Week4_Lecture 3 - CL2 2.1 Transfusion & Immunology.pdf

Transfusion & Immunology 1  Immunology and Transfusion Science Immunology studies the mechanisms which the body defends itself against microbial infections. Transfusions can be lifesaving for patients with various conditions such as: severe anemia, thro...

Transfusion & Immunology 1  Immunology and Transfusion Science Immunology studies the mechanisms which the body defends itself against microbial infections. Transfusions can be lifesaving for patients with various conditions such as: severe anemia, thrombocytopenia, or deficiency of plasma components. An immune response can be elicit in transfusion recipients as allogenic blood cells and plasma proteins are foreign substances Plasma contains immune mediators and antibodies that can react with recipient cells. 2  Transfusions Therefore, transfusion carries risks of immunologic reactions. Safe and effective red cell transfusions are based on the knowledge of red cell antigens and antibodies. Understanding the immune system is important to transfusion scientist 3  The Immune System & Immune Cells The immune system is a collection of cells and proteins that’s role is to protect the respiratory passages, intestinal tract, the skin and other areas from foreign antigens, such as microbes (bacteria, fungi, and parasites), viruses, toxins and cancer cells. Functions of the immune response 1. Detect infection 2. Contain infection and eliminate it if possible 3. Self-regulate 4. Protective immunity against recurrent disease. 4  Immune Response There are two components of the immune system 1. Innate immune response 2. Adaptive immune response Innate Immune Response: Present in most multicellular organisms First line mechanism Activated immediately or within hours of encountering an antigen Detects microbial components and damaged host cells Main function is to prevents, controls, eliminates infections Stimulates the adaptive immune response 5  Immune Response Adaptive immune response Present in vertebrates Activated by innate immune response (Only) Second line of defense Antigen-dependent and antigen-specific Capacity for producing memory cells Hosts innate and adaptive immunity are not mutually exclusive mechanisms, they are complementary, any defects in either system results in host vulnerability 6  Innate Immune Response Overview Nonspecific immunity, natural resistance Provides resistances through several physical, chemical and cellular approaches Microbes first encounter skin epithelial layers (physical barriers) and mucous membranes. Subsequent general defences include secreted chemical signals (cytokines), antimicrobial substances, fever, and phagocytic activity associated with the inflammatory responses. The phagocytes express cell surface receptors that can bind and respond to common molecular patterns expressed on the surface of invading microbes. 7  Cellular Components of Innate Immunity 1. Epithelial Barriers -Epithelial surfaces: physical barriers that produce anti- microbial chemicals, and with protective T lymphocytes -Physical: Tight junctions between epithelial cells, mucus secretions, fluid and electrolyte secretions - Chemical - Immune 8  Cellular Components of Innate Immunity 2. Phagocytes Phagocytes (mainly macrophages, neutrophils). Phagocytes defensive mechanisms: Neutrophil Mast cell Phagocytose and kill pathogens Produce cytokines (promote inflammation) Macrophages that repair damaged epithelial barrier Neutrophils (short lived)/ Marcrophages (long lived) Basophil Eosinophil Macrophages are Antigen Presenting Cells (APCs) 3. Dendritic cells (DCs) Recognition and effector roles in immunity Constitutively present in epithelia to detect invading microbes In Innate immunity: express multiple PRRs, detect PAMPs and DAMPs, release antiviral cytokines, interferons In adaptive immunity: APC to T cells to mediate adaptive immunity Dendritic 9  Cellular Components of Innate Immunity 4. Natural Killer (NK) Cells Lymphocytes that kill injured, damage or infected cells NK receptors distinguish between unhealthy and healthy cells through receptor expression balance. Activating receptors expressed by unhealthy cells (infected or tumor cells) Inactivating receptors expressed by healthy cells (class I major histocompatibility (MHC I) antigens). Activated NK cells release granule proteins near infected cells initiating apoptosis NK cells respond to cytokines (IL-12) from macrophages and release interferon (IFN-g) that activates macrophages to kill microbes 10  Cellular Components of Innate Immunity 5. T and B Lymphocytes with limited antigen receptor specificities Major mediators of adaptive immunity Subsets of T and B lymphocytes detect PAMPs 6. Mast cells Present in mucosal epithelial tissues e.g. intestine, skin Detect infection, release multiple substances (histamine, prostaglandins, cytokines) Defend against bacteria and parasites Responsible for allergic diseases symptoms 11 Soluble Effector Mediators of Innate Immunity Soluble (humoral) components bind to extracellular pathogens : 1.Acts as opsonins enhance the ability of macrophages, neutrophils, dendritic cells to phagocytose microbes 2. Promote inflammatory responses that recruit phagocytes to sites of infection Components soluble effector mediators: Natural antibodies, Compliment system, Pentraxins 1. Natural antibodies. Subsets of B cells that produce antibodies with limited specificity 2. Pentraxins Pentameric plasma proteins mediate innate immunity 12  Soluble Effector Mediators of Innate Immunity 2. Compliment system Plasma proteins that opsonize microbes, recruit phagocytes to sites of infection, and kill microbes. Three mechanisms detect molecules on microbial surfaces: Classical pathway: Plasma protein C1q detects antibodies bound to microbes. Alternate pathway: Plasma protein C3 directly binds to microbial surface molecules (e.g. LPS) Lectin pathway: Plasma protein mannose-binding lectin directly binds mannose residues of microbes Three pathways activates the complement system 13  ADAPTIVE IMMUNITY Initiated when innate immunity is fails to eliminate infectious agents and infection is established. Adaptive Immunity Primary functions: - Recognition of specific “non-self” antigens presented byAPCs - Generation of pathogen-specific immunologic effector pathways - Development of immunologic memory 14  MECHANISMS OF ADAPTIVE IMMUNITY Components of Adaptive Immunity: (characterized by the cells involved) 1. Humoral immunity. is the aspect of immunity that is mediated by secreted antibodies. (B cell) 1. Cellular immunity. is the aspect of immunity that involved T cells alone (T cell) 15  Types of Adaptive Immunity 1. Active immunity. 2. Passive immunity. 16 Adaptive Immunity  Cellular components of adaptive immunity 1. B lymphocytes (Humoral immunity) - Detect extracellular antigens - Differentiate into plasma cells. - Plasma cells will secrete Abs - Abs opsonize & neutralize the microbe - Opsins promotes phagocytosism & activates complement system. 2. T lymphocytes (Cell mediated Immunity) - Detect intracellular antigens presented on cell-surface MHC molecules activating cell mediated immunity T cell subtypes: 1. Helper T cells 3. Regulatory T cells 2. Cytotoxic T cells 4. Natural killer (NK) cells Antigen presenting cells (APCs) - Dendritic cells that engulf microbial antigens, migrate to lymphoid tissues and present the antigens to naïve T cells to initiate immune responses 17  Adaptive Immune Response to Infection (Phases) 1. Induction phase APCs ingest antigens (microbes, microbial secretions, vaccines) APCs digest and process antigens APCs present processed antigen via MHC molecules to CD4 or CD8 T cells in lymphoid tissues 2. Effector phase CD4 cells (Express IL-2 receptors & release IL-2) CD4 differentiation to Th0 (autocrine control) Th0 cells differentiate (presence cytokines) Th2 cells interact with B cells producing: Memory Bcells(MB) & Plasma cells (P) (secrete antibodies) Th1 & Th17 cells (activate macrophages) Treg cells (dampen the inflammatory response) CD8 cells (Express IL-2 receptors & release IL-2) Differentiate into cytotoxic T cells (CT) (autocrine control) CT cells (kill virally-infected cells) 18  Antibodies Structure and Function Structure: - Highly variable proteins - Generated in response to foreign antigens - Immunoglobulins: IgG, IgM, IgE, IgA, IgD Functions: 1. Membrane-bound antibodies act as antigen receptors 2. Secreted antibodies bind toxins in tissues, mucosal surfaces, circulation, neutralizing and eliminating pathogens 3. Activates complement cascade causing bacterial lysis 4. Promote phagocytosis of antibody-coated bacteria 5. Recruit immune cells to large pathogens e.g. recruit eosinophils to antibody- coated parasitic worms 6. Mast cells & basophils degranulation, releasing inflammatory mediators 19  Innate and adaptive inflammatory response chemical mediators 1. Cytokines – peptides and proteins released by immune cells during inflammation (Interleukins, Chemokines, Interferons) 2. Histamine 3. Eicosanoids 4. Platelet-activating factor, PAF 5. Bradykinin 6. Neuropeptides 7. Nitric oxide iNOS (inducible NO synthase) 20  Time in weeks vs serum levels of an antibody (Ab) generated by the adaptive immune response. 