1. Alloimmunization: A review and recent insights into pathogenesis
Today I’ll be talking to you about an interesting patient I encountered on multiple clinical pathology services and then exploring the topic of Alloimmunization: in the form of both a review and with a focus on some new insights into its pathogenesis.
Jessica Hata, MD
6/14/2013

2. 4 month old girl Previously healthy, full term baby
Admitted with hepatosplenomegaly, fever, and pancytopenia
No prior transfusions
Typed as A Rh-positive
WBC 3.5, Hgb 7.0, PCV 23, pts 119
received 5 U pRBCs and 1 U platelets
only child, with no significant birth history, family history or travel history

3. Day 14 Type and Screen blood type: A Rh-positive
Ab screen: positive, anti-K1 and anti-E (!)
DAT + (IgG (4+) and anti-C3d (1+))
Eluate: positive; Anti-Jka (!)
Segments tested: Jka, K1, E + RBCs
Molecular Genotype: Jka, K1, E -
No evidence of hemolysis
DDx: maternal-placental or Breast milk transmission vs neonatal alloimmunization vs false positive
antibody is coating her red cells in vivo. The eluate, where an acid elusion is performed to remove the antibody from the red cells and characterize it, shows an Anti-Jka!
Segments, the small plastic cords of blood which are retained from the unit,
Molecular genotype was performed, a phenotype would have been unreliable due to her recent transfusion,
Unlikely as the mother had two prior negative antibody screens (repeated for confirmation). Additionally, the half life of IgG is about 20 days, and should not be present in the child's circulation at 4 months of age. Although antibodies can be identified in breast milk (moms with FUT2 gene are secretors), the antibodies are degraded by the acid in the baby’s stomach and do not make it to the circulation. The tests were all run in triplicate to rule out a false positive.
Neonatal alloimmunization is exceedingly rare but seems to be a viable option in this case.

4. This is a timeline for our patient
This is a timeline for our patient. Antibodies developed on the 11th hospital day, 10 days after the first transfusion.

5. Blastomycosis antigen
Hospital Course
Urine Histoplasma/
Blastomycosis antigen
elevated
Soluble IL-2 Receptor significantly elevated
Treated with 4 weeks of Amphotericin B; Itraconazole therapy for 3 additional months
She was subsequently diagnosed with disseminated Histoplasmosis. I encountered her again on the bone marrow service. She had a reactive-appearing marrow with increased histiocytes. A GMS showed the narrow-based budding organisms.
IL-2 is an inflammatory cytokine, but it is also necessary for the growth, proliferation, and differentiation of T cells. A high IL-2R indicates ongoing significant T cell activation.
Bone Marrow Biopsy

6. Outline Alloimmunization Review
How do neonates/ infants differ?
Effects of immunogenetics on responder/ nonresponder status
Role of inflammation in red blood cell alloimmunization
Neonatal alloimmunization and a potential link to Histoplasmosis?

7. Basic Immunology
Many of you are very familiar with this diagram. As you know antibody generation in response to a foreign antigen (whether it is a red cell antigen or microbe) is a complicated process. This antigen is represented by the red ovals and can activate the immune system using one of two pathways. SO called dendritic cells can engulf and process the antigen to display as a flag on their cell surface. This antigen is recognized as foreign by T cells which can in turn activate B cells to induce antibody secretion. This can also happen independently of the T cells, as is also pictured here. The antibody then floats though the blood stream and soaks up/neutralizes antigen (if any remains from the innate immunity) like a sponge. When the immune system is exposed a second time, this is a much more rapid process and in the case of a foreign antigen on a red cell, alloantibodies can bind and cause intravascular or extravascular hemolysis.

