HTLA v. Dara v. PEG reactive Cold v. High Freq

[Callie1]

Does anyone have a procedure for pulling apart a panreactive weak IAT panel?

[Bet'naSBB]

What media are you starting in?

Is auto control pos?

DAT / monospecifics pos?

Edited November 16, 2023 by Bet'naSBB

[applejw]

Is your patient either taking Darzalex or Sarclisa or has taken it within the last 12 months?

[Mabel Adams]

This is from our ABID SOP (sorry the format is weird): 

a.    Warm Autoantibody Guidelines –(Expert judgment is required on a case-by-case basis to supplement these guidelines.  Contact supervisor or Reference Lab for advice) [return to top]

                        i.        Clues it’s a Warm Auto

1.    Reacts with most or all cells tested—usually at a fairly consistent strength.

2.    Positive DAT.

3.    If it is severe enough that there is hemolytic anemia, the patient would be anemic with a high LDH or bilirubin and high reticulocyte count.  The patient’s plasma sample may appear hemolyzed or icteric.  Haptoglobin would be low.

4.    Contact Blood Bank Supervisor for further consult if uncertain. 

5.    Patient has not taken anti-CD38 (daratumumab/Darzalex/DARA) or anti-CD47 drugs in prior 6 months.

                      ii.        Clues it’s NOT a Warm Auto

1.    Reactivity with most or all cells in gel, usually 2+ or less, varying strength could be HTLA-like antibody or antibody to gel diluent. It’s not uncommon for patients to have a positive DAT without having a detectable warm auto antibody so the positive DAT could be present coincidentally.

a.    To confirm antibody to gel diluent, convert 3% screen cells to 0.8% to run in gel. If negative, turn out the 3-cell gel screen and document situation in the patient record.

b.    An HLTA-like antibody will usually remain detectable (although sometimes weaker) in PEG and even saline techniques.

2.    Alloantibodies:

a.    If patient transfused in the prior 3 months, confer with the most expert person available before calling it a warm-auto.

b.    Elution may produce both a panagglutinin and another specificity or may show only a new alloantibody. The panagglutinin may be so strong a weak allo is undetectable.

c.    If the DAT looks mixed field (repeat IgG DAT in gel for clear-cut mixed field), it may be an allo.

3.    Drug-induced antibody:

a.    Research patient’s diagnoses for multiple myeloma, amyloidosis, or other autoimmune diseases. Look for recent surgeries or infections as a clue for cephalosporin treatment. Research their drug history for cephalosporins and anti-CD38 or anti-CD47 drugs.  There is variability whether these present with only a positive DAT (more common with cephalosporins) or with a negative DAT and positive screen or with both positive.

b.    Rarely, other drugs cause what looks like a warm autoantibody.

                     iii.        When the Auto control (AC) or DAT is positive, first check transfusion history.  The AC could be positive due to a delayed serologic/hemolytic transfusion reaction and NOT a warm auto.  See Positive autocontrol.

                     iv.        If not already done, perform a DAT (Direct Antiglobulin Test Procedure).  Warm autoantibody patients can have RBCs coated with IgG only or IgG and complement both.  Occasionally, only complement may be present.

                      v.        If not already done, perform an antibody panel in gel (or other primary testing method).

                     vi.        People who make warm autoantibodies are more likely to make allo-antibodies as well. We need to identify any allo-antibodies in the sample so we want to avoid detecting the autoantibody if possible but still be able to detect allo-antibodies (at least strong ones).

                    vii.        If reactivity in gel is < 1+ or there are some negative reactions, start a 3% tube PEG antibody screen.  If reactivity in gel is ≥ 1+, start a 3% Saline (no enhancement) 30-minute tube antibody screen.  If amount of specimen is minimal, skip PEG screen and only do saline or, if some negatives, rule out with negative reactions found in gel and run selected cells needed to complete ruling out the usual antibody specificities.

