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. 2026 Jan 30;10(2):103373. doi: 10.1016/j.rpth.2026.103373

A new, rapid, chemiluminescence enzyme assay for the detection of heparin-induced thrombocytopenia: a comparison between functional and immune heparin-induced thrombocytopenia assays

Annette Bowyer 1,∗, Kiyoko Kohama 2, Irfan Patel 3, Katsushi Kobayashi 2, Ryo Hasegawa 2, Ayano Mukaida 2, Andreas Greinacher 3,4, Steve Kitchen 1
PMCID: PMC12955639  PMID: 41783862

Abstract

Background

Heparin-induced thrombocytopenia (HIT) is a potentially fatal complication of therapy with unfractionated or low molecular weight heparins that can result in thrombocytopenia and thrombosis. Timely clinical and laboratory diagnoses are imperative to prevent further morbidity and mortality.

Objectives

To evaluate a rapid high-sensitivity chemiluminescence luminescence enzyme immunoassay (HISCL HIT IgG assay) compared with automated and ELISA antigen assays and heparin-induced platelet activation (HIPA) functional assays on samples healthy donors, patients suspected of having HIT, and other patient subgroups.

Methods

The HISCL HIT IgG, HemosIL AcuStar HIT-IgG, HemosIL HIT-Ab(PF4-H), Hyphen BioMed ZYMUTEST HIA IgG, and Immucor PF4 IgG assay kits were used to measure plasma and/or serum samples from healthy donors, patients suspected with HIT, and other groups, including patients receiving intensive care, and samples containing potentially interfering substances. All patients suspected with HIT and other HIT-positive results were compared to HIPA functional assay.

Results

Of the 67 HIPA-positive samples, 67 were positive by the HISCL HIT IgG assay. Of the 142 HIPA-negative samples, 104 were negative with the HISCL HIT IgG assay (specificity 73.2%), compared with 97 of 142 negative results with the HemosIL AcuStar HIT-IgG assay (specificity 68.3%). The other assays exhibited lower specificity. The HISCL HIT IgG Assay Kit had no false positive results in samples with potentially interfering substances.

Conclusions

The HISCL HIT IgG assay had 100% sensitivity and superior specificity compared with other antigen assays. Good correlation was observed between plasma and serum samples, and the time to result was ∼25% faster than the HemosIL AcuStar HIT-IgG assay.

Keywords: chemiluminescence, heparin-induced thrombocytopenia (HIT), heparin-induced platelet activation (HIPA), 4Ts score

Essentials

  • •

    Heparin-induced thrombocytopenia (HIT) is a potentially fatal complication of therapy with some blood thinners.

  • •

    This study evaluated a new rapid diagnostic test for HIT, HISCL HIT IgG, and compared results with existing assays.

  • •

    Results were obtained one-third faster than with similar assays.

  • •

    HISCL HIT IgG correctly identified all positive cases and was more accurate than other assays.

1. Introduction

Heparin-induced thrombocytopenia (HIT) is a potentially fatal complication of unfractionated or low molecular weight heparin therapy, causing thrombocytopenia and thrombosis. Timely diagnosis is essential to prevent further morbidity and mortality [[1], [2], [3], [4], [5], [6], [7], [8], [9], [10]]. Two types of HIT exist: type 1 HIT is a benign, non–immune-mediated, transient thrombocytopenia occurring in 10% to 30% of patients receiving heparins, without thrombosis or increased mortality [[2], [3], [4]]. Type 2 HIT is a rare autoimmune disorder affecting up to 8% of patients, resolving after drug discontinuation, with 20% to 64% developing arterial or venous thrombosis [[5], [6], [7], [8]]. HIT development increases early mortality by 50%, necessitating prompt diagnosis [9,10].

HIT results from IgG antibodies recognizing platelet factor 4 (PF4) complexes bound to heparin or polyanions such as polyvinylsulphate or polyvinylsulfonate [11]. Heparin-PF4 binding causes PF4 conformational change and antibody generation [12]. The heparin-PF4-IgG complex activates platelets via Fc receptor binding, releasing PF4 in a positive feedback loop leading to thrombocytopenia and procoagulant microparticle release increasing thrombin generation [13].

