia, Papaver somniferens, Paronychia capitata, Perilla frutescens, Pitacelia campanularia, Philodendron bipimatifidum, Phiomis samia (anti-A+ B), Phiomis viscosa (anti-A+ B), Phytolacca americana, Picnostachys urticifolia, Pueraria lobata, Quamoclit lobata, Rumex scutatus, Ruta graveolens, Sal via argentea(iinü-Tn), Salvia farinácea (anli-Tn and anú-Cad), Salvia haematodes (anti-Tn), Salvia horminum (anti-Tn and anti-Cad), Salvia officinalis, Salvia patens, Salvia pratensis (anti-Tn), Salvia sclarea (anti-Tn), Salvia sclarea turkestanica (anti-Tn), Salvia splendens, Sanguinia canadensis, Saponaria calabrica, Sarcococca con fusa, Schefflera digitata, Scutellaria alpina, Scutellaria baicalensis coelestina, Shepherdea argéntea, Silybum marianum, Sorbus acuparia, Stachys lanata, Swainsonia galeg ¡folia, Tcucrium chamaedris, Thermopsis caroliniana (anti-T), Thermopsis montana (anti-T), Thymus serpyllum, Torenia fournieri grandiflora, Verbascum bombyclferum, Visearía candida, Zea mays
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Human sera: ‘incomplete’ Rhesus antibodies e.g. anti-D with HEMPAS D + ve cells in saline
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Bird /W ingham Table V. Titre scores with various reagents
HEMPAS Control Anti-H reagents Anguilla anguilla (anti-H) Cerastium tomentosum (weak extract) Cytisus glabrescens Cytisus sessilifolius Evonymus europaeus (anti-B + H)
Human anti-H (Bombay plasma) Laburnum alpinum Lotus tetragonolobus (weak extract) Ulex europaeus
16 38 5 10 23 44 25 47 44 47 13 21 not tested, extract too weak 7 13 32 51
Miscellaneous 92 46
77 16
and malignant disorders of lymphoreticular tissues [11]. An explanation must await the full chemical characterization of I and i and elucidation of their biosynthesis. A relationship between I, i and H is already known [2]. Our observa tions show that in HEMPAS cells there is a reciprocal relationship be tween H and i. R ochant et al. [12], independently tested the red cells of a HEMPAS patient with an extract of Ulex europaeus seeds, with nega tive results, but observed that anti-H was not present in the patient’s ser um. We do not know whether the H antigen of their patient’s red cells was very much more depressed than those of our patients, or whether their Ulex extract was very weak. We confirmed the weak agglutination of HEMPAS cells by the Helix pomatia protectin reported by B izot and M onis [5]. The Helix hortensis protectin, however, reacted about equally with HEMPAS and control cells. Early in our investigation, we confirmed that HEMPAS cells were dif ferent from other known forms of polyagglutinable erythrocytes: T, Tk, Tn, which are acquired characters, and Cad (Cad 1) which is an inherited form of polyagglutination. The non-identity of HEMPAS with any of these forms of polyagglutination was demonstrated by cross-absorption studies and by the reactions of various seed reagents. For example, HEM PAS cells are not agglutinated by the lectin of A rachis hypogaea (anti-T)
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Maclura aurantiaca (syn. pomífera) Wistaria sinensis
HEMPAS Erythrocyte Antibodies
179
A cknow ledgem ents. We are grateful to Mrs. M arie C rookston for sending us HEMPAS bloods, for expert advice and much valuable information; and to Dr. A. G erbal for providing blood samples from HEMPAS patients.