8. RBC Alloimmunization
Potential occurrence with blood product transfusion, pregnancy, or organ transplantation
An infrequent event: % in large series
Rates of alloimmunization to all antigens are higher in certain disease states
Immunization rates to other sources of foreign antigen (including vaccines and microbial infections) often approaches 100%
Acute HTRs, delayed H/STRs, and costly, time-consuming evaluations in the blood bank!
Why do we care about alloimmunization? For as many red cell products which are transfused the number of patient who develop an alloantibody is actually surprisingly low. We do know that special populations such as patients with sickle cell disease have higher rates of antibody formation (rate of alloimmunization ranges from 18-47%) and the true cause is unknown. Specifically for sickle cell disease, patients are AA and the donor poor is largely made of caucasions, so the antigenic differences may be greater to provoke the immune system. It may also be that SCD has an underlying inflammatory condition. Dr Booth has some data to suggest that SCD patients with an additional inflammatory state (such as ACS) have extremely high levels of alloimmunization.
This last point is very curious. As you know there are hundreds of human blood group antigens and despite multiple antigenic differences between donor and recipient in each transfused unit of red blood cells alloimmunization to these antigens overall is an infrequent event ( %). These numbers exclude the highly immunogenic Rh protein antigen (RhD), which causes up to 80% alloimmunization in healthy recipients, and I’ll tell you the reason later in the talk.
Curr Opin Hematol 15:631–635
Arch Pathol Lab Med 1995;119:42–45
Br J Haematol 1995; 91:1000–1005

9. Neonatal Alloimmunization
In vitro studies show minimal Ig production
? poor interaction between T and B cells
Relative immunodeficiency of the neonatal state: an increased ratio of T suppressor cells to T helper cells
Exceedingly rare and reported in only isolated case reports
The exact pathophysiologic mechanism is unknown.
While the infant has normal adult numbers of circulating B lymphocytes bearing surface IgM and IgG, they have little to no endogenously produced IgG
The neonatal state is one of relative immunodeficiency with increased susceptibility to bacterial, viral, and protozoal infections; for these reasons, and for neonatal in vivo inhibition of maternal cell proliferation, an increased T suppressor ratio may be beneficial.
The exact pathophysiologic mechanism, with additional co-stimulatory or contributory factors, is unknown.

10. AABB Standards
And this observed phenomenon has shaped the way neonatal testing is done in the lab. This is taken from the AABB Standards. You can see that we don’t do a reverse type because neonates seldom make anti-A or anti-B, so there would be little to no reaction. If an initial antibody screen does not detect any unexpected antibodies, crossmatching is optional and another antibody screen does not need to be performed until the infant is 4 months of age!!
Recently, Shaikh and Sloan reported maternal isohemagglutinins to be present in 6.4% of infants one month old and younger, while none were detected in infants between 2-4 months of age. With this data, they suggest that type-specific blood may be safely given to infants 2 months and older without additional testing for maternal isohemagglutinins.

11. Neonatal Alloimmunization & Infection
Case reports
Age
Sex
Primary
Diagnosis
Alloantibody
Formation
Transfusion 1992; 32:
11 w M
Necrotizing
enterocolitis
Anti-E
Transfusion 1978; 18(2)
20 d F
E Coli sepsis
Anti-K
Transfusion 2013; 53 (1140)
4 mo
Disseminated
Histoplasmosis
Anti-Jka
I mentioned there are only a handful of case reports of neonatal alloimmunization and here they are. A very curious finding is that all the patient had severe concominent infection. It’s hard to know what that means at face value. Are they infected and sick enough to require multiple transfusions so it is a dosage effect? Or, as I will argue, is it possible the microbial infections reported in these studies stimulated an increased inflammatory state, with altered Th1/Th2 ratios, allowing for a more robust response to foreign RBC antigens?

12. Molecular Mimicry?
Alloimmunity can be induced through exposure to microbial antigens that mimic the 3D structure of the alloantigen
Microbial sequence data looks similar to human blood group antigens
And what about molecular mimicry? Could that play a role in these patients?
Microbial sequencing data show striking homology of selected bacterial species with epitopes of Kell, Duffy, and Kidd blood group antigens.
BLOOD, 2010; 115(19):

13. Outline Alloimmunization Review
How do neonates/ infants differ?
Effects of immunogenetics on responder/ nonresponder status
Role of inflammation in red blood cell alloimmunization
Neonatal alloimmunization and a potential link to Histoplasmosis?
As I mentioned before, despite multiple antigenic differences between donor and recipient in each transfused unit of red blood cells alloimmunization to these antigens overall is an infrequent event ( %). In the next few slides we will try o understand why.

