John J. Moulds mcmxliii–mmxi

Vo l u m e 27, N u m b e r 4, 2011

Immunohematology Volume 27, Number 4, 2011

CONTENTS

I n M e m o r i a m 117 John J. Moulds G.M. Meny

Tribute to John J. Moulds 118 S. Nance, J. Vincent, M.K.G. Moulds, J.M. Moulds, T.S. Casina, and C. Flickinger

R e v i e w 131 The ISBT 700 series of low-incidence and 901 series of high- incidence group M.E. Reid

R e v i e w 136 The LW blood group system: a review M.K.G. Moulds

O r i g i n a l R e p o r t 143 Occurrence of to low-incidence antigens among a cohort of multiply transfused patients with sickle cell disease P. Jack son

O r i g i n a l R e p o r t 146 Determination of optimal method for identification in a reference laboratory J.R. Haywood, M.K.G. Moulds, and B.J. Bryant

O r i g i n a l R e p o r t 151 Preoperative coagulation studies to predict blood component usage in coronary artery bypass graft surgery S. Josefy, R. Briones, and B.J. Bryant

C o m m u n i c at i o n 154 Letter from the editors Thank you to the contributors to the 2011 issues 155 A nnouncements 158 A dvertisements

Index 162 Volume 27, Nos. 1, 2, 3, and 4, 2011 166 I nstructions f o r A u t h o r s E d i to r - i n -C h i e f E d i to r i a l B oa r d Sandra Nance, MS, MT(ASCP)SBB Philadelphia, Pennsylvania Patricia Arndt, MT(ASCP)SBB Joyce Poole, FIBMS Pomona, California Bristol, M a n ag i n g E d i to r Cynthia Flickinger, MT(ASCP)SBB James P. AuBuchon, MD Mark Popovsky, MD Philadelphia, Pennsylvania Seattle, Washington Braintree, Massachusetts Martha R. Combs, MT(ASCP)SBB Marion E. Reid, PhD, FIBMS S e n i o r M e d i c a l E d i to r Durham, North Carolina New York City, New York Geralyn M. Meny, MD Geoffrey Daniels, PhD S. Gerald Sandler, MD Philadelphia, Pennsylvania Bristol, United Kingdom Washington, District of Columbia

Tec h n i c a l E d i to r Anne F. Eder, MD Jill R. Storry, PhD Dawn M. Rumsey, ART (CSMLT) Washington, District of Columbia Lund, Sweden Glen Allen, Virginia George Garratty, PhD, FRCPath David F. Stroncek, MD Pomona, California Bethesda, Maryland A s s o c i at e M e d i c a l E d i to r s Brenda J. Grossman, MD David Moolten, MD St. Louis, Missouri E m e r i t u s E d i to r Philadelphia, Pennsylvania Christine Lomas-Francis, MSc Delores Mallory, MT(ASCP) SBB Ralph R. Vassallo, MD New York City, New York Supply, North Carolina Philadelphia, Pennsylvania Paul M. Ness, MD E d i to r i a l A s s i s ta n t Baltimore, Maryland Sheetal Patel

C o p y E d i to r Mary L. Tod

P r o o f r e a d e r Lucy Oppenheim Immunohematology is published quarterly (March, June, September, and December) by the American Red Cross, National Headquarters, Washington, DC 20006.

P r o d u c t i o n A s s i s ta n t Immunohematology is indexed and included in Index Medicus and MEDLINE on the Marge Manigly MEDLARS system. The contents are also cited in the EBASE/Excerpta Medica and Elsevier BIOBASE/Current Awareness in Biological Sciences (CABS) databases.

E l ec t r o n i c P u b l i s h e r The subscription price is $40.00 (U.S.) and $50.00 (foreign) per year. Paul Duquette Subscriptions, Change of Address, and Extra Copies: Immunohematology, P.O. Box 40325 Philadelphia, PA 19106 Or call (215) 451-4902 Web site: www.redcross.org/immunohematology Copyright 2011 by The American National Red Cross ISSN 0894-203X

O n O u r C o v e r

Never forget the history . . . John J. Moulds July 29, 1943–June 13, 2011 I n M e m o r i a m John J. Moulds

G.M. Meny

John J. Moulds, the widely acclaimed leader in , died on June 13, 2011, after battling pancreatic cancer. He was 67. John was a 1961 graduate of Rapid City High School in Rapid City, South Dakota, and a 1965 graduate of Chadron State. He became a certified medical technologist after completing studies at St. John’s McNamara Hospital in Rapid City, South Dakota, and earned the specialist in blood banking from the American Society for Clinical Pathology after completing studies at the Minneapolis War Memorial . John was a longtime participant in the field of transfusion medicine. He published approximately 100 scientific papers, presented lectures on every continent except Antarctica, and was named to several technical advisory boards. He founded and directed the Serum, Cells, and Rare Fluids Exchange, whose membership has expanded to more than 150 immunohematologists. John received numerous awards in recognition of his achievements, including the 1983 AABB Ivor Dunsford Award, the 2003 AABB/National Blood Foundation Sally Frank Award/Lectureship, the 2007 University of Texas Medical Branch L. Jean Stubbins Memorial Lectureship, and the 2011 International Society for Presidential Award. Recently, the LifeShare Reference and Scientific Support Laboratories at Shreveport, Louisiana, were named for John. We are honored that John served on the editorial board of Immunohematology for 15 years, beginning in 1997. His knowledge and valuable insight will be missed. Survivors include his wife, Dr. Joann Moulds, and two daughters, Dr. Terri Moulds Bowen and Dr. Christie Moulds-Merritt.

Geralyn M. Meny, MD, MS Senior Medical Editor, Immunohematology

The following tributes to John Moulds include testimonials from colleagues as well as reiterations of presentations from the dedication of the LifeShare Reference and Scientific Support Laboratories in February 2011.

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S. Nance, J. Vincent, M.K.G. Moulds, J.M. Moulds, T.S. Casina, and C. Flickinger

John J. Moulds, MT(ASCP)SBB

John J. Moulds had a prolific career in immunohematology. For the 2011 South Central Association of Blood Banks (SCABB) His name is synonymous with analytic scientist. From my annual meeting, I nominated John Moulds for the Larry L. Trow Memorial Education Award. This award is for people who have perspective, John’s most admirable trait and one that we shown excellence in education in blood banking. The following is should all strive for was his genuine and incisive interest in a reiteration of my nomination of John for the award, which, by the case, whether presented by an esteemed colleague or a the way, he won. student. He endeavored to find the reason for the results and what it meant for the patient’s care. He was a truth seeker, John Moulds spent his career providing education to eagerly remembering the past to help solve today’s case! One the blood bank community in many different aspects. I first of my favorite recollections is talking at length (was there any met John while I was an SBB student when he was working other way with John?) about a very minute detail of a most at Gamma Biologicals in Houston, Texas. I do believe my complex case from the past, which of course was always the knowledge of the Lewis system and my philosophy of antibody clue that led to the resolution. He was always the one with the identification came from that short week with John. I will corporate memory; it was John who could tell us the family never forget him saying, “Don’t look for unicorns until you members by name and type, which laboratory studied the have ruled out the zebras.” case, the exact reactivity of the samples in each of the media, Besides his work with Gamma, he worked for Ortho as and the conclusion for the case. a Senior Research Fellow and more recently as the Director Immunohematology benefitted immeasurably from his of Scientific Support Services at LifeShare Blood Center. No experience and ideas, and we miss his presence. John served on matter what his job was, he always found time to teach at local, the editorial board of Immunohematology for many years, and national, and international meetings. In 1972, he founded and I and the rest of the editorial board members are truly grateful was director of the Serum, Cells, and Rare Fluid (SCARF) for his contributions and opinions. In one of his last attendances International Exchange. John has chaired committees for at the annual editorial board meetings, John offered to do the AABB and SCABB; he was a peer reviewer for Vox Sanguinis centerfold. Let me explain. Immunohematology has a new and Transfusion, and served on the editorial board for feature that will be used, when appropriate, to help educate our Immunohematology. staff and students. This is a page within the journal containing John’s association with my SBB program prompted me serologic, technical, or blood group information that can to nominate him for the Larry L. Trow Memorial Education be separated and posted for educational purposes, in other Award for Education. He spent one day with my SBB students words, a centerfold. Alas, John did not get to complete this. telling them how to become the best blood bankers ever, and But the first centerfold, with his beloved “orphan” antigens of he did this on his own time and with his own money! When the 700 series, appears in this issue. The tributes in this issue answering students’ questions, he would ask beforehand, “Do give credence to the intensity of his aura in the transfusion you want the long or the short version?” I do think his goal medicine science community. was to instill his love of antibodies in the future blood bankers. He had my students on the edge of their seats waiting for the Sandra Nance, MS, MT(ASCP)SBB next story, the next tidbit of information. He was a role model Editor-in-Chief, Immunohematology for teachers and for anyone who wanted to make antibody American Red Cross Biomedical Services identification their life’s work. He would pick up the phone Penn-Jersey Region and talk to somebody who was emailing about a problem. The Philadelphia, Pennsylvania email may have been a broad email that just happened to get to him, but he would take the time to actually pick up the phone and call. John taught each time he answered a question. He

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had a rare gift of being able to teach with a flair that left the specificities. One time he helped one of the physicians diagnose students in awe as they knew they were hearing words from a patient with lupus erythematosus from the butterfly rash on the master! the patient’s face. John has won the Ivor Dunsford Memorial Award from John applied and was accepted to Specialist in Blood AABB, the L. Jean Stubbins Award from SCABB, the A. Bank (SBB) school in 1968 at War Memorial Blood Bank (now Konugres Lectureship from MABB, and the Sally Frank known as Memorial Blood Center) in Minneapolis, Minnesota. Memorial Award from NBF/AABB, and was recently honored Here he is with the laboratory director, Eleanor Amberg, and at the 49th SCABB meeting with the Scientific Award Lecturer. his SBB instructor, Helen Arndt (Fig. 3). The medical director But to me the most important award is missing, and that is the of the blood center was Dr. Herbert Polesky. John’s favorite Larry Trow Memorial Award for Education. John was a true department was the reference laboratory, which was directed educator and should be the winner of every education award by Jane Swanson, one of the pioneers in the field of blood possible. Education has been his true calling. groups. She would be the one who fueled his passion for the many fascinating aspects of the rare blood groups, many of Janet Vincent, MS, SBB(ASCP) which were identified or investigated in that laboratory. This is Education Coordinator, SBB Program a picture of Jane and John from 1997 when she visited him at University of Texas Medical Branch Gamma Biologicals in Houston (Fig. 4). Galveston, Texas After graduation from SBB school, John was a research technologist at the blood center from 1969 to 1970 until he took over Jane’s position as supervisor of the reference laboratory. The American Association of Blood Banks (AABB) rare donor file was moved from Chicago to Minneapolis in 1974, John J. Moulds and his adventures in blood and John directed the activities until 1975. One of the first banking from the 1960s to 2011 units of blood distributed internationally from Minneapolis was a group O, D–, Vel– rare unit of blood for a patient in John James Moulds started his career in South Africa. John was also a member of the AABB Reference immunohematology and transfusion medicine in 1964 at St. and Rare Donor committee from 1972 to 1978; he chaired it John’s McNamara Hospital in Rapid City, South Dakota, as a from 1975 to 1978. Medical Technology (MT) student. The medical directors were Early on John became involved in education when he Dr. Geib and Dr. Frost, and his MT instructor was Mrs. Bonnie and I were part of a team that presented American Society of Fingerhut. It became obvious that blood banking was John’s Clinical Pathology (ASCP) paternity workshops. Drs. Herbert favorite department. Here he is as a student doing blood typing Polesky and Richard Walker gave the lectures, and John and (notice no gloves) (Fig. 1). There were three others in John’s I directed the “wet” workshops. Yes, “wet” and in hotels!! We class—Jo Ashburn, Mary Ramos, and Irene Coates (Fig. 2). also, of course, were the ones who prepared the samples. The One day, John and Jo were scheduled in the blood bank, and workshops were held in Boston, Massachusetts, in 1971 and the supervisor of that department failed to show up for work. Atlanta, Georgia, and San Francisco, California, in 1972. John and Jo just carried on, typing donor and patient blood, John founded the Serum, Cells, and Rare Fluids (SCARF) performing antibody screenings, and crossmatching and program in 1972. He contacted more than 60 reference issuing blood. Bonnie Fingerhut was rather upset when she laboratories, and approximately 30 responded. Each member found out they were doing all this without supervision! agreed to send 10 mL of a rare or unusual sample once a year John worked at St. John’s for a while after graduation, to each of the other members. John sent the first sample, which and in December 1965 he took a job in a small laboratory was blood from an Rh-null patient. It did not take long before at Chadron Community Hospital in Chadron, Nebraska, the other 30 or so laboratories that had not responded wanted where he had attended college. He was the only technologist to join. The Rh-null sample was from Dr. Alvin Lebeck, a for several months until I was hired to help out. A variety of veterinarian in Wisconsin who John had become friends with tests were performed, especially in the busy departments of when we went to Wisconsin on vacation to draw blood from , chemistry, and blood banking. Unfortunately, him and his family (Fig. 5). This led to John being invited there were no panels to identify antibodies, and they had to be to give a talk in 1973 at the annual seminar of the AABB, sent out. John was almost always right on his guesses as to the which that year was called “A Seminar on Recent Advances

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Figure 1. John Moulds as a Medical Figure 2. John Moulds with his classmates Figure 3. John Moulds with the laboratory Technology student at St. John’s McNamara in medical technology—Jo Ashburn, Mary director, Eleanor Amberg, and his instructor, Hospital in Rapid City, South Dakota, 1964. Ramos, and Irene Coates, circa 1964. Helen Arndt, at the Specialist in Blood Bank (SBB) school at War Memorial Blood Bank (now known as Memorial Blood Center) in Minneapolis, Minnesota, circa 1968. in Immunohematology.” This was his first major presentation. The title of the talk was “Rhnulls: amorphs and regulators.” While at the blood center, John was fortunate to work In this picture (Fig. 6) (left to right standing) are John Case on samples from many patients with rare blood types and (Director of Regulatory Affairs), Dr. Jacob Struck (Research antibodies. He learned a great deal from his mentor and and Development), Leah Walthers, Barbara Fryer, Mary Ann special colleague Jane Swanson, who always said that John Bridges, John Moulds, and (seated) myself and Pamela Lacey. was her “second son.” Some of these exciting cases included We were very fortunate to have the two gentlemen to turn to Rh-nulls, D, Dombrock, Colton, Cartwright, Gregory, Hy, for advice on rare cases and production of special solutions. We Knops, York, JMH, Chido, and Rodgers, to name a few. Several should also recognize two administrative assistants, Barbara early publications were (1) some observations on the T, Tn, (Babs) Smith and Rose Quiroz. and Sda antigens and antibodies that define them in 1972; (2) The consultation laboratory was certified by the state of blood group U on Rh-null leukocytes in 1974; and (3) Texas and eventually became a laboratory certified by the observations on the Gya and Hy antigens and the antibodies Clinical Laboratory Improvement Act (CLIA) and also an that define them in 1975. AABB Immunohematology Reference Laboratory (IRL). John was given the opportunity to become the Director Around this time SCARF expanded into two groups, which of Consultation and Education at Gamma Biologicals, Inc., in now included international members sharing samples from all Houston, Texas. The family moved in August 1975 from cold over the world. John also added a third group of small reference Minnesota to hot and humid Texas. I was offered the position laboratories in the to assist them in building up of Supervisor of Consultation and Education. On the first their collection of rare cells, serums, and fluids. SCARF helped day of work there were more than 75 samples waiting to be us to solve many blood group problems and build up one of tested. At that time, and until 2004 when the laboratory was the largest liquid nitrogen and plasma collections of rare and closed, Gamma Biologicals offered a service to their customers unusual samples in the world. It consisted of ABO subgroups, worldwide: a no-charge workup on difficult, unusual, and Rh typing discrepancies, polyagglutinable cells, high- and rare samples. Local SBB students had been invited to spend low-incidence antigens and antibodies, and, most importantly, a week at Gamma Biologicals to learn the various aspects family studies that would be very useful for molecular studies of reagent and (RBC) screening and panel in the future. It also helped approximately 150 laboratories production. Three students from St. Luke’s Hospital (Carl throughout the world solve unusual and difficult problems. As Northam, Pamela Lacey, and Barbara Fryer) caught our eye, many know, John rarely tossed an interesting sample, and to and shortly after the three graduated from MT school they this day, LifeShare Blood Centers in Shreveport, Louisiana, were offered positions at Gamma Biologicals. Carl became the under the leadership of the CEO Margaret Wallace, MT(ASCP) Supervisor of Quality Control, and Pam and Barb were hired SBB, continues to preserve these rare samples, even though it in the Consultation and Education Department. They were the can be very costly to maintain the rare cells in liquid nitrogen. first of several technologists to work with us on samples and An official Gamma Tutorial was started for Gamma publish results of our findings on many interesting cases. Biologicals customers in the United States that eventually

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Figure 4. John Moulds and Jane Swanson at Figure 5. Dr. Alvin Lebeck, a veterinarian in Figure 6. Staff at Gamma Biologicals,

Gamma Biologicals in Houston, Texas, 1997. Wisconsin who was of the Rhnull , Houston, Texas. Left to right standing are circa 1972. John Case (Director of Regulatory Affairs), Dr. Jacob Struck (Research and Development), Leah Walthers, Barbara Fryer, Mary Ann Bridges, John Moulds; seated are Marilyn would lead to International Tutorial programs. It began Grandstaff (Moulds) and Pamela Lacey. with two invited customers spending Monday through Friday working with the consultation staff on interesting samples and learning about blood groups. In 1980, a special probably one of his most favorite blood groups, and he was laboratory was built that would accommodate 12 participants involved from the very beginning of his career with many of (sometimes more would be added by John as he hated to turn the new findings in this blood group system. anyone away). There were several other blood group systems John had The first group of classes began in 1982, and six to eight a special interest in, one of which was Colton, in which the classes were held during the year. Lectures were given by the first Co(a–b+) was discovered in Minneapolis and a patient consultation staff and other departments in the morning, and sample studied in Houston by John and the staff was the first a “wet” laboratory was conducted in the afternoon, in which Co(a–b–). He was very proud to be included in publications participants worked on almost every type of unusual sample with Peter Agre, who received the Nobel Prize for Chemistry using various techniques. More than 1000 blood bankers for his work on 1 and Colton and also was the went through the Tutorial Program until it closed in 2006. recipient of the award from the AABB. Many of these participants are leaders today in the field of Cromer was another blood group system that John immunohematology, including several physicians. became involved with, when one of the staff worked on a As John read publications on various techniques, he would sample from the second example of IFC negative, which is the start making reagents that were needed to perform them. He null of Cromer. In collaboration with Cyril Levene in Israel our then gave them to the consultation staff to use on cases, and laboratory described Dr in the Cromer system, and later on would then publish the results. He even had his mother, Edith John would work with Bogdan Nowicki in Houston to describe Moulds, growing Vicia graminea seeds to make our own a of uropathogenic Escherichia coli recognizing lectin, and these details were outlined in a poster presented the Dr blood group antigen. at the AABB. Most of the homemade reagents eventually John was also very fortunate to be able to work with became commercially available from Gamma Biologicals, and the German scientist Wolfgang Dahr, who had a particular ultimately other reagent and RBC manufacturing companies interest in the MNS and Gerbich blood group systems. It was followed suit on some of these. This subject will be discussed interesting to see the two of them in the laboratory preparing by Tony Casina in this issue. membranes and testing unusual human and monoclonal As if this was not enough to keep John busy, he also traveled antibodies. nationally and internationally giving talks on blood group John was a member of several professional organizations findings, techniques, etc. John presented his second major talk (often chairing committees) and editorial boards. The in 1978 for the AABB Preconvention Seminar in Seminar on organizations included AABB, ASCP, South Central Perinatal Blood Banking. The topic was “Immunosuppression Association of Blood Banks (SCABB), International Society by Passive Antibody, Rh D Suppression—Rationale and Use of Blood Transfusion (ISBT), and Invitational Conference of and Antenatal and Postnatal Prophylactic Treatment.” Rh was Investigative Immunohematologists (ICII).

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Figure 7. John Moulds receiving the L. Jean Figure 8. John Moulds with his mother, Figure 9. John Moulds with his mother, Edith Stubbins Award from SCABB in 1979. Edith Moulds, and his daughter, Terri Moulds, his daughter, Christine Moulds- Moulds Bowen, PhD, upon receiving the Merritt, MD, FACS, and his grandsons, Daniel Distinguished Alumni Award from Chadron Bartley Merritt III (age 13) and Matthew State College, 2009. Ryan Merritt (age 9), at the dedication of the LifeShare laboratories in 2011.