21  Comparison between Innate and Adaptive Immunity Innate Adaptive Specificity Molecules shared by related Microbial and non-microbial microbes or generated by antigens damaged cells Diversity Germline encoded, limited Highly diverse Memory None Yes Non-reactivity to Yes: Healthy cells not Yes: Healthy cells not self recognised recognised (except in auto- immune disease) Major Cell types Macrophages, Neutrophils, T cells, B cells, and other Natural Killer Cells, Dendritic antigen presenting cells Cells, Basophils, Eosinophils Blood proteins Complement, others Antibodies Response Time Fast: Minutes or hours Slow: days 22  Introduction Blood Bank: Institution or place where blood & blood components are collected from eligible donors, blood is tested, processed, stored and issued to patients in hospitals. Blood transfusion: Requires the removal of blood from one human being (the donor) for infusion into another (the recipient). Transfusion might be for the: 1) whole blood or 2) for any of its components involving: packed red cells, fresh-frozen plasma, platelets or cryoprecipitate. 23  Blood groups Blood group antigens are clinically important for: ✓ Allogenic blood transfusion. ✓ Management of pregnancies at risk for hemolytic disease of newborn (HDN). ✓ Organ transplantation. 24  ABO blood system 25  ABO Blood system 26  How common is your blood type? Group Frequency O 45% A 43% B 9% AB 3% 27  ABO blood system There are 3 alleles or genes for blood type: A, B & O. Since we have 2 genes, there are 6 possible combinations. 28  ABO Antigens The A & B genes on chromosome 9 code for enzymes that can add: N-acetyl-galactosamine (NG) or D-galactose (DG) to H substance, converting it to A or B substance 29  ABO Antigens 30  ABO Antigens Genes that control ABO antigens code for enzymes. These enzymes add sugar molecules (carbohydrates) to a basic molecule called precursor substance (PS). The first sugar added to PS is L-Fucose (LF). An enzyme under the control of a gene called the H gene → adds L-Fucose to the precursor substance. The product of this reaction is called H substance H gene is not an allele at the ABO locus, but is located on chromosome 19. Most people are HH, very few are Hh (h is an amorph). Very very few are hh. 31  The ABO System https://transfusion.com.au/blood_basics/blood_groups/abo_rh 32  Rh system Rh system is highly complex & polymorphic, >50 antigens recognized. D Ag is the most important & clinically significant antigen. Other important Ags :C, c, E , e Rh negative→ no D Ag Rh positive→ presence of D Ag Expression of D Ag ranges from weak D & fully expressed D. Anti-D Anti-C Anti-c Anti-E Anti-e Blood phenotype R h negative (dce) R h positive (DE) 33  Rh system Weak D (Du): Weak expression of D Ag on red cells (Fewer in number than normal) Can not directly react with most anti-D reagents→ giving a weak or even negative agglutination. So each RhD negative for donors blood should be tested for the Du by the indirect antiglobulin test (IAT). For transfusion practice: Du positive donors should be considered as Rh positive. Du positive recipients should be considered as Rh negative. 34  Rh system Antibodies for Rh antigens are NOT naturally occurring (except for some anti-E) Immune formed that produced by transfusion or pregnancy. More than 80% of Rh negative people will develop anti-D after being transfused with Rh positive RBCs. 35  ABO, Rh system ABO system Rh system Antigens A, B, H Ag (forward typing) D, C, c, E, e (forward typing) Carbohydrate Protein 50% Expressed at birth Fully expressed at birth Present in all body cells & secretion Only present on RBCs “specific” Abs -Naturally occurring (reverse typing) -Not naturally occurring Ab (except Anti E), (No reverse typing) -Produced only from transfusion or -Influenced by (environmental substances) pregnancy (immune form) -IgM (Cold Abs), Pentamer, cant cross -IgG (Warm Abs), monomer so placenta & cause HDN, (Agglutinating Ab) cross placenta (HDN), (Sensitizing Optimal reactivity at 4˚C, can react at RT Ab) 36 Can activate Complement - BT (37˚C)  Other Blood Group Systems Blood System Antigens Antibody Lewis Lea Leb IgM , Kell K, k IgG MNSs M, N, S ,s Anti-M & Anti-N are IgM Anti-S & Anti-s are IgG Duffy Fya, Fyb IgG (mainly) Kidd Jka, Jkb IgG (mainly) Lutheran Lua, Lub IgM & IgG P P1, P IgM Ii I, i IgM 37  Basic terms to remember Clinical significance of Abs: Abs are associated with decreased RBC survival 1. Transfusion reactions 2. HDN Not clinically significant: antibodies that do not cause red cell destruction Cold reacting antibodies: agglutination best observed at or below room temp. Warm reacting antibodies: agglutination best observed at 37°C 38  Cold Antibodies (IgM) Naturally OccurringAntibodies: ABO system Lewis system Ii system P system MN system 39  Warm antibodies (IgG) Rh antibodies Kell Duffy Kidd S,s Lutheran 40  DAT DAT detects IgG antibody or complement coating the surface of RBCs (in- vivo sensitization). Perform that: RBCs are washed with saline carefully to remove unbound antibodies, then coombs reagent is added 41  DAT If negative, to confirm this result: Add one drop of Coombs cells (IgG coated red cells) to the mixture, Centrifuge again & re-examine for agglutination. Development of agglutination confirms the presence of active AHG in the mixture. DAT is used in the investigation of: 1. Auto-immune or drug-induced hemolytic anemia 2. Hemolytic Disease of Newborn (HDN) 3. Suspected Hemolytic Transfusion Reaction (HTR) 42  IAT IAT identified antibodies in the tested serum. Serum will be incubated with normal RBCs to allow in vitro sensitization of these cells, Cells are washed to remove unbound antibodies. Coombs reagent “AHG (anti-human globulin)” is added. 43  IAT IAT is used in many aspects: 1. Weak D (Du) identification. 2. Antibody screening. 3. Antibody identification (ID) Panel Test. 4. Cross matching XM. 44  Donor Identification & Inclusion Criteria The first step in blood donation is the proper registration and identification of the donor through filling a special form (Donation Card) Donation process should be ensured to be safe and cause no harmful side- effects on donor, and this can be accomplished through ensuring that the donor meets the following inclusion criteria 45  Blood Donation Selection of donors The most important consideration is the well-being of the donor. Donors must meet certain requirements to give a maximum of 525 ml blood including samples for testing (A unit of whole blood is approximately 450 ml + anticoagulant). These requirements are aimed at making sure that no harm comes to the donor or recipient. Selection Of Donors: 1. Physical 2. General health 3. Other reasons for exclusion 46  Selection Of Donors 1. Physical examination Donor weight:  50 kg & no recent weight loss of 2.5 kg or more. Normal temperature: at time of donation (37.5C). Pulse: 50-100 beats per min. Blood Pressure: 90 – 180 mm systolic; 50 - 100 mm diastolic No skin infections 47  Selection Of Donors 2. General Health Not pregnant No respiratory tract infection, cold, TB, etc. No asthma (allergy) No disease of heart, lungs or liver No epilepsy No history of Hepatitis No Malaria 3 years after disease. 6 months after being in an endemic area. Some Blood Banks may require 1 year. No recent (6 months) major surgery 48  Selection Of Donors No current medication (mild analgesics and oral contraceptives are OK) No alcohol No drug addiction No recent infection (less than 3 week recovery period) with measles, mumps, chicken pox, rubella) No recent history of blood transfusion (3 months) No recent history of tattooing, ear-piercing, acupuncture (12 months) ✓ A donor may be deferred based on history (drugs, medications, immunizations, medical conditions, travel, other) or physical exam. ✓ Confidential self-deferral opportunity should be offered before phlebotomy starts. 49  Collection Of Blood Registration ✓ Name in full ✓ Address ✓ Telephone numbers ✓ Date of birth ✓ Proof of identity ❖Details are required to be able to obtain donor again & to trace unit’s history Signed & witnessed consent form Allocation of a permanent registration number All future information, i.e. Group, antibodies, etc 50  Selection Of Donors Transmission of infectious diseases through transfusion of blood & blood products has become a major issue in the last decade. ✓ Spread of HIV & hepatitis viruses has made the pre-transfusion testing of blood a requirement for all blood banks. ✓ Questioning of donors is an important factor in reducing the risk of TTDs (Transfusion Transmitted diseases). Donors must be given the opportunity to exclude themselves by answering a list of questions, including their lifestyle & exposure to the previous diseases. Questions should include all the medical history & require an answer to: “Do any of these exclude you?” 51  TTDs (Transfusion Transmitted diseases) DISEASE ORGANISM PREVENTION TEST AIDS Virus - HIV Screening questions- IV drug use, sexual Antibody - HIV-1 Human Immunodeficiency Virus habits, etc. Antibody - HIV-2 Treated products. Antigen - HIV-1 p 24 HEPATITIS B Virus - Hepatitis B Screening questions (as for AIDS) Antibody –HBc (core) (HBV) Treated products Antigen – HBs Ag (surface) HEPATITIS C Virus - Hepatitis C Screening questions Antibody –HCV (HCV) Treated products T Cell Virus - HTLV – 1 - Antibody - HTLV-1/II LEUKAEMIA HTLV – II SYPHILIS Bacterium - Treponema pallidum Screening questions Antibody testing CYTOMEGALOVI Virus - CMV - Antibody - CMV RUS (CMV) Very widespread herpes virus Special cases only N5O2 T ROUTINE  TTDs (Transfusion Transmitted diseases) DISEASE ORGANISM PREVENTION TEST MALARIA Parasite: Plasmodium sp Transmission; Rare No practical serologic tests to Vector – mosquito Exclude donors who have been in a detect transmissible malaria in malarious area for 1 year asymptomatic donors TOXOPLASMOSIS Parasite: Toxoplasma gondii Transmission very rare NO TEST Vector - many animals Disease not considered to be a problem in routine transfusion practice BABESIOSIS Parasite: Babesia microti Disease and transmission, very rare - NO TEST Vector – ticks Exclude sufferers CHAGAS Parasite:Trypanosoma cruzi Transmission Rare NO TEST DISEASE Vector: Tsetse Fly Exclude sufferers (Trypanosomiasis) LEISHMANIASIS Parasite: Leishman donovani Very rare NO TEST Vector: Sand Fly Exclude sufferers 53  Selection Of Donors Frequency of donation: A. Donation of whole blood Minimum 8 weeks (usual 12 weeks)→ 2-3 months B. Donation of components by apheresis. Plasma - 5 days Platelets- 2 days Apheresis: Whole blood components are separated then the part wanted is kept while the rest returned. 