Diagnosis of HIT requires clinical assessment and laboratory testing. Clinical pretest probability algorithms such as the 4Ts score predict HIT likelihood and guide laboratory testing [14,15]. Scores ≤3 indicate low probability, scores 4 to 5 intermediate probability, and scores 6 to 8 high probability [15]. Laboratory tests include platelet activation assays or immunologic antigen assays. Functional assays such as heparin-induced platelet activation [16,17] or serotonin release assay (SRA) [18,19] are the gold standard but are difficult to perform and limited to specialist centers. Antigen-based assays include ELISAs [20], automated latex immunoassays (LIAs) [21], or chemiluminescence immunoassays (CLIAs) [22]. Qualitative or semiquantitative assays such as lateral flow [23] or flow cytometry [24] are less commonly used. High-sensitivity IgG antigen assays should be used as a first-line screening assay with negative results excluding HIT [20].

We evaluated the HISCL HIT IgG Assay Kit on the Sysmex CN-6500 analyzer and compared results to those of LIA, CLIA, and ELISA assays in samples from healthy donors, patients suspected with HIT, and patients treated in the intensive care unit (ITU) and samples with potentially interfering substances including antiphospholipid antibodies, rheumatoid factor, hemolysis, icterus, and lipemia. Functional heparin-induced platelet activation (HIPA) assays were performed on samples from all patients suspected with HIT and samples from other groups that were antigen-positive by any test method.

2. Methods

2.1. Sample selection

Residual plasma or serum samples were collected from patients suspected with HIT, stored frozen in aliquots at −70 °C for up to 24 months, and thawed at 37 °C before analysis. Local ethics approval was obtained for the use of residual samples in laboratory service development.

A total of 209 samples from patients suspected with HIT were identified: 87 plasma-only, 76 serum-only, and 46 matched plasma/serum samples. These included local cases and referrals from across the UK (108 males, 99 females, 2 with unavailable data; median age 66 years, range 1-99 years). Samples from patients with suspected vaccine-induced thrombotic thrombocytopenia were excluded. Control donor samples comprised 122 matched plasma and serum samples from 64 female and 58 male healthy adults.

Twenty-six matched plasma and serum samples were obtained from patients treated in the general ITU (13 females, 13 males; median age 66.5 years, range 25-85 years). The HemosIL HIT-Ab(PF4-H) assay was not performed on ITU samples.

Twenty-eight plasma samples from patients with antiphospholipid antibodies (13 males and 15 females; median age 41 years, range 19-80 years) were included. The median dilute Russell's viper venom time ratio was 1.39 (range 1.20-1.90). HISCL HIT IgG was performed only on these samples.

Thirty-nine plasma samples from patients with rheumatoid factor >100 IU/mL were tested with HISCL HIT IgG only.

Thirty-five plasma samples flagged by the Sysmex CS-5100 analyzer as significant (flag of 4-5) for hemolysis, 30 for icterus, and 25 for lipemia were analyzed. The levels of hemolysis, icterus, or lipemia were not measured in these samples. ELISA assays were not performed on these samples.

2.2. HISCL HIT IgG assay

The HISCL HIT IgG Assay Kit detects HIT IgG antibodies using a 2-step sandwich chemiluminescent enzyme immunoassay (CLEIA), as outlined in Figure 1. Human PF4-heparin conjugated to magnetic particles magnetic particles reacts with HIT IgG antibodies in the samples. After washing steps, mouse anti-human IgG monoclonal antibodies conjugated to alkaline phosphatase bind to HIT IgG on the magnetic particles. The alkaline phosphatase-labeled complex reacts with CDP-Star substrate, producing a luminescent signal proportional to antibody concentration. Luminescence signals were compared to 5-point calibration curves using the HISCL HIT IgG Calibrator containing known HIT IgG concentrations (manufacturer provided). Results are expressed in units per milliliter (U/mL). Time to first result is ∼25 minutes. Initially, a single calibration curve was used for both plasma and serum samples. Separate calibration curves were subsequently evaluated for plasma and serum samples. This assay was performed on Sysmex CN-6500 instrumentation.

Figure 1.

Figure 1

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Illustration of the steps in the HISCL HIT IgG chemiluminescence enzyme immunoassay (CLEIA). Step 1: patient plasma or serum is mixed with human PF4/heparin-conjugated magnetic particles; step 2: a BF separation washing step precedes addition of alkaline phosphatase-conjugated anti-human IgG antibody. A second BF separation then occurs; step 3-buffer solution and chemiluminescent substrate is added then the resulting luminescence intensity measured. ALP, alkaline phosphatase; BF, bound/free; HIT, heparin-induced thrombocytopenia; PF4, platelet factor 4.

2.3. Other HIT antigen assays

The HemosIL HIT-Ab(PF4-H) assay was performed on an ACL TOP 700 (Werfen) with plasma samples only (the other assays were suitable for both plasma and serum). The HemosIL AcuStar HIT-IgG (PF4-H) assay was performed on an ACL AcuStar (Werfen). Two ELISA assays were used: ZYMUTEST HIA IgG (Hyphen Biomed) and Immucor Lifecodes PF4 IgG (Immucor).

