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Use of an automated coagulation analyzer to perform heparin neutralization with polybrene in blood samples for routine coagulation testing: practical, rapid, and inexpensive.

Heparin contamination in blood samples can lead to false prolongation of routine coagulation tests: activated partial thromboplastin time (aPTT) and, to a lesser extent, prothrombin time (PT). Such contamination commonly occurs when blood is drawn through an indwelling catheter kept patent by heparin, (1) or from a needle or syringe previously rinsed with heparin. (2) It also arises from using the wrong order of blood collection tubes, for example, when a heparinized tube is filled before the citrated tube for coagulation tests.

This problem can be resolved by adding heparinase, an enzyme that degrades heparin without any influence on normal coagulation, to the sample with potential heparin contamination. (1,3,4) However, its cost is rather expensive, at 460 Thai Baht (US $13.80)/test (Dade Hepzyme, Dade Behring Marburg GmbH, Marburg, Germany), compared with the cost of coagulation tests charged by our laboratory: 60 Baht (US $1.80) for PTs and 70 Baht (US $2.10) for aPTTs. This method requires manual preparation by adding 1.0 mL, or at least 0.5 mL, of plasma to a vial of heparinase and needs 15 minutes for stabilization before transferring the plasma to a cup and then to the analyzer. In addition to the disadvantage of consuming more time per sample, the available plasma may not be sufficient when a minimal blood volume is obtained, especially in the case of pediatric patients.

Basic compounds, such as protamine sulfate or hexadimethrine bromide (polybrene), can neutralize heparin, which is an acidic glycosaminoglycan. (5,6) These substances are much cheaper than heparinase. However, they can also interfere with normal coagulation. Polybrene is considered superior to protamine because it has greater potency in neutralizing high doses of heparin, but it has less of an effect on coagulation tests. (6)

The use of polybrene to neutralize heparin contamination in blood samples for aPTT was described decades ago when manual methods were relied on. According to that study, plasma should be mixed with the aPTT reagent before adding polybrene to diminish its interference with the contact activation process. The recommended activators of aPTT reagents to be used with polybrene were kaolin, celite, or micronized silica but not ellagic acid. (7) This technique has not yet, to our knowledge, been used in routine coagulation laboratories during the era of automation.

Facing the problem of heparin contamination with economic constraints, we attempted to use polybrene for neutralization by manually mixing this reagent with heparin-contaminated plasma, and then performing aPTT analysis using an automated analyzer. (8) In that previous study, polybrene was added before the aPTT reagent because the analyzer could not be programmed to work with a different sequence of reagents chosen by users. Moreover, the activator of the aPTT reagent was ellagic acid, which was not recommended. Although the method helped differentiate between prolongation caused by heparin contamination and by factor deficiency, it was not practical because a volume of plasma had to be removed from the primary tube, mixed with polybrene, and then returned to the analyzer.

Nowadays, with new generations of automated coagulation analyzers, the sequence of the reagents used for reactions can be set by the users. Thus, we would like to introduce a novel procedure for heparin neutralization using polybrene by adapting the whole process so it may be performed rapidly with PT and aPTT tests on the analyzer. The objectives of this study were to find the optimal concentration of polybrene, to compare the effects of adding polybrene before and after contact activation of aPTT, and to apply this process of heparin neutralization to routine practice.

MATERIALS AND METHODS

This study was done with the permission of the Siriraj Institutional Review Board.

Study Population

Normal plasmas were obtained from 10 healthy volunteers who were not receiving any medication and had no history of any bleeding disorder. Seventy-six abnormal plasmas and 13 plasmas with normal baseline PT and aPTT results were derived from the residual plasmas sent for routine coagulation tests. Plasmas were prepared from blood samples collected in 2.7-mL evacuated tubes (Vacutainer, Becton, Dickinson and Company Ltd, Oxford, England) containing 0.3 mL of 3.2% sodium citrate for a 9:1 ratio of blood to anticoagulant. Plasma was separated by centrifugation at 1500g for 15 minutes, kept at -70[degrees]C and thawed only once at 37[degrees]C for 5 minutes when the experiments were performed.