He was on the ISBT Working Party on Terminology While at Gamma Biologicals, John went on to become for Red Cell Surface Antigens since 1980 and chaired the Chief Operations Officer and Executive Vice President, then committee from 1990 to 1994. He also was on the 3rd and President and Chief Science Officer. He helped automate 4th International Workshop and Symposium on Monoclonal various aspects of manufacturing and brought computers into Antibodies Against Red Blood Cells and Related the workplace. He was also a member of the board of directors program committees in 1995 to 1996 and 2000 to 2001. from 1992 to 1997. Both of these committees helped shape the organization and John left Gamma Biologicals in 1997 and worked for naming of antigens in the human blood groups and set criteria a year as a research associate in the Department of Medical for inclusion in a blood group system. Hematology, Baylor College of Medicine, in Houston, Texas, John received numerous honors and awards from and from there he joined Ortho Clinical Diagnostics in Raritan, various organizations for his contributions to the field of New Jersey, in various capacities in research. immunohematology, transfusion medicine, and education. John left Ortho in 2004 to become Director of Scientific The first of these was the L. Jean Stubbins Award from Support Services at LifeShare Blood Centers in Shreveport, SCABB in 1979, which he and I shared (Fig. 7). Next were Louisiana. One of the first items on the agenda was to attend the Ivor Dunsford Memorial Award from AABB in 1983, a workshop and read articles on monocyte monolayer assay Angelyn Konugres Lectureship from MABB in 2001, Sally (MMA) and develop the assay at LifeShare for assisting in Frank Award Lectureship from NBF/AABB in 2003, Kay determining the clinical significance of antibodies when Beattie Award Lectureship, Michigan Association of Blood patients needed to receive incompatible blood. Again, he led Banks in 2005, L. Jean Stubbins Memorial Lectureship from this laboratory and the staff to become a source of knowledge University of Texas Medical Branch in Galveston, Texas, in and expertise for the reference laboratory at LifeShare and 2007, SCABB 49th Annual Meeting Scientific Award Lecturer other laboratories throughout the blood bank community. from SCABB/CBBS in 2007, Award for Technical Excellence This Scientific Support Laboratory has also become one of the from America’s Blood Centers/Institute for Transfusion leaders in the field of DNA technology, led by Dr. Joann Moulds. Medicine in 2011, and the Larry L. Trow Memorial Education In February 2011, LifeShare honored John with a dedication Award from SCABB in 2011. ceremony at which the reference and scientific laboratories One honor that John received outside the field was were renamed the John J. Moulds Reference and Scientific recognition by Chadron State College in Chadron, Nebraska, Support Laboratories for John’s lifetime accomplishments. when he was presented with the Distinguished Alumni Award Many colleagues attended this dedication, and others sent in 2009 for his many accomplishments. Before the awards letters of their personal memories of John. ceremony he gave a talk to the various classes of premedical On a personal note, John has two daughters who went on students on how to succeed in whatever you set your sights on to college and got advanced degrees (there was no question and reach for the stars. in John’s mind that they would). He was very proud of them

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and their accomplishments. One, Terri Moulds Bowen, PhD (Fig. 8), received her advanced degree in biology from UCLA in California and loved working in the laboratory and research The antibodies that no one wanted to work on— (like her father). However, she has now found a new career in except John teaching, again like her father, who never turned down anyone who would ask him for advice or to give a talk, anywhere in When preparing the titles for our talks at the laboratory the world. His other daughter, Christine Moulds-Merritt, MD, dedication, one of the laypersons who organized the meeting FACS (Fig.9), who completed a surgery residency at Scott and read my title and commented, “Oh, you mean like little Orphan White in Temple, Texas, is a general surgeon and specializes in Annie Bodies.” And indeed, the HTLA group could have just as breast surgery. easily acquired this name had it not been for John’s persistence John’s two grandsons, Daniel Bartley Merritt III (age 13) to investigate them. Those of you who have heard me speak and Matthew Ryan Merritt (age 9) (Fig. 9), have also inherited know of my campaign to stomp out the term HTLA. However, some of John’s traits—his skill for seeing a problem and to understand where we are today means that we have to coming up with solutions and his sense of humor. They both appreciate the history behind the terminology—a history in seem to find science their favorite subject in school, so it will be which John Moulds was a key player. interesting to see what lies ahead for them in the future. They The beginning of the term HTLA goes back to the early also have gone deer hunting with their mother and father, just 1970s when the American Association of Blood Banks as John did with his dad, and the oldest grandson got his first (AABB) and American Red Cross reference laboratories buck last year. The younger one also shares John’s love for had joint meetings actually consisting of wet workshops! fishing. The participants would bring antibodies to high-incidence And lastly, John’s love of animals, especially dogs (Fig. antigens and antibodies to low-incidence antigens and try 10), has led to his rescuing them from numerous situations to pair them up or place them in a blood group system. But and giving them a home filled with kindness and love. there always seemed to be a group of antibodies that never What will be John’s legacy, you might ask? Some say fit into a known blood group system. These antibodies were it is the international SCARF program he founded, which notoriously weak and difficult to work with, hence the name is still active today. I say this and his love of blood groups, serum of inscrutable type proposed by John high- and low-incidence antigens, investigating unusual test Judd. The resulting acronym made some serologists snicker, results, preparing test solutions, his sharing of the knowledge whereas others were offended. So finally Jane Swanson, he gathered over the years, his joy in teaching, and his sense Delores Mallory, and John Moulds came up with a descriptive of pride in what he did throughout his career. He always name of high titer–low avidity, or HTLA, antibodies. reminded us to “never forget the history.” He loved the stories Technologists would come to automatically use this as an that went with the discoveries of the blood groups and the explanation for weak anti-human globulin (AHG)–reactive various antigens and antibodies. antibodies, and others would incorrectly use the name HTLA antigens. John was to live long enough to regret that name. Marilyn K. Grandstaff Moulds, BA, MT(ASCP)SBB In fact, he once wrote that “serologists frequently use slang Immunohematology Specialist or colloquial statements that are intended to be descriptive of John Moulds Reference Laboratory the general problem.… These slang terms are not intended to Shreveport, Louisiana define an antibody specificity, but rather to roughly describe the serological results.” So what are the serologic characteristics of the HTLA antibodies? Probably their hallmark is their weak and variable reactivity at the AHG phase of testing. The reactions can range from microscopic + to 1+ or sometimes 2+. Although they were initially believed to be of high titer (>64), not all exhibit this characteristic and often the titer is dependent on the indicator cell chosen for testing. In general, they are not enhanced with low-ionic strength saline or polyethylene glycol, and the effect Figure 10. John Moulds with Bodie. of enzymes varies with the individual specificities. Cord cells,

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as well as older stored red blood cells (RBCs), often give weaker Anti-Hy (Holley) was reported in an abstract in 1967, or negative reactions. Finally, the antibodies have not been and several more examples were published, all being found reported to bind complement or cause hemolytic transfusion in Blacks. The consultation laboratory that John directed reactions. The antibodies originally meeting these criteria at Gamma Biologicals studied a number of these unique included anti-JMH, -Yta, -Gya, -Hy, -Joa, -Ch, -Rg, -Kna, -Knb, antibodies and described a subdivision that they named Jca. -McCa, -McCb (McCc/McCd/McCe), -Sla, -Vil, -Yka, and -Csa. Although others thought that Jca was synonymous with Joa, Each of these has now been placed in a system recognized by John always believed that they were different, and there is the International Society of Blood Transfusion, and they are some emerging molecular data that may support his theory. described in subsequent sections. The exception is Csa, which John would also be the first to recognize an association remains in a collection of two antigens. between Hy and Gya as all Hy– Blacks were Gya weak. Finally an explanation came forth in 1995 when Banks reported that System 011-YT RBCs having the rare phenotype of Gy(a–), Hy–, Jo(a–) were also Do(a–b–), i.e., Gy(a–) was the null for this blood group YT (Cartwright) existed as a blood group system before system. This is a rare null phenotype occurring in less than the terminology HTLA gained favor; however, because of the 1:10,000. antibodies’ weak reactivity, many placed them into the HTLA The Dombrock antigens would later be determined to be group. Presently there are two known antigens, Yta and Ytb, and located on the adenosine 5′-diphosphate ribosyltransferase only one reported example of a true null phenotype Yt(a–b–). 4 , and the molecular mechanism for each would be John’s involvement with this system was at the population identified (Table 1). John was quick to remind technologists genetics level. Working with Dr. Cyril Levine in Israel he that although most HTLA antibodies are considered clinically studied Israeli and they showed that this ethnic group had insignificant, anti-Doa had caused delayed hemolytic a high incidence of Yt(b+), approximately 25 percent. The Ytb transfusion reactions. He believed that the availability antigen has also been found with increased incidence among of donors molecularly typed for Doa and Dob would be a Arabs but is almost nonexistent in Asians. Therefore, one of significant improvement in supplying safer blood for these the best sources for Yt(a–) donors is the Jewish population. antibody producers. In the early 1990s, Dr. Dave Anstee in Bristol, United Kingdom, and Dr. Marilyn Telen in Durham, North Carolina, System 017-Ch/Rg both reported that the YT system antigens were carried on a protein known as acetylcholinesterase. Thus, paroxysmal Table 1. Molecular background for the common DO antigens nocturnal hemoglobinuria type III RBC, which lack all Phenotype Nucleotide Exon Amino Acid glycophosphatidylinositol-linked proteins, will have the Do(a+) A 2 Asn265 acquired phenotype Yt(a–b–). A mutation at codon 322 Do(b+) G 2 Asp265 changes histidine (Yta) to arginine (Ytb). Using this known Hy+ G 2 Gly108 single nucleotide polymorphism, DNA-based methods have Hy– T 2 Val108 now been developed to accurately type for YT. Jo(a+) C 2 Thr117 Jo(a–) T 2 Ile117 System 014-DO John hated using “selected cells” to determine antibody John was destined to be involved with the Dombrock specificities. He thought that serologists should use their system because his mentor at Minneapolis War Memorial knowledge of the antigen biochemistry and be able to Blood Bank, Ms. Jane Swanson, was the first to report anti- manipulate the test medium. The remaining systems Doa. He was able to squirrel away probably the biggest stash of exemplify this opinion. After the identification of Chido anti-Doa on the continent, and he used it sparingly but wisely. (Ch) and Rodgers (Rg) antigens on the fourth component of Being the generous soul that he was, John shared some of this complement, specifically C4d, John theorized that one could antibody with Dr. Nakajima from , and they were able to enhance the antibody reactivity by increasing the amount of show that Doa was a low-incidence antigen in that population. C4 on the reagent RBC. In fact, that’s just what he did. In a Later, with Dr. Yoshida Okubo, he would report the first report in Transfusion, John Judd and John Moulds showed example of Gy(a–) with anti-Gya in the Japanese. that you could use a simple sucrose method to put more C4

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on the RBC, and with the increased number of antigen sites, observations from this work was that Blacks had higher levels anti-Ch or anti-Rg could react as a direct agglutinin. This, of C4B in their plasma, i.e., Chido substance, which was the in combination with the fact that ficin destroyed antibody result of a C4B duplication. This finding validated John’s reactivity, made it simple to identify anti-Ch or anti-Rg. The earlier observations that Black donors made better C4-coated fact that some Ch/Rg antibodies have caused anaphylactic RBCs for the manufacturing of Gamma Biological’s reagent reactions when transfusing plasma components makes it quality control kit. important to be able to at least get the HTLA antibody into the correct system. System 022-KN Working with Dr. Carolyn Giles, John and the staff at Gamma Biologicals helped define six Chido antigens and three Once again, John’s Minneapolis connection would Rg antigens. Later, Dr. Yung Yu would work with Carolyn to contribute greatly to his involvement in a blood group system, define all of these at the molecular level. The rare null for this this time Knops. Mrs. Knops was identified as having anti- system would be the total C4-deficient patient, of which there Kna, and one serologically compatible donor was found, i.e., were fewer than 20 known in the world. This fact would lead Margaret Helgeson. Margaret was a technologist at the blood to John’s next mark on the Ch/Rg system, i.e., mentoring a bank and found later that she was not only Kn(a–) but was, in struggling graduate student (Fig. 1). fact, the serologic null for the system. She became infamous Dr. Joann Moulds would go on to investigate two C4- when John made her a regular contributor on the international deficient (Ch–/Rg–) brothers as well as the association of the exchange he founded known as SCARF (Serum, Cells, and Rg– phenotype with systemic lupus erythematosus. Using Rare Fluid Exchange). monoclonal anti-Ch and anti-Rg, assays were developed When John and Marilyn Moulds went to Gamma to quantify these plasma proteins. One of the surprising Biologicals, they continued to work on the weak antibodies no one cared about and began adding new specificities to the KN system. These included the Hall serum (anti-Knb), anti- McCa, anti-Sla (Swain-Langley or Sl1), and anti-Vil (Villien or Sl2). The latter two were only presented in an AABB abstract because of a difference of opinion with another collaborator, Dr. Lyndall Molthan. Over John’s objections, Dr. Molthan continued to add these new specificities to the McCoy locus, i.e., McCc, McCd, McCe, etc. Molecular analysis would later prove the Gamma Biologicals group correct that these were separate mutations on the Knops protein. As if there were not enough Mouldses involved with the HTLA story, the new Dr. Moulds would take on Knops as one of her postdoctoral projects. Armed with many of the original antisera provided by John, she found that the Knops antigens resided on complement receptor type one (CR1). The variability in reactivity as well as the Helgeson phenotype could now be explained by the known inherited RBC expression polymorphism of the CR1 gene. But this was just the tip of the iceberg, and John’s collection of antigen-negative cells and Knops antisera would prove invaluable for the next set of investigations. Dr. Lou Miller (of Duffy blood group and malaria fame) was now studying Plasmodium falciparum malaria and the phenomenon known as rosetting. Because John had shown that certain Knops such as Sl(a–) and McC(a–) occurred more Figure 1. John the consummate mentor (for Dr. Joann Moulds and frequently in Blacks, Joann postulated that this may also be a many others). protective phenotype. So off to Africa (Mali) the two traveled

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to begin what would become a 6-year project. The data are these antigens may play in the modulation of natural killer cell too numerous to discuss here, but the conclusions were as function. follows: (1) the African McC(a–b+), Sl(a–), KCAM- negative results in reduced rosetting of infected RBCs and Summary less severe malaria; (2) in Asia the protective phenotype is the Helgeson type, which also results in reduced rosetting; and John’s dogged determination to work with and identify (3) the most virulent forms of P. falciparum use CR1 (Knops) the HTLA group of antibodies has certainly led to many as an alternative invasion pathway into the RBCs. So much important discoveries. From a medical technology student to good scientific information has come from those silly serologic a world-renowned immunohematologist, John Moulds has studies that John championed. come a long way (Fig. 2). And we, his students, friends, and collaborators, “stand up and cheer for this doer, this achiever, System 026-JMH the one who recognized the challenge and did something about it” (paraphrased quote from Vince Lombardi). We are Perhaps it is fitting that the last system arising out of the glad you did, and we will never forget your many scientific HTLA group would share part of John Moulds’ name, i.e., JMH contributions. (John Milton Hagen). This strange collection of antibodies went by many names: The Boys, The Cats, The Over 60s, etc. This Joann M. Moulds PhD, MT(ASCP)SBB was because many of the first examples were found in elderly Director, Scientific Support Services men or women who owned cats. The antibodies were rather LifeShare Blood Centers unexciting, not causing hemolytic disease of the newborn or Shreveport, Louisiana fetus or hemolytic transfusion reactions, and occasionally one would appear to be an autoantibody. But again, John would be involved in elevating this group to full system status. In 1991, he and Marilyn Telen published a paper in Blood locating JMH on a phosphatidylinositol-linked membrane protein. Later If a reagent can be made, John J. Moulds can this was identified as semaphorin A (SEMA7A or CD108). make it Using samples sent to him by Dr. Cyril Levine many years earlier, John collaborated with Dr. Axel Seltsam to expose the John J. Moulds has had a dramatic impact on blood molecular diversity of the JMH gene. Presently there are six bank technologists worldwide, making their lives easier when antigens identified from what was once believed to be a single, performing pretransfusion testing and problem solving. annoying antibody specificity. It remains to be seen what role John’s many contributions to the development of commercial reagents allow technologists to perform testing without the need for extensive preparation of reagents and without intensive method and technique applications. “Professionally developed and manufactured reagents for professional use that simplify the life of the blood banker …” On February 19, 2011, the John J. Moulds Reference and Scientific Support Laboratories were dedicated to John J. Moulds. This is a reiteration of the presentation, “If a reagent can be made, John J. Moulds can make it,” delivered at this ceremony. When I was asked in the fall of 2010 to participate as a speaker at the symposium and laboratory dedication of the John J. Moulds Reference and Scientific Support Laboratories at LifeShare Blood Centers, I was both humbled and proud Figure 2. John as a medical technology graduate (left) and as of that opportunity to honor both a great friend and mentor Director of Scientific Support Services at LifeShare Blood Centers for the many accomplishments he achieved throughout his (right). illustrious career. However, before I go into the focus of my

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presentation at that dedication, I want to share a personal Over the years, John and I encountered each other at story, which I shared at the dedication as well, about John’s blood bank meetings. Always open to speaking at local, state, influence on my career in the transfusion medicine industry. regional, national, and international meetings, John shared My first encounter with John was via the phone. I was his knowledge with the blood bank masses. a full-time employee at the Medical College of Pennsylvania In the early 2000s, I would get to work directly with John in Philadelphia, Pennsylvania, where I had just graduated at Ortho Clinical Diagnostics. During more than 30 years of from the medical technology internship program. My blood interactions with John, his willingness to share his knowledge, bank manager asked me to call Gamma Biologicals and ask to discuss cases, and to be a mentor has had a great impact for John Moulds to find out “what was wrong with these on my career. I am sure there are many out there who have a screening cells.” One of the antibody screening cells was similar story to share about John. showing reactivity with serum samples from a few patients; Many of John’s contributions to the development of subsequent antibody panel identification testing showed commercial reagents have either simplified or solved challenges results with reactivity that appeared to have no antibody for the transfusion service or reference laboratory medical specificity. John kindly took the time to explain to me that the technologist in applying testing methods and techniques. With particular antibody screening cell expressed the Bga antigen John’s guidance and insights, and the assistance of colleagues and was likely involved because other cells that were tested at Gamma Biologicals, significant contributions were made to on the antibody identification panel had “Bg” expression. the introduction of these reagents.1 As typical of the teacher in John, he patiently answered Now on to the contributions that John has made to the my questions and clarified what he could in light of the blood banking world from a commercial blood bank reagent conversation being a phone discussion. perspective that could be applied in testing by just about any I walked away from that call impressed. About 2 years medical technologist willing to follow instructions. So yes, “If later, the sales representative from Gamma Biologicals a reagent could be made, John J. Moulds can make it.” Figure happened by the hospital the week before the next meeting of 1 provides a timeline summary of the release of these various the local antibody club, the Delaware Valley Blood Bank Club, commercial blood bank reagents. which I had planned to attend and which featured John as the speaker. The representative made me an offer that I jumped at: after John was done speaking at the meeting, would I like to have a drink with him at a local “watering hole”? So after talking a little bit about the blood bank consultation service at Gamma and his travels talking about blood group serology and blood banking reagents, John asked if we could talk about something other than blood banking (I am sure that, as is the customary topic of conversation between sales representatives and customers, all John had done that day was talk about blood banking). So we talked about where we grew up, hobbies like hunting and fishing, and an old Corvette that he was trying to rebuild. Again, I walked away significantly impressed by the Figure 1. Adapted timeline for introduction of reagents.2 man and his career. Because of these encounters with John, my experiences with teaching students, working on just about every antibody 1970s that was received in the hospital laboratory, and attending local and state blood bank meetings, I was hooked on blood One of John’s first uniquely developed commercial reagents banking. So my fate was sealed. His influence fueled my desire in the early 1970s was stable complement-coated cells. Having to work for a blood group reagent manufacturing company, to prepare special reagents, select the right donor red blood cell focusing on blood group serology and the use of blood bank (RBC) to coat, and attempt to stabilize the complement coating reagents. In 1983, I started my career in the commercial on the RBC when preparing complement-coated RBCs are industry working for Biological Corporation of America (BCA) true challenges. The introduction of a commercially available in the Blood Bank Consultation Service. ready-to-use stabilized complement-coated cell to quality

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control anticomplement antiglobulin reagents certainly made John followed with two reagents formulated to help it easy to assure that anti-human globulin reagents containing enhance antibody detection and identification tests. The low- anticomplement activity were performing acceptably. No ionic-strength saline (LISS) solutions method, as described longer was it necessary to spend hours preparing materials, by Löw and Messeter,4 used RBCs that required the cells qualifying the “right” donor, coating the cells, and in the to be washed and suspended in the solution for use. John end having to quality control the prepared complement- helped develop two reagents, Gamma LO-ION and Gamma coated cells. N-Hance. The Gamma LO-ION product, an additive-based Proteolytic enzymes and their effects on RBC structure LISS reagent, used the traditional LISS solution along with a and antigens have been extensively studied. Use of enzymes in high-molecular-weight protein (polyvinylprolidine) to create blood group serology was limited to those experienced in their a popularly used tube-based LISS additive. Gamma N-Hance preparation and use. Variations in enzymes and treatment used the traditional LISS-based approach combined with approaches created variability in observed test results. In bovine serum albumin as part of the formulation. Both additive the late 1970s, John, along with the consultation service and methods allowed for any properly prepared RBC suspension to RBC production departments at Gamma, had prepared ficin- be tested easily without special preparation of the cells in a treated panels, and John shared them with other serologists LISS solution. in a few reference laboratories throughout the United States. After doing this several times during the year, John found 1980s the number of reference laboratories wanting to get in on the “enzyme panel” expanding. The first commercially The 1980s brought a plethora of new innovative available enzyme-treated panel was born. The Gamma ficin commercial reagents driven by John. Polyagglutinable panel became popular because it eliminated the need for the cells are generally easy to define and identify with the use special preparation required for enzymes, and for treatment of the right lectins that react and agglutinate certain RBC standardization and it had stability, so that the product had antigens. John enjoyed working with cells that demonstrated a longer shelf life compared with the self-made preparations. polyagglutination. Making life easier for technologists working Now not only reference laboratories but transfusion service with polyagglutinable cells, John worked to produce and laboratories could perform enzyme antibody identification standardize lectin reagents for manufacturing. An abstract panels using the commercial panel. Resolving some complex “Care and cultivation of Vicia graminea” by John and others in antibody problems with enzymes now became simpler and the early 1980s kept it all in the family, as even John’s mother, quicker, decreasing turnaround time. Edith, made a significant discovery.5 (Fig. 2.) V. graminea, The ability to remove the coating antibody from RBCs to the plant from which the seeds for the lectin are harvested, identify it is a very useful tool for solving antibody problems is actually in a dormant state when it looks as if the plant has associated with autoimmune hemolytic , hemolytic died. Edith found that the plant starts to grow again in the disease of the newborn and fetus, drug-induced , spring to produce seeds (personal communication, Marilyn and hemolytic transfusion reactions. Elution procedures for preparing eluates were often limited to a safe but ineffective method such as a 56°C heat elution or choosing a chemically oriented method with potential dangers in its use. The discovery that an acidic solution could be used to elute antibody from RBC stroma created a safe, effective way to produce an eluate.3 John took this information and created the first commercially available elution kit, Gamma Elu-Kit I, which used a series of reagents including a solution of digitonin, a wash solution, an acid solution, and a buffer solution to create a popular approach to performing elution. Later in the 1970s the introduction of the first licensed anti-Cob, with John’s leadership, led to a stream of new reagent antisera to “rare antigens” and to a variety of blood group Figure 2. Tony Casina and Edith Moulds at the dedication of the antigens during the next decade. LifeShare laboratories to John Moulds in 2011.

