Immunoadsorption, Intravenous Immunoglobulins and Rituximab (IIR): Successful New Treatment Approach for Severe Anti K- Alloimmunisation during Pregnancy
Authors: Capraru Annina , Raio Luigi , Müller Martin , Daskalakis Michael , Rovo Alicia , Henri Hustinx Christian , Jane McDougall , Zeerleder Sacha Sergio , Mansouri Behrouz Teleghani 
Published: November 2020

Background:
Fetal red blood cells (RBC), erythroid and megakaryopoietic progenitor cells start to express the Kell (K) antigen already during the 10th gestational week (GW). Maternal anti-Kell alloimmunization (allo-anti-K-Ab) causes early (&lt; 20th GW) and severe hemolytic disease of the fetus/newborn (HDFN) accompanied by erythroaplasia and potential thrombocytopenia. Therefore, prevention/delay of HDFN is highly relevant. Current treatment options include intra-uterine transfusions (IUT) of the fetus in case of suspected anemia, i.v. high-dose immunoglobulins (IVIG), plasma exchange and immunoadsorption (IA). Their efficacy often remains unsatisfactory and procedure related complications might occur.
We present our new triple-treatment approach, combining IA + IVIG + Rituximab (Rtx) (IIR), which we successfully applied in 2 high-risk patients with allo-anti-K-Ab.
Methods:
The first patient was a 24 y, para 1, gravida 2, with an allo-anti-K-AB titer of 1:2000 in the 15th GW, at first presentation. Duplex-sonography showed no signs of fetal anemia. The second patient was a 40 y para 1, gravida 2. She presented with an allo-anti-K-Ab titer of 1:8000 in the 20th GW. Duplex-sonography revealed signs of severe anemia of fetus, requiring immediate IUT.
Fetal K- genotyping (RT-PCR) using cell-free fetal DNA from maternal plasma confirmed a K- positive status in both fetuses. Duplex sonography monitoring was executed weekly, including the assessment of arteria cerebri media peak systolic velocity (ACM PSV).
After having received informed consent of patients, we started the treatment at 14th and 21st GW in our first and second patient, respectively. IIR treatment cycles (1 cycle = 21 days) comprised 6x IA (each processing 2x the patient's plasma volume, weeks 1 + 2, every other day), followed by IVIG (1g/kg body weight) and i.v. Rtx (375 mg/m2) after the last IA of each cycle. This resulted in a total of 6 and 5 cycles for the first and second patient, respectively. We omitted Rtx for the last cycles in both patients.
Results:
IIR treatment resulted in a significant reduction of maternal anti K-titer to minimum of 1:32 in both patients, with a fluctuating course.
In case 1 we performed a cordocentesis in the 30th GW because of an increased ACM PSV. The blood count of the fetus showed no anemia (Hb: 132 g/L) and only minimal Thrombocytopenia (145 G/L).
The fetus in case 2 had already duplex- sonographic signs for a severe anemia at first presentation. Diagnostic cordocentesis confirmed a severe anemia (Hb 47 g/L) and also severe thrombocytopenia (21 G/L). Fetal alloimmune thrombocytopenia was ruled out. The first IUT of RBC has been performed right before starting IIR. Two weeks later a second IUT of RBC was necessary (fetal Hb 54 g/L), but thrombocytopenia was completely reversed (Tc: 352 G/l). The third and last IUT of RBC was performed 5 weeks later (Hb 99 g/L). Thereafter, no other signs of fetal anemia were detected.
Patient one showed nausea/headache after her first IVIG-application and hypotonia/sinustachykardia during IA of the 5th cycle. IIR-treatment and IUT revealed no further adverse events.
We continued treatment in both cases until the 32nd GW. Both neonates were born healthy in the 38th and 37th GW, respectively. The neonate in case 1 presented a mild anemia (Hb: 131 g/l), but normalized his Hb from 2nd day onwards. Both newborns showed a positive direct antiglobulin test (DAT), elution verified an anti-K-Ab, and their RBC were serologically positive for K. Both cases had normal Bilirubin and LDH levels, but a haptoglobin &lt;0.1 g/l at birth. Both developed an icterus neonatorum during the first days after birth and needed phototherapy, Hb remained normal at all times. Flow cytometry of fetal lymphocytes showed a B-cell decrease (5.2%) in the first case and completely missing B-cells in the second case. Rtx level was low (2.13 µg/ml) in maternal plasma and neg. in the newborn of the first case. In the second case Rtx levels of the newborn and mother were 15,87 µg/ml and 4,5 µg/ml, respectively. In both cases, breastmilk showed no Rtx but positivity for anti-K-Ab (IgG). We observed no hemolysis of the newborns, while both were on breastfeeding.
Conclusion:
High-titer anti-Kell alloimmunisation causes early onset and severe HDFN and may be associated with severe thrombocytopenia. Treatment of affected pregnant women by using the Bern IIR-protocol was effective and safe in both severely affected patients.

Disclosures
Rovo: Novartis:Honoraria, Research Funding;CSL Behring:Research Funding;AG Alexion:Research Funding;Celgene:Honoraria, Other: financial support for congresses and conferences travel;BMS:Other: financial support for congresses and conferences travel;AstraZeneca:Other: financial support for congresses and conferences travel;Sanofi:Other: financial support for congresses and conferences travel;Amgen:Other: financial support for congresses and conferences travel;Roche:Other: financial support for congresses and conferences travel.

OffLabel Disclosure:
Rituximab is used for B-cell depletion in different malignant and non-malignant diseases. The off-label use concerns ist use in pregnant women with alloimmunisation.