3. Other reasons for exclusion: ✓ Inability to give consent - mental status ✓ Age 65 years ✓ Low hemoglobin 54  Donation of whole blood Donation of blood by apheresis 55  Collection Of Blood Situation (Collection of Blood) “Phlebotomy” ✓ Well-trained staff - supervised by doctor. ✓ Well-equipped, pleasant & friendly atmosphere ✓ Convenient location Information for donors ✓ Informed consent - chance to say no - signed consent ✓ Clear understanding for reasons for exclusion ✓ Explanation of procedure 56  Collection Of Blood 57  Collection Of Blood Pre-donation tests 1. Hemoglobin: must be  12.5 g /dl 2. Hematocrit: must be  0.38 l / l Post donation testing of donor’s blood All donor samples are tested to: 1. Ensure correct matching with recipients 1. ABO - Forward & reverse grouping 2. Rh - Rh D testing→ Weak D testing of Rh D negative blood 3. DAT test: to prevent post transfusion reaction in the patient (red cells) 4. Antibody screen: to detect presence of atypical antibodies (serum) 58  Testing Of Blood 2. Ensure freedom from disease-causing organisms: HBs Ag Hepatitis B surface antigen Anti-HBc Hepatitis B core antibody Anti-HCV Hepatitis C antibody Anti-HIV-1 Human Immunodeficiency Virus-1, antibody Anti -HIV-2 Human Immunodeficiency Virus-2, antibody HIV-1 p24 antigen Human Immunodeficiency Virus-1 antigen Anti-HTLV-1 Human T-cell Leukemia Virus antibody Syphilis Treponema pallidum - antibody 59  Adverse Donor Reactions 1-Nausea & vomiting: Make donor comfortable & instruct to breath deeply & slowly, cold compresses to forehead 2-Hematoma at phlebotomy site: Remove tourniquet & needle, apply firm pressure with sterile gauze for 7 to 10 minutes with arm above the heart level, may apply ice to about 5 minutes to site. 3- Arterial puncture suspected: If suspected, remove needle & apply firm pressure for 10 minutes with a pressure dressing afterward; check for a radial pulse & if no pulse palpable, call blood bank physician. 60  Adverse Donor Reactions 4- Syncope (Fainting, vasovagal syndrome): This is caused by psychologic or neurophysiologic response. Sometimes the skin feels cold & blood pressure falls as low as 50 mm Hg. Symptoms: weakness, sweating, dizziness, pallor, loss of consciousness or bowel & bladder control & convulsions. Treatment: cold compress to donor forehead or back of neck, donor lays on back with legs above head level, loosen tight clothes, assure appropriate airway, monitor blood pressure-pulse- respiration until recovery. Prolonged hypotension may respond to normal saline infusion with blood bank physician approval. 5- Convulsions: Call for help immediately, prevent donor from injuring themselves or others, assure adequate airway, notify blood bank physician 61  BLOOD PRODUCTS & COMPONENTS Sources Of Material Blood & blood products are obtained by: 1. Single unit donations A unit of blood is obtained from one person In the past, it was the only source of supply of blood products & components. Recently, the use of whole blood has been reduced to the point where, in most blood banks,  90% of donations are separated into individual components (red cells, platelets, leukocytes, plasma, FVIII, etc) for transfusion as individual products. The amount of blood taken is limited by the ability of the donor to replace the red cells that have been lost. 62  Sources Of Material 2. Apheresis Apheresis is a process in which donor blood is separated by mechanical means on a cell separator. The component that is needed is collected & the components that are not needed are returned to the donor. It is usual to return RBCs & collect platelets, WBCs or plasma. Because red cells are not collected, larger amount of the other components can be taken. The donors system can replace platelets, WBCs & plasma much more readily than it replaces RBC. 63  Sources Of Material 3. Recombinant DNA products The use of recombinant technology to produce specific plasma protein fractions in unlimited amounts has the potential to replace the use of donors for some blood products. How: 1. DNA codons for specific proteins are inserted into animal or bacterial cell DNA. 2. Cells are cultured in the lab & they secrete the protein into the culture liquid. 3. After purification, the protein can be used for therapeutic purposes. Recombinant FVIIIc is currently available & a number of other products are being investigated, e.g. FIX, antithrombin III. 64  Sources Of Material 4. Non-human products Early attempts to transfuse animal cells, understandably, met with fatal consequences. The human recipient’s immune system rejected them as foreign. The only successful products are animal FVIII concentrates from cows (bovine) & pigs (porcine). These have limited uses. 65  Methods Of Separation A. Single bag: for collection of WB donation B. Double bag: for removal of plasma - leaves red cells C. Triple bag: red cells, platelets & plasma D. Quadruple bag: red cells, platelets, plasma & cryoprecipitate E. Add-back preservative pack to preserve red cell concentrate 66  Methods Of Separation Plasma expressor SAGM: Saline Adenine glucose Mannitol 67  Blood donation/ Collection bags 68  Anticoagulant preservative solutions Whole blood from single unit donations & apheresis procedures are collected into plastic bags. Bags are manufactured with an anticoagulant - preservative solution in them to: 1. Prevent clot formation 2. Preserve blood components so that they remain useful during storage. Anticoagulant preservative solutions are mainly designed to preserve red cells, but the solutions have been found to preserve other cellular components and are suitable for preparation of plasma components. 69  Anticoagulant preservative solutions The solutions used all contain: 1. Tri-sodium citrate as an anticoagulant (removal of Ca+2 ) 2. Dextrose to provide the fuel for cellular metabolism during storage Solutions in use: ACD, CPD and CPDA-1 1. ACD→ (Acid Citrate Dextrose). A solution with these three components was used for many years. Red cells are stored at 4◦C Can be used up to 21 days after storage. At this time  75% of red cells are viable (useful) → i.e. survive to carry oxygen. 70  Anticoagulant preservative solutions 2. CPD→ (Citrate Phosphate Dextrose). A solution in which extra phosphate in the form of sodium phosphate is added. This extends the viability of cells to 28 days (USA still allows only 21 days). 3. CPDA-1→ (Citrate Phosphate Dextrose Adenine). A solution in which adenine is added to CPD is called CPDA-1 The added adenine allows red cells to synthesize ATP & prolongs storage life (viability) to 35 days at 4˚C. At this time  75% of red cells are viable. 71  Anticoagulant preservative solutions Limiting factors in cell viability during storage are: 1. Levels of ATP (adenosine triphosphate). ATP represents the energy reserve of cells. When it is depleted, the cells die. 2. Levels of 2, 3 - DPG (2, 3- diphosphoglycerate). For red cells to function as providers of oxygen, 2, 3- DPG is necessary. Without it, Hb can pick up oxygen but does NOT release it efficiently to the tissues. As the majority of red cells are used within 14 days, when 2, 3- DPG levels in CPDA-1 are  70% CPDA-1 is the solution of choice for red cell preservation. SAGM: extends storage life to 42 days. 72  Blood component preparation 73  Blood products & components available WHOLE BLOOD CELLULAR PLASMA COMPONENTS COMPONENTS RED BLOOD GRANULOCYTES PLATELETS CELLS 74  Blood products & components available 1) Cells a) Whole Blood b) RBCs products: 1. Packed Red Cells or Red Cell Concentrates 2. Leukocyte poor red cells 3. Plasma free red cells (washed red cells) 4. Frozen Red Cells c) Leukocytes d) Platelet Concentrates 2) Plasma components 75  Whole blood (WB) Use: To replace massive blood loss in acute bleeding. The use of packed red cells + platelets + fresh frozen plasma is preferable, why? Plasma components (some clotting factors) & platelets deteriorate rapidly when stored at 4C. Preparation & storage Single unit donations in CPDA-1 at 4C 35 days maximum. 