10.4081/btvb.2023.72
A syndrome occurring after adenoviral vector anti-SARS-CoV-2 vaccination, characterized by thrombocytopenia, venous thrombosis, and circulating anti-PF4 antibodies, known as vaccine-induced immune thrombotic thrombocytopenia (VITT), is well described. Data on the long-term course of this syndrome are lacking. Our aim is to report the clinical and laboratory features of a patient with VITT from diagnosis and during 21 months of follow-up. Cerebral venous thrombosis associated with elevated D-dimer, low fibrinogen, thrombocytopenia, and anti-PF4 antibodies positivity occurred in this patient after ChAdOx1 nCoV-19 vaccination. Cerebral thrombosis required a revascularization procedure and decompressive craniectomy. Upon dexamethasone and anticoagulant treatment initiation, the platelet count recovered. However, a persistently high anti-PF4 antibody titer, without thrombosis recurrence, was observed. Little is known about the long-term persistence of anti-PF4 antibodies, their clinical significance, and their possible role in guiding therapeutic decisions. In our patient, we decided to continue anticoagulant treatment beyond 21 months with parallel anti-PF4 antibody monitoring.
application/pdf
2785-5309
72
2
en
Anna Falanga
https://creativecommons.org/licenses/by-nc/4.0
Bleeding, Thrombosis and Vascular Biology
immune
Long-term persistence of high anti-PF4 antibodies titer in a challenging case of AZD1222 vaccine-induced thrombotic thrombocytopenia
Text.Serial.Journal
https://www.btvb.org/btvb
2
Case Reports
2023-06-28
2023-03-19
2023-04-30
2023-06-28
Bleed Thromb Vascul Biol
Anna Falanga
2023/06/28
10.4081/btvb.2023.72
Immunohematology and Transfusion Medicine and Thrombosis and Hemostasis Center, Papa Giovanni XXIII Hospital, Bergamo; School of Medicine and Surgery, University of Milan Bicocca, Milan
2785-5309
2
immune
en
Lundstrom K, Barh D, Uhal BD, et al. COVID-19 Vaccines and Thrombosis-Roadblock or Dead-End Street? Biomolecules. 2021;11(7).
Long-term persistence of high anti-PF4 antibodies titer in a challenging case of AZD1222 vaccine-induced thrombotic thrombocytopenia
https://www.btvb.org/btvb/article/download/72/75
2
                                                                                                                        CASE REPORT

Long-term persistence of high anti-PF4 antibodies titer in a challenging
case of AZD1222 vaccine-induced thrombotic thrombocytopenia
Luca Barcella,1 Chiara Ambaglio,1 Paolo Gritti,2 Francesca Schieppati,1 Varusca Brusegan,1 Eleonora Sanga,1
Marina Marchetti,1,3 Luca Lorini,2,3 Anna Falanga1,3

Immunohematology and Transfusion Medicine and Thrombosis and Hemostasis Center, and 2Intensive Care Unit, Papa Giovanni
1

XXIII Hospital, Bergamo; 3School of Medicine and Surgery, University of Milan Bicocca, Milan, Italy



ABSTRACT
    A syndrome occurring after adenoviral vector anti-SARS-CoV-2 vaccination, characterized by thrombocytopenia, venous throm-
bosis, and circulating anti-PF4 antibodies, known as vaccine-induced immune thrombotic thrombocytopenia (VITT), is well described.
Data on the long-term course of this syndrome are lacking. Our aim is to report the clinical and laboratory features of a patient with
                                                                        VITT from diagnosis and during 21 months of follow-up. Cere-
                                                                        bral venous thrombosis associated with elevated D-dimer, low
                                                                        fibrinogen, thrombocytopenia, and anti-PF4 antibodies positiv-




                                                                                                         ly
  Correspondence: Anna Falanga, Papa Giovanni XXIII Hospital,
                                                                        ity occurred in this patient after ChAdOx1 nCoV-19 vaccina-




                                                                                                 on
  Piazza OMS n. 1, 24127 Bergamo, Italy.                                tion. Cerebral thrombosis required a revascularization
  E-mail: annafalanga@yahoo.com                                         procedure and decompressive craniectomy. Upon dexametha-
                                                                        sone and anticoagulant treatment initiation, the platelet count