Reagents and Instrumentation

The PT and aPTT results were measured with a Thromborel S (Siemens AG, Erlangen, Germany) and Dade Actin FS (DadeBehring), respectively, using the CA-1500 (Sysmex, Kobe, Japan). The Actin FS contains ellagic acid as the contact activator. The reference range for PT was 10.0 to 12.7 seconds, and 24-32 seconds for aPTT.

Polybrene (Sigma-Aldrich, Milwaukee, Wisconsin) was diluted to 10 000 [micro]g/mL in 0.15M NaCl to prepare the stock solution, which was subsequently diluted with 0.15M NaCl to generate working solutions. Four concentrations--100, 250, 500, and 750 [micro]g/ mL--were prepared. With 5 [[micro]]L of each working solution added to 50 [micro]L of plasma by the analyzer, the final concentration of polybrene in plasma was 10, 25, 50, and 75 [[micro]]g/mL, respectively.

Three new tests were programmed on the menu of the analyzer. Prothrombin time was modified to be "PT with polybrene." The reagent sequence was as follows: plasma 50 [[micro]]L, incubated for 180 seconds; polybrene 5 [[micro]]L, incubated for 20 seconds; PT reagent 100 [mu]L. The other two were "aPTT with polybrene program 1" and "aPTT with polybrene program 2," which differed only in the sequence of reagents. The first program was plasma 50 [micro]L; aPTT reagent 50 [micro]L, incubated for 180 seconds; polybrene 5 [micro]L, incubated for 20 seconds; 0.025 mol/L Ca[Cl.sub.2] 50 [micro]L. The second program was similar to the first except that the plasma was incubated with polybrene before adding the aPTT reagent.

Four doses of unfractionated heparin (Heparin Leo, LEO Pharma A/S, Ballerup, Denmark) were used to simulate different concentrations of heparin contamination in blood samples. Heparin (5000 U/mL) was diluted in 0.15M NaCl to obtain working solutions of 20, 50, 100, and 150 U/mL; then, 4 [micro]L of these solutions was added to 400 [micro]L of plasma to obtain final concentrations of 0.2, 0.5, 1, and 1.5 U/mL, respectively.

Experimental Design

To Determine the Effect of Polybrene on Coagulation Testing and Its Proper Concentration for Heparin Neutralization. --Baseline PT and aPTT results were studied in 10 normal plasma samples. Different concentrations of polybrene were added to the plasma samples, and PT and aPTT tests were performed again to determine the effect of polybrene on the coagulation tests, using the 3 new tests already mentioned. Each result was compared with the baseline.

To study the neutralizing capability of polybrene, various concentrations of heparin were added to normal plasma before testing for PT and aPTT, and then, they were neutralized with each concentration of polybrene using the analyzer. The concentration of polybrene that minimally interfered with the coagulation tests, but most effectively neutralized heparin, was then chosen for routine use.

To Evaluate the Use of Polybrene in Eliminating Heparin Contamination in Abnormal Plasmas.--The selected concentration of polybrene from the earlier experiment was used to neutralize various concentrations of heparin in 76 plasmas with abnormal PT and/or aPTT results. The baseline PT and aPTT results were compared with those after neutralization.

To Determine the Stability of the Polybrene Working Solution.--The polybrene working solution was aliquoted and kept at either 2[degrees]C to 8[degrees]C or -20[degrees]C for 1 year, from January 2009 to January 2010. The stored working solutions and the freshly prepared reagents were used to neutralize 1 U/mL of heparin in plasmas from 13 patients with normal baseline PT and aPTT results. Lastly, their results were compared with one another.

To Study the Incidence of Heparin Contamination of In-Patient Blood Samples Sent for Routine Coagulation Tests During A 3-Month Period.--Samples suspected of contamination with heparin were tested during July 2009 to September 2009. The selection criteria were samples for screening coagulogram with (1) isolated, prolonged aPTT without a definite cause, for example, hemophilia, von Willebrand disease, or lupus anticoagulant; and (2) prolonged PT and aPTT without a relation to each other, such as samples with a PT of 17 seconds and an aPTT of 80 seconds. The exclusion criteria included samples from outpatient departments, patients receiving warfarin or heparin therapy, and samples with preanalytic errors, such as underfilled collection tubes or high hematocrits. Repeated samples from patients with no decline in aPTT value after neutralization were also excluded. The selected samples were neutralized with the proper concentration of polybrene from the first experiment. The following information was recorded: ward, route of specimen collection, whether the patient was receiving anticoagulant, and whether other specimens were collected in heparinized tubes at the same time. The conclusion of contamination was based on a reduction in aPTT results of more than 5% of the initial value because the coefficient of variation in abnormal aPTT results was less than 2%, denoting the between-run precision of our aPTT testing.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Statistical Analysis

