Br Heart J 1992;68:449-53 449 Streptokinase resistance: when might streptokinase administration be ineffective? Maurice B Buchalter, Ganesh Suntharalingam, Ian Jennings, Catherine Hart, Roger J Luddington, Ronjon Chakraverty, S Kim Jacobson, Peter L Weissberg, Trevor P Baglin Abstract patients after 24 months. Retreatment Objective-(a) To develop an assay for with streptokinase is likely to be sub- streptokinase resistance. (b) To deter- optimal even after 24 months. The fibrin mine the prevalence of streptokinase plate lysis assay detects resistance in resistance in patients presenting with patients with normal concentrations of acute myocardial infarction for the first streptokinase antibodies. Streptococcal time. (c) To determine the prevalence of infection is associated with a high streptokinase resistance in patients after incidence of streptokinase resistance. exposure to streptokinase or strepto- coccal infection. (Br Heart J 1992;68:449-53) Design-Open, prospective. Patients-30 healthy volunteers. 40 Thrombolysis has become an essential com- patients admitted to the coronary care ponent of the management of an acute unit at Addenbrooke's Hospital with sus- myocardial infarction. Large controlled trials pected acute myocardial infarction, 12 have shown that streptokinase compares patients 12 months after streptokinase favourably with other fibrinolytic agents in treatment, eight patients 24 months terms of efficacy, side effects, and cost.'-5 It is after streptokinase treatment, and sera therefore likely to remain the thrombolytic from 12 patients with raised anti- agent of choice in the United Kingdom for the streptolysin 0 (ASO) titres. foreseeable future. Because of its antigenic Methods-Three assays were used; a nature, however, the presence of neutralising dilution neutralisation a,ssay, an enzyme antibodies in some patients may reduce its linked immunosorbent assay (ELISA) effectiveness. Rapid development of immuno- for immunoglobulin G (IgG) anti- globulin G (IgG) antibodies to streptokinase streptokinase antibodies, and an in vitro has been reported,6 and antibodies have been fibrin plate lysis assay. All measurements detected up to eight months after treatment.7 were performed on venous blood Current recommendations advise against samples. retreatment with streptokinase within six Results-Neutralisation and IgG months and immediately after a streptococcal antibody titres were positively infection.89 The prevalence of antistrepto- correlated. Mean (SEM) Clinical antistrepto- kinase antibodies in the general population Pharmacology Unit, kinase concentrations in the 30 controls and in patients presenting to the coronary care School of Clinical were 87 (10) U/ml (neutralisation assay) unit for the first time is unknown, and their Medicine, University and 28 (6 3) U/ml (ELISA). Correspond- to neutralise a standard of Cambridge potential dose of M B Buchalter ing concentrations in patients before streptokinase has not been rigorously G Suntharalingam streptokinase were 68 (6-1) U/ml and 18 evaluated. R Chakraverty (4 5) U/ml with a mean fibrin plate assay To test whether the of anti- P L Weissberg development 117 (7-1)% that of controls. Resistance to bodies is associated with significant strepto- Clinical Microbiology streptokinase was detectable in and Public Health one kinase resistance we have developed an in Laboratory, patient after 72 hours and in all patients vitro fibrin plate lysis assay and correlated this Addenbrooke's by day 10. By day 10 concentrations were with both functional and immunological Hospital, Cambridge 4388 (919) 773 (109) and 17 concentrations of S K Jacobson U/ml, U/ml, streptokinase antibodies. A (5 4)%. At both 12 and 24 months resis- normal range for neutralising activity was Department of Haematology, tance was present in 75% of patients. established in a group of normal subjects. Addenbrooke's Similarly 66% of high ASO titre sera Patients admitted to a coronary care unit with Hospital, Cambridge showed resistance. The fibrin plate lysis suspected myocardial infarction were inves- I Jennings detected R J Luddington assay significantly reduced tigated on arrival and, to determine how soon T P Baglin streptokinase dependent fibrinolysis in and for how long neutralising activity was C Hart vitro in the absence of raised total con- present, blood samples were taken early (three Corrcspondencc to centrations of antistreptokinase anti- to ten days) and late (12 and 24 months) after M B Buchalter, Dcpartrnent of Clinical bodies. streptokinase treatment. We also tested sera Pharmacology, Conclusions-The prevalence of strep- from patients with raised antistreptolysin 0 F and G Block, Addenbrookcs Hospital, tokinase resistance in patients present- (ASO) titres for streptokinase neutralising Cambridgc CB2 2QQ. ing with their first myocardial infarction activity to confirm whether recent strepto- Acccpted for publication is low. Resistance develops early after coccal infection precluded administration of 21 April 1992 treatment and is still present in 75% of streptokinase. 