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Moulds). An anti-N lectin (V. graminea) was developed and specificities to the Gamma-clone line of monoclonal antibodies, a b commercialized through these efforts. In the early 1980s, the including anti-Mg, -M, -N, -Le , -Le , -P1, and -K. Gamma Lectin Kit, with its four lectins, Arachis hypogaea, Salvia sclarea, Salvia horminum, and Glycine max (soja), 1990s allowed the identification of the most common forms of polyagglutination. The kit delivered the convenience of a Unique new reagents continued appearing in the manufactured standardized set of lectins without all of the immunohematology testing market, flowing from a concept “grinding and refining.” in John’s mind to a manufactured commercialized reagent. The presence of antibody on RBCs can be quite impairing In the early 1990s John took a recently identified method when attempting to antigen type RBCs. The removal of for detecting and identifying blood group antibodies using antibody from direct antiglobulin (DAT)-positive RBCs had polyethylene glycol (PEG), a test that Nance and Garratty7 traditionally been attempted by gentle heat elution to effect published in the late 1980s, and produced a product that can removal of antibody from the RBCs. This method was time be used in tube-based tests for antibody detection that showed consuming and often did not work very effectively. The greater sensitivity with some blood group antibodies. The discovery that chloroquine diphosphate could elute antibody combination of PEG and LISS solution led to the Gamma PeG effectively from most RBCs without significantly affecting reagent that delivered the sensitivity of PEG in a convenient antigen structure led to John’s development of a manufactured commercially prepared reagent. reagent, Gamma-Quin. This reagent could be used to remove Later in the 1990s John revolutionized the way antibody from DAT-positive RBCs, which allowed the use antiglobulin reagents are produced, introducing the first of antiglobulin-based typing reagents to test these RBCs for monoclonal-based anti-IgG that eliminated the need to use other blood group antigens. rabbit-sourced anti-IgG. This murine monoclonal antibody In the mid-1980s, John drove innovative improvements had unique specificity in that it did not detect antibodies of to the Elu Kit I elution kit by introducing the capability of the IgG4 subclass, which have been shown to rarely have eluting antibody from intact RBCs. This innovation eliminated any clinical significance. Additionally, this monoclonal anti- the need to wash the stroma free of hemolysis created by the IgG allowed for the development of the only total monoclonal digitonin lysis of RBCs and further reduced the turnaround polyspecific anti-human globulin (anti-IgG, -C3d). time to obtain a quality eluate. Additionally, the remaining intact RBCs could be used for antigen typing in the appropriate 2000s circumstances. The middle 1980s brought two manufactured reagents, John brought his innovative thinking and problem-solving which addressed the needs of obstetrical patients: Gamma skills to Ortho Clinical Diagnostics in 2000. Applying those Fetal Bleed kit and Gamma r-set antibody screening cell. skills, he helped resolve challenges with immunohematology John and the consultation staff formulated the Fetal Bleed products used worldwide and contributed to the development Kit based on the Sebring-Polesky procedure6 for fetomaternal of new reagents. hemorrhage testing. This kit made the evaluation of It is obvious that John’s unique thinking and practicality fetomaternal hemorrhage an easy-to-perform blood bank afforded him the knowledge and wisdom to take complex test. The introduction of the Gamma r-set screening cells, technical processes and procedures and simplify them. of which John directed the design, simplified dealing with That ability has led to the many unique immunohematology patients, particularly obstetrical patients who had received reagents that are used by medical technologists throughout Rh immunoglobulin and had anti-D, eliminating the need to the world to perform test procedures that normally would not perform standard antibody identification in these patients. be available to them. This simplified the process of finding selected cells for testing I have only scratched the surface of the many reagents when anti-D is present. that John J. Moulds has had some hand in formulating into With the innovation in the 1980s of monoclonal antibody a medical technologist–friendly usable tool for immunohema- technology in blood group serology, John introduced the first tology testing and problem solving. Professionally developed monoclonal antiserum for blood group antigen testing, anti- and manufactured reagents for professional use that simplify He. He was instrumental in the introduction of additional the life of a blood banker …

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John will be missed by the many people he touched with low-incidence antigens led to his being asked to write a review his knowledge, wisdom, and mentorship. Personally, John was of these important but sometimes forgotten blood group a great friend and I will miss him tremendously. antigens. However, despite his intentions, time did not allow for this publication. I was honored to present John with a plaque Acknowledgments commemorating his many years of insight, inspiration, and service on the board of Immunohematology at the LifeShare I would like to thank Marilyn Moulds for her review and laboratory dedication (Fig. 1). John will be long remembered suggested improvements of this article. for his many contributions to the journal Immunohematology as well as to the field of transfusion medicine. References Cynthia Flickinger, MT(ASCP)SBB 1. 1998 Product Catalog Gamma Biologicals, Inc. 2. Blood Banking: The Niche Market of Immunohematology, Managing Editor, Immunohematology Timeline of Key Events in the Blood Bank Reagent Industry, American Red Cross Biomedical Services Gamma Biologicals Inc. Penn-Jersey Region 3. Kochwa S, Rosenfield RE. Immunochemical studies of the Philadelphia, Pennsylvania Rh system. I. Isolation and characterization of antibodies. J Immunol 1964;92:682–92. 4. Löw B, Messeter L. Antiglobulin test in low-ionic strength salt solution for rapid antibody screening and cross-matching. Vox Sang 1974;26:53–61. 5. Moulds M, Moulds E, Moulds JJ. Care and cultivation of Vicia graminea (abstract). Transfusion 1978;18:646. 6. Sebring ES, Polesky HF. Detection of fetal maternal hemorrhage in Rh immune globulin candidates. A rosetting technique using

enzyme-treated Rh2Rh2 indicator erythrocytes. Transfusion 1982;22:468–71. 7. Nance SJ, Garratty G. A new potentiator of red blood cell antigen-antibody reactions. Am J Clin Pathol 1987;87:633–5.

Tony S. Casina, MT(ASCP)SBB Marketing Manager Ortho Clinical Diagnostics World Wide Marketing Raritan, New Jersey Figure 1. Cindy Flickinger presenting commemorative plaque to John Moulds at LifeShare Dedication in 2011.

It was with great humility, admiration, and pleasure that I attended the symposium and dedication of the John J. Moulds Reference and Scientific Support Laboratories at LifeShare Blood Centers in Shreveport, Louisiana, on February 19, 2011. John had been a member of the editorial board of Immunohematology since 1997. During that time, John had contributed to the journal through his innovative input to the journal process and format at the annual journal breakfast meetings at the AABB conference as well as through his timely and thorough review of manuscripts submitted for possible publication. John’s suggestion of providing serologic information in removable centerfold format in the journal will be implemented in this issue. His interest in the antibodies to

130 IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 R e v i e w The ISBT 700 series of low-incidence and 901 series of high-incidence blood group antigens

M.E. Reid

The International Society of Blood Transfusion Working Party of respect for John, I write this review knowing that it is in no on Terminology for Red Cell Surface Antigens developed a way a substitute for what he would have imparted. terminology that brought order to the chaos of antigen names. They classified antigens into three categories: systems, collections, and series. This review summarizes the early decisions of the Order From Chaos Working Party with an emphasis on the 700 series of low- incidence antigens and 901 series of high-incidence antigens. At their first meeting in Montreal, at the ISBT biannual Immunohematology 2011;27:131–135. congress in 1980, the Working Party began to develop a scheme for naming blood group antigens that was both eye Key words: blood group antigens, common antigens, high- and machine readable, in keeping with the genetic basis incidence antigens, low-incidence antigens, private antigens, of blood groups, and infinitely expandable. The priority of public antigens, uncommon antigens the group was to develop a scheme to name and to assign a number for each blood group antigen—not to replace the By 1980, the number of blood group antigens that had original names, which are easier to remember, but to have been described and the multitude of creative names used were standard alternatives for use in computers. The Working Party becoming overwhelming. An antibody or antigen could be decided to follow the rules of the Human Gene Nomenclature found in more than one laboratory and, if an effort was not group and agreed to no longer use Greek letters, superscripts, made to perform cross testing, could be assigned a different and subscripts, in favor of using uppercase Roman alphabet name. Examples include the finding that Rla was the same letters and Arabic numerals in naming blood group antigens. as Lsa, and Lan was the same as Gna and So. Similarly, two The second meeting was held in New York City in 1981, after different antigens could bear the same name, e.g., Hughes which a preliminary report was published with recommended (Hga) is an antigen in the Diego system (DI12) and Hughes numbers for 28 antigens in nine systems.1 This ingenious is an antigen in the Lutheran system (LU13). Other examples scheme, albeit considerably expanded, is still in use today. of inadvertent, unfortunate symbol choices were (1) the alphabetically similar sD in the MNS system (MNS23) and the Scheme for Categorizing Antigens polyagglutination-related antigen Sda, and (2) the phonetically similar Emm (901008) and M (MNS1). The manner in By definition, all blood group antigens must be defined which an antibody or antigen was labeled could sometimes serologically by the use of a specific antibody. All antigens only be deciphered by the worker who wrote the label. It receiving ISBT numbers must have been shown to be was obvious that a system was needed to bring order to the inherited characters. After the sixth meeting, held in 1988 growing chaos. The principal force in the organization of an at the 20th ISBT Congress in London, the Working Party International Society of Blood Transfusion (ISBT) Working wrote a monograph providing their rationale and guidelines Party on Terminology for Red Cell Surface Antigens was the for the official numeration of new specificities.2 The Working ISBT Working Party on Automation and Data Processing. Party classified serologically determined antigens into three The first Working Party consisted of 32 eminent international categories: systems, collections, and series. Obviously, the investigators, one of whom was John Moulds. John was a classifications were made based on knowledge of the time and specialist in all blood groups but had a particular fondness were considered to be “carved in soap rather than in stone.”3 for the orphan “private” or “uncommon,” and “public” or “common” antigens. He had agreed to write a review for Blood Group Systems Immunohematology on this topic, but his premature death A blood group system is a discrete genetic entity under the prevents us from knowing what he had in mind to share. Out control of a single gene or by contiguous, largely homologous

IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 131 M.E. Reid

that had been differentiated from each other. The the obsolete numbers, antigen symbols and names, current different antigens within a system are encoded by alternative ISBT numbers, and the reason why each was made obsolete. forms of the gene. The number of discrete antigens in a system As of the last meeting of the Working Party in July 2010, 18 currently ranges from 1 to more than 50.4 A six-digit number for use in computers was devised; the first three numbers Table 1. 700 series of obsolete numbers, antigen symbols and represent the system (e.g., 001 for ABO, 002 for MNS) and the names, current numbers, and reasons why they were made obsolete remaining three the specificity (e.g., 001001 for A, 001002 for Obsolete no. Symbol Name Current no.* Reason why made obsolete B, 002001 for M, 002002 for N, 002003 for S).2 700001 Wra Wright 010003 In Diego system 700004 Swa Swann 010013 In Diego system Collections 700007 Lsa Lewis 020006 In Gerbich system Antigens that did not belong in a system, because 700008 Tra Traversu 010019 In Diego system their controlling genes were not known, were gathered 700009 Wb Webb 020005 In Gerbich system into “collections.” Antigens placed on one collection had a 700010 Bpa Bishop 010010 In Diego system serologic, biochemical, or genetic connection. This category 700011 Or Oriss 002031 In MNS system was considered a temporary classification, pending conclusive 700012 Gf Griffiths … Control reagent no longer extant evidence that the members of any one collection are controlled 700013 Wu Wulfsberg 010009 In Diego system by a single gene different from those of all of the systems. This 700014 Jna Jensen 010017 In Diego system was indeed the case, and many collections have been promoted 700015 Rd Radin 013004 In Scianna system to system status. To allow for expansion of systems (beyond all 700016 Heibel … Control reagent no longer expectations), numbers for collections commenced at 201; the extant first three numbers represent the collection and the last three 700020 Ana Ahonen 020007 In Gerbich system the specificity. For example, 201001 for Ge2, 201002 for Ge3, 700022 Moa Moen 010011 In Diego system 201003 for Ge4, 202001 for Cra, and 202002 for Tca.2 700023 Hey Hey … Control reagent no longer extant 700024 Rla Rosenlund … Same as Lsa Series 700025 Ina Indian 023001 Antithetical to Inb; in Indian Two series were formed to accommodate orphan antigens system of low or high incidence. The ISBT Working Party defines 700026 Fra Froese 010020 In Diego system a low-incidence antigen as one that occurs in less than 1 700027 Rba Redelberger 010006 In Diego system percent of people in most populations studied, whereas a high- 700029 Vga Van Vugt 010013 In Diego system incidence antigen is defined as one that occurs in more than 700030 Wda Waldner 010005 In Diego system 90 percent of people in most populations studied. Again, to 700031 Dha Duch 020008 In Gerbich system allow for expansion and to adequately separate the systems, 700032 POLL Pollio … Control reagent no longer extant collections, and series, numbers for the low-incidence series 700033 Osa 002038 In MNS system started with 700 and the numbers for the high-incidence series 700034 Hga Hughes 010012 In Diego system initially commenced at 900. The first three numbers represent 700035 Tcb 021003 In Cromer system the series and the last three the specificity. As a result of the 700036 Tcc 021004 In Cromer system formation of the collections category, the 700 series was only 700037 NFLD Newfoundland 010016 In Diego system slightly affected, but the 900 series was much depleted and 700038 Hov … Same as Wu 2 was replaced by the 901 series. Both the 700 and 901 series 700041 SW1 010021 In Diego system were considered temporary classifications that might be called 700042 WES (or 021008 In Cromer system holding files. WESa) 700043 Ola Oldeide 030002 In the RhAG system The 700 Series of Low-Incidence Antigens 700046 BOW Bowyer 010015 In Diego system 700048 FPTT 004050 In Rh system In keeping with the temporary nature of this classification, 700051 ELO 010008 In Diego system 36 of the 55 antigens have been upgraded from the 700 series 700053 LOCR 004055 In Rh system 700055 WARR Warrior 010007 In Diego system to collections or systems. When an antigen is promoted, its Some antigens were upgraded to collections before finally residing in a associated 700 series number is made obsolete. Table 1 lists system.

132 IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 ISBT 700 and 901 series of antigens: a review

antigens remain in the 700 series of low-incidence antigens 3. It must be an inherited character. (Table 2).4 For each of these antigens, this table lists the specific 4. Plasma containing the antibody and RBCs expressing 700 number, its symbol, and its name. the antigen must be available so that further examples can be identified. Table 2. Antigens of low incidence currently in the 700 series The 901 Series of High-Incidence Antigens ISBT number Symbol Name 700002 By Batty Ten of the 16 antigens in the 901 series of high-incidence 700003 Chra Christiansen 700005 Bi Biles antigens have been upgraded to collections or systems. Table 700006 Bxa Box 3 lists the obsolete numbers, antigen symbols and names, 700017 Toa Torkildsen current ISBT numbers, and the reason why each was made 700018 Pta Peters obsolete. As of the last meeting of the Working Party, in July 700019 Rea Reid 2010, eight orphan antigens remain in the 901 series of high- 4 700021 Jea Jensen incidence antigens (Table 4). For each of these antigens, this 700028 Lia Livesey table lists the specific 901 number, its symbol, and its name. 700039 Milne In January 2012, based on three reports in Nature Genetics,5–7 700040 RASM Rasmussen Jra and Lan antigens were provisionally upgraded to their own 700044 JFV blood group systems. 700045 Kg Katagiri Table 3. 901 series of obsolete numbers, antigen symbols and 700047 JONES Jones names, current numbers, and reasons why they were made obsolete 700049 HJK Obsolete no. Symbol Name Current no. Reason why made obsolete 700050 HOFM 901001 Vel 212001 In Vel collection 700052 SARA Sarah 901002 Lan Langereis 033001 Formed a new system 700054 REIT 901004 Joa Joseph 014005 In Dombrock system 901005 Jra 032001 Formed a new system How Low-Incidence Antigens Are Found 901006 Oka 024001 In Ok system An antibody to a novel low-incidence antigen is revealed 901007 JMH John Milton Hagen 026001 In John Milton Hagen system when red blood cells (RBCs) expressing that antigen are 901010 Wrb 010004 In Diego system agglutinated unexpectedly by plasma in one of the following 901011 MER2 025001 In Raph system scenarios. The antibody 901013 Duclos 030001 In RhAG system 1. causes hemolytic disease of the fetus and newborn 901015 ABTI 212002 In Vel collection (HDFN) 2. causes an incompatible crossmatch Table 4. Antigens of high incidence currently in the 901 series 3. is found in serum known to contain one or more ISBT number Symbol Name antibodies to low-incidence antigens—the so-called 901003 Ata August multi-low serum 901008 Emm 4. is found as a contaminant in a blood typing 901009 AnWj Anton reagent, giving an unexpected reaction during RBC 901012 Sda Sid phenotyping. 901014 PEL 901016 MAM Criteria for Inclusion in the 700 Series The following criteria are in effect for an uncommon antigen to join the 700 series: How High-Incidence Antigens Are Found 1. The antigen must have an incidence of less than 1 An antibody to a novel high-incidence antigen is revealed percent in most populations tested. when RBCs expressing that antigen are agglutinated 2. The antigen must be distinct from antigens of the blood unexpectedly by plasma in one of the following scenarios. The group systems, the collections, and the other numbered antibody low-incidence antigens of the 700 series. 1. causes an incompatible crossmatch

IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 133 M.E. Reid

2. is found during the investigation of a transfusion Concluding Remarks reaction 3. causes a positive direct antiglobulin test on fetal RBCs In 1992, John Moulds coauthored a review with Peter or mild HDFN. Issitt in which they described the salient features of the ISBT It is worth noting that, with the exception of anti-MAM, alternative terminology to assist readers of Transfusion who antibodies to antigens remaining in the 901 series have caused might encounter the then-new terms.9 At that time 36 antigens mild HDFN requiring nothing more than phototherapy. were in the 700 series and 12 antigens in the 901 series. In 20 years, four new antigens have been added to the 700 and Criteria for Inclusion in the 901 Series three to the 901 series. As predicted, many antigens in these The following criteria are in effect for a common antigen “holding tanks” of the ISBT series have been promoted to to join the 901 series: systems or collections; the number has dropped to 18 in the 1. The antigen must have an incidence of greater than 90 700 series and 6 in the 901 series. With knowledge of genes percent in most populations tested. and our ability to test DNA, it is likely that the remaining 2. The antigen must be distinct from antigens of the blood orphans will eventually find homes. group systems, the collections, and the other numbered John Moulds had an encyclopedic memory. He would high-incidence antigens of the 901 series. warmly remember each orphan antigen-antibody pair and 3. It must be an inherited character. regale colleagues with stories about who found the first (or 4. Plasma containing the antibody and RBCs lacking the first few) case(s), when and where it was found, how it was antigen must be available so that further examples can named, the clinical scenarios, and adventures of getting blood be identified. samples from family members, sometimes in far-flung parts of the world. He was always ready with a yarn and loved to Naming an Antigen share his knowledge. Until shortly before his death, John was convinced he would write a review about orphan antigens If you are lucky enough to find a new antigen, it is for Immunohematology. Indeed, his desk was piled high important to ask the appropriate member of the Working Party with reprints ready to be read. We thought it appropriate for a provisional ISBT number. This prevents duplication of to include a review, albeit short, to appear in this issue numbers, and the inadvertent, unfortunate use of a name as dedicated to him. Landsteiner has been called the “Father of described above should be avoidable. At the next biannual ISBT Immunohematology”; I like to think that John was the father Congress, when the Working Party meets, assuming there is of orphan antigens in the ISBT 700 series and 901 series. agreement, the number is ratified. In this way, a structured, See Table 5 for information on, including characteristics standard terminology is maintained. Instructions for obtaining of, the ISBT 700 series. This information has also been an ISBT number for an antigen and contact information are provided as a removable educational insert in the centerfold provided in the latest update report of the Working Party,4 in of this issue. more detail in the last full report,8 or at the ISBT Web site at this address: http://www.isbtweb.org/working-parties/red- Note: Information on the characteristics of the antigens cell-immunogenetics-and-terminology/. and antibodies in the ISBT 700 series has been provided as a removable educational insert in the centerfold of this issue. Cynthia Flickinger/Managing Editor

134 IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 ISBT 700 and 901 series of antigens: a review

References 7. Helias V, Saison C, Ballif BA, Peyrard T, Takahashi J, Takahashi H, Tanaka M, Deybach JC, Puy H, Le Gall M, Sureau C, Pham 1. [No authors listed]. ISBT working party on terminology for BN, Le Pennec PY, Tani Y, Cartron JP, Arnaud L. ABCB6 is red cell surface antigens: preliminary report. Vox Sang 1982; dispensable for erythropoiesis and specifies the new blood 42:164–5. group system Langereis. Nature Genetics 2012; E-pub doi: 2. [No authors listed]. Blood group terminology 1990. The ISBT 10.1038/ng.1069. Working Party on Terminology for Red Cell Surface Antigens. 8. Daniels GL, Fletcher A, Garratty G, et al. Blood group Vox Sang 1990;58:152–69. terminology 2004: from the International Society of Blood 3. Lewis M. Blood group terminology. Transfusion Today 1989; Transfusion committee on terminology for red cell surface 3:6 –7. antigens. Vox Sang 2004;87:304–16. 4. Storry JR, Castilho L, Daniels G, et al. International Society of 9. Issitt PD, Moulds JJ. Blood group terminology suitable for use Blood Transfusion Working Party on red cell immunogenetics in electronic data processing equipment. Transfusion 1992; and blood group terminology: Berlin report. Vox Sanguinis 32:677–82. 2011;101:77–82. 5. Zelinski T, Coghlan G, Xiao-Qing L, Reid ME. ABCG2 null Marion E. Reid, FIBMS, PhD, DSc (Hon), Head, Laboratory of define the Jr(a–) blood group phenotype. Nature Immunochemistry, New York Blood Center, 310 East 67th Street, Genetics 2012;E-pub doi:10.1038/ng.1075. New York, NY 10065. 6. Saison C, Helias V, Ballif BA, Peyrard T, Puy H, Miyazaki T, Perrot S, Vayssier-Taussat M, Waldner M, Le Pennec PY, Cartron JP, Arnaud L. Null alleles of ABCG2 encoding the breast cancer resistance protein define the new blood group system Junior. Nature Genetics 2012; E-pub doi:10.1038/ ng.1075.

