Current Topics in Immunohematology Presented by Jacie Mundahl, MLS (ASCP)cm

Objectives Upon completion of this presentation the participant will be able to recognize the effects of daratumumab in immunohematology patient testing and manage those patients in future visits. Upon completion of this presentation the participant will appreciate the need for Zika virus testing in the blood donor population. Upon completion of this presentation the participant will be able to define monoclonal B-cell lymphocytosis and state the risk it has been suspected to pose in blood donation and transfusion.

Daratumumab, What Is It? Daratumumab is an immunoglobulin G1 kappa human monoclonal antibody Produced in mammalian cell line (Chinese Hamster Ovary) using recombinant DNA technology Mechanism of Action CD38 is a transmembrane glycoprotein expressed on the surface of hematopoietic cells. Daratumumab binds to CD38 and inhibits the growth of CD38 expressing tumor cells by inducing apoptosis directly through Fc mediated crosslinking as well as by immune-mediated tumor cell lysis through complement dependent cytotoxicity(CDC), antibody dependent cell- mediated cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP). Brand Name: Darzalex

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Patient Treatment First approved for therapeutic use by the FDA in November, 2015 Daratumumab is used for multiple myeloma patients with dexamethasone and either bortezomib or lenalidomide in patients who have already received at least one other type of treatment. Or used alone in patients who have already received at least three other types of treatment, including a proteasome inhibitor and an immunomodulating agent. CD38 is highly expressed on myeloma cells Efficacy for treatment of other types of cancers is being researched.

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Effects On Transfusion Testing CD38 is weakly expressed on red blood cells Anti-CD-38 binds to CD38 on reagent RBCs causing panreactivity in vitro Positive indirect antiglobulin (IAT) tests Agglutination may occur in all media and all methods Saline, low ionic strength saline, polyethylene glycol Gel, tube, solid phase Reactions are usually weak (1+), but stronger reactions have been observed in solid phase (up to 4+) Anti-CD38 could mask a clinically significant alloantibody

Impact on Transfusion Testing ABORh Not affected Antibody Screen Panreactivity Crossmatch Immediate Spin: No interference AHG: Incompatible DAT Variable reactivity Autocontrol Adsorption Panreactivity not eliminated

Treatment of Red Blood Cells Dithiothreitol (DTT) Disrupts daratumumab binding, allowing detection of underlying atypical alloantibodies in patient plasma Test in conjunction with a k positive cell to confirm effectiveness of DTT treatment Antigens sensitive to DTT include; Kell blood group antigens, Yta, Doa/Dob Adsorptions using ZZAP and untreated RBC’s failed to remove interference Other methods of mitigating anti-CD38 interference Neutralization using recombinant soluble human CD38 or daratumumab idiotype antibody Neither reagent is widely available at this time Antigen-typed cord cells have been used for the antibody screen as an alternative to DTT-treated cells

Managing Patients on Daratumumab Anti-CD38 interference may cause delays in issuing RBCs Before a patient begins anti-CD38 treatment Perform baseline ABORh and antibody screen Perform baseline phenotype or genotype

Managing Patients on Daratumumb After a patient has begun anti-CD38 treatment ABORh performed normally Perform antibody screen and identification using DTT treated RBCs

Managing Patients on Daratumumb Crossmatch Antibody screen negative (using DTT-treated cells) IS or electronic crossmatch ABORh compatible, K matched RBCs Known alloantibody Give phenotypically similar RBCs May perform AHG crossmatch using DTT-treated donor cells Even if phenotypically similar RBCs are selected, AHG crossmatch will still be incompatible Transfusion emergently required: uncrossmatched ABORh compatible RBCs can be given per local transfusion service practices

Managing Patients on Daratumumab Hospitals establish procedures to inform the transfusion service whenever any patient is scheduled to begin taking daratumumab Set up notification in EMR when daratumamb is ordered by physician for ABORh, Antibody Screen, DAT, and genotyping testing to be ordered Daratumumab-mediated positive indirect globulin tests may persist for up to six months after the last daratumumab infusion

