EXPERIMENTAL PRODUCTION OF MICROANGIOPATHIC HÆMOLYTIC ANÆMIA IN VIVO

EXPERIMENTAL PRODUCTION OF MICROANGIOPATHIC HÆMOLYTIC ANÆMIA IN VIVO

1121 remained afebrile. Antibiotic control guineapigs remained well. Since the most probable route of infection in man was through skin, two guineapig...

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1121 remained afebrile. Antibiotic control guineapigs remained well. Since the most probable route of infection in man was through skin, two guineapigs were shaved and left for 72 hours to allow any minute skin lesions to heal. Infected guineapig blood was then smeared on to the skin and allowed to dry. One of these guineapigs became febrile 10 days later, the other has remained well. Two C. aethiops monkeys have been successfully infected I.P. (0-5 ml.) with infected guineapig blood (from the 9th passage). The agent was detected in the blood of both monkeys starting on the second day after infection, and they died 6 and 7 days after infection. It is too early to report on any other findings. The agent passes through a ’Gradocol ’ membrane of 790 m[t average pore diameter but not through a membrane of 340 m{1. pore diameter. Mice and hamsters inoculated with original material have not shown any signs of illness. Hamsters inoculated l.p. and i.c. with 5th passage guineapig blood became sick 5-10 days after infection and typical intracytoplasmic bodies were seen in their livers. Further passage is being carried out. None of the mice inoculated with guineapig passage material have shown any signs of illness. Nothing has been grown in any of the eggs inoculated with original or guineapig passage material. No cytopathic changes were seen in any of the tissue culture systems, but fluid removed from L-cell and BHK 21 cultures after 9 days incubation at 37°C produced illness in guineapigs when inoculated 1.P.

pigs

DISCUSSION

definite conclusion is yet possible about the nature of the infective agent, it is clear that the organism responsible for the disease has been isolated from three patients and transmitted through nine passages in guineapigs, resulting in fairly uniform lesions. Irrespective of the route of inoculation, the most prominent lesions were invariably found in the liver, spleen, and lung. All affected guineapig livers contained intracytoplasmic accumulations of structures very similar in shape and staining properties either to rickettsia or to organisms of the psittacosis-lymphogranuloma group. The negative bacteriological and virological findings, the filtration data, and the high infectivity of the monkey tissues for man seem to be reasonably consistent with one or other of these groups of organisms. The infectious agent may be an unknown organism, and further work is now required to elucidate the nature of the intracytoplasmic granules: whether they represent the actual pathogen or are formed within the cytoplasm of the liver cells as a reaction to the presence of an infective agent. Sera from a variety of monkeys in East Africa will shortly be tested for antibodies to this agent. The isolation of a further agent from laboratory monkeys only serves to emphasise the hazards of working with these animals. It is probably only because the majority of adventitious infections in monkeys are relatively nonpathogenic for man that laboratory infections associated with monkeys have not been more frequent. The kidneys from this particular consignment of monkeys were being used for tissue culture and might

Although

,



no

have been used for vaccine production. An adequate

test

will have to be devised to test all vaccines made with monkey tissue for the presence of this highly dangerous agent. We thank Prof. G. W. A. Dick, Bland-Sutton Institute, Middlesex Hospital, London, who brought back the blood and necropsy

to the U.K.; Dr. G. May (Frankfurt) and Prof. W. Hennessen (Marburg) for acute and convalescent blood-samples; Mr. W. R. Bale and Mr. E. J. Morris for help in the bacteriological work.

specimens

C. E. GORDON SMITH St. And., M.R.C.P., M.C.PATH. D. I. H. SIMPSON

M.D.

M.B.

Belf.

E. T. W. BOWEN Microbiological Research Establishment, Experimental Station, Porton, Salisbury, Wiltshire

F.I.M.L.T.

PH.D.

I. ZLOTNIK Edin., B.V.SC., M.R.C.V.S.

EXPERIMENTAL PRODUCTION OF MICROANGIOPATHIC HÆMOLYTIC ANÆMIA IN VIVO

Prolonged hæmolysis with red-cell fragmentation followed the production of acute defibrination in rabbits in which the fibrinolytic system Summary

had been inhibited. Red blood-cells adhered to fibrin strands within the blood and attached to the bloodvessels of these rabbits. It is suggested that hæmolysis and red-blood-cell fragmentation are brought about by the continuing interaction of red blood-cells and fibrin within the circulation, and that a similar process may occur in patients with microangiopathic hæmolytic anæmia. INTRODUCTION