Repeated-measures analysis of variance and pairwise comparison with baseline using the Bonferroni test were performed to evaluate the results from the first and third experiments. The Friedman test and post hoc testing compared with baseline with the Bonferroni test were used for the second experiment. Descriptive statistics and the Pearson correlation were used for the fourth experiment. The statistical software SPSS for Windows (version 16.0; SPSS Inc., Chicago, Illinois) was used for statistical analysis, with P < .05 considered significant.

RESULTS

The concentration of polybrene or of heparin, where mentioned in this section, and in the discussion, indicates the final concentration in the plasma.

Effect of Polybrene on Coagulation Tests and Its Proper Concentration

Figure 1 shows that polybrene significantly increased the PT results for 10 normal plasmas in a dose-dependent manner, with P values all < .001. However, when 25 [micro]g/mL of polybrene was added, the mean difference in the results from baseline was minimal, that is, 0.24 seconds (95% confidence interval, 0.12-0.36).

A heparin concentration of 0.2 U/mL had no significant effect on PT results (P = .11). The mean increase in PT results caused by a heparin concentration of 0.5 U/mL was only 0.72 seconds (P < .001), whereas those with 1.0 and 1.5 U/mL were 2.54 and 5.88 seconds, respectively (P < .001). Therefore, the data concerning heparin at 0.2 and 0.5 U/mL are not shown. The neutralizing capability of 10 [micro]g/mL polybrene did not differ significantly from that at 25 [micro]g/mL when tested against 1.0 U/mL heparin (Figure 2, A). However, when the concentration of heparin was increased to 1.5 U/mL (Figure 2, B), 25 [micro]g/mL was the most effective concentration. Using that concentration, the changes in PT from baseline after neutralization of either 1.0 U/mL or 1.5 U/mL heparin amounted to 0.5 seconds (95% confidence interval, 0.36-0.64) and 0.73 seconds (95% confidence interval, 0.48-0.99), respectively.

When polybrene was added before the aPTT reagent (program 2), aPTT was prolonged so strikingly (Figure 3) that only 2 concentrations of polybrene were tested. Polybrene, at concentrations of 10 and 25 [micro]g/mL added after the aPTT reagent, significantly shortened the aPTT results. The latter had less of an effect, with a mean difference from baseline of -0.72 seconds (95% confidence interval, -0.99 to -0.45). However, higher concentrations (50 and 75 [micro]g/mL) of polybrene increased the aPTT results.

[FIGURE 4 OMITTED]

Polybrene at a concentration of 25 [micro]g/mL was the most effective at neutralizing heparin in plasma for APTT, using the aPTT with polybrene program 1 method (Figure 4, A through D). The capability of polybrene to neutralize heparin was also tested with program 2, but the results were vastly inferior (data not shown). There was no significant difference between the aPTT results at baseline and those after neutralization of up to 0.5 U/mL heparin (Table 1). Although the mean differences were significant at higher heparin concentrations, they were less than 3 seconds.

The Use of Polybrene to Eliminate Heparin Contamination in Abnormal Plasmas

Polybrene at a concentration of 25 [micro]g/mL was selected for heparin neutralization in 76 abnormal plasmas (Figure 5, A and B). The effects of 0.2 and 0.5 U/mL heparin on PT results were not clinically significant; therefore, their data are not displayed in the figure. Both the PT and aPTT results decreased strikingly after neutralization but differed from the baseline (P < .001).

The Stability of the Polybrene Working Solution

Three types of polybrene working solutions, ie, freshly prepared, stored for 1 year at -20[degrees]C, and stored for 1 year at 2[degrees]C to 8[degrees]C, were used to neutralize 1 U/mL of heparin in plasmas from 13 patients with normal baseline PT and aPTT. There was no difference between the PT or aPTT results after neutralization (P = .50 and P = .14, respectively).