14. Rate of Additional Antibody Formation After Transfusion in Non-Chronically Transfused Alloimmunized Patients
1° Ab formation in non-chronically transfused patients ~1%
Repeat transfusion in alloimmunized patients >20%
15/653 patients experienced >11 additional transfusions without forming additional Ab
The formation of the first antibody identifies “high responders” against subsequent alloantigenic challenges
Most studies have been performed in chronically transfused patients, for example, hemoglobinopathies and hematologic malignancies. Alloimmunization in these groups has a reported incidence up to 60 percent, with an up to fourfold increased risk of multiple antibodies. This group performed a 20 yr retrospective study to investigate the rate of formation of additional alloantibodies in >600 patients who were not chronically transfused, but subsequently presented for another transfusion after receiving an initial one.
Less than the % average
>20% formed additional antibodies after repeat transfusion and MOST antibodies were formed after one additional transfusion episode with a median of 2 units. The same plateau can be seen with HLA antibodies in pregnant women.
This suggests a role for recipient factors. For example, patients who become immunized against one alloantigen (responders) are more likely to become subsequently immunized against other alloantigens. In contrast, patients who do not make alloantibodies to initial transfusions (nonresponders) tend not to make alloantibodies to subsequent transfusions.
Transfusion. 2006;46:

15. Advantages to Elucidating Risk Factors for Alloimmunization
Focused phenotypic /genotypic matching of RBCs for chronically transfused “responder” patients
Limit wasted resources matching “nonresponders”
Rational basis for development of therapeutic interventions to prevent RBC alloimmunization
OR use available drugs to block known inflammatory cascade signals around time of transfusion
Use drugs that are already available to block the cascade of signals needed to make an antibody

16. Effects of Immunogenetics on Responder/Nonresponder Status
Vast majority of blood group antigens
consist of a single AA polymorphism bet.
donor and recipient
Each MHC variant (determined by HLA)
will present different peptides from a
given protein
100% DRB1*04 in pts immunized to Fya (Transfusion 2006; 46:1328–1333)
Higher DRB1*01 and DQB1*05 in pts immunized to Jka (Transfusion 2005; 45:956–959)
Anti-K assoc with multiple DRB1 types (Transfusion 2006; 46:1328–1333)
Only a few RBC antigens consist of a protein present on donor but not recipient RBCs, most notably RhD. The human leukocyte antigen (HLA) encodes the major histocompatibility complex (MHC), which initiates adaptive helper T-cell immunity by presenting peptides from foreign antigens. The HLA is widely varied
in humans, particularly in sequences that determine which peptides fit in the MHC pocket. If a protein is highly foreign to a recipient, generally any HLA will be able to fit at least one (or more) peptides into the MHC. Thus, most RhD negative people should have an MHC that can present a peptide from RhD to T cells. Alloimmunization of RhD in healthy individuals is 80%! However, this is not the case when the foreign antigen varies only slightly from the recipient’s own proteins, as with most blood group antigens. The ability of the recipient to respond to the foreign antigen is variable, depending on whether a peptide containing the difference can fit into the pocket of the recipient’s MHC.
Kell is highly immunogenic, which in this context seems to make sense as multiple HLA types are able to respond.