                   viii.        If patient has not been recently transfused and usual specificities can’t be ruled out in tube testing, a PEG Autoadsorption should be considered.  PEG adsorptions should not be attempted with patients who have a strong complement coating.

                     ix.        If can’t rule out usual specificities in tube testing and the patient has been transfused in the past 3 months, send the specimen to the Reference Lab for allo-adsorptions to determine the presence/absence of underlying alloantibodies. See Red Cross (ARC) BloodHub/Connect--Standard Work.  If the patient is too critical to wait for the workup, contact the on-call Pathologist and Blood Bank supervisor.  Phenotypically matched units may be indicated. See Increased Risk Transfusion Release Form. 

                      x.        Turn out ABID results as Warm Autoantibody plus any other specificities detected.

                     xi.        Approach to crossmatching in the presence of warm auto-antibodies:

Situation	Pt Hemolyzing*	XM Methods	XM Results
Enough negs in gel to rule out all usual Ab specificities	Yes	Gel	Incompatible
Enough negs in gel to rule out all usual Ab specificities	No	Gel	Compatible
Can rule out all usual Ab specificities in PEG	Yes	Gel	Incompatible
Can rule out all usual Ab specificities in PEG	No	PEG	Compatible
Can rule out all usual Ab specificities in Saline	Yes	Gel	Incompatible
Can rule out all usual Ab specificities in Saline	No	Saline	Compatible
Alloantibodies identified or can’t rule out some Ab specificities in Saline	Yes	Saline** to assess compatibility with allos, then gel to turn out results	Incompatible
Alloantibodies identified or can’t rule out some Ab specificities in Saline	No	Saline	Compatible
Autoadsorption required and able to rule out usual Ab specificities	Yes or No	Gel with neat plasma	Incompatible
Autoadsorption required and alloantibodies identified or can’t rule out usual Ab specificities	Yes or No	With adsorbed sample** to assess compatibility with allos, then gel with neat sample to turn out results	Incompatible

 

*If patient is hemolyzing, no transfused unit will be truly compatible. Use “incompatible” XM result code in STTx, not “least incompatible” for these cases.

**If second XM method (that’s not to be turned out) is required, record on log sheet.

1.    Warm Auto Notes:

a.    The purpose of the PEG or Saline Antibody screen or PEG adsorption is not to be able to call the primary antibody screen negative, but to rule out underlying alloantibodies.  Generally, these tube ABSC’s will NOT be reported in STTx.

b.    Incubation in the presence of enhancement (gel/PEG) reagents may cause reactivity in the AC that is only an in vitro phenomenon. If the DAT is negative and the AC is positive, antibodies to enhancement constituent or autoantibodies reactive only in enhancement medium should be considered.  An Antibody Elution (Eluate) may help determine the presence/absence of warm autoantibody reactivity.

c.    If the patient is demonstrating active hemolysis, use gel or PEG to crossmatch units. The units still may suffer shortened red cell survival in vivo so calling them incompatible is justifiable.

d.    Consult with Blood Bank Supervisor about performing a full phenotype with the available monoclonal (non-AHG) antisera.  Consider giving phenotypically similar RBCs for transfusion.  If alloantibodies are ruled out in a current specimen, units that are only historically antigen-negative are acceptable.   If we must transfuse before alloantibodies can be ruled out, confirmed antigen-matched units are advised if time permits.

e.    Warm autoantibodies can be confirmed in one of two ways: demonstrate that EGA-treated (antibody removed) pre-transfusion autologous cells react with neat plasma or prove that the antibody reactivity is adsorbed out with pre-transfusion, autologous cells.

f.     Patients on daratumumab (Darzalex or DARA or other anti-cd38 drugs) may appear to have a warm auto antibody but it is actually the drug reacting with the cd38 antigens on the red cells. They may have either a negative or positive DAT and AC.  The only effective way to test these patients is to test against DTT treated cells, recognizing that this will miss antibodies to antigens destroyed by DTT like the Kell system.  These patients benefit from having a pre-treatment antibody screen run and possibly antigen typing for K (and if K positive, for k).  In most cases molecular genotyping may be indicated. See Dithiothreitol (DTT) Treatment and Anti-CD38 Drugs (daratumumab/Darzalex)--Blood Bank Testing.