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IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 135 R e v i e w The LW blood group system: a review

M.K. Grandstaff Moulds

The LW blood group system is unique in that it is the only Landsteiner and Wiener5 reported in 1940 on studies one named after the investigators (Landsteiner and Wiener) of immunizing rabbits and guinea pigs with RBCs of rhesus who first reported the antibody rather than the antibody monkeys which resulted in the production of an antibody producer, as is common in system naming. This blood group that reacted with 85 percent of Caucasian blood samples. The is also fascinating because of its association with the Rh blood antigen recognized was named Rhesus. group system, in particular D, which in the early years was Levine and Stetson6 investigated a case of erythroblastosis not clearly explained by serologic studies. Biochemical and fetalis a year earlier (1939) in which the maternal antibody molecular methods have led to a better understanding of reacted with about 80 percent of ABO-compatible individuals. the interdependence of LW and D. They are both part of the They believed a new system was found, but no name was given. Rh macromolecule complex, but LW (ICAM-4) is a member The animal and human antibodies appeared to be the of the intercellular adhesion molecule (ICAM) family. LW is same. Fisk and Foord7 were the first, in 1942, to demonstrate rarely clinically important in transfusion and pregnancy, but it that all cord cells were Rhesus positive, not 85 percent. At appears to play a role in sickle cell disease. that time some thought that the animal reagents were not My interest in LW began when I was employed, in the as reliable as the human ones. The Rhesus blood group was early 1970s, at the War Memorial Blood Bank (now Memorial defined by antibodies, and the Rh factor was termed D when Blood Center) in Minneapolis, Minnesota. Jane Swanson in Fisher devised the CDE nomenclature.8 Murray and Clark9 the reference laboratory investigated guinea pig anti-LW, some reported that both D– (Rh-negative) and D+ (Rh-positive) of the original alloanti-LW, and the first example of alloanti- RBCs stimulated apparent “anti-D-like” reactivity in guinea LWab. She also reported on some of the findings with adult pigs. This was confirmed by Levine et al.,10 who showed that and cord red blood cells (RBCs). Later on I moved to Houston adult D– RBCs did not react with D-like antibodies, but they in 1975, and at the Consultation and Education Services at would adsorb the anti-D-like activity to exhaustion. This Gamma Biologicals Inc. we were referred patient samples that was conclusive proof that the animal and human antibodies involved anti-LW. These included examples of suppression defined two different specificities. Levine11 in 1967 renamed of LW with subsequent production of anti-LW and warm the anti-D-like factor LW in honor of Landsteiner and Wiener. reactive autoantibodies with LW or Rh specificities. This There is a very interesting report on the “LW factor” by Wiener current review will summarize the early studies (primarily et al.12 that details the events leading up to the naming of the serologic) and give an update on recent biochemical and LW factor in humans and the recognition that it was not the molecular developments. same as the Rh (rhesus) factor.

History LW Antigens and Antibodies

There are several reviews leading up to 1992 that The first two human antibodies to the D-like or LW antigen summarize early discoveries of LW and D, which are were initially investigated in 1955 but were not reported by interrelated. Malcolm Beck1 prepared one in 1973 for an Tippett and Sanger until 1962.13 Two D+ individuals (G and American Association of Blood Banks preconvention seminar B) produced an apparent weak anti-D, not reactive by the and detailed the facts known on the subject to that time. antiglobulin test. The antibodies were mutually compatible Stillwell2 in 1979, Giles3 in 1980, and Storry4 in 1992 followed with each other’s D+ RBCs and could be adsorbed by D– RBCs. with summaries, primarily of the serologic aspects of LW The first example of the Rhnull phenotype was reported antigens and antibodies, which did include some enzyme and by Levine et al.14 in an Australian aborigine. The RBCs lacked chemical studies. However, the true nature of LW blood group all Rh antigens and were also not reactive with guinea pig antigens within the RBC membrane or their integral function anti-LW and the two human D-like antibodies of G and B. It was (if any) in RBC immunology was still not known. obvious that LW and D were two different antigens but were

136 IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 LW blood group system: a review

certainly linked. Swanson and Matson15 reported in 1964 on that the Nea antigen was stronger on D+ RBCs than on D– the third example of anti-LW (Mrs. VW). Her antibody reacted RBCs, but cord RBCs of either D type reacted equally well. strongly with D+ RBCs and only weakly with D– RBCs. Her Sistonen and Tippett demonstrated that eight LW3 individuals own RBCs and the RBCs of a D+ sister were compatible with in eight families as well as other family members were Ne(a+). the antibody. VW’s RBCs were nonreactive with the first two The RBCs of LW4 Big and her brother were Ne(a–). examples of human anti-LW and Levine’s guinea pig anti-LW. Sistonen and Tippett21 suggested that Nea be designated 16 b Levine in 1968 was one of the first to suggest anti-LW as LW and the high-incidence antigen of the RBCs of LW3 a 24 was composed of fractions, anti-LW1 and anti-LW2, analogous individuals as LW . Sistonen and associates performed family to anti-A and anti-A1. LW1 was the LW antigen on D+ RBCs studies and tested Finnish LW– blood donors and confirmed a b and LW2 on D– RBCs. the allelic relationship of LW and LW . They also showed that Two anti-LWs added further complexity to the LW story. the LW blood group system was independent of ABO, MNS, A very potent example of anti-LW in the serum of a woman P1, Kell, Secretor, Kidd, and Colton blood group systems. Table (Big), who had delivered her third child, was reported in 1971 1 lists the former and current LW phenotypes and genotyes.21,25 by deVeber et al.17 The antibody had a titer of 32,000 against Sistonen26 in 1984 studied the Finnish and other D+ RBCs and 400 against D– RBCs in the antiglobulin test. European populations and published the frequencies of LWa The RBCs of one brother were compatible, and eluates from and LWb. In a more recent study, Sistonen et al.27 in 1999 VW and Big were mutually compatible. Table 1. LW blood group phenotypes and The next anti-LW was investigated by Swanson et al.18 Phenotype in an inbred family (Wald) that had three LW– individuals. Blood Frequencies group Formerly Present Formerly Present in Finland, % The antibodies of G and B were compatible with the RBCs a a LW+ LW1 if D+ LW(a+b–) LWLW or LW LW or 93.9 of Wald, but the anti-LW of Big was incompatible. These LWlw LWaLW

a b workers proposed the notation of LW1 representing normal LW2 if D– LW(a+b+) LW LW 6.0 b b LW+ RBCs, LW2 the weaker LW antigen of VW and the Wald LW– LW3 LW(a–b+) lwlw LW LW 0.1 family, and LW3 the Big RBCs, and only Rhnull RBCs were LW4 LW(a–b–) LWLW very rare LW–. Vos et al.19 from their experiments in 1973 had proposed b something similar: LW1, D+ RBCs; LW2, D–; and LW3, Big. studied the Baltic population for the frequency of LW . The b They suggested variations of LW1, LW2, LW3, and LW4. Beck’s gene for LW was high in the Balts, around 6 percent among review in 1973 took the two suggestions and came up with Latvians and Lithuanians, very low among other western these combined notations: Europeans (0–0.1%), and apparently absent in Asians

D+ = LW1 Big type = LW4 and Africans. There was a steady decline in neighboring

D– = LW2 Rhnull = lw populations: 4.0 percent in Estonians, 2.9 percent in Finns,

Wald and VW type = LW3 2.2 percent in Vologda Russians, and 2.0 percent in Poles. White et al.20 reported in 1975 an elegant study of a white Along with these data and their other population studies, this b woman (SC) who produced alloanti-LW3 after receiving 5 group concluded that LW can be considered a “Baltic tribal units of blood. She had one compatible sibling. Her RBCs and marker,” and its presence in other populations is an indicator those of her compatible brother were tested with various anti- of the degree of Baltic genetic influence.

LWs. Their RBCs were not reactive with the human anti-LW3 One last antibody produced by the proband (AK) of VW and Wald and guinea pig anti-LW but were reactive important to the LW story is the only other known example ab 28 with the anti-LW4 of Big. From their extensive testing they of alloanti-LW , reported by Poole et al. in 1996. The man’s cautioned that if random donors were typed for LW using anti- RBCs were LW(a–b–) with anti-LWa, -LWb, -LWab, and mouse ab ab LW4, those that were LW3 would have been classified as LW+. monoclonal anti-LW and did not adsorb or elute anti-LW . These various notations proposed by the three groups are His sister’s RBCs were also LW(a–b–) and did not adsorb or important to those who might still have some of these early elute AK’s anti-LWab. antibodies in their collections of LW antisera. The designations would change when Sistonen and Tippett in 198221 showed that Nomenclature a new low-incidence antigen (Nea), reported by Sistonen et al.22 in 1981, was associated with LW. The latter report had shown Landsteiner-Wiener (LW) is the 16th blood group system that Nea was independent of Rh. Sistonen had also reported23 recognized by the International Society of Blood Transfusion

IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 137 M.K.G. Moulds

(ISBT) Committee on Terminology for Red Cell Antigens.29,30 LW Human and Monoclonal Antibodies The antigen numbers are 016005 (LWa), 016006 (LWab), and 016007 (LWb). LWab was described before LWb and thus was Alloanti-LWa is found in the sera of individuals whose given the antigen number 06 and LWb 07. The numbers LW1 RBCs are genetically LW(a–b+). Most examples were probably through LW4 became obsolete and were not used so as to stimulated by transfusion, one by pregnancy,3 and another by avoid confusion with the old designations. a male donor who had been immunized to produce anti-D.35 Alloanti-LWab has only been found in the sera of two LW Antigen Characteristics individuals who were genetically LW(a–b–) and had compatible siblings. Monoclonal anti-LWab has been described Early publications had reported that the LW antigens were by several investigators.36–38 Three were IgG1 and one was unaffected by treatment of RBCs with the proteases papain, IgM. Anti-LWb individuals have been found in Finland22,26 and ficin, trypsin, or chymotrypsin. Konigshaus and Holland31 in one in Canada.39 1984 found that an anti-LW did not react with ZZAP-treated RBCs. Branch and Petz32 had described in 1982 this reagent Transient Suppression of LW Antigens and that was composed of papain and dithiothreitol (DTT). Production of Anti-LW They had shown that ABO, Rh, and Kidd antigens were not destroyed, but Duffy, MNSs, and all Kell antigens tested were The first example of anti-LW produced by a pregnant affected. Because papain treatment of RBCs did not affect D– woman, whose RBCs initially typed LW– but 12 months LW antigens or even enhance them, Konigshaus and Holland after delivery typed LW+, was reported in 1967 by Giles tested RBCs treated with DTT to assess its effect on LW. They and Lundsgaard.40 Anti-D had been detected in her serum 3 demonstrated that several examples of anti-LWa, -LWab, and weeks before delivery, but she had no previous pregnancies or -LWb did not react with DTT-treated RBCs. transfusions. Anti-C and anti-LW were detected at delivery, in Lomas and Tippett reported in 198533 that the enzyme addition to the anti-D. pronase destroyed the LW antigen, unlike other proteolytic Three cases were described by Chown et al41: two were enzymes. They published the reactions obtained with anti- with pregnant women and one was attributable to transfusion. LW against the following enzyme-treated RBCs: papain, The women in the pregnancy-associated cases also were D– ficin, trypsin, chymotrypsin, and pronase. They studied and produced potent anti-D simultaneous to the production two examples of anti-LWa, two of anti-LWab, three of murine of anti-LW. These workers proposed that the antibodies monoclonal anti-LWab, and five of anti-D. D is not destroyed by were not anti-self like those seen in autoimmune hemolytic pronase—a useful tool for distinguishing anti-LW from anti-D. anemia (AIHA), but rather the individuals had a temporary They also showed that two examples of anti-LWb also did not LW– phenotype with production of anti-LW. As the titer of the react with pronase-treated LW(a–b+) RBCs. See Table 2 for antibody diminished, the antigen would reappear. The first these results. case of suppression of LW antigens and production of anti- Daniels34 reported in this journal in 1992 on destruction LW in Hodgkin’s disease was reported in 1977 by Perkins et or reduced LWa and LWab activity on RBCs by two sulfhydryl al.42 The patient’s RBCs typed LW– with all known examples reducing agents, DTT and 2-aminoethylisothioronium of anti-LW, and his serum contained anti-LW. The patient bromide, showing a requirement for intact disulfide bonds. donated an autologous unit of blood drawn 5 months later that was not typed for LW. He donated 3 months after that, and Table 2. Reactions of LW and D antibodies against enzyme- treated RBCs this unit typed LW+ and there was no antibody in the serum. D+ LW(a+b–) cells Treated with Anti-LWa Anti-LWab Anti-D AB serum Autoantibodies Papain ++++ ++++ ++++ 0 Ficin ++++ ++++ ++++ 0 LW autoantibodies are produced in the sera of individuals Trypsin ++++ ++++ ++++ 0 whose RBCs type LW+. Celano and Levine43 reported in 1967 Chymotrypsin ++++ ++++ ++++ 0 on autoanti-LW identified in direct testing or by adsorptions in Pronase 0 0 ++++ 0 six cases of AIHA. Five of the six cases also contained other Rh- Buffer ++++ ++++ ++++ 0 related autoantibodies. Vos et al. in 197319 described patients

RBCs = red blood cells; ++++ = strong positive reactions; 0 = negative. with AIHA and autoantibodies to LW and concurrent to Rh.

138 IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 LW blood group system: a review

Swanson et al.44 reported in 1990 an unusual case of donor- antibody-dependent cellular cytotoxicity and mononuclear derived antibodies to LW, Rh, and M found in a bone-marrow phagocyte assays did not indicate the antibody was hemolytic. transplant (BMT) chimera. Perrault45 screened the sera of The studies with technetium 99m (99mTc)-labeled LW(a+) RBCs 45,000 patient and donor samples, using an autoanalyzer, and showed a slight reduction in half-life (18 hour) compared with found ten examples of cold-reactive autoanti-LW reactive by a normal survival of autologous cells. The authors concluded that low-ionic strength Polybrene method. even though the in vivo study indicated moderate reduction in cell survival, they felt that in an emergency, transfusion of Rh– Clinical Significance LW+ blood could be given. Villalba et al.51 studied the anti-LWab of a female patient One of the original alloanti-LW was produced by a admitted for surgical removal of a breast mass. She had multiparous woman (Mrs. G), who had 13 pregnancies and never been transfused and had a history of five uneventful did not make anti-LW until she was transfused. Mrs. VW, who pregnancies. A monocyte monolayer assay showed a 54.9 a produced alloanti-LW3 (LW ), had eight children and none percent reactivity of monocytes for D+ LW+ RBCs and 23.2 showed evidence of hemolytic disease of the newborn. An percent for D– LW+ RBCs. Only results greater than 3 percent example of alloanti-LWab described in an LW– woman (Big) are associated with accelerated destruction of transfused by deVeber et al.17 was very potent, but her third baby had only incompatible RBCs. The authors concluded that anti-LW a weakly positive direct antiglobulin test (DAT) and minimal should not be considered clinically insignificant for transfusion evidence of active hemolytic disease. before in vitro or in vivo survival studies are performed. Little is published about the clinical significance of Several examples of anti-Nea (LWb) have been identified alloanti-LW in a genetically LW– individual in regard to in patients who had received at least 50 transfusions with no transfusion. Information is primarily on transfusion of LW+ history of transfusion reactions.22 These workers reported blood to patients with production of an apparent alloanti-LW on a patient with Nea who was given 2 mL of an intravenous when their RBCs are transiently LW(a–) or LW(a–b–). injection of freshly labeled Ne(a+) RBCs. The estimated half- Tregellas et al.46 in 1977 reported on a 14-year-old male life for the RBCs was approximately 2.5 hours. They concluded with anti-LW in his serum who was transfused with 50 units that this particular anti-Nea was capable of causing rather of “least incompatible” D–, LW+ blood without any untoward rapid RBC destruction. effects. The DAT was weakly positive during and after transfusion, but the RBCs typed LW– with two potent anti- LW Glycoprotein and the Gene That Encodes It LW. One year after the transfusion, the patient’s RBCs typed LW+. Moore in 198352 determined that the LW antigens reside Cummings et al.47 in 1984 reported on a patient who on an RBC membrane glycoprotein of about 40,000 Da, and had produced an apparent alloanti-LW. Chromium 51 (51Cr) Mallison et al.53 confirmed this in 1986 with immunoblotting survival studies and mononuclear phagocyte assays indicated studies using two murine monoclonal antibodies: B546 and low likelihood of significant RBC destruction. The patient B556. Bloy et al.54 in 1989, using these monoclonal antibodies, received 51Cr-labeled D– RBCs that had normal survival. Two showed that another protein was precipitated. Its molecular other reports48,49 also indicated that LW antibodies appeared weight of 31,000 Da corresponded to the molecular weight of to be clinically unimportant. the D antigen protein. They suggested that the LW component In sharp contrast, Herron et al.50 reported in 1986 on was linked to the Rh proteins in the RBC membrane in a an IgG3 anti-LWab in an 85-year-old man, with a diagnosis functional complex called the “Rh cluster.” Bloy et al.55 in 1990 of myelodysplastic anemia, that had the potential to be demonstrated that expression of LWa was inhibited by EDTA, hemolytic. The 53 percent 1-hour posttransfusion survival of and inhibition was reversible by addition of magnesium, radiolabelled LW+ RBCs fell short of the 70 to 85 percent range suggesting that it plays some role in RBC membrane function. as a minimum requirement for transfusion of incompatible In addition, Bloy et al.56 showed that LW, but not D, carries blood. N-linked carbohydrate chains. The LW map was different Napier and Rowe35 reported in 1987 on alloanti-LWa from the D map after deglycosylation, which confirmed that produced by a 30-year-old male volunteer from Latvia the D and LW polypeptides are different proteins. These whose RBCs typed LW(a–b+). His antibody was of the reports were among some of those published at the time of the potentially hemolytic class IgG1, although evidence from the review by Storry in this journal in 1992.4