Zika Virus First discovered in 1947 in a Rhesus monkey, named after the Zika forest in Uganda First human case was detected in 1952 RNA arbovirus from the Flaviviridae family Transmitted by Aedes species mosquitos (Ae. aegypti and Ae. albopictus) Modes of transmission Mosquito bite Pregnant woman to fetus Linked with birth defects Sexual contact Blood transfusions Not confirmed, but very likely 2 probable blood transfusion transmission cases in Brazil https://upload.wikimedia.org/wikipedia/commons/thumb/3/3e/Aedes_aegypti_feeding.jpg/250px-Aedes_aegypti_feeding.jpg

Zika Virus Most individuals infected with Zika virus will not show symptoms, or will only have mild symptoms Symptoms of infection Fever, rash, joint pain, red eyes, muscle pain, headache Majority do not show symptoms Similar to dengue and chikungunya infections May lead to Guillain-Barré syndrome Diagnosis Laboratory tests, travel history, symptoms Infection during pregnancy Microcephaly, miscarriages, brain defects At this time, there is no vaccine or medication for treatment A number of vaccines are in trial phase https://attachment.outlook.office.net/owa/jacie_mundahl@hotmail.com/service.svc/

Testing for Zika Virus Molecular Testing Method used for blood products and patients RNA NAT (Nucleic Acid Testing) RNA can be detected early in the course of illness Other tests available - Patient testing Serologic IgM detection Quantitative Zika IgM Antibody Capture Enzyme-Linked Immunosorbent Assay (Zika MAC- ELISA) Qualitative detection of IgM antibodies Plaque-reduction neutralization testing (PRNT) Performed by the CDC on positive, equivocal, or inconclusive IgM results Trioplex Real-time RT-PCR Assay Not FDA approved

Zika Virus Outbreaks 2007: First large outbreak in humans on the Island of Yap in Micronesia 73% of the island’s population was infected 2013-2014: 4 other Pacific Island groups have outbreaks 1,505 asymptomatic blood donors are reported to be positive for Zika by PCR. These findings alert authorities that Zika virus can be passed on through blood transfusion. 2015: Reference lab in Brazil reports circulating Zika virus (May) First case in the Americas Other South American countries begin reporting locally acquired infections Microcephaly cases increase Virus reported in Central American countries (October)

Zika Virus Outbreaks 2016 February: World Health Organization (WHO) Emergency Committee declares Zika Virus a Public Health Emergency of International Concern (PHEIC) Based on the large clusters of cases of microcephaly in Brazil and French Polynesia No longer a PHEIC, but long-term response mechanisms have been established to manage global responses July: First case of local or non-travel related transmission reported in Continental U.S. in Miami-Dade County, Florida

Zika Virus in The United States January 1st, 2015 – April 12th, 2017 5,234 Zika virus disease cases reported 4,935 cases in travelers returning from affected areas 223 cases acquired through presumed local mosquito-borne transmission in Florida (217) and Texas (6) 76 cases acquired through other routes, including sexual transmission, congenital infection, laboratory transmission, and person-to-person through an unknown route

Zika and the Blood Supply Donor Questionnaire Travel history or residence in the 4 weeks prior to donation to an area affected by Zika virus Zika virus-like symptoms Combination of rash or fever with muscle aches, headache, malaise, or pinkeye Sexual partners of men with confirmed or suspected Zika virus infection in the last 3 months Deferral for at least 28 days

Zika and the Blood Supply Recipient’s physician should be notified if Units have been transfused from a donor with a history of Zika Virus infection in the 4 weeks prior to the donation Units have been transfused from a donor who reports symptoms suggestive of Zika Virus infection within the two weeks after donation and have recently departed from an area with active transmission