76  Blood products & components available 77  Packed red cells or red cell concentrates RBCs products: Washed 1. Packed red cells or Red cell concentrates RBCs RBC Concentrate 2. Leukocyte poor/ reduced/ depleted red cells RBCs 3. Plasma free red cells (washed red cells) 4. Frozen Red Cells RBCs LR-RBCs frozen 78  Packed red cells or red cell concentrates Use: To increase oxygen-carrying capacity in anemia. Transfusion of excess plasma is avoided. Transfusion of cells with hematocrit of  80% (0.8 l/l) is difficult, due to increased viscosity. Preparation Blood is centrifuged in a refrigerated centrifuge & the supernatant plasma removed into a transfer pack. 79  Leukocyte-poor red cells Use: Transfusion of unwanted white cells can result in: 1. Febrile reactions due to HLA & leukocyte antigen/antibody reactions. 2. Sensitization to HLA antigens in potential transplant patients. 3. Transmission of leukocyte-borne viral diseases. (CMV) Filtration Special filters are available that remove leukocytes. They can be used before transfusion or at the time of transfusion. 80  Plasma free red cells (washed RBCs) Use: When anemic recipient is allergic to plasma components. Preparation Red cells are washed in the bag by: 1. Centrifugation to remove plasma that is replaced with cold sterile saline. 2. Centrifugation is then used to remove the saline. 3. Washing procedure is carried out three times. Storage Keep at 4C until used. Use within 24 hours. 81  Frozen Red Cells Use 1. Long term storage of rare groups (may survive for years in liquid nitrogen). 2. Long term storage of autologous blood. 3. Method of obtaining leukocyte-poor, plasma-poor blood. Preparation Use of glycerol to prevent hemolysis. Have to be washed when thawed to remove glycerol. Storage At temperatures below -65C. Stored for up to 3 years. Use as soon as thawed & washed. 82  Leukocytes Use Granulocytes (myeloid cells) for leukopenic patients with infection. Preparation 1. Filtration 2. Leukopheresis Storage: Use at once 83  Platelet concentrates Use: Thrombocytopenia due to: 1. Low production - e.g. cytotoxic drugs, etc 2. High use - e.g. Disseminated Intravascular Coagulation(DIC), bleeding 3. Immune destruction of plts - e.g. ITP Use ABO & Rh compatible platelet unit Storage 5 days at 22C (low temperature destroys aggregation). Constant, gentle movement: platelets form large aggregates (clumps) if allowed to stay still. 84  Platelet concentrates Preparation: 1. Single donor units Differential centrifugation of a unit of whole blood: ✓ Centrifuge WB at low speed → platelet rich plasma (PRP) ✓Centrifuge PRP at high speed → platelet poor plasma (PPP) + platelet concentrate Dose = 6 packs are pooled in I unit taken from 6 donors. 1 unit = 60 x 109 platelets in 50 ml plasma. 2. Plateletpheresis Dose: 300 x 109 platelets in 100 ml plasma from 1 donor. 85  2. Plasma components a) Fresh Frozen Plasma (FFP) b) Cryoprecipitate c) Single factor concentrates ✓ Albumin - liquid ✓ FVIII - freeze dried ✓ Vitamin K factors - freeze dried ✓ Gamma globulin - liquid ✓ Anti Rh (D) immune globulin - liquid ✓ Antithrombin III 86  Fresh Frozen Plasma (FFP) Use Contains all coagulation factors & other plasma components, e.g.,: 1. DIC 2. Liver failure 3. Vitamin K deficiency (Warfarin overdose) 4. Massive transfusion NOT used: for volume replacement Use ABO compatible 87  Fresh Frozen Plasma (FFP) Preparation Prepared from single unit donations within six hours of donation by centrifugation at high speed (5,000rpm, 5 min @ 5C) to remove all cells. Transfer cell- free plasma into transfer pack. Freeze at -65C immediately in dry ice - ethanol. Storage: Store at -20C for 1 year. To use: 1. Thaw at 37C in water bath with agitation. 2. Use at once - no longer than 2 hours after thawing. 88  Cryoprecipitate Preparation Fresh frozen plasma is thawed at 4C. A precipitate remains undissolved in the bags. Precipitate is cryo (cold) - precipitate :contains fibrinogen, FVIII, vWF & FXIII. The bag is centrifuged at high speed (5,000 rpm, 5 min 4C) & the supernatant taken off to leave 50 ml plasma + precipitate. 89  Cryoprecipitate Use: 1. Factor VIII replacement in Hemophilia A& von Willebrand's disease. 2. Factor VIII &/or fibrinogen replacement in DIC. 3. Fibrinogen replacement in liver disease. Use ABO compatible. Storage: Freeze & store at -20C - 1 year. To use: Redissolve plasma in bag at 37C in water bath with agitation, before use. 90  Single factor concentrates Preparation of these products is beyond the scope of regional Blood Banks & involves extraction procedures. The products are all capable of transmitting viral diseases unless treated during manufacture. Antiviral treatments available are: 1. Heat treatment @ 60C - destroys some labile factors: VIII & V. 2. Chemical “Detergent” treatment: to destroy the viral envelope. 91  Single factor concentrates Products available Use Albumin - liquid Fluid replacement - burns, etc Albumin loss in cirrhosis of the liver FVIII - freeze dried FVIII replacement Vitamin K factors - freeze dried FIX, VII, II, X replacement Gamma globulin - liquid Passive replacement of antibody in immunodeficiency - AIDS, congenital immunodeficiency, etc Anti Rh (D) immune globulin - liquid Prevention of Rh HDNB Antithrombin III Experimental 92  Differential Centrifugation Separation of components is based on the principle that different components have differentspecific densities. 1)WB is centrifuged at Soft (light) Spin, components are separated with the heaviest in the bottom(RBCs, WBCs, platelets, plasma). 2)After separating of RBCs from PRP, plasma is centrifuged again for longer time and harder spin (heavy spin). 3)Platelets settle to the bottom of the bag ,and plasma is drawn into the connected bag and platelets remaining in the first bag. 4) These components may be processed further to produce other products. 5)All products then properly labeled and stored under appropriate conditions until completion of testingand final distribution 93  Whole Blood Volume:450±50 ml Storage: 1-6 C Centrifugation rpm/min/20-24 C Packed Red Cells Plasma (PRP) Storage:1-6 C Centrifugation rpm/min/20-24 C Platelets Plasma(FFP) Storage:20-24C with agitation Storage: Frozen 94 95  Compatibility testing Definitions Recipient: The person who receives the blood Donor: The person from whom the blood was obtained Transfusion: The process of transferring blood from a donor to a recipient Donation: The blood from the donor Cross-matching: The test used to make sure that there is no reaction between donor & recipient blood 96  Compatibility Testing Compatibility Testing: all lab procedures utilized in the selection and testing of a donor unit to be transfused safely to the recipient These procedures include: 1. ABO & Rh D Typing: for both patient & donor 2. Ab screening: for patient 3. Ab ID: for patient 4. Cross-matching XM 5. Documentation procedures 97  Objectives of matching procedures The most frequently transfused blood component is red cells. They are transfused to replace oxygen carrying capacity lacking in the patient due to anaemia or acute blood loss. When blood is selected for transfusion, the tests used are chosen to detect anything in the donated blood that may cause harm to the recipient. 98  Possible causes of harm to the recipient 1. An antibody-antigen reaction between donor red cell antigens & recipient serum antibodies. ✓ Cross matching 2. The transmission of infection from donor to recipient. ✓ Testing to prevent transmission of infection ✓The tests used are done on all donor units before the blood is placed in hospital transfusion department. 99  Cross-matching: To detect Ag-Ab reactions Ag-Ab reactions that take place may be classified into 2 types: 1. Reactions between donor red cells & recipient antibodies These are the most important ✓Because the recipient may have large amounts of antibody in his/her serum & is capable of producing more antibody. ✓Rapid & fatal destruction of transfused red cells can result from transfusion of incompatible red cells. The test used to prevent such reactions is called “Major cross-match” 10 0  Cross-matching: To detect Ag-Ab reactions 2. Reactions between donor Abs in the serum & recipients red cells These reactions rarely cause serious problems Because a relatively small amount of Abs may be transfused & is absorbed by recipient antigens with little damage to the recipient’s cells. The test used to prevent such reactions is called: Minor cross-match. 10 1  Major cross-match The objectives of major cross-match procedure are: 1. To detect important antigens on donor cells that will either : I. react with recipient antibodies already present II. stimulate the production of atypical antibodies, if transfused. 2. To detect antibodies in the recipient’s serum that may react with donor cells. 