Allogeneic Red Blood Cell Adsorption for Removal of Warm Autoantibody

Allogeneic red blood cell adsorption for removal of warm autoantibody

C. Barron

Adsorption studies are usually required to confirm or rule out Reagents/Supplies the presence of underlying alloantibodies in samples containing warm autoantibody. Allogeneic adsorptions are necessary if the Treatment patient has been recently transfused. Most commonly, allogeneic Method Reagents Supplies adsorptions are performed using a trio of phenotyped reagent Papain • 1% cysteine-activated red blood cells to rule out clinically significant alloantibodies to papain common antigens. The adsorbing cells may be used untreated • Pipettes or treated with enzymes or with ZZAP before adsorption. Ficin • 1% ficin • Isotonic • 37°C water Adsorption may also be performed using enhancement such ZZAP • 1% cysteine-activated saline bath REVIEW METHOD SEROLOGIC as low-ionic strength saline or polyethylene glycol added to papain or 1% ficin • Allogeneic the mixture. Multiple adsorptions may be necessary to remove RBCs • Large-bore • 0.2 M DTT test tubes strongly reactive autoantibodies. Allogeneic adsorptions • Glycine max • Commercial LISS will not detect alloantibodies to high-prevalence antigens. var. soja • Calibrated enhancement reagent centrifuge Immunohematology 2014;30:153–155. • 20% PEG or commercial PEG enhancement Key Words: AIHA, adsorption, autoantibody, alloantibody, DTT = dithiothreitol; RBCs = red blood cells; LISS = low-ionic-strength allogeneic RBCs saline; PEG = polyethylene glycol.

The serum from patients with warm autoimmune Procedural Steps hemolytic anemia (WAIHA) typically demonstrates broad Preparation of RBCs for adsorption reactivity with all reagent red blood cells (RBCs). This • Select reagent RBCs for use as adsorbing cells. reactivity usually necessitates the use of incompatible blood for • Treat the adsorbing cells with the desired enzyme or ZZAP. transfusion. When blood is incompatible as a result of an RBC Adsorption autoantibody, transfused RBCs will survive at a rate similar • Add 1 to 2 volumes of treated adsorbing cells to 1 volume of to that of the patient’s own RBCs.1 However, autoantibody patient serum or plasma and 1 volume of enhancement reagent (if applicable). reactivity may mask the reactions of underlying clinically • Incubate for 30–60 minutes at 37°C without enhancement, or 15 significant alloantibodies to common antigens, which can minutes at 37°C with LISS or PEG enhancement. • Harvest the adsorbed serum or plasma and test for the presence of cause rapid RBC destruction. The rate of alloimmunization in underlying alloantibodies. patients with warm autoantibodies varies among investigators. RBCs = red blood cells. Reported alloimmunization rates range from 17.5 to 38 percent, with an average rate of 32 percent.2–7 Issitt et al.4 found that 47 percent of alloantibody specificities identified after adsorption were actually partially adsorbed autoantibodies autoantibody has been removed and whether alloantibody with mimicking specificities. Mimicking autoantibody is present.8 Although this may be useful as a screening tool, specificities were found most often when autologous adsorption this method has been shown to be unreliable in consistently was used to remove autoantibody reactivity. Identification of detecting alloantibody underlying autoantibody.9 Autologous alloantibody specificities underlying a warm autoantibody is adsorption, which uses the patient’s own RBCs to remove critical for a safe and effective transfusion. the autoantibody, is a preferred method. Using autologous Methods used to determine the presence of an alloantibody RBCs ensures that only autoantibodies are removed from the underlying an autoantibody include serum dilution, autologous serum, leaving intact any alloantibody the patient may have adsorption, and allogeneic adsorption. The dilution screening produced, including antibodies to high-prevalence antigens. method uses a 1:5 dilution of serum in phosphate-buffered However, autologous adsorptions may only be performed saline. The diluted serum is tested to determine whether the when the patient has not been recently transfused or pregnant,

