Immunohematology/ISBB/IH - Compatibility Testing PDF

IMMUNOHEMATOLOGY / ISBB / IH LESSON#1: COMPATIBILITY TESTING FINALS | A.Y. 2023 - 2024 | SIR JASON “AB” CHUA 1 COMPATIBILITY TESTING ○ WHAT IS COMPATIBILITY TESTING? Also called pretransfusion testing PURPOSE: ○ To select blood components that will not cause harm to the recipient and will have acceptable survival when transfused. If properly performed, compatibility tests will conﬁrm ABO compatibility between the component and the recipient, and will detect most clinically signiﬁcant unexpected antibodies. NOTE: Cross Matching and Compatibility testing ARE NOT THE SAME. ○ Compatibility testing ensures broader sense of SAFETY TO THE PATIENT Since it encompasses pre-transfusion down to post-transfusion testing Very important to avoid any transfusion rxn or any discrepancy in forward and reverse typing ○ Cross Matching is JUST A COMPONENT OF COMPATIBILITY TESTING ○ COMPATIBILITY TESTING There are several components of compatibility testing(5): 1) Proper specimen collection - make sure it is not hemolyzed to avoid erroneous result 2) Reviewing patient transfusion history 3) ABO, Rh, and antibody testing (screen/ID) 4) Crossmatching 5) Actual transfusion - only performed by nurses 3 CATEGORIES Can be divided into 3 categories: ○ PREANALYTICAL PROCEDURES ○ SEROLOGICAL TESTING/ANALYTICAL ○ POSTANALYTICAL PROCEDURES PRE ANALYTICAL PHASES More common to error PATIENT IDENTIFICATION ○ Must conﬁrm recipient’s ID from bracelet ON the patient Full patient name and hospital number Name of physician ○ Patient misidentiﬁcation is the MOST COMMON ERROR SPECIMEN COLLECTION ○ Collected in tube with EDTA, PINK TOP or no additives(RED TOP). ○ If the venipuncture causes hemolysis, the sample may be rejected. ○ True hemolysis in the patient is the result of complement activation. Complement activation = agglutination ○ Samples are labeled at the bedside (PRE-LABELING is NOT RECOMMENDED) ○ A record of individuals who collect (or test) the specimens should be documented in order to “backtrack” in case of an error. (ie. Initials of MT should be on the tube) ISBB - LEC If the sample is drawn from an IV line or both arms of the px have an IV line, the IV infusion should be stopped by the nurse 5-10 minutes prior to blood drawing and the ﬁrst 10mL discarded Collecting from an IV line leads to a diluted sample -> discrepant results in forward & reverse. First 10mL extracted should be discarded d/t contamination from the IV. The MT can also opt to extract from other body sites. Testing should be performed on samples less than 72 hours or else complement dependent antibodies may be missed (complement can become unstable) NOTE: Manual washing takes about 1 hour Gel tech results obtained in a span of 30 minutes since it does not require washing anymore SAMPLE IDENTIFICATION ○ The sample should also have; Full patient name Hospital number Physician Date and time of collection Phlebotomist’s initials ○ All of this should be on the request form and the sample. NOTE: Clerical Error is common in Blood Bank ○ Happens either during the input of information in the computer or the penmanship is unreadable – that’s why proper penmanship is strictly imposed SPECIMEN TUBES ○ Hemolyzed sample MUST BE REJECTED We have the right to reject even though we’re still an intern (pa-interna mi Lord please) RECALL: Hemolysis in the sample activates complement thus agglutination is triggered Most preferred tube anticoagulant in blood bank - PINK TOP Alternative if pink top is not available: ○ EDTA ○ RED TOP - serum REVIEW OF PATIENT HISTORY ○ GETTING THE HISTORY Look at the recipient's records for any prior unexpected antibodies formed from previous transfusion. Note for any past transfusion reactions(ie. Allergies) There are some px that develop antibodies during the second transfusion. MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 1 1 ANALYTICAL PHASES SEROLOGICAL TESTING 3 tests: ○ ABO/Rh Pretest First performed before crossmating ○ Antibody detection or identiﬁcation Routinely performed in US ○ Crossmatch 1 ABO/Rh TYPING In the ABO typing, the forward and reverse MUST match. In the Rh typing, the control must be negative. Both of these will indicate what type of blood should be given. FIRST PERFORMED BEFORE CROSSMATCHING 2 RECALL: Most common discrepancy in ABO typing - Type 1(reverse) ANTIBODY SCREEN AND/OR IDENTIFICATION The antibody screen will detect the presence of any unexpected antibodies in patient serum If antibodies are detected, identiﬁcation should be performed using the panel cells (with an autocontrol). Autocontrol (px red cells + px serum) ○ IS (immediate spin phase; IgM) ○ 37°C (LISS; IgM & IgG) ○ AHG (IgG) If an antibody is present, negative units for the antigen must be given Proceed to the crossmatch 3 CROSSMATCHING PURPOSE: ○ Prevent transfusion reactions (severe reactions → death) ○ Increase in vivo survival of red cells ○ Double checks for ABO errors (since forward and reverse typings are done along the process of crossmatching) ○ Another method of detecting antibodies HISTORY OF CROSS MATCHING 1900 Landsteiner ABO system 1907 Major and minor XM begin 1950s Antibody screens began 1976 Minor XM optional 1980s Immediate spin XM CROSSMATCH PHASES PHASE PURPOSE IS (Immediate Spin) Detection of IgM cold agglutinin (Clinically insigniﬁcant) Note: Lewis & M are mixed (IgG/IgM) 37°C/ LISS/ INCUBATION PHASE/ THERMOPHASE IgG and IgM Abs Potent Rh Abs AHG/COOMBS PHASE/ IAT IgG Abs Rh, Kell, Duffy, Kidd, Ss, Lub OCC AHG absence, (O Check Cells) inadequacy, or Sensitized RBCs (optional) neutralization LISS - low ionic strength saline | AHG - antihuman globulin 3 Main Phase: ○ IS ➡ IgM Recall: IgM is Malaki, therefore can easily react and visible reaction after 15s centri ○ 37°C, LISS ➡ IgG and IgM Abs Recall: both are detected b/c of wide range thermal ability Phase usually read only macroscopically If No agglutination - unsure