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57 Bailly et al. reported in 1994 that the LW protein for αv(CD50) integrins on nonhemopoietic cells. Antibodies to had sequence similarities (approximate 30% identity) with LW gp particularly inhibit adhesion of unfractionated sickle ICAM -1, -2, and -3 that are counterreceptors to lymphocyte RBCs to tumor necrosis factor α-activated human umbilical function-associated antigen (LFA-1). Bailly et al.58 also showed vein endothelial cells. LW gp is upregulated on sickle RBCs like the RBC LW blood group glycoprotein is an ICAM that binds Lu gp. It is suggestive that LW gp also may contribute to the to leukocyte-specific integrins CD11a/CD18, CD11b/CD18, abnormal adhesion of sickle RBCs to endothelium. Elevation of and CD11c/CD18, which are involved in leukocyte adhesion. Lu and LW on sickle RBCs suggests all cells in the circulation Hermand et al.59 reported on their studies of the LW blood of sickle patients have the potential to participate in adhesive group locus and summarized the work of others. Landsteiner- interactions and not only the subpopulations of reticulocytes Wiener (LW) blood group antigens reside on a 42-kDa that were previously identified as being most adhesive under erythrocyte membrane glycoprotein. The LW locus has been flow conditions. Spring et al.63 continued their studies of LW assigned to chromosome 19p13.3 by in situ hybridization. gp (ICAM-4); in 2001 they showed that ICAM-4 is the first Rh proteins are not the biochemical precursor of LW. The ICAM family member shown to be a ligand for integrins other Gln70Arg substitution is responsible for the blood group than those of the β2 family, and data suggested that ICAM- LWa/LWb polymorphism. The LW gene is not rearranged in 4 has a novel integrin-binding site(s). These findings suggest 60 LW(a–b–) and Rhnull cells. Hermand et al. in 1996 reported a role for ICAM-4 in normal erythropoiesis and may also be that a deletion of 10 bp in exon 1 of the LW gene was identified relevant to the adhesive interactions of sickle cells. in the genome of an LW(a–b–) individual (Big) deficient for Delahunty et al. in 200664 reviewed the role of the LW blood LW antigens but carrying a normal Rh phenotype. The 10- group antigen glycoprotein, part of the Rh macromolecular bp deletion generates a premature stop codon and encodes a complex, as a member of the ICAM family. They noted that truncated protein without transmembrane and cytoplasmic “LW is likely to contribute to RBC adhesion in a variety domains. They suggested that this truncated protein most of settings, including hematopoiesis, as well as vascular likely is either secreted or rapidly degraded, thus explaining disorders. The best documentation of a pathophysiological the absence of the LW protein in the RBC membrane. In role for LW in human disease is in sickle cell disease, where their summary they said that major questions concerning it contributes to RBC adhesion to endothelial cells and the the modulation of LW expression and biosynthesis of the Rh development of vaso-occlusion, the hallmark of that disease. membrane complex remain unresolved. LW may also contribute to other intravascular processes, such as both venous and arterial thrombosis, because of its ability LW and Disease to interact with both activated as well as leukocytes. The evidence that LW itself can undergo activation on RBCs Parsons et al.61 in 1999 reported on the Lutheran holds promise that pharmacotherapeutic maneuvers may glycoprotein (Lu gp) and LW glycoprotein (LW gp), both be found to prevent such pathophysiological interactions. members of the immunoglobulin superfamily (IgSF). Levels of Understanding how LW is activated and how to prevent such Lu gp and LW gp expression on sickle RBCs are greater than activation may well contribute to better therapeutic modalities normal RBCs, and sickle RBCs adhere to alpha 5-containing for these disorders in the future.” This review by these authors laminins. Their data suggested that Lu and LW molecules elegantly summarizes what was known in 2006 about LW may contribute to the vasoocclusive events associated with biochemistry, LW function, and LW as an activatable receptor episodes of acute pain in sickle cell disease. with a pathophysiologic role. Spring and Parsons62 in 2000 reviewed the structure, Toivanen et al. in 200865 reviewed molecular studies on tissue distribution, and ligand-binding properties of the major blood groups, with particular focus on LW (ICAM-4). different cell adhesion molecules (CAMs) expressed on A key finding in their experiments was that phagocytosis of erythroid cells and their function in erythropoiesis. The 45- senescent RBCs is in part ICAM-4/β2-integrin dependent. kDa LW gp belongs to the ICAM family and is designated And recently, Mohandas and Chasis in 201066 summarized ICAM-4. Each ICAM family has a characteristic tissue several of their previous reports on some of the findings on distribution. LW gp has the most restrictive distribution and molecular processes occurring within erythroblast islands. is expressed only on erythroid cells and weakly expressed on They found a receptor-counterreceptor interaction between . LW gp is a ligand for the β1 family integrin, VLA- erythroid ICAM-4 (blood group antigen LW) and macrophage

4 (α4β1, CD 49d/CD29) on hemopoietic cells and is a ligand αv integrin that appears to maintain erythroid island integrity.

140 IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 LW blood group system: a review

A 70 percent decrease in islands is seen when ICAM-4/αv 11. Levine P, Celano MJ. Agglutinating specificity for LW factor binding is blocked with αv synthetic peptides. The investigators in guinea pig and rabbit anti-Rh serum. Science 1967;156: 1744–6. studied whether ICAM-4/αv binding plays a role in stress 12. Wiener AS, Moor-Jankowski J, Brancato GJ. LW factor. erythropoiesis. They found that the reticulocyte response is Haematologia 1969;3:385–93. different in ICAM-4 null mice compared with control, wild- 13. Tippett PA, Sanger R. Observations on subdivisions of the Rh type mice, using erythropoietin as a stress inducer. They antigen D. Vox Sang 1962;7:9–13. speculated that this may be a reflection of the baseline decrease 14. Levine P, Celano MJ, Vos GH, Morrison J. The first human blood ___/___, which lacks the ‘D-like’ antigen. Nature in island number in the ICAM-4 null mice. 1962;194:304–5. 15. Swanson J, Matson GA. Third example of a human ‘D-like’ Summary antibody or anti-LW. Transfusion 1964;4:257–61. 16. Levine P. Rh and LW factors. Int Convoc on Immunol. Buffalo, NY;1969:140-3. LW and Rh were discovered at the same time, LW is 17. deVeber LL, Clark GW, Hunking M, Stroup M. Maternal anti- stronger on D+ RBCs than on D– RBCs, and LW is absent LW. Transfusion 1971;11:33–5. on Rhnull RBCs that lack all Rh antigens. Both LW and Rh are 18. Swanson JL, Azar M, Miller J, McCullough JJ. Evidence for part of the IgSF but are two distinct proteins and arise from heterogeneity of LW revealed in a family study. Transfusion 1974;14:470–4. unrelated genes on separate chromosomes. LW blood group 19. Vos GH, Petz LD, Garratty G, Fudenberg HH. Autoantibodies protein (or ICAM-4) is a member of the IgSF subfamily known in acquired with special reference to the LW as ICAMs. system. Blood 1973;42:445–53. The role of LW (ICAM-4) in RBC adhesion contributing to 20. White JC, Rolih S, Wilkinson SL, Hatcher BJ, Issitt PD. A new vasoocclusion in sickle cell disease and other vascular events example of anti-LW and further studies on the heterogeneity of the system. Transfusion 1975;15:368–72. is currently under investigation. Hopefully, in the future our 21. Sistonen P, Tippett P. A ‘new’ giving further insight into understanding of LW activation could lead to better therapy the LW blood group system. Vox Sang 1982;42:252–5. for these disorders. 22. Sistonen P, Nevanlinna HR, Virtaranta-Knowles K, et al. Nea, a new blood group antigen in Finland. Vox Sang 1981;40: 352–7. References 23. Sistonen P. A phenotypic association between the blood group a 1. Beck ML. The LW system: a review and current concepts. In: antigen Ne and the Rh antigen D. Med Biol 1981;59:230–5. Walker RH, Block UT, eds. A seminar on recent advances in 24. Sistonen P, Green CA, Lomas CG, Tippett P. Genetic immunohematology. Washington, DC: American Association polymorphism of the LW blood group system. Ann Hum Genet of Blood Banks, 1973:83–100. 1983;47:277–84. 2. Stillwell GF. The LW blood group system. Canadian Society of 25. Daniels G. Human blood groups. 2nd ed. Oxford, UK: Blackwell Laboratory Technologists (CSLT) Bulletin 1979;1:17–25. Science, 2002:405. 3. Giles CM. The LW blood group: a review. Immunol Commun 26. Sistonen P. The LW (Landsteiner-Wiener) blood group system: 1980;9:225–42. elucidation of the genetics of the LW blood groups based on the 4. Storry JR. Review: the LW blood group system. finding of a ‘new’ blood group antigen. PhD thesis, University Immunohematology 1992;8:87–93. of Helsinki, 1984. 5. Landsteiner K, Wiener AS. An agglutinable factor in human 27. Sistonen P, Virtaranta-Knowles K, Denisova R, Kucinskas V, blood recognized by immune sera for rhesus blood. Proc Soc Ambrasiene D, Beckman L. The LWb blood group as a marker Exp Biol Med 1940;43:223–4. of prehistoric Baltic migrations and admixture. Hum Hered 1999;49:154–8. 6. Levine P, Stetson RE. An unusual case of intra-group . JAMA 1939;113:126–7. 28. Poole J, Ford D, Tozer R, Banks J, Mallinson G, Marosszeky S. A case of LW(a–b–) in Papua New Guinea. 24th Cong Int Soc 7. Fisk RT, Foord AG. Observations on the Rh agglutinogen of Blood Transfusion 1996:144. human blood. Am J Clin Pathol 1942;12:545–52. 29. [No authors listed]. Blood Group Terminology 1990. The ISBT 8. Fisher RA. Cited by Race RR. An ‘incomplete’ antibody in Working Party on Terminology for Red Cell Surface Antigens. human serum. Nature 1944;153:771–2. Vox Sang 1990;58:152–69. 9. Murray J, Clark EC. Production of anti-Rh in guinea pigs from 30. Reid ME, Lomas-Francis C. Blood group antigens and human erythrocyte extracts. Nature 1952;169:886–7. antibodies: A guide to clinical relevance and technical tips. 10. Levine P, Celano M, Fenichel R, Pollack W, Singher HA. A New York, NY: Starbright Books, 2007:6. “D-like” antigen in rhesus monkey, human Rh positive and 31. Konigshaus GJ, Holland TI. The effect of dithiothreitol on the human Rh negative red blood cells. J Immunol 1961;87: LW antigen. Transfusion 1984;24:536–7. 747–52. 32. Branch DR, Petz LD. A new reagent (ZZAP) having multiple applications in immunohematology. Am J Clin Pathol 1982; 78:161–7.

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33. Lomas CG, Tippett P. Use of enzymes in distinguishing anti- 53. Mallinson G, Martin PG, Anstee DJ, et al. Identification and LWa and anti-LWab from anti-D. Med Lab Sci 1985;42:88–9. partial characterization of the human erythrocyte membrane 34. Daniels G. Effect of enzymes on and chemical modifications component(s) that express the antigens of the LW blood-group of high-frequency red cell antigens. Immunohematology 1992; system. Biochem J 1986;234:649–52. 8:53–7. 54. Bloy C, Blanchard D, Hermand P, Kordowicz M, Sonneborn 35. Napier JAF, Rowe GP. Transfusion significance of LWa allo- HH, Cartron JP. Properties of the blood group LW glycoprotein antibodies. Vox Sang 1987;53:228–30. and preliminary comparison with Rh proteins. Mol Immunol 36. Oliveira OLP, Thomas DB, Lomas CG, Tippett P. Restricted 1989;26:1013–19. expression of LW antigen on subsets of human B and T 55. Bloy C, Hermand P, Blanchard D, Cherif-Zahar B, Goossens D, lymphocytes. J Immunogenet 1984;11:297–303. Cartron JP. Surface orientation and antigen properties of Rh 37. Sonneborn H-H, Ulhemann H, Tills D, Lomas CG, Shaw MA, and LW polypeptides of the human erythrocyte membrane. J Tippett P. Monoclonal anti-LWab. Biotest Bull 1984;2:145–8. Biol Chem 1990;265:21482–7. 38. Sonneborn H-H, Ernst M, Voak D. A new monoclonal anti-LW 56. Bloy C, Hermand P, Dherif-zahar B, et al. Comparative analysis (BS87) (abstract). Vox Sang 1984;67(Suppl 2);114. by two-dimensional iodopeptide mapping of the RhD protein and LW glycoprotein. Blood 1990;75:11;2245–9. 39. Anderson C, Green CA. Unpublished observations 1982. In: Daniels G, ed. Human blood groups. Oxford, UK: Blackwell 57. Bailly P, Hermand P, Callebaut I, et al. The LW blood group Science, 1995:520. glycoprotein is homologous to intercellular adhesion molecules. Proc Natl Acad Sci U S A 1994;91:5306–10. 40. Giles CM, Lundsgaard A. A complex serological investigation involving LW. Vox Sang 1967;13:406–16. 58. Bailly P, Tontti E, Hermand P, Cartron JP, Gahmberg CG. The red cell LW blood group protein is an intercellular adhesion 41. Chown B, Kaita H, Lowen B, Lewis M. Transient production of molecule which binds to CD11/CD18 leukocyte integrins. Eur anti-LW by LW-positive people. Transfusion 1971;11:220–2. J Immunol 1995;25:3316–20. 42. Perkins HA, McIlroy M, Swanson J, Kadin M. Transient 59. Hermand P, Gane P, Mattei MG, Sistonen P, Cartron JP, Bailly LW-negative red blood cells and anti-LW in a patient with P. Molecular basis and expression of the LWa/LWb blood group Hodgkin’s disease. Vox Sang 1977;33:299–303. polymorphism. Blood 1995;86:1590–4. 43. Celano MJ, Levine P. Anti-LW specificity in autoimmune 60. Hermand P, LePennec PY, Rouger P, Cartron JP, Bailey P. acquired hemolytic anemia. Transfusion 1967;4:265–8. Characterization of the gene encoding the human LW blood 44. Swanson J, Scofield T, Krivit W, Ramsey N, Kersey J, Whitley group protein in LW+ and LW– phenotypes. Blood 1996:7: C. Donor-derived LW, Rh and M antibodies in a post BMT 2962–7. chimera (abstract). Joint ISBT/AABB Conference, Los Angeles, 61. Parsons SF, Spring FA, Chasis JA, Anstee DJ. Erythroid cell CA: Nov. 10-15, 1990. adhesion molecules Lutheran and LW in health and disease. 45. Perrault R. ‘Cold’ IgG autologous anti-LW. An immunological Baillieries Best Pract Res Clin Haematol 1999;12:729–45. comparison with immune anti-LW. Vox Sang 1973;24:150–64. 62. Spring FA, Parsons SF. Erythroid cell adhesion molecules. 46. Tregellas WM, Moulds JJ, South SF. Successful transfusion Transfus Med Rev 2000;14:351–63. of a patient with anti-LW with LW-positive blood (abstract). 63. Spring FA, Parsons SF, Ortlepp S, et al. Intercellular adhesion Transfusion 1977;18:384. molecule-4 binds (alpha)4(beta)1 and (alpha)V-family 47. Cummings E, Pisciotto P, Roth G. Normal survival of Rho (D) integrins through novel integrin-binding mechanisms. Blood negative, LW(a+) red cells in a patient with allo-anti-LWa. Vox 2001;98:457–66. Sang 1984;46:286–90. 64. Delahunty M, Zennadi R, Telen MJ. LW protein: a promiscuous 48. Chaplin H, Hunter VL, Rasche ME, Shirey RS. Long-term in integrin receptor activated by adrenergic signaling. Transfus vivo survival of Rh(D)-negative donor red cells in a patient Clin Biol 2006;13:44–9. with anti-LW. Transfusion 1985;25:39–43. 65. Toivanen A, Ihanus E, Mattila M, Lutz HU, Gahmberg CG. 49. Pope M, Sullivan C, Kline WE, Bowman RJ. Investigation of Importance of molecular studies on major blood groups— Rho(D) negative donor RBC survival in a patient with allo-anti- intercellular adhesion molecule-4, a blood group antigen LWab. (abstract) Transfusion 1986;26:547. involved in multiple cellular interactions. Biochim Biophys 50. Herron R, Bell A, Poole J, Clark M, Smith DS, Hamblin TJ. Acta 2008;1780:456–66. Reduced survival of isotyope-labelled Rh(D)-negative donor 66. Mohandas N, Chasis JA. The erythroid niche: molecular red cells in a patient with anti-LWab. Vox Sang 1986;51: processes occurring within erythroblastic islands. Transfus 314 –17. Clin Biol 2010;17:110–11. 51. Villalba R, Ceballos P, Fornés G, Eisman M, Gómez Villagrán JL. Clinically significant anti-LWab by monocyte monolayer assay. Vox Sang 1995;68:66–7. Marilyn K. Grandstaff Moulds, BA, MT(ASCP)SBB, Immunohematology Specialist, John Moulds Reference Laboratory, 52. Moore S. Identification of red cell membrane components associated with rhesus blood group antigen expression. In: 8910 Linwood Avenue, Shreveport LA 71106; Correspondence to SJ Cartron J-P, Rouger C, Salmon C, eds. Red cell glycoconjugates 47 Lake Cherokee, Henderson, TX 75652. and related genetic markers. Paris: Librairie Arnetter; 1983: 97–106.

142 IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 O r i g i n a l R e p o r t Occurrence of antibodies to low-incidence antigens among a cohort of multiply transfused patients with sickle cell disease

P. Jackson

Data from an immunohematology reference laboratory were transfusing RBCs that are at least partially phenotypically compiled retrospectively to determine the occurrence of the matched to their patients with SCD. Afenyi-Annan et al.4 formation of alloantibodies to low-incidence antigens associated found that the majority (78%) of NIH Comprehensive Sickle with the African American population (AA-LIAs) among patients with sickle cell disease (SCD). The AA-LIAs under study were V, Cell Centers match such patients. Although the rate is lower VS, Jsa, and Goa. The records from 137 recurrently transfused when considering all blood centers, results from a study by patients with SCD were selected on the basis of transfusion Osby and Shulman5 of 1182 North American laboratories activity from the 2009 calendar year. We found that 13 patients (9.49%) exhibited one or more antibodies to AA-LIAs, and a total showed that 27.9 percent of blood banks transfuse RBC units of 17 antibodies to these AA-LIAs have been developed by these phenotypically matched beyond ABO and D. Both scenarios patients. The occurrence of antibody formation to AA-LIAs is of transfusing phenotypically matched RBCs (whether before significantly greater than that of alloantibodies to low-incidence or after antigen exposure and subsequent alloimmunization) antigens in the general population. Considering the possibility of the presence of antibodies to AA-LIAs in multiply transfused expose these patients to a greater number of African American patients with SCD is warranted. Immunohematology donors as a result of transfusion therapy over the course of 2011;27:143–145. their disease. Consequently, exposure to the low-incidence antigens V, VS, Jsa, and Goa is more likely. Key Words: sickle cell disease, low-incidence antigens, A secondary goal of this study was to determine the rate of alloantibodies alloimmunization against low-incidence antigens commonly associated with the African American population (AA-LIA)— The purpose of this study is to describe the occurrence namely V, VS, Jsa, and Goa—among recurrently transfused of the formation of alloantibodies to low-incidence antigens patients with SCD. The risk of forming antibodies to low- among a group of patients with sickle cell disease (SCD) who incidence antigens normally is low, ranging between 0.3 and are transfused recurrently. Of concern are the antigens V, VS, 10 percent.3 A large study conducted by Schonewille et al.,6 Jsa, and Goa. The occurrence of these antigens is 30 percent, using transfusion and antibody identification records collected 26 to 40 percent, 20 percent, and 2 percent, respectively, over a period of 23 years, found a prevalence of antibodies within the African American population.1 Consequently, to low-incidence antigens of 5 percent. Data collected for when compared with the general red cell donor pool, which the present study were analyzed to determine whether the is overwhelmingly Caucasian,2 these antigens are encountered rate of formation of alloantibodies to AA-LIAs among our more frequently when using red blood cell (RBC) units from recurrently transfused SCD patient population was congruent African American donors. This fact has importance when with the low-incidence alloimmunization rate for the general considering the transfusion needs of patients with SCD. population. From a collection of 12 reports, Garratty3 found the mean and median rates of alloimmunization among patients with Materials and Methods SCD to be 25 percent. The most common alloantibodies formed in this population were anti-E (21%), -C (14%), -K (14%), -Fya Data used for this study were compiled retrospectively (7%), -Jkb (5%), and -S (4%).3 Once these alloantibodies have from records in an immunohematology reference laboratory been formed, compatible RBC units are most often found from of the American Red Cross. A retrospective examination of African American donors. Additionally, in an effort to prevent the presence of alloantibodies to the low-incidence antigens V, alloimmunization, some transfusion centers (including VS, Jsa, and Goa was conducted using those patients with SCD our own) have adopted the practice of prophylactically who are part of a therapeutic transfusion program of a client

IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 143 P. Jackson

10 hospital. It is the policy of this client hospital to provide units 9 AA-LIAs = African American Antibodies to AA-LIAs that are prophylactically matched for the antigens C, c, E, e, K, low-incidence antigens in SCD patients 8 Fya, Fyb, Jka, and Jkb. Selected for the study were all patients SCD = sickle cell disease 9.49% to whom we provided partially phenotypically matched RBC 7 units during the 2009 calendar year. The antibodies under 6 5 study were formed prior to or during 2009. Review of this Antibodies to low-incidence timeframe produced a total of 137 such patients comprising 4 antigens in general population 5.0% both male and female, and representing childhood, adolescent, 3 and adult ages. The sickle cell diagnosis was reported by the 2 client hospital. Our lab supplied phenotypically matched RBC 1 0 units primarily for scheduled transfusions. It is not uncommon Rate for patients to receive transfusions at other institutions in Figure 1. Alloimmunization rate for low-incidence antigens in urgent situations or when traveling out of area. We provided general and AA-LIAs in SCD populations more than 3000 total units for these patients during that year.

