1. Interesting case conference
Mary-Margaret Allen, MD
February 24, 2015

2. Case 1 58 yo male with a history of CLL (2008)
Later developed AIHA 2/2013
Prednisone taper (Feb to May 2013)
Recurrent 9/2013: rituxan and quick steroid taper
Recurrent 12/2014: rituxan x4
Most recent DAT: IgG 4+ positive and weakly positive for complement fixation
LDH of 207 unit/L (Ref: <=225) and a PCV 40%
Stable labs with no indication of clinically significant hemolysis

3. Background
Autoimmune cytopenias frequently complicate CLL, most commonly warm AIHA (7-10%)
IgG autoAb coat the RBCs for accelerated clearance by the spleen and liver
In CLL, 90% of AIC are due to polyclonal high- affinity IgG Ab produced by non-malignant B-cell clones
Malignant clones may produce autoAb but typically of the IgM subclass
Am J Hematol Nov;89(11):

4. Background CLL contains actively proliferating cells
CLL cell’s ability to proliferate is strongly influenced by the mutational status of the immunoglobulin heavy chain variable (IGHV) gene
IGHV mutational status defines two subgroups
U-CLL: No or very few somatic mutations: – worse outcomes and higher prevalence of AIC
BCR is able to bind (auto)Ag and respond, thus promoting their survival and proliferation
20% of CLL patients exhibit closely homologous “stereotyped” heavy chain complementary-determining region 3 sequences
This suggests that clones sharing stereotyped BCRs may have expanded through stimulations by a restricted set of epitopes
They have shown that stereotyped BCR configurations are associated with occurrence of ITP and AIHA
M-CLL : Several somatic mutations
BCR is quiescent and unable to transmit external signals
The role of Ag stimulation on BCR configuration and activity is highlighted by the evidence that more than
Am J Hematol Nov;89(11):

5. Background
CLL cells themselves may act as APC, inducing the formation of auto-reactive T helper cells
T-regs are impaired in patients with CLL, diminishing the key role of preventing autoimmune phenomena
Interaction between neoplastic B-cells and T- regs through CD27-CD70 lowers T-regs susceptibility to apoptosis and confers functional impairment
TLR are also found to be abnormally expressed in CLL. Pts with reduced TLR4 had increased risk for autoimmune complications
Am J Hematol Nov;89(11):

6. AIHA Diagnostic Criteria
Rule out alternatives
common causes of anemia (iron, vitamin deficiencies, occult bleeding, inflammatory conditions, sepsis, etc)
Bone marrow failure, hypersplenism, and recent chemotherapy should be excluded
Criteria
Hgb levels lower than or equal to 11 g/dL
One or more lab signs of hemolysis (increased indirect/unconjugated bilirubin, elevated LDH, reduced haptoglobin)
Caveat – LDH can be altered due to disease progression, haptoglobin may be influenced by underlying inflammation, bilirubin may be elevated secondary to chemotherapy
Either reticulocytosis or positive DAT
Am J Hematol Nov;89(11):

7. AIHA treatment First line therapy Second line therapy
Prednisone 1 mg/kg per day given for 3-4 weeks, which is slowly tapered over 1-2 months
Alternative if high dose dexamethasone (40 mg /d for 4 d)
IVIg may be administered in patients with massive hemolysis (1 g/kg on days 1 and 2)
Second line therapy
Cyclophosphamide, low dose cyclosporine, mycophenolate mofetil, azathioprine
Monoclonal Ab
Valid option, although as a single agent may not achieve a durable remission
Rituximab is dosed as for the treatment for lymphoma
One study cited, steroid refractory AIHA secondary to CLL tx w/rituximab only, CR was achieved in 22% and PR was achieved in 50%
Am J Hematol Nov;89(11):

8. Prognostic Implication of AIC Is Unknown
Using the Rai and Binet classification systems from 30 years ago, cytopenias are associated with poor prognosis in CLL
The cause of cytopenias were not considered, including reversible causes
Major reasons of uncertainty
time of AIC presentation
variability of cytotoxic treatments and their interplay with tumor/AIC response
the lack of consistency in the way that survival is measured (from dx of CLL or onset of AIC)
Multiple studies show that cytopenias secondary to AIC (AIHA, ITP, PRCA, AIG) had improved survival compared to cytopenias related to bone marrow failure
Two studies showed that patients who had AIC had significantly worse overall survival (stage C of Binet) compared to stage A (only lymphocytosis)—one study included more recent therapeutic approaches using the monoclonal antibodies
Am J Hematol Nov;89(11):

