Resolving Blocked Antigen Phenomenon in Hemolytic Disease of the Fetus and Newborn Due to Anti-K

Journal Pre-proof Resolving Blocked Antigen Phenomenon in Hemolytic Disease of the Fetus and Newborn Due to Anti-K

Mitchell Moosavi, Yao Ma, Janet Baez, Rebecca Jeffreys, Dawn C. Ward, Alyssa Ziman, Andrea M. McGonigle PII:

S0887-7963(20)30013-4

DOI:

https://doi.org/10.1016/j.tmrv.2020.02.002

Reference:

YTMRV 50606

To appear in:

Transfusion Medicine Reviews

Please cite this article as: M. Moosavi, Y. Ma, J. Baez, et al., Resolving Blocked Antigen Phenomenon in Hemolytic Disease of the Fetus and Newborn Due to Anti-K, Transfusion Medicine Reviews(2020), https://doi.org/10.1016/j.tmrv.2020.02.002

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Journal Pre-proof Resolving Blocked Antigen Phenomenon in Hemolytic Disease of the Fetus and Newborn Due to Anti-K Mitchell Moosavia, Yao Maa, Janet Baeza, Rebecca Jeffreysa, Dawn C. Warda, Alyssa Zimana, Andrea M. McGoniglea a

From the Wing-Kwai and Alice Lee-Tsing Chung Transfusion Service, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

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Correspondence: Andrea M. McGonigle Department of Pathology and Laboratory Medicine, 10833 Le Conte Ave., Los Angeles, CA, 90095, USA E-mail: [email protected]

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Abstract

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Keywords: HDFN, antigen blocking, blocked antigen, heat elution, prozone, anti-K.

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High-titer antibodies are a cause of false-negative reactions in red blood cell (RBC) antigen

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phenotyping; an event referred to as “blocked antigen phenomenon (BAP).” In hemolytic disease of the fetus and newborn (HDFN), BAP complicates laboratory workups as fetal phenotype is helpful in confirming the responsible antibody. Acid elution techniques, techniques utilizing ethylenediaminetetraacetic-acid-glycine-acid (EGA), as well as those utilizing chloroquinediphosphate (CDP) have been used to resolve BAP, however EGA destroys K-antigen expression and CDP is not always effective. We report a case of severe HDFN from anti-K where a modified gentle heat elution resolved BAP. Although infrequently considered with isolated reports in the literature, heat elution is simple, effective, and involves readily available materials in most blood banks.

Journal Pre-proof Introduction Hemolytic disease of the fetus and newborn (HDFN) is a potentially life-threatening situation in which the fetus or neonate’s red blood cells (RBCs) undergo antibody mediated destruction secondary to maternal antibodies that cross the placental barrier. 1,2 Most cases of HDFN, caused by naturally formed ABO antibodies, are generally lead to minimal or mild symptoms. In contrast, clinically significant cases of HDFN are more frequently seen in mothers

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who have been alloimmunized prior to pregnancy.1, 2 The spectrum of HDFN can be quite

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variable, from inconsequential to severe, and can include severe anemia, hydrops fetalis,

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erythroblastosis fetalis and neonatal death at the time of delivery.1, 2 While RhD alloimmunization was historically the most frequent source of HDFN, numerous other antibodies

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have been implicated.3

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The Kell antigen system is a family of proteins, encoded by the KEL gene, which

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function in linking membrane proteins to disulfide bonds. 3 K and k, are the most common antigens from the Kell system.4 The K antigen has a relatively low frequency of approximately

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9% in Caucasian populations.5 Antibodies to the K antigen typically manifest as IgG antibodies

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and rank as the second most common cause of minor red cell antigen related HDFN after anti-D5, . HDFN secondary to anti-K is expected to increase in relative frequency as immunoprophylaxis

against anti-D continues.6

Blocked antigen phenomenon (BAP) is a rare cause of false-negative reactions/results in RBC antigen phenotyping.3, 7 In the case of BAP in HDFN, high-titer maternal RBC antibodies (titer of 256 or greater in most cases involving antibodies to the K antigen) can cross the placenta and bind their corresponding antigen(s) on fetal RBCs. 1 Subsequent attempts to type the fetal RBC antigens with reagent anti-sera produces false-negative results, as all antigen sites are saturated by antibody and therefore are unavailable to react with anti-sera.1 The blocking