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or Salvia sclarea (anti-Tn), Salvia horminum (anti-Tn + anti-Cad) or Dolichos biflorus (anti-A1; anti-Tn and anti-Cad) [1, 4], C rookston and C rookston [8] have shown that HEMPAS is different to T, B izot and M onis [5] that it is different to Cad, and R ochant et al. [12] that it is not T, Tn or Cad. The En(a-) character, in which there is a red cell membrane defect, was also distinguished from HEMPAS. En(a-) cells are not polyagglutinable. They are agglutinated in saline by ‘incomplete’ Rhesus antibodies and by the lectin of Sophora japonica [9]. We found that HEMPAS cells (M. F.) were not agglutinated either by ‘incomplete’ Rhesus antibodies in saline or by the Sophora japonica lectin which reacts strongly with En(a-) cells or those of En(a+) heterozygotes; this was first observed by Sanger [13] who also observed that these cells in saline do not react with incom plete anti-D, unlike En(a-) or En(a+) heterozygous cells, that they react ed normally with rabbit or human anti-Ena and reacted more strongly than a normal NN control with Vicia graminea and Bauhinia purpurea lectins. Furthermore, the Maclura aurantiaca lectin, which reacts relative ly poorly with En(a-) cells [3], reacts more strongly with HEMPAS cells (table V). Brief note on a ‘new’ anti-H lectin. Because of the difficulty sometimes experienced in obtaining seeds of various plants for the preparation of blood-grouping reagents, it is always useful to learn of new ones. In March 1973, during one of our periodic searches for ‘new’ lectins, we found an anti-H agglutinin in the seeds of Cytisus glabrescens. Extracts of the seeds reacted on red cells in the following descending order of avidity: O, A2, B, A,. Oh cells were not agglutinated. Titre scores were as under: O = 56, A2 = 52, B = 25, A, = 10. The avidity and titration results are typical of anti-H [2]. The agglutinin was inhibited by secretor, but not non-secretor, saliva. L-fucose did not inhibit, IV-acetylglucosamine inhibit ed only slightly, but salicin neutralized the lectin (titre: 32). Thus the Cy tisus glabrescens anti-H agglutinin is of the Cytisus sessilifolius type, not the Lotus tetragonolobus eel serum type of anti-H, which is neutralized by L-fucose. It is quite a good anti-H and would be suitable for routine laboratory use. The Cytisus glabrescens agglutinin, as did other anti-H re agents, reacted relatively poorly with HEMPAS cells (table V).
180
Bird /W ingham
References 1 Bird , G. W. G.: Erythrocyte polyagglutination. Nouv. Revue fr. Hémat. 11: 885-896 (1971). 2 Bird , G. W. G. and W ingham , J.: Anti-H from Cerastium tom entosum seeds. A comparison with other seed anti-H agglutinins. Vox Sang. 19: 132-139 (1970). 3 Bird , G. W. G. and W ingham , J.: The action of seed and other reagents on En(a-) erythrocytes. Vox Sang. 24: 48-57 (1973). 4 Bird , G. W. G. and W ingham , J.: Haemagglutinins from Salvia. Vox Sang. 26: 163-166 (1974). 5 Bizot , M. et M onis, M.: Un cas de dysérythropoïèse congénitale de type II. Re vue fr. Trans. 17: 147-150 (1974). 6 C rooskton, J. H.; Crookston , M. C.; Burnie, K. L.; F rancombe, W. H.; D acie, J. V.; D avis, J. A., and L ew is , S. M.: Hereditary erythroblastic multinuclearity with a positive acidified serum test: a type of congenital dyserythropoietic anae mia. Br. J. Haemat. 17: 11-26 (1969). 7 C rookston, J. H.; C rookston , M. C., and R osse, W. F.: Red cell abnormalities in HEMPAS (hereditary erythroblastic multinuclearity with a positive acidified serum test). Br. J. Haemat. 23: 83-91 (1972). 8 C rookston, J. H. and C rookston , M. C.: Hereditary anemia with multinuclear erythroblasts (HEMPAS). Birth Defects 8: 15-19 (1972). 9 F uruhjelm , U.; M yllyla, G.; N evanlinna, H. R.; N'ordling , S.; P irkola, A.; G avin, J.; G ooch , A.; Sanger, R., and T ippett , P.: The red cell phenotype En(a-) and anti-Ena; serological and physicochemical aspects. Vox Sang. 17: 256-278 (1969). 10 G iblett , E. R. and C rookston , M. C.: Agglutinability of red cells by anti-i in patients with thalassaemia major and other haematological disorders. Nature, Lond. 201: 1138-1139 (1966). 11 R ochant, H.; T on T hat , H.; N’G o M inh , M.; L efaou, J.; H enri, A. et D rey fus , B.: Etude quantitative des antigènes érythrocytaires I et i en pathologie. Nouv. Revue fr. Hémat. 13: 307-318 (1973). 12 R ochant, H.; N’Go M inh , M.; T on T hat, H.; H enri, A.; Basch, A.; Sultan, C. et D reyfus, B.: Un nouveau cas de dysérythropoïèse congénitale de type II. Caractères immunologiques et transmission héréditaire de l’antigène de polyagglutinabilité distinct des antigènes T, Tn et Cad. Nouv. Revue fr. Hémat. 13:
Dr. G. W. G. Bird , Regional Blood Transfusion Service, Birm ingham , B15 2SG (England)
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649-660 (1973). 13 Sanger, R.: Personal communication to M. C. C rookston (1969). 14 V ervilghen , R. L.; L ew is , S. M.; D acie, J. V.; Crookston , J. H. and Crook ston , M. C.: HEMPAS: congenital dyserythropoietic anaemia (type II). Q. J1 Med. 42: 257-278 (1973).
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