Manufacturer-recommended cutoff limits determined HIT positivity: HISCL HIT IgG ≥0.60 U/mL, HemosIL IgG HIT-Ab(PF4-H) ≥1.00 U/mL, and AcuStar HIT-IgG ≥1.00 U/mL. Upper limits were 128.00 U/mL for plasma and 108.03 U/mL for serum in the HISCL HIT IgG. The AcuStar HIT-IgG upper limit was 128.00 U/mL for both sample types, and HIT-Ab(PF4-H) was 29.90 U/mL, measured in plasma only.

ELISA antigen assays used the following optical density (OD) cutoffs: ZYMUTEST OD 0.30 to 0.50 was considered weakly positive and >0.50 positive; Immucor OD ≥0.40 was considered positive.

Paired plasma and serum samples were analyzed in parallel in the same assay runs, and there was no bias in the order of testing of automated or ELISA assays. Antigen assays were performed at the Royal Hallamshire Hospital, Sheffield.

2.4. Functional assays

The HIPA assay was performed on all samples from patients suspected of HIT and samples from other groups that were positive in any antigen assay. HIPA testing was performed at the Institute for Transfusion Medicine, Greifswald University Hospital, Greifswald, Germany using previously published methodology [16]. HIPA-positive results indicated HIT, and negative results excluded HIT. Greifswald investigators were blinded to clinical and laboratory test information.

2.5. Statistical analysis

All statistical analyses were performed using Graphpad Prism V10. Data comparison was conducted using Wilcoxon matched-paired signed rank tests. Kruskal-Wallis nonparametric analysis of variance was used to compare results of different tests. Results with P > .05 were considered statistically significant. Spearman correlation coefficient was used to assess correlations between results.

3. Results

3.1. HISCL HIT IgG assay characteristics

The initial 5-point calibration curve was constructed in duplicate using the HISCL HIT IgG Calibrator with values of 0, 1.0, 4.0, 16.0, and 128.0 U/mL. Results exceeding the calibration curve upper limit were reported as >128.0 U/mL (plasma/serum). Ten calibration curves were performed over a 5-month period. The percentage coefficient of variation (CV) between luminescence results at each calibration point was <6.5%. Sysmex Corporation evaluated separate calibration curves for plasma and serum samples (Supplementary Figure S1). All serum data were back-calculated against the serum curve to offset anticoagulant dilution effects and ensure comparable HIT results between sample types. A subsequent study of paired plasma and serum samples using respective calibration curves (HITp and HITs) validated 2-curve use with excellent correlation (Spearman correlation coefficient r = 0.9918) (Supplementary Figure S2).

Within-run variation was established by running 10 consecutive samples of 2 HISCL HIT IgG quality control (QC) levels: Level 1 (Lv.1) and Level 2 (Lv.2). Lv.1 yielded a mean HIT of 2.95 U/mL with CV of 2.6%. Lv.2 yielded a mean HIT of 0.49 U/mL with CV of 1.7%. Lv.1 and Lv.2 were performed daily. Lv.1 was tested 20 times with mean HIT of 3.04 U/mL and CV of 3.9% Lv.2 was tested 20 times with mean HIT of 0.51 U/mL and CV of 5.5% (quality control data not shown).

3.2. Establishment of cutoff for positivity with HISCL HIT IgG

Different cutoff values for HISCL HIT IgG positivity were explored at study start by comparing HIPA and HISCL HIT IgG results. Using ≥1.00 U/mL cutoff, 3 false-negative HISCL HIT IgG results (0.66, 0.89, and 0.92 U/mL) occurred in HIPA-positive samples. Sensitivity was 92.5% and specificity 81.7%. Reducing the cutoff to ≥0.80 U/mL increased sensitivity to 97.5%, with specificity reduced to 77.4%. The positivity cutoff was then reduced to ≥0.60 U/mL, achieving 100% sensitivity with 73.2% specificity. The ≥0.60 U/mL cutoff for HISCL HIT IgG positivity was used throughout statistical analysis.

3.3. Healthy donors

All automated HIT antigen assays were negative in the control donor group (median HIT 0 U/mL for the HISCL HIT IgG and AcuStar HIT-IgG assays, 0.10 U/mL for HIT-Ab(PF4-H)). The ZYMUTEST ELISA assay was weakly positive in 1 serum sample (OD 0.32) and positive in a single donor with both plasma (OD 0.96) and serum (OD 1.05) samples. Immucor ELISA showed positivity in 4 plasma samples (OD 0.54-1.31) and 7 serum samples (OD 0.41-1.59). The 4 positive plasma samples were also positive in matched serum samples. Positive samples underwent HIPA testing; all were negative (Table 1).

