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Dilithium Sequestrene As Ananticoagulant

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Dilithium Sequestrene As Ananticoagulant J Clin Pathol: first published as 10.1136/jcp.12.3.254 on 1 May 1959. Downloaded from J. clin. Path. (1959), 12, 254. DILITHIUM SEQUESTRENE AS AN ANTICOAGULANT BY L. S. SACKER,* K. E. SAUNDERS,t BERYL PAGE, AND MARGARET GOODFELLOW From the Departments of Pathology, Dulwich and Lewisham Hospitals, London (RECEIVED FOR PUBLICATION SEPTEMBER 20, 1958) The variety of different anticoagulants used for Ethylene diamine tetra-acetic acid (E.D.T.A.), 29.2 blood samples has increased, but there is still need g., and 7.4 g. of lithium carbonate were intimately for a suitable anticoagulant for the routine mixed. Then 200 ml. of water was added and solution estimation of sodium and potassium by flame took place with vigorous evolution of carbon dioxide. photometry. So far the only satisfactory substance for this purpose has been the expensive calcium Method 2 heparin (King and Wootton, 1956). Disodium sequestrene (Proescher, 1951 ; Hadley E.D.T.A., 29.2 g., was dissolved in 200 ml. of normal and Larson, 1953) and dipotassium sequestrene lithium hydroxide solution (41.96 g. LiOH.H20 per are the most valuable of litre). (Hadley and Weiss, 1955) In both cases the resulting solution contained the the recently introduced anticoagulants for routine dilithium salt. haematological procedures because they preserve Dilithium sequestrene was obtained from these morphology of leucocytes for short periods the solutions after filtration to remove a small quantity copyright. better than the ammonium and potassium oxalate of amorphous debris, by precipitation on the addition mixture or heparin, and they prevent platelets of an equal volume of absolute methyl alcohol and clumping and preserve them. The disodium cooling. Dilithium sequestrene separated out as a sequestrene is less satisfactory than the dipotassium fine white crystalline substance which was recovered salt because of its lower solubility in blood. by filtration on a buchner funnel and washed four was prepared and its times with 10 ml. of absolute methyl alcohol. The Dilithium sequestrene salt was dried at atmospheric pressure over anhydrous properties as an anticoagulant were investigated. calcium chloride to a constant weight. http://jcp.bmj.com/ It was found to have the properties of dipotassium The usual yield by either of these methods was sequestrene in routine haematological work but 29 g., namely, 85% of the theoretical yield. could also be used in many biochemical procedures, including flame photometry. This salt had previously been investigated (Hadley and Physical Properties Weiss, 1955) and found to be unsatisfactory The substance produced by either method was because of its low solubility. identical. It was a white micro-crystalline solid on September 25, 2021 by guest. Protected which had a solubility in water of 16 g. per 100 Preparation ml. at 18° C. and was freely soluble in 10% v/v Dilithium sequestrene can be prepared from methyl alcohol in water but insoluble in absolute sequestric acid by reaction with the theoretical methyl alcohol. The melting point was 300° C. amounts of either lithium carbonate or lithium with decomposition. The theoretical lithium hydroxide. content of the dilithium salt is 4.08 % and the Method 1 estimated lithium content was 4.16% using the CH2N (CH2COOH) CH2COOH EEL flame photometer. This confirms that the + Li2CO3 dilithium salt has been produced. CH2N (CH2COOH) CH2COOH Carbon and hydrogen estimations were per- 292.24 73.89 formed on the dilithium sequestrene and the CH2N (CH2COOH) CH2COOLi results obtained were hydrogen content 5.4% and .2H20 + H20 + C02 carbon content 37.08%. The theoretical values CH2N (CH2COOH) CH2COOLi for dilithium sequestrene with two molecules of 340.14 water of crystallization is hydrogen 5.33 % and *Present address: Kingston Hospital, Hull. tPresent address: Brook Hospital, Woolwich, London, S.E.18. carbon 35.3%. J Clin Pathol: first published as 10.1136/jcp.12.3.254 on 1 May 1959. Downloaded from DILITHIUM SEQUESTRENE AS AN ANTICOAGULANT 255 Distribution as an Anticoagulant blood anticoagulated with either calcium heparin Distribution of dilithium sequestrene from or dilithium sequestrene. Plasma chloride was aqueous solution. was unsatisfactory as the estimated by the method of Schales and Schales deposit was difficult to dissolve in blood. (1941). The most efficient method of distributing Blood urea was estimated by the method of dilithium sequestrene was by placing the Archer and Robb (1925) and plasma bicarbonate appropriate amount of a solution containing 2.1 g. in the volumetric Van Slyke apparatus. per 100 ml. in 10% v/v methyl alcohol in water The electrolyte results obtained from 100 into each universal container. These containers comparable samples of plasma are tabulated in were placed in a hot air oven at 1600 C. for 30 Table I. minutes. This procedure did not affect the TABLE I