g.    Additional anti-CD38 drug therapeutics are in clinical trials in addition to Daratumumab (Janssen Biotech) include MOR202 (MorphoSys), Sarclisa -Isatuximab (Sanofi-Aventis), and TAK-079 (Takeda) for treatment of systemic lupus erythematosus, Amyloidosis, or other autoimmune diseases. Daratumumab and Sarclisa are approved for treating multiple myeloma.

h.    CD47 is a glycoprotein expressed on all cells including RBCs and platelets, which usually signals to prevent phagocytosis.  Anti-CD47 blocks this signal targeting cells for destruction. Samples from patients taking anti-CD47 drugs (Hu5F9-G4 or avelumab) will react with everything like a warm auto and the reverse type may be affected like a cold auto. Anti-CD47 interferes with all RBC and platelet serological tests performed including ABO reverse typing. False positive reactions can be seen in all phases of testing (immediate spin, 37°C, and IAT) and with all forms of IAT testing (i.e., tube, gel, solid phase). Reactions with D negative cells may be stronger than with other Rh phenotypes. False negative phenotyping test results can occur due to RBCs heavily coated by anti-CD47. DATs may be falsely negative due to a “blocking effect” caused by high levels of antibody present, but eluates are strongly positive. Plasma interference and strong panreactive eluates are observed as soon as 1 hour after drug infusion. CD47 antigens cannot be denatured with DDT or other common denaturing agents.  It is highly recommended to perform pretransfusion testing, including blood type, antibody screen and extended phenotype (either serological or predictive genotype) before initiating treatment. Using monoclonal Gamma-clone Anti-IgG in indirect antiglobulin testing (which does not detect IgG4 immune classes like anti-CD47) may avoid most of the interference in AHG testing.  Giving antigen-matched units may be an option if full phenotyping is available.  [return to top]

i.      Additional CD47 drug therapies are also in clinical trials and include the CD47 targeting antibodies CC9002 (Celgene), which, like Hu5F9, is also an IgG4 antibody, and the human monoclonal SRF231 (Surface Oncology). CD47 agonists are also in clinical trials. These include TT1-621 (Trillium)31 and ALX148 (ALX Oncology), which are fusion proteins with the Fc region of IgG1 antibody fused to the CD47-binding domain of SIRPα with the goal of interrupting the CD47-SIRPα survival signal. Unlike CD47-targeting antibodies, TT1-621 appears to bind only minimally to human RBCs and interference in pretransfusion testing has not been observed or reported to date.

[Callie1]

Hi, Everyone:

Sorry I am so late responding. Too much going on. Yes - this question was regarding procedures and not a particular case. Wow - that is a fantastically detailed SOP. I don't have time tonight but will give it a good read tomorrow. Thank you for sharing :).

Callie

 

[Mabel Adams]

On 12/2/2023 at 9:21 PM, Callie1 said:

Hi, Everyone:

Sorry I am so late responding. Too much going on. Yes - this question was regarding procedures and not a particular case. Wow - that is a fantastically detailed SOP. I don't have time tonight but will give it a good read tomorrow. Thank you for sharing :).

Callie

 

My detailed ABID SOP prevents a certain number of midnight phone calls.   It also reminds me what the heck I decided last time this came up.

[butlermom]

Mabel,

That's a significantly detailed SOP, well written! How often do you repeat the workup on a patient taking Darzalex who is getting frequent transfusions? Since we're only transfusing phenotypically matched units in these cases, it seems we would not necessarily have to repeat everything every 3 days when we collect a new sample. We don't perform the Darzalex workup in house and would have to send it to our reference lab. It doesn't seem logical or feasible to do that every 3 days.  Is there any guidance about how often the DTT treatment should be performed?

Thanks for sharing your SOP.

Kathryn