Table 1.

Summary of HIT data: mean, median, and range of results from each antigen assay in different sample groups.

Group, n HISCL HIT IgG (U/mL) mean, median (range)
 AcuStar HIT-IgG (U/mL) mean, median (range)
 HIT-Ab(PF4-H) (U/mL) mean, median (range)
 ZYMUTEST (OD) mean, median (range)
 Immucor (OD) mean, median (range)
Plasma Serum Plasma Serum Plasma Plasma Serum Plasma Serum
Measurement range 0-128 0-108 0-128 0-128 0-29.9 0- 0- 0- 0-
Cutoff for positivity ≥0.6 ≥0.6 ≥1.0 ≥1.0 ≥1.0 >0.5 >0.5 >0.4 >0.4
Healthy donors, 122 0, 0 (0-0.05) 0, 0 (0-0.06) 0.3, 0 (0-0.56) 0.3, 0
0-0.54		 0.12, 0.10 (0-0.50) 0.10, 0.09 (0.01-0.96) 0.12, 0.09 (0.01-1.05) 0.15, 0.11 (0.05-1.31) 0.17, 0.12 (0.03-1.59)
ITU, 26 0.01, 0 (0-0.19) 0.01, 0 (0-0.18) 0.06, 0 (0-0.45) 0.07, 0 (0-0.54) ND 0.11, 0.09 (0.03-0.27) 0.12, 0.10 (0.03-0.29) 0.13, 0.12 (0.05-0.32) 0.15, 0.15 (0.05-0.37)
APS, 28 0.02, 0 (0-0.34) ND ND ND ND ND ND ND ND
RF, 39 0.01, 0 (0-0.07) ND ND ND ND ND ND ND ND
Hemolysis, 35 0.01, 0 (0-0.16) ND 0.06, 0 (0-0.89) ND 0.17, 0.1 (0-0.7) ND ND ND ND
Icterus, 30 0, 0 (0-0.02) ND 0.02, 0 (0-0.18) ND 0.49, 0.3 (0-5.6) ND ND ND ND
Lipemia, 25 0.03, 0 (0-0.18) ND 0.12, 0 (0-2.02) ND 0.46, 0.4 (0-1.20) ND ND ND ND
Patients suspected with HITa, 209 16.46, 0.22 (0 to >128.0) 14.86, 0.82 (0 to >128.0) 12.93, 0.96 (0 to >128.0) 13.66, 01.80 (0 to >128.0) 6.02, 1.1 (0-29.9) 1.08, 0.78 (0.01-2.77) 1.20, 0.94 (0.03-2.75) 0.95, 0.43 (0.05-3.21) 1.03, 0.51 (0.05-3.32)
Patients suspected with HIT, paired samples, 46 13.46, 0.54 (0 to >128.0) 13.50, 0.55 (0 to >128.0) 8.34, 0.98 (0-78.1) 8.75, 0.79 (0-80.3) ND 1.10, 0.91 (0.03-2.65) 1.16, 0.91 (0.04-2.68) 0.85, 0.43 (0.06-2.80) 0.88, 0.46 (0.06-2.78)
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Ab, antibody; APS, antiphospholipid antibody positive samples; HIT, heparin-induced thrombocytopenia; ITU, general intensive care samples; ND, not done; OD, optical density; RF, rheumatoid factor positive samples.

a

All 209 suspected HIT samples were tested—87 were plasma only, 76 were serum only, and 46 were matched plasma/serum.

Correlation between healthy donor plasma and serum samples was calculated by Spearman correlation coefficient, ranging from r = 0.7649 to 0.9396 (AcuStar HIT-IgG and ZYMUTEST, respectively). Wilcoxon matched-pairs signed rank test showed statistically significant differences in all antigen assays performed in healthy donor samples: P = .001 for AcuStar-IgG HIT, P = .001 for HISCL HIT IgG, and P < .0001 for both ZYMUTEST and Immucor ELISAs (Table 2).

Table 2.

Statistical comparison of plasma and serum matched paired samples by Wilcoxon nonparametric matched test and Spearman correlation.