since it cannot be determined if IgG or IgM is present ○ AHG ➡ IgG Can serve as a conﬁrmatory test from the negative result of LISS phase. Read microscopically for agglutination Agglutination - IgG antibodies present Optional: ○ OCC (O Check Cells) Additional in AHG Added to all negative tests and must produce positive results Computer XM ○ Data crossmatching ○ Prone to clerical error XM-cross match 1990s 2 TYPES MAJOR DR-PS (Donor Red cell-Px Serum) MINOR DS-PR (Donor Serum-Px Red cell) ISBB - LEC Routinely performed in labs Detects if px has antibodies against the donor Not routinely by AABB since 1976 Detects if antigen of px reacts to the antibodies of the donor (+) agglutination → transfuse with negative antigen, same blood type, but PACKED RED BLOOD CELL ONLY DETECTS (Antibodies) Most ABO incompatibilities P1, I, MN Lewis(Lea, Leb) Lua MAJOR VS MINOR CROSSMATCH Why is the minor crossmatch unnecessary? ○ Recall : Patient’s RBC + Donor’s serum ○ Donated units are tested for antibodies. In minor , the one being tested is the antibodies of the donor against the patient’s RBC antigen. Supposedly it is much more essential to test if the patient has antibodies against the donor’s RBC (perform in major XM). thus it is not routinely required by the AABB since 1976 In the Ph labs some perform both, some do not ○ Most blood is transfused as packed cells, having little antibodies. CROSSMATCHES The AABB and FDA develop the standards for blood banking. According to the AABB Standards(Association for the Advancement of Blood & Biotherapies) : ○ The crossmatch “shall use methods that demonstrate ABO incompatibility and clinically signiﬁcant antibodies to red cell antigens, and shall include an antiglobulin phase.” Should perform forward and reverse Antiglobulin phase- AHG ,IAT , Coombs phase MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 2 INCOMPATIBLE ○ AGGLUTINATION → antigen - antibody interaction COMPATIBLE ○ NO AGGLUTINATION → no antigen - antibody interaction THE PROCEDURE MAJOR (DR + PS) O MINOR (DS + PR) O REPORTING Compatible + + Incompatible + O Incompatible O + Compatible (transfuse packed RBC only) In the Ph, is considered as incompatible already , but theoretically it is compatible (O) = no agglutination | (+) = agglutination LABORATORY SETTING SETUP Remember: in blood bank , “First IN , ﬁrst OUT “ Donor cells are taken from segments that are attached to the unit itself. Segments (encircled) are a sampling of the blood and eliminate having to open the actual unit. ○ Seal ﬁrst before cutting ○ The blood in the cut portion or the “segment” is placed in the donor’s red cell tube (refer to the tube rack picture) ○ If insuﬃcient cut another segment ○ 2 inches long would suﬃce Patient’s tubes - closer to the MT (front row of tube rack) ○ Packed red cells ○ Plasma ○ Red cell suspension ○ Forward & reverse typing Donor’s tubes - farther from the MT (3rd row of tube rack) ○ Packed red cells ○ Plasma ○ Red cell suspension ○ Forward & reverse typing Major & Minor tubes & autocontrol (placed in the middle in between) ○ Autocontrol (Px red cell + Px Serum) - to determine if px’s own antibodies is reactive to own antigens ○ Major (Donor Red cell + Px Serum) ○ Minor (Donor Serum + Px Red cel) PROCEDURE(CROSSMATCHING) - only major, minor, autocontrol tubes 1) Immediate spin phase Centrifuge major, minor, autocontrol tubes ~15secs Dislodge, read, interpret Agglutination - stop, incompatible No agglutination - proceed to LISS phase 2) LISS phase Add LISS to the 3 tubes then incubate(37^C)~10mins Centrifuge ~15secs Dislodge, read, interpret Agglutination - stop, incompatible No agglutination - proceed to AHG phase 3) AHG phase Wash 3x Add AHG Centrifuge ~15secs Dislodge, read, interpret Agglutination - incompatible No agglutination - compatible ISBB - LEC UNITS OF WHOLE BLOOD WITH SEGMENTS ATTACHED (encircled) Units of blood should be arranged orderly from nearly expiring blood units up to the newly acquired blood units. OBTAINING BLOOD BANK UNIT: ABO/Rh typing is FIRST PERFORMED Antibody Screen is performed next If antibodies are NOT DETECTED: ○ Only immediate spin (IS) is performed using patient serum and donor blood suspension(Major). ○ This fulﬁlls the AABB standard for ABO incompatibility. ○ This is an INCOMPLETE CROSSMATCH If antibodies ARE DETECTED: ○ Antigen negative units found and X-matched ○ All phases are tested: IS, 37°C, AHG ○ This is a COMPLETE CROSSMATCH ANTIBODY SCREEN CROSSMATCH IMMEDIATE SPIN PHASE AGGLUTINATION INCOMPATIBLE = STOP NO AGGLUTINATION PROCEED TO THERMOPHASE THERMOPHASE AGGLUTINATION INCOMPATIBLE = STOP NO AGGLUTINATION PROCEED TO AHG PHASE ANTIHUMAN GLOBULIN (AHG) PHASE AGGLUTINATION INCOMPATIBLE NO AGGLUTINATION COMPATIBLE MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 3 CROSSMATCHES WILL Verify donor cell ABO compatibility. Detect most antibodies against donor cell. WILL NOT Guarantee normal survival of RBCs. Prevent patient from developing an antibody. Detect all antibodies. Prevalent delayed transfusion reactions Detect ABO/Rh errors. The card containing the microtubes is then centrifuged at a controlled speed for 10 minutes. At the start of centrifugation the cells are separated from the serum; then they meet the AHG contained in the microtube. Finally the cells are trapped by the gel (if agglutinated) or pellet to the bottom of the tube. INCOMPATIBLE CROSSMATCHES ANTIBODY SCREEN CROSSMATCH CAUSE RESOLUTION + - Antibody directed against antigen on screening cell ID antibody, select antigen negative blood. - + + + Antibody directed against antigen on the donor cell which ID antibody, select may not be on antigen negative screening cell OR blood OR perform donor unit may have DAT on donor unit IgG previously attached. Antibodies directed Antibody ID, select against both antigen negative screening and donor blood cells ADDITIONAL INFORMATION ON TYPES OF COMPATIBILITY TESTS: Manual (IS and IAT) Gel Technology