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A simple functional assay for heparin-induced thrombocytopenia/thrombosis syndrome using a full blood count analyser.

Introduction

Heparin-induced thrombocytopenia (HIT) is a serious complication from heparin use, arising from the binding of antibody against heparin-platelet factor 4 (PF4) complexes onto platelets. This activates the platelets, resulting in a prothrombotic state with the risk of arterial or venous thrombosis, as well as thrombocytopenia from the platelet consumption (1).

The Heparin-PF4 antibodies (HIT antibodies), which are mainly of IgG class, usually develops 5 to 10 days in susceptible patients after heparin exposure. The incidence of HIT is between 0.5-5% of patients treated with heparin (2). It is higher in surgical patients than medical patients, and with unfractionated heparin compared to low molecular weight heparin. If untreated, 30-50% of patients with HIT develop severe thrombotic problems including skin necrosis, venous gangrene, limb complication, or even death. The treatment is to stop the heparin and use alternative anticoagulants such as direct thrombin inhibitors or direct-Xa inhibitors (3,4).

Danaparoid was used previously with success, but this product is no longer available. While the newer anticoagulants can be effective to stop the haemostatic activation, experience in their use is limited. Other disadvantages include difficulty monitoring the therapeutic effect, lack of effective antidotes for anticoagulant reversal in case of bleeding from overdose, and higher cost. Early diagnosis and management of HIT is therefore essential to prevent the morbidity or even death resulting from the uncontrolled thrombosis, and to avoid the unnecessary use of alternative anticoagulants.

The HIT diagnosis is based on clinical and laboratory criteria (5). The laboratory testing of HIT falls into immunological or functional groups (6). The immunological tests include enzyme-linked immunosorbant assays (ELISA) and particle-gel immunoassays (PaGIA). They are used to detect the presence of HIT antibodies. They are sensitive and can provide the results relatively rapidly. ELISA typically can produce the results in two to four hours, whereas the PaGIA has a turn-around time of within one hour. Both tests, however, have low specificity for the diagnosis of HIT, as they cannot distinguish between heparin-PF4 antibodies that are non-platelet-activating and hence innocuous, or pathological platelet-activating antibodies. The percentage of patients on heparin who develop HIT antibody is much higher than the actual incidence of clinical HIT. This difference is seen in all patient groups, but the cause is not known (2). A classic example is after cardiopulmonary bypass operations in which up to 50% of patients develop HIT antibodies by the fifth post-operative day, but clinical HIT is seen in 2-3% of post-cardiopulmonary patients only.

The laboratory diagnosis therefore frequently requires functional assays to demonstrate the platelet activation by the HIT antibody. The current functional tests include serotonin release assay, heparin-induced platelet aggregation test using platelet-rich plasma (PRP), and the emerging whole-blood impedence platelet aggregometry (7). They are specific but time consuming and require specialised skills and equipment. They are therefore usually done in specialised/referral centres only, and the results are not available for immediate patient management.

We aimed to develop a simple functional assay which can be done in all clinical laboratories with a modern blood cell counter with the result available rapidly, to facilitate the rapid confirmation of HIT for optimal patient management. The assay we have devised is based on the pathological mechanism of HIT: that that true pathogenic HIT antibody will activate platelets. One of the consequences would be platelet clumping, leading to a drop in the platelet count. We hypothesise that this drop in the platelet count in a donor blood specimen after exposure to heparin and the plasma from a patient suspected of HIT can be used to demonstrate the presence of the pathological HIT antibody.

Methods

Stored plasmas from patients with suspected HIT were used to develop the test. These plasmas were anonymised after the original clinical tests and stored as controls for HIT testing; therefore only a limited number of these samples were available. The HIT positive plasmas were confirmed by heparin-induced-platelet aggregation test (HIPAT) using light aggregometry and normal platelet-rich plasma. The negative plasmas were negative with a Particle-Gel-Immunoassay (IDPF4/heparin antibody test (Diamed AG, Cressier, Switzerland).