Journal Pre-proof phenomenon is suspected to result in part from steric hindrance of the C1 component of human complement.3 A variety of methods have been reported to facilitate accurate antigen typing when RBC antigen sites are blocked by antibody. 1, 3, 8-13 Treating cord RBCs with ethylenediaminetetraacetic glycine acid (EGA) or acid elution will remove the interfering antibody causing BAP and maintain expression of most antigens. 3, 10, 12, 14 Chloroquine-diphosphate (CDP) has also been

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used to resolve BAP. 7, 8, 12 Gentle heat elution at 45° C has been shown to remove antibodies

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saturating RBCs and allow accurate phenotyping, including cases caused by anti-ABO, anti-K,

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and other antibodies with a positive direct antiglobulin test (DAT).3,10-13

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Despite the availability of these techniques, accurate RBC phenotyping in BAP due to anti-K presents unique challenges. While EGA is effective at removing BAP in general, it

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denatures the K antigen. 10,12 In the two reported cases identified in the literature where CDP was

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used to resolve BAP due to anti-K, it was only effective in one of the cases.7,12 Although molecular genotyping can confirm phenotype in cases of BAP, results are not always readily

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available. While gentle heat elution has been shown to be effective at removing anti-K

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antibodies, to our knowledge, it has not been published for the use of resolving BAP. Here, we report a case of severe HDFN with BAP due to anti-K where initial typing indicated the neonate to be K antigen negative, but a modified gentle heat elution was effectively utilized to obtain accurate phenotyping results. Case Presentation A 36-year-old multiparous (G4P3) female presented at 33 weeks and 2 days by last menstrual period from an outside hospital for higher level care for a pregnancy complicated by high-titer anti-K antibodies (1:1024) and low-titer anti-Jka antibodies (1:4). Her prior three

Journal Pre-proof pregnancies were uncomplicated per outside hospital records. Per clinical history, all four children have the same biologic father, whom was typed as K antigen positive based on an outside laboratory report. The father’s Jka phenotype was unknown to our transfusion service. The mother also had a history of transfusion of two units of RBCs, received during separate hospitalizations for anemia secondary to severe bleeding from uterine fibroids, both of which were transfused following her third pregnancy

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Fetal ultrasound, at the time of presentation, showed pericardial effusion, dilation of the

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right ventricle, a dilated four-chamber view, biventricular hypertrophy, moderate patency of the

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ductus arteriosus, and holosystolic tricuspid regurgitation. Intermittent sinusoidal fetal heart

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tones were also detected. Middle cerebral artery (MCA) flow Doppler ultrasonography, a highly sensitive method for predicting moderate to severe fetal anemia, was normal on admission at

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1.08 multiples of the median (MoM). 14 MCA flow quickly rose over the next three days to 1.58

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MoM (generally considered abnormal when greater than 1.5 MoM). This constellation of findings prompted suspicion of worsening fetal anemia and decision to process with immediate

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delivery via cesarean section.

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The delivery of a male neonate was performed at 33-weeks and 5 days via cesarean section. The neonate’s birth weight was 2.34 kg. The neonate was noted to be pale and jaundiced immediately after delivery. Apgar scores were reported as 8 and 9 at one and five minutes, respectively, with points deducted for skin appearance. There was clinical and laboratory evidence of severe anemia, hyperbilirubinemia, and reticulocytopenia (Table 1). Cord blood testing demonstrated a positive DAT for IgG with an eluate was positive for anti-K. Phenotyping of neonatal RBCs were negative for the K and Jka antigens. Given the clinical and laboratory findings, concern for a false negative K antigen typing prompted performance of

Journal Pre-proof repeat antigen phenotyping after acid elution of neonatal RBCs, which was also negative (Table 2). Additional phenotyping performed following gentle heat elution confirmed the neonatal RBCs to be K antigen positive. These findings were consistent with HDFN and prompted performance of an emergent double volume neonatal RBC exchange transfusions followed by IVIG and triple phototherapy. The neonate’s anemia and hyperbilirubinemia initially resolved following a double

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volume exchange transfusion with reconstituted whole blood performed at six hours of life

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(Table 1). At twelve hours of life, the patient again demonstrated hyperbilirubinemia. At twenty-

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four hours of life, blood cultures obtained immediately post-delivery revealed growth of

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coagulase-negative Staphylococcus organisms; the corresponding complete blood count and differential demonstrated increased band neutrophils. Blood pressure remained depressed and

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bilirubin remained elevated. Antibiotic therapy was initiated to address possible sepsis. At

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approximately thirty-six hours of life, after completion of triple phototherapy, the neonate’s hyperbilirubinemia resolved. Following initial correction of anemia post-exchange transfusion,

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the neonate’s hematocrit continued to decline (nadir of 33.8 g/dL). At 84 hours of life,

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hemoglobin assessments demonstrated gradual spontaneous recovery; no transfusion support was required. An echocardiogram at this time revealed significantly improved cardiac function. The neonate was discharged fourteen days following the emergent delivery.

