RADIALLY ORIENTED FIBRIN CRYSTALLISATION

21

Preliminary Communication RADIALLY ORIENTED FIBRIN CRYSTALLISATION A New Test for Endotoxin Sensitivity in Man

Department of Dermatology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, U.S.A

Radially oriented

acicular crystalline could be induced by incubaaggregates bacterial endotoxins. These blood with ting heparinised in blood of 37 healthy the aggregates did not appear volunteers but were observed in the blood of 130 patients, predominantly those with vasculitis, psoriasis, and bacterial infections. Study of these asteroid structures, which resemble "sunbursts", led to the view that they are oriented crystals of fibrin radiating from a central platelet mass undergoing lysis. INTRODUCTION

CRYSTAL formation is one of the most precise and predictable of physicochemical phenomena, reflecting as it does the symmetric and periodic internal alignment of atoms. In contrast, the deposition of crystals appears to be a random, haphazard event in nature. Only rarely is there an ordered pattern. We report here a remarkable example of a patterned deposition of crystals in a biological system. The phenomenon, first observed in the blood of a psoriatic patient, appears as a "sunburst" of long acicular crystals radiating from a central amorphous mass. The present study discloses the nature of this crystalline formation, the factors causing its appearance, and its potential significance as a test for endotoxin sensitivity. METHODS

Fasting

venous

heparinised (14.4 units/ml) blood-samples

taken from 264 patients with cutaneous diseases and 37 healthy volunteers were mixed in 1 ml aliquots with 0.01 ml of the following killed bacterial suspensions (1000 million/ml, Hollister-Stier Laboratories): Aerobacter aerogenes, Corynebacterium acnes, Diplococcus pneumonia, Escherichia coli, Hœmophilus influenzce, Klebsiella pneumonia, Neisseria catarrhalis, Proteus vulgaris, Pseudomonas ceruginosa, Salmonella spp., enteritidis, typhosa, and dysenterice, Shigellaflex-

neri, Staphylococcus

aureus,

hœmolyticus, lysate extracts (Becton-Dickinson)

Morphology Typically, the object observed was a stellate crystalline spherulite measuring 0.05 to 0-2 mm in diameter (see figure). The overall appearance was that of a "sunburst" or "starburst". It was best seen under phase, phase interference, or dark-field microscopy, being indistinct under regular lighting. In each instance, there was a central globose refractile body of an amorphous nature. In some early forms, one or two white cells could be

the centre. However, the distinctive feature the presence of dozens of fine long straight needlelike crystals radiating from this central nucleus. These long crystals had the appearance of fibrin and could be selectively stained with anti-fibrin flourescent antibody. Neither randomly crossed crystals with no visible centre nor amorphous nuclei with a short stubble of crystals were considered examples of the phenomenon we are seen at

was

describing. The number of these star formations was variable, in some instances exceeding several hundred per mm3 of plasma. Responses were graded on the following scale: 0-5 stars/mm3 = 0; 6-14 1+; 15-29 2+; 30-59 3+; and over 60 = 4+. =

=

=

Induction Factors The critical induction factor was bacterial. Although the phenomenon was discovered when killed Streptococcus pyogenes was used, it was found that the whole organism was not essential. In a study of 18 patients, streptococcal lysate extracts (10-4) of group C, D, F, and G induced "stars" in 12, 11, 11, and 9 patients respectively, group F producing the greatest number of "stars" (table i). In contrast, both group-A extracts and acetylglucosamine were without effect. In 2 of the 301

