Can Rh-positive FFP induce anti-D alloimmunization in an Rh-negative recipient?

A transfusion medicine physician in Maryland submits the following case for discussion. The patient is a group O Rh negative, 65 year-old male patient who spent over 5 weeks in a hospital, during which time he received 14 units of group O Rh negative RBCs and 13 units of group O Rh positive FFP. The patient had several negative antibody screening test results during the course of his hospitalization until anti-C was exhibited at 37C and AHG, while anti-D was seen only at AHG. These antibodies were initially detected after 11 FFP's and 12 RBC's had been given. Two RBC and two FFP units were transfused AFTER the anti-C and anti-D were identified. Of note, the direct antiglobulin test was negative. Realizing that the emerging antibodies might actually be anti-G (rather than anti-D+C), the following workup was done to rule out anti-G.

• The patient's serum reacted with an R2R2 cell with a titer of 8
• The patient's serum reacted with an r'r cell with a titer of 4
• The serum was absorbed with a C-D+ donor cell. The adsorbed serum did not react with C-D+ cells but did react with a C+D- cell
• The neat (unabsorbed) serum did not react with an expired rGr cell.

The expired rGr cell is from an Immucor Panocell-10 lot#12365 exp 3 Apr 03, TC (donor T12). This same panel cell was used to test a recent CAP survey sample (CAP J-survey set 2003 J-A, educational challenge) that contained anti-G+C. They tested the patient's sample with the rGr cell twice and used a saved sample from the recent CAP transfusion survey sample as a simultaneous positive control. The rGr cell gave a strong 2+ reaction with the CAP sample and was negative with the patient's sample, seemly ruling out anti-G as being in the patient's sample.

Since the inquiring physician appears to have ruled out anti-G as one of the newly detected alloantibodies, and since the patient only received Rh negative RBCs, she is wondering if the transfusion of the Rh positive FFP might be responsible for the development of the anti-D that the patient developed. She is writing to find out what is the experience of others with D alloimmunization in patients who have received only Rh negative RBCs but who received Rh positive FFP.

Specifically, she wants to know:

• How extensively would others investigate the Rh type of each donor RBC unit that the patient received, to ensure that alloimmunization was not due to an Rh positive unit that was mistakenly labeled as Rh negative? She has been reassured that all of the Rh negative units the patient received were tested according to AABB and FDA standards. Would it be prudent to repeat the Rh typing for all of the implicated donors whose RBCs were given to the patient in this case?
• Does anyone offer anti-D immunoprophylaxis to Rh negative patients who receive large volumes (or any volume for that matter) of Rh positive FFP? The inquiring physician has found three case reports of alleged RBC antigen alloimmunization attributed to the administration of FFP. McBride et al (Transfusion 1983;23352-354) include a recommendation that one should consider Rh immunoprophylaxis when transfusing Rh negative patients with "large volumes" of Rh positive FFP.

The following responses were received.

ADDENDA April 22, 2003

1. A colleague has asked for clarification regarding this case report. She writes (verbatim): "Did any of the RBC donor units have anti-D/anti-C in the plasma? We have seen a 3+ anti-D in the serum of a patient who was transfused with several units of O Rh negative red cells stored in Adsol. One of the units had anti-D identified in the plasma. The anti-D persisted for several weeks. (See "Anti-D antibody in the serum of an Rh-positive Oriental woman" Shahidi-Asl M, Kirkley K, Kao Y, in Laboratory Medicine, January 2003, no.1 vol 34.)"
   
   
2. In response to the above request for clarification, the Maryland physician who initially reported the case under discussion responds that the report of Shahidi-Asl and colleagues is very interesting, especially if the transfusion of an Adsol-preserved RBC unit can be responsible for enough passive transfer of an antibody that the antibody is detectable in the recipient at such strong reactivity. The Maryland physician adds that it is her expectation that the passive transfer of an antibody as the explanation for this case's findings would be no more likely than if one of the RBC donors had been mistyped as Rh negative, when in fact it was really a weak D positive individual. She is of the opinion that her blood suppliers perform antibody screens for blood donors in compliance with the AABB standards. Thus, she adds the following regarding the donors of the RBC units that the patient in question received (verbatim): "However, this information does raise the question of whether it would be reasonable to request that the supplier re-test both the D typing AND the antibody screen test results."
   
   
3. A colleague from the University of Ulm comments that the differential diagnosis of the case report under discussion was discussed in Transfusion 40(2000):429. He adds the following insights: "In the above case report from New Zealand a weak D RBC unit was identified as the cause of the anti-D immunization. In the meantime a second - quite unexpected - explanation for inadvertent anti-D alloimmunization was identified - microchimerism in donors carrying small populations of Rh-positive red cells. These cells escape even sophisticated AABB & FDA compliant serologic detection but are capable of anti-D immunization in every transfusion (BMC Genet 2(2001)10, online) . Without commenting on anti-D immunization induced by small RBC contamination in FFP, I recommend attempting to exclude the possibility of an exposure to antigen D by one of the 12 RBC units administered before anti-D detection; this would require a detailed work-up (including molecular techniques) of the involved 12 donors. The inquiring physician did not report the transfusion anamnesis (history), which may provide a clue to a secondary anti-D immunization event that is more likely to occur following exposure to minute amounts of antigen D."