Group, n HISCL HIT IgG
 AcuStar HIT-IgG
 ZYMUTEST
 Immucor
Spearman r Wilcoxon P Spearman r Wilcoxon P Spearman r Wilcoxon P Spearman r Wilcoxon P
Healthy donors, 122 .88 .001a .76 .01a .93 <.0001a .93 <.0001a
ITU, 26 .76 >.99 .88 .01a .98 .001a .96 <.0001a
Patients suspected with HIT, 46 .99 <.009a .97 .26 .98 <.0001a .98 .015a
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HIT, heparin-induced thrombocytopenia.

a

P <.05 is considered statistically significant.

3.4. ITU and potentially interfering substances groups

All automated and ELISA results were HIT negative in general ITU patient samples. HIT-Ab(PF4-H) was not performed due to inability to conduct plasma/serum comparison. No statistically significant difference between plasma and serum samples was observed for HISCL HIT IgG (P > .99), but statistical significance was found for other assays studied (P = .01 to <.0001). Spearman correlation r ranged from 0.7643 (HISCL) to 0.9811 (ZYMUTEST) (Tables 1 and 2).

Plasma samples containing antiphospholipid antibodies or high rheumatoid factor levels were tested only with the HISCL HIT IgG assay and were HIT negative (HIT <0.34 U/mL); this contrasts with the AcuStar HIT IgG assay, which does report overestimation of the test result in the presence of rheumatoid factor [25]. Plasma samples containing hemolysis, icterus, or lipemia were tested with 3 automated antigen assays. All samples tested negative with the HISCL HIT IgG assay. Mean and median values from the HIT-Ab(PF4-H) assay were higher than those obtained with HISCL HIT IgG or AcuStar HIT-IgG assays (Table 1). One icteric sample was positive with HIT-Ab(PF4-H) (5.6 U/mL). Three separate lipemic samples were HIT positive; 1 with AcuStar HIT-IgG (2.02 U/mL) and 2 with HIT-Ab(PF4-H) (both 1.20 U/mL). All these antigen assay positive samples were negative by HIPA, thus considered HIT negative. Kruskal-Wallis test calculated statistically significant differences between hemolyzed plasmas tested by 3 automated assays (P = .0008), whereas icteric and lipemic samples were both P < .0001 (Tables 1 and 2).

3.5. Patients suspected with HIT

3.5.1. All patient data

A total of 209 samples from patients suspected with HIT were included in the study: 87 plasma-only, 76 serum-only, and 46 matched plasma/serum samples (totaling 133 plasma and 122 serum samples). The overall mean plasma HIT results were positive for all HIT antigen and ELISA assays; however, median HIT results were negative with the HISCL HIT IgG and AcuStar HIT-IgG assays but positive for the remaining assays (Table 1). Mean and median serum HIT results were positive for each assay. Friedman nonparametric test was calculated for 3 automated HIT assays plasma results (P = .056, not statistically significant), whereas Wilcoxon matched pairs nonparametric test was calculated for the remaining assay pairs: HISCL HIT IgG/AcuStar HIT-IgG serum samples (P = .61), ZYMUTEST/Immucor plasma samples (P = .005), and ZYMUTEST/Immucor serum samples (P = .007).

3.5.2. Paired plasma/serum results

Forty-six patients suspected with HIT had both plasma and serum samples available: 13 HIPA-positive and 33 HIPA-negative (Tables 1 and 2; Figure 2). Spearman correlation coefficients between plasma and serum results ranged from r = 0.9745 (AcuStar HIT-IgG) to r = 0.9991 (HISCL HIT IgG) (Figure 1). Wilcoxon matched-pairs signed rank test showed statistically significant differences in HISCL HIT IgG (P = .009), ZYMUTEST (P < .0001), and Immucor (P = .052) but not AcuStar HIT-IgG (P = .26), but these would not change clinical management of the patient. An additional group of 35 paired matched plasma and serum samples validated separate HISCL HIT IgG curves for both matrices. Spearman correlation coefficient r was 0.9918 for these samples. Comparison of original single curve and separate plasma/serum curves is shown in Supplementary Figure S1, and correlation in the 35 samples is shown in Supplementary Figure S2.

Figure 2.

Figure 2

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Matched plasma and serum results in 46 samples suspected with HIT. Correlation coefficient r is shown for each assay. HIT, heparin-induced thrombocytopenia; OD, optical density.

3.5.3. HIPA results

Sixty-seven patients (27 plasma-only, 27 serum-only, and 13 both) were HIPA positive (Table 3). All antigen assays, except HIT-Ab(PF4-H), correctly identified all HIPA-positive patients with 100% sensitivity. One HIPA-positive patient repeatedly tested negative by HIT-Ab(PF4-H) (208 and 204 monoclonal antibodies, 0 U/mL) but was HIT-positive with HISCL HIT IgG (3.13 U/mL), AcuStar HIT-IgG (2.64 U/mL), ZYMUTEST (OD 2.10), and Immucor (OD 1.74).