Journal Pre-proof Materials and Methods Serologic testing Routine ABO/Rh typing, antibody screening, and indirect antiglobulin testing were performed on both maternal and newborn samples using automated testing (Immucor Inc., Norcross GA). Neonatal samples were collected via cord blood sampling; in our institution cord blood is routinely collected into a 5mL test tube that is filled completely or near completely,

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following delivery. All manual serologic tests including DAT using polyclonal antihuman

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globulin (AHG) reagent, antibody titer panels, acid elution and phenotyping were performed

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according to standard operating procedures as outlined in the AABB Technical Manual. 3 A

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modified gentle heat elution method, in which equal parts packed RBCs and 6% albumin were heated for 30 minutes at 45°C (instead of the standard eluate temperature of 56°C), was used to

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dissociate blocking K antibodies from RBCs. Repeat K phenotyping was performed after 5-

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minute room temperature incubation, with agglutination assessed macroscopically and microscopically. Confirmatory genotyping was performed by a reference laboratory using the

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Genetic testing

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BioArray platform (Immucor Inc., Norcross GA).

DNA typing for human RBC antigens was performed at the American Red Cross National Molecular Laboratory using HEA BeadChip Technology on the BioArray platform (Immucor Inc., Norcross GA). RBC selection O-negative, K-negative, and Jka-negative packed RBCs reconstituted in AB-plasma to a hematocrit of 55% were supplied for exchange transfusion.

Journal Pre-proof Results Maternal blood testing demonstrated high titers of anti-K at 1:1024, and anti-Jka at a titer of 1:4 on initial serologic testing. The cord blood DAT was positive, and cord blood eluate was positive for anti-K. Initial phenotyping of neonatal RBCs was non-reactive with both Jka and K anti-sera (Table 2). Repeat phenotyping performed on acid eluted neonatal RBCs was also non-reactive

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with K anti-sera. Following gentle heat elution, macroscopic assessment of agglutination with K-

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antisera was read as negative. However, agglutination with K-antisera was re-assessed

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microscopically due to suspicion that it was weakly positive macroscopically, the clinical history

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and the knowledge that even gentle heat elution may weaken the strength of serologic reactivity. Phenotyping of neonatal RBCs after gentle heat elution demonstrated strongly positive

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microscopic agglutination with anti-K reagent.

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Discussion

While nearly all reported cases of BAP in the literature are due to RhD antibodies, it is

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important to recognize that other less common antibodies may also participate in this

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phenomenon.3 In this case, recognition of BAP as a possible cause of initial negative neonatal K antigen typing encouraged further testing, which was helpful for consultation with the clinical team. Though HDFN was strongly suspected, the differential diagnosis for anemia and hyperbilirubinemia in a neonate is broad.16 K phenotyping following modified heat elution was helpful in aligning laboratory findings with the evolving clinical picture. Demonstration of K antigen on neonatal RBCs supported the diagnosis of HDFN while excluding other causes of neonatal jaundice and encouraged completion of phototherapy and IVIG treatment despite an alternative, albeit less likely, differential diagnosis of neonatal sepsis (given concurrent neonatal blood cultures

Journal Pre-proof demonstrating growth of coagulase negative Staphylococcus). Additionally, the immunohematology findings provide an explanation as to the prolonged duration of the neonate’s anemia and the continued decline in hematocrit following the exchange transfusion. Suppression of K antigen positive erythroid progenitor cells in-utero by anti-K antibodies is a well-known phenomenon and are consistent with the clinical and laboratory findings in this case. 6

Although molecular genotyping ultimately confirmed the neonate was K antigen positive; these

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results were not available until after discharge and therefore did not assist in confirmation of the

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clinical diagnosis in real time.

