Manual 512402

คู่มอื การฝึกปฏบิ ตั งิ าน Laboratory Practice in Transfusion Science (512402) แขนงวชิ าวทิ ยาศาสตร์การบรกิ ารโลหติ ภาควชิ าเทคนิคการแพทย์ คณะเทคนิคการแพทย์ มหาวทิ ยาลยั เชยี งใหม่ พ.ศ. 2562

คาํ นํา การปรบั ปรุงหลกั สูตรวทิ ยาศาสตรบณั ฑติ สาขาวชิ าเทคนิคการแพทย์ 2558 มกี ารควบรวม กระบวนวชิ าฝึกปฏบิ ตั งิ านสองกระบวนเขา้ ด้วยกนั เป็นหนึhงกระบวนคอื การฝึกปฏบิ ตั งิ านทาง วทิ ยาศาสตร์การบรกิ ารโลหติ (Laboratory Practice in Transfusion Science 512402) แขนงวชิ าฯ จงึ ปรบั ปรุงรูปแบบการเรยี นการสอนและการฝึกปฏบิ ตั งิ านใหม่ ประกอบด้วย การฝึกปฏบิ ตั งิ านภายใน หอ้ งปฏบิ ตั กิ ารแขนงวชิ าฯ ด้วยตวั อย่างผูป้ ่ วยซึhงได้รบั ความอนุเคราะห์จากหน่วยงานธนาคารเลอื ด โรงพยาบาลมหาราชนครเชยี งใหม่ และการปฏบิ ตั กิ ารเทคนิคพเิ ศษในงานวทิ ยาศาสตร์การบรกิ ารโลหติ และกําหนดช่วงเวลาใหม้ กี ารฝึกปฏบิ ตั งิ านในสถานทีhจรงิ ณ หน่วยงานธนาคารเลอื ด โรงพยาบาล มหาราชนครเชยี งใหม่ และกาชาดภาคทhี 10 จงั หวดั เชยี งใหม่ เพhอื ใหน้ ักศกึ ษาได้ประสบการณ์จรงิ ในการ ปฏบิ ตั งิ านร่วมกบั นักเทคนิคการแพทย์ นอกจากนrี นักศกึ ษายงั ได้ฝึกทกั ษะการอ่าน เตรยี มสhอื และการ นําเสนอหวั ขอ้ ทhไี ด้รบั มอบหมายเป็นภาษาองั กฤษ ซhงึ เน้นใหผ้ ูเ้ รยี นได้มสี ่วนร่วมในการวพิ ากษ์ในชนัr เรยี น มากยงhิ ขนrึ คู่มอื ฉบบั นีrรวบรวมความรูค้ วามเขา้ ใจเนืrอหาในเรืhอง Blood donation eligibility guidelines, Blood drawing, ABO and H blood group systems and secretor status, Pathogen inactivation, Molecular applications in transfusion Science, Donor processing, Blood component preparation Pretransfusion test, Transfusion reaction และ Transfusion reaction investigation จากหนังสอื ด้าน วทิ ยาศาสตร์การบรกิ ารโลหติ ภูมคิ ุม้ กนั โลหติ วทิ ยา และ American Association of Blood Bank (AABB) ซhึงเป็นมาตรฐานการปฏบิ ตั งิ านด้านวทิ ยาศาสตร์การบรกิ ารโลหติ ทhไี ด้รบั การยอมรบั ทวั h โลก โดยรวบรวม จากต้นฉบบั ภาษาองั กฤษ เพืhอใหน้ ักศกึ ษาได้ทบทวนความรู้ วเิ คราะห์ สงั เคราะห์ และสามารถสรุป เนrือหานําเสนอได้อย่างถูกต้อง โดยมคี ณาจารย์ร่วมวพิ ากษ์และใหข้ อ้ เสนอแนะ รศ. ดร. ปรยี านาถ วงศ์จนั ทร์ ผูร้ วบรวม

สารบญั หน้ า Chapter 1: Blood donation eligibility guidelines 1 Chapter 2: Blood drawing 15 Chapter 3: ABO and H blood group systems and secretor status 20 Chapter 4: Molecular applications in Transfusion Science 50 Chapter 5: Pathogen inactivation 61 Chapter 6: Noninfectious complications of blood transfusion 67 Chapter 7: Case studies in Transfusion Science Chapter 8: Protocols in elution techniques 104 Report form 118 Memo pages 128 136

CHAPTER 1: BLOOD DONATION ELIGIBILITY GUIDELINES GENERAL GUIDELINES To give blood for transfusion to another person, you must be healthy, be at least 17 years old or 16 years old if allowed by state law, weight at least 110 lbs, and not have donated blood in the last 8 weeks (56 days). “Healthy” means that you feel well and can perform normal activities. If you have chronic conditions such as diabetes or high blood pressure, “healthy” also means that you are being treated and the condition is under control. Other aspects of each potential donor’s health history are discussed as part of the donation process before any blood is collected. Each donor receives a brief examination during which temperature, pulse, blood pressure and blood count (hemoglobin or hematocrit) are measured. Making donations for your own use during surgery (autologous blood donation) is considered a medical procedure and the rules for eligibility are less strict than for regular volunteer donations. Acupuncture Hepatitis, Jaundice Age Hepatitis exposure Allergy, stuffy nose, itchy eyes, dry cough Herpes (see Sexually Transmitted Diseases) Antibiotics HIV, AIDS Aspirin Hormone Replacement Therapy (HRP) Asthma HPV (see Sexually Transmitted Diseases) Birth control Hypertension, High blood pressure Bleeding disorders Immunization, Vaccination Blood pressure, High Infections Blood pressure, Low Insulin (Bovine) Blood transfusion Intravenous Drug Use Cancer Malaria Chronic illnesses Medications Cold, Flu Organ/Tissue Transplants Creutzfeldt-Jacob disease (CJD) Piercing (ears, body), Electrolysis Creutzfeldt-Jacob disease, Variant (vCJD); Pregnancy, Nursing “Mad Cow disease” Sexually Transmitted Diseases Dental procedures Sickle cell Donation intervals Syphilis/Gonorrhea Heart diseases Tattoo Heart Murmur, Heart Valve Disorders Tuberculosis Hemochromatosis Travel outside of U.S., Immigration Hemoglobin, hematocrit, blood count Vaccination Venereal diseases Weight 1

Acupuncture Donors who have undergone acupuncture treatments are acceptable as long as the donor can confirm that the needles used in the treatment were sterile. Donors who cannot confirm that sterile needles were used in the acupuncture treatment are deferred from donating for 12 months. Age You must be at least 17 years old to donate to the general blood supply, or 16 years old if allowed by state law. There is no upper age limit for blood donation as long as you are well with no restriction or limitation to your activities. Allergy, Stuffy nose, Itchy eyes, Dry cough Acceptable as long as you feel well, have no fever, and have no problems breathing through your mouth. Antibiotics A donor with an infection should not donate. The reason for antibiotics used must be evaluated to determine if the donor has a bacterial infection that could be transmissible by blood. Acceptable after finishing antibiotics for an infection (bacterial or viral). Acceptable if you are taking antibiotics to prevent an infection, for example, following dental procedures or for acne. Antibiotics for acne do not disqualify you from donating. If you have a temperature above 99.5 oF, you may not donate. Aspirin See “Medications” Asthma Acceptable as long as you are not having difficulty breathing at the time of donation and you otherwise feel well. Medications for asthma do not disqualify you from donating. Birth control Women taking birth control (pills or injections) are acceptable. Blood pressure, High Acceptable as long as your blood pressure is below 180 systolic (first number) and below 100 diastolic (second number) at the time of donation. Medications for high blood pressure do not disqualify you from donating. Blood pressure, Low Acceptable as long as you feel well when you come to donate. If your blood pressure normally runs low, it may be more difficult for your body to adjust to the volume loss following donation, especially if you are dehydrated. Drinking extra water before and after donation is important. 2

Blood transfusion Wait for 12 months after receiving a blood transfusion from another person in the US. You may not donate if you received a blood transfusion since 1980 in the UK (England, Wales, Scotland, Northern Ireland, Channel Islands, Isle of Man), Gibraltar or Falkland Islands. This requirement is related to concerns about variant CJD, or “Mad Cow Disease”. You may not donate if you received a blood transfusion in certain countries in Africa since 1977. This requirement is related to concerns about rare strain of HIV that are not consistently detected by all current test methods. Cancer Eligibility depends on the type of cancer and treatment history. If you had leukemia or lymphoma, including Hodgkin’s Disease, you are not eligible to donate. Other types of cancer are acceptable if the cancer has been treated successful and it has been at least 5 years since treatment was completed and there has been no cancer recurrence in this time. Some low-risk cancer including squamous or basal cell cancers of the skin do not require a 5-year waiting period. Precancerous conditions of the uterine cervix do not disqualify you from donation if the abnormality has been treated successfully. You should discuss your particular situation with the health historian at the time of donation. Chronic illnesses Most chronic illnesses are acceptable as long as you feel well. The condition is under good control, you have an adequate hemoglobin level and your temperature is normal when you come to donate, and you meet all other eligibility requirements. Bleeding condition If you have a history of bleeding problems, you will be asked additional questions. If your blood does not clot normally, you should not donate since you may have excessive bleeding where the needle was placed. For the same reason, if you are taking any “Blood thinner” (such as coumadin or heparin) you should not donate. If you are on aspirin, it is OK to donate blood. However, you must be off of aspirin for at least 48 hours in order to donate platelets by apheresis. Cold, Flu Wait if you have a fever or a productive cough (bringing up phlegm). Wait if you do not feel well on the day of donation. Wait until you have completed antibiotic treatment for sinus, throat or lung infection. Creutzfeldt-Jacob disease (CJD) If you ever received a dura mater (brain covering) transplant or human pituitary growth hormone, you are not eligible to donate. Those who have a blood relative Creutzfeldt-Jacob disease are also not eligible to donate. 3

Creutzfeldt-Jacob disease, Variant (vCJD); “Mad Cow disease” See under Travel outside of US. Dental procedures and Oral Surgery Acceptable after dental procedures as long as there is no infection present. Wait until finishing antibiotics for a dental infection. Wait for 3 days after having oral surgery. Insulin Those who since 1980, received an injection of bovine (beef) insulin made from cattle from the UK are not eligible to donate. This requirement is related to concerns about vCJD, or Mad Cow disease. Donation intervals Wait at least 8 weeks between whole blood (standard) donations. Wait at least 3 days between plateletpheresis donations. Wait at least 16 weeks between double red cell (automated) donations. Heart disease In general, acceptable as long as you have been medically evaluated and treated, have no current (within the last 6 months) heart related symptoms such as chest pain and have no limitation or restriction on your normal daily activities. Wait at least 6 months following an episode of angina. Wait at least 6 months following a heart attack. Wait at least 6 months after bypass surgery or angioplasty. If you have a pacemaker, you may donate as long as your pulse is between 50 and 100 beats/minute with no more than a small number of irregular beats, and you meet the other heart disease criteria. You should discuss your particular situation with the health historian at the time of donation. Heart Murmur, Heart Valve Disorder Acceptable if you have a heart murmur as long as you have been medically evaluated and treated and have not had symptoms in the last 6 months, and no restriction on your normal daily activities. Hemochromatosis (Hereditary) American Red Cross does not accept individuals with hemochromatosis as blood donors for other persons at this time. However, we are currently planning a pilot program for hemochromatosis donors in one of our regions that will be evaluated for possible system wide implementation. The pilot program is expected to be completed by the end of 2005. 4

Hemoglobin, Hematocrit, Blood Count Acceptable if you have a hemoglobin at or above 12.5 g/dL. Acceptable if you have a hematocrit at or above 38%. Hepatitis, Jaundice If you had hepatitis (inflammation of the liver) caused by a virus, or unexplained jaundice (yellow discoloration of the skin), since age 11, you are not eligible to donate blood. This includes those who had hepatitis with Cytomegalovirus (CMV), or Epstein-Barr Virus (EBV), the virus that causes Mononucleosis. Acceptable if you had jaundice or hepatitis caused by something other than a viral infection, for example: medications, Gilbert’s disease, bile duct obstruction, alcohol, gall stones or trauma to the liver. If you ever tested positive for hepatitis B or C, at any age, you are not eligible to donate, even if you were never sick or jaundiced from the infection. Hepatitis exposure If you live or have had sexual contact with a person who has hepatitis, you must wait 12 months after the last contact. Persons who have been detained or incarcerated in a facility (juvenile detention, lockup, jail, or prison) for more than 72 consecutive hours (3 days) are deferred for 12 months from the date of last occurrence. This includes work release programs and weekend incarceration. These persons are at higher risk for exposure to infectious diseases. Wait 12 months after receiving a blood transfusion (unless it was your own “autologous” blood), non-sterile needle stick/body piercing or exposure to someone else’s blood. Wait 12 months following a human bite, if it broke the skin. HIV, AIDS You should not give blood if you have AIDS or have ever had a positive HIV test, or if you have done something that puts you at risk for becoming infected with HIV. You are at risk for getting infected if you: § have ever used needles to take drugs, steroids, or anything not prescribed by your doctor § are a male who has had sexual contact with another male even once, since 1977 § have ever taken money, drugs or other payments for sex since 1977 § have had sexual contact in the past 12 months with anyone described above § received clotting factor concentrates for a bleeding disorders such as hemophilia § were born in, or lived in, Cameroon, Central Africa Republic, Chad, Congo, Equatorial Guinea, Gabon, Niger, or Nigeria, since 1977 § since 1977, received a blood transfusion or medical treatment with a blood product in any of these countries, or § had sex with anyone who, since 1977, as born in or lived in any of these countries. 5