Table 3.

Assay results from samples from patients suspected with HIT.

Assay Patient status HIPA + HIPA − Total Sensitivity (HIPA+, Ag+) Specificity (HIPA−, Ag−)
HISCL HIT IgG + 67 38 105 100%
− 0 104 104 73.2%
Total 67 142 209
AcuStar HIT-IgG + 67 45 112 100%
− 0 97 87 68.3%
Total 67 142 209
HIT-Aba + 39 28 67 97.5%
− 1 65 66 69.8%
Total 40 93 133
ZYMUTEST HIT + 67 59 126
− 0 83 83 100% 58.5%
Total 67 142 209
Immucor PF4 + 67 50 117
− 0 92 92 100% 64.5%
Total 67 142 209
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A comparison of antigen assays with HIPA. N = 209; 67 were HIPA-positive, 142 were HIPA-negative. In 46 matched serum and plasma samples, only plasma results were included in this table.

Ab, antibody; Ag, antigen; HIPA, heparin-induced platelet activation; HIT, heparin-induced thrombocytopenia; PF4, platelet factor 4.

a

Only plasma samples were tested.

Of the patients, 142 (60 plasma-only, 49 serum-only, and 33 both) were HIPA negative (Table 3). In this HIPA-negative group, each antigen assay had varying proportions of HIT-positive results ranging from 26.8% (HISCL HIT IgG) to 41.5% (ZYMUTEST).

3.5.4. 4Ts score

4Ts score was available for 118 of 209 patients: 32 HIPA positive, 86 HIPA negative. The median 4Ts score was 5 (range 2-8, mode 6). Low probability of HIT (4Ts ≤3) scores were obtained for 11 patients: 9 HIPA negative, 2 HIPA positive. Intermediate probability of HIT (4Ts 4-5) scores were obtained for 59 patients: 50 HIPA negative, 9 HIPA positive. High probability of HIT (4Ts 6-8) scores were found in 47 patients: 27 HIPA negative, 21 HIPA positive. Summary data is shown in Table 4.

Table 4.

Sex, age, and 4T score distribution in HIPA positive and negative samples from patients suspected with HIT.

Patient details HIPA positive
 HIPA negative
Females Males Females Males
No. of participants 39 28 62 80
Median age (y) 69 71 62 66
HISCL positive, n 21 10 3 10
HISCL negative, n 0 0 23 28
4Ts score 0-3, n 1 1 4 5
4Ts score 4-5, n 7 2 14 36
4Ts score 6-8, n 13 8 15 12
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4Ts scores were available for 118 patients, of whom 32 were HIPA-positive (21 females and 11 males) and 86 were HIPA-negative (33 females and 53 males).

4. Discussion

This study evaluated the HISCL HIT IgG rapid automated CLEIA assay for detection of IgG antibodies reacting with heparin-PF4 complexes, comparing sensitivity and specificity with other commercially available HIT antigen assays. The HIPA functional assay served as the reference standard for determining HIT positivity and comparing results with the HISCL HIT IgG assay and other antigen assays.

The cutoff value for HISCL HIT IgG positivity was established at ≥0.60 U/mL in plasma and serum samples. No false-positive results were observed in samples from healthy donors and general ITU patients or in samples containing potentially interfering substances. The HISCL HIT IgG assay demonstrated equivalent sensitivity and superior specificity compared with the other antigen assays tested. The manufacturers’ thresholds for positivity were used for the other automated and ELISA assays. The 510(k) submission to the US Food and Drug Administration for the AcuStar HIT-IgG assay reports the assay cutoff was assessed by comparison with SRA in 87 citrated patient samples, and the HIT-Ab(PF4-H) cutoff was assessed in 63 patient plasma samples and also compared to SRA [25,26]. ZYMUTEST HIA IgG cutoffs were evaluated in HIT-negative hospital patients, and healthy donors. Cutoff was established as mean +3SD in hospital patients, which corresponded to +5SD in healthy donors. Specificity was assessed by comparison to SRA in 174 samples [27]. Data is not readily available for the Immucor assay.

4.1. Plasma vs serum samples

To enhance laboratory flexibility, HIT assays should accommodate both citrated plasma and serum samples. There is a potential matrix effect between plasma and serum samples linked to the presence of liquid trisodium citrate in plasma tubes, which is not in serum tubes. HIT IgG antibody levels in citrated plasma may be ∼85% of those obtained with serum samples because of the effect of liquid anticoagulant, and the patient hematocrit will also have an impact. The AcuStar HIT-IgG assay uses a single calibration curve with an algorithm correcting plasma results relative to serum results, reporting no difference in HIT positivity between sample anticoagulant [28].

