1281 The results show little significant change between 1969 and 1977 in the relative calibration constant of 67/40, 68/434, and NL/73/528. Some prolongation of the clotting-time is observed, but different test conditions (pH, concentration, and ionic strength of citrate solution) were used during the eightyear period. The test conditions now used in the Dutch reference laboratory give longer clotting-times (2-5% longer with 67/40, 4-7% longer with 68/434 and NL/73/528) compared with non-buffered samples.3 Differences in test conditions (especially molarity of citrate) unquestionably lead to different prothrombin-timesbut differences of pH and ionic strength of the test solutions do not influence the calibration constant;3 these are points of prime importance in the assessment of reference
preparations of thromboplastins.
These results show that the estimates of stability determined after real-time storage at -20 ° C accord closely with the predictions based on accelerated (thermal) degradation studies. The two reference thromboplastins 67/40 and 68/434 were freeze dried and ampouled using the specific conditions recommended for the preparation of international biological standards4 and have since been stored at -20°C in the dark. One of them (67/40) is the W.H.O. International Reference Preparation of thromboplastin (Human, Combined).s Similar stability studies should be done with other reference thromboplastins-e.g., "plain" thromboplastin (containing no added factors) prepared from human brain (69/233) and from rabbit brain (70/178), ampouled and stored under the same conditions. On a priori grounds one may expect their long-term stability to be not much different from that of 67/40 and 68/434. The stability of a biological standard may be investigated6 by reference to its activity in a control population, by direct comparison with other similar preparations, or by accelerated degradation. The first method (and to some extent the second) assumes that the test system remains stable so that any change may be attributed to a change in the material. Poller et al. use these methods without demonstrating the validity of this assumption. Furthermore, they give levels of statistical significance which seem to be based on comparisons of results obtained over periods of time chosen on the grounds that they support the claim that 67/40 and 69/223 are unstable. Such a procedure inevitably leads to a falsely high level of significance. Indeed, the intrinsic variability of their data is quite large enough to explain the apparent trends that they take as evidence of instability. Although thromboplastins are ex-
tremely heterogeneous in composition, biological materials, particularly in the freeze-dried state, do not show the kind of variability of behaviour described by Poller et al. Unless contrary data are available, a more likely explanation of these results is that they relate to changes in reagents, procedure, equipment, or staff. We consider that the international reference preparation is stable enough for its purpose, and certainly no less stable than any other reference thromboplastin we know of. Many different types of thromboplastins are manufactured in the world and every hmmatologist has his preference; what is urgently needed is a scheme to relate test results obtained with such diverse types to a single scale. The system for control of oral anticoagulation treatment recommended in the 28th report of the W.H.O. Expert Committee on Biological Standardisation could
at
,
S!R,—Iwas astonished to see Dr Poller and his colleagues questioning the stability of the international reference preparation of thromboplastin (67/40). The W.H.O. took a great deal of advice on the suitability of this preparation before it was established as an international reference preparation, and there had been a number of contacts with Dr Poller informing him of the intended establishment of this preparation as well as keeping him informed of its subsequent establishment. I was surprised, therefore, to see that he referred to the preparation as being "considered" as an international reference preparation when it was established over a year ago.
F. T. PERKINS, Chief, Biologicals
World Health Organisation, 1211 Geneva 27, Switzerland
HYPOLIPIDÆMIC AGENTS: WHAT ARE THEY GOOD FOR?