You should not give blood if you have any of the following conditions that can be signs or symptoms of HIV/AIDS: § unexplained weight loss (10 lbs or more in less than 2 months) § night sweats § blue or purple spots in your mouth or skin § white spots or unusual sores in your mouth § lumps in your neck, armpits, or groin, lasting longer than 1 month § diarrhea that won’t go away § cough that won’t go away and shortness of breath, or § fever higher than 100.5 oF lasting more than 10 days. Hormone Replacement Therapy (HRP) Women on HRP for menopausal symptoms and prevention of osteoporosis are eligible to donate. Hypertension, High blood pressure See “Blood pressure, High” Immunization, Vaccination Acceptable if you were vaccinated for influenza, tetanus, or meningitis, providing you are symptom-free and fever-free. Wait 4 weeks after immunization for German Measles (Rubella), MMR (Measles, Mumps and Rubella) and Chicken Pox. Wait 2 weeks after immunization for Red Measles (Rubella), Mumps, Polio (By mouth), and Yellow Fever vaccine. Wait 7 days after immunization for Hepatitis B as long as you are not given the immunization for exposure to hepatitis B § Smallpox vaccination and did not develop complications Wait 8 weeks (56 days) from the date of having a smallpox vaccination as long as you have had no complications. Complications may include skin reactions beyond the vaccination site or general illness related to the vaccination. § Smallpox vaccination and developed complications Wait 2 weeks after all vaccine complications have resolved or 8 weeks from the date of having had the smallpox vaccination whichever the longer of time. You should discuss your particular situation with the health historian at the time of donation. Complications may include skin reactions beyond the vaccination site or general illness related to the vaccination. § Smallpox vaccination-close contact with someone who has had the smallpox vaccine in the last 8 weeks and you did not develop any skin lesions or other symptoms Eligible to donate 6

§ Smallpox vaccination-close contact with someone who has had the smallpox vaccine in the last 8 weeks and you did not develop any skin lesions or other symptoms Wait 8 weeks from the date of the first skin lesion or sore. You should discuss your particular situation with the health historian at the time of donation. Complications may include skin reactions or general illness related to the exposure. Infections If you have a fever or an active infection, wait until the infection has resolved completely before donating blood. Wait until finished taking antibiotics for an infection (bacterial or viral). Those who have had antibiotics with Chagas Disease or Babesiosis are not eligible to donate. Intravenous drug use Those who have ever used IV drugs that were not prescribed by a physician are not eligible to donate. This requirement is related to concerns about hepatitis and HIV. Malaria Wait 3 years after completing treatment for malaria. Wait 12 months after returning from a trip to an area where malaria is found. Wait 3 years after living in a country where malaria is found. Medications In almost all cases, medications will not disqualify you as a blood donor. Your eligibility will be based on the reason that the medication was prescribed. As long as the condition is under control and you are healthy, blood donation is usually permitted. There are a handful of drugs that are special significance in blood donation. Persons on these drugs have waiting periods following their last dose before they can donate blood: § Accutane, Amnesteem, Claravis or Sotret (Isoretinoin), Proscar (Finasteride), and Propecia (Finasteride) – wait 6 months from the last dose. § Avodart (Dutasteride) – wait 6 months from the last dose. § Aspirin, no waiting period for donating blood. However, you must wait 48 hours after taking aspirin or any medication containing aspirin before donating platelets by apheresis. § Clopidogrel – wait 7 days after taking this medication before donating platelets by apheresis. § Coumadin (Warfarin), heparin or other prescription blood thinners – you should not donate since your blood will not clot normally. If your doctor discontinues your treatment with blood thinners, wait 7 days before returning to donate. § Hepatitis B Immune globulin–given for exposure to hepatitis - wait 12 months after exposure to hepatitis. 7

§ Human pituitary-derived growth hormone at any time – you are not eligible to donate blood. § Plavix – wait 7 days after taking this medication before donating platelets by apheresis. § Soriatane (Acitretin) – wait 3 years. § Tegison (Etretinate) at any time – you are not eligible to donate blood. § Ticlid – wait 7 days after taking this medication before donating platelets by apheresis. § Ticlopidine – wait 7 days after this medication before donating platelets by apheresis. If you ever took Tegison (Etretinate), you are not eligible to donate blood. If you ever took human pituitary-derived growth hormone, you are not eligible to donate blood. If you take aspirin, you can donate blood. However, you must wait 48 hours after taking aspirin or any medication containing aspirin before donating platelets by apheresis. If you take Ticlid or Plavix, wait 7 days after taking these medications before donating platelets by apheresis. If you are taking prescription blood thinners such as Coumadin or heparin, you should not donate since your blood will not clot normally. If your doctor discontinues your treatment with blood thinners, wait 7 days before returning to donate. Organ/tissue transplants Wait 12 months after receiving a kidney transplant or tissue transplant from another person. If you are taking medications to prevent rejection of the kidney or tissue you are not eligible to donate. You are not eligible if you have had any other type of organ transplant regardless of medications to prevent rejection. If you ever received a dura mater (brain covering) transplant, you are not eligible to donate. This requirement is related to concerns about the brain disease, CJD. Piercing (ears, body), Electrolysis Acceptable as long as the instruments used were sterile, one time used. Wait 12 months if there is any question whether or not the instrument used were sterile and free of blood contamination. This requirement is related to concerns about hepatitis. Pregnancy, nursing Persons who are pregnant are not eligible to donate. Wait 6 weeks after giving birth. 8

Sexually transmitted diseases Wait 12 months after treatment for syphilis or gonorrhea. Acceptable if it has been more than 12 months since you completed treatment for syphilis or gonorrhea. Clamydia, venereal warts (human papilloma virus), all genital herpes are not a cause for deferral if you are feeling healthy and well and meet all other eligibility requirements. Sickle cell Acceptable if you have sickle cell trait. Those with sickle cell disease are not eligible to donate. Skin disease, rash, acne Acceptable as long as the skin over the vein to be used to collect blood is not affected. If the skin disease has become infected, wait until the infection has cleared before donating. Taking antibiotics to control acne does not disqualify you from donating. Syphilis/gonorrhea Wait 12 months after being treated for Syphilis or gonorrhea. Tattoo Wait 12 months after a tattoo if the tattoo was applied in a state that does not regulate tattoo facilities. This requirement is related to concerns about hepatitis. Learn more about hepatitis and blood donation. Acceptable if the tattoo was applied by a state-regulated entity using sterile technique. Only a few states currently regulate tattoo facilities, so most donors with tattoo must wait 12 months after tattoo application before donating blood. You should discuss your particular situation with the health historian at the time of donation. Tuberculosis If you have active tuberculosis or are being treated for active tuberculosis you should not donate. Acceptable if you have a positive skin test, but no active tuberculosis, or if you are receiving antibiotics for a positive TB skin test only. If you are being treated for a tuberculosis infection, wait until treatment is successfully completed before donating. Travel outside of US., Immigration Wait 12 months after travel in an area where malaria is found. Wait 3 years after living in a country where malaria is found. Persons who have spent long period of time in countries where “mad Cow Disease” is found are not eligible to donate. This requirement is related to concerns about vCJD. 9

Persons who were born in or who lived in certain countries in Western Africa, or who have had closed contact with persons who were born in or who lived in certain West Africa countries are not eligible to donate. This requirement is related to concerns about HIV group O. Venereal diseases See also “sexually transmitted disease” Wait 12 months after treatment for syphilis or gonorrhea. Clamydia, venereal warts (human papilloma virus), or genital herpes are not a cause for deferral if you are feeling healthy and well and meet all other eligibility requirements Weight You must wait at least 110 lbs to be eligible for blood donation for your own safety. Blood volume is in proportion to body weight. Donors who weigh less than 110 lbs may not tolerate to removal of the required volume of blood as well as those who weight more than 110 lbs. there is no upper weight limit as long as your weight is not higher than the weight limit of the donor bed/lounge you are using. You can discuss any upper weight limitations of beds and lounges with your local health historian. 10

Full-Length Donor History Questionnaire Yes NO Are you 1. Feeling healthy and well today? 2. Currently taking an antibiotic? 3. Currently taking any other medication for an infection? Please read the Medication Deferral List 4. Are you now taking or have you ever taken any medications on the Medication Deferral List? 5. Have you read the educational materials? In the past 48 hours 6. Have you taken aspirin or anything that has aspirin in it? In the past 6 weeks I am male 7. Female donors: have you been pregnant or are you pregnant now? (Males: check “I am male”) In the past 8 weeks have you 8. Donated blood, platelets or plasma? 9. Had any vaccinations or other shots? 10.Had contact with someone who had a smallpox vaccination? In the past 16 weeks 11. Have you donated a double unit of red cells using an apheresis machine? In the past 12 months have you 12. Had a blood transfusion? 13. Had a transplant such as organ, tissue, or bone marrow? 14. Had a graft such as bone or skin? 15. Come into contact with someone else’s blood? 11

Full-Length Donor History Questionnaire (cont.) 16. Had an accidental needle-stick? 17. Had sexual contact with anyone who has HIV/AIDS or has had a positive test for the HIV/AIDS virus? 18. Had sexual contact with a prostitute or anyone else who takes money or drugs or other payment for sex? 19. Had sexual contact with anyone who has ever used needles to take drugs or steroids, or anything not prescribed by their doctor? 20. Had sexual contact with anyone who has hemophilia or has used clotting factor concentrates? 21. Female donors: had sexual contact with a male who has ever had sexual contact with another male? I am (Males: check “I am male”) male 22. Had sexual contact with a person who has hepatitis? 23. Lived with a person who has hepatitis? 24. Had a tattoo? 25. Had ear or body piercing? 26. Had or been treated for syphilis or gonorrhea? 27. Been in juvenile detention, lockup, jail, or prison for more than 72 hours? In the past 3 years have you 28. Been outside the US or Canada? From 1980 through 1996 29. Did you spend time that adds up to 3 months or more in the UK? (Review list of countries in the UK) 30. Were you a member of the US military, a civilian military employee, or a dependent of a member of the US military? From 1980 through the present, did you 31. Spend time that adds up to 5 years or more in Europe? (Review list of countries in Europe.) 32. Receive a blood transfusion in the UK? (Review list of countries in Europe.) 12

Full-Length Donor History Questionnaire (cont.) From 1977 through the present, have you 33. Received money, drugs, or other payment for the sex? 34. Male donors: had sexual contact with another male, I am even once? (Female: check “I am female”) female Have you EVER 35. Had a positive test for the HIV/AIDS virus? 36. Used needles to take drugs, steroids, or anything not prescribed by your doctor? 37. Used clotting factor concentrates? 38. Had hepatitis? 39. Had malaria? 40. Had Chagas’ disease? 41. Had Babesiosis? 42. Received a dura mater (or brain covering) graft? 43. Had any type of cancer, including leukemia? 44. Had any problems with your heart or lung? 45. Had a bleeding condition on a blood disease? 46. Had sexual contact with anyone who was born in or lived in Africa? 47. Been in Africa? 48. Have any of your relatives had CJD disease? Source: AABB last modified on 4/12/2006 13

Full-Length Donor History Questionnaire Yes No Use this area for additional questions http://www.aabb.org/documents/Donate_Blood/Donor_History_Questionnaire/udhqmeddef05.dochttp://aabb.org/ Content/Donate_Blood/Donor_History_Questionnaire/dhq.htm ……………………………………………………………………… Reference Silva MA, ed. Standards for blood banks and transfusion services. 23rd edition. Bethesda, MD. AABB 2005. 14