9. Prognostic Implication of AIC Is Unknown
One study looked at clinical, therapeutic, and prognostic features of AIHA in CLL and reported no independent effect on survival
One study showed that later onset of AIC has a more negative effect on outcome compared to early onset
Inferior survival of CLL patients may be explained by the higher risk of infections, bleeding, and cardiovascular complication associated with AIC onset and treatment
Or AIC may represent a clinical expression of a more aggressive CLL clone
Evidence that both AIHA and ITP were consistently associated with U-CLL, higher CD38 and ZAP-70 expression and unfavorable CG (de11q23 and del17p13) in several independent series
Am J Hematol Nov;89(11):

10. Case 2
41 yo female w/postpartum cardiomyopathy s/p LVAD, recently admitted for OHT
Previous antibody testing showed non-specific reactions with +IgG DAT with panagglutinin in eluate
Phenotyped: K1 and E negative
Current T&S: O positive with anti-E alloantibody and with positive IgG DAT
In solid phase, testing revealed anti-E alloantibody and ? of anti-c autoantibody
In LISS, anti-c was ruled out
Eluate showed panagglutinin
HCT 37%
Resident was paged for instructions on what to release since the testing showed an anti-E alloAb and an anti-c autoAb

11. Background Alloimmunization rate
General transfused population - approximately 1- 4%
Hospitalized, non-oncology patients %
The likelihood of alloimmunization is highly variable and related to the patient’s circumstances
Probability of exposure to a foreign antigen
Immunogenicity of the antigen
Analytic factors that influence antibody detection
Antibody screening testing methods
Persistence of the antibody detection
Transfus Med Hemother Nov;41(6):420-9.

12. Transfus Med Hemother. 2014 Nov;41(6):420-9.

13. Transfus Med Hemother. 2014 Nov;41(6):420-9.

14. Genetics
A recent study showed that RBC alloimmunization persisted at high rates even using Rh-matched minority donors
There may be a genetic basis for responder status
Maybe, the HLA system determines an individual’s ability to present a given blood group antigen
Class II HLA alleles have been associated with alloimmunization to some minor RBC antigens
Alloimmunization to Diego, Mia, Fya, Jka, K, E, and S have been shown to be dependent on recipient HLA class II polymorphisms
Transfus Med Hemother Nov;41(6):420-9.

15. Inflammation
Hypothesis that increased systemic inflammation increases tendency of immune system to “see” foreign antigen
Study showed that treatment of mice with inflammatory RNA increased immune response to blood group antigens
Believed that viral-type inflammation enhances trafficking of transfused RBCs to splenic dendritic cells
Immunity appears suppressed when inflammatory source is LPS (bacterial mediator of inflammation)
Also, it has been found that patients with autoimmune disease have a high rate of formation of Ab to low-incidence and low-immunogenicity Ag
Transfus Med Hemother Nov;41(6):420-9.

16. Age Extremes of age seem to alloimmunize less frequently
>77 yo has been reported to be associated with a decreased rate of alloimmunization
Neonates and young children are generally considered poor-responders
Transfus Med Hemother Nov;41(6):420-9.

17. Solid Organ Transplant
The immunosuppressive regimens are believed to substantially reduce the incidence of alloimmunization, even to Rh(D)
Development of alloantibodies may be evidence of incomplete immunosuppression or of the reappearance of a previously formed antibody
Transfus Med Hemother Nov;41(6):420-9.

18. Clinically significant?
Hemolytic transfusion reaction and HDFN
One study, 8% of 110 mothers had >1:16 anti-E titer
Donor pool:
70% neg for E
90% neg for K1
20% neg for c
1 K1, E, and c antigen negative unit  must screen 8 units (need 2, screen 16, etc)

19. Clinically significant?
Do we need to give Rh(c) antigen negative units to avoid potential hemolysis from autoAb to little c? (only 20% little c negative)
We would not have given antigen negative units if only tested patient’s plasma in LISS because we would not have seen the specificity for the autoAb
Do we need to give antigen negative units for all known antigens after phenotyping?
To provide antigen matched blood in addition to giving units that are negative for antigens for which she has antibodies could make finding units impossible
What is her risk for additional alloimmunization?

20. VUMC WAIHA SOP
8.1 Perform T&S and DAT
If panagglutinin present in plasma, test in LISS
If LISS negative, report as negative
8.2 If panagglutinin persists, adsorptions may be necessary
Notify clinician of additional time for testing
Medical director will order phenotyping after clinical evaluation in rounds
Note: If blood is needed before rounds contact the blood bank resident

21. Resources
Visco C, Barcellini W, Maura F, Neri A, Cortelezzi A, Rodeghiero F. Autoimmune cytopenias in chronic lymphocytic leukemia. Am J Hematol Nov;89(11):
Gehrie EA, Tormey CA. The Influence of Clinical and Biological Factors on Transfusion-Associated Non-ABO Antigen Alloimmunization: Responders, Hyper- Responders, and Non-Responders. Transfus Med Hemother Nov;41(6):420-9.
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