                                                                                           e
  Citation: Barcella L, Ambaglio C, Gritti P, et al. Long-term persist- recovered. However, a persistently high anti-PF4 antibody titer,
  ence of high anti-PF4 antibodies titer in a challenging case of
                                                                        without thrombosis recurrence, was observed. Little is known
  AZD1222 vaccine-induced thrombotic thrombocytopenia. Bleeding,
  Thrombosis, and Vascular Biology 2023;2:72.                                      us
                                                                        about the long-term persistence of anti-PF4 antibodies, their
                                                                        clinical significance, and their possible role in guiding thera-
  Key words: anti-SARS-CoV2 vaccine, thrombosis, thrombocy-             peutic decisions. In our patient, we decided to continue antico-
                                                                              al
  topenia, anti-PF4 antibody, immune.                                   agulant treatment beyond 21 months with parallel anti-PF4
                                                                    ci

  Contributions: LB, CA, PG, FS, VB, MM collected the data, con-
                                                                        antibody monitoring.
    tributed to the interpretation of the results and wrote the manu-
                                                                  er


    script. MM and ES performed laboratory testing. AF wrote and
    critically revised the manuscript.
                                                                 m



                                                                              Introduction
    Conflict of interest: the authors declare no potential conflict of in-
                                                      om




    terest.                                                                        Several reports are currently available on ChAdOx1 nCoV-
                                                                              19 (AZD1222, AstraZeneca) vaccinated patients, who developed
    Funding: the study was partially supported by the Fondazione
    ARTET Onlus-Bergamo, Italy.                                               thrombocytopenia, venous thrombosis at unusual sites, and ele-
                                               -c




                                                                              vated anti-platelet factor 4 (PF4) antibody levels with platelet-ac-
    Availability of data and materials: data and materials are available      tivating capacity, which characterize a specific medical
                                      on




    by the authors.                                                           emergency called vaccine-induced thrombotic thrombocytopenia
    Informed consent: written informed consent for publication was            (VITT).1-3
                                N




    obtained from the patient.                                                     While the first cases presented high mortality rates, awareness
                                                                              to encourage early presentation combined with a clearer pathway
    Acknowledgments: the authors thank the Fondazione ARTET                   for diagnosis and immediate treatment had reduced mortality to
    Onlus-Bergamo for its support.
                                                                              less than 5%, as described in Australian patients (https://www.tga.
    Received: 18 March 2023.                                                  gov.au/periodic/covid-19-vaccine-weekly-safety-report/current).
    Accepted: 1 June 2023.                                                         Although diagnosis and acute-phase treatment recommenda-
                                                                              tions have been released,4-7 data on the long-term course and man-
    Publisher’s note: all claims expressed in this article are solely those
    of the authors and do not necessarily represent those of their affili-    agement of this syndrome is lacking. The evolution of antibodies
    ated organizations, or those of the publisher, the editors and the re-    and their ability to activate platelets remains not well-defined.
    viewers. Any product that may be evaluated in this article or claim            Early experience suggests that anti-PF4 antibodies persist at
    that may be made by its manufacturer is not guaranteed or endorsed        least for several months;8 the National Institute for Health and
    by the publisher.
                                                                              Care Excellence guidelines suggests anti-PF4 antibody measure-
    ©
     Copyright: the Author(s), 2023                                           ments weekly for 4 weeks and then monthly for 6 months.9
    Licensee PAGEPress, Italy                                                      A decline over time of pathogenic antibodies against PF4 has
    Bleeding, Thrombosis and Vascular Biology 2023; 2:72                      been recently described.10-12 In a German case series, only five pa-
    doi:10.4081/btvb.2023.72
                                                                              tients (7.5%) showed persistent platelet-activating antibodies and
    This work is licensed under a Creative Commons Attribution-               high IgG anti-PF4/heparin antibody levels for more than 11
    NonCommercial 4.0 International License (CC BY-NC 4.0).                   weeks. In a UK cohort of patients, the median duration of posi-
                                                                              tivity of the PF4 assay is 87 days with 72% of patients remaining