Citrated blood was collected from volunteer laboratory staff not on medication affecting platelet function and whose platelets had previously shown to be sensitive to platelet-activating HIT antibodies. Normal platelet-rich plasma (NPRP) was prepared by centrifuging at 170g for 10 minutes at 20[degrees]C and rested for 30 minutes at room temperature prior to testing.

After the initial optimisation, the test protocol was established as follows:

* Platelet count was performed on normal donor PRP using a Sysmex XE5000[TM] (Sysmex, Kobe, Japan).

* Four plastic centrifuge tubes were labelled and the following added:

Tube 1: 200 uL NPRP + 100 uL test plasma + 20 ul 8 IU/mL heparin.

Tube 2: 200 uL NPRP + 100 uL HIT negative (normal donor) plasma + 20uL8 IU/mL heparin

Tube 3: 200 uL NPRP + 100 uL test plasma + 20 uL 1,600 IU/mL heparin.

Tube 4: 200 uL NPRP + 100 uL test plasma + 20 uL saline.

* The tubes were inverted 5 times to gently mix the contents, incubated in a 37[degrees]C water-bath for 15 minutes, with gentle mixing every 5 minutes.

* After incubation, the platelet counts in the tubes were determined, and the results compared to the original platelet count of the NPRP.

[FIGURE 1 OMITTED]

Tube 1 was the test tube, Tube 2 the main control tube and Tubes 3 and 4 were additional control tubes. The test was done on the only remaining HIT positive plasma, six HIT negative plasma and the HIT-positive control provided in the PaGIA kit by the manufacturer.

Smears from Tube 1 of the HIT positive specimen before and after incubation were also made and stained with May-Grunwald-Giemsa stain to check for platelet aggregates.

Results

HIT-positive patient specimen

The platelet counts of the normal PRP and the mixture in the tubes after incubation for the HIT-positive case are summarised in Table 1 and Figure 1. The only sample that showed a significant drop in platelet count was the HIT positive plasma with 8 IU/mL heparin. The platelet count dropped from 384 x [10.sup.9]/L pre-incubation to 40 x [10.sup.9]/L post-incubation, with a posttest/normal PRP platelet count ratio of 0.10. The May-Grunwald-Giemsa stained smears confirmed that the drop in the platelet count was due to platelet aggregation (Figure 2).

The platelet counts in the control tubes (Tubes 2 to 4) also dropped after incubation but this could be explained by dilution and the mild platelet clumping due to the charge effect from the heparin (8). The post-test/normal PRP platelet count ratios in the control tubes were much higher (range 0.61-0.69).

The results can also be expressed by the platelet count ratio in the tubes compared to the main control (Tube 2 in the test protocol which contains HIT-negative, normal donor plasma with therapeutic concentration of heparin). This ratio is 0.15 for the HIT-positive plasma and around 1.0 for the other control tubes (Table 1).

[FIGURE 2 OMITTED]

HIT-negative patient specimen and commercial positive control

The range of the platelet count ratio of test/control tube for the HIT-negative specimens were 0.91 to 1.06, and for the commercial HIT-positive control was 0.08 (Table 2).

Discussion

This simple assay using a modern blood cell counter and based on the pathological mechanism of HIT syndrome shows great promise as a functional test for the laboratory diagnosis of HIT. The large drop in platelet counts in the HIT-positive patient specimen and commercial control but not in the HIT-negative specimens suggests this observation is genuine and not a spurious, chance finding.

Because of the constraint of the availability of HIT positive and negative specimens, however, the present results are only a proof-of-principle finding. As only one HIT positive specimen was available, the cut-off of the test/control platelet ratio in order to call a result positive cannot be determined from this study.

Further work is required to refine the test procedure, to validate the result on a large number of HIT positive and negative specimens, to understand its performance characteristics including the sensitivity, specificity and predictive values for HIT, and to define its role in conjunction with the rapid antigenic test for HIT diagnosis.