CHAPTER 2: BLOOD DRAWING Donation of 450 mL limits loss to 13% of blood volume individuals weighing 50 kg or more. The area for venipuncture, typically an antecubital vein, must be prepared by a two-step scrub shown to remove bacteria from the area. Vein selection should ensure that a fairly straight, smooth, pliable, resilient vein, preferably in the center, is chosen (Figure 2.1). Figure 2.1 Superficial veins of the left upper limb The skin may be marked, but the vein can only be palpated before the area is prepared. Iodine compounds are the most effective for preparation. The area is scrubbed for a set period of time with a set period of time for drying to ensure antibacterial action. A skin preparation validated for removing bacteria from the venipuncture site should be used. The vein is distended by tourniquet and donor squeezing action. Air contamination is prevented by clamping the tubing before removing the exterior cap. The clamp is removed after needle is inserted and the cap cannot be replaced after removal. The needle should be handled gently to avoid removal of the anticoagulant filling the tubing, a safe-guard against contamination by organism in the air. A two- part venipuncture in which the needle is inserted at a 30o angle 1/2 inch below the intended point of vein entry under the skin, and then realigned for a second thrust into the vein prevents skin cores from developing. Collection of blood should be accomplished in approximately 10 minutes, with periodic agitation to prevent clotting. Long donation should be marked “difficult” so that coagulation components are not made from them. The tubing is reclamped before the needle is removed so that air is not drawn into the blood bag. Samples are generally taken into pilot tubes for testing. Undiluted sampling system, straight-line tubing, or in-line needles are used. Then the 15

tubing is scaled, and the needle is disposed of without recapping. The donor holds firm pressure over the venipuncture for some time before a pressure bandage is applied (3-5 minutes), and the donor spends approximately 15 minutes in a canteen with liquid refreshment and nourishment to replace blood volume. Post donation instructions include increased fluid for 24-48 hours, maintaining pressure dressing for at least 5 hours, lying down if dizzy and reapplying pressure if bleeding occurs. Criteria for vein selection Size § Diameter must be equivalent to or greater than a 16-guage needle. Location § Center veins are normally the first choice for blood collection. These veins are usually deeper, well anchored and provide a better blood flow than the superficial side vein. § Veins on the outer aspect of the arms may lack tissue support, making it difficult to position the needle. § Veins that lie diagonally over the inner aspect of the arm may also lack good tissue support and may lie over bone and muscle. § Remember the most visible vein is not necessarily the best vein. Condition § Determine condition by palpating along the length of the vein § Veins should feel smooth, pliable and resilient § The vein should feel firm, having adequate turgor to support blood flow. Turgor can be evaluated by pressing firmly along the vein and feeling resilience or a “bounding back” sensation. § Sclerotic veins feel core-like and roll easily § Bumpy veins may indicate that the vein is thrombosed or extremely valvular. Pushing a needle through a valve may cause the donor pain. Direction § The vein should be fairly straight so it can accept the insertion of the needle. Adverse donor reaction Most donors tolerate giving blood very well, but adverse reactions occur occasionally. Personnel must be trained to recognize adverse reactions and to provide initial treatment. Donor room personnel should be trained in cardiopulmonary resuscitation (CPR). Special equipment to handle emergency situation must be available. Syncope (fainting or vasovagal syndrome) may be caused by the sight of blood. By watching others give blood or by individual or group excitement; it may also happen for unexplained reasons. Whether caused by psychologic factors or by neurophysiologic response to blood donation, the symptoms may include weakness, sweating, dizziness, pallor, loss of consciousness, convulsions, and involuntary passage of feces or urine. On occasion, the skin feels cold and blood pressure falls. Sometimes, the systolic blood 16

pressure levels fall as low as 50 mmHg or cannot be heard with the stethoscope. The pulse rate often slows significantly. This can be useful indistinguishing between vasovagal attack and cardiogenic or hypovolemic shock, in which cases the pulse rate rises. This distinction, although characteristic, is far from absolute. Rapid breathing or hyperventilation may cause the anxious or excited donor to lose excessive amount of carbon dioxide. This may cause generalized sensation of suffocation of anxiety, or localized problems such as tingling or twitching. The donor center physician must provide written instruction for handling donor reactions, including a procedure for obtaining emergency medical help. Sample instruction might be as follows: 1. General a) Remove the tourniquet and withdraw the needle from the arm if signs of a reaction occur during the phlebotomy. b) If possible, remove any donor who experiences and adverse reaction to an area where he or she can be attended in privacy. c) Apply the measures suggested below and, if they do not lead to rapid recovery, call the blood bank physician or the physician designated for such purposes. 2. Fainting a) Apply cold compresses to the donor’s forehead or the back of the neck. b) Place the donor on his or her back, with their legs raised above the level of the head. c) Loosen tight clothing d) Be sure the donor has an adequate airway. e) Monitor blood pressure, pulse, and respiration periodically until the donor recovers. Note: some donors who experience prolonged hypotension may respond to an infusion of normal saline. The decision to initiate such therapy should be made by the donor center physician either on a case-by-case basis or in a policy stated in the facility’s SOP manual. 3. Nausea and vomiting a) Make the donor as comfortable as possible. b) Instruct the donor who is nauseated to breathe slowly and deeply. c) Apply cold compresses to the donor’s forehead and/or back of neck. d) Turn the donor’s head to the side. e) Provide a suitable receptacle if the donor vomits and have cleansing tissue or a damp towel ready. Be sure the donor’s head is turned to the side because of the danger of aspiration. f) After vomiting has ended, give the donor some water to rinse out his or her mouth. 17

4. Twitching or muscular spasms. Extremely nervous donor may hyperventilate, causing faint muscular twitching or titanic spasm of their hands or face. Donor room personnel should watch closely for these symptoms during and immediately after the phlebotomy. a) Divert the donor attention by engaging in conversation to interrupt the hyperventilation pattern. b) Have the donor cough if he or she is symptomatic. Do not give oxygen. 5. Hematoma during or after phlebotomy a) Remove tourniquet and the needle from the donor’s arm. b) Place 3 or 4 sterile gauze squares over the venipuncture site and apply firm digital pressure for 7-10 minutes, with the donor’s arm held above the heart level. An alternative is to apply a tight bandage, which should be removed after 7-10 minutes to allow inspection. c) Apply ice to the area for 5 minutes, if desired. d) Should an arterial puncture be suspected, immediately withdraw needle and apply firm pressure for 10 minutes. Apply pressure dressing afterward. Check for the presence of a radial pulse. If pulse is not palpable or is weak, call a donor center physician. 6. Convulsions a) Call for help immediately. Prevent the donor from injuring himself or herself. During severe seizures, some people exhibit great muscular power and are difficult to restrain. If possible, hold the donor on the chair or bed; if not possible, place the donor on the floor. Try to prevent injury to the donor and to yourself. b) Be sure the donor has an adequate airway. A padded device should separate the jaws after convulsion has ceased. c) Notify the donor center physician. 7. Serious cardiac difficulties a) Call for medical aid and/or an emergency care unit immediately. b) If the donor is in cardiac arrest, begin CPR immediately and continue it until help arrives. The nature and treatment of all reactions should be recorded on the donor’s record or a special incidence report form. This should include a notation of whether the donor should be accepted for future donation. 18

The medical director should decide what emergency supplies and drugs should be in the donor area. The distance to the nearest to the emergency room or emergency care unit heavily influences decisions about necessary supplies and drugs. Most donor centers maintain some or all of the following: 1. Emesis basin or equivalent 2. Towels 3. Oropharyngeal airways, plastic or hand rubber 4. Oxygen and mask 5. Emergency drugs: drugs are seldom required to treat a donor’s reaction. If the donor center physician wishes to have any drugs available, the kind and amount to be kept on hand must be specified in writing. In addition, the medical director must provide written policy starting when and by whom any of the above medical supplies or drugs may be used. …………………………………………………………………………. Reference Silva MA, Ed. Allogeneic donor selection and blood collection. Standards for Blood Banks and Transfusion Services. 23rd Ed. Bethesda, MD: AABB, 2005; 107-109. 19

OVERVIEW OF THE MAJOR BLOOD GROUPS PART II ABO and H Blood Group Systems 4  and Secretor Status CHAPTER OUTLINE Human Anti-A,B from Group O Individuals Anti-A1 SECTION 1: HISTORICAL OVERVIEW OF ABO BLOOD SECTION 5: ABO BLOOD GROUP SYSTEM AND GROUP SYSTEM SECTION 2: ABO AND H BLOOD GROUP SYSTEM TRANSFUSION ANTIGENS Routine ABO Phenotyping Selection of ABO-Compatible Red Blood Cells and General Characteristics of ABO Antigens Plasma Products for Transfusion Inheritance and Development of A, B, and H Antigens SECTION 6: RECOGNITION AND RESOLUTION OF ABO Common Structure for A, B, and H Antigens Development of H Antigen DISCREPANCIES Development of A and B Antigens Technical Considerations in ABO Phenotyping ABO Subgroups Sample-Related ABO Discrepancies Comparison of A1 and A2 Phenotypes Additional Subgroups of A and B ABO Discrepancies Associated with Red Cell Testing Importance of Subgroup Identification in Donor ABO Discrepancies Associated with Serum or Plasma Testing SECTION 3: GENETIC FEATURES OF ABO BLOOD Testing GROUP SYSTEM SECTION 4: ABO BLOOD GROUP SYSTEM ANTIBODIES SECTION 7: SPECIAL TOPICS RELATED TO ABO AND H General Characteristics of Human Anti-A and Anti-B BLOOD GROUP SYSTEMS Immunoglobulin Class Classic Bombay Phenotype Hemolytic Properties and Clinical Significance Secretor Status In Vitro Serologic Reactions LEARNING OBJECTIVES 11. Describe the ABO blood group system antibodies with regard to immunoglobulin class, clinical significance, and On completion of this chapter, the reader should be able to: in vitro serologic reactions. 1. Define a blood group system with regard to blood group 12. Discuss the selection of whole blood, red blood cell, and antigens and their inheritance. plasma products for transfusions. 2. Explain Landsteiner’s rule. 13. Define the terms universal donor and universal recipient 3. List the cells, body fluids, and secretions where ABO as they apply to red blood cell and plasma products. antigens can be located. 14. Apply concepts of ABO compatibility in the selection of 4. Describe the relationships among the ABO, H, and blood products for recipients. Se genes. 15. List the technical errors that may result in an ABO 5. Differentiate between type 1 and type 2 oligosaccharide discrepancy. structures, and state where each is located. 16. Define the acquired B antigen and the B(A) phenotypes; 6. Describe the formation of the H antigen from the interpret the ABO discrepancies that would result from these phenotypes and methods used in resolving these gene product and its relationship to ABO antigen discrepancies. expression. 7. List the glycosyltransferases and the immunodominant 17. List reasons for missing or weakly expressed ABO sugars for the A, B, O, and H alleles. antigens, and identify the test methods used to resolve 8. Compare and contrast the A1 and A2 phenotypes with these discrepancies. regard to antigen structure and serologic testing. 9. Compare and contrast serologic testing among A3, Ax, 18. Describe ABO discrepancies caused by extra reactions in and Ael subgroups. serum testing and how they can be resolved. 10. Predict the possible ABO genotypes with an ABO phenotype. 20 77

78 PART II  n  Overview of the Major Blood Groups 19. Illustrate the Bombay phenotype with 21. Identify and resolve ABO typing regard to genetic pathway, serologic discrepancies from ABO typing results. reactions, and transfusion implications. 22. Apply concepts to solve case studies with 20. Define the terms secretor and nonsecretor. ABO discrepant information. Blood group antigens This chapter begins a section of the textbook dedicated to the basic understanding form part of the red cell of blood group systems and their significance in the practice of transfusion medicine. membranes. Antigens differ A blood group system is composed of antigens that are produced by alleles at a single depending on inheritance of genetic locus or at loci so closely linked that genetic crossing over rarely occurs.1 blood group genes as Blood group antigens are molecules located primarily on the red cell membrane. These described in Chapter 3. molecules can be classified biochemically as proteins and as carbohydrates linked to either a lipid (glycolipid) or a protein (glycoprotein) as shown in Fig. 4-1. With adequate immunologic exposure, a blood group antigen may elicit the production of its correspond- ing antibody in individuals who lack the antigen. During transfusions, the recipient is exposed to many blood group antigens. Patients receiving transfusions may produce alloantibodies in response to the exposure to blood group antigens not present on their own red cells. Because the terminology for red cell antigens is inconsistent, the International Society of Blood Transfusion (ISBT) created a Working Party on Terminology for Red Cell Anti- gens in 1980 to standardize blood group systems and antigen names. The committee’s goal was not to create replacement terminology, but rather to provide additional termi- nology suitable for use with computer software. The ISBT Working Party has assigned genetically based numeric designations for red cell antigens and presently has defined 30 blood group systems (Table 4-1).2,3 According to ISBT criteria, genetic studies and sero- logic data are required before an antigen is assigned to a blood group system. The ABO blood group system has been assigned the ISBT number 001 and includes four antigens, whereas the H blood group system is ISBT number 018 with one antigen. This textbook addresses blood group systems with commonly used names and includes ISBT symbols and numbers. Cell surface GPA*1(MNS) GPB*2(MNS) Carbohydrates (ABO) Band 3 (Diego) (Lewis) Rh Polypeptide Rh Glycoprotein Lipid bilayer 4.2 Ankyrin 4.1 Actin P55 Spectrin tetramer *1 Glycophorin A Cell content *2 Glycophorin B Fig. 4-1  Model of red cell membrane that carries blood group antigens from blood group systems and collections. The red cell antigens are molecules that form part of the red cell membrane’s lipid bilayer or extend from the surface of the red cell. (Redrawn from Reid ME, Lomas-Francis C: The blood group antigen facts book, ed 2, San Diego, 2004, Elsevier Academic Press.) 21

CHAPTER 4  n  ABO and H Blood Group Systems and Secretor Status 79 TABLE 4-1  ISBT Blood Group System Assignments BLOOD SYSTEM NAME ISBT GENE NAME ISBT NUMBER ABO ABO 001 MNS MNS 002 P1PK P1 003 Rh RH 004 Lutheran LU 005 Kell KEL 006 Lewis LE 007 Duffy FY 008 Kidd JK 009 Diego DI 010 Cartwright (Yt) YT 011 Xg XG 012 Scianna SC 013 Dombrock DO 014 Colton CO 015 Landsteiner-Wiener LW 016 Chido/Rodgers CH/RG 017 Hh H 018 Kx XK 019 Gerbich GE 020 Cromer CROM 021 Knops KN 022 Indian IN 023 Ok OK 024 Raph RAPH 025 JMH JMH 026 I IGNT 027 Globoside GLOB 028 Gil GIL 029 Rh-associated glycoprotein RHAG 030 ISBT, International Society of Blood Transfusion. SECTION 1  Landsteiner’s rule: rule stating that normal, healthy individuals HISTORICAL OVERVIEW OF ABO BLOOD GROUP SYSTEM possess ABO antibodies to the ABO blood group antigens absent The discovery of the ABO blood group system by Landsteiner4 in 1900 marked the begin- from their red cells. ning of modern blood banking and transfusion medicine. In a series of experiments designed to show serologic incompatibilities between humans, Landsteiner recognized different patterns of agglutination when human blood samples were mixed in random pairings. He described the blood groups as A, B, and O. Several years later, Landsteiner’s associates, von Decastello and Sturli,5 added group AB to the original observations. In his investigations, Landsteiner noted the presence of agglutinating antibodies in the serum of individuals who lacked the corresponding ABO antigen. He observed that group A red cells agglutinated with the serum from group B individuals. This observation has been termed Landsteiner’s rule (or Landsteiner’s law). Landsteiner’s rule established that 22