Electronic (Computerized) Cross match Red cell Aﬃnity Column Technology (ReACT) Solid Phase Adherence Assays (SPAA) MANUAL (IS AND IAT) IS (Immediate Spin) ○ detect Room Temp reactive antibodies (Auto, Alloantibody, Naturally occurring) Auto - self Allo - antibodies from a diff indiv, same spp. IAT (Indirect Antiglobulin Test) ○ detect IgG antibodies (Auto & alloantibody) GEL TECHNOLOGY Patient serum, and 1% of suspended RBCs in LIM/LISS are dispensed into the microtube and incubated at 37°C for 15 minutes. ○ Drop red cell → drop serum/plasma → Incubate for 15 minutes → Centrifuge for 10 minutes → Result no washing involved Principle: Separation of red blood cells via speciﬁc gravity ○ Gel acts like a sieve (salaan) ○ Agglutination = stay on top ○ No agglutination = will go down (0 or negative) ○ (1+, 2+, 3+) = will stay in the middle ISBB - LEC Refer to the picture: left to right ○ The gel (yellow) acts like a sieve ○ Slow reaction will stay in the gel (3rd & 6th) ○ No reaction will settle at the bottom (1st, 2nd, 4th, & 5th) LIMITATIONS OF PRETRANSFUSION TESTING Hemolytic transfusion reaction, if the patient's antibody is too weak to be detected. Standard antibody detection methods such as the indirect antiglobulin test require several 100 antibody molecules per red cell to produce detectable reactions. A hemolytic transfusion reaction due to patient misidentiﬁcation. ○ For example, group A red cells (meant for transfusion to a group O recipient) will be compatible in vitro tests with an incorrect specimen drawn from a group A person. Hemolytic transfusion reaction if donor red cells are inadvertently hemolyzed before entering the patient. ○ e.g., red cells hemolyzed by an improperly functioning blood warmer or red cells hemolyzed by contact with an ice pack in a transport container Non hemolytic transfusion reaction such as: ○ allergic, febrile and other reactions. Pretransfusion tests ○ meant to detect only red cell antibodies. INCOMPATIBLE CROSSMATCH ABO INCOMPATIBILITY ○ Recheck patient and blood unit ABO group Check forward or reverse Clinically signiﬁcant antibody ○ Check in AHG, Coomb's, DAT & IAT POST ANALYTICAL PHASES Involves labeling, inspecting, and issuing the blood unit. Labeling form/tag includes patient’s full name, ID number, ABO/Rh of patient and unit, donor #, compatibility results, and tech ID. Form is attached to the donor unit and only released for the recipient. The unit is visually inspected for abnormalities, such as bacterial contamination, clots, etc. BACTERIAL CONTAMINATION This unit shows bacterial contamination and should NOT be given to the patient. The plasma in the segments is ﬁne, but the plasma in the unit shows heavy hemolysis from bacteria. RECALL: Most common blood bag contaminant ○ Yersinia enterocolitica (Green coloration in blood bag) MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 4 ISSUING BLOOD When it's time to release a blood product to the nurse or physician, a few "checks" must be done ○ Requisition form ○ Comparing requisition form → donor unit tag → blood product label ○ Name of persons issuing and picking up blood ○ Date and time of release ○ Expiration date WHAT IF THE UNIT IS UNUSED? Blood can be returned if it is not needed for transfusion ○ Given that it was not transfused or opened Unit closure has to remain unopened Storage temperature must have remained in the required range (1° to 10°C for RBCs) If not at correct temp, unit must be returned within 30 minutes of issue DEVICES MASSIVE TRANSFUSION Deﬁned as a transfusion approaching or exceeding the recipient's own blood volume (about 5 liters or 10-12 units in an adult male) within 24 hour period. ○ Not only 1 unit is transfused The original sample no longer represents the patient's condition. ○ Too many units transfused to the patient Complete crossmatch is not necessary (if no antibodies were detected originally). ○ In cases of massive transfusion and absence of antibodies, immediate spin will suﬃce ○ Reporting identical units of ABO is necessary to identify the antibodies to be safe Give ABO identical units: ○ If antibodies were identiﬁed, continue to give antigen negative units APPROPRIATE UNITS TO GIVE ABO compatible should always be given ﬁrst PATIENT’S TYPE 1st CHOICE O O NONE A A O B B AB Infusion device Blood warmer Infusion device: where blood bags are hung Blood warmer: Maintain the temperature, to avoid hemolysis SPECIAL CIRCUMSTANCES OTHER CHOICES O 2nd AB A ➡ B3rd➡ O4th Group O individual are “universal donors”, they can donate to any blood group because they have no A or B antigens Group AB individuals are “universal recipients”, they can receive blood from any group because they do not have A or B antibodies RED BLOOD CELL COMPATIBILITY TABLE EMERGENCY RELEASE In an emergency (ER or OR), there may not be enough time to test the recipient's sample(no time for XM). O(universal donor) Transfer only packed red cells Safest to release since it does not contain any antigens AB (universal recipient) Transfer plasma only Does not contain any antibodies In this case, blood is released only when signed by the physician (O negative). The tag must indicate it is not crossmatched. Segments should be retained for X-matching Every detail is documented (names, dates..) Once the specimen is received, ABO/Rh typing and antibody screening should be performed. Crossmatching the segments from the released unit should be done. In addition, the lab may crossmatch additional units as a precaution if more blood is needed. If death should occur, testing should be complete enough to show that the death was unrelated to an incompatibility. Summary of red blood cell compatibility PLASMA COMPATIBILITY TABLE WHAT CAN BE GIVEN IN AN EMERGENCY? Group O Rh(-)negative red cells or AB plasma ○ Should only be issued if: Emergency release Women below or of childbearing age Or if in doubt Group O Rh(+)positive red cells ○ Used as a substitution ○ Male or