Results hypothesis, and an alternative hypothesis that the rate of alloantibody formation (y = 13) to AA-LIAs for our patients A total of 17 antibodies to AA-LIAs have been developed (n = 137) would be greater than that of the general population, by the patients who were active during 2009. The distribution a one-tailed binomial probability distribution resulted in a for each antibody is shown in Table 1. Of the 137 patients who probability value of 0.0089. P-values of less than or equal to were active in 2009, 13 exhibited an antibody to an AA-LIA, 0.05 are typically considered significant. This finding provides or 9.49 percent. Seven of those patients also have other (non strong evidence that for multiply transfused patients with SCD AA-LIA) alloantibodies, or 5.1 percent. Figure 1 illustrates receiving RBCs from the African American population, there the difference in rate of alloimmunization for the general is increased likelihood of forming alloantibodies to AA-LIAs. population (as reported by Schonewille et al.6) compared with Table 2. Patients with antibodies to AA-LIAs* the patients in this study. The number of patients who exhibited Patient Antibody(ies) Patient Antibody(ies) more than one AA-LIA is 2.19 percent. Table 2 shows the 1 VS, Goa, Jsa 8 Goa distribution of AA-LIAs by patient. It is of interest to note that 2 V 9 Goa the percentage of our study population with anti-Jsa exceeds 3 V 10 Goa, Jsa the alloimmunization rates of the common alloantibodies Jkb 4 Jsa 11 Jsa (5%) and S (4%) as indicated by Garratty.3 5 Jsa 12 Jsa Table 1. Antibodies to African American low-incidence antigens 6 V, Jsa 13 Goa a Percent with antibodies to 7 Js AA-LIAs Number of antibodies AA-LIAs (n = 137) *African American low-incidence antigens V 3 2.19 VS 1 0.73 Jsa 8 5.84 Discussion Goa 5 3.65 AA-LIAs = African American low-incidence antigens The findings of this study support the assertion that multiply transfused patients with SCD will often form The secondary purpose of this study was to test our antibodies to the low-incidence antigens that are most hypothesis that our patients with SCD form alloantibodies commonly associated with those found in the African to the low-incidence antigens in question (AA-LIAs) at a rate American population. Additionally, the rate of formation of higher than the general population’s formation of antibodies antibodies to AA-LIAs outpaces that of antibody formation to to low-incidence antigens. A binomial probability distribution low-incidence antigens among the general population. These was used to test that hypothesis. Using the 5 percent rate of findings are particularly salient when one considers the nature general population patients with antibodies to low-incidence of the AA-LIAs that were under study. The V, VS, and Goa antigens as determined by Schonewille et al.6 as the null antigens are part of the Rh system; Jsa is a member of the Kell

144 IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 Study of antibodies to low-incidence antigens among patients with SCD

system. Consequently, all antibodies against these antigens alloimmunization to the low-incidence antigens associated are capable of causing hemolytic transfusion reactions.7 with African American phenotypes and Linda Simmons, Another consideration, as has been mentioned previously, MT(ASCP)SBB, for support during the course of the study. is the proposition that transfusing recipients with RBCs from donors of the same ethnic background will decrease the References formation of alloantibodies. When attempting to meet the 1. Reid ME, Lomas-Francis C. The blood group antigen factsbook. requirement of phenotypically matched RBCs for our SCD 2nd ed. San Diego, CA: Academic Press; 2004. population, the likelihood of fulfilling the patients’ needs with 2. Sharma UK, Schreiber GB, Glynn SA, et al. Knowledge of RBC units from donors not of African descent is very low. HIV/AIDS transmission and screening in United States blood donors. Transfusion 2001;41:1341–50. According to the study by Vichinsky et al.8, alloimmunization 3. Garratty G. Severe reactions associated with transfusion of among patients with SCD is attributable in part to the patients with sickle cell disease. Transfusion 1997;37:357–61. differing prevalence of phenotypes in ethnically dissimilar 4. Afenyi-Annan A, Willis MS, Konrad TR, Lottenberg R. Blood donor and patient populations. The most frequently identified bank management of sickle cell patients at comprehensive sickle cell centers. Transfusion 2007;47:2089–97. alloantibodies in patients with SCD are anti-C, -E, -K, -Fya, b b 8 5. Osby M, Shulman IA. Phenotype matching of donor red blood -Fy , and -Jk . Patients with SCD have the lowest frequency cell units for nonalloimmunized sickle cell disease patients: a of the antigens associated with those alloantibodies.8 The survey of 1182 North American laboratories. Arch Pathol Lab findings of this study demonstrate that while the occurrence Med 2005;129:190–93. of alloantibodies to common antigens may decrease when 6. Schonewille H, van Zijl AM, Wijermans PW. The importance of antibodies against low-incidence RBC antigens in complete donor and recipient are from the same ethnic population, and abbreviated cross-matching. Transfusion 2003;43: the occurrence of antibodies to low-incidence antigens may 939–44. increase. 7. Roback JD, Combs MR, Grossman BJ, Hillyer, CD. AABB technical manual. 16th ed. Arlington, VA: American These results support the idea that consideration Association of Blood Banks; 2008. of the presence of antibodies to AA-LIAs is appropriate 8. Vichinsky EP, Earles A, Johnson RA, Hoag MS, Williams when attending to the transfusion needs of patients with A, Lubin B. Alloimmunization in sickle cell anemia and SCD. Furthermore, considering these antibodies in patient transfusion of racially unmatched blood. New Eng J of Med 1990;322:1617-21. populations other than SCD with transfusion histories and needs similar to those with SCD (i.e., antigen profile of Pamela Jackson, MLT(ASCP), Reference Technologist, transfused RBCs) may also prove to be prudent. Immunohematology Reference Laboratory, American Red Cross, Alabama Central Gulf Coast Region, 1130 22nd Street South, Acknowledgments Birmingham, AL 35205.

The author wishes to acknowledge Gerald Sapp, MT(ASCP), for inspiring our initial concerns involving

IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 145 O r i g i n a l R e p o r t Determination of optimal method for antibody identification in a reference laboratory

J.R. Haywood, M.K.G. Moulds, and B.J. Bryant

Methods commonly used for antibody identification are method for the detection and identification of all clinically hemagglutination (tube), column agglutination (gel), and solid- significant antibodies. phase red cell adherence. Our AABB immunohematology reference laboratory (IRL) conducted a study to determine which antibody identification testing method was optimal for Materials and Methods detecting all clinically significant antibodies. Patient specimens were sent to our IRL from August 2008 to September 2009. Patient specimens used in the study were sent to our Routine testing was performed by tube method and then by manual gel and manual solid-phase methods. Of the 254 samples reference laboratory from August 2008 until September 2009. tested, 115 showed agreement in antibody identification with all The specimens came from hospitals, clinics, and dialysis three methods. The tube method identified all but six clinically centers that use our laboratory for antibody workups. Most significant antibodies. The gel method did not identify 59 samples referred to our IRL had incomplete or inconclusive clinically significant antibodies. Fifty-six clinically significant antibodies were not identified by solid-phase testing. Tube testing antibody identifications. The majority of the referring identified 27 clinically insignificant antibodies, primarily cold laboratories used automated gel methodology (Ortho ProVue, autoantibodies. Gel and solid-phase methodologies identified two Ortho Clinical Diagnostics, Inc., Rochester, NY) and others and three cold autoantibodies, respectively. Solid-phase testing used automated solid-phase testing (Immucor Galileo or failed to detect 12 examples of anti-K. No identifiable pattern of reactivity was found in 13 samples using gel testing compared Galileo Echo, Immucor Gamma, Norcross, GA). The least with 6 for solid-phase and none for tube methodologies. common method used by the referring laboratories was Hemagglutination tube method was the best choice for our IRL tube testing. Routine testing in our IRL was performed by because it missed the fewest number of clinically significant alloantibodies. Benefits also included the ability to use various the tube method, and an aliquot of serum or plasma was potentiating factors, incubation times, and temperature phases saved for manual gel and manual solid-phase testing (aliquot to enhance antibody identification. The tube method provided refrigerated for testing within 24 hours or frozen for later critical data for determining antibody clinical significance. testing). Immunohematology 2011;27:146–150. Tube testing was performed using a modified tube Key Words: antibody identification methods, reference testing methodology introduced by John Moulds when he laboratory, hemagglutination, gel testing, solid-phase red cell joined LifeShare Blood Center, Shreveport, Louisiana, in adherence 2004 as Director of Scientific Support. Two drops of patient’s serum or plasma was incubated with 1 drop of reagent red The three methods commonly used in the United States blood cells (RBC) for 5 minutes at room temperature (RT). for pretransfusion testing and antibody identification are The tubes were centrifuged and read. Two drops of low ionic hemagglutination (tube), column agglutination (gel), and solid- strength saline (LISS) additive (LO-ION, Immucor Gamma) phase red cell adherence.1 All are effective, but usually one is was added to each tube; the tubes were incubated at RT selected by a laboratory as the primary method. Of 58 AABB for an additional 10 minutes before being centrifuged and immunohematology reference laboratories (IRLs) surveyed at read. The tubes were then incubated at 37°C for 30 minutes, the time of our study, 49 used tube, 7 used gel, and 2 used centrifuged, and read. Tubes were washed four times with solid-phase methodologies.2 Our IRL used the tube method 0.9 percent sodium chloride, and the last wash was decanted. as the primary method for antibody determination, although One to two drops of anti-IgG (Gamma-Clone Anti-IgG, most facilities we received samples from used gel or solid- Immucor Gamma) was added to each tube, centrifuged, and phase testing for detection, and in some cases preliminary read. Additional enhancement methods were used as needed identification, of antibodies before sending the specimens for depending on the initial test results, i.e., saline 60 minutes complete identification. The aim of this study was to evaluate incubation at 37°C without enhancement, or a polyethylene tube, gel, and solid-phase test results to determine the optimal glycol (PEG) additive (PeG, Immucor Gamma) to enhance

146 IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 Optimal antibody identification method

Table 2. Solid-phase and gel methods agree weak reactions or rule out alloantibodies, or enzyme and chemical testing. Solid-phase and gel results Tube results One cell positive Anti-E The manual solid-phase testing used 14-cell antibody All cells positive except one Anti-C, warm autoantibody identification panels (Capture-R Ready-ID, Immucor Gamma). Anti-D Anti-D, cold autoantibody Manual gel antibody identification panels used gel cards (IgG Anti-D Anti-D, other cells positive gel cards, Ortho Clinical Diagnostics, Inc.) with commercial Anti-D, C Anti-D RBCs (Panocell 10-panel, Immucor Gamma) diluted to 0.8 Anti-D, K Anti-D, K, Jka percent concentration. Solid-phase and column agglutination Anti-E Anti-E, McCa (gel) methods were performed per manufacturers’ Anti-E, Jka Anti-E, Jka, cold autoantibody a a recommendations. Anti-E, K, Fy Anti-E, K, Fy , cold autoantibody Anti-E, other cells positive Anti-E, Jsa, M Antibody identification results of each specimen were Anti-K Anti-C, K, Jkb sorted into one of five groups based on the findings for the Anti-K Anti-K, cold autoantibody three testing methods: all methods agree, none agree, tube Anti-K Anti-K, M and gel methods agree, tube and solid-phase methods agree, No pattern Anti-C and solid-phase and gel methods agree. No pattern Anti-C, E No pattern Anti-D, K Results No pattern Anti-E No pattern No reactivity No pattern No reactivity In a 13-month period, a total of 254 specimens were No reactivity All positive in PEG only processed by all three methods. The three methods No reactivity Anti-C demonstrated the same antibody identification results in 115 No reactivity Anti-D samples (45% of the total tested). Table 1 lists the antibodies No reactivity Anti-E No reactivity Anti-E, cold autoantibody Table 1. Tube, gel, and solid-phase methods agree No reactivity Anti-K

Antibodies detected Occurrences Antibodies detected Occurrences No reactivity Anti-K, cold autoantibody No reactivity Anti-Leb Anti-C 1 Anti-e 1 No reactivity Anti-M Anti-c 1 Anti-E, Fya 2 No reactivity Anti-M Anti-c, E, s 1 Anti-E, K 2 No reactivity Anti-P1 Anti-D 13 Anti-Fya 3 No reactivity Cold autoantibody a Anti-D, C 2 Anti-Jk 2 No reactivity Cold autoantibody Anti-D, C, E 1 Anti-K 5 No reactivity Cold autoantibody Anti-D, C, Fya, Jka 1 Anti-M 1 No reactivity Cold autoantibody Anti-D, C, V 1 No antibodies 58 No reactivity Cold autoantibody Anti-E 10 Warm autoantibody 10 No reactivity Cold autoantibody No reactivity Cold autoantibody No reactivity Cold autoantibody identified by all three methods and the number of times these No reactivity Cold autoantibody antibodies were found. No reactivity Cold autoantibody Solid-phase and gel methods agreed, but the tube method No reactivity Cold autoantibody did not, in 51 samples (20%). There were 16 clinically significant No reactivity Cold autoantibody antibodies detected by the tube method that were missed by No reactivity Cold autoantibody both the solid-phase and gel methods. These included anti-D, No reactivity Cold autoantibody No reactivity Cold autoantibody -C, -E, -K, -Jka, and -Jkb. There was only one sample in which No reactivity Cold autoantibody with N specificity the tube method missed a clinically significant antibody, No reactivity Enhancement related (all positive) anti-C, which was detected by the solid-phase and gel methods No reactivity HTLA-like (Table 2). No reactivity Weak reactivity The results of the tube and gel methods agreed, but the No reactivity Weak reactivity solid-phase missed 15 clinically significant antibodies in 29 Warm autoantibody No reactivity samples (12% of the samples tested). The clinically significant HTLA = high-titer, low-avidity antibody; PEG = polyethylene glycol.

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Table 3. Tube and gel methods agree Table 4. Tube and solid-phase methods agree

Tube and gel results Solid-phase results Tube and solid-phase results Gel results All positive All positive except one cell Anti-c, E, K, S, Ch (all cells positive) 3 cells negative; no pattern Anti-D Anti-D, one additional cell positive Anti-C, S Anti-S Anti-D No reactivity Anti-C, warm autoantibody No pattern Anti-D No pattern Anti-D 4 of 5 D+ cells positive Anti-D, C, Jka Anti-D, C Anti-D, C, Jka, warm autoantibody No pattern Anti-E No reactivity Anti-e 1 e+ cell negative Anti-E 2 of 3 E+ cells reactive Anti-E, Fya No pattern Anti-E No reactivity Anti-E, Fya All cells positive Anti-e All cells positive Anti-E, K, Jkb Anti-E, K, few other cells positive (not Jkb) Anti-E, K One cell positive (E, K neg) Anti-Jkb Anti-Jkb, one additional cell positive Anti-E, U One cell positive Anti-K Anti-K, other cells positive Anti-Jkb No pattern Anti-K Anti-K, other cells positive Anti-K No reactivity Anti-M One cell positive Anti-K No pattern Cold autoantibody No pattern Anti-K 1 of 3 K+ cells positive Cold autoantibody No pattern Anti-McCa No reactivity Cold autoantibody No reactivity Anti-McCa No reactivity No reactivity Weak reactivity Anti-S No reactivity No reactivity 8 of 11 cells positive Anti-S No reactivity No reactivity 1 cell positive Anti-Sla No reactivity No reactivity (prev Anti-E, K) Anti-E Cold autoantibody No pattern Warm autoantibody 6 of 11 cells positive Cold autoantibody (strong) All positive except one cell Warm autoantibody 7 of 11 cells positive Cold autoantibody (strong) No pattern Warm autoantibody 8 of 11 cells positive No reactivity Anti-Jka No reactivity No pattern No reactivity No pattern No method captured every clinically significant antibody. No reactivity Weak reactivity The gel method missed 59 antibodies and seemed to give the Weak reactivity No reactivity most results that showed no pattern. Solid-phase testing missed Weak reactivity No reactivity 56 antibodies. The tube method missed only 6 antibodies, but also identified the most insignificant antibodies, primarily antibodies missed by the gel method included anti-D, -E, -e, cold autoantibodies. -K, -S, -Jka, -Jkb, and -U. However, in one sample, anti-Jka was identified in solid-phase, but not in tube or gel methods Discussion (Table 3). Results from tube and solid-phase testing, but not gel, Several studies have compared various antibody detection revealed the same antibodies in 23 samples (9%). Gel testing and identification methods in search of the best method missed 19 significant antibodies: anti-D, -C, -c, -E, -K, -S, for pretransfusion serologic testing.3–5 Our study differed -Fya, -Jka, -Jkb, and warm autoantibody. One sample revealed from these previous comparison studies in that it sought to anti-E identified by gel when it was not detectable in tube or determine the optimal routine testing methodology for the solid-phase testing methods (Table 4). identification of antibodies referred to our IRL. Referring None of the methods agreed in 36 samples (14%). In laboratories used various methods of pretransfusion testing; this sample cohort, the tube method missed 3 significant most used automated gel or solid-phase methods. Antibody antibodies (one anti-C and two anti-E). The solid-phase workups referred to our IRL included detected but not method missed 24, and gel missed 23. Gel was the only identified antibodies, incomplete antibody identifications, method demonstrating hemolysis with two samples tested. and antibody workups with weak inconclusive results. The There were many panels that were inconclusive, which referring laboratories depended on the IRL to accurately and complicated data analysis (Table 5). completely identify the detected antibodies. All three manual

148 IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 Optimal antibody identification method

Table 5. None of the testing methodologies agree Tube Method Tube results Solid-phase results Gel results Tube method testing missed the fewest clinically Anti-C Anti-C, other cells positive Anti-C, E significant antibodies (6). This was because of the many ways Anti-C All positive 3 of 4 C+ cells positive that this method could be manipulated, such as temperature, Anti-c 10 of 11 c+ cells positive 5 of 7 c+ cells positive time, and enhancement media. The tube method detected not Anti-C Anti-C, other cells positive Anti-C, E only clinically significant antibodies, but also cold-reactive and Anti-C (PEG) No reactivity Anti-C, one other cell positive insignificant antibodies. This method demonstrated the best Anti-c, E, Fya, 2 No reactivity 3 of 10 cells positive sensitivity by detecting 188 clinically significant antibodies. other cells positive However, the tube method did detect 49 insignificant Anti-D Anti-D, one other cell Anti-D, C positive antibodies, 30 of which were cold autoantibodies. Identification Anti-D, C, M Anti-D Anti-D, C, and M (dosage) of cold autoantibodies is time and labor intensive, although in Anti-e 8 of 14 cells positive 1 cell positive some instances, cold autoantibody identification by the tube Anti-e 12 of 13 e+ cells positive No reactivity method provided the cause for unexplained reactions obtained Anti-E Anti-E, K, other cells Anti-E by the referring laboratory. positive Anti-E, Jka Anti-E, 9 of 10 Jka+ cells All positive (one E, Jka Solid-Phase Method positive negative cell) Solid-phase testing failed to detect 6 examples of Knops Anti-e, McCa No reactivity Hemolyzed Anti-E, warm Anti-E, one other cell Anti-E, 6 of 9 other cells system antibodies. Two of these 6 antibodies were detected autoantibody positive positive by gel testing, and all 6 were detected by tube method. In Anti-Jka Anti-Jka (dosage) No pattern one sample, anti-Jka was identified by solid-phase method Anti-Jka, Yka Anti-Jka (homozygous only) No pattern whereas tube and gel testing showed no reactivity. Solid-phase Anti-Jsb, C, K, Fya Anti-K Anti-C, K, Fya testing has also been shown not to detect most cold-reactive Anti-K 1 of 3 K+ cells positive No reactivity antibodies, although 3 were identified. Anti-K was missed 12 Anti-K No reactivity 1 of 3 K+ cells positive times by solid-phase testing in this study, whereas gel testing Anti-K No reactivity Anti-K, other cells positive missed 9 and tube testing missed 1. Solid-phase testing was a a Anti-K, Jk No reactivity Anti-K and Jk (dosage) also inconsistent in identifying Rh antibodies. Anti-K, P1 Anti-K No pattern Anti-K, warm No reactivity No pattern Gel Method autoantibody Anti-M 2 cells positive No reactivity As with the solid-phase method, gel testing did not detect Anti-M Anti-M (homozygous cells No reactivity most cold-reactive antibodies, although two were identified. only) Anti-E reacted strongly in gel, but was still missed in some Cold autoantibody No reactivity Hemolyzed samples in which the tube method identified it. Gel testing gave Cold autoantibody No reactivity 7 of 10 cells positive the most results that had no specific pattern. Many antibody Cold autoantibody 9 of 14 cells positive No reactivity specificities were suggested but not confirmed because not No reactivity All positive No pattern (5 cells positive) every antigen-positive cell was reactive. Gel was the only No reactivity Anti-Jkb (dosage) No reactivity method that demonstrated unexplained hemolysis during No reactivity Anti-e All positive testing with two specimens. No reactivity Anti-D No pattern One factor to consider in this analysis of multiple methods Warm autoantibody No reactivity Possible anti-C of antibody identification is the number of passively acquired Warm autoantibody Looks like anti-f (patient No pattern (PEG) is f+) anti-D antibodies resulting from Rh immune globulin (RhIG) Warm autoantibody No reactivity Hemolyzed administration versus true allo-anti-D. Based on antibody (PEG) screens from hospitals, RhIG is detected uniformly in both Weak reactivity All positive 3 cells positive automated gel and solid-phase methods. In tube testing, PEG = polyethylene glycol. anti-D from RhIG is detected weakly, if at all. This study did not attempt to evaluate the difference in testing modalities used at our IRL and evaluated in this study antibody identification results obtained by manual versus had unique performance profiles, which are summarized in automated solid-phase and gel testing. Many of the referring the following sections. facilities used automated solid-phase and gel methods for

IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 149 J.R. Haywood et al.

antibody detections and initial antibody identifications, where References our reference laboratory used only manual methods. The 1. Downes KA, Shulman IA. Pretransfusion testing. In: Roback impact of this variation is unknown. J, Combs MR, Grossman B, Hillyer C, eds. Technical manual. In conclusion, this study demonstrated that the 16th ed. Arlington, VA: American Association of Blood Banks, hemagglutination tube method was the best choice for 2008:437–63. 2. AABB accredited Immunohematology Reference Labs antibody identification, as it missed the fewest number Proficiency Data; March 2009. of clinically significant alloantibodies compared with the 3. Casina TS. In search of the Holy Grail: comparison of antibody other two methods. A reference laboratory is responsible for screening methods. Immunohematology 2006;22:196–202. identifying all antibodies present, whether clinically significant 4. Bunker ML, Thomas CL, Geyer SJ. Optimizing pretransfusion antibody detection and identification: a parallel, blinded or not. Major benefits of the tube method are identification comparison of tube PEG, solid phase, and automated methods. of all antibodies present and the ability to enhance testing Transfusion 2001;41:621–6. using various potentiating factors, incubation times, and 5. Voak D. The status of new methods for the detection of red cell temperature phases. The tube method provides critical data agglutination. Transfusion 1999;39:1037–40. for determining antibody clinical significance. However, it is beneficial for a reference laboratory to have gel and solid- Jennifer R. Haywood, MLS(ASCP)SBB, Reference Technologist, Immunohematology Reference Laboratory, and Marilyn K. phase methodologies available for comparison because many Grandstaff Moulds, BA, MT(ASCP)SBB, Immunohematology referring hospitals use these methods for initial antibody Specialist, Immunohematology Reference Laboratory, LifeShare detection. Overall, the tube method was the most reliable Blood Centers, Shreveport, Louisiana; and Barbara J. Bryant, MD method for antibody identification in our reference laboratory. (corresponding author), Associate Professor, Pathology, Associate Director, Blood Bank Division, Director, Coagulation Consult Service, Acknowledgments Director, Clinical Pathology Residency Program, University of Texas Medical Branch, 301 University Boulevard, JSA 2.148, Galveston, TX 77555-0717. The authors would like to acknowledge and thank the staff of the Immunohematology Reference Laboratory— John Moulds Reference and Scientific Support Laboratory at LifeShare Blood Centers for their technical support.