80 PART II  n  Overview of the Major Blood Groups None Anti-B Anti-B Plasma ABO antibodies Anti-A Anti-A Red cells B A B ABO antigens A BO A ABO phenotype AB Fig. 4-2  Relationship between ABO antigens and antibodies. ABO antigens are located on the red cells. Group AB possesses both A and B antigens; group A possesses A antigens; group B possesses B antigens; group O lacks both A and B antigens. ABO antibodies are located in plasma. Group AB lacks ABO antibodies; group A possesses anti-B; group B possesses anti-A; group O possesses anti-A and anti-B. (Modified from Immunobase, Bio-Rad Laboratories, Inc., Hercules, CA.) Acute hemolytic transfusion normal, healthy individuals possess ABO antibodies to the ABO blood group antigens reaction: complication of lacking on their red cells. Individuals with group A red cells possess the A antigen and transfusion associated with lack the B antigen. Therefore, these individuals possess anti-B antibodies. Individuals with intravascular hemolysis, group B red cells possess the B antigen and lack the A antigen. Therefore, these individu- characterized by rapid onset als possess anti-A antibodies. Antigens and antibodies associated with each ABO pheno- with symptoms of fever, chills, type are illustrated in Fig. 4-2. Four major phenotypes are derived from the two major hemoglobinemia, and antigens (A and B) of the system. These phenotypes are group A, group B, group AB, hypotension; major complications and group O. include irreversible shock, renal failure, and disseminated The first blood group system to be described, the ABO blood group system, remains intravascular coagulation. the most important blood group system for transfusion purposes. Accurate donor and recipient ABO types are fundamental to transfusion safety because of the presence of ABO antibodies in individuals with no previous exposure to human red cells. The trans- fusion of ABO-incompatible blood to a recipient can result in intravascular hemolysis and other serious consequences of an acute hemolytic transfusion reaction. SECTION 2  ABO AND H BLOOD GROUP SYSTEM ANTIGENS GENERAL CHARACTERISTICS OF ABO ANTIGENS ABO antigens are widely distributed and are located on red cells, lymphocytes (adsorbed from plasma), platelets (adsorbed from plasma), most epithelial and endothelial cells, and organs such as the kidneys.6 Soluble forms of the ABO blood group system antigens can also be synthesized and secreted by tissue cells. As a result, ABO blood group system antigens are found in association with cellular membranes and as soluble forms. Soluble antigens are detected in secretions and all body fluids except cerebrospinal fluid.6 ABO blood group system antigens, which are intrinsic to the red cell membrane, exist as either glycolipid or glycoprotein molecules, whereas the soluble forms are primarily glycoproteins. ABO antigens are detectable at 5 to 6 weeks in utero. A newborn possesses fewer antigen copies per red cell compared with an adult. For example, adult red cells carry 610,000 to 830,000 B antigens; whereas newborn red cells carry 200,000 to 320,000 B antigens.7 Newborns’ red cells also lack the fully developed antigen structures of adults’ 23

CHAPTER 4  n  ABO and H Blood Group Systems and Secretor Status 81 red cells. In cord blood samples, ABO antigens have fewer numbers and partially devel- Cord blood: whole blood oped antigen structures and may demonstrate weaker ABO phenotyping reactions. obtained from the umbilical vein Antigen development occurs slowly until the full expression of adult levels is reached at or artery of the fetus. about 2 to 4 years of age. When phenotyping cord blood The worldwide frequency of ABO phenotypes within the white population has been samples, blood grouping well documented. Group O and group A individuals constitute 45% and 40% of whites. reagents, anti-A and anti-B, These two blood groups are the most common ABO phenotypes, followed by group B may show weaker with an 11% frequency and group AB with a 4% frequency.8 ABO phenotype frequencies agglutination reactions. differ in selected populations and ethnic groups. For example, the group B phenotype has a higher frequency in blacks and Asians compared with whites (Table 4-2). INHERITANCE AND DEVELOPMENT OF A, B, AND H ANTIGENS A discussion of the inheritance and formation of ABO antigens requires an understanding of the H antigen, which is inherited independent of the ABO blood group system antigens. The production of H antigen is genetically controlled by the H gene, which is located on a different chromosome from the ABO genetic locus. In addition to the ABO and H genes, the expression of soluble ABO antigens is influenced by inheritance of the Se gene (see the section on Secretor Status later in this chapter). The Se gene genetically influences the formation of ABO antigens in saliva, tears, and other body fluids. Consequently, occurrence and location of the ABO antigens are influenced by three genetically indepen- dent loci: ABO, H, and Se. ABO antigens are assembled on a common carbohydrate structure that also serves as the base for the formation of the H, Lewis, I/i, and P1 antigens. Consequently, this common carbohydrate structure is capable of antigen expression for more than one blood group system (Fig. 4-3). This common structure is analogous to an antigen building block. Because of the interrelationship between the common antigen building block and multiple blood group systems, it is important to recognize that the action of genes of one blood group system can affect the expression of antigens in another system. TABLE 4-2  Frequency Distributions of ABO Phenotypes (U.S. Population) ABO PHENOTYPE WHITE (%) BLACK (%) ASIAN (%) A 40 27 28 B 11 20 27 AB 4 4 5 O 45 49 40 H antigen Lewis Blood group I/i antigens system: antigens common carbohydrate structure P1 antigen tahir99-VRG & vip.persianss.ir Fig. 4-3  Antigens in several blood group systems are formed from the same carbohydrate precursor structure. 24

82 PART II  n  Overview of the Major Blood Groups Gal Protein Gal GlcNAc Gal Protein GlcNAc R or R or 1→ 4 linkage 1→ 3 Gal 1→ 3 Lipid Lipid 1→ 3 linkage Type 1: Body Fluids and Secretions Type 2: Red Blood Cells* Body Fluids and Secretions Fig. 4-4  Type 1 and type 2 oligosaccharide chain structures. Gal, Galactose; GlcNAc, N-acetylglucosamine; *, most type 2 chains are located on the red cells. (Modified from Brecher ME, editor: Technical manual, ed 15, Bethesda, MD, 2005, AABB.) Oligosaccharide chain: Common Structure for A, B, and H Antigens chemical compound formed by a small number of simple The common structure (antigen building block) for A, B, and H antigens is an oligosac- carbohydrate molecules. charide chain attached to either a protein or a lipid carrier molecule. The oligosaccharide chain comprises four sugar molecules linked in simple linear forms or in more complex structures with a high degree of branching. The two terminal sugars, D-galactose and N-acetylglucosamine, may be linked together in two different configurations. When the number 1 carbon of D-galactose is linked with the number 3 carbon of N-acetylglucosamine, the linkage is described as β1→3. Type 1 oligosaccharide chains are formed. When the number 1 carbon of D-galactose is linked with the number 4 carbon of N-acetylglucosamine, the linkage is described as β1→4. Type 2 oligosaccharide chains are formed (Fig. 4-4). Type 2 structures are associated primarily with glycolipids and glycoproteins on the red cell membrane, and type 1 structures are associated primarily with body fluids. Some type 2 glycoprotein structures are located in body fluids and secretions.9 Transferase: class of enzymes Development of H Antigen that catalyzes the transfer of a chemical group from one molecule The H antigen is the only antigen in the H blood group system. This blood group system to another. has been assigned to a locus on chromosome 19 and is closely linked with the Se locus. The H locus has two significant alleles: H and h. The H allele is a dominant allele with Glycosyltransferase: enzyme high frequency (>99.99%), whereas the h allele is classified as an amorph with rare fre- that catalyzes the transfer of quency. Genes encode for the production of proteins, and the gene product of the H allele glycosyl groups (simple is a protein classified biochemically as a transferase enzyme. Transferase enzymes promote carbohydrate units) in biochemical the transfer of a biochemical group from one molecule to another. A glycosyltransferase reactions. enzyme catalyzes the transfer of glycosyl groups (simple carbohydrate units) in biochemi- cal reactions. Immunodominant sugar: sugar molecule responsible for In the formation of H antigen, a glycosyltransferase enzyme transfers a sugar molecule, specificity. L-fucose, to either type 1 or type 2 common oligosaccharide chains. The biochemical name for this enzyme is L-fucosyltransferase (FUT-1). The L-fucose added to the terminal galac- Bombay phenotype: rare tose of the type 1 and type 2 chain is called the immunodominant sugar for H antigens phenotype of an individual who because the sugar confers H specificity (Fig. 4-5).10 Formation of the H antigen is the end genetically has inherited the h product of an enzymatic reaction. This formation is crucial to the expression of A and B allele in a homozygous manner; antigens because the gene products of the ABO alleles require that the H antigen be the the individual’s red cells lack H acceptor molecule. The FUT-1 gene adds galactose to both oligosaccharide chains on red and ABO antigens. cells and in secretions. The precursor of A and B This section previously described the h allele as an amorph with no detectable gene antigens is the H antigen. product. The red cells from an h homozygote (hh) are classified as the Bombay phenotype. These rare individuals lack both H antigen and ABO antigen expression on their red cells. The Bombay phenotype is discussed in more detail at the end of this chapter. Development of A and B Antigens Genetic control of A and B antigens has been mapped to chromosome 9. Three major alleles exist within the ABO locus: A, B, and O. The A and B alleles, similar to the H 25 tahir99-VRG & vip.persianss.ir

CHAPTER 4  n  ABO and H Blood Group Systems and Secretor Status 83 H antigen GlcNAc Gal Protein R or Gal 1→ 4 1→ 3 Lipid Gene product: 1→ 2 L-Fucosyltransferase Immunodominant sugar: L-Fucose Fuc A antigen GlcNAc Gal Protein Gene product: R or N-Acetylgalactosaminyltransferase Gal 1→ 4 1→ 3 GalNAc Lipid 1→ 3 Immunodominant sugar: 1→ 2 N-Acetylgalactosamine Fuc B antigen GlcNAc Gal Protein Gene product: R or D-Galactosyltransferase Gal 1→ 4 1→ 3 Gal Lipid 1→ 3 Immunodominant sugar: 1→ 2 D-Galactose Fuc Fig. 4-5  Biochemical structures of the H, A, and B antigens. Gal, d-Galactose; GlcNAc, N-acetylglucosamine; Fuc, l-fucose; GalNAc, N-acetylgalactosamine. (Modified from Brecher ME, editor: Technical manual, ed 15, Bethesda, MD, 2005, AABB.) allele, are glycosyltransferases. The A allele produces N-acetylgalactosaminyltransferase, The specificity of A and which transfers the sugar N-acetylgalactosamine to an oligosaccharide chain; the chain B antigen is defined by was previously converted to H antigen. The B allele produces D-galactosyltransferase, immunodominant sugars: which transfers the sugar D-galactose to an oligosaccharide chain; the chain was previ- N-acetylgalactosamine ously converted to H antigen (see Fig. 4-5).10 N-acetylgalactosamine is the immunodomi- (A antigen) and d-galactose nant sugar for A specificity, and D-galactose is the immunodominant sugar for B (B antigen). specificity. A and B antigens are not The O allele is considered nonfunctional because the resulting gene product is an primary gene products. enzymatically inactive protein. As a result, group O red cells carry no A or B antigens but are rich in unconverted H antigens. Adult group O red cells have about 1.7 million H-antigen copies per red cell and possess the greatest concentration of H antigens per red cell.6 Other ABO phenotypes have fewer copies of H antigens because the H antigen is the acceptor molecule for the A and B enzymes. Group A1B phenotype possesses the lowest number of unconverted H sites. Fig. 4-6 illustrates the variation of H-antigen concentration in ABO phenotypes. Yamamoto et al11 defined the molecular basis of the ABO phenotypes. These investiga- tors discovered that a few mutations exist in the glycosyltransferase gene at the ABO locus. On the molecular level, the A and B glycosyltransferases differ slightly in their nucleic acid compositions. Additionally, the nucleic acid composition of the O allele has revealed that it does not produce an enzymatically active protein capable of acting on the H-antigen precursors. Readers are encouraged to review the article by Poole and Daniels in the Suggested Readings for greater detail on the molecular basis of the ABO phenotypes. 26 tahir99-VRG & vip.persianss.ir

84 PART II  n  Overview of the Major Blood Groups O A2 B A2B A1 A1B Most Fewest H antigens H antigens HHH H H H H HH H H H HH H H H H H H HH H HH Fig. 4-6  Variation of H-antigen concentrations in ABO phenotypes. Group O red cells possess the most H antigens; group A1B red cells possess the fewest H antigens. A1 Most A antigens A2 A3 Ax Ael Fewest A antigens Fig. 4-7  Gradient of the subgroups of A: number of A antigen sites per red cell. Red cells classified as subgroups of A possess fewer A antigens than the A1 phenotype. ABO SUBGROUPS Comparison of A1 and A2 Phenotypes The ABO phenotypes can be divided into categories termed subgroups. Subgroups differ in the amount of antigen expressed on the red cell membrane, representing a quantitative difference in antigen expression (Fig. 4-7). Some evidence also exists to support the theory of qualitative differences in antigen expression. Some subgroups possess more highly branched, complex antigenic structures, whereas others have simplified linear forms of antigen.12 The group A phenotype is classified into two major subgroups: A1 and A2. These gly- cosyltransferase gene products, which are genetically controlled by the A1 and A2 genes differ slightly in their ability to convert H antigen to A antigen. The A1 phenotype, encoded by the A1 gene, exists in about 80% of group A individuals. In the A1 phenotype, A antigens are highly concentrated on branched and linear oligosaccharide chains. The A1 gene effectively acts on the H antigens in the production of A antigens. The A2 phe- notype, encoded by the A2 gene, constitutes about 20% of group A individuals. In the A2 phenotype, A-antigen copies are fewer than in the A1 phenotype. This phenotype is assembled on the simplified linear forms of the oligosaccharide chains. An alloantibody, anti-A1, can be detected in 1% to 8% of A2 individuals and in 22% to 35% of A2B individuals. 27 tahir99-VRG & vip.persianss.ir