INTRAVASCULAR hxmolysis develops in rabbits during acute defibrination produced by the injection of the purified coagulant fraction of the venom of the Malayan

pit-viper, Agkistrodon rhodostoma, (‘ Arvin ’).1 Although few morphological changes were observed in the red blood-cells and the exact mechanism of haemolysis was uncertain, we thought that the red blood-cell damage was sustained in the initial phase of microthrombus formation. (" Microthrombus is used to describe the aggregates of fibrin and cells which result from venom defibrination; when specific reference is made to small aggregates freely circulating, the term " microclot " is used.) We describe here experiments in which the hxmolysis has been prolonged, and we have produced definite and sometimes striking red-blood-cell fragmentation. "

MATERIALS AND METHODS

Fibrinogen prepared as described by Regoeczi and Walton2 and iodinated at low substitution levels using 1251 monochloride.3 Plasma haemoglobin was determined by the benzidine method.4 59Fe-labelled red blood-cells were obtained from a donor rabbit which received 560 C of lal-elled ferric chloride after repeated venesections to ensure m", nal was

incorporation. White New Zealand rabbits weighing 1-2-1-9 kg. were injected with 125I-fibrinogen and the initial values of circulating radioactivity and plasma hxmoglobin were obtained. Before arvin and at 4-hourly intervals during the duration of the 500 mg. per kg. of e-aminocaproic acid (E.A.C.A.) and 5 mg. per kg. of soya-bean trypsin inhibitor (S.B.T.I.) were injected. Then 3-0-40 g. of arvin per kg. body-weight was

experiment,

injected. One group of animals was rendered thrombocytopenic less than 40,000 per c.mm.) by whole-body irradiation with 525 rad 6-8 days before the study. Another group of animals was anticoagulated by the injection of 5000 l.u. heparin per kg. immediately before an experiment or by the injection of 2 mg. per kg. of warfarin. daily for 4 days. 1. Regoeczi, E., Rubenberg, M. L., Brain, M. C. Lancet, 1967, i, 601.

(platelet-count

2. 3. 4.

Regoeczi, E., Walton, P. L. Thrombos. Diath. hœmorrh. 1967, 17, McFarlane, A. S. J. clin. Invest. 1963, 42, 346. Crosby, W. H., Furth, F. W. Blood, 1956, 11, 380.

237.

1122 the role of platelets and of activation of the clotting system on hxmolysis, experiments were performed in rabbits rendered thrombocytopenic by irradiation and in animals anticoagulated by both warfarin and heparin. These measures enabled the rabbits to withstand larger doses of arvin and more rapid rates of defibrination. Both phases of haemolysis were enhanced and more striking changes in red-cell morphology were provoked. Examination by phase-contrast microscopy of wet preparations of peripheral blood obtained during defibrinaTo

assess

intrinsic

Fig. 1-Ha’moglobinaemia in a rabbit pretreated with E.A.C.A. and S.B.T.I. as measured by benzidine method and by 6BFe activity released from labelled red blood-cells which were injected 140 minutes after arvin. The disappearance of plasma-125Ifibrinogen is also shown.

tion revealed the presence of microclots in which red blood-cells adhered to each other and to the microclots by fibrin strands. Movement of the blood caused distortion of these cells. Similar fibrin aggregates could be seen in stained sections of arteries, arterioles, and venules of the lung and liver. Enmeshed in these microthrombi were fragments of red blood-cells. The presence of fibrin on the surface of red blood-cells has been confirmed by us. DISCUSSION

The 59Fe-labelled red cells were injected at 140 minutes after arvin administration into animals which had already received 125I-fibrinogen. 59Fe and 1251 were measured in a gamma

spectrometer. RESULTS

The injection of arvin into rabbits resulted in a rapid fall in the whole-blood 1251 radioactivity. There was no subsequent rise in non-clottable radioactivity in animals pretreated with E.A.C.A. and S.B.T.I. as lysis of the microthrombi was prevented. As in our previous experiments,1 the appearance of haemoglobin in the plasma coincided with the fall in 1251 radioactivity, but haemoglobinaemia now persisted. In many instances a second peak of haemoglobinaemia occurred 3-5 hours after defibrination had been achieved, and at a time when no clottable fibrinogen could be detected (fig. 1). The second peak of hxmoglobinxmia was accompanied by the appearance of fragmented red blood-cells (fig. 2). These abnormal cells were observed for as long as the experiments continued, and were present in several animals 24 hours after the start of the experiment. The injection of 59Fe-labelled red cells 2 hours after complete defibrination was followed by the release of 59Fe haemoglobin into the plasma indicating the persistence of intravascular haemolysis (fig. 1).