elderly females Young - O(-) ISBB - LEC MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 5 BLOOD TYPES COMPATIBILITY TYPE & SCREEN Used to conserve blood inventory. On average, a surgical procedure uses about 1 unit of RBCs, however, many times the units are on "hold" in the lab and will not be needed (reducing inventory). For this reason, only a type & screen are performed and if any blood is needed, the sample can be retrieved for crossmatching (only the Immediate Spin phase is required). If antibodies are identiﬁed, then antigen negative blood is reserved or crossmatched. NEONATAL TRANSFUSION A neonate who is 72 hours Tattoos or permanent makeup (unless applied by state regulated facility with sterile needles and ink that is not reused) Transfusion of blood, components, human tissue, plasma-derived clotting factor concentrates Human diploid cell-rabies vaccine after animal bite ISBB - LEC NAMES OF TRANSCRIBERS PHYSICAL EXAMINATION General Appearance: Appears Healthy Age: 18 years old & above (can donate) ○ if below 18 years old or minor must have parent’s consent Weight: >50kg or 110 lbs ○ If donor did not reach 50 kg, still allowed to donate provided that calculations regarding blood volume to be extracted will be computed prior to donation Practiced in US Hematocrit: ○ Allogeneic donor: Women: >38% Men: >39% ○ Autologous donor: >33% Hemoglobin: ○ Allogeneic donor: Women: >12.5 g/dL Men: : >13.0 g/dL ○ Autologous donor: >11.0 g/dL Temperature: bone marrow not induced to produce packed RBC -> anemia, shortness of breath ○ Premature infants ○ Patients with sickle cell disease Impaired oxygen carrying capacity of the cell hence would need packed RBC transfusion Can be modiﬁed into additional products needed for speciﬁc patient requirements. (discussed below) RED BLOOD CELLS (LEUKO-REDUCED) Storage life: 24 hours Storage temp: 1-6oC Removal of leukocytes via ﬁltration ○ Leukocytes could also remain in RBCs during component separation. Not all of them are removed, rather they are retained in the blood bank with the packed RBCs. ○ These leukocytes are implicated in adverse transfusion reactions such as chills and/or fever Leukocytes also have HLA antigens on the surface which could lead to the immunization of the recipient to such antigens in w/c later on, they will produce antibodies against them. ISBB - LEC Leukocytes also produce cytokines which are proteins that are responsible for febrile transfusion reactions. WBCs produce these proteins more upon storage. Best performed by the use of commercially available leukocyte removal or leukocyte reduction ﬁlters (third generation) ○ These ﬁlter are able to remove leukocytes but not platelets ○ The same ﬁlter is also applied when there is transfusion of platelet concentrates so that only platelets will be transferred to the px Most often performed prior to storage ○ The problem is that it will be an open system (viability will only be within 24 hours) ○ Can be performed bedside but is not preferred During transfusion, a ﬁlter is attached before the blood is going to reach the vein of the px. Although it is able to remove WBC, it is unable to remove cytokines leading to febrile HTR Should retain 85% of RBCs and reduce WBC to less than 5 x 106 Nowadays, Leukoreduction is done with the use of sterile connector devices to maintain sterility of procedure ○ From one blood bag, close system is maintained and could be preserved up to 21 or 35 or 42 days if additive solution is present Indications for use: ○ Avoid adverse transfusion reactions and immunization to leukocyte antigens ○ To avoid transmission of CMV Cytomegalovirus resides in the cytoplasm in WBC Image of leukoreduction ﬁlter Attached below the packed red cell is the leukoreduction ﬁlter ○ As blood or PRBC passes through, it will be ﬁltered. WBC is left in the ﬁlter RBC easily pass through and go to other blood bag Image of 3rd gen leukoreduction ﬁlter Sterile connective device maintain sterility of PRBC as it is going to be transferred to another blood bag BEST PERFORMED PRIOR TO STORAGE ○ The moment PRBC are separated from plasma, leukoreduction can be performed ○ Somehow, it can still be performed during storage as long as it is not exceeding 3 days from the time it was ﬁrst stored If it exceeds, it will be known as Post-leukoreduced PRBC MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 6 ○ 1. 2. The lesser time that the WBCs are stored along with RBCs, there will be LESS PRODUCTION OF CYTOKINES UNLESS if blood bag is not fresh whole blood is transfused in px, there will be cytokines present produced by WBCs and will not be ﬁltered by leukoreduction ﬁlter Could still lead to Febrile hemolytic transfusion reaction APHERESIS RED BLOOD CELLS Involve use of machines that ﬁlter out necessary components to be collected. The rest of the blood component will be transfused back to the donor. ○ 2 packs can be retrieved from the donor It is collected every 16 weeks ○ After 4 months from the previous apheresis to the next apheresis Beneﬁts of the method/Advantages: ○ reduced risk of potential viral exposure to the recipient ○ less exposure of recipient to red cell antigens Disadvantages: ○ takes longer time than whole blood donation ○ donor is required to be on the target hemoglobin and hematocrit value in order to be accepted as apheresis RBC donor Saline is used to replace ﬂuid lost to minimize volume depletion to the donor For directed donation Storage life: depends on the anticoagulant used Indications for use: same with RBC components ○ The only difference is the procedure in retrieving the blood component This procedure is speciﬁc to the needs of patient Additive solutions can be added MANUALLY or can be incorporated in apheresis machine ○ Since it is packed RBCs FROZEN RED BLOOD CELLS RBC can be frozen to maintain an inventory of rare units or extend availability of autologous units ○ Once it is expired, it can be rejuvenated or subjected