CHAPTER 4  n  ABO and H Blood Group Systems and Secretor Status 85 AA A AA A A A AA A A AA A A A A A A A A A AA AAA A1 Phenotype A2 Phenotype Branched A antigens Linear A antigens 2 million A antigens/adult red cells 500,000 A antigens/adult red cells Positive with anti-A Positive with anti-A Positive with anti-A1 lectin Negative with anti-A1 lectin Fig. 4-8  Comparison of A1 and A2 red cells. (From Issitt PD, Anstee DJ: Applied blood group serology, ed 4, Durham, NC, 1998, Montgomery Scientific Publications.) In routine ABO phenotyping, both A1 and A2 red cells agglutinate with commercially Dolichos biflorus: plant lectin available anti-A reagents. These red cells can be distinguished in serologic testing only with specificity for the A1 antigen. with a reagent called Dolichos biflorus lectin. This lectin is extracted from the seeds of A1 and A2 phenotypes the plant Dolichos biflorus and possesses anti-A1 specificity. When properly diluted, the demonstrate 3+ to 4+ Dolichos biflorus lectin (anti-A1 lectin) agglutinates A1, but not A2, red cells. The anti-A1 reactions with commercial lectin is not used in routine ABO testing of donors and recipients because it is unneces- anti-A reagents. sary to distinguish between the A1 and A2 phenotypes for transfusion purposes. This Anti-A1 lectin agglutinates reagent is useful in resolving ABO typing problems and identifying infrequent subgroups only A1 red cells. This lectin is of A. Fig. 4-8 compares the A1 and A2 phenotypes. used to distinguish the A1 and A2 phenotypes in resolving Additional Subgroups of A and B ABO typing problems when A2 phenotypes develop anti-A1. Subgroups of A Mixed-field agglutination: Although more infrequent than A1 and A2, other A subgroups have been described that agglutination pattern in which a involve reduced expression of A antigens. The decreased number of A antigen sites per population of the red cells has red cell result in weak or no agglutination when tested with commercial anti-A reagents. agglutinated and the remainder of The subgroups are genetically controlled by the inheritance of rare alleles at the ABO the red cells are unagglutinated. locus and collectively occur at less than 1% frequency. Because these subgroups occur so Ulex europaeus: plant lectin infrequently, they are mainly of academic interest. The A subgroups have been classified with specificity for the H antigen. as Aint, A3, Ax, Am, Aend, Ael, and Abantu, based on the reactivity of red cells with human anti-A and anti-A,B. Historically, human polyclonal-based anti-A,B contained an anti- body with specificity toward both the A and the B antigens that could not be separated into anti-A and anti-B components. This reagent possessed an enhanced ability to detect weaker subgroups compared with anti-A. Current anti-A,B monoclonal antibody reagents blend anti-A and anti-B clones for formulations to detect the weaker subgroups. Weak or no agglutination with commercial anti-A monoclonal antibody reagents is a key factor in recognizing a subgroup in this category. Murine monoclonal blends of com- mercial anti-A have been formulated to enhance the detection of these weaker subgroups in ABO phenotyping. These monoclonal antibody anti-A reagents are blended to ensure that some subgroups of A are readily detected. Some subgroups may characteristically demonstrate mixed-field agglutination patterns (e.g., A3 subgroup) or possess anti-A1 in the serum (e.g., A3, Ax, and Ael subgroups).13 Some subgroups of A continue to react weakly or not react with murine monoclonal blends of anti-A. In these circumstances, saliva studies for the detection of soluble forms of A and H antigens and testing with anti-H lectin (Ulex europaeus) may provide additional information. The amount of H 28 tahir99-VRG & vip.persianss.ir

86 PART II  n  Overview of the Major Blood Groups TABLE 4-3  Serologic Characteristics of A3, Ax, and Ael Subgroups RED CELL AGGLUTINATION WITH SUBGROUP ANTI-A HUMAN ANTI-H ANTI-A1 SOLUBLE ANTIGENS ANTI-A1 IN ANTI-A,B LECTIN* LECTIN† IN SALIVA‡ SERUM 0 to ++§ A3 ++mf ++mf +++ 0 A and H 0 to ++§ 0 to ++§ Ax weak/0 + to ++ ++++ 0H Ael 00 ++++ 0H mf, Mixed field. *Ulex europaeus. †Dolichos biflorus. ‡If secretor. §Variable occurrence of anti-A1. Adsorption: immunohematologic antigen present on the weak subgroups of A is usually equivalent to group O red cells technique that uses red cells (3+ to 4+ reactions). Special techniques of adsorption and elution may also be necessary (known antigens) to remove red to demonstrate the presence of the A antigen (e.g., Ael subgroup). However, these tech- cell antibodies from a solution niques are not performed routinely. (plasma or antisera); group A red cells can remove anti-A from The serologic classification of rare A subgroups is determined by the following: solution. • Weak or no red cell agglutination with anti-A and anti-A,B commercial reagents • No agglutination with anti-A1 Elution: process that dissociates • Presence or absence of anti-A1 in the serum antigen-antibody complexes on • Strong agglutination reactions with anti-H red cells; freed IgG antibody is • Presence of A and H in saliva tested for specificity. • Adsorption and elution studies Table 4-3 provides information regarding the serologic characteristics of A3, Ax, and Ael subgroups.10 Weak subgroups of A are difficult to classify using serologic techniques. Usually the phenotype is described as A subgroup or A subgroup B. For definitive clas- sification, molecular techniques are available to characterize the genotype, if necessary. For an in-depth presentation of the other subgroups, the reader is referred to the Sug- gested Readings. Subgroups of B B subgroups are rarer than the A subgroups. The criteria for the recognition and differ- entiation of these subgroups are similar to criteria of the A subgroups. Typically, these subgroups demonstrate weak or no agglutination of red cells with anti-B reagents. Importance of Subgroup Identification in Donor Testing Although subgroups of A and B are considered to be of academic interest, the failure to detect a weak subgroup could have serious consequences. If a weak subgroup is missed in a recipient (the individual receiving the transfusion), the recipient would be classified as group O. Classification as a group O rather than a weak subgroup would probably not harm the recipient because group O red cells would be selected for transfusion and can be transfused to any ABO phenotype. However, an error in donor phenotyping and the subsequent labeling of the donor unit as group O (rather than group A) might result in the decreased survival of the transfused cells in a group O recipient. Group O recipients would possess ABO antibodies capable of reacting with the weak subgroup antigens in vivo, resulting in the decreased survival of these transfused red cells in the recipient’s circulation. SECTION 3  GENETIC FEATURES OF ABO BLOOD GROUP SYSTEM Inheritance of genes from the ABO locus on chromosome 9 follows the laws of Mendelian genetics. An individual inherits two ABO genes (one from each parent). The three major 29 tahir99-VRG & vip.persianss.ir

CHAPTER 4  n  ABO and H Blood Group Systems and Secretor Status 87 alleles of the ABO blood group system are A, B, and O. The A gene subsequently can be divided into the A1 and A2 alleles. The A and B genes express a codominant mode of inheritance, whereas the O allele is recessive. The A1 allele is dominant over the A2 allele, and both alleles are dominant over the O allele. The major ABO phenotypes and possible corresponding genotypes for the phenotypes are outlined in Table 4-4. Correct use of terminology regarding the ABO blood group system should be mentioned. When reference is made to the alleles A1 and A2, the numbers are always indicated as superscripts. In references to the A1 and A2 phenotypes, the numbers are always indicated in a subscript format. Because the O allele is recessive, it is not always possible to determine the ABO geno- type from the corresponding phenotype without family studies or molecular analysis. Red cells can be phenotyped only for the presence or absence of antigens and cannot be geno- typed. Unless a family study has been performed with conclusive results, a genotype is only a probable interpretation of a phenotype. Deduction of the genotype from a family study is illustrated in Fig. 4-9. The Generation I female’s phenotype is group B with pos- sible genotypes of BB or BO, and the male’s phenotype is group A with possible genotypes of AA or AO. The parental genotypes may be deduced only after phenotyping the off- spring. The four offspring’s phenotypes are presented in Generation II. To produce a group O offspring, both parents must have passed on the O allele. Therefore their geno- types must be AO and BO. TABLE 4-4  ABO Phenotypes and Possible Genotypes PHENOTYPE POSSIBLE GENOTYPES Group A1 A1A1 Group A2 A1A2 A1O Group B A2A2 Group A1B A2O Group A2B Group O BB BO A1B A2B OO I. Phenotype: A B Punnett square diagram- Genotype: AO BO ming the pedigree chart shown at left II. A O B AB BO Phenotype: AB O BA O AO OO Genotype: AB OO BO AO Fig. 4-9  Practical application: ABO inheritance patterns. Group A and group B phenotypes may produce offspring with group AB, O, B, and A phenotypes if the parents’ genotypes are AO and BO. 30 tahir99-VRG & vip.persianss.ir

88 PART II  n  Overview of the Major Blood Groups Non–red blood cell SECTION 4  stimulated: immunologic stimulus for antibody production ABO BLOOD GROUP SYSTEM ANTIBODIES is unrelated to a red cell antigen. As Landsteiner recognized in his early experiments, individuals possess the ABO antibody Titers: extent to which an in their serum directed against the ABO antigen absent from their red cells. Landsteiner’s antibody may be diluted before it rule remains an important consideration in the selection of blood products given that loses its ability to agglutinate with ABO antibodies exist in healthy individuals. These ABO antibodies, present in individuals antigen. with no known exposure to blood or blood products, were originally thought to be “naturally occurring.” The current hypothesis is that biochemical structures similar to A and B antigens are present in the environment in bacteria, plants, and pollen. As a result of this environmental exposure to these similar forms of A and B antigens, individuals respond immunologically to these antigens and produce ABO antibodies detectable in plasma and serum.14 Consequently, the term naturally occurring is a misnomer because an immunologic stimulus is present for antibody development. The term non–red blood cell stimulated is more appropriate for describing the ABO antibodies. Newborns do not produce their own ABO antibodies until they are 3 to 6 months of age. ABO antibodies detected prior to this time are maternal in origin. Maximal ABO titers have been reported in children 5 to 10 years old. As a person ages, the ABO titers tend to decrease and may cause problems in ABO phenotyping. In addition to newborns and older patients, other situations exist where ABO antibody titers may be weak or not demonstrable in testing (Table 4-5).15 Recognition of these circumstances can assist in resolving ABO phenotyping problems discussed later in this chapter. Clinical significance: antibodies GENERAL CHARACTERISTICS OF HUMAN ANTI-A AND ANTI-B capable of causing decreased survival of transfused cells as in a Immunoglobulin Class transfusion reaction; have been The anti-A produced in group B individuals and the anti-B produced in group A individu- associated with hemolytic disease als contain primarily antibodies of the IgM class along with small amounts of IgG. In of the fetus and newborn. contrast, anti-A and anti-B antibodies found in the serum of group O individuals are composed primarily of IgG class. Congenital hypogammaglobulinemia: Hemolytic Properties and Clinical Significance genetic disease characterized by IgG and IgM forms of anti-A and anti-B are capable of the activation and binding of reduced levels of gamma globulin complement and eventual hemolysis of red cells in vivo or in vitro. Because of their ability in the blood. to activate the complement cascade with resultant red cell hemolysis, the ABO antibodies are considered of clinical significance in transfusion medicine. An antigen-antibody reac- Acquired tion between a recipient’s ABO antibody and the ABO phenotype of the transfused red hypogammaglobulinemia: cells can cause activation of complement and destruction of the transfused donor red lower than normal levels of cells, precipitating the clinical signs and symptoms of an acute hemolytic transfusion gamma globulin in the blood reaction. For example, a group A recipient has circulating anti-B antibodies in serum. If associated with malignant this individual is transfused with group B or AB donor red cells, the circulating anti-B diseases (chronic leukemias and would recognize the B antigen on the donor red cells and combine with the antigens. The myeloma) and immunosuppressive therapy. TABLE 4-5  Reduction in ABO Antibody Titers Congenital Age-Related agammaglobulinemia: genetic disease characterized by the Newborn absence of gamma globulin and Elderly antibodies in the blood. Pathologic Etiology Acquired agammaglobulinemia: absence Chronic lymphocytic leukemia of gamma globulin and antibodies Congenital hypogammaglobulinemia or acquired hypogammaglobulinemia associated with malignant Congenital agammaglobulinemia or acquired agammaglobulinemia diseases such as leukemia, Immunosuppressive therapy myeloma, or lymphoma. Bone marrow transplant Multiple myeloma 31 tahir99-VRG & vip.persianss.ir