ADDENDA April 23, 2003

4. A colleague in Saudi Arabia informs the e-network forum about a few other reports of FFP-induced alloimmunization to RBC antigens in the literature. One involved anti-D immunization after 4 units of FFP by de la Rubia, Garcia et al. Vox Sang 1994;66:297-8 (letter).
   Although the following references report on non-anti-D antibodies allegedly "induced" by FFP transfusions, they may still be of interest.
   • Ching, Poon, Neurath, Ruether AM J Clin Pathol 1991;96:201-2. Red blood cell alloimmunization complicating plasma transfusion. (The authors report on a patient who developed a strong (IgM+IgG) anti-E and a weak IgG anti-Jk(a) 15 days after FFP transfusion.)
   • Wolfowitz & Shechter, Transfusion 1984;24:544 (letter). (More about alloimmunization after transfusion of fresh-frozen plasma.)
   At the responding colleague's institution in Saudi Arabia, they do not currently recommend anti-D Rh immune prophylaxis when transfusing Rh-D pos FFP to RhD negative recipients (which happens frequently due to inventory constraints).
   
   
5. As a follow-up to the possibility that one of the donor units might have been the source of anti-D, one of the blood providers that provided blood products for the patient's case under discussion reported that they express as much plasma as possible to pack the cells prior to adding the Optisol. They routinely perform antibody screens on all units that they manufacturer, and these tests were negative. They also believe that this is standard practice for other manufacturers whose units were implicated.
   
   
6. A colleague in a major city in Pennsylvania reports that her institution recently had seen a sickle cell patient (group O Rh negative), who had only received group O Rh negative red cell units in their institution. This patient had never received FFP. Interestingly, this patient developed an anti-D alloantibody. They suspect the most likely explanation for this alloimmunization is that the patient may have received a red cell unit that was actually weak D positive, although labeled as D negative. The Pennsylvanian physician would be interested to learn if there are data from donor centers to show how often a weak D donor is 'mistyped' as D-negative, and are there any statistics derived from repeat typing of blood donors with subsequent donations that address this question?

ADDENDA April 24, 2003

7. The colleague from the University of Ulm wishes to reply to the comment of April 23: (verbatim) "At our institution, routine molecular testing for the RHD gene was established for first-time donors who serologically type D-negative, effective January 2002. For recent statistics on weak D and other Rh positive donors among 'mistyped' D negative donors see discussion in BMC Genet 2(2001)10. Blood group serologists might note the observation of 5 D-positive donors among 8,442 allegedly Rh-negative blood donors tested with disturbance. ... Immunizations caused by units of such donors will generally be missed, because the occurrence of an anti-D in a patient is usually not further investigated. For example, the two anti-D immunizations induced by units of the chimerical donor of this study were found only in a lookback triggered by our molecular screen. Chimeras in the Rh system have repeatedly been observed and chimeras may be a more widespread phenomenon than anticipated. However, checking D-negative samples, especially those occurring with a C or E or both, for RHD specific sequences by nucleic amplification techniques may become practical in the near future."
   
   More on Anti-D immunization: "A flow cytometry study of the RBC from the D+/- chimera revealed 94% D-negative RBC and an admixture of 6% D-positive RBC (Fig. 7). This chimera was confirmed following a 3-month investigation. The 24 year-old donor was healthy and had no twin. A lookback performed on the units from this donor revealed 13 that had been issued as D-negative. Two D-negative recipients were traced and available for antibody screen testing, both of whom were found to be anti-D immunized." If these results were representative, this incidence would amount to about one anti-D immunization per 50,000 donations (4 immunizations per 200,000 donations in 1 year at our institution). We believe the cost-efficiency of molecular typing is therefore established.
   
   
8. Dr. Breanndan Moore of the Mayo Clinic (attribution used with permission) thinks it is a good idea to reconsider the possibility that the antibodies were passively transmitted from the plasma of one of the donor units; perhaps antibody(-ies) existed, but were not detected in the initial screening. Apparently, his facility had such a case many years ago. They performed all conceivable testing until a resident physician (!!!) suggested the idea of re-screening the donors' sera for previously undetected alloantibodies.

ADDENDA April 25, 2003

9. A colleague in North Carolina reports that the question of RBC alloimmunization to plasma was covered in the AABB Collected Questions & Answers, 7th edition, 2001, edited by Mark E. Brecher, M.D., pages 57-59. In brief, this discussed that plasma is not a truly cell-free product and very small RBC residues are found in fresh liquid plasma.¹ Although there are a few cases of "alloimmunization" to plasma reported in the literature, primary alloimmunization due solely to frozen plasma has not been conclusively reported (summary table and references -PDF).
   In addition, there are reports of at least four other cases of secondary alloimmunization, two D and two Fy(a) to plasma (type not specified).⁶
   It was concluded that RBC alloimmunization:
   • to plasma is rarely reported.
   • following exposure solely to frozen plasma appears to be a secondary immune response.
   • can occur as a primary immune response following transfusion of liquid stored plasma.
   While the there is sufficient intact D antigen to cause a secondary (anamnestic) response, it is generally thought to be insufficient to cause a primary alloimmunization.

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