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

The Incidence of Heparin Contamination

During a 3-month period, 81 of 4921 samples (1.6%) from inpatients who had not received anticoagulant therapy were suspected of heparin contamination according to the criteria and were selected for neutralization. Of those 81 samples, 68 (84.0%) had aPTT results with a 5% or greater decrease after neutralization, whereas the others did not decrease but tended to increase in coagulation time (the changes ranged from -0.2 to 14.5 seconds, with a median of 2.8 seconds). Therefore, the incidence of heparin contamination was 1.4% (68 of 4921). The aPTT results from these samples varied from 35.5 seconds to more than 220 seconds (Table 2). The reductions in the aPTT results ranged from 2.6 seconds to more than 160 seconds. Figure 6 shows a strong correlation between the initial aPTT results that were in the detectable range and their reductions. The reasons for contamination were blood drawn from heparinized catheters (52 of 68; 76%); and wrong tube order for the collection process (15 of 68; 22%). There was one sample with an undetermined source of contamination because it was noted on the request form that the sample was collected by venipuncture. However, that patient had a line for central venous pressure monitoring, flushed with heparin. The reduction in the aPTT result in that case was 16% (from 72.3 to 60.4 seconds), and the PT was 35 seconds. The patient had liver cirrhosis, and during the previous 10 days, her PT and aPTT varied from 23 to 34.4 and 31.3 to 54.4 seconds, respectively, because of plasma transfusion.

COMMENT

Our experimental results demonstrate that polybrene prolongs the results of PT in a dose-dependent manner (Figure 1). It suppresses extrinsic coagulation by down-regulating tissue factor-dependent factor VII activation. (9) The anticoagulant effect of polybrene on aPTT is much less when the contact-activation process is stimulated before, rather than after, adding polybrene to plasma (Figure 3). Our finding that polybrene shortened the aPTT results at low concentrations but prolonged them at higher concentrations is similar to that in a previous study, (10) in which less than 50 [micro]g/mL polybrene had clot-promoting activity. Polybrene was also found to shorten the clotting time of the functional assay for factor VIIa. (11) However, these effects of polybrene on coagulation were insubstantial at a concentration of 25 [micro]g/mL; the changes in the PT and aPTT results of plasmas induced by polybrene were less than 1 second compared with baseline results.

The most effective concentration of polybrene for neutralizing heparin was 25 [micro]g/mL, higher than that in our previous study, (8) which relied on 10 [micro]g/mL because the plasmas were manually mixed with polybrene before aPTT testing, allowing greater interference with the contactactivation process. In a study (7) using micronized silica as a contact activator, the maximal final concentration of polybrene was 50 [micro]g/mL, which could neutralize up to 2.0 U/mL heparin. The aPTT reagent was less sensitive to heparin and excess polybrene than was the reagent with ellagic acid. The aPTT results from plasmas with heparin at 1.5 U/mL were still in the reportable range, (7) whereas, in our study, heparin at 1 U/mL prolonged the clotting time of the aPTT test beyond the limit of the detectable range.

One approach to interpreting this neutralization method is to consider the results of PT and aPTT together. Even though their results after neutralization were statistically different from baseline at higher doses of heparin, that might not be clinically significant for either normal (Figure 2; Table 1) or abnormal plasmas (Figure 5). When all tests fall within the reference range after neutralization, there is no need for repetition. Repeat samples may be necessary for plasmas with results that are still borderline for prolongation after neutralization. If the aPTT result becomes normal after neutralization, even though the PT of the sample is still clearly prolonged, there is no need for repeat blood collection. Because the effect of heparin on PT is much less than it is on aPTT, a prolongation of PT that is not corrected to normal should be due to factor deficiency. At least 29 of 68 samples (43%) had normal aPTTs after neutralization (Table 2), and repeat blood collections could be omitted. If the result of the aPTT test after neutralization does not decrease into the reference range, then whether the PT result is within the reference range should be considered. If the PT result is normal (initial result or after neutralization), the isolated, prolonged aPTT may be due to a bona fide factor deficiency or to an imperfect result after heparin neutralization. If the PT result is abnormal, for example, 20 seconds, whereas the aPTT result decreases from 130 to 60 seconds, the baseline of that patient is abnormal, and the exact value of the aPTT is not necessary unless the patient has hereditary factor VII deficiency, in which case, the aPTT result should be normal. The exact aPTT result has less influence on the differential diagnosis for common acquired disorders that cause both prolonged PT and aPTT, for example, liver cirrhosis, disseminated intravascular coagulation, and vitamin K deficiency; because factor VII has the shortest half-life, PT will be prolonged first. The minimal benefit of this method is to confirm that the prolongation is caused by heparin contamination, avoiding unnecessary further investigation or treatment.