for freezing for it to be viable up to 10 years Thaw the blood prior to transfusion to the px BUT washing is NECESSARY Blood is drawn into an anticoagulant preservative. ○ Plasma is removed and glycerol is added Expiration: ○ If frozen, still viable for: 10 years ○ After deglycerolization(open system) it is viable for: 24 hours The units are transferred on polyoleﬁn or polyvinyl chloride bags placed on metal canisters. ○ Placement into a metal canister ensures that the blood bag does not break from very low temperatures Indications for use: ○ Special purposes: E.g Intrauterine Transfusion Preserve autologous units For rare phenotypes 2 TYPES OF CRYO PRECIPITATING AGENT Penetrating cryo precipitating agent Glycerol Non-penetrating cryoprotective agents HYDROXYETHYL STARCH DIMETHYL SULFOXIDE ISBB - LEC PENETRATING CRYO PRECIPITATING AGENT: GLYCEROL Cross plasma membrane in which each osmotic forces prevent water from migrating outward Small and can enter the phospholipid bilayer Surround the inner portion of the red cell membrane so water cannot go out ○ prevent dehydration and ice crystal formation of red cells Ice crystals lead to hemolysis of red cells Commonly used for mature red cells Glycerolization of red cells has 2 types: LOW GLYCEROL HIGH GLYCEROL 40% of total volume is glycerol Storage temp: -65°C (common freezer) Most widely used due to its ADVANTAGES: Makes use of large volumes of washed solution to totally remove glycerol Equipment to be utilized is very easy to use 20% of total volume of PRBC is glycerol (Reduced use of glycerol) Storage temp: -120°C (initial freezing is -196°C) Requires rapid freezing and makes use of liquid nitrogen NON-PENETRATING CRYO PRECIPITATING AGENT: HYDROXYETHYL STARCH and DIMETHYL SULFOXIDE Made of LARGER molecules ○ Cannot pass through plasma membrane Forms shell around the red cell to preventing water loss and dehydration Commonly used to freeze hematopoietic progenitor cells DEGLYCEROLIZED RED BLOOD CELLS Deglycerolization: frozen RBCs are thawed, glycerol is removed ○ Thawing using a water bath set at 37^C. ○ Utilizes a blood cell processor in which the red cell are washed in a series of saline solutions with decreasing osmolality ○ 12% > 1.6% > 0.9% with 0.2% dextrose Used to draw the glycerol out of the cell ○ Disadvantages: It is an open system (Should be transfused within 24hrs) Storage life: 24 hours Storage temperature: 1-6oC WASHED RBCs Prepared by using a machine (blood processor) which washes the cells 3 times with saline to remove WBCs 1000mL of 0.9% saline is used for washing ○ Upon washing, it is expected that there will be a loss of 10% or 20% of the total volume of red cells unintentionally when decanting. Storage life: 24 hours Storage temperature: 1-6oC Indications for use: ○ Patients who react to small amount of plasma proteins that remain on RBCs (REACTIONS: anaphylactic, febrile, allergic) MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 7 Ineffective method for removing leukocytes Effective in removing residual plasma IgA deﬁciency with clinically signiﬁcant Anti-IgA Infant or Intrauterine transfusions ○ ○ IRRADIATED RED CELLS Purpose: Irradiation prevents transfusion-associated graft vs. host disease(TA-GVAD) Transfused blood containing viable T-cells is actually responsible for the occurrence of TA-GVAD ○ because of the presence of t-cells from the donor’s blood that is now attacking the body of the recipient ○ Cannot reject the t cells transfused by the donor: Due to the degree of immunodeﬁciency of the recipient. Higher propensity of having the same HLA type (Most common problem if the donor is a relative of the recipient) Method: gamma irradiation of cellular blood components to prevent proliferation of T cells. ○ Irradiators: can be a form of linear accelerators UV rays Safer option: x-rays (non-radioisotope equipment) Gamma irradiator: cesium 137 or cobalt 60 radioisotopes Disadvantage: causes RBC membrane damage ○ Causing hemolysis leading to: Increased: Hgb, K, decreased: ATP and 2,3-DPG Storage life: 28 days ○ For instance, the blood is stored in CPDA1 which has a shelf life of 35 days. If the blood product has been irradiated, the expiry date will be the 28th day after the collection. ○ For instance, the blood has a shelf life of 21 days, and was irradiated. The blood will truly expire on the 21st day after the collection, not the 28th Storage temperature: 1-6oC ISBB - LEC MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 8 —--------------------------------------------------PART 2-----------------------------------------------------BLOOD COMPONENT PREPARATION AND THERAPY PART 2 PLATELET CONCENTRATE Prepared from a single unit of whole blood. ○ Or through apheresis Due to storage at RT it is the most likely component to be contaminated with bacteria. Therapeutic dose for adults is 6 to 10 units ○ number of platelet concentrate units to be transfused will always be by the doctors discretion Expiration: ○ Pooled(open system): 4 hours ○ Single unit (closed system): 5 days Storage temperature: 20-24oC with CONSTANT AGITATION ○ Use agitators to reduce clumping CCI-Corrected Count Increment| BSA-Body surface area FORMULA used to assess patient’s platelet count improvement ○ Corrected Count Increment can detect platelet refractoriness ○ Unit=10^11 (measures plt refractoriness) (+) refractoriness = px unresponsive to plt transfusion(d/t immune or non immune cause) ○ Immune causes due to HLA autoantibodies, platelet autoantibodies ○ Non-immune causes include splenomegaly, medications, sepsis, internal bleeding, disseminated intravascular hemolysis, DIC, high fever SAMPLE PROBLEM: Random Donor - CCI is 7200 ○ Px BSA = 1.2m2 ○ Precount: 2000/uL ○ Postcount = 29,000/ uL ○ Platelet units transfused: 4.5 x 1011 Interpretation ○ 1 hour CCI >7500 indicates successful transfusion (platelet transfusion refractoriness unlikely) Indications for use/functions: ○ maintenance of