p. 808) and othersl-5 have pointed there is a growing awareness of the inverse relationship between high-density lipoproteins (H.D.L.) and coronary heartdisease (C.H.D.). On the other hand, the role of lipids in the pathogenesis of C.H.D. has been strongly opposed,6,7 mainly because of the discouraging results of hypolipidwmic drug interventions in improving life-expectancy8,9 despite reductions in plasma-lipid concentrations. Our own findings prompt us to ask-Are we justified in looking for improvements in lifeexpectancy when we use hypolipidsemic drugs the way we do? We gave clofibrate to 52 hyperlipidaemic patients (25 type IIA, 20 HB, and 7 type iv) over a 6-month period at a daily dose of 1500 mg. Before clofibrate the patients had plasma-cholesterols above 7.7mmol/1, triglycerides above 2.0 mmol/1, or both. Lipids were measured twice before clofibrate and every 4 weeks during therapy. Lipoproteins were analysed by agarose-gel electrophoresis using sudan-black staining. Beta, prebeta, and alpha lipoproteins were considered to be equivalent
SiR,-As you (Oct. 15,
out
1. Barr, D. P., Russ, E. M., Eder, H. A. Am. J. Med. 1951, 11, 480. 2. Nikkilä, E. Scand. J. clin. Lab. Invest. 1953,5, suppl. 8. 3. Lancet. 1976, ii, 131. 4. Gordon, T., Castelli, W. P., Hjortland, M. C., Kannel, W.
B., Dawber, T. R. Am. J. Med. 1977, 62, 707. 5. Miller, N. E., Førde, O. H., Thelle, D. S., Mjøs, O. D. Lancet, 1977, i, 965. 6. McMichael, J. Eur. J. Cardiol. 1977, 5/6, 447. 7. Mann, G. C. New Engl. J. Med. 1977, 297, 644. 8. Oliver, M. F. Br. med. J. 1971, iv, 775. 9. Coronary Drug Project Research Group. J. Am. med. Ass. 1975, 231, 360.
last effect this and deserves every support.
National Institute for Biological Standards and Control, London NW3 6RB
D. R. BANGHAM
National Laboratory for
Anticoagulant Control, University Hospital, Leiden, Netherlands
E. A. LOELIGER
Plasma-lipoprotein concentrations in 2S type-IIA hyperlipidaemic patients before and after clofibrate therapy for 6 months. 4. W.H.O./B.S./77.1142. Unpublished. 5. W.H.O. Expert Committee on Biological Standardisation Tech. Rep. Ser. Wld Hlth Org. 1977, no. 610. 6. Jerne, N. K., Perry, W. L. M. Bull. Wld Hlth Org., 1956, 14, 165.
The mean pre and post treatment values for the beta lipoproteins ’ 5-64 and 4-53 g/l (p
preparations of thromboplastins.
These results show that the estimates of stability determined after real-time storage at -20 ° C accord closely with the predictions based on accelerated (thermal) degradation studies. The two reference thromboplastins 67/40 and 68/434 were freeze dried and ampouled using the specific conditions recommended for the preparation of international biological standards4 and have since been stored at -20°C in the dark. One of them (67/40) is the W.H.O. International Reference Preparation of thromboplastin (Human, Combined).s Similar stability studies should be done with other reference thromboplastins-e.g., "plain" thromboplastin (containing no added factors) prepared from human brain (69/233) and from rabbit brain (70/178), ampouled and stored under the same conditions. On a priori grounds one may expect their long-term stability to be not much different from that of 67/40 and 68/434. The stability of a biological standard may be investigated6 by reference to its activity in a control population, by direct comparison with other similar preparations, or by accelerated degradation. The first method (and to some extent the second) assumes that the test system remains stable so that any change may be attributed to a change in the material. Poller et al. use these methods without demonstrating the validity of this assumption. Furthermore, they give levels of statistical significance which seem to be based on comparisons of results obtained over periods of time chosen on the grounds that they support the claim that 67/40 and 69/223 are unstable. Such a procedure inevitably leads to a falsely high level of significance. Indeed, the intrinsic variability of their data is quite large enough to explain the apparent trends that they take as evidence of instability. Although thromboplastins are ex-
tremely heterogeneous in composition, biological materials, particularly in the freeze-dried state, do not show the kind of variability of behaviour described by Poller et al. Unless contrary data are available, a more likely explanation of these results is that they relate to changes in reagents, procedure, equipment, or staff. We consider that the international reference preparation is stable enough for its purpose, and certainly no less stable than any other reference thromboplastin we know of. Many different types of thromboplastins are manufactured in the world and every hmmatologist has his preference; what is urgently needed is a scheme to relate test results obtained with such diverse types to a single scale. The system for control of oral anticoagulation treatment recommended in the 28th report of the W.H.O. Expert Committee on Biological Standardisation could
at
,
S!R,—Iwas astonished to see Dr Poller and his colleagues questioning the stability of the international reference preparation of thromboplastin (67/40). The W.H.O. took a great deal of advice on the suitability of this preparation before it was established as an international reference preparation, and there had been a number of contacts with Dr Poller informing him of the intended establishment of this preparation as well as keeping him informed of its subsequent establishment. I was surprised, therefore, to see that he referred to the preparation as being "considered" as an international reference preparation when it was established over a year ago.