10 2  Detection of important Ags 1. ABO typing of donor & recipient Because Abs to ABO Ags are naturally occurring, they are usually present in the serum of people who do not have the Ag. Selection of ABO compatible blood is the most important matching procedure Group of recipient Possible donor group O O A A and O B B and O AB AB, A, B and O 10 3  Detection of important Ags NOTE: The transfusion of dangerous amounts of anti-A or anti-B when a whole unit of O blood is used, is avoided by: 1. Removing plasma from the donated cells (packed red cells) 2. Testing the plasma of the O donation→ to detect dangerous levels of anti-A or anti-B 10 4  2. Rh D Typing D Ag of Rh system is the most antigenic RBCs Ags other than ABOAgs. It is capable of stimulating the production of a strong Ab, anti-D, that causes: 1. Severe transfusion reactions (severe HTR) 2. Haemolytic disease of the newborn (HDN) Transfusion of Rh D positive RBCs to Rh D negative recipients who do not have anti-D in their serum is unlikely to cause IMMEDIATE damage. It may however stimulate the production of anti-D in the recipient & a SECOND transfusion of Rh D positive cells will result in a reaction. 10 5  2. Rh D Typing Women of child-bearing age who are Rh D negative can produce anti-D that can cross the placenta & cause HDNB. The damage may occur years after the first transfusion. Testing for weak D is not necessary in recipients. If they are typed as negative & receive negative blood, no harm will be caused. Donors are tested for weak D. Any weak D positive donors are called Rh D positive→ as weak D Ag may cause anti-D production in Rh D negative persons. Rh D type of recipient Possible Rh D Type of donor Rh D negative Rh D negative Rh D positive Rh D negative Rh D positive & weak D 10 6  2. Rh D Typing If no Rh D negative blood unit is available & the recipient has no detectable anti-D in their serum Rh D positive blood can be given to Rh D negative recipients when: 1. A transfusion of red cells is unavoidable. 2. The recipient is a male, or a female no longer able to bear children. From birth to menopause, Rh D Negative females should never receive Rh D positive blood 10 7  3. Typing for Rh Ags other than D Most blood banks try to avoid transfusing blood that is Rh E &/or Rh C positive to recipients that do not have these antigens. Full Rh typing & matching of donors & recipients is NOT practical or necessary in most cases. However, it is general practice to issue blood for donation labelled as Rh negative only when it is of the genotype: cde/cde (rr) i.e. when it is Rh C & Rh E negative as well as Rh D negative. Donations are tested for antigens C & E as well as the D & weak D antigens. 10 8  3. Typing for Rh Ags other than D The reasoning is as follows: Antigens C & E are next in ability to produce antibody (antigenic potency) after D. c & e are not as potent. Most people who are Rh D negative are also C & E negative & may produce antibodies to these antigens if they are transfused with C &/or E positive. Recipients are called Rh negative when they are Rh D negative. Donors are called Rh negative when they are C, E, D & weak D negative 10 9  4. Typing for other Ags Antigens other than ABO & Rh are NOT tested for as a routine. However, there are some circumstances when it is necessary. 1)When a recipient is known to have an atypical antibody capable of causing a reaction, donor blood is typed for that antigen before transfusion. e.g.: Recipient has an anti-K antibody - transfuse only K antigen negative blood. Recipient has an anti-Fya - transfuse Fya antigen negative blood. 11 0  4. Typing for other Ags 2) When it is known that a patient will require red cell transfusions over a long period of time (e.g. -thalassemia, aplastic anemia). Some blood banks attempt to prevent atypical antibody production by: Typing the patient & donors to attempt as close a match as possible. e.g. Patient is: R1r (CDe/cde) Kell negative (K) Duffy negative (Fya ) Attempt to transfuse R1r, K negative, Fya negative red cells, whenever possible. 11 1  Detection of atypical antibodies 1) Screening of recipient serum (Ab screen) 1. Antenatal studies 2. Suspected transfusion reaction 3. Pre-transfusion testing of recipient’s sera 4. Screening of blood donor units 2) Identification of atypical antibodies (Panel test, Ab ID) 1. If Ab screen test is positive 2. If crossmatching test is positive 11 2  Antibody screening test AIM: to detect the presence of clinically significant, unexpected antibodies. Clinically significant antibodies: are Abs previously reported to cause: 1. Hemolytic disease of newborn (HDN). 2. Hemolytic transfusion reactions (HTRs). All clinically significant antibodies are reactive in vitro at 37°or by IAT. 11 3  Antibody screening test Unexpected antibodies: antibodies other than naturally occurring anti-A or anti-B. Alloantibodies: antibodies DO NOT react with RBC's self- antigens. Autoantibodies: antibodies react with RBCs' self- antigens. 11 4  Antibody screening test Recipient’s serum is screened to detect atypical antibodies. This is done by testing patient’s serum using a variety of techniques with at least two (preferably three) screening cells that carry the important blood group antigens. 11 5  Panel test, Ab ID If atypical antibodies are detected, they must be identified, to decide whether they are significant. This will allow selection of blood for transfusion that does not have the corresponding antigen on red blood cells. This is achieved by testing the serum with a minimum of 8 (preferably 12) group of O cells fully typed for all the significant antigens. The pattern of reactivity with this panel should allow the identification of theAb. 11 6  Panel test, Ab ID Choice of Panel cells (reagent): 1. A commercial available panel is usually used 2. They have 8-12 vials 3. O adult red cells 4. Fully typed for all the significant RBCs antigens Autocontrol is always set up to test for autoantibodies in patient’s serum. Sample for panel test: Fresh serum/ plasma (EDTA) samples (Aged collected < 72 hr). Serum may be stored at 40C for up to 48 hours or -200C for up to 7 days. Note: It is good practice to retain serum samples at -200C for up to 1 month following pre transfusion testing, to allow for possible investigation of delayed transfusion reactions. 11 7  Direct matching Selection of blood of the correct type for transfusion can be based on the: ABO, Rh D type & any antibodies detected. e.g. Patient is group A Rh D negative with an anti-K antibody in the serum. Select: Group ARh D negative, antigen K negative blood. 1)As a final check before transfusion when no antibodies have been detected in the recipient’s serum. Confirm ABO & Rh D type of donor units & recipient. Add 1 drop 4% donor cells to 2 drops recipient serum, centrifuge & read. This acts as a final check on compatibility. 11 8  Direct matching 2)As a final check before transfusion when antibodies have been detected in the recipient’s serum. Confirm ABO & Rh D type of donor units & recipient. Confirm donor cells are negative for antigens corresponding to antibody detected. Use saline RT, LISS & Coombs techniques to cross-match donor cells with recipient serum. 3) Use of routine cross-match before transfusion. Most Blood Banks still use Coombs cross-matching techniques before transfusion even when no antibody has been detected in the serum. When reliable screening cells are not available, a Coombs cross-match must be carried out. 11 9  Direct matching (Summery) Testing of donor units. (on donor units, focus on antigens) All donor units are tested for ABO type & Rh D type, with a weak D detection for all Rh D negativeblood on routine testing. Rh D negative units are tested for C & E. Any C or E positive units are classified as Rhpositive. All donor units are tested for atypical antibodies by the Central Blood Bank. ABO & Rh D type must be re-checked in the hospital blood bank before transfusion. 12 0  COMPATIBILITY TESTING FOR BLOOD TRANSFUSION Remember: all these tests are performed at central blood bank 12 1  COMPATIBILITY TESTING FOR BLOOD TRANSFUSION 2. REC RED CELLS SER ABO forward grouping Reverse ABO grouping Rh D type Antibody screen (Coombs antiglobulin min 12 2  Policies for matching pre-tested patients needing transfusion 1. Group & screen procedure Check ABO & Rh D type of donor & recipient. Screen patient’s blood for clinically significant antibodies. Issue compatible blood. 2. Group, screen and ABO check procedure Check ABO & Rh D type of donor & recipient. Screen patient’s blood for clinically significant antibodies Immediate spin of donor cells & recipient’s serum as final compatibility check. Issue compatible blood. 12 3  Policies for matching pre-tested patients needing transfusion 3. Group, screen & cross-match procedure Check ABO & Rh D type of donor & recipient. Screen patient’s blood for clinically significant antibodies Cross-match donor cells with recipient serum by saline at room temperature, LISS at 37C, and coombs. Then issue ABO & Rh D compatible blood. 12 4  Adverse Reactions Of Blood Transfusion An adverse transfusion reaction: is any adverse outcome from a transfusion of blood or blood product that does harm rather than good. 10% of the recipients experience an adverse effect. Any adverse reaction to a unit of blood or one of its components should be considered a potentially life-threatening reaction until clinical observations and/or laboratory results establish otherwise. 