                                                Bleeding, Thrombosis and Vascular Biology 2023; 2:72
                                                                                                                                  25
Long-term persistence of high anti-PF4 antibodies titer in a challenging case of AZD1222 vaccine-induced thrombotic thrombocytopenia




positive after a median follow-up of 105 days. The rate of relapse       and D-Dimer level >35,000 ng/mL (Table 1). The direct Coombs
was 12.6% with all cases exhibiting persistently positive PF4 an-        test was negative, and no schistocytes were observed in the pe-
tibodies and falling platelet count, in one case with extension of       ripheral blood smear. Molecular and antigenic nasal swab tests
thrombosis.13                                                            for SARS-CoV-2 were negative. A non-contrast brain computed
    Here we report on the clinical and laboratory features of a          tomography (CT) scan showed a temporal intracerebral hemor-
patient with VITT at diagnosis and after 21 months of follow-            rhage and a left subdural hematoma, while a contrast brain CT
up, showing a long-term persistence of high titer of anti-PF4 an-        scan showed thrombosis of the left transverse and sigmoid sinuses
tibodies.14                                                              and jugular vein (Figure 1A-C), subsequently confirmed by digital
                                                                         angiography (Figure 1D).
                                                                              In the suspicion of VITT, anticoagulation was started with low
Case Report                                                              dose fondaparinux 2.5 mg daily, and the patient was transferred
                                                                         to the Intensive Care Unit. A blood sample for anti-PF4 IgG anti-
     A 61-year-old woman with a personal history of SARS-CoV2            body testing was immediately sent to our specialist laboratory
infection in March 2020 received her first dose of the AZD1222           (Immunohematology and Transfusion Medicine). The enzyme
vaccine on May 5th, 2021. Known comorbidities were arterial hy-          linked immunosorbent assay, which detects IgG against
pertension, nephrolithiasis, and hyperuricemia, all treated by spe-      PF4/polyanions complexes (ELISA, PF4 Enhanced, IMMU-
cific medications. Nonspecific antinuclear antibody positivity was       COR), was strongly positive [2.810 Optical Density (OD); normal
also known since 2019. Twelve days after vaccination, she devel-         values <0.400 OD] (Table 1 and Figure 2). The multiple-electrode




                                                                                           ly
oped an acute frontal headache unresponsive to analgesics, and           aggregometry (MEA), performed to verify whether these antibod-




                                                                                    on
subsequent disorientation and drowsiness.                                ies have platelet-activating properties, showed that the patient’s
     The Out-of-Hospital Emergency Service was alerted due to            plasma could induce the aggregation of platelets from normal con-
the severe clinical conditions of the patient, who was urgently          trol subjects, an effect that was potentiated in the presence of a
transferred to the Emergency Department of our Hospital. Her




                                                                              e
                                                                         low-dose of heparin (0.5 IU/mL) and inhibited at a high heparin
complete blood cell counts were normal except for thrombocy-             dose (200 IU/mL). High titer (IgG 4+) of anti-SARS-CoV2 IgG
topenia (44×109/L). Prothrombin time ratio and partial thrombo-
plastin time ratio were normal, fibrinogen level was 70 mg/dL,
                                                                       uswas detected by the chromatographic immunoassay qSars-CoV-
                                                                         2 IgG Cassette Rapid Test (Cellex). This positivity was further
                                                                 al
                                                   ci
                                                 er
                                                m
                                      om
                                -c
                        on
                   N




Figure 1. Non-contrast brain computed tomography (CT) scan showing left temporal intracerebral hemorrhage (A) and subdural
hematoma (B). CT venogram revealed a filling defect in the left transverse sigmoid sinus (C), while digital angiography confirmed
thrombosis of the left transverse and sigmoid sinuses and jugular vein (D) that was partially resolved at the end of endovascular treatment
(E). Non-contrast brain CT scan showing left decompressive craniectomy of the patient (F).