Initial HISCL HIT IgG results were compared using a single calibration curve, revealing ∼10% difference between paired plasma and serum samples. Sysmex Corporation subsequently evaluated separate calibration curves for plasma and serum samples with good correlation between sample types. HISCL HIT IgG results from Sheffield using the single calibration curve were back-calculated against appropriate separate curves. This backfitting did not alter test result positivity levels. Good agreement was observed between matched plasma and serum samples from healthy donors, ITU patients, and HIT-suspected patients across the HISCL HIT IgG, AcuStar HIT-IgG, ZYMUTEST, and Immucor assays.

4.2. Samples from patients in ITU or containing potentially interfering substances

Thrombocytopenia is common in ITU settings, with reports estimating 30% to 50% of patients experiencing episodes of thrombocytopenia and thrombosis that are unrelated to heparin therapy [29,30]. Cardiac surgery patients, exposed to heparin during cardiopulmonary bypass, exhibit higher HIT positivity rates than other patient groups [9,31]. This study included only general ITU patients, who have been reported to have lower HIT incidence [9]. All antigen assay results were HIT-negative in this patient group.

Laboratory assays may be interfered with by lipids (lipemia), bilirubin (icterus), free hemoglobin (hemolysis), and antiphospholipid antibodies. False-positive LIA HIT results have been reported in cases of severe hyperbilirubinemia [32]. Elevated rheumatoid factor interferes with animal IgG antibodies in immunoassays, causing falsely high results [33]. Autoimmune disorders producing excessive antibodies, such as antiphospholipid antibody syndrome (APS), can also lead to false positive immunoassay results [34]. The HISCL HIT IgG assay was negative in samples containing hemolysis, icterus, lipemia, high rheumatoid factor levels, and APS. Some false positive HIT results occurred with HIT-Ab(PF4-H) and AcuStar HIT-IgG assays in lipemic or icteric samples.

4.3. Assay sensitivity/specificity

Accurate and timely laboratory diagnosis of HIT reduces morbidity and mortality in type 2 HIT. Treatment of false-positive HIT is to use nonheparinoid anticoagulants, which are associated with an increased bleeding risk and add an estimated $25,000 to patient stay costs [35,36]. Conversely, false-negative laboratory results can be fatal, with 30% to 50% of patients with untreated HIT developing thrombosis and have a 5% mortality risk [37]. False-positive HIT incidence with HIT-Ab(PF4-H) has been reported at 5.5% in a single US laboratory but up to 95% in other studies [38,39]. Functional assays represent gold standard tests offering greater specificity than antigen assays. SRA shows ∼95% sensitivity and specificity [40], whereas HIPA demonstrates slightly higher sensitivity than SRA for recognizing platelet-activating antibodies [41]. Due to functional assay complexity, antigen assays are more widely available and used as screening tools until confirmation by SRA or HIPA is possible. Lower antigen assay specificity results in significant false-positive rates.

The HISCL HIT IgG assay sensitivity matched that of AcuStar HIT-IgG, ZYMUTEST, and Immucor (all 100%) and exceeded that of HIT-Ab(PF4-H) (98%). Specificity was higher with the HISCL HIT IgG (73.2%) assay than the other assays studied due to lower false-positive rates. AcuStar HIT-IgG, HIT-Ab(PF4-H), and ZYMUTEST assay specificity was lower in our study than previously published (68.3%, 69.8%, and 87.5% respectively) [42]. The AcuStar HIT-IgG CLIA has been reported to have 98% sensitivity and 98% specificity [22]. The HemosIL HIT-Ab(PF4-H) LIA demonstrated lower sensitivity (∼95%) and specificity (95%) compared with SRA and polyspecific enzyme immunoassay [21] (Supplementary Table S1). Other commercial HIT IgG assays are globally marketed but were not included in this study due to issues with local availability. This could be considered as a limitation to the study.

Some authors have proposed the use of a high threshold value that would enable a specificity of 100% with the ELISA assays (OD >2.0) or AcuStar HIT-IgG CLIA (>10 U/mL) [43,44]. In our HIPA-negative samples, 15 HISCL HIT IgG samples gave positive results as high as 32 U/dL, 14 AcuStar HIT-IgG samples were positive up to 85 U/mL, and 15 ZYMUTEST and 10 Immucor samples had ODs >2.0. Our data does not support the use of an upper threshold value for these assays.