12 5  Classification of adverse transfusion reaction Transfusion reactions may be: 1. Acute TR→ occurs during or within 1 to 2 hours after transfusion 2. Delayed TR→ may not be evident for days, weeks, months, or years after transfusion Acute and delayed TR are classified as: 1. Immunologic TR→ mediated by antibody-antigen reaction 2. Non-Immunologic TR 12 6  Classification of adverse transfusion reaction 12 7  Acute Occurs within few hours of transfusion Immune Nonimmune HTR Noncardiogenic pulmonary Physical/ Bacterial Circulatory chemical contamination overload hemolysis FNHTR Allergic 12 8  Acute Hemolytic Transfusion Reaction (Acute HTR) Defined as hemolysis of the donor or the recipient RBCs by performed alloantibodies. Rare (incidence of 1:25,000). It’s one of the most dangerous TR. Life-threatening type, most commonly due to ABO-incompatible blood (Intravascular hemolysis) Mediators of AHTR: IgM (ABO) & complement 12 9  Signs & Symptoms of AHTR Fever Sudden onset of fever, chills, facial flushing, chest Chill pain or low back pain, hypotension & dyspnea. Hemoglobinemia Shock Hemoglobinuria Nausea Anemia Vomiting Renal failure Substernal pain DIC Lightheadedness Uncontrollable bleeding Pain at infusion site The reaction happens during first few ml of transfused blood 13 0  Pathogenicity of AHTR Main complications of AHTR: 1. Hypotension 2. Renal failure (36%) 3. DIC (10%) Management/ Prevention: 1. Decrease opportunity of errors 2. Treat renal failure & DIC 13 1  Febrile Non Hemolytic Transfusion Reaction 2- Febrile (fever causing): Defined by a rise in temperature of 1oC more above the baseline level during transfusion with no other explanations (no other signs)..(Fever & chills) The reaction will occur within an hour or less. Common (1:200 transfusions) Mediators: Abs to HLA class 1 antigens. Due to a reaction between HLA Class I antigens on transfused blood & antibodies in previously alloimmunized recipients. Management/ Prevention: Antipyretics & Leukocyte depleted products (filtration) 13 2  Allergic TR Defined as: During or after transfusion, patient develops Erythema urticaria (skin rash) & sometimes mild shock. Urticaria Reaction usually happens within minutes after transfusion. Signs: urticaria, erythema, itching, anaphylaxis. Mediators: Plasma proteins (mild reaction) & antibodies to IgA Management/ Prevention: 1. Antihistamines 2. Treat symptoms 3. Transfusion IgA deficient components (washed packed RBCs) 13 3  Noncardiogenic pulmonary TR Defined as: Occlusion of pulmonary vasculature, leads to Cyanosis damage to the lungs (potentially fatal) Mediators: Donor/ recipient WBCs Abs Signs & symptoms: Fever, chills, cyanosis, hypotension, noncardiogenic pulmonary edema & acute respiratory distress. Management/Prevention: Vigorous respiratory support & steroids, filtrated components 13 4  Non-immuonologic acute TR 1- Bacterial contamination also called “Warm”: Defined as: Fever, with symptoms of septicaemia usually after transfusion of whole blood, red cells or platelets contaminated with bacteria. Either from the donor venipuncture site or during component preparation. Mediators: endotoxins produced by gram negative bacteria Signs & symptoms: Fever, shock & hemoglobinuria (renal failure) & DIC/ death. Management/ Prevention: IV antibiotic, treat hypotension. 2- Hemolysis due to physical or chemical Means: Mediators: exogenous destruction of RBCs. Signs & symptoms: hemoglobinuria Management/Prevention: Document & role out hemolysis due to the other causes, treat DIC. 13 5  Non-immuonologic acute TR 3- Circulatory overload: (Hypervolemia) Defined as: Too much fluid transfused in a short time causes overload of the heart. Mediators: fluid volume Signs & symptoms: coughing, cyanosis, orthopnoea, severe headache, peripheral edema, difficulty breathing, hypertension or CHF (congestive heart failure). Management/Prevention: Administer subsequent transfusions slowly & in small volumes Transfusion patients at greater risk of developing circulatory overload may include: 1. Pediatric & Geriatric 2. Chronic anemias: Thalassemia major & sickle cell disease. 3. Cardiac disease (cardiopulmonary compromises are susceptible) 13 6  Delayed TR Occurs after 24hr of transfusion Immune Non-immune Hemolytic TR Transfusion TA-GVHD Transmitted Haemosiderosis (iron overload) Disease Post Transfusion (TTD) Purpura (PTP) 13 7  Delayed haemolytic reactions Defined as: Destruction of RBCs by antibodies, extravascularly in RES after 24 hr. Mechanism: ✓If a recipient has been previously immunized by transfusion or pregnancy, Ab may at a very low level & the Coombs test carried out pre-transfusion may not detect this low-level Ab→ (Ab screen –ve) ✓However, when the corresponding antigen is transfused again, a delayed secondary response may occur which may destroy the red cells. The patient starts to develop anaemia & fever about one week after transfusion. e.g. anti-Jka & anti-Jkb may show this type of reaction. Causes: patients develop IgG Abs against Ags other thanABO system leading to extravascular hemolysis 13 8  Leukocytosis Delayed haemolytic reactions Clinical Features: Within 5-10 days after transfusion Ab affects RBC life period (shorter than expected), gradually causing the following symptoms: ✓ Fever; ✓ Leukocytosis; ✓ Jaundice; Jaundice ✓ Renal impairment (6% of the cases). 13 9  Delayed haemolytic reactions Investigation: ✓ Hemoglobin: ↓ ✓ Blood film: shows spherocytes & polychromasia. ✓ Serum Unconjugated bilirubin: ↑ ✓ Serum LDH: ↑ ✓ Serum haptoglobin: ↓ or absent. ✓ Renal function Monitoring. ✓ DAT: positive with IgG &/or C3 coating on red cells. ✓ Ab screen & Ab ID test: to detect the antibody. 14 0  Delayed haemolytic reactions Prognosis & complication: Rarely life-threatening Treatment: Treatment for antibodies, such as: intravenous immunoglobulin. Prevention/ Management: 1. Pre-transfusion testing & Post-transfusion testing, including Ab screen to detect any Ab that may cause a reaction with the donor cells. 2. Choose appropriate antigen negative blood for the detected Ab in the recipient, especially for chronic transfusion patients (sickle cell, thalassemia). 14 1  Graft Versus Host Reaction (GVHD) Defined as: If live-lymphocytes (immunocompetent WBCs) are transfused to (immunosuppressed recipients or infants, fetus in utero; risk people), they can multiply & start damaging host’s tissues (BM, skin, liver & GI) by T cells cytokine effect. Prognosis: fatal (mortality rate of > 90%). Clinical Features: appear in 8-10 days post transfusion: Fever, Vomiting, Skin rash, Pancytopenia, Abnormal liver function (e.g. hepatitis), Diarrhea, Enterocolitis. Investigation: Histological & molecular analysis of CD3+, CD8+ lymphocytes from affected tissue. Management/Prevention: Gamma irradiation of cellular blood products (WB, RBCs, platelets, granulocytes) for risk patients “irradiated blood” 14 2  Graft Versus Host Reaction (GVHD) Risk group: ✓ Patients with congenital cellular immunodeficiency. ✓ Patients receiving granulocyte transfusions. ✓ Patients with Hodgkin's Disease. ✓ Patients with aplastic anemia receiving immunosuppression, such as treated with purine analogue drugs. ✓ Recipients of blood from biologically related (directed) or HLA matched donors ✓ Intrauterine & all subsequent transfusion & exchange transfusion recipients. 14 3  Post Transfusion Purpura (PTP) Defined as: Delayed transfusion reaction of platelet destruction due to an immune response. Mediators: antiplatelet Abs against plateletAgs. Signs/symptoms: appear within 1-2 weeks after transfusion: Abs affect platelet function life, causing thrombocytopenia, purpura, bleeding (vaginal bleeding), bruising & hematuria. Treatment: Intravenous immune globulin (IVIG), plasmapheresis with FFP replacement (Therapeutic plasma exchange) & splenectomy. Prognosis/ Complication: Self-limited (≤ 2 weeks) Management/Prevention: Select platelet from matched donors once Ab has been identified. 14 4  Delayed (Non-immunologic) TR 1- Hemosiderosis (Iron overload) Fatigue Defined as iron accumulation in patient’s blood & tissue due to several red cell transfusions One unit of red cells contains 250 mg of iron; while maximum daily Fe excretion is 1 mg Mediators: Iron overload Signs & symptoms: Saturating of RES of iron storage resulting to: ✓ Organ toxicity such as heart, liver & endocrine after only 10-15 units of RBC→ tissue damage & fatigue Management/ Prevention: Fe chelation therapy “desferrioxamine” 14 5  Delayed (Non-immunologic) TR 2- Transmission of disease (transfusion transmitted disease TTDs) Bacteria, viruses & parasites can be transmitted to recipients in donated blood products. The effects may take months or years to appear. Caused by: blood-borne pathogen such as: Hepatitis B, HCV; HIV, HTLV-1, CMV; Malaria. Clinical Features (Window period): Clinical features related to underlying disease Investigation: Microbiological/ serological testing of donor blood Treatment: related to underlying disease Management/Prevention: Careful selection of donors & post donation testing 14 6  Investigation of reported transfusion reaction Incidence About 4% of all transfusions of blood products result in some observable adverse reaction at the time of transfusion. Most of these are febrile or allergic reactions that are NOT serious. Only 3 in every 1,000 transfusions results in a haemolytic reaction; of these 3, most are NOT serious → do not cause permanent harm or death. It has been estimated that where good blood bank practice is followed,  1 in every 5,000 transfusions results in problems & most of these are due to: 1. Wrongly labelled samples 2. Wrongly labelled units 3. Failure to check documentation 14 7  Causes of reported reactions Because the signs of febrile reactions, allergic reactions, bacteriogenic reactions & haemolytic reactions can be confused, the request for investigation is common. Because haemolytic reactions can be fatal, their investigation must be given priority. So, the investigation is designed to detect haemolytic reactions & eliminate other causes. Causes associated with Adverse Transfusion Reaction: # Patient misidentification. # Sample error. # Wrong blood issued. # Technical error. # Storage error. 