A Compliance and QI Supervisor at a hospital in Kansas reports that in 2004 they discontinued performing antiglobulin testing to detect weak D expression of apparent Rh negative patients with the exception of infants born to Rh negative mothers. At the same time that they implemented the aforementioned policy, they rewrote their Rh typing procedure to include a 15 minute room temperature incubation with a "monoclonal blend" anti-D. Validation testing has showed that patients who formerly tested as weak D positive by an indirect antiglobulin method also reacted in the 15 minute room temperature Rh typing method. They are currently validating typing for the D antigen using the Ortho ID-MTS™ Gel Test™ technology and the Ortho ProVue® system. Up to this date the results of the validation show similar results for the 15 minute incubation method uising a tube test and the Gel-based test results. In fact, the inquiring colleague comments that in general, the reactions appear to be at least as strong or stronger in the Gel Test™ than in the tube test. She asks if any other laboratories are using a Gel-based Rh typing system for detection of weak D status of infants born to Rh negative mothers. If so, was it necessary to request a variance from the 24th Edition of the AABB Standards [5.20.2(3)] which states that when considering Rh immunoprophylaxis for an Rh negative woman, if a test for D on her neonate is negative... "Weak D testing is required when the test for D is negative." She comments that her reading of the standard is that it does not define how the Weak D testing must be done (antiglobulin versus direct agglutination), but the package insert of the anti-D Monoclonal IgM typing card states on page 3 of 4 that "In instances where confirmation of D-negative status is required; negative D reactions obtained with the Anti-D card should be retested with an Anti-D reagent licensed for antiglobulin phase testing."

The following comments have been received.

ADDENDA Feb. 13, 2007

1. In regards to the statement by the Compliance and QI Supervisor at a hospital in Kansas in which she says

"they rewrote their Rh typing procedure to include a 15 minute room temperature incubation with a 'monoclonal blend' anti-D"

and that ...

"Validation testing has showed that patients who formerly tested as weak D positive by an indirect antiglobulin method also reacted in the 15 minute room temperature Rh typing method."

Sheryl A. Kochman of CBER/OBRR/DBA, Chief, Devices Review Branch (attribution used with permission) reminds us of the following points:

• "It is important to understand that the term weak D includes cells that have a quantitative difference in D antigen status, i.e., fewer D antigen sites than 'average,' AND cells with a qualitative difference in D antigen status, i.e., partial D (formerly D mosaic)."
• "While the 15-minute room temperature incubation with a monoclonal blend Anti-D might coax cells with a quantitative difference in D antigen status to react prior to the AHG phase, it might not do the same with partial D’s, especially D VI cells"

ADDENDA Mar. 27, 2008

2. A South Florida Blood Bank Physician is interested in a response by Sheryl A. Kochman of CBER/OBRR/DBA, Chief, Devices Review Branch to the following questions with regards to her statement of Feb. 13, 2007 that "While the 15-minute room temperature incubation with a monoclonal blend anti-D might coax cells with a quantitative difference in D antigen status to react prior to the AHG phase, it might not do the same with partial D’s, especially D VI cells".

Q: Are there any references available that provide support for the use of a 15 minute room temp incubation with monoclonal blend Anti-D to distinguish weak D vs partial D VI?

A: [Response of Sheryl A. Kochman] Not as far as I know. We have only anecdotal information about this.

Q: Is the GEL detecting the weak D or the partial D?

A: [Response of Sheryl A. Kochman] The package insert lists the following Limitation of the Procedure “Very weak expressions of the D antigen may not be detected. The partial DVI epitope variant of the D antigen has not been found positive with this reagent. Other rare cells with very low copy numbers of the D antigen may need to be tested with antiglobulin and will be negative with this Anti-D reagent. The package insert also lists this Specific Performance Characteristic “Very weak expressions of D may not be detected by the MTS Anti-D (Monoclonal) (IgM) Card. The partial DVI epitope variant of the D antigen will not be detected with this reagent.”

Q: Our blood banks are in the process of looking at policies for handling discrepant results for D testing between tube testing and GEL methodology. In our current policy, we perform the immediate spin D test. Patients are reported Rh negative, transfused Rh-negative products, and are eligible for Rhogam if this test is negative. We are in the process of switching to Provue and have discovered that these patients test positive using this methodology. What is the best way to manage these discordant results?"

A: [Response of Sheryl A. Kochman] You should determine if the reaction is a valid positive reaction, at least for some period after switching to the ProVue. If it is, the patient might be a weak D or a partial D. You can chose to do (or have done for you) further testing to characterize the D antigen, e.g., monoclonal antibody studies, molecular testing. Many facilities refer these kinds of samples to a reference lab. Alternatively, you can assume they are weak D or partial D and may be capable of eliciting a response, in which case you should administer Rh negative blood and/or RhIg.

Editor’s note: Sheryl Kochman (attribution used with permission) reminds us that her aforementioned comments (responses) are consistent with 21 CFR 10.85(k) and constitute an informal communication that represents her best judgment at this time, but does not constitute an advisory opinion, does not necessarily represent the formal position of FDA, and does not bind or otherwise obligate or commit the agency to the view expressed.

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Ira A. Shulman, MD
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