spp., fcecalis, nonwell as streptococcalgroups A, C, D, F, and

as

A. du P., Van den Berg, D. J., Potgieter, G. M., Retief, F. P. Thromb. Diath. hœmorrh. 1974, 32, 417. 19. Constantinides, P. Adv. Cardiol. 1970, 4, 67. 20. Slater, T. F. Free Radical Mechanisms in Tissue Injury. London, 1972. 21. Glavind, J., Hartmann, S., Clemmersen, J., Hessen, K. E., Dam, H. Acta path. microbiol. scand. 1952, 30, 1 22. O’Brien, J. R. Lancet, 1968, i, 894. 23. Quick, A. J.Am.J. med. Sci. 1966, 252, 265. 24. Elwood, P. C., Cochrane, A. L., Burr, M. L., Sweetnam, P. M., Williams, G., Welby, E., Hughes, S. J., Renton, R. Br. med. J. 1974, i, 436. 25. Boyd, A. M., Ratcliffe, A. H., Jepson, R. P., James, G. W. H. J. Bone Jt Surg. 1949, 31, 325. 26. Boyd, A. M. Angiology, 1963, 14, 198. 27. Toone, W. M. New Engl. J. Med. 1973, 289, 979.

18.

RESULTS

Streptococcus

pyogenes, and viridans,

cell

conjugated antihuman-fibrinogen antibody (goat), as well as antihuman immunoglobulins G (rabbit), A (goat), M (rabbit), and D (goat) (Cappel Laboratories).

WALTER B. SHELLEY

LENNART JUHLIN

Summary

G and lipopolysaccharides type B (E. coli, 0128:BI2) and type W (E. coli, 0127:B8) (Difco). Buffy coats were prepared,’ dispersed in plasma, drawn into rectangular capillary tubes (Vitro Dynamics), sealed (’Crito-Seal’, Arthur H. Thomas), and read under phase-contrast microscopy after 24 h at 37°C. Immunofluorescent studies were done with fluorescein-isothiocyanate-

Heyns,

Crystalline "sunburst" aggregate induced by endotoxin (lipopolysaccharide W) in vitro in blood of vasculitis patient. spines proved to be fibrin crystals, and the centre appears to platelet aggregate undergoing lysis. Red cells give size relationships. Phase contrast. Radial

be

a

22 TABLE I-EFFECTIVENESS OF KILLED BACTERIAL CELLS IN

INDUCING ORIENTED FIBRIN FORMATIONS IN

18

1/20 000)

PATIENTS

did

not

produce

star

formations by itself, but by bacterial cells or

it increased the number induced extracts.

The phenomenon did not occur on addition of the endotoxin to buffy coat in serum, to platelet-rich plasma in the absence of white cells, or to red-cell suspensions in platelet-rich plasma. Fractionation studies indicated that the lymphocyte-rich upper buffy coat was essential, whereas the neutrophil-rich lower buffy coat did not play a part in star formation. Association with Disease

.Concentrations: bacteria 10 000/mm3 blood; saccharide 1/10 000. See Results: Morphology.

lysate 1/10 000 ; lipopoly-

limited forms of psoriasis showed little or no star formation. Likewise, clinical improvement was associated with a progressive decrease in star formation.

TABLE II—ORIENTED FIBRIN FORMATIONS INDUCED BY PSEUDOMONAS AERUGINOSA

BLOOD)

Table II presents an example of the response to Pseudomonas in patients with cutaneous diseases. Findings were similar in each of 16 other bacterial tests. In patients with positive findings the number of star formations often correlated with the clinical severity of the disease process. Thus, blood from patients with mild

(104 KILLED ORGANISMS/MM3

IN CUTANEOUS DISEASES

DISCUSSION

The

* Negative: aphthae, dandruff, elastic-tissue deficit, erythema multitorme, mastocytosis, melasma, morbilliform drug eruption, morphoea,

pityriasis rubra pilaris, polymorphous light eruption, telogen hair-loss, trichotillomania, vitiligo. Some positive: actinic keratoses, basal-cell carcinoma, granuloma annulare, lichen planus, parapsoriasis, progressive telangiectasia, rheumatoid arthritis, rosacea, sarcoidosis. individuals studied the phenomenon

was seen

trol-i.e., in the absence of added bacteria

in the

con-

or extract.

