Heparin Assay and Protamine Titration

Heparin Assay and Protamine Titration GUILLERMO ANIDO, M.D. AND DONNA JEAN FREEMAN, MT, H(ASCP) Anido, Guillermo and Freeman, Donna Jean: Heparin assay and protamine titration. Am J Clin Pathol 76: 410-415,1981. By utilizing the fact that heparin and protamine sulfate directly neutralize each other, it is possible to quickly detect excess levels of protamine (anti-heparin activity) by back-titrating a plasma specimen with predetermined amounts of heparin. The approach suggested allows for the simultaneous deflnitiori of low levels of heparin, and incorporates an equally rapid and accurate measurement of high heparin concentrations. The methodology presented employs heparin assays using the "Protopath"* technic. The concept appears to be applicable to other test systems currently designed to monitor heparin activity. (Key words: Protopath; Heparin; Protamine; Thrombin; Synthetic substrates; Antithrombin III.) THE MAJORITY of requests received by this laboratory for heparin assays are generated by in-house surgical teams performing cardiovascular procedures which require the administration of high levels of heparin, followed post-surgically by neutralizing doses of protamine. Depending upon when the specimen is obtained, three distinct conditions can be encountered: 1) a "highly heparinized" specimen obtained during the course of surgery, 2) a "slightly heparinized" specimen obtained after partial neutralization with protamine, or 3) a "minus heparin" specimen that contains no residual heparin but, instead, excess levels of protamine. Our objective has been to develop an approach which would allow us to respond quickly and accurately to any of these three circumstances. The Protopath heparin assay is based upon the inhibition of thrombin by heparin as measured by using a synthetic fluorescent substrate. 2 The assay includes the following steps, and all reagents are supplied with the test kit: 1) patient plasma is added to diluted pooled normal plasma (Citrated Plasma Reagent = CPR) to eliminate effects of possible variations in levels of Antithrombin III (ATIII), 2) a standard amount of thrombin is added to the plasma dilution and the mixture is incubated for a specific length of time, 3) the mixture is added to a fibrinogen analogue, thrombin-sensitive, synthetic substrate (D-phenylalanine- Received January 16, 1981; received revised manuscript and accepted for publication March 2, 1981. Address reprint requests to Dr. Anido: Department of Laboratory Medicine, Broward General Medical Center, Fort Lauderdale, Florida 33316. * Dade Division, American Hospital Supply Corporation; Miami, Florida. Department of Laboratory Medicine, Broward General Medical Center, Fort Lauderdale, Florida proline-arginine-5-"aminoisophthalic acid dimethyl ester" = "AIE"), and 4) the fluorescence of AIE released by the action of thrombin is read in the Protopath. Equipment calibration establishes a reading of 100% thrombin activity when no detectable heparin is present. Dade recommends preparation of standard curves for each test kit lot number and each source of heparin (beef lung or porcine intestinal mucosa) as well as for each manufacturer. Heparin concentrations of 0, 0.1, 6.2, 0.4, and 0.8 U/ml heparin are assayed and results plotted on linear graph paper (See Fig. 1). Heparin concentrations in patient plasmas are read from the standard curve, relative to fluorescence. As set forth by Dade, the assay accurately measures therapeutic heparin levels commonly employed in the treatment of deep vein thrombosis, pulmonary emboli, etc., and the protocol includes brief suggestions for adapting the technic to measuring levels of heparin encountered during cardiovascular surgery. The following are the modifications and applications used in our laboratory. Materials and Methods Between June and December, 1980, 75 patient specimens were assayed for heparin activity. Of these, 50 specimens were "post-protamine" and were assayed for heparin and protamine. The other 25 were specimens from patients being heparinized for various conditions. Although we do use our technic to measure therapeutic levels of mucosal heparin, our most frequent assay is for beef lung heparin at either low or high concentrations. Beef lung heparin calls for the use of longer incubation times to stabilize assay results. Longer times, unfortunately, decrease the sensitivity of the curve at the low heparin concentrations. The first step of our investigation was to modify the protocol for constructing standard curves to suit our needs. Preparation of Standard Curve Heparin reference points of 0.2, 0.4, 0.6, and 0.8 U/ ml are assayed. The results are plotted onto three cycle 0002-9173/81/0010/0410 $00.80 American Society of Clinical Pathologists 410

100 90 80 o z z < oo o cc 70 60 50 *»0 30 20 i.. 10 0.1 0.2 0.3 O.k 0.5 HEPARIN, USP U/ml 0.6 0.7 0.8 FIG. 1. Typical heparin standard curve obtained following the protocol established by the manufacturer of the Protopath. Plotting on regular graph paper fails to show straight linear relationship of remaining thrombin with heparin concentration.