injection of arvin there is an early phase of the haemolysis, degree of which is closely related to the rate of defibrination.5 This initial hsemolytic phase is probably due to the firm trapping of red blood-cells in microthrombi which form in the circulation and adhere to the endothelium of the heart and blood-vessels and collect in narrowing portions of the circulation. In normal animals these microthrombi are rapidly lysed and the release of haemoglobin ceases as cells are no longer subjected to further damage. When the fibrinolytic system is blocked by pretreatment of the animal with E.A.C.A. and s.B.T.l., the microthrombi persist. The clot formed by venom is friable and more porous than one formed by thrombin. This is probably due to slower polymerisation during the venom-initiated fibrinogen-fibrin transition.Venom has been demonstrated to have no effect on platelets in vitro. In-vivo haemolysis with the release of red-blood-cell adenosine diphosphate and activation of the intrinsic clotting system by released red-blood-cell thromboplastins would, however, cause platelets to adhere to fibrin strands and change the structure of the venom-formed loose fibrin meshwork. It seems probable that red-blood-cell fragmentation and persistent haemolysis were produced by the continuing direct interaction between circulating red blood-cells and the loose fibrin meshwork within blood-vessels. Under these circumstances physical forces fragment and lyse red blood-cells adherent to fibrin strands. Thrombocytopenia, or failure of activation of platelets by interference with the intrinsic clotting system, prevented attachment of platelets to fibrin strands and subsequent plugging of the fibrin clot. Thus blood was allowed to flow through and around the clots and permitted continued interaction between red blood-cells and fibrin. Interaction of red blood-cells and intravascular fibrin provides an explanation for the occurrence of red-bloodcell fragmentation in the late phase of haemolysis after The same mechanism may also venom defibrination. explain the fragmented red blood-cells seen in microangiopathic haemolytic anaemia in man. Thus fragmented red blood-cells in patients with intravascular coagulation,’7 and accelerated fibrinogen catabolism in microangiopathic After the

5.

Fig. 2-Peripheral-blood film defibrination. x 700.

obtained

5

hours

after

arvin

6. 7.

Rubenberg, M. L., Regoeczi, E., Bull, B. S., Dacie, J. V., Brain, M. C. Br. J. Hœmat. (in the press). Regoeczi, E. Z. Tropenmed. Parasit. 1966, 17, 144. Brain, M. C., Baker, L. R. I., McBride, J. A., Rubenberg, M. L., Dacie, J. V. Br. J. Hœmat. (in the press).

1123

hsemolytic anarmia8 have been observed. The disturbance of platelet function in urmia,9 and the presence of fibrin split products, may contribute to the production of a loose fibrin meshwork 10 which fails to undergo effective platelet occlusion and retraction. Persistence of haemolysis will maintain intravascular coagulation and fibrin deposition through release of red-blood-cell thromboplastins. Peritoneal dialysis with consequent improvement in platelet function and the use of heparin to prevent further intravascular coagulation and fibrin deposition will enable the vicious circle

to

be broken.

We thank Miss Marion Scales for her skilled technical assistance and Twyford Laboratories Ltd. for their generosity in supplying Arvin. Requests for reprints should be addressed to M. C. B.

M. L. M.R.C. Group for the Study of

M.D.

Hæmolytic Mechanisms, Department of Medical School, London W.12 Biophysics Division, National Institute for Medical Research, Mill Hill, London

M.D.

Loma Linda

E. REGOECZI Tuebingen J. V. DACIE

M.D.

M.R.C. Group for the Study of Hæmolytic Mechanisms, Department of Hæmatology, Royal Postgraduate

School, London

Washington

B. S. BULL*

Hæmatology, Royal Postgraduate

Medical

RUBENBERG*†

W.12

Lond., F.R.C.P., F.R.S. M. C. BRAIN D.M. Oxon., M.R.C.P.

M.D.

RED-BLOOD-CELL FRAGMENTATION IN MICROANGIOPATHIC HÆMOLYTIC ANÆMIA: IN-VITRO STUDIES Red-blood-cell

fragments Summary when normal red blood-cells

were

produced forced,

were

at arterial flow velocity, through a loose mesh of fibrin or other synthetic fibres. These fragments were morphologically indistinguishable from those which are found in the microangiopathic hæmolytic anæmia syndrome. Fragmentation resulted when membrane tears occurred in an arrested red blood-cell as a result of buffeting from rapidly flowing cells. The shape of the fragment formed depended on the position in which the red blood-cell was arrested and the site of membrane tears. INTRODUCTION