vascular integrity ○ Initial arrest of bleeding by formation of platelet plug ○ Stabilization hemostatic plug ○ Cancer patients undergoing chemotherapy/radiation therapy Pancytopenia d/t mature arrest from chemo ○ Px with postoperative bleeding D negative patients should be transfused with D negative platelets due to the presence of a small number of RBC. ○ D antibodies are very potent Storage life: 5 days ○ 5 days only d/t stored @RT w/ constant agitation. Storage temperature: 20-24oC ○ Some agitators are incorporated into incubators in cold areas PLATELET CONCENTRATE (RANDOM DONOR PLATELETS) Should contain at least 5.5 x 1010 platelets per unit ○ Reduced using the leukocyte content Prevent nonhemolytic febrile reactions, HLA immunization of the recipient, and avoid transmission of cytomegalovirus (CMV) Should elevate platelet count of a 75 kg patients for 5000uL ISBB - LEC Refer to the photo above: ○ Remember: Platelet concentrate is derived from unit of whole blood ○ After ﬁrst spin (light spin): we were able to retrieve RBCs (ﬁrst bag), we also have plasma that is compressed to go to the satellite bag which will be our platelet-rich plasma (second bag) ○ Second bag (Platelet-Rich plasma): undergo heavy spin to harvest platelets Upon separation we have now the PLATELET CONCENTRATE and PLASMA (Platelet-Poor) POOLED PLATELETS Open system ○ 6-10 units transferred into one bag (bigger blood bag) ○ Joined together (Pooled Platelets) ○ Not using of sterile connective device reduction of storage life in this particular blood component Dose: 1 unit per 10 kg of px’s weight ○ number of units to be transfused depends on the doctor’s discretion ○ Also depends on the platelet count of the patient ○ Dose of normal individual is 6-10 units depending on the weight of the patient E.g. the patient is 60 kg with low platelets 1 unit per 10 kg which means [60/10=6] 6 units of platelet concentrate will be pooled together for it to be transfused In some cases, the doctors will not perform pooling of platelets but rather speciﬁc or individual units of random platelet concentrates will be administered to the patients. Not together, only one unit is transfused and then computed for the CCI. If the CCI result is not okay, then they will transfuse another unit until there is a good increment of the platelet count of the patient. ○ Remember: when doing platelet transfusion, it is necessary that the units to be transfused must be type speciﬁc due to the presence of some RBCs. could sensitize the recipient Storage life: 4 hours ○ instead of 5 days (normal) Storage temperature: 20-24oC (room temperature) WITH CONSTANT AGITATION (avoid clumping of platelet) MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 9 APHERESIS PLATELETS (SINGLE DONOR PLATELETS) Single Donor Platelets ○ effective method for harvesting therapeutic dose of platelets from one individual alone Should contain a minimum of 3 x 1011 platelets Storage temperature is 20-24C with constant agitation Volume: 250mL ○ In one large blood bag, you have an approximate of 6-8 random donor platelets contained in one pack. 1. 2. Two types of storage/frozen plasma : PF24 & FFP Stored at freezing temp ( -18oC) Differentiated according to content & storage ○ Higher than -18 oC Factors V & VIII do not store well Purpose: Frozen to preserve all coagulation factor Fresh Frozen Plasma (FFP) ○ frozen within 8 hours of collection ○ has normal levels of all labile factors Plasma Frozen within 24 hours(PF24) ○ frozen w/in 24 hours of collection factor VIII reduced factor V normal EXPIRATION PLATELETS LEUKOCYTES REDUCED Must have less than 5 x 106 ○ Performed using leukoreduction ﬁlters or via Apheresis Reduction must be done prior to storage ○ To prevent formation of cytokines from WBC that may be included in the platelet conc Quality control: Leukocytes in 95% of units tested must have less than 5 x 10^6 conc. ○ In random plt concentrate, WBCs must be reduced to prevent febrile non-hemolytic reax, HLA immunization of recipient, & prevent CMV transmission(CMV residing @RBC cytoplasm) Storage temperature: 20-24oC WITH CONSTANT AGITATION FROZEN 1 year stored at less than -18oC FROZEN 7 years stored at less than -65^C Apheresis machine ○ Donor’s blood connected to machine ➡ ongoing separation of blood components inside machine ➡ collect target blood component ➡ non-target components transfused back to the donor FROZEN PLASMA Plasma contains all coagulation factors such as labile factors: ○ V and VIII Indications for use: ○ Used to replace labile and non-labile coagulation factors in massively bleeding patients ○ Treat bleeding associated with clotting factor deﬁciencies when factor concentrate is not available Nowaday there is “Speciﬁc clotting factor therapy” , but when clotting factor concentrate is not available → transfused is the frozen plasma(2nd choice) ISBB - LEC Water bath→ stores at 1-6^C (ref temp.) Once thawed, transfuse within 24 hours (ideally) In case plasma that has been thawed cannot be transfused within 24 hours, label “thawed plasma” and stored at 1-6oC and will only remain viable for 5 days. THAWED Prior to transfusion, FFP and PF24 are thawed at 30-37oC for 30-45 minutes using water baths with agitators ○ During thawing, blood bags must be placed in a protective overwrap to avoid contamination. often utilized are plastics Lifespan: 5 days from the day it is thawed Dose for coagulation factor requirement: 10-20mL/kg ○ Ex. a 50kg patient presents to the hospital. This patient will then require 1000mL of coagulation factors 50kg x 20mL/kg =1000mL If blood bag volume is 250ml, For 1000mL, you need 4 units of FFP to achieve the coagulation factor requirement needed 250ml x 4 =1000ml Volume of 200-250mL PLASMA, LIQUID PLASMA, RECOVERED PLASMA AND SOURCE PLASMA Plasma that is left unfrozen → utilized to treat patients w/ stable clotting deﬁciency 1) Recovered Plasma ○ Prepared by separating the plasma from the RBCs on