Heparin Resistant Recalcifying Solution, a simple and quick recalcifying reagent that can identify and neutralize heparin contamination.

 

Overview:

Antihepca™ is to be used as a heparin neutralising agent in the recalcifying step of APTT, surface activated clotting tests (SACT, KCT) and other tests. It is dyed light blue to avoid confusion with regular 0.025M calcium chloride. It is currently available in 5 x 10ml vials. The Antihepca™ is stable for at least 5 years stored 2-8⁰C

Often plasma samples are not identified as containing heparin and may be present as an unexpected contaminant. Thus, the reason for prolonged APTT or KCT tests may not be apparent and laboratory testing may not be simple. Laboratories may find it useful to have a simple method for confirming the presence of heparin before going on to other investigations as may be required.

Measurement Principle:

The antihepca™-HRRS can be substituted for the usual M/40 calcium chloride solution in APTT and KCT tests to virtually eliminate the effect of unfractionated heparin concentrations up to 1u/ml in normal plasma.

Method Principle

Test plasmas giving prolonged APTT, SACT or KCT results with regular 0.025M calcium chloride may contain heparin or heparin-like substances which may be responsible for the test prolongation. Such plasmas will usually give shorter results when the APTT, SACT or KCT is repeated using Antihepca™. The concentration of anti-heparin agent is enough to neutralize up to 0.5u/ml heparin in most cases but is not so high that it affects APTT results prolonged by other abnormalities or defects.

Polybrene, protamine or similar cationic heparin neutralizing agents should not be added to APTT reagents or test plasmas because they can interfere with the contact activation by negatively charged surfaces occurring in the APTT preincubation stage.

Test Procedure

APTT and SACT tests are usually carried out by preincubating 0.1ml of test plasma with 0.1ml of a contact activating (APTT) reagent for 3-5minutes at 37⁰C, then adding 0.1ml of 0.025M calcium chloride and timing to a clotting endpoint. Proportionally smaller volumes are often used in automated clotting instruments.

To check for heparin in a test sample with a prolonged APTT the test should be repeated but using Antihepca in place of the regular 0.025M CaCl2.

Key Features and Benefits

  • Compatible with APTT on an analyzer
  • Streamlined process, easy to implement
  • Simple and unique product which can play a regular role in coagulation research
  • Less expensive than methods to remove heparin including heparinase enzyme
  • Easier than adsorbing agents such as ECTEOLA cellulose and specially treated filters
  • Adding HRRS in the calcium chloride solution after the contact activation stage in APTTs or KCTs is more reliable than polymers to neutralize heparin, which in excess, they can interfere with contact activators used in most APTT reagents.

5 x 10 mL Ready-to-use stable liquid reagent, 5 years shelf life.

 

Table 1 shows results obtained on test plasmas with various LAC APTT resistant reagents used with 0.025M calcium chloride or Antihepca™
 

Test plasma

Intrinsin LR (HTX) Cephascreen (Stago) Triniclot S (Stago) Actin FS (Siemens) Hemosil SynthFax (IL)
CaCl2 Antihepca CaCl2 Antihepca CaCl2 Antihepca CaCl2 Antihepca CaCl2 Antihepca
Normal PNP.HTX 30.7 30.1 33.6 31.3 33.9 35.0 29.8 29.3 24.4 27.8
Anticoags aa + Heparin 0.4IU/ml 114 34.5 71.8 35.2 83.0 40.9 70.3 30.6 51.7 30.4
PNP + Clexane 0.2IU/ml 51.2 36.0 46.4 32.6 58.0 41.3 48.1 30.4 38.6 28.0
Rivaroxaban 400ng.ml 52.2 52.1 50.8 52.8 58.4 58.0 54.3 52.1 38.2 42.9
aa + Heparin 0.2IU/ml 131 52.4 92.8 53.3 112 70.0 115 49.1 68.5 46.3
Dabigatran 200ng.ml 60.2 60.8 60.0 59.5 79.8 74.7 58.6 58.2 45.5 57.3
aa + Heparin 0.2IU/ml 119 61.9 86.3 60.7 125 81.8 94.3 52.3 68.1 50.8
Adsorbd Plas.+ 30%NP 77.8 76.3 85.8 86.4 100 91.3 92.8 83.0 86.9 88.5
aa + Heparin 0.2IU/ml >200 77.8 >200 95.3 >200 120 >200 94.0 >200 116
Defic. 20% Factor IX 40.8 39.2 38.1 38.9 44.2 43.7 41.0 34.6 29.5 31.9
aa + Heparin 0.2IU/ml 75.3 42.5 51.7 42.0 58.7 46.1 51.4 32.0 39.5 33.7
20% Factor XI 43.8 42.6 44.0 44.8 50.8 44.0 42.6 43.6 38.1 40.4
aa + Heparin 0.2IU/ml 85.9 46.5 64.0 45.7 77.0 50.0 73.3 37.1 59.1 41.1
Other D-Dimer (2µg/ml) 77.9 74.0 67.1 66.2 77.6 70.2 75.1 66.1 47.9 53.0
aa + Heparin 0.2IU/ml >200 77.7 102 69.8 130 75.9 158 67.7 71.0 56.5
PNP + Polybrene 0.001% 76.3 85.1 35.3 35.3 42.9 47.0 68.3 88.0 >200 >200
LAC LAC pos.#168 31.3 30.7 37.0 37.1 55.9 55.3 29.0 26.1 25.3 28.0
aa + Heparin 0.2IU/ml 51.1 32.6 49.2 39.5 82.8 62.8 40.0 26.5 31.4 27.4
Weak LAC (PBL) 36.1 34.4 38.9 36.2 54.2 50.1 32.3 29.5 29.0 30.9
LAC pos.(PBL) 35.5 34.5 42.1 40.1 62.5 59.6 30.8 28.5 30.5 32.7