412 semi-log paper which yields a linear relationship between the four points. Our subsequent assay approach is adjusted to utilize this linearity. A measured 1.0 ml specimen of pooled normal plasma is assayed using 5 /A to obtain a zero reference point (this point is not actually included in the curve). Then 5 /u.1 of a 40 U/ml heparin-saline preparation is added to the plasma specimen for a heparin concentration of 0.2 U/ml in exactly 1.0 ml volume; 5 /ul of the specimen are then used to assay that concentration. Another 5 IJL\ of the 40 U/ml heparin-saline preparation is then added to the specimen, resulting in a heparin concentration of 0.4 U/ml in the 1.0 ml volume. As before, 5 jxl are used to assay the 0.4 U/ml heparin concentration. This cycle of removing 5 p\ from the specimen for assay and then adding 5 fi\ of the heparinsaline preparation to it is repeated for the remaining two reference points of 0.6 and 0.8 U/ml heparin. The 40 U/ml heparin-saline preparation is stored at 2-8 Centigrade for later use. The resulting standard curve is illustrated in Fig. 2. Assay of "Highly Heparinized" Specimens Upon receipt of a highly heparinized specimen (obtained during surgery), a 1.0 ml volume of a 1:10 dilution of the specimen is prepared in normal pooled plasma (according to diluent requirements set forth by Dade) and assayed using 5 /ul of the dilution. If the original specimen contains between 2 and 6 U/ml heparin, the assay of the dilution reads between 0.2 and 0.6 U/ml on the standard curve prepared for that particular heparin. Multiplication by the dilution factor 10 yields the final result. A specimen dilution reading greater than 0.6 U/ml (indicating that the original specimen contained greater than 6 U/ml heparin concentration) would be further diluted 1:2 to obtain a reliable reading with which to calculate a final result. Usually the one step 1:10 dilution is sufficient. Should a specimen dilution read less than 0.2 U/ml, the final result would be obtained using the definitive assay described below. Assay of "Slightly Heparinized" Specimens For a specimen suspected of having a low heparin level, usually post-surgically, therapeutically, or "rebound," the assay is first run "as is" for confirmation using 5 /xl from a 1.0 ml volume. A definitive assay is then performed as follows: 5 (JL\ of the appropriate 40 U/ml heparin-saline preparation is added to the plasma - specimen, restoring exactly the 1.0 ml volume and increasing the specimen heparin concentration by 0.2 U/ml. 5 /u.1 of this specimen is then assayed for the cumulative heparin result. ANIDO AND FREEMAN A.J.C.P.. October 1981 If the original specimen contains, for instance, 0.06 U/ml heparin, the "as is" results falls below our lower curve limit of 0.2 U/ml. However, upon the addition of 0.2 U/ml heparin, the cumulative result is 0.26 U/ml, and that concentration can be read accurately from the curve. The final result issued for the patient is 0.26-0.20 = 0.06 U/ml heparin. The addition of 0.2 U/ml heparin to the initial specimen shifts the readings into our standard curve where small changes in concentration can be reliably defined. Assay of "Minus Heparin" Specimens All post-protamine specimens are assayed as described in our protocol for "slightly heparinized" specimens. The "as is" result, under "minus heparin" conditions, will undoubtedly read below the 0.2 U/ml point on the standard curve. The definitive assay result, after the addition of 0.2 U/ml heparin, determines whether or not there is an excess of protamine, reported as "antiheparin activity." That is, if, after the addition of heparin, the specimen fails to read at a level of 0.2 U/ml or, in fact, stays near the zero level, the presence of excess protamine is confirmed. By adding incremental amounts of 0.2 U/ml heparin to the specimen and assaying it after each addition, it is possible to "back-titrate" the specimen to a reading at or above the 0.2 U/ml reference point. By deducting the accepted heparin reading from the amount of heparin added, a quantitative result can be issued in terms of "anti-heparin activity" demonstrated by the original specimen. For instance, if a total of 0.4 U/ml heparin is added to bring the specimen to a reading of 0.25 U/ml, the "anti-heparin activity" in the original specimen is 0.40-0.25 = 0.15 U/ml. As in the procedure described for constructing a standard curve, an exact volume of 1.0 ml specimen volume is maintained throughout the process. Considerations 1. All results are confirmed by performing an assay after the addition of a known amount of heparin. Levels of less than 0.05 U/ml heparin or antiheparin activity are reported as "none detected." 2. Excess levels of protamine interfere with the coagulation process and thus act as an anticoagulant. It is our experience that protamine does not give an apparent (but false) heparin reading using this fluorescent technic. 3. The terminology "anti-heparin activity" is employed for reporting results of this nature because the assay is not a direct assay of protamine, but rather it is an assay of overall anti-heparin activity.