The stability of the working solution is at least 1 year and it can be kept at either -20[degrees]C or 2[degrees]C to 8[degrees]C. The duration of stability testing should be further examined, especially at -20[degrees]C.

The incidence of heparin contamination may differ in each report because of population selection. In a study (12) using heparinase to treat samples with aPTT results of 45 seconds or more, a complete correction of the aPTT results was found in 32 of 83 samples (39%) selected from 4136 samples. However, 17 of 83 samples (20%) had significant decreases in aPTT after treatment with heparinase, but without normalization. If those samples, suspected of simultaneous factor deficiency and heparin contamination, were included, the rate of heparin contamination would be 59% (49 of 83) in samples with aPTT higher than 45 seconds; the overall incidence would be 1.2% (49 of 4136 samples). (12) Although the detection rate of our selection criteria for heparin neutralization was higher--84% (68 of 81 samples)--the incidence of heparin contamination is comparable (1.4%). All isolated, prolonged aPTT results (of unknown cause) were included. We did find samples with aPTTs of less than 45 seconds that converted to normal results after neutralization (Table 2). That implies that our criteria were sensible. Individual reductions in aPTT results strongly correlate with the initial aPTT results (Figure 6), meaning that whenever the initial aPTT value is inflated because of heparin, polybrene tends to neutralize the heparin and proportionally reduces the aPTT. This relationship suggests that the fixed concentration of polybrene provides a wide range of neutralization.

The most common cause of contamination is blood drawn from catheters previously rinsed with heparin (76%). To avoid that error, the proper method for drawing blood from catheters should be emphasized.

The polybrene neutralization method would also be beneficial for other laboratories. The PT and aPTT reagents used in this study are the most common reagents used in Thailand, around 76% and 72%, respectively. (13) This study ended in early 2010; however, it has been used in our routine coagulation laboratory since then (2009-2013; 4 years), providing evidence of its practicality.

The limitations of this study include the effect of polybrene on thrombin time, which is more sensitive to heparin than PT and aPTT are; this was not evaluated in this study because only PT and aPTT are performed in our laboratory. According to a study in 2007, around 33% (37 of 112) of laboratories in Thailand were performing thrombin time. (14) In addition, we did not have an anti-Xa assay to measure the level of heparin when this study was started. Therefore, neither the level of spiked heparin nor the heparin in the presumably heparin-contaminated samples could be confirmed. However, the conclusion of heparin contamination was based on the aPTT testing in our laboratory being precise; the coefficient of variation for the internal quality control for abnormal results was usually less than 2% each month. It was very rare to find a repeat aPTT that became shorter by more than 5% without reason. Moreover, causes of contamination could be identified in all cases, except one, in which the route of blood collection was questionable, and the aPTT results from samples determined not to have contamination did not decrease after adding polybrene but actually increased. Finally, we did not find out how many repeat samples, apart from those with normal aPTT results after neutralization, were omitted because the clinicians used the results after neutralization instead.

Polybrene neutralization must not be performed in samples from patients on heparin therapy. We either use data in the request form to identify patients on heparin therapy or request an aPTT ratio, which is only reported in patients receiving unfractionated heparin. The current request form, to inform clinicians, contains the following: in case heparin contamination is suspected by the laboratory staff, polybrene neutralization will be performed; the result after neutralization may not be equal to the true result without heparin; do not request polybrene neutralization for a patient receiving heparin therapy. In cases with no clinical information, we suggest repeating the sample with polybrene. If the aPTT result decreases, then contact the clinician or a nurse on the ward to find out whether the patient is receiving heparin. If the result remains the same or increases, report the result before neutralization.

