Current Perspectives on Diagnostic Assays and Anti-PF4 Antibodies for the Diagnosis of Heparin-Induced Thrombocytopenia

Figures & data

Figure 1 Guide to decision-making for HIT from suspicion to diagnosis, with advantages and disadvantages of the assays.

Table 1 Clinical scoring systems and variables used, with advantages and disadvantages

	Factors	Advantages	Disadvantages	Scoring system
4T score Lo et al^Citation19	Thrombocytopenia Timing of platelet count fall Thrombosis or other sequelae Other causes of thrombocytopenia	A commonly used screening test in the diagnostic pathway to HIT diagnosis		≤3 points: low probability for HIT (≤5% in original study, <1% in meta-analysis) 4–5 points: intermediate probability (~14% probability of HIT) 6–8 points: high probability (~64% probability of HIT)
HEP score (HIT-expert probability) Cuker et al^Citation20	Magnitude of platelet-count fall Timing of platelet-count fall Nadir platelet count Thrombosis Skin necrosis Acute systemic reaction Bleeding Other causes of thrombocytopenia	A cutoff value of 5 has similar predictive value to 4T score	HEP score is more complex and may be more time-consuming than 4T score	Score ranging from –3 (inconsistent with an HIT diagnosis) to 3 (consistent with an HIT diagnosis)
HIT score in cardiopulmonary bypass^a Lillo-Le Louët et al^Citation21	Platelet-count time course Time from CPB to index date CBP duration	Designed to determine probability without knowledge of laboratory-test results (except platelet counts) to avoid delays	Only applicable to assess the probability of HIT in patients following cardiopulmonary bypass surgery Requires more validation in larger prospective studies Lower sensitivity may be inappropriate as a clinical screening test	<2 = low probability of HIT ≥2 = high probability of HIT
Simple clinical scoring system^a Messmore et al^Citation23	Presence or absence of Heparin therapy for 5 days preceding platelet-count drop Platelet-count fall by >30% or not Other cause for thrombocytopenia present or not Development of new thrombosis or not		Requires more validation in larger prospective studies	0 = low probability of HIT 1 = possible HIT

Notes: ^aThis scoring has not been prospectively validated and is not yet recommended for clinical use. Nevertheless, it offers another option for guiding clinical decision-making.

Figure 2 The five phases of HIT, ranging from cases of suspected HIT without confirmatory laboratory tests; acute HIT, a highly prothrombotic phase that persists until platelet-count recovery through subacute and remote HIT, where functional and immunologic assays return to negative.

Figure 3 Timeline from discovery of platelets, HIT, and subsequent diagnostic milestones.

Table 2 Variables posing challenges while conducting functional assays and interpreting results

Factors	Variables
Activation end point	Radioactive signal vs flow-based vs light-transmission detection
Technology used	Local availability
Platelet-donor selection	Individual, pooled, standardized or not
Platelet suspension	Washed platelets, platelet-rich plasma, or whole blood
Patient sample	Serum or plasma
Heparin used	Type and concentration
Interlaboratory variability	Lack of standardization
Optical density values	Cutoffs vary for different kit manufacturers

Table 3 Functional and immunologic assays: end points and methodological principles

Functional
	Methodology	End point
Serotonin release-based assays
^14C SRA	β counter	Detects % release of ^14C labeled serotonin release from activated platelets
EIA SRA (type of functionalized immunoassay)	ELISA-based	Serotonin release from activated platelets
HPLC SRA	HPLC	Serotonin release from activated platelets
Flow cytometry–based assays
FCA-intraplatelet serotonin assay	Flow cytometry	Detecting loss of intraplatelet serotonin stores within activated platelets
FCA-membrane GP	Flow cytometry	Detecting increased surface expression of activation markers like P-selectin or anionic phospholipids
PMPGA	Flow cytometry	Detection of PMPs
Platelet aggregation detection–based assays
HIPA	Visual assessment	Visual detection of platelet aggregation
PAT	Using platelet aggregometer	Detecting change in light transmittance due to aggregation of activated platelets
HIMEA	Multiple-electrode aggregometry	Detecting change in impedance due to aggregation of activated platelets on electrodes
HIT-mimicking monoclonal antibody–based assays
5B9, IgG1 antibody	—	Formation of a 5B9–PF4 complex
HemosIL HIT-Ab (PF4-H) assay using KKO, an IgG2bκ antibody	Latex particle–enhanced immunoturbidimetry	Inhibition of agglutination of KKO-coated latex beads to PF4–polyvinylsulfonate complexes in the presence of human anti-PF4/heparin antibodies
Miscellaneous
ATP-release assay	Lumiaggregometery	Detecting ATP release from activated platelets
TGA	Fluorometer	Detecting fluorescence or light emitted from generation of thrombin
FcγRIIa-proteolysis assay	Western blot/densitometry	Detecting proteolysis of FcγRIIa
LIA	Turbidimetry	Opposite to conventional immunoassays in that presence of HIT antibodies results in inhibition of platelet agglutination through competition with an HIT-mimicking monoclonal antibody
DT40-Luc, a cell-based functional assay	Luciferase assay luminometry	Detecting luciferase activity when human FcγRIIa-transfected chicken cell line coupled to a luciferase reporter is activated by HIT antibodies
Immunologic assays
Immunologic assay	Methodology	End point
Optical density-based assay
ELISA	OD	Semiquantitative test, assesses the intensity of color change by measuring OD, which is proportional to the concentration of bound antibodies
Visual assessment–based assay
PaGIA	Visual assessment	Visual assessment of agglutination
Particle-immunofiltration assay (PIFA)	Size-dependent filtration	Inability of matrixed microparticles (formed as a result of antigen–antibody interaction) to move through the filter, with resulting color change indicating a positive test
Lateral-flow immunoassay (STiC)	Immunochromatography/lateral flow	Based on capillary action, which induces a flow of the test sample (patient serum) along a solid phase (gold nanoparticles coated with an antiligand); visible line upon addition of buffer containing biotinylated human PF4–polyanion (PA) complex indicates a positive reaction
Miscellaneous
Chemiluminescent immunoassay (AcuStar HIT-IgG)	Detection of emitted light produced by the reaction utilizing H202 and catalyst	The tracer (isoluminol-labeled antibody) binds specifically to the PF4–antibody complex and subsequently emits light when an oxidizer and a catalyst are added, indicating positive result
Latex-agglutination assay	Agglutination/clumping of latex beads	Patient sample mixed with latex beads coated with a PF4 antigen; clumping of latex beads (agglutination) indicates the presence of HIT antibodies

Abbreviations: ATP, adenosine triphosphate; ¹⁴C, carbon 14; EIA, enzyme immunoassay; DT40-Luc, DT40 luciferase; FCA, flow-cytometry assay; GP, glycoprotein; HPLC, high-pressure liquid chromatography; HIT, heparin-induced thrombocytopenia; HIMEA, heparin‐induced multielectrode aggregometry; HIPA, heparin-induced platelet activation; KKO, murine HIT–like monoclonal antibody; LIA, latex immunoturbidimetric assay; OD, optical density; PF4, platelet factor 4; PAT, platelet-aggregation assay; PMPGA, platelet microparticle–generation assay; PRP, platelet-rich plasma; SRA, serotonin-release assay; TGA, thrombin-generation assay; PaGIA, particle-gel immunoassay.

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