Transfusion medicine

Blood bank testing

Adsorption studies


Editorial Board Member: Mrigender Singh Virk, M.D.
Deputy Editor-in-Chief: Patricia Tsang, M.D., M.B.A.
Sarah Kesterson, M.D.
Melissa R. George, D.O.

Last author update: 8 August 2023
Last staff update: 8 August 2023

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PubMed Search: Adsorption studies

Sarah Kesterson, M.D.
Melissa R. George, D.O.
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Cite this page: Kesterson S, Potochny EM, George MR. Adsorption studies. PathologyOutlines.com website. https://www.pathologyoutlines.com/topic/transmedadsorptionstudies.html. Accessed April 14th, 2024.
Definition / general
  • Adsorption (or adsorption studies) is an advanced serologic testing method used to separate warm autoantibodies from serum in order to appropriately identify underlying alloantibodies
  • Antibodies can be strategically removed from the serum by allowing them to adsorb onto the surface of red blood cells (RBCs) that express the target antigen
  • Adsorbed serum can then be tested for remaining alloantibody reactivity
Essential features
  • Warm autoantibodies may mask alloantibodies on routine serologic testing
  • Adsorption allows for the identification of any underlying alloantibodies, an essential step in patient transfusion safety
Terminology
  • Warm autoantibody (WAA): antibody directed against high incidence antigens on patient's own RBCs, optimally bound at or near body temperature (~37 °C)
  • Cold autoantibody: antibody directed against high incidence antigens on patient's own RBCs, optimally bound at colder temperatures (room temperature or 4 °C)
  • Drug related WAA: many medications have the potential to cause hemolysis in vivo; routine serologic testing of these samples look similar to WAA
  • Elution: testing technique that dissociates bound IgG from RBCs
    • Multiple methods available (e.g., acid elution kits, heat, freeze - thaw)
    • Eluate can then be tested against reagent red cells to delineate WAA from drug related WAA
  • Autoadsorption: adsorption technique used if the patient has no recent (within 3 months) transfusion history; patient RBCs can be used to adsorb the WAA while alloantibodies, if present, will remain in serum
  • Alloadsorption: adsorption technique used if the patient has recent transfusion history (within 3 months), patient cells are not used in the adsorption testing as the sample would contain native and transfused cells, the latter which may adsorb alloantibodies (Immunohematology 2018;33:1)
  • Adsorbed serum (or plasma): serum remaining after each adsorption process, may contain additional antibodies
  • Cold adsorption: similar adsorption technique performed at cold temperatures, the temperature at which the antibody is optimally bound should be used for incubation
  • Reference: Harmening: Modern Blood Banking & Transfusion Practices, 7th Edition, 2018
Pathophysiology
  • Autoantibodies are directed against RBCs
  • Can cause destruction (autoimmune hemolytic anemia [AIHA]) of circulating RBCs
  • Phagocytosis of autoantibody coated RBCs occurs in the spleen
  • WAA present challenges in pretransfusion compatibility testing
    • Optimally reactive at 37 °C
    • May mask the presence of clinically significant alloantibodies by agglutinating most or all RBCs tested
    • Incidence of clinically significant alloantibodies is higher in patients with WAAs than in multiply transfused patients without AIHA
  • Reference: Cohn: Technical Manual, 20th Edition, 2020
Clinical features
  • WAAs may be associated with a variety of clinical conditions (e.g., autoimmune illnesses including systemic lupus erythematosus, lymphomas) or may be idiopathic
  • Certain drugs may also elicit a warm autoantibody reaction (e.g., methyldopa, penicillin, quinolones)
  • Elution is positive in WAAs but is normally negative in drug related WAA reactivity
  • WAAs may be associated with decreases in hemoglobin and hematocrit and therefore related symptoms (noted below)
  • ~7% of WAA cases suffer from Evans Syndrome, the combined entity of warm AIHA and immune thrombocytopenia (NIH: Evans Syndrome [Accessed 9 May 2023], J Clin Med 2020;9:3851)
Symptoms
Peripheral smear images