HEPARIN ASSAY AND PROTAMINE TITRATION 413 \ ^ \ \ Vw o.; 1 0. h 0. 6 0. 8 1.0 HEPARIN, USP U/ml FIG. 2. Typical heparin standard curve obtained following our protocol. Linear relationship of remaining thrombin with heparin concentration is obtained by plotting on three cycle semi-log paper. Extrapolation to zero heparin concentration is eliminated.

414 ANIDO AND FREEMAN A.J.C.P. October 1981 In-vivo contribution of Platelet Factor 4 is reflected in the assay. 4. Heparin or anti-heparin activity is reported with the assay requisition which identifies the heparin given to the patient. In our laboratory, "backtitration" is routinely performed using the heparinsaline standard preparation. A result for either heparin concentration or anti-heparin activity corresponds to, for instance, beef lung heparin from a certain manufacturer and lot number. 5. The 0.2 U/ml lower limit for reliability was chosen because normal plasma (containing no detectable heparin) may give somewhat different results when incubated with thrombin for 90 sec (an assay specification for beef lung heparin). Random normal specimens yield similar results upon the addition of 0.2 U/ml heparin. Our graph is sufficiently accurate at 0.2 U/ml to allow the detection of small changes in heparin concentration above this point. Also, this point is far enough removed from the zero point to avoid erroneous interpretations of low level heparin; and, while the slope of the curve at this point allows for minor variations, the heparin concentration primarily determines the outcome. 6. The reference point of 0.8 U/ml on the standard curve is assayed and plotted; however, this point serves primarily to reaffirm the linearity of the previous points. 7. A statement of the exact time a specimen was obtained must accompany all assay reports. Delayed responses must take into consideration metabolic clearance of heparin or protamine. After an initial 10 min incubation of the substrate cuvettes, each assay is completed within 5 min. The possibility of specimen contamination at the time of collection should be considered by those interpreting results. 8. The plasma diluent used in the assay (CPR) supplies sufficient ATIII to combine with heparin concentrations referenced on the standard curve. Thus, the results obtained from the assay represent the percent thrombin remaining when the heparin present functions at maximum reactivity. It is understood that, by artificially introducing a full complement of ATIII into the test system, a purely in-vitro measurement of heparin results. Patients on high doses of heparin demonstrate rapid saturation and resulting depletion of their own ATIII level. This must be considered when interpreting heparin assay results, or evaluating concurrent coagulation studies dependent upon clotting endpoints. The assay is of particular advantage if results are to be used for calculating neutralizing doses of protamine. 9. The assay uses 5 ul of plasma which is first diluted with 200 ul CPR, then further diluted with 2,000 ul substrate. This offers two advantages: 1) replicate determinations can be made from the 1.0 ml specimen volume without seriously disturbing the heparin concentration, and 2) hemolysis, often encountered during by-pass procedures, does not interfere with the accuracy or reproducibility of the assay. 10. The Protopath methodology for performing heparin assays requires meticulous technic. Incubation times, reagent specifications and other assay parameters are strictly standardized. A heparin control is provided with the test kit; and, for internal standardization, the heparin-saline preparation to be used in an assay is first assayed using known normal plasma to confirm the accuracy and reproducibility of the standard curve. Discussion Although heparin management during and after cardiovascular surgery is pertinent, the appearance of post-surgical bleeding can be due to a variety of causes. For appropriate patient follow-up, tests are provided to identify individual clotting factor deficiencies and inhibitors, ATIII levels, fibrin degradation products, plasminogen levels, etc. The methodology presented here introduces an approach to: distinguish the presence of protamine, define low levels of heparin; and rapidly determine high heparin concentrations. Results obtained using heparin as the titrating agent in this context are within 0.02 to 0.05 U/ml of the calculated heparin level. We have performed additional studies on the Protopath using protamine to titrate heparin of low, moderate, and high concentrations. Results correlate with calculated values within 0.05 to 0.10 U/ml heparin. Other methods for heparin assay include: 1) Activated Partial Thromboplastin Time (APTT), 4 2) Thrombin Time (TT), 5 3) Activated Coagulation Time (ACT), 1 and 4) Hepcon/System A-10* 3 an instrument utilizing protamine to measure heparin concentrations via an adaptation of the ACT. All four procedures are dependent upon clot production and prolonged times. In addition, the Hepcon/System A-10 is temperature dependent. The concept employed in our approach is simple and perhaps widely applicable. The addition of an internal standard of heparin to a specimen will either: 1) add to the effects of pre-existing heparin, 2) yield predictable results in patients having no detectable heparin, or 3) demonstrate an opposite or decreased effect from * HemoTec, Inc.

