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Factors that Influence the Measurement of Unfractionated and Low Molecular Weight Heparin with the APTT Test

By Roy Speck.

Abstract: This study demonstrates that the APTT test has sensitivity sufficient to measure the anticoagulant activity of both unfractionated and LMW heparin, if an activator which targets the anti-Xa function of the plasma and a low phospholipid concentration is used. Only platelet depleted plasma should be used as a sample. Platelet poor plasma contains residual platelets which contribute phospholipid and platelet factor 4 to the reaction. This results in unacceptable loss of sensitivity.

Introduction: Heparin is a mixture of sulfated polysaccharides whose structure is not precisely known. The molecular weight ranges from 3,000 to 35,000. It is prepared by a complicated process from tissue. The main commercial sources are bovine lung and porcine intestinal mucosa. The anticoagulant activity depends on its ability to bind with antithrombin III (ATIII). Unfractionated heparin has two fractions, an active and inactive fraction. The active fraction consists of 1/3 of the total (1). The remainder is essentially inactive. The active fraction must not only possess a binding site for AT III, but also a polysaccharide chain of at least 15 residues. The ratio for anti Xa to anti IIa activity is approximately 1.0. Preparations of LMW heparin are fragments of standard heparin produced by enzymatic or chemical depolymerization. LMW heparin have average molecular weights ranging from 4,000 to 6,000. The chief difference between standard heparin and LMW heparin is the inhibitory effect on Xa and thrombin (IIa). Standard heparin binds both AT III and IIa forming a ternary complex. This can only occur if there is a polysaccharide chain with at least 15 residues (2). Only a small fraction of LMW heparin binds to both Xa and IIa; most of the molecules lack a chain of sufficient length. LMW heparin preferentially inhibits Xa. The inhibition ratio for Xa to IIa is about 4 for most LMW heparin preparations (3). LMW heparin can inactivate platelet bound Xa and resists inhibition by platelet factor 4 (PF4). Heparin induced antibodies are less common with LMW heparin. The plasma half-life for LMW heparin is 2 - 4 times longer than that for standard heparin when measured as inhibiting Xa.

The APTT has been the most commonly used to monitor heparin activity. Shapiro et al (4) found variations among commercial reagents. Stevenson et al (5) studied the reliability of the APTT methods relationship to lipid composition and ultra-structure. D'Angelo et al (6) studied the effect of different clot detection methods for the APTT test when monitoring heparin activity. Shojania et al (7) found variations in heparin sensitivity with different lots of APTT reagents from the same manufacturer. Ofosu et al (8) reported that platelets, brain phospholipid and tissue factor inhibited heparin activity. I performed a new study in which I considered the factors which influence the measurement of anti-coagulant activity of unfractionated and LMW heparin in the APTT test. These factors are: l) Sample preparation. 2) Concentration of phospholipid. 3) Type of activator.

Study Plan:

1. Determine response of different commercial APTT reagents to unfractionated heparin, 6,000 MW heparin, and 3,000 MW heparin using platelet depleted plasma (PDP) samples.
2. Determine the effect of different concentrations of phospholipid have on the APTT sensitivity in measuring LMW heparin.
3. Determine the effect of different intrinsic pathway activators on the sensitivity of APTT sensitivity in measuring unfractionated heparin activity.

Materials.

1. Micronized silica. Sigma Chemical Co. St. Louis, Mo.

2. Celite. Sigma Chemical Co. St. Louis, Mo.

3. Kaolin. Sigma Chemical Co. St. Louis, Mo.

4. Propyl gallate. Sigma Chemical Co. St. Louis, Mo.

5. Ellagic acid. Sigma Chemical Co. St. Louis, Mo.

6. Purified phosphatidylethanolamine. American Lecithin Co. Danbury, Ct.

7. HEPES buffer, 0.02 M, pH 7.4. Research Organic Cleveland, Ohio

8. Unfractionated heparin, porcine intestinal mucosa, sodium salt. Sigma Chemical Co. St. Louis, Mo.

9. Low molecular weight heparin, MW = 6,000, porcine intestinal mucosa. Sigma Chemical Co. St. Louis, Mo.

10. Low molecular weight heparin, MW = 3,000, porcine intestinal mucosa. Sigma Chemical Co. St. Louis, Mo.

11. 0.02 M calcium chloride. Analytical Control Systems, Inc. Fishers, IN.

12. ACS Activated partial thromboplastin time reagent, propyl gallate activator (9). Analytical Control Systems, Inc. Fishers, IN.

13. Actin-FSL. Dade International. Miami, FL.

14. APTT-LS. Organon Teknika. Durham, NC.

15. Disposable membrane filter, 0.2 um, Product no. 4187, Gelman Sciences, Ann Arbor, MI.

16. BD Vacutainer tube no. 366415, 0.105 M buffered sodium citrate. Becton Dickinson Franklin Lakes, NJ.

17. Automatic pipette with 100 ul tips.

18. Fibrometer coagulation instrument. BBL Fibrosystems. Franklin Lakes, NJ.

Methods.