In conclusion, polybrene can be used to neutralize heparin contamination in blood samples for PT and aPTT testing. The results after neutralization may not be equal to the true results without heparin, but the differences may not be clinically significant. This method is inexpensive (the cost of polybrene per test is less than US $0.01) and can be performed rapidly with PT and aPTT on an automated coagulation analyzer, which makes it easy to use. There is no need for extra plasma volumes because the neutralization process is integrated into these tests. However, before applying this method to other PT and aPTT reagents, the optimal concentration must be verified, and the customized properties of the coagulation analyzer should be considered.

Caption: Figure 1. Prothrombin time (PT) results with 10 normal plasmas after adding polybrene to various final concentrations.

Caption: Figure 2. Prothrombin time (PT) results with 10 normal plasmas before and after adding heparin (Hep) to (A) 1.0 U/mL plasma, and (B) 1.5 U/mL plasma, and those after neutralization with polybrene (PB) at various final concentrations ([micro]g/mL).

Caption: Figure 3. Results of activated partial thromboplastin time (aPTT) testing with 10 normal plasmas after adding polybrene to different final concentrations, using 2 different reagent sequences: polybrene was added after (A) or before (B) the aPTT reagent. Only 2 concentrations of polybrene were tested for the latter sequence.

Caption: Figure 4. The ability of different concentrations of polybrene (PB) to neutralize heparin (Hep) in 10 normal plasmas (Hep, 0) and in plasmas containing Hep at final concentrations of (A) 0.2 U/mL, (B) 0.5 U/mL, (C) 1.0 U/mL, and (D) 1.5 U/mL. Heparin [greater than or equal to] 1.0 U/mL in plasma prolonged activated partial thromboplastin time (aPTT) beyond the limit of the detection range. Therefore, only data after neutralization are displayed. The PB was added after the aPTT reagent.

Caption: Figure 5. Heparin (Hep) neutralization with polybrene (PB) at a final concentration of 25 [mu]g/mL in 76 abnormal plasmas. The median results for (A) prothrombin time (PT) and (B) activated partial thromboplastin time (aPTT) in each group are displayed in the upper part of the box plot. Abbreviations: N/A, not applicable because some results fell outside the limits of detection; ([dagger]) aPTT results from 29 plasmas with 0.5 U/mL heparin fell outside the range of detection.

Caption: Figure 6. Correlation between those initial activated partial thromboplastin time (aPTT) results that were in the detectable range (n = 53; 78%) and their final, reduced values. The other 15 samples (22%) with aPTT results over the detectable range had reductions of more than 160 seconds.

This study was supported by the Siriraj Research Fund.

References

(1.) Keller FG, DeFazio J, Jencks F, Steiner M, Rogers J, Ritchey AK. The use of heparinase to neutralize residual heparin in blood samples drawn through pediatric indwelling central venous catheters. J Pediatr. 1998;132(1):165-167.

(2.) Brown JM, Dimeski G. Contamination of coagulation tests with heparin from blood gas samples. Br J Anaesth. 2001;87(4):628-629.

(3.) Hutt ED, Kingdon HS. Use of heparinase to eliminate heparin inhibition in routine coagulation assays. J Lab Clin Med. 1972;79(6):1027-1034.

(4.) van den Besselaar AM, Meeuwisse-Braun J. Enzymatic elimination of heparin from plasma for activated partial thromboplastin time and prothrombin time testing. Blood Coagul Fibrinolysis. 1993;4(4):635-638.

(5.) Grann VR, Homewood K, Golden W. Polybrene neutralization as a rapid means of monitoring blood heparin levels. Am J Clin Pathol. 1972;58(1):26-32.

(6.) Kikura M, Lee MK, Levy JH. Heparin neutralization with methylene blue, hexadimethrine, or vancomycin after cardiopulmonary bypass. Anesth Analg. 1996;83(2):223-227.

(7.) Israels ED. Partial thromboplastin time in the presence of heparin: a rapid polybrene neutralization method. Am J Clin Pathol. 1982;77(3):321-324.