We also tried using EDTA whole blood as the source of platelets and incubating blood group 'O' donor blood with the test mixtures at room temperature. If successful this could expand the pool of donor platelets instead of relying on voluntary donors, and obviate the need to prepare platelet rich plasma. Our initial experiment however resulted in spontaneous non-specific platelet aggregation. Further optimisation could not be achieved due to difficulty obtaining more HIT positive plasma.

In conclusion, this simple assay using a routine blood cell analyser shows great promise as a new functional laboratory test for HIT testing. Because of its simplicity, even with platelet-rich plasma as the indicator system, the test can be done in all contemporary haematology laboratories, and the result is available rapidly. It can be particularly useful in cases where the specialised functional tests are not readily available. Even if further validation studies show this test does not reach the sensitivity and specificity to be used as a stand-alone diagnostic test, provided that it still has reasonably high positive and negative predictive values, it may still be useful clinically in conjunction with a rapid immunoassay such as PaGIA for rapid exclusion or confirmation of HIT in many patients, and reduce the need for the complex diagnostic functional tests.

References

(1.) Warkentin TE. Heparin-induced thrombocytopenia: pathogenesis and management. Br J Haematol 2003, 121: 535-555.

(2.) Cuker A, Clines DB. How I treat heparin-induced thrombocytopenia. Blood 2012; 119: 2209-2218.

(3.) Arepally GM, Ortel TL. Clinical practice. Heparin-induced thrombocytopenia. N Engl J Med 2006; 355: 809-817.

(4.) Krauel K, Hackbarth C, Furll B, Greinacher A. Heparin-induced thrombocytopenia: in vitro studies on the interaction of diabigatran, rivaroxaban, and low-sulphated heparin, with platelet-factor 4 and anti-PF4/heparin antibodies. Blood 2012; 119: 1248-1255.

(5.) Warkentin TE. Greinacher A, Gruel Y, Aster RH, Chong BH; Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis. Laboratory testing for heparin-induced thrombocytopenia: a conceptual framework and implications for diagnosis. J Thromb Haemost 2011; 9: 2498-2500.

(6.) Griffiths E, Dzik WH. Assays for heparin-induced thrombocytopenia. Transfus Med 1997; 7: 1-11.

(7.) Galea V, Khaterchi A, Robert F, Gerotziafas G, Hatmi, M, Elalamy I. Heparin-induced multiple electrode aggregometry is a promising and useful functional tool for heparin-induced thrombocytopenia diagnosis: confirmation in a prospective study. Platelets 2013; 24: 441-447.

(8.) Franchini M. Heparin-induced thrombocytopenia: an update. Thromb J 2005; 3: 14.

Author information

George TC Chan, MBBS FRCPath FRCPA FHKCPath, Consultant Haematologist

Sophie HS Lee, MBChB FRACP FRCPA, Haematology Registrar

Simon DF Jones, DipMLT RMLS, Technical Specialist, Haematology Department, LabPlus, Auckland City Hospital

Corresponding author: Dr George Chan, Haematology Department, LabPlus--Auckland City Hospital, Grafton Road, Auckland 1023, New Zealand. E-mail: georgec@adhb.govt.nz
Table 1. Platelet counts and post-test/normal PRP platelet count
ratio

                   Normal                 Post test
                    PRP
                            Tube 1   Tube 2   Tube 3   Tube 4

Platelet count      384       40      266      234      240
(x[10.sup.9]/L)

Ratio of Post-       --      0.10     0.69     0.61     0.63
test/Normal-PRP
platelets

Ratio of             --      0.15      1       0.89     0.90
platelet count
compared to the
control (Tube 2)

Table 2. Platelet count ratio of HIT-positive and HIT-negative
specimens

                HIT-        HIT-        HIT-
              positive    positive    negative
              specimen   commercial   specimens
                          control      (n = 6)

Platelet        0.15        0.08      0.91-1.06
count ratio
of Test/
Control
tubes
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Author:Chan, George T.C.; Lee, Sophie H.S.; Jones, Simon D.F.
Publication:New Zealand Journal of Medical Laboratory Science
Date:Aug 1, 2014
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