450 Buchalter, Suntharalingam, Jennings, Hart, Luddington, Chakraverty, Jacobson, Weissberg, Baglin Patients and methods lyse the clot was used to calculate the strepto- PATIENTS AND CONTROLS kinase neutralising titre. Patient samples were Over a six week period, venous blood samples diluted in normal plasma until clot lysis was were taken before administration of strepto- achieved. The neutralising activity (U/ml) was kinase, from 40 patients admitted to the calculated as:- highest concentration of strep- coronary care unit of Addenbrooke's Hospital tokinase failing to lyse clot x dilution of test for the first time with a suspected acute myo- plasma in normal plasma/10. A concentration cardial infarction. Further samples were taken > 2 SDs above the mean ofthe normal controls on days three to four and on the day of was defined as raised. discharge (days seven to 10) from 15 patients who had received 1-5 million units of intra- FIBRIN PLATE LYSIS ASSAY venous streptokinase (Streptase, Hoechst). Fibrinogen (Diagen) was dissolved in None of the patients had previously received imidazole buffer to a concentration of650 mg/l. streptokinase. Venous blood was also taken Fibrin plates were prepared by clotting 10 ml from (a) 12 patients who had received strepto- fibrinogen solution with 10 U bovine thrombin kinase 12 months previously, (b) eight patients (Diagen) and 25 mM calcium chloride in petri who had received streptokinase 24 months dishes (Sterilin). Streptokinase was added to previously, (c) 12 patients with raised anti- the plasma samples to achieve a concentration streptolysin 0 titres, (d) 30 healthy normal of 625 U/ml. This was the calculated plasma controls with no documented history of strep- concentrations of streptokinase that would tococcal infection. All patients gave informed result from a dose of 1-5 million units to a 70 kg verbal consent, and the study was approved by subject with a packed cell volume of 45%. the local ethics committee. Twenty five p1 of each plasma sample were placed on a fibrin plate and incubated at 37°C MEASUREMENT OF STREPTOCOCCAL ANTIBODIES for 24 hours. The area of lysis was calculated as Immunoglobulin G antibodies to streptokinase the product of two diameters at 900 to each were measured by an enzyme linked immuno- other. The mean area of lysis of the 30 normal sorbent assay (ELISA) incorporating solid controls was 1394 mm2. All areas oflysis caused phase streptokinase. Neutralising antibodies by patients plasma with added streptokinase were measured by a dilutional clot lysis assay. were then reported as percentages of this value. (a) ELISA assay: 100 p1 of 1000 U/ml A reduction in streptokinase dependent in vitro streptokinase (Streptase, Hoechst) was added fibrinolysis >2 SDs below the mean normal to the wells of a microtitre plate (Nunc, activity was regarded as abnormal. Denmark). Plates were sealed, incubated over- night at room temperature, and washed three ASO TITRES times in 0-01 mol/l phosphate buffered saline Titres of ASO were measured by a standard (PBS) and 0-5 ml/l Tween. One hundred p1 of sheep red cell haemolysin assay (Wellcome plasma, diluted one in 40 with phosphate Diagnostics). buffered saline (PBS)/0- 1% bovine serum albumin, was then added to each well and STATISTICAL ANALYSIS incubated for two hours at room temperature. Comparisons between study groups and the After three washes the wells were incubated normal controls were performed with the Wil- with 100 p1 peroxidase-conjugated rabbit anti- coxon's ranking test for unpaired data (Mann- human IgG (Dako) diluted 1/5000 in PBS/ Whitney test). The same test was used for Tween for two hours at room temperature. comparisons in the group tested on several days After washing, 100 p1 substrate solution (3 mg/ as the numbers tested on each day varied. ml orthophenylenediamine in 0-01 mol/l citrate phosphate buffer, with 0 01% hydrogen perox- ide was added and the reaction stopped by the Results addition of 150 p1 sulphuric acid. Absorbance The mean age of the 40 patients (28 men) at 492 nm was read with a Titertek Multiscan admitted to the coronary care unit was 58 plate reader. A standard curve was derived (range 38-71) years. All patients received 1-5 from doubling dilutions ofnormal plasma from million units of streptokinase by a one hour 1/20, assigned 100 U/ml and patient samples intravenous infusion. None of the patients were sufficiently diluted to enable a concentra- suffered an allergic reaction. Venous blood tion of antibody to be found from the standard samples were taken from 15 patients on day 3 curve. The concentration of antibody in the and on the day of discharge (days 7/8 in seven patient sample was then multiplied by the patients and days 9/10 in eight patients). appropriate dilution factor. A concentration The ELISA assay and the neutralisation titre > 2 SDs above the mean of the normal controls assay produced comparable results and were was defined as raised. positively correlated (r = 0-55; p < 0-001). (b) Neutralisation assay: streptokinase Overall neither assay was correlated with the neutralising activity was measured by a method fibrin plate lysis assay.