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commonly defined as having no transfusions or pregnancy in specificity corresponding to an antigen that is destroyed by the prior 3 months. Alloantibody may be removed from the enzymes (e.g., anti-Ge2) or ZZAP (e.g., anti-LWa or Kell system test system if autologous adsorptions are performed using antibodies). In these cases, adsorption with untreated reagent a sample from a recently transfused patient. Laine et al.10 RBCs is necessary. showed that even small amounts of transfused RBCs are The addition of enhancement media to the adsorbing capable of removing all alloantibody reactivity. Allogeneic mixture has also become commonplace and can be used in RBC adsorption is the method of choice for removal of warm place of RBC pretreatment. The addition of low-ionic-strength autoantibody reactivity in patients with recent transfusions. saline (LISS) or polyethylene glycol (PEG) reduces the Note that the term “serum” is used to denote the patient sample processing time and may decrease the number of adsorptions throughout this article; however, either serum or plasma may required to remove the autoantibody.9,11,12 However, some be used for testing. investigators caution that some underlying alloantibodies may not be detected when PEG adsorptions are performed.13,14 Principle Indications Allogeneic adsorptions are used to remove broadly reactive autoantibody from a patient’s serum to allow for detection of Allogeneic adsorptions should be used when a patient has underlying clinically significant alloantibodies to common been recently transfused or when autologous cells are of limited antigens. The adsorbing cells will remove autoantibody along supply. Adsorption eliminates the reactivity of the warm with any additional antibody specificities to antigens that are autoantibody, allowing for the identification of alloantibodies present on the RBC. When performing allogeneic adsorptions, in the adsorbed serum. the goal is to eliminate or confirm the presence of those alloantibodies directed at the common antigens D, C, E, c, e, Procedure K, S, s, Fya, Fyb, Jka, and Jkb. Traditional allogeneic adsorptions use a set of three Select cells for adsorption. If the patient’s RBC phenotype is adsorbing cells with known phenotypes: R1R1, R2R2, and rr. known, select a phenotypically similar reagent RBC that lacks At least one of the adsorbing cells should be negative for K and the same common RBC antigens as the patient’s RBCs. If the Jka or Jkb. In addition, at least one of the reagent cells should patient’s RBC phenotype is unknown, select a trio of reagent a b also lack S or s and Fy or Fy unless the cells are pretreated RBCs: R1R1, R2R2, and rr. Additionally, at least one cell of the to denature the antigens. A separate aliquot of patient serum set should be negative for K and Jka or Jkb so that all common is incubated with each adsorbing cell. Once all autoantibody alloantibodies can be detected using the adsorbed serum. If reactivity is removed, the three adsorbed serums are tested to adsorptions will be performed using untreated reagent RBCs, eliminate or prove the presence of any underlying alloantibody. at least one cell should be negative for S or s and Fya or Fyb as Each aliquot of adsorbed serum contains alloantibody well.15–18 corresponding to those antigens for which the adsorbing cell If desired, treat the adsorbing cells with enzymes (ficin was negative. or papain) or ZZAP (ficin or papain and 0.2 M DTT). This If a patient’s RBC phenotype is known, it may be possible pretreatment will typically enhance the adsorbing process. to adsorb using a single phenotypically similar reagent RBC. Following pretreatment, wash the adsorbing cells, For instance, if a patient is only negative for E, K, S, and Fya, centrifuge, and remove all excess saline before use. Perform one reagent RBC negative for those antigens can be used for the adsorption by mixing one to two volumes of allogeneic adsorption. After adsorption, the adsorbed serum can be RBCs with one volume of patient serum at 37°C for 30 to 60 tested to confirm or eliminate the presence of anti-E, -K, -S, minutes. and/or -Fya. Another option is to perform the adsorption by mixing Various methods are used for performing the adsorption. one volume of patient serum and one volume of LISS or PEG19 Treatment of the adsorbing cells using enzymes or ZZAP may with one to two volumes of allogeneic reagent RBCs at 37°C enhance the uptake of the autoantibody.9 These cell treat- for 15 minutes.15–18 ments will also destroy some antigens, which may be After the adsorption, centrifuge the mixture and harvest advantageous when selecting cells for adsorption and testing the adsorbed serum. Test the adsorbed serum to determine the adsorbed serum. Rarely, autoantibodies may demonstrate whether adsorption is complete. This may be accomplished