                                         Bleeding, Thrombosis and Vascular Biology 2023; 2:72
26                                                                L. Barcella et al.




confirmed by the measurement of specific anti-Spike IgG (anti-S                 July 2nd, 2021, she was transferred to the rehabilitation Institute,
SARS-Cov2 IgG, Abbott). The diagnosis of VITT was made and                      where she switched to oral anticoagulant therapy with warfarin
dexamethasone (40 mg per day for 4 days) was started.                           targeting a PT-INR of 2.5 (therapeutic range 2.0-3.0).
     Due to the severity of the intracerebral hemorrhage and the                    In September 2021, after 4 months, she was discharged from
risk of intracranial hypertension, the patient underwent a cerebral             rehabilitation in good general conditions, with notes of fluent
thrombosis revascularization procedure (Figure 1D,E), followed                  aphasia (Wernicke's aphasia) in marked improvement and a
by left decompressive craniectomy (Figure 1F). After these inva-                Barthel Index of 95/100.
sive procedures, anticoagulation was resumed by intravenous (i.v.)                  At follow-up visits at the outpatient Hemostasis and Throm-
continuous infusion argatroban, targeting an aPTT ratio of 2.0,                 bosis Clinics in December 2021 and July 2022 (respectively
range 1.5-2.5, as per ISTH-SSC recommendations.4 The platelet                   +198 and +413 days from the onset of the VITT) the patient was
counts markedly improved in the subsequent days. After 27 days,                 in good health. As shown in Table 1, blood cell counts, including
she underwent autologous cranioplasty and a week later she                      platelets, were normal, but anti-PF4 antibodies resulted persist-
stopped i.v. argatroban and switched to subcutaneous fonda-                     ently positive.
parinux at the dose of 7.5 mg once daily. About 2 weeks later, on                   In October 2022, she underwent urgent cholecystectomy for
                                                                                acute emphysematous cholecystitis; anticoagulant therapy with
                                                                                warfarin was suspended and fondaparinux 2.5 mg/day was in-
                                                                                troduced postoperatively. After a week Fondaparinux, oral anti-




                                                                                                                ly
                                                                                coagulant therapy was resumed with apixaban 2.5 mg every 12
                                                                                hours. At follow-up visits at the outpatient Hemostasis and




                                                                                                    on
                                                                                Thrombosis Clinics in January 2023 (+610 days from VITT
                                                                                onset), blood cell counts, including platelets, were normal, how-
                                                                                ever anti-PF4 antibodies resulted still positive. We decided to




                                                                                                e
                                                                                continue apixaban 2.5 mg bid.

                                                                                       us
                                                                                    Written informed consent for publication was obtained from
                                                                                the patient.
                                                                              al
                                                                                Discussion
                                                              ci

                                                                                    ChAdOx1 nCov-19 vaccine against SARS-CoV2, currently
                                                            er


                                                                                named as AZD1222, employs an Adenovirus derived from the
                                                                                chimpanzee. The added sequence encodes for the full-length S
                                                           m



Figure 2. Modifications in anti-PF4 IgG antibodies and their
platelet activating capacity over time.                                         protein with a tissue plasminogen activator signal sequence. Start-
At onset, the high titer of anti-PF4 antibodies (by ELISA) dis-
                                                 om




                                                                                ing from late February 2021, some reports documented the ap-
playing a full platelet activating capacity by multiple-electrode
aggregometry (MEA) contributed to the development of the                        pearance in healthy individuals of complications such as
thrombotic event. After diagnosis, the continuous administration                thrombocytopenia and thrombosis at atypical sites (in particular,
                                           -c




of an anticoagulant therapy together with the progressive decrease              cerebral and/or splanchnic) within 2 weeks from ChAdOx1 nCov-
in the platelet activating capacity of anti-PF4 antibodies, in the              19 vaccination. This new syndrome, named VITT, has been asso-
absence of the triggering factor (i.e., the vaccine), was effective
                                  on




                                                                                ciated with AZD1222 or Ad26.COV2.S vaccines.15
in the prevention of a thrombotic recurrence in this patient.
                                                                                    In the first reports, patients were primarily younger than 60
                             N