Contributed by Melissa R. George, D.O.
Spherocytes

Spherocytes

Spherocytes and polychromasia

Spherocytes and polychromasia

Screening
  • WAAs can be detected when
    • Antibody screen / panel demonstrates panagglutination at 37 °C and antihuman globulin (AHG) phases
    • Autocontrol is positive
    • Direct antiglobulin test (DAT) is positive at IgG with or without complement C3
    • Elution is panreactive (if elution is negative, a drug related antibody is suspected)
    • Peripheral blood smear demonstrates increased spherocytes, polychromasia, anisopoikilocytosis
    • pRBC units selected for transfusion may be incompatible
  • Decisions about future adsorption studies can be made based upon changes in strength of DAT
    • If DAT strength increases, suspect either new alloantibody or increase in strength of WAA
      • If DAT reactivity has become stronger, consider adsorption to rule out alloantibody
    • If DAT strength decreases, suspect WAA has become weaker and there is likely not an underlying alloantibody
    • If DAT reactivity is the same or weaker, may be able to avoid adsorption and continue with least incompatible blood
  • References: Cohn: Technical Manual, 20th Edition, 2020, Hematology Am Soc Educ Program 2022;2022:96
Blood donor testing
  • Donor testing includes antibody screening, auto or alloantibodies should be detected at this stage
Donor deferral
  • Donors with positive antibody screens are generally deferred, except in particular circumstances
Laboratory
  • Blood bank testing leading up to the need for adsorption studies often includes
    • In the case of autoantibodies
      • Panreactive antibody screen and panel
      • Positive autocontrol
      • Positive DAT
      • Panreactive elution
    • 2 types of adsorption
      • Autoadsorption if patient has not been transfused recently (within past 90 days)
      • Alloadsorption if patient has been transfused (within past 90 days)
    • Autoadsorption involves using the patient's own RBCs to adsorb autoantibodies from serum
    • Alloantibodies are left in plasma
    • Adsorbed serum is tested against a panel to identify alloantibodies
    • Traditional allogeneic adsorptions use a set of 3 adsorbing cells with known phenotypes: R1R1, R2R2 and rr
      • At least 1 of the adsorbing cells should be negative for K and Jka or Jkb
      • At least 1 of the reagent cells should also lack S or s and Fya or Fyb unless the cells are pretreated to denature the antigens
      • A separate aliquot of patient serum is incubated with each adsorbing cell
      • Once all autoantibody reactivity is removed, the 3 adsorbed serums are tested to eliminate or prove the presence of any underlying alloantibody
      • Each aliquot of adsorbed serum contains alloantibody corresponding to those antigens for which the adsorbing cell was negative
      • Once adsorption is complete, test the adsorbed serum against reagent RBCs to rule out the presence of underlying alloantibodies
        • For instance, if the adsorbing cell is R1R1, S-, K-, Jka-, then the following alloantibodies can be ruled out in that aliquot of adsorbed serum: anti-E, -c, -S, -K and -Jka
    • References: Immunohematology 2014;30:153, Hematology Am Soc Educ Program 2022;2022:96
Case reports
  • 10 month old boy with AIHA, allogeneic adsorption and heat elution identified auto-anti-f, auto-anti-Jk(a) (Transfus Apher Sci 2020;59:102762)
  • 30 year old man with hyperhemolysis syndrome and sickle cell disease, allogeneic adsorption identified possible anti-Fy3 / Fy5 (Transfusion 2022;62:1447)
  • 78 year old man, adsorption elution studies identified anti-C, anti-D, anti-G (Ann Lab Med 2018;38:280)
Treatment
  • In the case of strong WAAs, when underlying alloantibodies have been ruled out, least incompatible pRBC units may be the only option and transfusion should not be postponed if the patient requires blood components
  • In the case of weaker WAAs, crossmatch compatible units may be identified using additional serologic techniques
  • When 1 or more alloantibodies are identified, the patient should receive only antigen negative units corresponding to the alloantibodies present
  • Infrequently, autoantibodies have specificity to particular antigens
    • Occasional specificity to simple Rh antigens but also specificities in the LW, KEL, JK, FY and DI systems have been reported (Cohn: Technical Manual, 20th Edition, 2020)
    • In this case, antigen avoidance may not be advantageous, particularly if this exposes the patient to an antithetical antigen they do not possess, creating the potential formation of an alloantibody to that antithetical antigen
    • Antigen avoidance in these scenarios should be determined on a case by case basis
  • Strategies
    • Transfuse least incompatible nonphenotypically matched RBCs
    • Transfuse least incompatible RBCs that are phenotypically matched for either the full phenotype or only for the common antigens in the Rh blood group system and for K
  • Prevalence is low when indirect antiglobulin test is negative (< 1%)
  • Significant portion of patients with WAA form new RBC alloantibody (~15%); however, the use of prophylactic antigen matched (PAM) approach for RBC selection does not appear to be protective against new alloimmunization (Vox Sang 2020;115:515)
Sample assessment & plan
  • Assessment: John Doe is a 66 year old man with O, Rh(D) positive blood type and history of acute myelogenous leukemia who presented with neutropenic fever. A type and screen performed by the hospital transfusion service identified a warm autoantibody; panreactivity was identified at AHG phase with polyethylene glycol (PEG) enhancement. Testing was also positive using 1 hour saline technique. The autocontrol was positive and the DAT was positive for IgG. An elution was panreactive (1+ grade of reaction). No prior history of antibodies. His last transfusion was 1 year ago.
  • Plan:
    • If red cell transfusion is required, O, Rh(D) positive or negative, least incompatible units using saline technique will be issued.
    • Recommend additional specimen (X # of red top and X # of lavender top tubes) be sent to the blood bank for autologous adsorption studies to exclude underlying alloantibodies.
    • An addendum will be issued with results of the adsorption studies.
Differential diagnosis
Board review style question #1
A 74 year old man with relapsed acute myeloid leukemia (AML) currently on chemotherapy had a complicated clinical course following COVID-19 infection, with subsequent fungal pneumonia and septic shock. He had known red blood cell antibodies against E and P1. During his hospital course, he developed new reactivity. Anti-Jka was identified and about a month later, a peripheral blood smear was reviewed on the patient. The slide signed out by the clinical pathologist on service noted: "moderate anisopoikilocytosis with spherocytes, occasional ovalocytes and teardrop cells... The findings are consistent with patient's history of chemotherapy for AML. The presence of spherocytes, positive autocontrol, positive DAT at IgG phase and panreactive elution, is suggestive of a warm autoantibody."