1. Preparation of micronized silica activator. Add 500 mg micronized silica to one liter HEPES buffer and suspend particles by vigorous shaking.
2. Preparation of celite activator. Add 500 mg celite to one liter HEPES buffer and suspend particles by vigorous shaking.
3. Preparation of kaolin activator. Add 500 mg kaolin to one liter HEPES buffer and suspend particles by vigorous shaking.
4. Preparation of ellagic acid activator (9). Prepare according to the procedure outlined in U. S. patent no. 3,486,981.
5. Preparation of propyl gallate activator. Prepare according to U. S. patent no. 5,451,509 (10).
6. Preparation of micronized silica APTT reagent. Add 50 mg phosphatidylethanolamine and 500 ml micronized silica activator to a Waring blender. Blend at full speed for three minutes. Store at 2-8⁰C. for up to one week.
7. Preparation of celite APTT reagent. Add 50 mg phosphatidylethanolamine and 500 ml celite activator to a Waring blender. Blend at full speed for three minutes.Store at 2-8^°C. for up to one week.
8. Preparation of kaolin APTT reagent. Add 50 mg phosphatidylethanolamine and 500 ml kaolin activator to a Waring blender. Blend at full speed for three minutes. Store at 2-8^°C. for up to one week.
9. Preparation of ellagic acid APTT reagent. Add 50 mg phosphatidylethanolamine and 500 ml ellagic acid activator to Waring blender. Blend at full speed for three minutes. Store at 2-5^oC. for up to one week.
10. Preparation of propyl gallate APTT reagent. Add 50 mg phosphatidylethanolamine and 500 ml propyl gallate activator to a Waring blender Blend at full speed for three minutes. Store at 2-8° C. for up to one week.
11. Preparation of platelet poor plasma (PPP). Draw venous blood without hemostasis using vacutainer TM tubes no. 366415. Discard first tube to eliminate tissue thromboplastin contamination. Centrifuge for 20 minutes at 3,000 rcf. Discard any tubes showing hemolysis. Transfer plasma using plastic transfer pipettes to plastic tubes. Store at room temperature. Do not freeze. Pool plasma from at least five donors.
12. Preparation of platelet depleted plasma (PDP). Filter PPP using a 0.2 um membrane filter without delay into plastic tube. Store at room temperature.

Results:

1. See Table I for response of different commercial APTT reagents to unfractionated heparin, 6,000 MW heparin, and 3,000 MW heparin using PDP samples. Table I also shows the difference between PPP and PDP samples when the APTT is used to measure heparin anticoagulant activity.
2. See Table II for the effect of different concentrations of phospholipid has on the APTT sensitivity in measuring LMW heparin.
3. See Table III for the effect of different intrinsic pathway activators on the sensitivity of APTT test measuring unfractionated heparin activity.

Discussion:

Table I shows the variation in heparin sensitivity when commercial APTT reagents are used. This is mainly due to a combination of factors. These factors are improper sample preparation, various levels of platelet substitute and difference in the sensitivity of the activator used. It must also be remembered that in the case of liquid APTT reagents, there is a loss of phospholipid during the shelf life of the product. This results in a change of sensitivity for the reagent. APTT reagents which mainly target the antithrombotic function of heparin are unsuitable for measuring the anticoagulant activity of LMW heparin because of its low antithrombotic activity. Table II demonstrates that for maximum sensitivity in measuring heparin, a low concentration of phospholipid is necessary. Table III indicates that the choice of activator is important. In fact, kaolin and celite are so poor their use should be excluded from APTT reagents.

In review, in order to have the maximum sensitivity for an APTT reagent, it is necessary to properly prepare the sample, use a reagent which contains a low phospholipid concentration and an activator which targets the anti-Xa function of the patient's plasma, rather than the anti-IIa function. If these principles are carried out, the APTT reagent can be used to measure the anticoagulant activity of both unfractionated heparin and LMW heparin.

References.

1. Barrowcliffe TW, Mulloy B, Johnson EA, Thomas DP The anticoagulant activity of heparin: Measurement and relationship to chemical structure. J Pharm and Biomed Analysis 1989; 7:217-26.
2. Verstraete M, Pharmacotherapeutic aspects of unfractionated and low molecular weight heparins. Drugs 1990; 40:498-530.
3. Schafer AT, Low molecular weight heparin--An opportunity for home treatment of venous thrombosis. NEJM 1996; 334:724-725.
4. Shapiro GA, Huntzinger SW, Wilson JF Variations among commercial activated partial thromboplastin reagents in response to heparin. Am J Clin Pathol 1977; 67:477-80.
5. Stevenson KJ, Easton AC, Curry A, Thomson JM, Poller L
6. D'Angelo A, Seveso MP, Gilardoni F, Dettori AG, Bonini P Effect of clot detection methods and reagents on activated partial thromboplastin time (APTT). Implications in heparin monitoring by APTT. Am J Clin Pathol 1990; 94:297-306.
7. Shojania AM, Tetreault J, Turnbull G The variations between heparin sensitivity of different lots of activated partial thromboplastin time reagents produced by the same manufacturer. Am J Clin Pathol 1988; 89:19-23.
8. Ofusu FA, Cerhus AL, Hirsch J, Smith LM, Modi GI, Blajchman MA The inhibition of the anticoagulant activity of heparin by platelets, brain phospholipids, and tissue factor. Br J Haematol 1984; 57:229-238.
9. Speck RE Substance relating to blood coagulation. U.S. Patent no. 3,486,981. Issued Dec. 30, 1969.
10. Speck RE Coagulation assays and reagents. U.S. Patent no. 5,451,509. Issued Sep. 19, 1995.

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