17. Outline Alloimmunization Review
How do neonates/ infants differ?
Effects of immunogenetics on responder/ nonresponder status
Role of inflammation in red blood cell alloimmunization
Neonatal alloimmunization and a potential link to Histoplasmosis?
Emerging data suggests infection and inflammatory conditions help promote alloimmunization

18. Paradigm of Humoral Immunity
Patient must be antigen negative and exposed to that antigen
Patient must encode an HLA capable of presenting a representative peptide
Additional environmental stimuli  ? Role of inflammation
tNuts and bolts of humoral immunity as applied to alloimmunization.
By transfusion, pregnancy, or organ trasplantation
Yet we know that NOT all people who carry the correct HLA and are exposed to a given RBC antigen become alloimmunized

19. Sources of Inflammation in RBC Transfusion
Few inflammatory activators of microbial origin
Prestorage leukoreduction decreases the risk of donor cytokines
Synthetic chemicals (i.e. plastic of bag) or breakdown products of senescent RBCs
Inflammatory status of the recipient
10 mo with JRA developed anti-E, Fya, and Jka following transfusion of one unit (N Engl J Med 2007; 357:2092–2093)
16-yr with JRA and macrophage-activation syndrome developed anti-Cw, E, K, Jkb , S, M, and Lea following the transfusion of two units (Transfusion 2007; 47)
Pt with SLE developed 5 alloantibodies after a single transfusion (Mollison’s blood transfusion in clinical medicine. 11th ed.)
Sickle cell disease?
Ideally, a donor unit should contain few inflammatory activators of microbial origin if it was collected from a clinically ‘well’ donor and processed and stored properly. Deferral of donors with symptoms or history of illness decreases the likelihood of inflammatory cytokines of donor origin.
The effect of leukoreduction on RBC alloimmunization remains controversial, although a retrospective study reported decreased alloimmunization in AML patients over the time period during which leukoreduction was first introduced.
it is theoretically possible that synthetic chemicals (i.e. plasticizers in bag) or breakdown products of senescent RBCs can introduce an inflammatory signal; however, there is currently no evidence to support this possibility.
Thus, transfusion under optimal conditions accomplishes exposure to foreign antigen in the absence of inflammation, which often fails to induce immunity. So we need to consider the high degree of inflammation associated with the underlying disease of these patients I will call your attention to some more recent cases which illustrate this point.
However, both JRA patients also received infliximab [a tumor necrosis factor (TNF)-a-neutralizing antibody] soon after transfusion, which may have increased antibody production by decreasing T helper (Th)1 and increasing Th2 responses. Rates of alloimmunization in patients with sickle cell disease (SCD) are substantially higher than in other patient populations There is an ongoing debate as to whether this is simply due to larger antigenic disparities between donor and recipient populations than are found in non-SCD patients who receive chronic transfusion, or if the underlying biology of SCD promotes RBC alloimmunization due to an intrinsic inflammatory baseline

20. Mouse Model
RBCs transfused from mHEL donor mice into wild-type recipients
100 μL of leukoreduced RBCs was injected via tail vein
3-6 hours before transfusion, recipients were injected intraperitoneally with either poly(I:C) in buffer or buffer alone
This group tried to address the question of recipient inflammation in mice with an expieriemtn looking at transfusion in non-inflammed and artifically-inflammed animals. Wild-type mice do not express HEL antigen, but there lysozyme similar in structure to HEL so transfusion of mHEL RBCs into wild-type mice crosses a small antigenic barrier, similar to many human blood group antigens.
Poly(I:C)=synthetic double-stranded RNA molecule that induces viral-like inflammation
Current Opinion in Hematology 2008, 15:631–635
Transfusion 2006; 46:1526–1536.

21. Pretreatment with poly(I:C) significantly increased alloimmunization to transfused RBCs
Column (Gel) Agglutination was used to screen for antibodies, similar to what is done in the blood bank.
Microtubules filled with gel particles and anti-IgG
RBCs and plasma added to top, incubated, and centrifuged
Gel particles separate red cell clusters by size (larger clumps composed of antibody stuck on red cells don’t migrate and individual cells pass freely
In four of four experiments transfusion of mHEL RBCs in the absence of inflammation resulted in a weak anti-HEL IgG signal for some animals. This response was significant in 4 of 18 mice as defined by exceeding two standard deviations of Control Groups 3 and 4. In contrast, pretreatment with poly(I:C) resulted in a significant enhancement of anti- HEL IgG 90% of mice responding
Thus, pretreatment with poly(I:C) significantly increased both the frequency and the magnitude of alloimmunization to transfused RBCs.
Current Opinion in Hematology 2008, 15:631–635
Transfusion 2006; 46:1526–1536.