or before the 5th day after expiration of the whole blood. ○ Ex. whole blood suspended in preservative (CPDA1), you have 35 days storage life → recovered plasma can be obtained during 30th day of the storage of WB. Separate plasma from WB thru centrifugation → PRBC & plasma → recovered plasma stored @-18^C ○ Storage : Freeze -18oC for 5 years Since whole blood is stored at 1-6 oC , which is higher than -18oC, thus this plasma has no labile factors anymore(VIII & V), only stable clotting factor 2) Liquid plasma ○ If recovered plasma is not frozen→ labeled as “liquid plasma”, ○ store at 1-6oC for up to 5 days after expiration of Whole blood Whole blood can be expired ﬁrst, but liquid plasma is up to 5 days since expiration of Whole blood MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 10 3) 4) Plasma ○ ○ Indications for use: ○ Deﬁciency of Factor VIII and ﬁbrinogen(I) Acquired or congenital ﬁbrinogen (Dysﬁbrogenemia) Thawed at 1-6C, formation of insoluble precipitate is observed ○ Contains vWF, F VIII, Fibrinogen, ﬁbronectin, F XIII ○ Within one hour from its collection, should be refrozen at -18C and to be stored for a year ○ Day one is the time of collection for whole blood, not the formation of insoluble precipitates. The remaining plasma is now termed as CPP (cryo-poor plasma) ○ Can still be refrozen within 24 hours from the time it was thawed and cryoprecipitate was collected ○ Storage temperature -18C ○ Storage time 1 year storage time will start @collection of WB ○ Indicated for people with TTP(thrombotic thrombocytopenic purpura) Because CPP contains abundant ADAMTS 13 TTP - lacks ADAMTS 13 ○ Should be transfused to the patient within 24 hours (If not: Store at 1-6C storage life of 5 days, but labeled as thawed plasma, not CPP.) Aka FFP ( fresh frozen plasma ) Once FFP is 1 year in the freezer → redesignate as plasma expiration is 5 years store at -18oC Source Plasma ○ Plasma taken from apheresis procedure ○ Apheresis can be subjected to freezing or transfused right away to a patient. GRANULOCYTES Prepared by hemapheresis Can only be harvested with the used of apheresis Rare and uncommon used Granulocyte concentrate tend to deteriorate rapidly → transfuse right away as soon as collected Indications for use: ○ Primary use is for patients with neutropenia ○ Px who have gram negative infections documented by culture, but are unresponsive to antibiotics This therapy is continued until the granulocyte increases or infection is resolved. ○ Why is it rarely requested? The therapeutic eﬃcacy and indications for granulocyte transfusions are not well deﬁned ○ Not very successful with adults because there are better antimicrobial agents and use of granulocyte and macrophage colony stimulating factors best for adults. ○ Success with this component has been with babies ○ Daily transfusions are necessary ○ We often process granulocytes to irradiation to prevent graft vs. host disease because most individuals who are indicated for this blood product is immune compromised Storage time: 24 hours but best to infuse ASAP Storage temperature: 20-24oC without agitation ○ until it is going to be transfused There are instances that you need to perform ﬁltration of RBC, leaving behind the granulocytes. ○ In transfusing granulocyte concentrate , a cross match is necessary because of the presence of RBC in the granulocyte concentrate that could be >2 ml CRYOPRECIPITATE Cold insoluble precipitate that forms when a unit of fresh frozen plasma is thawed between 1-6C of refrigerated temperature Contains most of the coagulation factors that are found in fresh frozen plasma. Contents: ○ vWF - needed for platelet adhesion to damaged endothelium ○ Fibrinogen (150mg) ; cleaved to ﬁbrin in the presence of thrombin in order to attain clot formation ○ Factor VIII; procoagulant activating factor that is deﬁcient in individuals w/ hemophilia80 IU ○ Fibronectin; ○ Factor XII/XIII Once cryoprecipitate is separated from the ﬂuid plasma, it can be refrozen within 1 hour of its preparation. Storage life: 1 year ○ Day 1 starts @ collection of the WB Storage temperature: -18oC therapeutic dose for an adult: 6 to 10 units ISBB - LEC Expiration ○ Can be stored frozen for 1 year ○ Once thawed: Must be kept at room temperature and transfused within 6 hours ○ Pooled: 4 hours storage life Because it is an open system Best to be ABO compatible but not important due to small volume Volume: 15mL or (25mL presence of plasma) Fibrin Sealant from Cryoprecipitated AHF ○ Used for topical hemostatic solution ○ Contains 1-2 units of Cryo mixed with thrombin and applied to the bleeding site. Thrombin will stimulate ﬁbrinogen to form ﬁbrin. = clot formation. PLASMA DERIVATIVES Coagulation factors/plasma derivatives can be harvested from pooled plasma by manipulating pH, alcohol content, and temperature. ○ Can be inactivated by solvent detergent treatment such as nanoﬁltration. MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 11 A greenish hue need not cause a unit to be rejected d/t contraceptive pills Resolution: remove plasma & retain PRBC → washing to ensure no residual plasma Sulfonamides (arthritis medication) Resolution: same w/ contraceptive pills Bacterial contamination (Pseudomonas spp.) reject ○ Inspect platelets for aggregates Move blood bag back & forth to view clumpings Inspect FFP and CRYO for signs of thawing, evidence of cracks in bag, or unusual turbidity in CRYO or FFP (i.e, extreme lipemia) ○ Thawed blood units most likely indicate that the thermometer was not calibrated or storage equipment is broken ○ Cracks in bag could lead to contamination -reject ○ Unusual turbidity in CRYO or FFP that could indicate extreme lipemia If a unit’s appearance looks questionable to the following ○ Quarantine unit until disposition is decided ○ Gently mix, allow to settle and observe appearance Gentle mixing allows it to settle and questionable appearances can be thought to be d/t