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While anti-K is involved in approximately 10% of reported cases of HDFN, few cases of

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BAP due to anti-K have been reported in the literature. 6, 7, 12, 14 In three cases, acid elution, EGA and CDP were the methods of choice to remove blocking anti-K. Acid elution was effective in

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removing BAP and allowing K antigen phenotyping in one published case. 3 When we initially

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attempted acid elution to resolve BAP in our case, repeat phenotyping of eluted neonatal RBCs remained K-antigen negative. While both CDP and EGA are well-known RBC-preserving

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elution methods, CDP would be expected to preserve K antigen, unlike EGA. 10, 12, 14 In the two

7,12

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cases that reported use of CDP to resolve BAP due to anti-K, it was effective in only one case. Given the broader utility of EGA in immunohematology workups, our lab no longer keeps

CDP in stock. Heat elution is another technique which preserves the RBC membrane and prevents denaturation of the K antigen.3, 6, 10-13 Modified gentle heat elution, a heat-based elution performed at a low temperature with long incubation, was not an obvious choice for resolution of BAP. It is uncommonly utilized in our laboratory and must be performed carefully to prevent denaturing the underlying antigen. One study had demonstrated that heat elution removed coating antibodies on RBCs causing a positive DAT six times more frequently than CDP. 11, 14

Journal Pre-proof Our laboratory performed a modified gentle heat elution which effectively preserved K antigen expression and removed blocking antibodies to allow for accurate K phenotyping. Conclusion Although infrequently considered, heat elution is useful in resolving BAP, particularly if CDP or acid elution are not available or fail to resolve BAP. It is simple, effective and readily

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available in most hospital laboratories. We report a case in which a modified gentle heat elution was effective in resolving BAP in a case of severe HDFN with anti-K when other methods

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failed.

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Support

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This research did not receive any specific grant from funding agencies in the public, commercial,

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or not-for-profit sectors.

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Conflict of interest statement

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The authors have no conflicts of interest to report.

Journal Pre-proof References 1. Sulochana PV, Rajesh A, Mathai J, Sathyabhama S. Blocked D phenomenon, a rare condition with Rh D haemolytic disease of newborn - a case report. Int J Lab Hematol, 2008; 30(3): 244-7. 2. Delaney M, Wikman A, van de Watering L, Schonewille H, Verdoes JP, Emery SP, et al. Blood Group Antigen Matching Influence on Gestational Outcomes (AMIGO) study. Transfusion. 2017; 57(3):525-532.

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3. Fung MK, Grossman BJ, Hillyer CD, Westhoff CM. Technical Manual. 18th ed. Bethesda, MD: AABB; 2014.

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4. Daniels G. Human blood groups.3rd ed. Chichester, West Sussex (UK): John Wiley & Sons; 2013.

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5. Reid ME, Lomas-Francis C. The Blood Group Antigen Facts Book. 3rd Ed. New York: Academic Press; 2007.

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6. Vaughn JI, Manning M, Warwick RM, Letsky EA, Murray NA, Roberts IA, et al. Inhibition of Erythroid Progenitor Cells by Anti-Kell Antibodies in Fetal Alloimmune Anemia. N Engl J Med. 1998; 338(12): 798-803.

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7. Lee E, Redman M, Owen I. Blocking of fetal K antigens on cord red blood cells by maternal anti-K. Transfus Med. 2009; 19:139–40.

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8. Judd WJ, Steiner EA, O’Donnell DB, Oberman HAl. Discrepancies in reverse ABO typing due to prozone. Transfusion. 1988; 28:334-38.

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9. Judd WJ. Elution—dissociation of antibody from red blood cells: Theoretical and practical considerations. Transfus Med Rev, 1999;13:297-310. 10. Judd WJ, Johnson ST, Storry JR. Judd’s Methods in Immunohematology. 3 rd ed. Bethesda, MD. AABB; 2008. 11. Katharia R, Chaudhary RK. Removal of antibodies from red cells: Comparison of three elution methods. Asian J Transfus Sci. 2013;7(1):29-32. 12. Hannon J, Clarke G, Caruk B, Button E. Blocking phenomenon due to anti‐Kell in post‐ natal investigation. Transfus Med, 2007; 17: 233 (Abstract 26). 13. South SF, Rea AE, Tregellas WM. An evaluation of 11 red cell elution procedures. Transfusion. 1986; 26:167-70. 14. Manfroi S, Velati C. K-antigen blocking in a case of haemolytic disease of the foetus and newborn. Blood Transfus. 2017;15(6):585–586.

Journal Pre-proof 15. Sohsman M, Yuan S, Ziman A, Lu Q. Prenatal diagnosis and management of hemolytic disease of the fetus and newborn. ASCP, Transfus Med. 2010;53(4):39-49. 16. Flerlage J, Engorn B. The Harriet Lane Handbook: A Manual for Pediatric House Officers, 20th ed. Philadelphia, PA. Elsevier; 2015. 17. American Academy of Pediatrics Subcommittee on Hyperbilirubinemia. Management of hyperbilirubinemia in the newborn infant 35 or more weeks of gestation. Pediatrics, 2004;114: 297–316. 18. Hughes-Jones NC, Gardner B. The Kell System Studied with Radioactively-Labelled Anti-K. Vox Sang. 1971; 21: 154–158.