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150 IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 O r i g i n a l R e p o r t Preoperative coagulation studies to predict blood component usage in coronary artery bypass graft surgery

S. Josefy, R. Briones, and B.J. Bryant

Bleeding remains a serious complication of cardiac surgery. available fibrinogen in the body plasma at a given time. Because Studies indicate that preoperative fibrinogen concentration is fibrinogen is not stored in any great quantity in any cells and it an independent predictor of blood loss during coronary artery is a consumable component of , fibrinogen’s major bypass graft (CABG) surgery. This study evaluates whether fibrinogen concentration is a better predictor of blood usage than reserve is in the plasma. Fibrinogen constitutes approximately the prothrombin time (PT) and activated partial thromboplastin 2 percent of the total plasma protein concentration.2,3 time (aPTT) tests. Patients not taking clopidogrel bisulfate who The prothrombin time (PT) and the activated partial underwent CABG surgery during a 3-month period at a 350-bed community hospital were included in this prospective study. thromboplastin time (aPTT) are routine coagulation tests The parameters evaluated included patient’s age, preoperative used to determine whether patients have abnormalities in coagulation test results (PT, international normalized ratio [INR], blood clotting. The PT value is a measure of the extrinsic aPTT, fibrinogen), and number of blood components transfused. pathway of clotting, whereas the aPTT is a measure of the A probability value of less than 0.05 was deemed significant. Thirty-five patients were included in this study. Mean blood usage intrinsic pathway of coagulation. Both the intrinsic and was 6 units. Patient’s age approached significance as a predictor extrinsic systems include the common pathway of clotting and of blood usage, and fibrinogen levels trended toward significance terminate with the successful formation of a fibrin clot.2–5 more than the other coagulation parameters. In this study, the The aim of this study was to determine whether the increased age of the patient and low plasma concentrations of fibrinogen were associated with increased blood usage. Although preoperative fibrinogen concentration was a better predictor no indicators clearly demonstrated statistical significance, of blood component usage during hospitalization than the the vast difference in the probability values for patients’ ages traditional PT and aPTT tests in patients not taking clopidogrel and fibrinogen levels indicated that there was a trend toward significance in blood usage for CABG patients. Further studies with bisulfate (Plavix) and undergoing CABG surgery. larger patient populations are indicated. Immunohematology 2011;27:151–153. Materials and Methods

Key Words: blood usage, coagulation studies, coronary This study was conducted at a 350-bed community artery bypass graft surgery hospital serving a midsized city and surrounding communities with a population of greater than 100,000 inhabitants. Hemostasis continues to be a challenge in coronary artery Patients undergoing CABG surgery and not taking clopidogrel bypass graft (CABG) surgery. Preoperative predictors of blood bisulfate were eligible to be included in this prospective usage have been elusive. A patient’s preoperative coagulation study. Clopidogrel bisulfate is an inhibitor of adenosine status has been touted as a potential gauge of intraoperative diphosphate–induced aggregation, thus preventing and postoperative bleeding. Recent studies indicate that platelet aggregation or clot formation. Clopidogrel bisulfate is preoperative plasma fibrinogen levels are independent associated with increased bleeding tendencies, and therefore predictors of blood loss in patients undergoing CABG surgery.1 patients taking this drug were excluded from the study. Plasma fibrinogen plays a key role in hemostasis after The data analysis occurred during a period of 3 months. platelet activation and aggregation. Fibrinogen is the key to No consideration was given to patient’s sex or race. This study platelet stabilization and strengthening of the thrombus to did not distinguish between the types of blood components ensure that bleeding stops and the healing process begins. used and included , platelets, fresh- Fibrinogen’s major contribution to clotting is in the common frozen plasma, and cryoprecipitated AHF. pathway of coagulation, in which it forms a stable fibrin The parameters evaluated in this study included patient’s clot.2–5 The fibrinogen test is a measure of the concentration of age, preoperative coagulation test results (PT, international

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normalized ratio [INR], aPTT, and fibrinogen), and total blood components used during hospitalization. All coagulation testing was performed on a Stago Compact Analyzer (Diagnostica Stago, Inc., Parsippany, NJ).

Statistics Total Blood Products Used Outcomes were compared using the t test, and correlation between parameters and blood usage, r values, were calculated. A probability value of less than 0.05 was deemed statistically significant.6,7 Fibrinogen (mg/dL) Figure 2. Analysis of total blood components transfused by patient’s preoperative fibrinogen conentration. Results

A total of 35 patients (n = 35) with a mean age of 67 years (range, 52 to 85 years) undergoing CABG procedures and not on clopidogrel bisulfate were included in this study. The mean number of blood components transfused was 6 units (range, 0 to 22 units). Linear regression showed that a relationship existed between patient age and blood component usage (Fig. 1). An Total Blood Products Used inverse relation existed between fibrinogen level and blood component usage (Fig. 2). The PT, INR, and aPTT results compared with the blood components transfused did not PT (seconds) demonstrate a relationship (Figs. 3–5). Figure 3. Analysis of total blood components transfused by patient’s preoperative prothrombin time (PT) value. Total Blood Products Used Total Blood Products Used

INR Figure 4. Analysis of total blood components transfused by patient’s Age in years preoperative international normalized ratio (INR) value. Figure 1. Analysis of total blood components transfused by patient age. significance as a predictor of blood usage, and fibrinogen levels trended toward significance more than the other coagulation The correlation and probability values for each indicator parameters. evaluated (age, PT/INR, aPTT, and fibrinogen levels as compared with blood component consumption) are as follows: Discussion age (r = 0.3191; p = 0.062), PT (r = –0.0223; p = 0.898), INR (r = –0.0120; p = 0.945), aPTT (r = 0.0060; p = 0.973), and The results of this study indicate that increased patient age fibrinogen (r = –0.2541; p = 0.141). Although the PT/INR and a low preoperative plasma fibrinogen level were indicators and aPTT levels did not show any significance in predicting associated with increased blood component consumption blood component consumption, patient’s age approached during and after CABG surgery. According to this data analysis,

152 IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 Predicting blood usage in CABG surgery

2. O’Malley BA. Primary hemostasis. In: McKenzie SB, Williams JL, eds. Clinical laboratory hematology. 2nd ed. Upper Saddle River, NJ: Pearson Education Inc., 2010:612–38. 3. O’Malley BA. Secondary hemostasis and fibrinolysis. In: McKenzie SB, Williams JL, eds. Clinical laboratory hematology. 2nd ed. Upper Saddle River, NJ: Pearson Education Inc., 2010: 639–70. 4. Harmening DM, Escobar CE, McGlasson DL. Introduction to Total Blood Products Used hemostasis. In: Harmening DM, ed. Clinical hematology and fundamentals of hemostasis 5th ed. Philadelphia, PA: FA Davis Co., 2009:543-76. 5. Ehsan A, Herrick JL. Introduction to thrombosis and PTT (seconds) anticoagulant therapy. In: Harmening DM, ed. Clinical hema- Figure 5. Analysis of total blood components transfused by patient’s tology and fundamentals of hemostasis 5th ed. Philadelphia, preoperative activated partial thromboplastin time (aPTT) value. PA: FA Davis Co., 2009:660-5. 6. Bluman AG. Hypothesis testing. In: Weiss NA, ed. Elementary statistics. 6th ed. New York, NY: McGraw-Hill, 2007:391–453. there were no indicators clearly demonstrating statistical 7. Bluman AG. Correlation and regression. In: Weiss NA, ed. significance. However, the vast difference in the probability Elementary statistics. 6th ed. New York, NY: McGraw-Hill, values for patient age and fibrinogen level as compared with 2007:527–75. those for the other tests suggests that there is a relationship 8. Karkouti K, Wijeysundera DN, Beattie WS, et al. Variability and predictability of large-volume red blood cell transfusion predicting blood and blood component usage in CABG in cardiac surgery: a multicenter study. Transfusion 2007;47: patients. Increased patient age and blood usage approached 2081–8. significance, and decreasing fibrinogen levels and blood usage 9. Tinmouth AT. The value of a clinical prediction rule for trended toward significance. allogeneic transfusion in cardiac surgery. Transfusion 2006;46: 1072–4. The prediction of blood usage in CABG surgery has been 10. Alghamdi A, Davis A, Brister S, Corey P, Logan A. Development an area of interest for more than 25 years. Numerous groups and validation of Transfusion Risk Understanding Tool have developed algorithms and prediction rules for use as (TRUST) to stratify cardiac surgery patients according to their blood transfusion needs. Transfusion 2006;46:1120–9. tools to predict the probability of clinical bleeding in CABG 8–13 11. Covin R, O’Brien M, Grunwald G, et al. Factors affecting patients. Methods of testing coagulation parameters have transfusion of , platelets, and red blood been evaluated to determine which method was the most cells during elective coronary artery bypass graft surgery. Arch useful in given populations of patients.14,15 Several of these Pathol Lab Med 2003;127:415–23. studies were contradictory; experiences, accuracy of test 12. Karkouti K, O’Farrell R, Yau TM, Beattie WS; Reducing Bleeding in Cardiac Surgery Research Group. Prediction of results, and clinical usefulness were variable. massive blood transfusion in cardiac surgery. Can J Anaesth In part, this study supports the observations of Karlsson 2006;53:781–94. and colleagues that preoperative fibrinogen concentrations 13. Despotis G, Avidan M, Eby C. Prediction and management of bleeding in cardiac surgery. J Thromb Haemost 2009;7 can be used to identify patients with an increased likelihood (Suppl 1):111–17. 1 of severe bleeding after CABG surgery. Multicenter studies 14. Shih RL, Cherng YG, Chao A, Chen JT, Tsai AL, Liu CC. with larger populations of CABG patients are warranted to Prediction of bleeding diathesis in patients undergoing determine whether a significant correlation exists among cardiopulmonary bypass during cardiac surgery: viscoelastic measures versus routine coagulation test. Acta Anaesthesiol these parameters and whether these findings can be Sin 1997;35:133–9. generalized. Subset analyses by specific blood components 15. Wang JS, Lin CY, Hung WT, et al. Thromboelastogram fails may provide additional insight to potential predictors of to predict postoperative hemorrhage in cardiac patients. Ann transfusion. Prediction of blood usage in CABG cases would Thorac Surg 1992;53:435–9. assist physicians and the blood bank in proper preparation Shana Josefy, MT(ASCP)SBBCM and Ricardo Briones, MT(ASCP)CM, and support of patients undergoing CABG surgery. United Regional Health Care System, Wichita Falls, TX, and Barbara J. Bryant, MD (corresponding author), University of Texas Medical References Branch, Department of Pathology, Blood Bank Division, 301 University Boulevard, Route 0717, Galveston, TX 77555-0717. 1. Karlsson M, Ternström L, Hyllner M, Baghaei F, Nilsson S, Jeppsson A. Plasma fibrinogen level, bleeding, and transfusion after on-pump coronary artery bypass grafting surgery: a prospective observational study. Transfusion 2008;48:2152–8.

IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 153 C o mm u n ic at i o n Letter from the editors

To Contributors to the 2011 Issues

The journal depends on readers, authors, editorial board, peer reviewers, and our Penn-Jersey staff. We wish we could thank all of you personally, but doing so is not practical. Instead, we thank each of you as members of an honored group. First and foremost, we thank the authors for their reviews, scientific articles, case reports, book reviews, and letters to the editors that come not only from the United States but from many countries of the world. This has given the journal an international flavor. Our editorial board is a prestigious one and we depend on them, not only for peer reviews, but for guidance in policy and suggestions for improvements. Special thanks go to our medical editors, who review every article for medical content, and to our technical editors, who read every article for technical content. The current board is listed by name in the front of each issue of the journal. Our peer reviewers did a wonderful job in 2011. They are listed below; our thanks to each.

Roy A. Alther Jr., MT(ASCP)SBB Monique Mohammad Joan Boyd, MT(ASCP)SBB Ruth Mougey, MT(ASCP)SBB Karen Byrne, MT(ASCP)SBB John J. Moulds, MT(ASCP)SBB Lilian Castilho, PhD Pamela Nickle, MT(ASCP)SBB Joann Christensen, MT(ASCP)SBB Dorris Neurath, BScPharm, ART, MBA Laura Cooling, MD John Nobiletti, MD Geoffrey Daniels, PhD Joyce Poole, FIBMS David F. Friedman, MD Thierry Peyrard, PhD Richard Haspel, MD NurJehan Quraishy, MD Trina Horn, MS Jorje Rios, MD Courtney Hopkins, MD Jill R. Storry, PhD, FIBMS Nanette Johnson, MT(ASCP)SBB Susan Shirey, MS Julie Karp, MD Paul Schmidt, MD Hallie Lee-Stroka, MT(ASCP)SBB Chelsea Sheppard, MD Lisa McLaughlin, MD Ira Shulman, MD Dolores Mallory, MT(ASCP)SBB Sunitha Vege, MS Claire Meena-Leist, MD Kathleen Weber, MT(ASCP)SBB

We also want to thank Marge Manigly, our production assistant, and Sheetal Patel, our editorial assistant, for their help in preparing the journal for press. We also thank Dawn Rumsey, our technical editor; Mary Tod, our copy editor; Lucy Oppenheim, our proofreader; and Paul Duquette, our electronic publisher. Finally, thanks go to our readers, whose enthusiasm and interest in the journal make it all worthwhile.

Sandra Nance Editor-in-Chief

Cindy Flickinger Managing Editor

154 IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 A n n o u n c e m e n t s

Specialist in Blood Bank (SBB) Program The Department of Transfusion Medicine, National Institutes of Health, is accepting applications for its 1-year Specialist in Blood Bank Technology Program. Students are federal employees who work 32 hours/week. This program introduces students to all areas of transfusion medicine including reference serology, cell processing, HLA, and infectious disease testing. Students also design and conduct a research project. NIH is an Equal Opportunity Organization. Application deadline is December 31, 2012, for the July 2013 class. See www.cc.nih.gov/dtm > education for brochure and application. For further information contact Karen M. Byrne at (301) 451-8645 or [email protected]

Manuscripts The editorial staff of Immunohematology welcomes manuscripts issues are the first weeks in November, February, May, and pertaining to blood group serology and education for consideration August, respectively. For instructions for scientific articles, case for publication. We are especially interested in case reports, reports, and review articles, see Instructions for Authors in every papers on platelet and white cell serology, scientific articles issue of Immunohematology or on the Web at www.redcross.org/ covering original investigations, and papers on new methods for immunohematology. Include fax and phone numbers and use in the blood bank. Deadlines for receipt of manuscripts for e-mail address with all manuscripts and correspondence. consideration for the March, June, September, and December E-mail all manuscripts to [email protected]

Free Classified Ads and Announcements

Immunohematology will publish classified ads and announcements (SBB schools, meetings, symposia, etc.) without charge. Deadlines for receipt of these items are as follows:

Deadlines 1st week in January for the March issue 1st week in July for the September issue 1st week in April for the June issue 1st week in October for the December issue

E-mail or fax information to [email protected] or phone (215) 451-2538

IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 155 Announcements, cont.

156 IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 Announcements, cont.

Masters (MSc) in Transfusion and Transplantation Sciences at The University of Bristol, England

Applications are invited from medical or science graduates for the Master of Science (MSc) degree in Transfusion and Transplantation Sciences at the University of Bristol. The course starts in October 2012 and will last for 1 year. A part-time option lasting 2 or 3 years is also available. There may also be opportunities to continue studies for PhD or MD following the MSc. The syllabus is organized jointly by The Bristol Institute for Transfusion Sciences and the University of Bristol, Department of Pathology and Microbiology. It includes: • Scientific principles of transfusion and transplantation • Clinical applications of these principles • Practical techniques in transfusion and transplantation • Principles of study design and biostatistics • An original research project

Application can also be made for Diploma in Transfusion and Transplantation Science or a Certificate in Transfusion and Transplantation Science.

The course is accredited by the Institute of Biomedical Sciences.

Further information can be obtained from the Web site: http://ibgrl.blood.co.uk/MSc/MscHome.htm

For further details and application forms please contact:

Dr Patricia Denning-Kendall University of Bristol Paul O’Gorman Lifeline Centre Department of Pathology and Microbiology Southmead Hospital Westbury-on-Trym, Bristol BS10 5NB, England Fax +44 1179 595 342, Telephone +44 1779 595 455, e-mail: [email protected].

IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 157 A dv e r t i s e m e n t s

Reference and Consultation Services IgA/Anti-IgA Testing

Antibody identification and problem resolution IgA and anti-IgA testing are available to do the

HLA-A, B, C, and DR typing following:

HLA-disease association typing • Identify IgA-deficient patients • Investigate anaphylactic reactions Paternity testing/DNA • Confirm IgA-deficient donors Our ELISA for IgA detects protein to 0.05 mg/dL. For information, contact:

Mehdizadeh Kashi For additional information contact:

at (503) 280-0210 Janet Demcoe at (215) 451-4914,

or e-mail: or write to: [email protected],

Tissue Typing Laboratory or write to: American Red Cross Biomedical Services American Red Cross Biomedical Services

Pacific Northwest Region Musser Blood Center 700 Spring Garden Street 3131 North Vancouver Philadelphia, PA 19123-3594 Portland, OR 97227 ATTN: Janet Demcoe CLIA licensed, ASHI accredited CLIA licensed

National Reference Laboratory Donor IgA Screening for Blood Group Serology • Effective tool for screening large volumes of donors Immunohematology Reference Laboratory • Gel diffusion test that has a 15-year proven track record: AABB, ARC, New York State, and CLIA licensed Approximately 90 percent of all donors identified as 24-hour phone number: IgA deficient by this method are confirmed by the more (215) 451-4901 Fax: (215) 451-2538 sensitive testing methods

American Rare Donor Program For additional information: 24-hour phone number: (215) 451-4900 Kathy Kaherl Fax: (215) 451-2538 at (860) 678-2764 [email protected] e-mail: Immunohematology [email protected] Phone, business hours: (215) 451-4902 or write to: Fax: (215) 451-2538 Reference Laboratory [email protected] American Red Cross Biomedical Services Quality Control of Cryoprecipitated–AHF Connecticut Region Phone, business hours: 209 Farmington Ave. (215) 451-4903 Farmington, CT 06032 Fax: (215) 451-2538

CLIA licensed

158 IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 Advertisements, cont.

IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 159 Advertisements, cont.