CHAPTER 4  n  ABO and H Blood Group Systems and Secretor Status 89 complement system is readily activated, causing a decreased survival of transfused red Anti-A and anti-B react in cells. immediate-spin phases (direct agglutination reactions). In Vitro Serologic Reactions ABO antibodies directly agglutinate a suspension of red cells in a physiologic saline envi- ronment and do not require any additional potentiators. They are optimally reactive in immediate-spin phases at room temperature (15° C to 25° C). The agglutination reactions do not require an incubation period and react without delay on centrifugation. HUMAN ANTI-A,B FROM GROUP O INDIVIDUALS Human anti-A,B is detected in the serum of group O individuals and possesses unique activities beyond mixtures of anti-A and anti-B antibodies. Activity of human anti-A,B is regarded as a specificity that is cross-reactive with both A and B antigens. A cross-reactive antibody is capable of recognizing a particular molecular structure (antigenic determi- nant) common to several molecules. This distinguishing characteristic enables the anti- body to agglutinate with red cells of group A, B, and AB phenotype because this antibody recognizes a structure shared by both A and B antigens. Human anti-A,B also manifests the property of agglutinating red cells of infrequent subgroups of A, particularly Ax. Before the advent of monoclonal reagents, human anti-A,B was widely used to detect these infrequent subgroups in routine ABO typing. Monoclonal antibody reagents have since replaced the use of human anti-A,B in ABO phenotyping. ANTI-A1 Incompatible crossmatches: occur when agglutination or In accordance with Landsteiner’s rule for expected ABO antibodies, sera from group O hemolysis is observed in the and B individuals contain anti-A antibodies. The anti-A produced by group O and B crossmatch of donor red cells and individuals can be separated by adsorption and elution techniques into two components: patient serum, indicating a anti-A and anti-A1. Anti-A1 is specific for the A1 antigen and does not agglutinate A2 red serologic incompatibility. The cells. The optimal reactivity of this antibody is at room temperature or lower. Anti-A1 is donor unit would not be not considered clinically significant for transfusion purposes. Anti-A1 becomes a concern transfused. when it causes problems with ABO phenotyping results and incompatible crossmatches on immediate spin. Anti-A2 does not exist because the A2 phenotype possesses the same Donor blood samples are A antigens as A1 phenotype but in reduced quantities. Individuals with A1 phenotype do routinely typed at the time of not respond immunologically when exposed to A2 red cells. donation. The ABO-labeled red blood cell (RBC) donor units SECTION 5  are confirmatory typed on ABO BLOOD GROUP SYSTEM AND TRANSFUSION receipt at the hospital transfusion service. ROUTINE ABO PHENOTYPING The procedure for ABO A fundamental procedure of immunohematologic testing is the determination of the ABO phenotyping is presented in phenotype. The procedure is straightforward and is divided into two components: testing the Laboratory Manual that of the red cells for the presence of ABO antigens (or forward grouping) and testing of accompanies this textbook. serum or plasma for the expected ABO antibodies (or reverse grouping). According to the Standards for Blood Banks and Transfusion Services, donor and recipient red cells Reverse grouping (serum or must be tested using anti-A and anti-B reagents. Donor and recipient serum or plasma plasma testing) is not required must be tested for the expected ABO antibodies using reagent A1 and B red cells.15 Neither for confirmatory testing of human anti-A,B nor the monoclonal blend anti-A,B is required in ABO typing. Testing labeled, previously typed of cord blood and samples from infants younger than 4 months requires only red cell donor RBCs and in infants testing in ABO phenotyping because ABO antibody levels are not detectable. younger than 4 months of age. The ABO phenotype is determined when the red cells are directly tested for the pres- ence or absence of either A or B antigens. Serum testing provides a control for red cell ABO discrepancy: occurs when testing because ABO antibodies would reflect Landsteiner’s rule. Table 4-6 shows the ABO phenotyping of red cells expected reactions observed in ABO phenotyping. An ABO discrepancy occurs when red does not agree with expected cell testing does not agree with the expected serum testing. Any discrepancy in ABO serum testing results for the testing should be resolved before transfusion of recipients or labeling of donor units. particular ABO phenotype. 32 tahir99-VRG & vip.persianss.ir

90 PART II  n  Overview of the Major Blood Groups Review the sections in TABLE 4-6  ABO Phenotype Reactions Chapter 2 on ABO typing reagents. RED CELL REACTIONS WITH SERUM OR PLASMA REACTIONS WITH Agglutination reactions for ABO typing are usually 3+ to PHENOTYPE ANTI-A ANTI-B A1 CELLS B CELLS 4+ in strength. 0 Group A + 0 + Group B 0 + 0 Group O 0 + 0 ++ 00 Group AB + + +, Agglutination; 0, no agglutination. TABLE 4-7  Practical Application: ABO Compatibility for Whole Blood, Red Blood Cells, and Plasma Transfusions RECIPIENT WHOLE BLOOD DONOR PLASMA ABO PHENOTYPE Group A RED BLOOD CELLS Groups A, AB Group A Group B Groups A, O Groups B, AB Group B Group AB Groups B, O Group AB Group AB Group O Groups AB, A, B, O Groups O, A, B, AB Group O Group O Universal donors: group O SELECTION OF ABO-COMPATIBLE RED BLOOD CELLS AND PLASMA donors for RBC transfusions; these PRODUCTS FOR TRANSFUSION RBCs may be transfused to any ABO phenotype because the cells In routine transfusion practices, donor products (RBCs and plasma) with identical ABO lack both A and B antigens. phenotypes are usually available to the recipient. This transfusion selection is referred to as providing ABO-identical (ABO group–specific) blood for the intended recipient. In Universal recipients: group AB situations where blood of identical ABO phenotype is unavailable, ABO-compatible (ABO recipients may receive transfusions group–compatible) blood may be issued to the recipient. of RBCs from any ABO phenotype; these recipients lack circulating For RBC transfusions, ABO compatibility between the recipient and the donor is ABO antibodies in plasma. defined as the serologic compatibility between the ABO antibodies present in the recipi- ent’s serum and the ABO antigens expressed on the donor’s red cells. For example, a group A recipient who concurrently demonstrates anti-B in serum would be compatible with either group A or group O donor red cells because serum anti-B would not react with either the group A or the group O red cells in vivo. However, if this individual receives a transfusion with either group B or group AB donor red cells, recipient anti-B antibodies would recognize the B antigens present on the red cells. Antigen-antibody complexes form, may activate the complement cascade, and result in the signs and symp- toms of an acute hemolytic transfusion reaction. ABO compatibility applies to RBC transfusions but not to transfusions of whole blood. When whole blood is transfused, ABO-identical donor units must be provided because both plasma and red cells are present in the product. The concepts of ABO compatibility for whole blood and RBC transfusions are outlined in Table 4-7. Persons with group O red cells are called universal donors because the RBC product lacks both A and B antigens and could be transfused to any ABO phenotype. Group O donor RBCs can be used in times of urgency for emergency release of donor units. Conversely, group AB recipients are considered universal recipients because these individuals lack circulating ABO antibodies and can receive RBCs of any ABO phenotype. When plasma products are transfused, the selection of an ABO-identical phenotype is the ideal situation. When identical ABO phenotypes are unavailable, the rationale for compatible plasma transfusions is the reverse of RBC transfusions. In this case, the 33 tahir99-VRG & vip.persianss.ir

CHAPTER 4  n  ABO and H Blood Group Systems and Secretor Status 91 donor’s plasma must be compatible with the recipient’s red cells. This concept translates Universal donor for RBC to the serologic compatibility between the ABO antibodies in the donor unit with the transfusions is group O; ABO antigens present on the recipient’s red cells. Group A recipients needing plasma universal donor for plasma would be compatible with group A and AB plasma products. Because group A plasma transfusions is group AB. contains anti-B, and group AB has no ABO antibodies, these plasma products do not recognize the A antigen on recipient red cells. No adverse antigen-antibody reaction Universal recipient for RBC would ensue. For the transfusion of plasma, group AB is considered the universal donor transfusions is group AB; and group O is the universal recipient (see Table 4-7). universal recipient for plasma transfusions is group O. SECTION 6  RECOGNITION AND RESOLUTION OF ABO DISCREPANCIES The recognition and resolution of ABO discrepancies are challenging aspects of problem solving in the blood bank. As defined in a previous section, an ABO discrepancy is an ABO phenotype in which the results of the red cell testing do not agree with the results of expected serum testing. Discrepancies may be indicated when the following observa- tions are noted in the results of ABO phenotyping: • Agglutination strengths of the typing reactions are weaker than expected. Typically, the reactions in ABO red cell testing with anti-A and anti-B reagents are 3+ to 4+ agglutination reactions; the results of ABO serum testing with reagent A1 and B cells are 2+ to 4+. • Expected reactions in ABO red cell testing and serum testing are missing (e.g., group O individual is missing one or both reactions in serum testing with reagent A1 and B cells). • Extra reactions are noted in either the ABO red cell or serum tests. The source of these discrepancies can be either technical or sample-related problems. The first step in the resolution of an ABO discrepancy is to identify the source of the problem. Is the discrepancy a technical error in testing, or is the discrepancy related to the sample itself? TECHNICAL CONSIDERATIONS IN ABO PHENOTYPING Several types of technical errors can transpire in ABO typing and lead to erroneous results. An awareness and recognition of these technical errors can assist in the resolution of an ABO discrepancy. These technical errors can be classified into several categories, including identification and documentation errors, reagent and equipment problems, and standard operating procedure errors. By following the guidelines outlined in Table 4-8, technical TABLE 4-8  Practical Application: Guidelines for Investigating ABO Technical Errors Identification or Documentation Errors Correct sample identification on all tubes Results are properly recorded Interpretations are accurate and properly recorded Reagent or Equipment Errors Daily quality control on ABO typing reagents is satisfactory Inspect reagents for contamination and hemolysis Centrifugation time and calibration are confirmed Standard Operating Procedure Errors Procedure follows manufacturer’s directions Correct reagents were used and added to testing Red blood cell suspensions are at the correct concentration Cell buttons are completely suspended before grading the reaction 34 tahir99-VRG & vip.persianss.ir

92 PART II  n  Overview of the Major Blood Groups sources of error can be pinpointed more readily. A new sample can be obtained to elimi- nate possible contamination or identification problems. In addition, red cell suspensions prepared from patient samples can be washed three times before repeated testing. When a technical error is discovered and corrected, the ABO discrepancy can be quickly resolved with repeated testing. If the discrepancy still exists after repeated testing, the possibility of a problem related to the sample itself (e.g., related to the patient or donor) should be considered. SAMPLE-RELATED ABO DISCREPANCIES Sample-related problems can be divided into two groups: ABO discrepancies that affect the ABO red cell testing and discrepancies that affect the ABO serum or plasma testing. Is the problem associated with the patient or donor red cells, or is it associated with patient or donor antibodies? A logical approach to solving these sample- related problems is to select the side of the ABO test (red cell testing or serum or plasma testing) believed to be discrepant and to focus on the problem from this angle. The observed strengths of agglutination reactions in the testing of both the red cells and the serum or plasma are keys in determining whether to focus problem solving on a red cell or a serum/plasma problem. For success with this approach, a working knowledge of the multitude of potential problems relating to ABO red cell and serum testing is mandatory (Table 4-9). The most commonly encountered ABO discrepancies in the immunohematology laboratory are discrepancies relating to weak or missing ABO anti- bodies in serum/plasma testing. ABO discrepancies associated with red cell testing are reviewed first followed by discussion of discrepancies associated with serum/plasma testing. ABO Discrepancies Associated with Red Cell Testing ABO discrepancies that affect the testing of red cells (forward grouping) can be classified into three categories: extra antigens present, missing or weak antigens, and mixed-field reactions. Extra Antigens Present ABO red cell typing results may demonstrate unexpected positive agglutination reactions with commercial anti-A or anti-B reagents. Extra reactions are present in red cell testing or forward grouping. For the purposes of this textbook, the scope of the discussion on TABLE 4-9  Overviews of ABO Discrepancies PROBLEMS WITH RED CELL TESTING PROBLEMS WITH SERUM/PLASMA TESTING Extra antigens Extra antibodies   Group A with acquired B antigen   A subgroups with anti-A1   B(A) phenotype   Cold alloantibodies   Polyagglutination   Cold autoantibodies   Rouleaux   Rouleaux   Hematopoietic progenitor cell transplants   IVIG Missing or weak antigens Missing or weak antibodies   ABO subgroup   Newborn   Pathologic etiology   Elderly   Transplantation   Pathologic etiology   Immunosuppressive therapy for Mixed-field reactions   Transfusion of group O to group A, B, or AB transplantation   Hematopoietic progenitor stem cell transplants   A3 phenotype IVIG, Intravenous immunoglobulin. 35