 

Table 2 shows results obtained similarly using several LAC sensitive APTT reagents.
 

Test plasma

Intrinsin LS (HTX) Triniclot HS (Stago) Hemosil Synthasil (IL) Actin FSL (Siemens) Hemosil SP (IL)
CaCl2 Antihepca CaCl2 Antihepca CaCl2 Antihepca CaCl2 Antihepca CaCl2 Antihepca
Normal PNP.HTX 35.3 35.4 32.6 32.7 34.5 38.4 29.3 28.2 32.6 36.9
Anticoags aa + Heparin 0.4IU/ml 77.1 36.2 103 38.7 109 46.7 69.0 34.7 83.5 42.4
PNP + Clexane 0.2IU/ml 58.6 38.6 55.3 38.9 57.3 48.2 46.8 37.6 56.6 43.6
Rivaroxaban 400ng.ml 53.5 54.5 50.5 53.9 58.0 64.2 48.9 53.8 58.0 61.0
aa + Heparin 0.2IU/ml 97.8 49.0 116.0 57.3 119 65.8 83.1 49.8 121 65.1
Dabigatran 200ng.ml 71.4 73.9 61.9 63.9 65.6 78.7 56.4 56.8 68.4 68.8
aa + Heparin 0.2IU/ml 109 64.5 119 67.0 130 81.3 86.0 58.5 113 73.7
Adsorbd Plas.+ 30%NP 64.0 56.5 70.6 77.9 63.7 76.6 75.5 75.1 76.2 75.8
aa + Heparin 0.2IU/ml 156 60.5 >200 85.8 150 80.5 184 77.7 >200 82.0
Defic. 20% Factor IX 42.5 40.9 44.6 43.6 54.8 58.4 37.2 37.5 45.1 43.7
aa + Heparin 0.2IU/ml 61.8 42.9 76.8 48.9 125 71.9 51.5 39.9 73.1 48.0
20% Factor XI 44.8 38.8 46.4 44.8 54.5 58.1 39.4 40.5 44.6 44.3
aa + Heparin 0.2IU/ml 68.1 44.1 91.0 50.5 118 75.1 58.6 43.1 78.8 48.6
Other D-Dimer (2µg/ml) 76.0 73.7 72.7 70.9 82.5 82.6 81.7 82.3 73.7 72.9
aa + Heparin 0.2IU/ml 158 72.1 172 81.0 186 90.3 181 88.1 157 80.5
PNP + Polybrene 0.001% 153 >200 37.7 36.8 >200 >200 40.7 42.5 37.8 37.0
LAC LAC pos.#168 105 93.2 53.8 54.7 52.9 63.8 32.0 36.0 59.3 63.5
aa + Heparin 0.2IU/ml >200 93.5 95.7 59.8 102 72.9 49.1 40.3 89.3 66.4
Weak LAC (PBL) 94.8 93.8 48.5 47.5 69.2 74.4 33.8 34.2 77.4 74.0
LAC pos.(PBL) 108 105 55.9 55.7 71.6 79.3 38.4 41.5 104 99.9