A

study of 24 different killed bacterial organisms showed star formation to be most commonly induced in 46 patients by gram-negative organisms. The most active were Pseudomonas œruginosa, Proteus vulgaris, and Klebsiella pneumoniœ. In contrast, Staphylococcus’ aureus, as well as Candida albicans, house dust, and saline were witheffect. Purified lyophilised lipopolysaccharides (10-4) from E. coli were as effective as the killed bacterial cells in inducing the asteroid phenomenon. Lower concentrations produced fewer stars. ’/ Star formation was not rapid but required 18-24 h at 37°C. Very few stars formed at room temperature, and none at 4°C. The stars proved stable, remaining for days in the sealed capillary tubes with no evidence of lysis. Readings made at 48 h revealed a significantly greater number of stars than at 24 h. However, in the tubes with no stars at 24 h, stars did not form subsequently. The asteroid phenomenon could be induced only in heparinised blood. It was not seen when E.D.T.A., oxalate, citrate, or acid-citrate dextrose was used as an anticoagulant. Furthermore, increasing the amount of heparin to 34.4 units/ml reduced the number of star formations. The addition of protamine (salmine sulphate out

discovery of a hitherto undescribed patterned crystalline formation in blood served as the basis of the present study. The formations, best described as "sunbursts", appeared only when four factors were present-namely, killed bacterial cells or extract, lymphocyte fraction of buffy coat, platelets, and heparinised plasma. The phenomenon was seen only in certain groups of patients and could not be induced in the blood of healthy volunteers. The "sunbursts" were found to be focal clotting, the fine radiant needles staining as fibrin. The fact that the phenomenon could not be produced in serum is in keeping with the view that a source of fibrinogen is essential. The observation that it cannot be induced in the cold indicates that the crystals are not heparin-precipitable fibrinogen or the fibrin peptides seen in association with intravascular clotting.2 The central amorphous nucleus seems best identified as a platelet aggregate which has lost its characteristic appearance as a result oflysis;3 We postulate that the "sunbursts" sighted by us develop in a system in which the primary event is platelet aggregation and activation. This in turn leads to the release of coagulation factors, possibly basic proteins4 which polymerise radial fibrin crystals locally out of a plasma in which coagulation is incipient. Thus, salmine favours star formation, and calcium depletion prevents it. There is apparently a role for the white cells of the lymphocyte-rich upper buffy coat, again a source of procoagulants.5 The role of the bacterial endotoxin is intriguing. Here is a compound which activates the complement system by the alternative pathway,6 produces platelet aggregation, and releases coagulants. The fact that our initial studies reveal no immunoglobulins in the star formation favours the absence of an antigen-antibody reaction in the process. Yet the nature of the specificity of the finding for certain patients remains unexplained, be it immune or not.

finding that streptococci as well as their lysates produce the same effect as the endotoxin apparently rests on the fact that the streptococcal cell-wall contains peptidoglycans (mucopeptides) with endotoxin-like properties.7 The fact that the streptococcal group-A lysate The

/

23 was ineffective in producing star formation suggests that it may not contain this peptidoglycan. No specific disease seems associated with the phenomenon, but it occurred most often in vasculitis, psoriasis, and infections-all processes with histological evidence of fibrin deposition in the clinical lesions. The relevance of the phenomenon we have described should thus extend to other diseases in which fibrin formation has an evi-

FREQUENCIES

OF Gm PHENOTYPES IN NEUROBLASTOMA PATIENTS

AND IN NORMAL BLOOD-DONORS

dent role-e.g., disseminated intravascular coagulation9 and endotoxwmia.10 Technical assistance was provided by Mrs Lynne Todoroff and Miss Inger Pihl and photographic assistance by Mr Edward Glifort. This study was supported by the Walter H. Annenberg Fund, the Thomas B. McCabe Fund, and the Swedish Medical Research Council. REFERENCES 1. Shelley, W. B. Am. J. clin. Path. 1963, 39, 433. 2. Lee, L., McCluskey, R. T. J. exp. Med. 1962, 116, 611. 3. Bessis, M. Living Blood Cells and Their Ultrastructure. New York, 1973. 4. Müller-Berghaus, G. Thrombosis Res. 1976, 8, 725. 5. Rickles, R. F., Hardin, J. A., Pitlick, F. A., Hoyer, L. W., Conrad, M. E. J. clin. Invest. 1973, 52, 1427. 6. Fearon, D. T., Ruddy, S., McCabe, W. R., Schur, P. H. ibid. 1974, 53, 23a 7. Rotta, J. Z. Immun. Forsch. 1975, 149, 230. 8. Soter, N. A., Mihm, M. C., Gigli, I., Dvorak, H. F., Austen, K. F. J. invest.