Vol. 76 No. 4 HEPARIN ASSAY AND PROTAMINE TITRATION 415 that normally found, due to the presence of protamine. It appears logical that, by using a heparin "back-titration" technic, laboratories now performing standard coagulation studies to monitor heparin activity could provide a test for excess protamine. Limited investigations using this concept with an APTT test system have been performed in this laboratory. The following is an example of the data obtained: Specimen Concentrations: Protamine Heparin 0.005 mg/ml None 0.005 mg/ml 0.2 U/ml 0.005 mg/ml 0.4 U/ml None 0.2 U/ml None 0.4 U/ml APTT (Normal Range = 21 to 29 seconds) 36.5 seconds 35.2 seconds 31.9 seconds 58.9 seconds 86.3 seconds Although heparins of different types neutralize protamine to various degrees, a general ratio of 1 mg protamine to 100 units of heparin could be applied to the above data such that 0.005 mg/ml protamine might be expected to neutralize 0.5 U/ml heparin. As shown, the addition of heparin to the specimen containing protamine yields APTT results correspondingly more normal. While the magnitude of the change is not great, the direction of the change is steady. It seems likely that, if 0.6 U/ml heparin were added to a specimen containing 0.005 mg/ml protamine, a "pass through" phenomenon might occur whereby the protamine present would be neutralized and the residual heparin would yield a prolonged result. Yet, if compared with the effects of adding that same amount of heparin to a known normal plasma, the difference in results would be readily apparent. In fact, it appears possible to estimate the amount of protamine present by identifying the heparin concentrations necessary to achieve such a "pass through." Quantitative limitations would include those of the test system utilized. Specific plasma clotting factors, especially ATIII levels, hemolysis, etc., may affect the findings in studies of this type. Yet, the feasibility of determining whether an initially prolonged finding is due to one of the other medications is upheld. Conclusions Heparin assay using a synthetic substrate eliminates the dependency upon clot production as an end point. Coagulation factor abnormalities and inhibitor levels do not affect the assay results. The use of a heparin internal standard and a linear standard curve in all our determinations increase the accuracy and reproducibility of results. 3. This system allows for the simultaneous determination of either heparin or protamine. 4. The reverse titration technic represents a simple approach to determine the concentration of excess protamine. 5. The Protopath heparin assay in conjunction with the adaptations we suggest offer the advantages of speed, accuracy, reproducibility, and specificity. References I. Bull BS, et al: Heparin therapy during extracorporeal circulation. J Thorac Surg 69:674-684, 1975 >. Dade Heparin Synthetic Substrate Assay Package Insert CO 56-7908 (Revised 6/80). I. HemoTec, Inc. Hepcon/System A-10. Heparin Analyzer Product Information Bulletin. 1980 L Langdell RD: Partial thromboplastin time technics. Thrombosis and bleeding disorders. Edited by NV Bang, FK Beller, E Deutsch, et al., New York, Academic Press, 1971 5. Sirridge MS: Laboratory evaluation of hemostasis, Philadelphia, Lea and Febiger, 1967.