14 8  Procedure for investigating reported transfusion reactions Severe reactions are due to: 1. Immune haemolysis (HTR): Transfusion of RBCs that are destroyed by an Ab in recipient’s plasma 2. Non-immune causes of haemolysis: (Physical/ chemical means of hemolysis and Bacteraemia) The investigation is designed to: 1. Demonstrate haemolysis 2. Demonstrate an antibody-antigen reaction 3. Look for causes other than antigen-antibody reactions 14 9  What to do when a reaction is reported ✓ Ensure that the transfusion is stopped. ✓ Keep the intravenous line open with normal saline. ✓ At the patient's bedside, check all labels, forms, & patient identification. ✓ Report the suspected transfusion reaction to blood bank personnel immediately. ✓In BB, check documentation on units concerned & all other units in the “issue” refrigerator → if one patient has the wrong blood, so may another. 15 0  What to do when a reaction is reported ✓ Collect or retrieve; 1. Pre transfusion samples from recipient. 2. Post transfusion samples from recipient: i. Clotted blood 20 ml → Plain tube ii. EDTA – 5 ml iii. First urine sample after reaction & all others for 24 hr. iv. Blood cultures. v. Used packs (units of blood transfused & unit causing problem) vi. Citrate samples for coagulation screen→ to check for DIC 15 1  What to do when a reaction is reported ✓ The following tests are carried out. ❖First step: Examine post transfusion EDTA plasma & Pre - Tx Post - Tx urine for haemoglobin→ haemolytic or not!! ❖ Post-transfusion blood sample from the patient: a. Carry out DAT “Direct coombs test”→ to detect HTR b. Carry out ABO & Rh group c. Carry out antibody screen d. Identify any antibodies detected e. Carry out cross-match with all units of blood 15 2  What to do when a reaction is reported ❖ Pre-transfusion blood sample from the patient: a. Repeat ABO & Rh group b. Repeat the antibody screen c. Identify any antibodies detected d. Repeat cross-match of all units of blood ❖ Donor blood units: a. Check for correct labelling of units of blood b. Check ABO & Rh group c. Carry out antigen typing for any antibodies identified in the pre- or post- transfusion samples from the patient ✓ Send the offending unit of blood for microbiological testing→ “Bacteraemia” 15 3  What to do when a reaction is reported ✓ Check for evidence of DIC in the patient: a. Blood film - Fragmentation (spherocytosis) b. Platelet count c. Coagulation tests ✓ If the donor was Group O & the recipient was Group A or Group B: a. Anti-A or anti-B in the donor serum is titrated. b. Titres  1 in 64 are associated with ‘minor’ incompatibility & could be the cause of haemolysis. ✓ Inspection of donor units for evidence of pre-transfusion haemolysis. →Physical/chemical hemolysis Causes of haemolysis other than Ab-Ag reactions must be considered: a. Thermal shock - freezing or heating b. Mechanical - high pressure infusion 15 4  Febrile Or Allergic No evidence of Ab-Ag reaction, or other causes of haemolysis of RBCs, the reaction is probably febrile or allergic. Febrile reactions are most often due to leukocyte &/or platelet antibodies. The use of leukodepleted blood reduces this risk. 15 5  Emergency blood transfusion ▪ Emergency situations: In some situations patients may require life-saving transfusion before the usual compatibility tests can be performed. ▪ In these situations it is necessary to issue incompletely processed blood, at the same time the blood bank must not increase the morbidity of patient. ▪ The most frequently encountered clinical situations where emergency transfusion is required are: massive obstetric hemorrhage. pediatric trauma. massive gastro-intestinal bleeding. 15 6  Emergency blood transfusion ▪ When emergency release of blood is requested, the blood bank personnel should do the following: Issue uncross matched blood , ABO and Rh compatible should be given if there is time to test a current patient specimen. If there is not sufficient time to perform ABO and Rh testing on the patient's current blood sample, group O RBCs must be given. ▪ Donor RBCs, should be negative for any antigens that corresponds to a clinically significant antibodiesin the serum of the recipient. In case of whole blood transfusion, only the same type as the recipient can be administration. Unless it is not available, Rh-negative RBCs should be given, especially if the patient is a female of childbearing age. If it is not available, Rh-positive RBCs can be given to an unimmunized Rh-negative 15 7  Emergency blood transfusion ▪ It is important that patient identification procedures are strictly followed in emergency situation. ▪ Blood bank records should contain a signed order from the physician stating the reason for emergency release. ▪ Blood must be clearly labeled indicating that the pretransfusion compatibility has not been completed. Records must include all of the following: Positive identification of the patient. The units number, ABO groups, Rh typing of the blood issued. Identify of personnel who issued the blood. 15 8  Emergency blood transfusion Information required by Blood Bank: Reason blood required. Time required for blood. Number of units required. Authorizing Doctor's name (responsible for resuscitation). Patient identification, if available. Destination and direct contact number. 15 9  Massive transfusion Definition ▪ Defined as the replacement by transfusion of more than 50 percent of a patient's blood volume in 12 to 24 hours. Or loss of 1.5 ml of blood per kg of body volume within 3 hours. In a normal adult, this is 10-12 units. ▪ Can occur in variety of clinical settings such as: Cardiac surgery. Liver transplant. Massive trauma. Involves the selection of the appropriate amounts and types of blood components to be administered. May be associated with a number of hemostatic and metabolic complications. 16 0  Massive transfusion ▪ Requires consideration of a number of issues including: Volume status. Tissue oxygenation. Management of bleeding and coagulation abnormalities. Changes in ionized calcium potassium, and acid-base balance. 16 1  Some possible adverse physiologic consequences of massive transfusion Coagulopathy: dilutions, shock, DIC, hypothermia. Acid base balance: acidic pH of stored blood. Citrate toxicity: components of anticoagulants. Immunosuppression: mechanism unclear. Hypocalcaemia: citrate in coagulant. Respiratory distress: microaggregates in stored blood Decrease O2 delivery to tissues: reduced 2,3 DPG. Hypothermia: administration of cold components and fluids. 16 2  Haemolytic Disease of New-born (HDNB) Definition Also known as→ Erythroblastosis fetalis Definition: Blood disorder that occurs when the blood types of a mother & baby are incompatible. It may occur because of ABO or Rh incompatibility Also because of atypical antibodies Only IgG produced from the mother can involve → it crosses the placenta 16 3  Mechanism of the Disease  Caused by the destruction(‫)تدمير‬ of the RBCs of the fetus by antibodies produced by the mother  Only IgG class → transported across the placenta  Maternal antibodies are directed against antigens on fetal RBCs that were inherited from the father. 16 4  Causes Caused→ incompatible blood type between the mother and baby Three types of HDNB based on antibody specificity: 1. ABO-HDNB → ABO incompatible → Most frequent 2. Rh D-HDNB → Rh D incompatible → Most Severe 3. HDNB of atypical antibodies → Due to Abs other than anti D, anti A & anti B 16 5  Types of HDNB 1 ABO hemolytic disease: 65% of HDNB is because of ABO incompatibility. Mild anemia and Jaundice start mainly after delivery. Can occur in the first pregnancy or any pregnancy. Occurs almost in→ infants of blood group A or B born to O group mothers. Why? Because IgG anti A or anti B, occurs more commonly in group O than group A or B individuals (high titer of IgG) 16 Rare cases→ reported in group A2 mothers with high titer anti B.(baby A2B or B) 6  Types of HDNB 2 Rh D hemolytic disease:  33% of HDN is caused by Rh incompatibility  HDN due to IgG anti-D Abs crossing the placenta  Sensitization normally occurs very late in pregnancy or in delivery time  It’s also due to pregnancy complications, miscarriage, abortion, prenatal testing..etc.  There is a variation in the clinical severity of Rh-HDN  Rh(D) positive women→ no affect 16 7 http://www.scribd.com/doc/55960591/4/Phototherapy  Types of HDNB 3- Due to antibodies other than anti D, anti A and anti B: Atypical antibodies: (Any IgG Ab) Study from Norway→ about one third of the new cases→ were found in Rh (D) positive women and included anti C & anti E.  