CHAPTER 4  n  ABO and H Blood Group Systems and Secretor Status 93 extra antigens in red cell testing is limited to the illustration of the group A with acquired Group A with acquired B B antigen and the B(A) phenotype. antigen: group A1 individual with disease of the lower Examples 1 and 2 follow to demonstrate extra antigens present with the acquired B gastrointestinal tract, cancer of and B(A) phenotype. the colon and rectum, intestinal obstruction, or gram-negative EXAMPLE 1 septicemia who acquires reactivity with anti-B reagents in ABO red cell testing and appears as group AB. Group A with Acquired B Antigen B(A) phenotype: group B individual who acquires reactivity ABO Testing Results with anti-A reagents in ABO red cell testing; in these individuals, Patient Red Cells with Patient Serum with Reagent Red Cells the B gene transfers trace amounts of the immunodominant Anti-A Anti-B A1 B sugar for the A antigen and the 4+ 1+ 0 4+ immunodominant sugar for the B antigen. EVALUATION OF ABO TESTING RESULTS 1. The agglutination of the patient’s red cells with anti-A is strong (4+). 2. The agglutination of the patient’s red cells with anti-B is weaker (1+) than usually expected (3+ to 4+). 3. These red cells react as the phenotype group AB. 4. The results of serum testing reactions are typical of a group A individual. CONCLUSION These reactions are typical of individuals possessing the acquired B antigen. In group A acquired B antigen, a group A individual possesses an extra antigen in red cell testing (notice the weaker agglutination with anti-B reagents). Anti-B is observed in the serum testing. Serum testing reactions are typical for a group A individual. BACKGROUND INFORMATION Deacetylating: removal of the Usually only group A1 individuals with diseases of the lower gastrointestinal tract, cancers acetyl group (CH3CO–). of the colon and rectum, intestinal obstruction, or gram-negative septicemia express the acquired B antigen. The most common mechanism for this phenotype is usually associated with a bacterial deacetylating enzyme that alters the A immunodominant sugar, N-acetylgalactosamine, by removing the acetyl group. The resulting sugar, galactosamine, resembles the B immunodominant sugar, D-galactose, and cross-reacts with many anti-B reagents.16 In the early 1990s, an increase in the detection of acquired B antigen with certain monoclonal anti-B blood grouping reagents licensed by the U.S. Food and Drug Admin- istration was observed.17 The observation was linked to the use of ES-4 monoclonal anti-B clone at pH levels of 6.5 to 7.0. If the formulation of the clone was acidified to pH 6.0, the acquired B antigen was not observed. Red cells agglutinated strongly by anti-A and weakly by anti-B in combination with a serum containing anti-B suggest the acquired B antigen. These patients should receive units of group A red cells for transfusion purposes. RESOLUTION OF ABO DISCREPANCY Autologous: pertaining to self. 1. Determine the patient’s diagnosis and transfusion history. The first step in the resolu- tion of any ABO discrepancy is to obtain more information about the patient. This information may provide additional clues about the root cause of the ABO discrepancy. 2. Test the patient’s serum against autologous red cells. Anti-B in the patient’s serum does not agglutinate autologous red cells with the acquired B antigen. 3. Test red cells with additional monoclonal anti-B reagents from other manufacturers that are documented not to react with the acquired B antigen or a source of human polyclonal anti-B. 36

94 PART II  n  Overview of the Major Blood Groups EXAMPLE 2 B(A) Phenotype ABO Testing Results Patient Red Cells with Patient Serum with Reagent Red Cells Anti-A Anti-B A1 B 1+ 4+ 4+ 0 Polyagglutination: property EVALUATION OF ABO TESTING RESULTS of cells that causes them to 1. The agglutination of the patient’s red cells with anti-A is weak (1+). be agglutinated by naturally 2. The agglutination of the patient’s red cells with anti-B is strong (4+). occurring antibodies found in 3. The results of serum testing are typical of a group B individual. most human sera; agglutination occurs regardless of blood type. CONCLUSION These reactions are characteristic of a possible B(A) phenotype. In the B(A) phenotype, Wharton’s jelly: gelatinous a group B with an apparent extra antigen reaction is observed with anti-A in red cell tissue contaminant in cord blood testing. samples that may interfere in immunohematologic tests. BACKGROUND INFORMATION The B(A) phenotype has been observed as a result of the increased sensitivity of potent monoclonal antibody reagents for ABO phenotyping.18 These reagents can detect trace amounts of either A or B antigens that are nonspecifically transferred by the glycosyl- transferase enzymes. In the B(A) phenotype, the B gene transfers trace amounts of the immunodominant sugar for the A antigen (N-acetylgalactosamine) and the immunodomi- nant sugar for the B antigen (D-galactose) to the H-antigen acceptor molecules. The trace amounts of A antigens are detected with certain clones from the monoclonal antibody reagents. A similar mechanism can cause an A(B) phenotype analogous to the acquired B antigen. RESOLUTION OF ABO DISCREPANCY 1. Determine the patient’s diagnosis and transfusion history. 2. Test red cells with additional monoclonal antibody anti-A reagents from other manu- facturers or a source of human polyclonal anti-A. Other potential explanations for extra antigens in ABO red cell testing include the following: a. Polyagglutination of red cells by most human sera as a result of the exposure of a hidden antigen on the red cell membrane because of a bacterial infection or genetic mutation. ABO discrepancies caused by polyagglutination are rarely detected because of the routine use of monoclonal antibody reagents, which have replaced human- derived ABO antisera. b. Nonspecific aggregation of serum-suspended red cells because of abnormal concen­ trations of serum proteins or Wharton’s jelly in cord blood samples (false-positive agglutination) Missing or Weakly Expressed Antigens In the category of ABO discrepancies concerning missing or weakly expressed antigens, patient or donor red cells demonstrate weaker than usual reactions with reagent anti-A and anti-B or may fail to demonstrate any reactivity. Phenomena associated with this category include the following: • ABO subgroups • Weakened A and B antigen expression in patients with leukemia or Hodgkin’s disease Example 3 illustrates missing or weakly expressed antigens by presenting an ABO discrepancy typically observed with a subgroup of A. 37

CHAPTER 4  n  ABO and H Blood Group Systems and Secretor Status 95 EXAMPLE 3 Subgroup of A ABO Testing Results Patient Red Cells with Patient Serum with Reagent Red Cells Anti-A Anti-B A1 B 0 0 0 3+ EVALUATION OF ABO TESTING RESULTS 1. No agglutination of the patient’s red cells with both anti-A and anti-B reagents is observed. The individual appears to be a group O phenotype. 2. The results of serum testing are typical of a group A individual. Agglutination of anti-B with reagent B red cells is strong (3+). CONCLUSION These reactions are characteristic of a missing antigen in the red cell testing. The serum testing results are those expected in a group A individual. Anti-A, found in group O individuals, is absent in the serum testing. BACKGROUND INFORMATION As previously discussed in this chapter, weak or missing reactions with anti-A and anti-B reagents correlate with subgroups of A and B. Subgroups of A represent less than 1% of the group A population, and the subgroups of B are even rarer. Inheritance of an alterna- tive allele at the ABO locus results in a quantitative reduction of antigen sites per red cell and in weakened or missing reactions with anti-A and anti-B reagents. RESOLUTION OF ABO DISCREPANCY 1. Determine the patient’s diagnosis and transfusion history. 2. Repeat the red cell testing with extended incubation times and include human poly- clonal anti-A,B or monoclonal blend anti-A,B. The extended incubation time may enhance the antigen-antibody reaction. Additional Testing Results Additional Testing Results Anti-A,B Anti-A,B Patient red cells 1+ Patient red cells 0 Conclusion: Probable subgroup of A Next Step: Perform adsorption and elution studies with anti-A; these studies assist in determining the presence of A antigens on the patient’s red cells Mixed-Field Reactions Hematopoietic progenitor cell transplant: replacement of Mixed-field reactions can occur in red cell testing with either anti-A or anti-B reagents. hematopoietic stem cells derived As noted earlier, a mixed-field reaction contains agglutinates with a mass of unaggluti- from allogeneic bone marrow, nated red cells. Usually a mixed-field reaction is due to the presence of two distinct cell peripheral stem cells, or cord populations. For example, testing red cells from a patient recently transfused with non– blood to treat certain leukemias, ABO-identical RBCs (group O donor RBCs to a group AB recipient) can yield mixed-field immunodeficiencies, and observations. In addition to the transfusion of group O RBCs to group A, B, or AB indi- hemoglobinopathies. viduals, recipients of hematopoietic progenitor transplants, individuals with the A3 38

96 PART II  n  Overview of the Major Blood Groups Tn-polyagglutinable red cells: phenotype, and patients with Tn-polyagglutinable red cells can demonstrate mixed-field type of polyagglutination that reactions. Example 4 illustrates an ABO discrepancy showing mixed-field reactions. occurs from a mutation in the hematopoietic tissue, EXAMPLE 4 characterized by mixed-field reactions in agglutination testing. Group B Patient Transfused with Group O RBCs The transfusion of red cell ABO Testing Results donor units or stem cell transplants can cause Patient Red Cells with Patient Serum with Reagent Red Cells mixed-field reactions. They are called artificially induced Anti-A Anti-B A1 B chimerisms. 4+ 0 0 2+mf mf, Mixed field. EVALUATION OF ABO TESTING RESULTS 1. The strength of the agglutination reaction with anti-B is weaker than expected for group B individuals. 2. The anti-B mixed-field grading of reactivity is a 2+ reaction with a sufficient number of unagglutinated cells. 3. The results of serum testing are typical of a group B individual. CONCLUSION These results demonstrate a group B individual possibly transfused with group O RBCs. BACKGROUND INFORMATION In certain situations, ABO-identical RBC products might not be available for transfusion, and group O RBC products are transfused. If many group O donor RBC units are trans- fused in respect to the recipient’s total body mass, mixed-field reactions may appear in the ABO red cell testing. Cold alloantibodies: red cell RESOLUTION OF ABO DISCREPANCY antibodies specific for other 1. Determine the patient’s diagnosis and recent transfusion history. human red cell antigens that 2. Determine whether the patient is a recent hematopoietic progenitor cell recipient. typically react at or below room 3. Investigate pretransfusion ABO phenotype history, if possible. temperature. ABO Discrepancies Associated with Serum or Plasma Testing Cold autoantibodies: red cell antibodies specific for autologous ABO discrepancies that affect serum or plasma testing (reverse grouping) include the antigens that typically react at or presence of additional antibodies other than anti-A and anti-B or the absence of expected below room temperature. ABO antibody reactions. The most commonly encountered ABO discrepancies involve the absence of expected ABO antibody reactions. Additional Antibodies in Serum or Plasma Testing This section of ABO discrepancies addresses the detection of anti-A1, cold alloantibodies, cold autoantibodies, and rouleaux in ABO typing. Example 5 is an illustration of group A2 with anti-A1. Example 6 illustrates a cold autoantibody and cold alloantibody. Example 7 illustrates rouleaux. In all of these situations, the ABO discrepancy manifests as addi- tional antibodies in serum or plasma testing. EXAMPLE 5 Group A2 with Anti-A1 ABO Testing Results Patient Red Cells with Patient Serum with Reagent Red Cells Anti-A Anti-B A1 B 4+ 0 2+ 4+ 39

CHAPTER 4  n  ABO and H Blood Group Systems and Secretor Status 97 EVALUATION OF ABO TESTING RESULTS 1. The agglutination pattern with anti-A and anti-B reagents is typical of a group A individual. 2. The results of serum testing with reagent A1 and B red cells indicate a group O individual. CONCLUSION These results demonstrate an extra reaction in the serum testing with the reagent A1 red cells (2+). Possible explanations for the extra reaction include an anti-A1, a cold alloan- tibody, a cold autoantibody, or rouleaux. This example illustrates an ABO discrepancy resulting from group A2 with anti-A1. RESOLUTION OF ABO DISCREPANCY 1. Determine the patient’s diagnosis and transfusion history. 2. Test the patient’s red cells with anti-A1 lectin to ascertain whether a subgroup of A is present. Additional Testing Results Patient Red Cells Tested with Anti-A1 Lectin Conclusion 0 Subgroup of A; suspect anti-A1 antibody 3. Test the patient’s serum with three examples of A1 and A2 reagent red cells to confirm the presence of anti-A1 antibody. Additional Testing Results Patient Serum Tested with A1 Cells A1 Cells A1 Cells A2 Cells A2 Cells A2 Cells 2+ 2+ 2+ 0 0 0 CONCLUSION Agglutination is observed with A1 red cells providing the evidence for anti-A1. The serum does not agglutinate with A2 red cells. Anti-A1 may be present in 1% to 8% of the group A2 phenotype. EXAMPLE 6 Cold Autoantibody and Cold Alloantibody in Serum/Plasma Testing ABO Testing Results Patient Red Cells with Patient Serum with Reagent Red Cells Anti-A Anti-B A1 B 4+ 4+ 0 1+ EVALUATION OF ABO TESTING RESULTS 1. Strong agglutination reactions are observed in red cell testing and are consistent with a group AB individual. 2. The results of serum testing with reagent B red cells demonstrate a weaker extra reac- tion (1+). This serum testing appears to be consistent with a group A individual. CONCLUSION These results indicate a possible extra reaction in the serum testing with the reagent B red cells. Example 6 illustrates the presence of a cold alloantibody or a cold autoantibody. 40