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19. Mari G, Deter RL, Carpenter RL, Rahman F, Zimmerman R, Moise KJ, et al. Noninvasive diagnosis by Doppler ultrasonography of fetal anemia due to maternal redcell alloimmunization. Collaborative Group for Doppler Assessment of the Blood Velocity in Anemic Fetuses. N Engl J Med. 2000;342(1):9-14.

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20. Masouredis SP, Sudora E, Mahan LC, Victoria EJ. Immunoelectron microscopy of Kell and Cellano antigens on red cell ghosts. Haematologia. 1980;13(1-4):59-64.

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21. Moiz B, Salman M, Kamran N, Shamsuddin N. Transfusion medicine illustrated: blocked D phenomenon. Transfusion. 2008; 48:1545–6.

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22. Parsons SF, Gardner B, Anstee, DJ. Monoclonal antibodies against Kell glycoprotein: serology, immunochemistry and quantification of antigen sites. Transfus Med. 1993; 3:137–142. 23. Rudmann SV. Serologic Problem-solving: A Systematic Approach for Improved Practices. 1st ed. Bethesda, MD. AABB; 2005.

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24. Rudmann SV. Textbook of Blood Banking and Transfusion Medicine, 2 nd ed. Philadelphia, PA. Saunders; 2008. 25. Verma A, Sachan D, Bajpayee A, Elhence P, Dubey A, Pradhan M. RhD blocking phenomenon implicated in an immunohaematological diagnostic dilemma in a case of RhD-haemolytic disease of the foetus. Blood Transfus. 2013;11(1):140-142.24.

Journal Pre-proof Table 1. Laboratory Screening Tests for Jaundice.

Proce N dure A HCT, 16. 2 %

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12

18

ET/ TP T 49.0 42.7 *

HCT refer ence range

TPT 36.6

3 7. 1

0.9 7%

33.8

2 2 7

3 5. 3

3 7. 6

N A

41.053.0%

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Retic Coun t Retic Coun t refer ence range Calc ulate d Retic Index Calc ulate d

36.0

5.8

5. 1

<8. <8. 0 0

40

75

95

40

75

th

th

th

th

th

95

N A

8.4

7. 5

40

75

95

40

75

95

th

th

th

th

th

th

%t %t %t %t ile ile ile ile 7. 8. 11 9. 0 9 .1 6

%t %t ile ile 12 15 .6 .2

9. 5

<2.0

NA

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th

%t %t ile ile 5. 6. 6 9

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%t %t %t %t ile ile ile ile 4. 5. 6. 4. 0 0 5 9

5.7

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7.4

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6.2

†

1 6 8

45.0-56.0%

#

10. 7

8 4

NA

42.0-51.0%

T bili, mg/d L T bili refer ence range

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0

Hours of life 3 36 0

NA

0.0 15 NA

≥2.0 indicates normal RBC proliferation. <2.0 indicates hypoproliferation

6. 1

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Retic Index refer ence range ET: Exchange transfusion; TPT: triple phototherapy; NA: Not any; HCT: hematocrit, T bili: total bilirubin, %tile: percentile, Retic: reticulocyte. *Post-exchange values # Hematocrit reference range for full term infants (> 37 wks).16 † Total bilirubin reference range for full term infants (> 37 wks).16, 17

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Table 2. Neonate pre-exchange transfusion testing results. O, RhD negative

Antibody Screen

Anti-K

DAT IgG

2+

DAT C3

3+

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Blood type

Anti-K

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Eluate

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Initial Jka Phenotype

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Initial Kell Phenotype

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Kell Phenotype following modified gentle heat elution

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Kell Genotyping

Negative Negative

K+*

K+

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*The untreated neonatal specimen was non-reactive with commercial anti-sera against K. Following attempt elution by heat-dissociation to remove suspected K-blocking antibodies from the RBCs, strongly positive microscopic agglutination with K anti-sera was observed.

Journal Pre-proof Highlights

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High-titer antibodies cause false-negatives in red blood cell antigen phenotyping. Accurate RBC phenotyping is important in hemolytic disease of the fetus and newborn. Resolving false-negative phenotyping from high-titer anti-K is uniquely challenging. We present a method to resolve false-negative phenotyping due to high-titer anti-K.

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