Zika and the Blood Supply - FDA Guidance February 16th, 2016 Recommendation to screen whole blood and blood components, use pathogen- reduction devices, or halt blood collection from areas with active Zika virus transmission Obtain whole blood and blood components from areas without active Zika virus transmission Guidance for donor screening/questionnaire to reduce the risk of transmission of Zika virus by human cells, tissues, and cellular and tissue-based products August 26th, 2016 Recommendation that all states and U.S. territories screen individual units of donated whole blood and blood components with an FDA approved testing method November 18th, 2016 Joint statement to the FDA presented by AABB, America’s Blood Centers, and the American Red Cross regarding concerns of cost and ability to develop and implement testing for Zika Virus in donors. Blood community was not consulted on any guidances issued by the FDA.

Zika and the Blood Supply September 19th, 2016 In accordance with the FDA guidance issued on August 26th United Blood Services, Blood Centers of the Pacific, and Inland Northwest Blood Center begin testing all donations for Zika virus Units labeled as “Neg for ZIKA by investigational NAT” Cost of the required test is passed on to the hospital customer Approximately $6 per unit, depending on transfusion service November 2016 American Red Cross begins testing all donations for Zika virus by NAT

Monoclonal B-Cell Lymphocytosis (MBL) Healthy older adults may have small numbers of monoclonal B cells detectable in their peripheral blood Low count MBL: MBL clones have an immunophenotype resembling typical CLL and represent a small number (<500/µL) of circulating B cells Little, to no risk of progression to CLL High count (clinical) MBL: MBL type cells present in peripheral blood in a larger number (>500/µL) Increased risk of progression to CLL

SLL = Small lymphocytic leukemia http://www.bloodjournal.org/content/123/9/1281?sso-checked=true

Monoclonal B-Cell Lymphocytosis and Blood Donation One study suggested that blood transfusions may be associated with an increased risk for developing B-cell malignancies due to the higher than expected detection of MBL in the donor population (2014) In a Midwestern US regional blood center MBL was detected in 149 out of 2098 donors ages 45 years or older between 2010-2011 In contrast, a study conducted in Denmark and Sweden suggested no association between donors with MBL and the recipient developing CLL (2015) Analyzed both country’s entire computerized transfusion data from over more than 30 years Database contained 1.5 million donors and 2.1million recipients

Monoclonal B-Cell Lymphocytosis and Blood Donation Further investigation needed to reach conclusion about the risk to recipients of transfused cells from a donor with MBL MBL has not been reported posttransfusion, although there is an increased risk for development of B-cell neoplasia MBL transfer from donor to recipient has been reported in allogeneic stem cell transplants for CLL Studies have been conducted to investigate the association between allogeneic blood transfusions and non-Hodgkin lymphoma Mixed results, but an increased risk for non-Hodgkin lymphoma was reported Risk was significantly higher for CLL/small lymphocytic lymphoma than other non-Hodgkin lymphoma subtypes

Other News in Immunohematology Labeling of Red Blood Cell Units With Historical Antigen Typing Results Draft guidance issued by the FDA on January 3rd, 2017 FDA guidance recommendation to indicate that antigen typing results are historical AABB, ABC, and ARC submitted comments on how this would benefit the recipient Comments also made regarding language used in the guidance Predicted phenotype rather than likely phenotype

Other News in Immunohematology Bacterial Risk Control Strategies for Blood Collection Establishments and Transfusion Services to the Safety and Availability of Platelets for Transfusion Draft guidance issued by the FDA in March 2016 Calls for use of pathogen reduction technology (PRT) on platelets previously bacterially-cultured before transfusion on days 4 or 5 Most likely to be performed by the transfusion service AABB, ABC, and ARC submitted comments on the impact on transfusion services and platelet inventory, suggested a longer timeline for implementation