National Platelet Serology Reference Laboratory National Neutrophil Serology Reference Laboratory

Diagnostic testing for: Our laboratory specializes in granulocyte antibody detection • Neonatal alloimmune thrombocytopenia (NAIT) and granulocyte antigen typing. • Posttransfusion purpura (PTP) • Refractoriness to Indications for granulocyte serology testing • Heparin-induced thrombocytopenia (HIT) include: • Alloimmune idiopathic thrombocytopenia purpura (AITP) • Alloimmune neonatal neutropenia (ANN) Medical consultation available • Autoimmune neutropenia (AIN) • Transfusion-related acute lung injury (TRALI) Test methods: • GTI systems tests Methodologies employed: — detection of glycoprotein-specific platelet antibodies • Granulocyte agglutination (GA) — detection of heparin-induced antibodies (PF4 ELISA) • Platelet suspension immunofluorescence test (PSIFT) • Granulocyte immunofluorescence by flow cytometry (GIF) • Solid phase red cell adherence (SPRCA) assay • Monoclonal antibody immobilization of neutrophil antigens • Monoclonal immobilization of platelet antigens (MAIPA) (MAINA) • Molecular analysis for HPA-1a/1b TRALI investigations also include: For further information, contact: • HLA (PRA) Class I and Class II antibody detection Platelet Serology Laboratory (215) 451-4205 For further information, contact: Janet Demcoe (215) 451-4914 Neutrophil Serology Laboratory (651) 291-6797 [email protected] Sandra Nance (215) 451-4362 Randy Schuller (651) 291-6758 [email protected] [email protected]

American Red Cross Biomedical Services American Red Cross Biomedical Services Musser Blood Center Neutrophil Serology Laboratory 700 Spring Garden Street 100 South Robert Street Philadelphia, PA 19123-3594 CLIA licensed St. Paul, MN 55107 CLIA licensed

160 IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 Advertisements, cont.

Becoming a Specialist in Blood Banking (SBB)

What is a certified Specialist in Blood Banking (SBB)? • Someone with educational and work experience qualifications that successfully passes the American Society for Clinical Pathology (ASCP) board of certification (BOC) examination for the Specialist in Blood Banking. • This person will have advanced knowledge, skills, and abilities in the field of transfusion medicine and blood banking.

Individuals who have an SBB certification serve in many areas of transfusion medicine: • Serve as regulatory, technical, procedural, and research advisors • Perform and direct administrative functions • Develop, validate, implement, and perform laboratory procedures • Analyze quality issues preparing and implementing corrective actions to prevent and document nonconformances • Design and present educational programs • Provide technical and scientific training in transfusion medicine • Conduct research in transfusion medicine

Who are SBBs? Supervisors of Transfusion Services Executives and Managers of Blood Centers LIS Coordinators Educators Supervisors of Reference Laboratories Research Scientists Consumer Safety Officers Quality Assurance Officers Technical Representatives Reference Lab Specialists

Why become an SBB? Professional growth Job placement Job satisfaction Career advancement

How does one become an SBB? CAAHEP-accredited SBB Technology program or grandfather the exam based on ASCP education and experience criteria. Fact: In recent years, a greater percentage of individuals who graduate from CAAHEP-accredited programs pass the SBB exam compared to individuals who grandfather the exam. The BEST route for obtaining an SBB certification is to attend a CAAHEP-accredited Specialist in Blood Bank Technology Program. Which approach are you more compatible with?

Contact the following programs for more information: Additional information can be found by visiting the following Web sites: www.ascp.org, www.caahep.org, and www.aabb.org

Onsite or Program Contact Name Phone Contact Email Contact Website Online Program

Walter Reed Army Medical Center William Turcan 202-782-6210 [email protected] www.militaryblood.dod.mil/fellow Onsite American Red Cross, Southern California Region Michael Coover 909-859-7496 [email protected] none Onsite ARC-Central OH Region Joanne Kosanke 614-253-2740 x 2270 [email protected] none Onsite Blood Center of Wisconsin Lynne LeMense 414-937-6403 [email protected] www.bcw.edu Onsite Community Blood Center/CTS Dayton, Ohio Nancy Lang 937-461-3293 [email protected] www.cbccts.org/education/sbb.html Online Gulf Coast School of Blood Bank Technology Clare Wong 713-791-6201 [email protected] http://giveblood.org/index.php?page=sbb-distance-program Online Hoxworth Blood Center/University of Cincinnati Susan Wilkinson 513-558-1275 [email protected] www.hoxworth.org Onsite [email protected] Indiana Blood Center Jayanna Slayten 317-916-5186 [email protected] www.indianablood.org Online Johns Hopkins Hospital Lorraine Blagg 410-502-9584 [email protected] http://pathology.jhu.edu/department/divisions/tranfusion/ Onsite sbb2.cfm Medical Center of Louisiana Karen Kirkley 504-903-3954 [email protected] none Onsite NIH Clinical Center Department of Transfusion Medicine Karen Byrne 301-496-8335 [email protected] www.cc.nih.gov/dtm/research/sbb.html Onsite Rush University Veronica Lewis 312-942-2402 [email protected] www.rushu.rush.edu/catalog/acadprograms/chs/cls/ Online clssbbcert.html Transfusion Medicine Academic Center at Florida Blood Marjorie Doty 727-568-5433 x 1514 [email protected] www.fbsblood.org Online Services University Health System and Affiliates, San Antonio Linda Myers 210-731-5526 [email protected] www.sbbofsa.org Onsite University of Texas Medical Branch at Galveston Janet Vincent 409-772-3055 [email protected] www.utmb.edu/sbb Online University of Texas SW Medical Center LeAnne Hutson 214-648-1780 [email protected] http://utsouthwestern.edu/mls Online Revised August 2009

IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 161 Subject index Immunohematology—Volume 27, Nos. 1, 2, 3, 4, 2011

Autoantibodies SC antibodies—summary of published Autoanti-I—acute hemolytic transfusion reports ...... 27(2):41 reaction ...... 27(3):101 SC antibodies—use of recombinant Autoanti-LW—clinical significance ...... 27(4):136 reagents for detection ...... 27(2):41 SC autoantibodies ...... 27(2):41 XG antibodies—clinical characteristics . . . . . 27(2):68

Autoimmune hemolytic anemia Blood group systems/antigens SC autoantibodies ...... 27(2):41 AnWj—association with Indian blood group system ...... 27(3):89 Use of SPRCA and gel microcolumn in eluate testing ...... 27(1):1 Blood group collections ...... 27(4):131 Fya/Fyb matching of donors and patients— Biochemistry statistical analysis ...... 27(1):12 B30.2 (PRYSPRY) cytoplasmic domain— High-incidence antigens—discovery ...... 27(4):131 role in erythropoiesis ...... 27(2):41 Indian blood group system ...... 27(3):89 ERMAP—functional role ...... 27(2):41 ISBT scheme for categorizing antigens . . . . .27(4):131 ERMAP—member of butyrophilin-like Jka/Jk b matching of donors and patients— family ...... 27(2):41 statistical analysis ...... 27(1):12 Ina and Inb—CD44 molecule ...... 27(3):89 K/k matching of donors and patients— LW glycoprotein ...... 27(4):136 statistical analysis ...... 27(1):12 Xga and CD99 ...... 27(2):68 Low-incidence antigens—discovery ...... 27(4):131 LW and Rh—interdependence ...... 27(4):136 Blood components LW antigens—characteristics ...... 27(4):136 Blood preservation—early studies LW blood group system ...... 27(4):136 by Charles R. Drew ...... 27(3):94 MNSs matching of donors and patients— Blood usage in CABG surgery—predictors . . . 27(4):151 statistical analysis ...... 27(1):12 Use of group A donors as universal donors . . . 27(2):61 Radin ...... 27(2):41 Blood group antibodies SC antigens—nomenclature ...... 27(2):41 SC variants—STAR, SCER, and SCAN ...... 27(2):41 Alloantibodies to low-frequency antigens— a occurrence in sickle cell patients ...... 27(4):143 Xg and CD99 antigens ...... 27(2):68 a a Anti-AnWj—characteristics ...... 27(3):89 Xg /Yg /CD99—relationship ...... 27(2):68 Anti-AnWj—complement dependent Xg(a+)/Xg(a-)—phenotype prevalences . . . . . 27(2):68 in lymphoproliferative disease ...... 27(3):83 XG antigens—nomenclature ...... 27(2):68 Anti-Jsa—frequency in a patient population . . 27(3):104 XG antigens—characteristics ...... 27(2):68 Anti-Kpa—possible suppression of 700 series of low-incidence antigens ...... 27(4):131 fetal erythropoiesis ...... 27(2):58 700 series of low-incidence antigens— Indian antibodies—characteristics ...... 27(3):89 criteria for inclusion ...... 27(4):131 Indian antibodies—clinical significance . . . . 27(3):89 901 series of high-incidence antigens ...... 27(4):131 LW antibodies—clinical significance . . . . . 27(4):136 901 series of high-incidence antigens— SC antibodies—characteristics ...... 27(2):41 criteria for inclusion ...... 27(4):131 SC antibodies—clinical significance ...... 27(2):41

162 IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 Subject index, contd.

Case report Hemolytic transfusion reactions (HTRs) Acute hemolytic transfusion reaction Caused by autoanti-I ...... 27(3):101 caused by autoanti-I ...... 27(3):101 Use of SPRCA and gel microcolumn in Anti-AnWj—complement dependent— eluate testing ...... 27(1):1 in lymphoproliferative disease ...... 27(3):83 Laboratory management Anti-Kpa—possible suppression of fetal erythropoiesis ...... 27(2):58 Comparison of gel, tube, and SPRCA for antibody identification ...... 27(4):146 Disease associations Routine use of Js(a+) screening cells . . . . . 27(3):104 Anti-AnWj and lymphoproliferative disease . . . 27(3):83 Titering group A donors for use as XG and genetic disorders ...... 27(2):68 universal donors ...... 27(2):61

Donors Methods Analysis of pooled D-/CE+ donor samples Determination of optimal antibody by PCR-SSP to detect RHD alleles . . . . . 27(1):25 identification method ...... 27(4):146 Predicted Rh phenotype results of Mayo Gel microcolumn—use in eluate testing ...... 27(1):1 Clinic donors ...... 27(1):12 RHD alleles—reactivity with monoclonal Use of group A plasma donors as universal reagents ...... 27(1):6 donors ...... 27(2):61 Routine screening for anti-Jsa ...... 27(3):104 Weak D type 42 cases in Caucasians of Serologic testing to determine D variants . . . . .27(1):6 European descent ...... 27(1):20 SPRCA—automated—use in eluate testing . . . . .27(1):1 Genetics Titering group A donors for use as universal donors ...... 27(2):61 D variants in Brazilians ...... 27(1):6 Use of phenotypes/plasma dilutions to ERMAP gene—expression of SC antigens . . . . 27(2):41 identify specificity/thermal amplitude ERMAP variants—global population of cold antibody ...... 27(3):101 frequencies ...... 27(2):41 Use of recombinant reagents for detection a b In /In —genetics and inheritance ...... 27(3):89 of SC antibodies ...... 27(2):41 Indian phenotypes—incidence in different populations ...... 27(3):89 Molecular LW gene ...... 27(4):136 D variants and polymorphism—molecular RH genotyping in multi-generation basis ...... 27(1):6 Caucasian samples ...... 27(1):20 ERMAP—polymorphisms in coding region . . . 27(2):41 RHD*DOL and RHCE*ce(818T)— ERMAP—transcripts, exon structure, prevalence in two populations ...... 27(2):66 and variants ...... 27(2):41 SC—genetics and inheritance ...... 27(2):41 Genomic DNA analysis for cases of weak D type 42 ...... 27(1):20 SCnull ...... 27(2):41 Xga/Xg—genotype frequencies ...... 27(2):68 Ina and Inb—CD44 ...... 27(3):89 LW gene ...... 27(4):136 Hemolytic disease of the fetus and newborn (HDFN) Multiplex PCR to detect RHD gene hybrid Anti-Kpa—possible suppression of fetal alleles ...... 27(1):6 erythropoiesis ...... 27(2):58 Partial D types and associated Determination of D phenotypes to prevent in Brazilians ...... 27(1):6 anti-D–related HDFN ...... 27(1):6 PCR—to determine D variants ...... 27(1):6 Use of SPRCA and gel microcolumn in RBC phenotype matching for various eluate testing ...... 27(1):1 ethnic groups ...... 27(1):12

IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 163 Subject index, contd.

RHD alleles—detection by analysis of pooled Serology D-/CE+ donor samples by PCR-SSP . . . . 27(1):25 RHD alleles—reactivity with monoclonal RH genotyping in multi-generation reagents ...... 27(1):6 Caucasian samples ...... 27(1):20 Serologic studies of D variants in Brazilians . . . .27(1):6 SCnull phenotypes—molecular basis ...... 27(2):41 Use of phenotypes/plasma dilutions to Sequencing—to determine D variants ...... 27(1):6 identify specificity/thermal amplitude Weak D types and associated haplotypes of cold antibody ...... 27(3):101 in Brazilians ...... 27(1):6 Use of recombinant reagents for detection XG and the sex chromosomes—molecular of SC antibodies ...... 27(2):41 basis ...... 27(2):68 Transfusion Practices Reviews Anti-Jsa occurrences in a patient Indian blood group system ...... 27(3):89 population ...... 27(3):104 LW blood group system ...... 27(4):136 Blood transfusion and evolution Scianna blood group system ...... 27(2):41 of blood banking ...... 27(3):94 XG blood group system ...... 27(2):68 Combining serology and molecular testing 700 series of low-incidence antigens ...... 27(4):131 to determine D-antigen status ...... 27(1):6 901 series of high-incidence antigens ...... 27(4):131 Correlation between patient age and blood usage in CABG surgery ...... 27(4):151 Rh blood group system RBC phenotype matching for various D variants in Brazilians ...... 27(1):6 ethnic groups ...... 27(1):12 Partial D and weak D phenotypes Testing and transfusion strategy in Brazilians in Brazilians ...... 27(1):6 with D variants ...... 27(1):6 RHD*DOL and RHCE*ce(818T)—prevalence Use of group A donors as universal donors . . . 27(2):61 in two populations ...... 27(2):66 Use of fibrinogen concentration to predict Rh matching of donors and patients— blood usage in CABG surgery ...... 27(4):151 statistical analysis ...... 27(1):12 STEM antigen ...... 27(2):66 Weak D type 42 in French Canadian population ...... 27(1):20

164 IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 Contributor index Immunohematology—Volume 27, Nos. 1, 2, 3, 4, 2011

A Haywood, J.R...... 27(4):146 P, Q, R Anderson, M.A...... 27(3):83 Heaton, W.A...... 27(3):104 Pellegrino, J. Jr...... 27(1):6 Hue-Roye, K...... 27(2):58 Rahman, S...... 27(3):101 B Reid, M.E...... 27(2):58 I Badjie, K.S.W...... 27(1):12 Reid, M.E...... 27(2):66 Imlay, S...... 27(2):58 Boctor, F.N...... 27(3):104 Reid, M.E...... 27(4):131 Isaak, E.J...... 27(2):61 Borosak, M...... 27(3):83 Richard, M...... 27(1):20 Briones, R...... 27(4):151 J Brunker, P.A.R...... 27(2):41 S Jack son, P...... 27(4):143 Bryant, B.J...... 27(4):146 Slapak, C...... 27(2):61 Jenkins, S...... 27(1):12 Bryant, B.J...... 27(4):151 Smith, C...... 27(1):12 Johnson, N.C...... 27(2):68 Smith, D...... 27(2):61 C Josefy, S...... 27(4):151 St-Louise, M...... 27(1):20 Casina, T.S...... 27(4):118 K Stockman, D...... 27(2):58 Castilho, L...... 27(1):6 Stubbs, J.R...... 27(1):12 Kalal, L...... 27(2):61 Castilho, L...... 27(2):66 Kazem, E...... 27(2):58 Condon, J...... 27(3):83 T, U, V Khalife, G...... 27(2):61 Costa, D.C...... 27(2):66 Tauscher, C...... 27(1):12 Koval, K...... 27(2):58 CÔté, M...... 27(1):20 Tchorz, K.M...... 27(2):61 Credidio, D.C...... 27(1):6 L Teng, S...... 27(1):1 Tuson, M...... 27(2):58 D, E Lang, N...... 27(2):61 Van Buskirk, C...... 27(1):12 Londero, D...... 27(1):25 Davis, R.J...... 27(1):1 Vincent, J...... 27(4):118 Long, A...... 27(1):20 De Angelis, V...... 27(1):25 Lu, Q...... 27(1):1 Desai, R...... 27(2):58 W Éthier, C...... 27(1):20 M Ward, S...... 27(3):101 Willis, M.S...... 27(3):94 F Martone, J...... 27(3):104 Wilson, B.A...... 27(3):94 McCarthy, M.C...... 27(2):61 Finck, R.H...... 27(1):1 Win, N...... 27(3):101 Meny, G.M...... 27(4):117 Fiorino, M...... 27(1):25 Wong, C...... 27(1):12 Miotti, V...... 27(1):25 Flegel, W.A...... 27(2):41 Wood, E...... 27(3):83 Moulds, J.M...... 27(4):118 Flickinger, C...... 27(4):118 Moulds, M.K.G...... 27(4):118 X, Y, Z G Moulds, M.K.G ...... 27(4):136 Xu, Q...... 27(3):89 Moulds, M.K.G...... 27(4):146 Garg, G...... 27(2):58 Yuan, S...... 27(1):1 Gold, P...... 27(3):101 N, O Zanette, A...... 27(2):66 Goldfinger, D...... 27(1):1 Ziman, A.F...... 27(1):1 Nance, S...... 27(4):118 Green, K...... 27(3):83 Needs, M...... 27(3):101 Grigoriadis, G...... 27(3):83 Nikolis, N.M...... 27(3):104 H O’Connor, W.G...... 27(3):94 Halter Hipsky, C...... 27(2):66 Omoto, R...... 27(2):66 Hamilton, J...... 27(2):58

IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011 165 Immunohematology Journal of Blood Group Serology and Education Instructions for Authors

I. GENERAL INSTRUCTIONS b. Use short headings for each column needed and capitalize first letter of first Before submitting a manuscript, consult current issues of Immunohematology for style. word. Omit vertical lines. Double-space throughout the manuscript. Number the pages consecutively in the upper c. Place explanation in footnotes (sequence: *, †, ‡, §, ¶, **, ††). right-hand corner, beginning with the title page. 8. Figures II. SCIENTIFIC ARTICLE, REVIEW, OR CASE REPORT WITH a. Figures can be submitted either by e-mail or as photographs (5 ×7ʺ glossy). b. Place caption for a figure on a separate page (e.g. Fig. 1 Results of…), ending LITERATURE REVIEW with a period. If figure is submitted as a glossy, place first author’s name and A. Each component of the manuscript must start on a new page in the following figure number on back of each glossy submitted. order: c. When plotting points on a figure, use the following symbols if possible: 1. Title page l l s s n n. 2. Abstract 9. Author information 3. Text a. List first name, middle initial, last name, highest degree, position held, 4. Acknowledgments institution and department, and complete address (including ZIP code) for all 5. References authors. List country when applicable. 6. Author information 7. Tables III. EDUCATIONAL FORUM 8. Figures A. All submitted manuscripts should be approximately 2000 to 2500 words with B. Preparation of manuscript pertinent references. Submissions may include: 1. Title page 1. An immunohematologic case that illustrates a sound investigative approach with a. Full title of manuscript with only first letter of first word capitalized (bold clinical correlation, reflecting appropriate collaboration to sharpen problem solving title) skills b. Initials and last name of each author (no degrees; all CAPS), e.g., M.T. JONES, 2. Annotated conference proceedings J.H. BROWN, AND S.R. SMITH B. Preparation of manuscript c. Running title of ≤40 characters, including spaces 1. Title page d. Three to ten key words a. Capitalize first word of title. 2. Abstract b. Initials and last name of each author (no degrees; all CAPs) a. One paragraph, no longer than 300 words b. Purpose, methods, findings, and conclusion of study 2. Text 3. Key words a. Case should be written as progressive disclosure and may include the a. List under abstract following headings, as appropriate 4. Text (serial pages): Most manuscripts can usually, but not necessarily, be divided i. Clinical Case Presentation: Clinical information and differential diagnosis into sections (as described below). Survey results and review papers may need ii. Immunohematologic Evaluation and Results: Serology and molecular individualized sections testing a. Introduction — Purpose and rationale for study, including pertinent iii. Interpretation: Include interpretation of laboratory results, correlating background references with clinical findings b. Case Report (if indicated by study) — Clinical and/or hematologic data and iv. Recommended Therapy: Include both transfusion and nontransfusion- background serology/molecular based therapies c. Materials and Methods — Selection and number of subjects, samples, items, v. Discussion: Brief review of literature with unique features of this case etc. studied and description of appropriate controls, procedures, methods, vi. Reference: Limited to those directly pertinent equipment, reagents, etc. Equipment and reagents should be identified in vii. Author information (see II.B.9.) parentheses by model or lot and manufacturer’s name, city, and state. Do not viii. Tables (see II.B.7.) use patient’s names or hospital numbers. IV. LETTER TO THE EDITOR d. Results — Presentation of concise and sequential results, referring to pertinent tables and/or figures, if applicable A. Preparation e. Discussion — Implication and limitations of the study, links to other studies; if 1. Heading (To the Editor) appropriate, link conclusions to purpose of study as stated in introduction 2. Title (first word capitalized) 5. Acknowledgments: Acknowledge those who have made substantial contributions 3. Text (written in letter [paragraph] format) to the study, including secretarial assistance; list any grants. 4. Author(s) (type flush right; for first author: name, degree, institution, address 6. References [including city, state, Zip code and country]; for other authors: name, degree, a. In text, use superscript, Arabic numbers. institution, city and state) b. Number references consecutively in the order they occur in the text. 5. References (limited to ten) 7. Tables 6. Table or figure (limited to one) a. Head each with a brief title; capitalize the first letter of first word (e.g., Table 1. Results of…) use no punctuation at the end of the title. Send all manuscripts by e-mail to [email protected]

166 IMMUNOHEMATOLOGY, Volume 27, Number 4, 2011

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