Derm. 1976, 66, 344. 9. Müller-Berghaus, G.Thromb. Diath. hœmorrh. 1969, 36, suppl. p. 45. 10. Caridis, D. T., Reinhold, R. B., Woodruff, P. W. H., Fine, J. Lancet, 1972

1, 1381.

EVIDENCE FOR AN ASSOCIATION BETWEEN UNCOMMON Gm PHENOTYPES AND NEUROBLASTOMA A. MORELL

H. KÄSER F. SKVARIL

R. SCHERZ

Institute for Clinical and Experimental Cancer Research of the University, and Central Laboratory of the Blood Transfusion Service of the Swiss Red Cross, Berne, Switzerland

The uncommon Gm phenotype Gm(a+ f+ g- b+) occurred significantly more often in sera from 68 patients with neuroblastoma than in sera from normal blood-donors.

Summary

INTRODUCTION

CLINICAL and experimental observations suggest that the growth of malignant neuroblastoma in man may be influenced by immunological mechanisms.’ IgG antibodies which form immune complexes with membrane antigens of neuroblastoma cells and thus inhibit the cytotoxic action of lymphocytes were found in the sera of neuroblastoma patients.2 Jose and Skvaril reported that such "blocking antibodies" were mainly confined to the subclasses IgGl and IgG3.3 We determined the genetic factors of IgG-i.e., Gm allotypes in the sera of 68 neuroblastoma patients. Gm allotypes or allotypic variations of IgG molecules are coded for by the structural genes for the four IgG subclasses. Population and family studies have shown a close linkage between genes coding for different subclasses, and the chromosomal region carrying this linked group of genes has been called the Gm gene complex.4 Gm allotypes in normal sera constitute a Gm phenotype which is composed of two haplotypes, both of them coded for by one parental chromosome. Three common or frequently occurring phenotypes are found in more

than 97% and several rare or uncommon 2 to 3% of the sera of caucasians.1

phenotypes

in

PATIENTS AND METHODS

Patients and sera.-Serum samples from 68 patients with clinically, biochemically, and histologically confirmed neuroblastoma, from 4 relatives of 1 patient (case A), and from 2773 normal blood-donors were studied. Patients were aged from 2 days to 13 years two months. In 42 of these children, tumour activity was present at the time of the study. In the other 26 patients, no signs of tumour activity could be detected. Remission after therapy in these 26 patients had lasted from two months to six years at the time of the study. Patients with active disease and patients in remission were combined for statistical analyses, since the distribution of Gm phenotypes did not differ between the two groups. Determinations of Gm phenotypes.-The allotypes Gm(a), Gm(j), Gm(g), and Gm(b) were determined by a passive haemagglutination-inhibition assay.6 The combinations of these four Gm allotypes in individual sera were designated as their Gm phenotypes. RESULTS

In normal blood-donors, the and of both of the two

common

frequency uncommon

of all three Gm pheno-

types resembled reported values’ (see accompanying

table). However, when the neuroblastoma group was compared with the normal blood-donors, the increased frequency of uncommon Gm phenotypes in the neuroblastoma patients became evident. X2 tests with Yates’ correction were used for statistical analysis. Frequencies of the uncommon phenotype Gm(a+ f+ g- b+) in the neuroblastoma group and in the blood-donor population

tree of patient. Uncommon Gm phenotype and presumed genotype are present in the mother, the patient, and one healthy sister of the patient.

Family

’