The patient was transfused several packed red blood cell and platelet units in the past 90 days. What would be the next best step in pretransfusion compatibility testing?

  1. Allogeneic adsorption
  2. Autologous adsorption
  3. Cold adsorption
  4. Dithiothreitol (DTT) treatment
Board review style answer #1
A. Allogeneic adsorption. In the setting of a warm autoantibody in a patient who already has alloantibodies and has been transfused within the past 90 days, allogeneic adsorption is the most appropriate next step. Answer B is incorrect because recent transfusion precludes autologous transfusion. Answer C is incorrect because the reactivity is seen at IgG phase and therefore at 37 °C, cold adsorption is not indicated. Answer D is incorrect because dithiothreitol (DTT) is useful for decreasing CD38 on red blood cells and therefore reducing / eliminating interference from daratumumab therapy often used in plasma cell myeloma and sometimes for pure red cell aplasia / delayed engraftment status post-bone marrow transplant. However, there is no indication from the clinical history that the patient is receiving this therapy.

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Reference: Adsorption studies
Board review style question #2
A type and screen in a 56 year old woman with newly diagnosed Waldenström macroglobulinemia reveals panreactivity at immediate spin phase only. The autocontrol is positive at immediate spin phase and the direct antiglobulin test (DAT) is positive for complement. No reactivity is seen at antihuman globulin (AHG) phase using polyethylene glycol (PEG) enhancement; the autocontrol at AHG phase is also negative. The patient has no history of transfusions and no prior history of red cell antibodies. What is the most likely next step(s) for the technologist to exclude any underlying alloantibody?

  1. No additional testing is necessary
  2. Repeat the DAT; the results are erroneous given the absence of a DAT positive for IgG
  3. Send out for allogeneic adsorption studies
  4. Send out for autologous adsorption studies
Board review style answer #2
A. No additional testing should be necessary. The panel described above is most compatible with a cold autoantibody or cold agglutinin, likely secondary to the patient's diagnosis of Waldenström macroglobulinemia. Cold agglutinins that do not show reactivity at AHG phase are unlikely to be clinically significant (i.e., cause in vivo hemolysis). Even if there were an underlying cold alloantibody, this would be unlikely to be clinically significant in the absence of reactivity at AHG phase; therefore, no additional testing is needed. AHG crossmatch compatible units of RBCs can safely be issued to this patient. Answers B - D are incorrect because these tests are not indicated in this case.

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Reference: Adsorption studies
Board review style question #3
A 60 year old woman was recently admitted to the hospital. Below are pertinent laboratory and blood bank results.

Parameter Patient results Normal range
Hemoglobin 10.2 g/dL 11.7 - 15.0 g/dL
Hematocrit 30.5% 35 - 44%
Lactate dehydrogenase (LDH) 175 U/L 135 - 250 U/L
Total bilirubin 1.1 mg/dL 0.0 - 1.2 mg/dL
Haptoglobin 76 mg/dL 30 - 200 mg/dL
  
Parameter Patient results
Antibody screen IS: negative; AHG: panreactivity (1+)
Antibody panel IS: negative; AHG: panreactivity (1+)
Autocontrol Positive
DAT IgG: 1+; C3: 0
Elution Panreactivity (2+)
IS: immediate spin; AHG: antihuman globulin


Based on these results, which of the following is true?

  1. Alloantibody to a high prevalence antigen is present
  2. Cold autoantibody is present
  3. Warm autoantibody is present
  4. Without evidence of hemolysis, a warm autoantibody can be ruled out
Board review style answer #3
C. Warm autoantibody is present. These serologic results indicate a warm autoantibody and the diagnosis of a warm autoantibody can be made based on these results alone. While warm autoantibodies have the potential to cause in vivo hemolysis, not all do. The diagnosis of warm autoimmune hemolytic anemia (WAHA), however, requires the presence of a warm autoantibody and evidence of in vivo hemolysis (e.g., drop in hemoglobin / hematocrit, elevated bilirubin, elevated LDH, decreased haptoglobin, peripheral smear findings). Answer D is incorrect because a warm autoantibody cannot be ruled out in the absence of hemolysis. Answer B is incorrect because immediate spin results were negative, which is not consistent with a cold autoantibody. Answer A is incorrect because in the case of an alloantibody or even multiple alloantibodies, panreactivity is common but the autocontrol would likely be negative.

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Reference: Adsorption studies
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