(8.) Tientadakul P, Wongtiraporn W, Cheunsomboon N, Opartkiattikul N. Elimination of heparin contamination with hexadimethrine in blood samples for coagulation testing. J Lab Med Qual Assur. 2004;26(suppl 3):S541-S548.

(9.) Chu AJ, Rauci M, Nwobi OI, Mathews ST, Beydoun S. Novel anticoagulant activity of polybrene: inhibition of monocytic tissue factor hypercoagulation

following bacterial endotoxin induction. Blood Coagul Fibrinolysis. 2002;13(2): 123-128.

(10.) Cumming AM, Jones GR, Wensley RT, Cundall RB. In vitro neutralization of heparin in plasma prior to the activated partial thromboplastin time test: an assessment of four heparin antagonists and two anion exchange resins. Thrombosis Res. 1986;41(1):43-56.

(11.) Cardigan RA, Mackie IJ, Machin SJ. The effect of heparin and its neutralisation on functional assays for factor Vila, factor VII and TFPl. Thrombosis Res. 1996;84(4):237-242.

(12.) Newman RS, Fagin AR. Heparin contamination in coagulation testing and a protocol to avoid it and the risk of inappropriate FFP transfusion. Am J Clin Pathol. 1995;104(4):447-449.

(13.) Tientadakul P, Opartkiattikul N, Wongtiraporn W. Improvement of coagulation laboratory practice in Thailand: the first-year experience of the national external quality assessment scheme for blood coagulation. Arch Pathol Lab Med. 2009;133(1):72-77.

(14.) Tientadakul P, Opartkiattikul N, Wongtiraporn W. A survey of coagulation laboratory practice in Thailand: the first step to establish a National External Quality Assessment Scheme (NEQAS) for blood coagulation. J Med Assoc Thai. 2007;90(12):2616-2623.

Panutsaya Tientadakul, MD Chulalak Kongkan, BSc Wimol Chinswangwatanakul, MD, PhD

Accepted for publication February 7, 2013.

From the Department of Clinical Pathology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.

The authors have no relevant financial interest in the products or companies described in this article. Presented in part at the 23rd Annual Meeting of the International Symposium on Technological Innovations in Laboratory Hematology; May 10-12, 2010; Brighton, England.

Reprints: Panutsaya Tientadakul, MD, Department of Clinical Pathology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Prannok Rd, Bangkoknoi, Bangkok 10700, Thailand (e-mail: siptd@ mahidol.ac.th).
Table 1. Differences in Activated Partial
Thromboplastin Time (aPTT) Between Baseline
and After Heparin Neutralization With Polybrene at a
Final Concentration of 25 [micro]g/mL in 10 Normal
Plasmas

                       Mean (SD)             Mean Difference
Heparin,               aPTT After             From Baseline
U/mL               Neutralization, s             (95% CI)

0 (baseline)      28.74 (2.07),                    N/A
                  no neutralization
0.2               28.41 (2.04)           -0.33 (-0.88 to 0.22)
0.5               29.21 (2.22)            0.47 (-0.28 to 1.23)
1.0               29.78 (2.24)            1.04 (0.30-1.78)
1.5               30.61 (2.23)            1.87 (1.14-2.60)

Abbreviations: SD, standard deviation; CI, confidence interval;
N/A, not applicable.

Table 2. Samples Classified With Heparin
Contamination According to Their Initial Activated
Partial Thromboplastin Time (aPTT) Results and
Samples That Decreased to Within Reference Range(a)
After Neutralization With Polybrene

                      Samples With            Samples Within
                      Heparin               Reference Range (a)
                      Contamination,      After Neutralization,
Initial aPTT, s       No.                        No. (%)

35-44                 8                           8 (100)
45-54                 6                           4 (67)
55-64                 8                           3 (38)
65-74                 5                           2 (40)
75-84                 3                           1 (33)
85-94                 1                           0 (0)
95-104                1                           0 (0)
105-220               21                          8 (38)
>220                  15                          3 (20)

Total                 68                         29 (43)

(a) PTT reference range, 24-32 seconds.
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Author:Tientadakul, Panutsaya; Kongkan, Chulalak; Chinswangwatanakul, Wimol
Publication:Archives of Pathology & Laboratory Medicine
Article Type:Report
Geographic Code:9THAI
Date:Nov 1, 2013
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