anticoagulant properties of the salt or in any way COMPARABLE ELECTROLYTE RESULTS lower its solubility in blood or cause its decomposition. All traces of methyl alcohol were Range of Variation (mEq. per Litre) also removed. ±2 ±4 I>±4 Mean The optimal amount of dilithium sequestrene i4 >i4 Error Sodium 75 25 0 +0 04 for routine haematological and chemical estima- Chloride 84 15 1 + 1 tions was found to be 0.2 ml. of this solution for Bicarbonate 99 1 0 +0-08 5 ml. of blood, i.e., 0.84 mg. per ml. The minimum mEq. per litre amount to prevent coagulation is mg. per 0.52 ml. ±0-2 ±04 >±04 The tolerance for haematological procedures, Potassium 95 4 1 -0-015 which ensures retention of good cell morphology, is between 1.05 mg. and 0.66 mg. per ml. of blood. Of The tolerance for blood urea and electrolytes, the 100 comparative urea estimations, 99 except plasma chloride, is between the minimum were within 10% of the calcium heparin standard copyright. amount necessary to prevent coagulation, i.e., 0.52 and one only was outside this range. The figures mg., and 4.2 mg. per ml. of blood. in this case were 43 mg. per 100 ml. for the calcium heparin and 38 mg. for the sequestrene For chlorides between 0.52 and 1.4 mg. per ml. estimation. is satisfactory. Sequestrene plasma was also found to be Dipotassium and disodium sequestrene have equally effective for the estimations of bilirubin, been distributed into universal containers using the cholesterol, and the phosphatases. http://jcp.bmj.com/ method previously described. The solubility of Blood sugar estimations performed by either the these salts in blood was as good as the solubility Hagedorn and Jensen or Folin and Wu methods of dilithium salt under similar conditions. were unsatisfactory when compared with blood Comparative chemical estimations were made containing fluoride, as there was a rapid loss of on samples of the same blood taken into dilithium reducing substance. It was, however, noted that sequestrene and calcium heparin. the loss in the first one and a half hours after Comparative haematological investigations were collection was about 5 %, so that estimations on September 25, 2021 by guest. Protected made on samples of the same blood taken into performed on blood within this time or when dilithium sequestrene, dipotassium sequestrene, fluoride was added within this time were satis- and the ammonium and potassium oxalate factory. mixture. Haematological Investigations.-In the inves- Results tigation of dilithium sequestrene as an Chemical Investigations.-The wavelength of anticoagulant for haematological investigations a the emission spectrum for lithium is 6708 A, for comparison was made between samples of the sodium 5890 A and 5896 A, and for potassium same blood taken into the ammonium and 7655 A. Using the EEL flame photometer with a potassium oxalate mixture, which were estimated coal gas/air flame no interference was detected after contact with the anticoagulant for one hour, due to lithium on the intensity of the sodium or and dipotassium sequestrene and dilithium potassium emission spectra, even when using 10 sequestrene, which were estimated after one and times the amount of lithium sequestrene normally four hours' contact with the anticoagulant. present. On each sample of blood taken in the Sodium and potassium were estimated, using the ammonium and potassium oxalate mixture after flame photometer, on plasma from samples of one hour and each sample in dipotassium or J Clin Pathol: first published as 10.1136/jcp.12.3.254 on 1 May 1959. Downloaded from 256 L. S. SACKER AND OTHERS dilithium sequestrene after one and four hours Platelet counts on sequestrene blood were the following investigations were performed: satisfactory. All duplicate platelet counts varied (1) Haemoglobin using the oxyhaemoglobin within a range of + 10%, which is satisfactory. method reading in a photo-electric colorimeter It was noted occasionally that the platelets using an Ilford 625 filter, (2) blood sedimentation clumped in dilithium sequestrene if the anti- rate (Wintrobe method); (3) packed cell volume coagulant had not been heated correctly at 1600 C. using an M.S.E. haematocrit centrifuge governed for 30 minutes. to 3,000 r.p.m. for 30 minutes; (4) platelet count (5) White Cell Count.-The maximum variation using direct counting in blood diluted either 1 in noted in wet counts was well within 20% limits 20 in Barr's fluid or 1 in 100 in formol citrate (Dacie, 1956). (Dacie, 1956); (5) total white cell count ; (6) blood (6) Blood Films.-(a) No significant difference films had differential white cell counts performed noted and were also examined for red cell morphology, in the differential white cell counts was platelet clumping and morphology, and the effect between the ammonium and potassium oxalate on leucocyte morphology. anticoagulant after one hour and dipotassium and of the anticoagulants dilithium sequestrene after one and four hours' Increased red cell destruction after contact with contact time.
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