98 PART II  n  Overview of the Major Blood Groups Autocontrol: testing a person’s BACKGROUND INFORMATION serum with his or her own red Donors and patients may possess antibodies to other blood group system red cell antigens cells to determine whether an in addition to those of the ABO blood group system. These alloantibodies may appear autoantibody is present. as additional serum antibodies in ABO typing as one of the following specificities: anti-P1, anti-M, anti-N, anti-Lea, and anti-Leb. Because they react at or below room temperature, these antibodies are sometimes referred to as cold. Reagent A1 and B red cells used in ABO serum testing may possess these antigens in addition to the A and B antigens. Screening cells, which are group O reagent red cells, are used to detect an alloantibody because they lack A and B antigens. Any serum reactivity caused by an existing ABO antibody would be eliminated in the reaction with group O cells. It is logical to conclude that screening cells are valuable in distinguishing between ABO antibodies and alloantibodies. Patients and donors may also possess serum antibodies directed toward their own red cell antigens. These antibodies are classified as autoantibodies. If autoantibodies are reac- tive at or below room temperature, they are also called cold. Cold autoantibodies usually possess the specificity of anti-I or anti-IH and react against all adult red cells, including screening cells, A1 and B cells, and autologous cells. An autocontrol (autologous control) is tested to differentiate a cold autoantibody from a cold alloantibody. If the autocontrol is positive, the reactions observed with the A1 and B cells and screening cells are probably the result of autoantibodies. See Chapter 7 for additional information on cold autoantibody test methods and techniques useful in negating their reactivity in ABO typing tests. RESOLUTION OF ABO DISCREPANCY 1. Determine the patient’s diagnosis and transfusion history. 2. Test the patient’s serum with screening cells and an autocontrol at room temperature. This strategy helps distinguish whether cold alloantibody or cold autoantibody is present. Interpretation of Testing Results Screening Cells Autologous Red Cells Conclusion Cold alloantibody Patient serum Pos* Neg Cold autoantibody Patient serum Pos Pos *Positive reaction if the corresponding antigen is present on the screening cell. 3. If an alloantibody is detected, antibody identification techniques can be performed (see Chapter 7). 4. If an autoantibody is detected, special techniques to identify the antibody (a mini-cold panel) and remove antibody reactivity (prewarming techniques) can be used (see Chapter 7). EXAMPLE 7 Rouleaux ABO Testing Results Patient Red Cells with Patient Serum with Reagent Red Cells Anti-A Anti-B A1 B 4+ 4+ 2+ 2+ EVALUATION OF ABO TESTING RESULTS 1. Strong agglutination reactions are observed in red cell testing and are consistent with the expected results of a group AB individual. 2. Serum testing results are consistent with those of a group O individual. 41

CHAPTER 4  n  ABO and H Blood Group Systems and Secretor Status 99 CONCLUSION Consider the possibility of extra reactions in serum testing with the reagent red cells because of an alloantibody, an autoantibody, or rouleaux. The phenomenon of rouleaux is demonstrated in this example. BACKGROUND INFORMATION Multiple myeloma: malignant Rouleaux can produce false-positive agglutination in testing. The red cells resemble neoplasm of the bone marrow stacked coins under microscopic examination. Increased concentrations of serum proteins characterized by abnormal can affect this spontaneous agglutination of red cells. Diseases associated with rouleaux proteins in the plasma and urine. include multiple myeloma and Waldenström’s macroglobulinemia. In addition to creating problems with the serum testing in ABO phenotyping, rouleaux can create extra reactions Waldenström’s in the ABO red cell typing if unwashed red cell suspensions are used. macroglobulinemia: overproduction of IgM by the RESOLUTION OF ABO DISCREPANCY clones of a plasma B cell in 1. Determine the patient’s diagnosis and transfusion history. response to an antigenic signal; 2. Wash red cell suspension and repeat the phenotyping. increased viscosity of blood is 3. Perform the saline replacement technique to help distinguish true agglutination from observed. rouleaux (Fig. 4-10). Saline replacement technique: test to distinguish rouleaux and true agglutination. Missing or Weak ABO Antibodies in Serum or Plasma Testing Missing or weak ABO antibodies in serum/plasma ABO antibodies may be missing or weakened in certain patient-related situations and testing are the most may result in an ABO discrepancy. Example 8 illustrates this type of ABO discrepancy commonly encountered ABO in serum/plasma testing. discrepancies. Rouleaux present in the sample? Following incubation of test serum and red blood cells, centrifuge for 1 minute, and remove serum with a pipette. Replace test serum with an equal volume of saline. Mix. Centrifuge for 15 seconds and resuspend the cell button gently. No agglutination Agglutination rouleaux true agglutination Fig. 4-10  Saline replacement technique. Rouleaux causing false-positive reactions can be distinguished from agglutination through the use of this simple technique. (Modified from Mallory D: Immunohematology methods and procedures, Rockville, MD, 1993, American Red Cross.) 42

100 PART II  n  Overview of the Major Blood Groups EXAMPLE 8 Missing or Weak ABO Antibodies in Serum or Plasma Testing ABO Testing Results Patient Red Cells with Patient Serum with Reagent Red Cells Anti-A Anti-B A1 B 0 0 0 0 EVALUATION OF ABO TESTING RESULTS 1. The agglutination pattern with anti-A and anti-B reagents is typical of a group O individual. 2. The results of serum testing with reagent A1 and B red cells indicate a group AB individual. CONCLUSION Consider approaching this problem from the angle of missing serum reactions with reagent A1 or B cells. BACKGROUND INFORMATION An investigation of the patient’s history, including age, diagnosis, and immunoglobulin levels, provides clues to explaining the missing reactions in the serum testing. The patient’s age is an important factor because the concentrations of ABO antibodies are reduced in newborns and elderly adults. Knowledge of the patient’s diagnosis is essential; reduced immunoglobulin levels are also associated with several pathologic states (see Table 4-5). In conjunction with the patient’s diagnosis, the immunoglobulin levels and serum protein electrophoretic patterns are helpful data in the resolution and identification of the root cause for this ABO discrepancy. RESOLUTION OF ABO DISCREPANCY 1. Determine the patient’s diagnosis, age, and immunoglobulin levels, if available. 2. Incubate serum testing for 15 minutes at room temperature, and then centrifuge and examine for agglutination. This simple incubation step often solves the problem. If the results are still negative, place the serum testing at 4° C for 5 minutes with an autolo- gous control. The autologous control validates the test by ensuring that positive reac- tions are not attributable to a cold autoantibody. Interpretation of Additional Testing Results 4° C A1 Red Cells B Red Cells Autologous Red Cells Conclusion Patient serum Pos Pos Neg Group O Patient serum Cold autoantibody Pos Pos Pos SECTION 7  SPECIAL TOPICS RELATED TO ABO AND H BLOOD GROUP SYSTEMS CLASSIC BOMBAY PHENOTYPE The classic Bombay phenotype is an unusual genetic occurrence associated with the ABO and H blood group systems. A 1952 report describing a family living in Bombay, India, is the source of the descriptive term for the phenotype.1 This family’s red cells were unusual because they lacked H antigens and subsequently any ABO antigen expression. Both red cells and secretions were deficient in H and ABO antigen expression. The red cell reactions were characteristic of the group O phenotype in routine ABO testing. Serum testing demonstrated reactions similar to group O individuals. Another related antibody, 43

CHAPTER 4  n  ABO and H Blood Group Systems and Secretor Status 101 anti-H, was detected in the family’s serum in addition to the ABO antibodies of anti-A, Bombay individuals have anti-B, and anti-A,B. The anti-H in the Bombay phenotype is of clinical significance group O phenotype in routine because this antibody is capable of high thermal activity at 37° C and complement activa- ABO typing. These individuals tion with resulting hemolysis. are not compatible with group O donor units because their More than 130 Bombay phenotypes have been reported with a relatively greater inci- serum possesses anti-H. dence in India.19 Genetic family studies have identified the genotype required for this phenomenon. An individual who is homozygous for the h allele (hh) expresses the Bombay phenotype (the H and h genes of the H locus were previously described in this chapter). The hh genotype does not produce the L-fucosyltransferase necessary to transfer the immunodominant sugar, L-fucose, to the acceptor oligosaccharide chain to form the H antigen. As a result, the H antigen is not assembled on the red cells. Because H antigen is the building block for the development of the A and B antigens, A and B glycosyltrans- ferases cannot act on their substrate to produce the corresponding antigen structures, even though the ABO alleles are inherited. The resulting phenotype lacks expression of both H and ABO antigens. Transfusion for these individuals presents an especially difficult problem because they are compatible only with the Bombay phenotype. If transfusion is necessary, stored autologous units, siblings, and rare donor files are potential options. SECRETOR STATUS Secretor: individual who inherits Se allele and expresses soluble The interrelationship of the secretor locus with the expression of ABO antigens in body forms of H antigens in secretions. fluids has been mentioned several times throughout this chapter. There are two allelic genes at this locus: Se and se. The gene product of the Se allele, FUT2, is an Nonsecretor: individual who L-fucosyltransferase that preferentially adds L-fucose to type 1 oligosaccharide chain inherits the genotype sese and structures in secretory glands. The FUT2 gene may also act on type 2 chains in the secre- does not express soluble H tory glands. The H gene, FUT1, preferentially adds fucose to type 2 chains. substance in secretions. The FUT2 gene is directly responsible for regulating the expression of soluble A, B, Secretor studies may be and H antigens on the glycoprotein structures located in body secretions such as saliva. helpful in identifying a An individual who inherits the Se allele in either a homozygous (SeSe) or a heterozygous subgroup of A or B antigens. (Sese) manner is classified as a secretor. About 80% of the random population inherits the Se allele and is classified as secretors. These individuals express soluble forms of H antigens in secretions that can be converted to A or B antigens by the A and B glycosyl- transferases. These soluble antigens are found in saliva, urine, tears, bile, amniotic fluid, breast milk, exudate, and digestive fluids. An individual with the genotype sese is classi- fied as a nonsecretor. About 20% of the random population can be considered nonsecre- tors. The allele, se, is an amorph. A homozygote does not convert glycoprotein antigen precursors to soluble H substance and has neither soluble H antigens nor soluble A or B antigens present in body fluids. Fig. 4-11 illustrates the genetic interaction of the ABO, H, and Se loci. Example 1 Genes inherited Antigen expression AB HH SeSe RBC Saliva A, B, H A, B, H AB HH sese A, B, H None Example 2 Antigen expression Genes inherited RBC Saliva HH OO HH Sese OO HH sese H None Fig. 4-11  Practical application: interaction of ABO, H, and secretor genes in the expression of soluble antigens in saliva. RBC, Red blood cell. 44

102 PART II  n  Overview of the Major Blood Groups CHAPTER SUMMARY The major concepts of ABO antigens and ABO antibodies presented in this chapter are summarized in the following table. Important Facts: ABO and H Blood Group System Antigens Widespread antigen distribution Blood cells, tissues, body fluids, secretions Biochemical composition Common structures Glycolipid/glycoprotein Gene products Immunodominant sugars Type 1 and type 2 oligosaccharide chains Antigen expression Glycosyltransferases Genetic loci H antigen: l-fucose Major alleles A antigen: N-acetylgalactosamine B antigen: d-galactose Bombay phenotype Secretor status Cord blood cells: weak Landsteiner’s rule ABO blood group system: chromosome 9 Antibody production H system: chromosome 19 A1, A2, B, O Antibody immunoglobulin class H, h In vitro reactions Genotype hh; no H or ABO antigens Complement binding Se allele; soluble H and ABO antigens Clinical significance Serum possesses the ABO antibody directed toward the A or B antigen that is absent from red cells No antibodies detectable in first few months of life; decreases in elderly adults IgM and IgG At or below room temperature Yes; some hemolytic Yes CRITICAL THINKING EXERCISES EXERCISE 4-1 Case Study RT, a 37-year-old woman, is a first-time donor at your blood center. She is a healthy donor with an unremarkable medical history and is not taking any medications. Initial ABO phenotyping results indicate an ABO discrepancy. ABO Testing Results Donor Red Cells with Donor Serum with Reagent Red Cells Anti-A Anti-B A1 B 0 4+ 3+ 1+ 1. Evaluate the ABO phenotyping results. Is the discrepancy associated with the red cell testing or the serum testing? State the reasons for this selection. 2. How would you classify the category of ABO discrepancy shown in this problem? 3. What are the potential causes of an ABO discrepancy in this category? Additional Testing No technical errors were found. The donor’s red cells were washed, and the ABO phe- notyping was repeated. Red cell testing results were identical to the first set. In addition to A1 and B reagent red cells, commercial screening cells and an autologous control were tested with the donor’s serum. The results of the testing are depicted in the following table. 45

CHAPTER 4  n  ABO and H Blood Group Systems and Secretor Status 103 Additional Testing Results Donor’s Serum Testing with A1 Red Cells B Red Cells Screening Cells Autologous Red Cells 3+ 1+ 1+ 0 4. What conclusions can be drawn from the results of additional serum testing? 5. What additional steps are required to resolve this ABO discrepancy? EXERCISE 4-2 What are the possible ABO phenotypes of offspring with parents possessing the genotypes A1A2 and BO? EXERCISE 4-3 Group O individuals are considered universal donors for the transfusion of RBCs and universal recipients for plasma transfusions. Provide an explanation for this statement. EXERCISE 4-4 Create a diagram to illustrate the genetic pathways for ABO antigen production and the Bombay phenotype. EXERCISE 4-5 For a Bombay phenotype encountered in the immunohematology laboratory: 1. Predict the agglutination reactions of the patient’s red cells with the following reagents: anti-A, anti-B, anti-A,B, and Ulex europaeus. 2. Predict the agglutination reactions of the patient’s serum sample with the following reagent red cells: A1, B, and O. EXERCISE 4-6 Why does an individual with the genotyping of AB, HH, and sese possess A, B, and H antigens on red cells but not have any soluble forms of these antigens in the saliva? STUDY QUESTIONS 1. Given the following ABO typing results, what conclusion can be drawn from these results? ABO Testing Results Patient Red Cells with Patient Serum with Reagent Red Cells Anti-A Anti-B A1 B 4+ 4+ 1+ 0 a. expected results for a group O individual b. expected results for a group AB individual c. discrepant results; patient has A antigen on red cells with anti-A in serum d. discrepant results; patient has B antigen on red cells with no anti-B in serum 2. What are the gene products of the A and B genes? a. glycolipids b. glycoproteins c. oligosaccharides d. transferase enzymes 46