Table 1. Laboratory parameters of the patient at hospital admission (day 0) and during the 21 months of follow-up
Days        Hb, g/L         WBC, 109/L Platelets, 109/L Fibrinogen,L               D-dimer,        CRP, mg/dL        Anti-PF4      MEA, AUmin
           (120-160)         (4.2-9.4)    (150-400)         mg/d                    ng/mL           (0.0-0.5)        IgG, OD         (<50)
                                                          (150-400)                 (< 500)                          (<0.400)
0              133               7.46               44                70            > 35,000             1.5              -                -
+1             114               6.52               38               184                 -                -            2.810            1,120
+2              87               4.79               72               132               17,882            3.3           3.070            1,256
+8              88               6.53              217               507               4,312             5.0           2.387             431
+15            100               4.41              210               547                 -               4.5           1.960             343
+18            101               4.94              205               584                 -               2.1           3.140             461
+23            101               6.35              262               501                 -               11.2          2.565             320
+128           138               3.89              213                -                  -                -            1.550             220
+413           149               5.43              221                -                  -               0.1           1.388               -
+610           144               4.07              214                -                <190               -            1.370             210
Hb, hemoglobin; WBC, white blood cells; OD, Optical Density; CRP, C-reactive protein; MEA, multiple-electrode aggregometry; AU, arbitrary units.


                                            Bleeding, Thrombosis and Vascular Biology 2023; 2:72
                                                                                                                                  27
Long-term persistence of high anti-PF4 antibodies titer in a challenging case of AZD1222 vaccine-induced thrombotic thrombocytopenia




years and were mostly females. None had received heparin earlier,
and few had other known risk factors for thrombosis. Many of the        References
patients were critically ill by the time thrombosis with thrombo-        1. Greinacher A, Thiele T, Warkentin TE, Weisser K, Kyrle PA,
cytopenia was diagnosed, and mortality rate was high.16 The path-           Eichinger S. Thrombotic thrombocytopenia after ChAdOx1
ogenic mechanisms of VITT have been linked to the production                nCov-19 vaccination. N Engl J Med 2021;384:2092-101.
of IgG autoantibodies targeting PF4 which activate platelets via         2. Schultz NH, Sorvoll IH, Michelsen AE, et al. Thrombosis and
their FcgRIIa receptors, as occurring in autoimmune heparin-in-             thrombocytopenia after ChAdOx1 nCoV-19 vaccination. N
duced thrombocytopenia (HIT).17 In HIT, PF4/heparin complexes               Engl J Med 2021;384:2124-30.
progressively decrease in circulation with parallel reduction and        3. Scully M, Singh D, Lown R, et al. Pathologic antibodies to
negativization of the antibody titer in the following 7-12 weeks,           platelet factor 4 after ChAdOx1 nCoV-19 vaccination. N Engl
upon interruption of the heparin treatment. Less is known in the            J Med 2021;384:2202-11.
VITT setting. One study, in 35 patients with VITT followed-up            4. Nazy I, Sachs UJ, Arnold DM, et al. Recommendations for
for a median time of 11 weeks (range, 4-19), showed that in more            the clinical and laboratory diagnosis of VITT against COVID-
than 90% of patients, pathologic, platelet-activating anti-PF4 an-          19: communication from the ISTH SSC Subcommittee on
tibodies disappeared within 12 weeks, while the serological re-             platelet immunology. J Thromb Haemost 2021;19:1585-8.
version (i.e., negative ELISA result) occurred only in 3 patients.12     5. Arepally GM, Ortel TL. Vaccine-induced immune thrombotic
     Our patient, after 21 months, still shows a persistently high          thrombocytopenia: what we know and do not know. Blood
titer of antibodies, but a normal platelet count. According to the          2021;138:293-8.




                                                                                          ly
above theory, the presence of anti-PF4 antibodies is the underlying      6. Oldenburg J, Klamroth R, Langer F, et al. Diagnosis and man-




                                                                                  on
condition causing VITT, however, our case of positive anti-PF4              agement of vaccine-related thrombosis following AstraZeneca
                                                                            COVID-19 vaccination: guidance statement from the GTH.
titer in clinical remission phase suggests that other factors may
                                                                            Hamostaseologie 2021;41:184-9.
intervene in the acute setting to determine the activation of coag-
                                                                         7. Gresele P, Marietta M, Ageno W, et al. Management of cere-