STRIKING red-blood-cell fragmentation characterises the peripheral blood of some patients with malignant hypertension, uraemia, thrombotic thrombocytopenic purpura, carcinomatosis, polyarteritis, and renal cortical necrosis. Intravascular coagulation has been proposed as an intermediary mechanism in several of these diseases,!1 and similar red-cell fragmentation develops in rabbits after the induction of intravascular coagulation with thrombin or venom. 11 13 Animal work strongly supports the idea that

intravascular fibrin is

one

of the

causes

of red-cell

fragments are only observed when intravascular coagulation results in the persistent

fragmentation,

since these

presence of fibrin in the vascular tree.14 We have set out * Special fellows, U.S. Public Health Service. † Present address: department of hematology, Maimonides Hospital of Brooklyn, New York. 8. Baker, L. R. I., Rubenberg, M. L., Dacie, J. V., Brain, M. C. ibid. 9. Stewart, J. H., Castaldi, P. A. Q. Jl Med. 1967, 36, 409. 10. Bang, N. U., Fletcher, A. P., Alkjaersig, N., Sherry, S. J. clin. Invest. 1962, 41, 935. 11. McKay, D. G. Disseminated Intravascular Coagulation. New York, 1965.

12. 13. 14.

Brain, M. C., Esterly, J. R., Beck, E. A. Br. J. Hœmat. 1967, 13, 868. Regoeczi, E., Rubenberg, M. L., Brain, M. C. Lancet, 1967, i, 601. Rubenberg, M. L., Bull, B. S., Regoeczi, E., Dacie, J. V., Brain, M. C. ibid. p. 1121.

discover whether the interaction of red blood-cells and fibrin in vitro can produce fragmentation. to

MATERIALS AND METHODS

Blood, anticoagulated with sodium citrate, was rendered poor platelets and white blood-cells by centrifugation. A 2-5% suspension of red blood-cells in plasma was used for experiin

requiring visual observation or photography in the slide chamber. For experiments done in the ring circuit a 20-25% suspension of blood was used. Coagulation was induced by recalcifying the citrated blood or by adding 0-1unit amounts of thrombin (Parke Davis Co.) or venom (’Arvin’, Twyford Laboratories Ltd.). ments

Slide Chamber This consisted of a microscope slide with a coverslip and polyethylene catheter cemented onto its surface so that blood could be forced through the space between slide and coverslip. Fibrin was deposited in the chamber by filling it with recalcified plasma. Blood was then introduced into the chamber and the response of the red blood-cells, forced at high speed through the fibrin mesh, was observed and photographed. The quantitation of fragmentation and hxmolysis required the use of larger quantities of blood than could be accommodated in the slide chamber. For these experiments a ring circuit was constructed. Circuit This circuit was formed from a 20-cm. length of siliconerubber tubing, and incorporated a filter chamber containing either glass beads or a perforated stainless-steel disc. Blood was pumped around the circuit by a peristaltic pump at various flow-rates. Fibrin was deposited on the glass beads or on the holes in the disc when thrombin or venom was added to the blood in the ring circuit. Blood in the circuit was forced to perfuse this fibrin during and after deposition. The haemolysis which resulted was determined by measurement of plasma haemoglobin with benzidine. The degree of red-blood-cell fragmentation was expressed as a percentage. The response of red blood-cells to non-fibrin fibres was investigated by the insertion of glass or nylon filter discs into the filter chamber of the ring circuit. Anticoagulated blood was " " pumped through these artificial clots and the haemolysis and fragmentation were measured.

Ring

RESULTS

Slide-chamber Observations

Red blood-cells, which encountered fibrin strands while traversing the observation chamber, were arrested and bent double by the force of the blood-flow, and the buffeting of other red blood-cells. The sequence of events which ensued depended upon the strength of the red-cell membrane, the speed of blood-flow, and whether or not fibrin polymerisation was still happening. If the force of blood-flow was not great or if the red-cell membrane was strong the cell eventually slipped off the obstruction apparently undamaged. When blood velocity was greater and/or the cell membrane was weaker, fragmentation often resulted. Fragmentation or complete haemolysis almost invariably happened when fibrin polymerisation was still occurring. Under these circumstances the red blood-cells became permanently attached to the obstructing strand and were thus subject to continual buffeting from unattached cells in the bloodstream.

The shape of the red-cell fragment formed depended on the position and plane in which the red blood-cell was folded about the fibrin strand, and on the site at which the membrane was torn or broken (fig. 1).

Ring Circuit

Fragmentation and haemolysis also happened when was pumped through a fibrin clot formed on the

blood