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by testing the adsorbed serum against the adsorbing cells 4. Issitt PD, Combs MR, Bumgarner DJ, Allen J, Kirkland or by testing the adsorbed serum against a reagent RBC A, Melroy-Carawan H. Studies of antibodies in the sera of patients who have made red cell autoantibodies. Transfusion phenotypically similar to the adsorbing cell. If necessary, 1996;36:481–6. repeat the adsorption using a new aliquot of adsorbing cells. 5. Laine EP, Leger RM, Arndt PA, Calhoun L, Garratty G, Once adsorption is complete, test the adsorbed serum Petz LD. In vitro studies of the impact of transfusion on the against reagent RBCs to rule out the presence of underlying detection of alloantibodies after autoadsorption. Transfusion 2000;40:1384–7. alloantibodies. For instance, if the adsorbing cell is R1R1, S–, 6. Young PP, Uzieblo A, Trulock E, Lublin DM, Goodnough LT. K– Jk(a–), then the following alloantibodies can be ruled out Autoantibody formation after alloimmunization: are blood in that aliquot of adsorbed serum; anti-E, -c, -S, -K, and -Jka. transfusions a risk factor for autoimmune hemolytic anemia? Transfusion 2004;44:67–72. Note: Test the adsorbed serum from the LISS or PEG 7. Maley M, Bruce DG, Babb RG, Wells AW, Williams M. The enhancements using four drops of the serum/enhancement incidence of red cell alloantibodies underlying panreactive reagent mixture. warm autoantibodies. Immunohematology 2005;21:122–5. 8. Øyen R, Angeles ML. A simple screening method to evaluate Limitations the presence of alloantibodies with concomitant warm autoantibodies. Immunohematology 1995;11:85–7. 9. Leger RM, Garratty G. Evaluation of methods for detecting Allogeneic adsorption will not rule out the presence of alloantibodies underlying warm autoantibodies. Transfusion alloantibodies to high-prevalence antigens. The adsorbing cells 1999;39:11–16. are presumed to be positive for all high-prevalence antigens; 10. Laine EP, Leger RM, Arndt PA, Calhoun L, Garratty G, Petz LD. In vitro studies of the impact of transfusion on the therefore, an alloantibody to a high-prevalence antigen would detection of alloantibodies after autoadsorption. Transfusion be adsorbed along with the autoantibody. 2000;40:1384–7. When a patient’s serum contains a very potent warm 11. Chiaroni J, Tousinssi M, Mazet M, De Micco P, Ferrera V. Adsorption of autoantibodies in the presence of LISS to detect autoantibody, multiple adsorptions are needed to remove the alloantibodies underlying warm autoantibodies. Transfusion broad reactivity. Generally, for each grade of reactivity, at least 2003;43:651–5. one adsorption will be necessary (a serum sample with an 12. Cheng CK, Wong M, Lee AW. PEG adsorption of autoantibodies autoantibody that reacts 2+ will require a minimum of two and detection of alloantibodies in warm autoimmune hemolytic anemia. Transfusion 2001;41:13–17. adsorptions). Multiple adsorptions have the potential to dilute 13. Barron CL, Brown MB. The use of polyethylene glycol (PEG) the serum. Increasing the ratio of adsorbing cells to serum to enhance adsorption of autoantibodies. Immunohematology may enhance the removal of the autoantibody. 1997;13:119–22. The use of PEG adsorption may cause weakening or loss 14. Judd WJ, Dake L. PEG adsorption of autoantibody causes loss of concomitant alloantibody. Immunohematology 2001;17: of antibody reactivity in some samples. The use of six drops of 82–5. the serum/PEG mixture in antibody identification tests may 15. Judd J, Johnson ST, Storry JR. Judd’s methods in minimize this effect. immunohematology. 3rd ed. Bethesda, MD: American Association of Blood Banks, 2008;469–72. Quality Control 16. Fung MK. Technical manual. 18th ed. Chapter 17: Positive DAT and immune-mediated hemolysis. Bethesda, MD: American Association of Blood Banks, 2014;425–51. To determine whether the adsorbing cells are adequately 17. Beatie K, Crawford M, Martin J, et al. Immunohematology treated with enzymes, test the cells with Glycine max var. soja methods. Rockville, MD: American Red Cross, 1993;47–51. lectin. Enzyme-treated RBCs should demonstrate positive 18. Fung MK. Technical manual. 18th ed. Method 4–9: Adsorbing warm-reactive autoantibodies using allogeneic red cells. reactivity with G. max var. soja and untreated RBCs should Bethesda, MD: American Association of Blood Banks, 2014. be nonreactive. 19. Fung MK. Technical manual. 18th ed. Method 4–10: Polyethylene glycol adsorption procedure. Bethesda, MD: References American Association of Blood Banks, 2014.

1. Petz LD. A physician’s guide to transfusion in autoimmune Christina Barron, MT(ASCP)SBB, Manager, Immunohematology haemolytic anaemia. Br J Haematol 2004;124:712–16. Reference Laboratory, American Red Cross, Missouri-Illinois 2. Wallhermfechtel MA, Pohl BA, Chaplin H. Alloimmunization Region, 4050 Lindell, St. Louis, MO 63108. in patients with warm autoantibodies. Transfusion 1984;24: 482–5. 3. Laine ML, Beattie KM. Frequency of alloantibodies accompanying autoantibodies. Transfusion 1985;25:545–6.

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