Other News in Immunohematology Transfusion Medicine Journal Article on the Distribution/Utilization Trends of Red Blood Cells in the United States Significant mismatch found between the demand for O neg RBC’s and available O neg RBC’s 6.3% of first time donors were O neg while 10.5% of all RBC’s distributed were O neg Recommendation to use O positive RBC’s in emergent situations for adult males and females of non-reproductive potential (</=50 or 55, based on institution’s policy) Explore ways to shorten ABORh TAT, use type-specific whenever possible, collect and monitor usage data

Other News in Immunohematology Oldest fossilized red blood cells found by Oregon State University entomologist Found in a 30 million year old tick fossilized in amber in the Dominican Republic Tick also was carrying Babesia microti http://cdn.zmescience.com/wp-content/uploads/2017/04/orn86evrwzudrrdrfetn.jpg

References AABB. (2016). Mitigating the Anti-CD38 Interference with Serologic Testing. Beckman, Y. (2016). Blood banks meet the paradox of Gabriel's Horn: what are the options to maintain supply as demand decreases? Transfusion Medicine, 170-6. Blood Systems. (2016, September 20). Blood Service Notice: Universal donor testing for Zika Virus. Centers for Disease Control. (2017, April 13). Zika Virus. Retrieved April 15, 2017, from www.cdc.gov: https://www.cdc.gov/zika/ Chapuy, C. (2015). Resolving the daratumumab interference with blood compatibility testing. Transfusion, 1545-54.

References Food and Drug Administration. (2016). Bacterial Risk Control Strategies for Blood Collection Establishments and Transfusion Services to the Safety and Availability of Platelets for Transfusion. Food and Drug Administration. (2016). Donor Screening Recommendations to Reduce the Risk of Transmission of Zika Virus by Human Cells, Tissues, and Cellular and Tissue-Based Products. Food and Drug Administration, U.S. Department of Health and Human Services, Rockville. Food and Drug Administration. (2016, August 26). FDA advises testing for Zika virus in all donated blood and blood components in the US. United States. Food and Drug Administration. (2016). Questions and Answers Regarding "Recommendations for Donor Screening, Deferral, and Product Management to Reduce the Risk of Transfusion-Transmission of Zika Virus: Guidance for Industry". Food and Drug Administration, U.S Department of Health and Human Services, Rockville.

References Hjalgrim, H. (2015). No evidence of transmission of chronic lymphocytic leukemia through blood transfusion. Blood. Janssen Biotech, Inc. (2015). Darzalex package insert. Horsham, PA: Janssen Biotech. Rosecrans, D. (2016, February). Anti- CD38/Daratumumab/Darzalex/Dara Lab Testing Process. Fargo, ND. Rosecrans, D. (2016). DTT Treatment Red Cells- Transfusion. Fargo, ND. Shim, Y. (2014). Monoclonal B-cell lymphocytosis in healthy blood donors: an unexpectedly common finding. Blood. Stetler-Stevenson, M. (2014). Monoclonal B-cell lymphocytosis in donors. Blood. Stramer, S. (2016). A Joint Statement to the Food and Drug Administration's Blood Product Advisory Committee. AABB, America's Blood Centers, American Red Cross.

References The Smithsonian. (2017, April 4). 30-Million-Year-Old Tick Full of Monkey Blood Found in Ancient Amber. Retrieved April 17, 2017, from www.smithsonianmag.com: http://www.smithsonianmag.com/smart-news/first-fossilized- mammal-blood-found-amber-encased-tick-180962784/ World Health Organization. (2017). The History of Zika Virus. Retrieved April 15, 2017, from www.who.int: http://www.who.int/emergencies/zika-virus/history/en/ World Health Organization. (2016). Fifth meeting of the Emergency Committee Under the International Health Regulations (2005) regarding microcephaly, other neurologic disorders and Zika virus. World Health Organization. (2016, February 19). Zika virus and safe blood supply: Questions and answers. Retrieved April 15, 2017, from www.who.int: http://www.who.int/features/qa/zika-safe-blood/en/