During this study, manufacturer-recommended cutoff OD limits were used for comparator assays. ELISA false-positive rates exceeded those of automated assays. Several publications recommend a higher cutoff OD in HIT ELISAs to enhance positive predictive values and reduce overdiagnosis [[45], [46], [47], [48], [49]]. In our cohort, false HIT-positive results in healthy donor samples occurred with ELISA antigen assays (2 with ZYMUTEST and 7 with Immucor PF4). Using a cutoff ≥1.0 OD reduced this to 0 false positives with ZYMUTEST and 2 false positives with Immucor. Similarly, in HIPA-negative patients, 59 false positives with ZYMUTEST would be reduced to 43 with ≥1.0 U/mL cutoff and 61 false positives with Immucor would be reduced to 32.

One HIPA-positive patient repeatedly tested negative with HIT-Ab(PF4-H) (208 and 204 monoclonal antibodies, 0 U/mL) but was positive with HISCL HIT IgG (3.13 U/mL), AcuStar HIT-IgG (2.64 U/mL), ZYMUTEST (OD = 2.10), and Immucor (OD = 1.74). This patient was a 70-year-old postoperative female with lung cancer who received low molecular weight heparin and was switched to anti-IIa drug argatroban (Exembol; Ethypharm). The HIT-Ab(PF4-H) assay is a LIA containing human PF4 and polyvinylsulfonate, but the reason for negative results remains unclear. Previous studies have also reported false-negative HIT with the HemosIL HIT-Ab(PF4-H) LIA [21].

4.4. 4Ts score

UK and US guidelines recommend pretest scores such as the 4Ts score to aid HIT diagnosis [20,50]. The 4Ts score shows >90% sensitivity and ≤38% specificity for HIT [[51], [52], [53]]. Scoring variation due to clinician experience is a disadvantage. Low 4Ts scores show high negative predictive value, but intermediate scores (4-5) have low positive predictive value. Some studies question the reliability of low 4Ts scores for excluding HIT, with our data supporting this [54,55]. A 4Ts score was not available for all patients suspected of HIT because some samples were provided by distant hospitals, representing a study limitation. Of the HIPA-positive patients, 6.3% had low 4Ts scores, and 31.3% of HIPA-negative patients had high probability 4Ts scores.

5. Conclusion

This study focused solely on using the HISCL HIT IgG Assay Kit for type 2 HIT diagnosis. Further research is warranted to determine if HISCL HIT IgG assay applications are appropriate for other PF4 disorders such as autoimmune HIT, vaccine-induced thrombotic thrombocytopenia, and spontaneous HIT.

The HISCL HIT IgG assay is a rapid, automated HIT CLEIA demonstrating 100% sensitivity and higher specificity than other commercially available immunoassays studied, evidenced by lower false-positive HIT rates. The assay is insensitive to common interfering substances including hemolysis, bilirubin, lipids, rheumatoid factor, and APS. Importantly, the assay requires no additional specialist instrumentation investment.

Acknowledgments

The authors would like to thank Ulrike Strobel, formally of Institut für Transfusionsmedizin, Universitätsmedizin Greifswald, Greifswald, Germany for performing the HIPA analysis and Sean Platton at the Royal London Hospital, UK for contributing some patient samples for this study.

Funding

Reagents used in this study were funded by Sysmex Corporation, Japan.

Author contributions

A.B. performed the testing, analyzed the data, and wrote the first draft. K. Kohama and I.P. devised the study, analyzed the data, and edited the final draft. S.K., K. Kobayashi, R.H., and A.M. devised the study and edited the final draft. A.G. supervised the HIPA analysis and edited the final draft. All authors read and approved the final version of the paper.

Relationship Disclosure

K. Kohama, I.P., K. Kobayashi, R.H., and A.M. are employees of Sysmex UK or Sysmex Corporation. A.B. and S.K. have received honoraria and funding for conference attendance from Sysmex. A.G. reports grants and nonfinancial support from Aspen, Boehringer Ingelheim, MSD, Bristol Myers Squibb (BMS), Paringenix, Bayer Healthcare, Gore Inc, Rovi, Sagent, and Biomarin/Prosensa; personal fees from Aspen, Boehringer Ingelheim, MSD, Macopharma, BMS, Chromatec, and Werfen; and nonfinancial support from Boehringer Ingelheim, Portola, Ergomed, and GTH e.V. outside the submitted work.

Footnotes

Handling Editor: Professor Michael Makris

The online version contains supplementary material available at https://doi.org/10.1016/j.rpth.2026.103373.

Supplementary material

Supplementary Figure 1.

Supplementary Figure 1

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Supplementary Figure 2.

Supplementary Figure 2

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