Examples: Anti c, K, e, E, Fya Criteria that are necessary for HDN to develop: 1. Blood types of a mother (no Ag) & baby (new Ag) are incompatible. 2. Mixing between these incompatible blood (in delivery time ) 3. Producing IgG Ab from the mother (memory cell)  The severity of HDNB is related to the amount of antibody crossing the placenta 16 8  Signs and Symptoms of Rh HDN  Pale skin and Jaundice: Yellowing of the amniotic fluid, umbilical cord, skin and eyes.  Kernicterus (brain damage) →Hyperbilirubinemia.  Enlarged liver or spleen  Severe swelling of the body The level of severity: 1. Mild form: moderate anemia & jaundice 2. Moderate form: severe anemia & jaundice 3. Severe form: Hydrops fetailis (mental damage & death)  Hydrops fetalis: Fetal condition with low Hb, pale skin, hepatosplenomegaly, swallon baby jaundice (high bilirubin) http://www.shjmschools.com/ which leads to kernicterus (brain damage) & stillbirth,. 16 (Hemolytic Disease of the Newborn,n.d.) 9  Diagnosis 1. Prenatal testing: (During pregnancy): A- Maternal testing: ABO + Rh (D) type + Ab screening (AHG) a) No antibodies: - Rh D positive→ Ab screen at 34 wks & at delivery (not necessary) - Rh D negative + previously transfused women→ screen at 24 wks, 34 wks & at delivery. b) Antibodies detected: - Identify Ab + Titration (IgG titer)→ Check Ab titer every month (ifincreasing) - Titer more than 1 in 32 (Anti D more than 4 IU is indicative of risk), especially before wk 13 ⟶ Helpful to predict HDNB is likely & how severe it might be 17 0  Diagnosis B- Paternal testing:  Husband’s blood group and phenotype provide useful information to predict the likelihood of a fetus carrying the relevant red cell antigen. 17 1  Diagnosis Other tests conducted during pregnancy may include: 1. CBC for the mother 2. Ultrasound; detect bilirubinemia 3. Amniocyte testing: Might cause FMH “fetomaternal hemorrhage” o Amniocytes “Chorionic villus sampling” can be tested as early as 10-12 weeks gestation to detect the gene for the D antigen & any other antigens. http://endometabol.com o Amniotic fluid examination→ Bilirubin level (high) 17 2  Why testing during pregnancy? After confirming the development of HDN, prenatal testing to assess the degree of severity which might be needed for: 1. Premature delivery 2. Intrauterine transfusion 17 3  Diagnosis  Postnatal testing: (After birth), tests may include: 1- Ab screening: * Negative: “No antibodies”: a. Rh D positive→ no need for postnatal test b. Rh D negative: Mother: ABO + Rh D typing + antibody screen Baby: use cord blood: ABO + Rh D typing + DAT * Antibodies detected: a. Mother: ABO + Rh D typing + antibody screen + Ab Identification b. Baby: use cord blood: ABO + Rh D typing + DAT → if DAT is positive: identify the Ab + CBC: Hb (low) and bilirubin (high) 17 4  Treatment  Treatment during pregnancy may include: 1. Blood / Exchange transfusion 2. Early delivery of the baby, if severe complications arise & baby’s lungs are mature  After birth, treatment may include: 1. Blood transfusion 2. Oxygen or mechanical breathing machine 3. Exchange transfusion→ to replace baby's damaged blood with fresh blood (next slide) 4. Phototherapy→ exposure to artificial light or sunlight to reduce jaundice (UV light source will speed up the break down of bilirubin) 17 5  Exchange transfusion Replace baby’s blood with fresh blood This may be performed as a prenatal or postnatal procedure. http://www.scribd.com/doc/5596 0591/4/Phototherapy Donor blood selected for exchange transfusion: a) Fresh: Less than 5 days old Aim of exchange transfusion: b) Hematocrit of approximately 0.80 I/I to 0.90 1. Free Abs or sensitized RBCs are I/I diluted or removed (reduce c) Blood should be irradiated (inactivate donor severity) lymph) 2. Hb is increased to normal level d) Blood for transfusion is checked for (improve anemia) compatibility by cross-matching with the 3. Bilirubin level is lowered. maternal serum. 17 4. Preventing further risk or damage 6  http://www.scribd.com/doc/55960591/4/Phototherapy 17 7  Prevention of Rh D HDN Prevention: Avoid/ stop the disease before it happens Check ABO-Rh grouping: (Mother: Rh negative) Rh-D negative Prenatal– detects mothers at risk: (Ab screen test neg for Anti D) mothers should be Prenatal: Give RhoGAM “anti D” for all Rh negative mothers followed by with Ab screen test neg for Anti D at 28, 34 weeks of pregnancy. their doctors Postnatal: RhoGAM given after birth for Rh positive babies (≤ 72 hr), only if Ab screen test is negative. How does “RhoGAM” Prophylactic Anti D work? Attaches to fetal cells in mother circulation & remove them by maternal RES, preventing mother immune system to be sensitized and produced memory cells. 17 8  Management Rho–Ig or RhoGAM or Prophylactic anti D (to prevent Rh D HDNB) 1. Is given at 28th weeks of pregnancy 2. Again within 72 hrs after birth When to give these injections: 1. During every pregnancy 2. If they have a miscarriage or abortion 3. After prenatal tests→ amniocentesis & chorionic villus biopsy 4. After injury to the abdomen during pregnancy Two types of doses: 1. 300 mg → for each 15 ml of fetal cell “Fetal-maternal hemorrhage occurs” 2. 100 mg (500 IU)→ for 4 ml fetal cells “standard dose” 17 9  Kleihauer Betke (KB) Test  Blood test that measures the amount of fetal hemoglobin that has transferred from a fetus to the mother’s bloodstream  To determine (fetal–maternal hemorrhage) FMH and dose of RhoGAM Acid elution technique of Kleihauer; Fetal RBCs stain with eosin (appear dark),  Normal Ranges: Adult RBCs do not stain (appear as ghosts). This maternal blood smear contained 11.2 % 1. Full-term newborns: Hb F cells are > 90% fetal RBCs, representing a transplacental hemorrhage of about 45,0 mL of fetal blood. 2. Normal adults Hb F cells are < 0.1%. 18 0  Management/ Prevention - Tests both mother & baby to follow the disease & evaluate the risk to baby. - Start preventative treatment (Prophylaxis) When the titer indicates an affected baby, do the following: 1. Tests to check severity. 2. Perform premature delivery, intrauterine transfusion to reduce harm. 18 1  Statistics Anti-D is still one of the most common antibodies found in pregnant women, followed by anti-K, anti-c, and anti-E. Kell antigen accounts for 10% of severely affected fetuses. ABO incompatibility frequently occurs during the first pregnancy and is present in approximately 12% of pregnancies. Less than 1% of births are associated with significant hemolysis. Affecting approximately 4,000 babies a year in the United States. 18 2  Ethics and Transfusion Anxiety about the risks of blood transfusion A clear and sympathetic discussion with a well-informed nurse, transfusion practitioner or doctor may help in calming the concerned patient. Clear information provided to the patient on the risks and benefits of transfusion and, where appropriate, alternatives to transfusion, is a key component of obtaining informed consent. 18 3  Ethics and Transfusion Mental competence and refusal of transfusion An adult (aged 16 or over) has full legal capacity to make decisions for themselves (right to autonomy) No one can give consent on behalf of a patient with mental capacity. In critical ill patients with temporary incapacity e.g. altered consciousness after accident , clinicians must provide life-saving treatment, including blood transfusion, unless there is clear evidence of prior patient refusal Documented. In these situations, patient record should document the indication for transfusion and the patient shouldbe informed of the transfusion when they regain their mental capacity If parents or legal guardians (children under 16 years old) refuse blood transfusion and the treating clinician disagrees to this decision, as they see transfusion as a life-saving or essential for the well-being of the child,a Specific Issue Order can be quickly obtained from court. 18 4  Ethics and Transfusion Jehovah’s Witnesses and blood transfusion Religious community who decline transfusion of specific blood components. Decision not based on risks of transfusion but is a scriptural stand based on biblicaltexts Individuals vary in their opinions and it is important to clearly understand the patientspreference Most Jehovah’s Witnesses refuse transfusions of whole blood and the primary blood components – redcells, platelets, white cells and unfractionated plasma. Many Witnesses accept the transfusion of derivatives of primary blood components such as albuminsolutions, cryoprecipitate, clotting factor concentrates (including fibrinogen concentrate) and immunoglobulins. 18 5  References Harmening, D.M, (2018) Modern Blood Banking Transfusion Practices 7th, Ed. ISBN 9780803668881 Overfield/ Dowson/ Hamer (2007) Transfusion science (2nd Revised ed.) Scion Publishing ISBN: 9781904842408 Dacie/Lewis. Practical Haematology. 9th ed. Churchill Livingstone. 2001. ISBN: 0443063788 Bryant, N. Introduction to immunohaematology. 3 rd ed. W.B Saunders Company. 1994. ISBN: 072163883X Normansell D. Principles and practice of diagnostic immunology. VCH Publishing. 1994. ISBN: 0560815345 https://www.transfusionguidelines.org/transfusion-handbook/5-adverse-effects-of-transfusion https://transfusion.com.au/blood_basics/blood_groups/abo_rh 18 6