22. Outline Alloimmunization Review
How do neonates/ infants differ?
Effects of immunogenetics on responder/ nonresponder status
Role of inflammation in red blood cell alloimmunization
Neonatal alloimmunization and a potential link to Histoplasmosis?

23. Question and Study Design
Do patients with Histoplasmosis have a higher rate of alloimmunization?
A comprehensive review of the standard derivative deidentified medical record database (from 1998 to present) at Vanderbilt University Medical Center was undertaken to identify patients with Histoplasmosis and concomitant alloantibodies.

24. Patients with Histoplasmosis have a 5% alloimmunization
Multiple antibodies were made in 2 of 5 cases. 4 of 5 patients developed at least one antibody to the RhCE or RhD blood groups
Patients with Histoplasmosis have a 5% alloimmunization
rate compared to healthy adults

25. Conclusions
Alloimmunization is rare overall and even more rare in neonates
Severe inflammation and infection may induce alloantibody formation
Patients with Histoplasmosis have a 5% alloimmunization rate compared to healthy adults ( %)
Conservative transfusion practices, including offering phenotypically matched RBCs to patients with clinical or laboratory evidence of a high inflammatory state, may be prudent.

26. References
Hoeltge GA, Domen RE, Rybicki LA, Schaffer PA. Multiple red cell transfusions and alloimmunization. Experience with 6996 antibodies detected in a total of 159,262 patients from 1985 to Arch Pathol Lab Med 1995;119:42–45.
Heddle NM, Soutar RL, O’Hoski PL, et al. A prospective study to determine the frequency and clinical significance of alloimmunization posttransfusion. Br J Haematol 1995; 91:1000–1005.
DePalma, L et al; Presence of the red cell alloantibody anti-E in an 11 week old infant; Transfusion 1992; 32:
Schonewille H, van de Watering LMG, Brand A. Additional red blood cell alloantibodies after blood transfusions in a nonhematologic alloimmunized patient cohort: is it time to take precautionary measures? Transfusion. 2006;46:
Noizat-Pirenne F, Tournamille C, Bierling P, et al. Relative immunogenicity of Fya and K antigens in a Caucasian population, based on HLA class II restriction analysis. Transfusion 2006; 46:1328–1333
Reviron D, Dettori I, Ferrera V, et al. HLA-DRB1 alleles and Jk(a) immunization. Transfusion 2005; 45:956–959.
Tyler LN, Harville TO, Backall DP. Multiple alloantibodies after transfusion in an infant treated with Infliximab. N Engl J Med 2007; 357:2092–2093

27. References cont.
Zantek ND, Abdullah N, Pary PP, et al. Development of multiple red blood cell alloantibodies in a pediatric patient with juvenile rheumatoid arthritis and macrophage activation syndrome. Transfusion 2007; 47 (Supplement):136A
Mollison’s blood transfusion in clinical medicine. 11th ed. Klein HG, Anstee DJ, editors. Blackwell Publishing; 2005.
Zimring JC, Hendrickson JE. The role of inflammation in alloimmunization to antigens on transfused red blood cells. Current Opinion in Hematology 2008, 15:631–635
DePalma L.; Review: red cell alloantibody formation in the neonate and infant: considerations for current immunohematologic practice; Immunohematology 1992; 8(2)
Marsh et al. Naturally occuring anti-Kell stimulated by E Coli enterocolitis in a 20 day old child. Transfusion.1978; 18(2):
Hendrickson JE, Desmarets M, Deshpande SS, et al. Recipient inflammation affects the frequency and magnitude of immunization to transfused red blood cells. Transfusion 2006; 46:1526–1536
Hata JL, Johnson MS, Booth GS. Neonatal alloimmunization: a rare case of multiple alloantibody formation in a patient with disseminated histoplasmosis. Transfusion 2013; 53 (1140)