                                                                            e
ulation, mainly in the cerebral or splanchnic venous districts. Al-
                                                                            bral and splanchnic vein thrombosis associated with throm-
ternatively, we can assume that anti-PF4 antibodies are less
reactive to platelets over time, as demonstrated by the decrease in
the platelet activating capacity by the functional assay, or that the
                                                                        us  bocytopenia in subjects previously vaccinated with Vaxzevria
                                                                            (AstraZeneca): a position statement from the Italian Society
                                                                            for the Study of Haemostasis and Thrombosis (SISET). Blood
                                                                   al
patient is protected by the anticoagulant therapy.                          Transfus 2021;19:281-3.
     We believe in the protective role of anticoagulation and in-        8. Lindhoff-Last E, Schoenborn L, Piorkowski M, et al. Hetero-
                                                    ci

deed we kept our patient on long-term anticoagulation, without              geneity of Vaccine-Induced Immune Thrombotic Thrombo-
bleeding complications or thrombotic recurrences. Differently
                                                  er


                                                                            cytopenia after ChAdOx1 nCoV-19 Vaccination and Safety
from our case, a complicated VITT case with extremely high                  of Second Vaccination with BNT162b2. Thromb Haemost
anti-PF4-IgG titers over three months was characterized by re-
                                                 m



                                                                            2022;122:304-7.
current thrombocytopenia despite treatment with i.v. IG and an-          9. NICE. COVID-19 rapid guideline: vaccine-induced immune
                                       om




ticoagulation and ended with a massive hemorrhage in the left               thrombocytopenia and thrombosis (VITT). National Institute
parietal and temporal lobe without signs of new thrombosis.11               for Health and Care Excellence: London; 2022.
This supports the relevance of deciding the optimal timing and          10. Thaler J, Jilma P, Samadi N, et al. Long-term follow-up after
                                                                            successful treatment of vaccine-induced prothrombotic im-
                                 -c




dosing of anticoagulant therapy in VITT patients with persisting
high titer anti-PF4 antibodies.                                             mune thrombocytopenia. Thromb Res 2021;207:126-30.
                                                                        11. Gunther A, Bramer D, Pletz MW, et al. Complicated long
                         on




                                                                            term vaccine induced thrombotic immune thrombocytopenia-
                                                                            a case report. Vaccines (Basel) 2021;9.
Conclusions
                   N




                                                                        12. Schonborn L, Thiele T, Kaderali L, Greinacher A. Decline in
     Little is known about long-term follow up of patients who              pathogenic antibodies over time in VITT. N Engl J Med
survived to VITT. The case here described shows how rapid                   2021;385:1815-6.
recognition and treatment of VITT is crucial for the short and          13. Craven B, Lester W, Boyce S, et al. Natural history of PF4
long-term clinical outcomes of this severe syndrome. Also, labo-            antibodies in vaccine-induced immune thrombocytopenia and
                                                                            thrombosis. Blood 2022;139:2553-60.
ratory monitoring of anti-PF4 IgG antibody and their platelet ac-
                                                                        14. Schonborn L, Thiele T, Kaderali L, et al. Most anti-PF4 anti-
tivating capacity, together with platelet count, over time, can be
                                                                            bodies in vaccine-induced immune thrombotic thrombocy-
of importance in these patients, as anticoagulant therapy might
                                                                            topenia are transient. Blood 2022;139:1903-7.
continue until the pathogenic mechanism is active. The disappear-       15. Cari L, Alhosseini MN, Fiore P, et al. Cardiovascular, neuro-
ance of pathogenic antibodies is an important criterion also for            logical, and pulmonary events following vaccination with the
deciding for the subsequent vaccination with a mRNA vaccine.                BNT162b2, ChAdOx1 nCoV-19, and Ad26.COV2.S vac-
However, when no platelet-activation assay is available, a sub-             cines: an analysis of European data. J Autoimmun 2021;125:
stantial decline (more than 50%) in the optical density of anti–            102742.
PF4–heparin IgG ELISA has been suggested as a decision                  16. Pavord S, Scully M, Hunt BJ, et al. Clinical features of vac-
criterion.12                                                                cine-induced immune thrombocytopenia and thrombosis. N
     Whether the presence or not of high titer anti-PF4 IgG anti-           Engl J Med 2021;385:1680-9.
bodies may be a tool for deciding on duration of anticoagulation        17. Lundstrom K, Barh D, Uhal BD, et al. COVID-19 vaccines
and/or the necessity of additional immunosuppression remains a              and thrombosis-roadblock or dead-end street? Biomolecules
challenge and is to be established by large studies.                        2021;11.


                                          Bleeding, Thrombosis and Vascular Biology 2023; 2:72