F. T. PERKINS, Chief, Biologicals
World Health Organisation, 1211 Geneva 27, Switzerland
HYPOLIPIDÆMIC AGENTS: WHAT ARE THEY GOOD FOR?
p. 808) and othersl-5 have pointed there is a growing awareness of the inverse relationship between high-density lipoproteins (H.D.L.) and coronary heartdisease (C.H.D.). On the other hand, the role of lipids in the pathogenesis of C.H.D. has been strongly opposed,6,7 mainly because of the discouraging results of hypolipidwmic drug interventions in improving life-expectancy8,9 despite reductions in plasma-lipid concentrations. Our own findings prompt us to ask-Are we justified in looking for improvements in lifeexpectancy when we use hypolipidsemic drugs the way we do? We gave clofibrate to 52 hyperlipidaemic patients (25 type IIA, 20 HB, and 7 type iv) over a 6-month period at a daily dose of 1500 mg. Before clofibrate the patients had plasma-cholesterols above 7.7mmol/1, triglycerides above 2.0 mmol/1, or both. Lipids were measured twice before clofibrate and every 4 weeks during therapy. Lipoproteins were analysed by agarose-gel electrophoresis using sudan-black staining. Beta, prebeta, and alpha lipoproteins were considered to be equivalent
SiR,-As you (Oct. 15,
out
1. Barr, D. P., Russ, E. M., Eder, H. A. Am. J. Med. 1951, 11, 480. 2. Nikkilä, E. Scand. J. clin. Lab. Invest. 1953,5, suppl. 8. 3. Lancet. 1976, ii, 131. 4. Gordon, T., Castelli, W. P., Hjortland, M. C., Kannel, W.
B., Dawber, T. R. Am. J. Med. 1977, 62, 707. 5. Miller, N. E., Førde, O. H., Thelle, D. S., Mjøs, O. D. Lancet, 1977, i, 965. 6. McMichael, J. Eur. J. Cardiol. 1977, 5/6, 447. 7. Mann, G. C. New Engl. J. Med. 1977, 297, 644. 8. Oliver, M. F. Br. med. J. 1971, iv, 775. 9. Coronary Drug Project Research Group. J. Am. med. Ass. 1975, 231, 360.
last effect this and deserves every support.
National Institute for Biological Standards and Control, London NW3 6RB
D. R. BANGHAM
National Laboratory for
Anticoagulant Control, University Hospital, Leiden, Netherlands
E. A. LOELIGER
Plasma-lipoprotein concentrations in 2S type-IIA hyperlipidaemic patients before and after clofibrate therapy for 6 months. 4. W.H.O./B.S./77.1142. Unpublished. 5. W.H.O. Expert Committee on Biological Standardisation Tech. Rep. Ser. Wld Hlth Org. 1977, no. 610. 6. Jerne, N. K., Perry, W. L. M. Bull. Wld Hlth Org., 1956, 14, 165.
The mean pre and post treatment values for the beta lipoproteins ’ 5-64 and 4-53 g/l (p