hemolysis, plasma discoloration, or blood clots If a bacterial contamination is suspected, the unit should be cultured and a gram stain performed ○ Should be worked up and identify contamination ○ Most common contaminant is Yersinia pestis, survive in refrigerated temperature Most common cause of sepsis associated with blood transfusion ○ Most common platelet contaminant is S. epidermidis, due to improper disinfection of patient’s arm during collection Platelets are stored at room temperature which provides an ample environment for S. epidermidis to survive Positive blood cultures usually indicative of: Inadequate donor arm preparation Recall: in blood donation disinfection is70% alcohol -> iodine -> 70% alcohol to ensure no fungal organism contaminates blood bag Improper pooling technique If the pooling device is not properly connected or is not sterile, it could allow air or contamination to enter. Pooled blood products should be transfused right away Health of donor - bacteremia in donor If one component is contaminated, other components prepared from the same donor unit may be contaminated also. ○ REJECT ALL COMPONENTS! ○ Activated factor VII Factor VIII Concentrates Patients with hemophilia A; derived from plasma or with the use of recombinant DNA technology, and pooled plasma Developed in 3 ways: ○ Monoclonal antibody puriﬁcation ○ Pooled plasma absorbed by barium sulfate or Aluminum hydroxide ○ Recombinant F IX methods For those with autosomal bleeding disorder associated with a characteristic pattern of neonatal hemorrhage and lifelong bleeding diathesis Indicated for patients with immunodeﬁciency disorders, those with idiopathic thrombocytopenia purpura(ITP), post transfusion purpura, HIV related thrombocytopenia, and also neonatal alloimmunization thrombocytopenia Recovered plasma which is going to be pooled and to be extracted with the use of cold alcohol Indicated for patients with hypovolemia and hypoproteinemia indicated for patients in shock, burn patients, but not indicated for those with dehydration unless they are going to be transfused with plasma expanders Prepared in the same manner as normal serum albumin but have fewer puriﬁcation stages this is contraindicated to patients with cardiopulmonary bypass surgeries Factor IX Concentrates Factor XIII Concentrates Immune Serum Globulin Normal Serum Albumin (NSA) Plasma Protein Fractions Rho Immune Globulin Prevent occurrence of HDFN Indicated for ITP Two forms ○ Colloids dextran hydroxyethyl starch ○ Crystalloids Ringers lactate & NSS Colloids indicated for hemorrhagic shock and crystalloids could also be applied to burn patients Used if No plasma available Source for antithrombin concentrated is pooled plasma administered to patients with thromboembolism Synthetic Volume Expanders Antithrombin Concentrates Indicated to patients with hemophilia a or b who are found to have circulating antibodies or inhibitors Also to individuals with congenital factor VII deﬁciency and glanzmann thrombasthenia DONOR BLOOD INSPECTION AND DISPOSITION It is required that donor units be inspected periodically during storage and prior to issuing to patient/taken out of the blood bank. The following may indicate an unacceptable unit ○ Red cell mass looks purple or clots are visible Any form of agglutination, big or small, so long as it is visible Could be from air insertion or bacterial contamination or due to improper collection and storage ○ Zone of hemolysis is observed just above RBC mass, look for hemolysis in springs, especially those closest to the unit Discoloration of the plasma & hemolysis in springs = hemolysis during storage ○ Plasma or supernatant plasma appears murky, purple, brown or red (Indicator of severe hemolysis) ISBB - LEC Reissuing of blood - returning blood bag to the blood bank facility after release. Reissuing blood cannot be done unless the following criteria is met: ○ Container closure must not have been penetrated or entered in any manner ○ Most facilities set 30 minutes time limit for accepting units back, warming above 6-10oC, even with subsequent cooling, increases RBC metabolism producing hemolysis and permitting bacterial growth MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 12 Platelets must be continually agitated. Discontinuation of agitation is only allowable up to 1 hour. Platelets are stored at room temperature. BUT if platelets exposed to temperatures outside the facility (beyond 20-24 C) for 30 minutes will not be acceptable for return. ○ Blood must have been kept at the appropriate temperature ○ Ice packs during transport (whole blood, plasma and packed red cells) Reject if not kept in ice beyond 30 mins ○ One sealed segment must remain attached to the container. These segments serve as the source of samples when re-typing and cross matching ○ Records must indicate that blood has been reissued and inspected prior to reissue DISTRIBUTION AND ADMINISTRATION ISBT 128 - International standard of labeling that incorporates barcoded labels ○ Computer readable by blood centers and transfusion services around the world ○ Labeling of blood components is a process that involves ﬁnal review of record including quality control, donor testing, modiﬁcations, and labels attached to unit. ○ Approved by USFDA by 2000s ○ Mandated to be implemented by all blood centers in May 1, 2008 ○ Donor identiﬁcation number is a special or unique combination of numbers that cannot be repeated in less than 100 years RECORDS Must be made concurrently with each step of component preparation, being as detailed as possible for clear understanding Must be legible and indelible Must include dates of various steps and person responsible ADMINISTRATION OF BLOOD COMPONENTS For safe blood transfusion: 1. Positive identiﬁcation 2. A system to avoid and detect clerical errors 3. Direct observation of transfused px ○ Must not leave bedside of px ﬁrst 15 mins of after infusion ○ Must return within 15mins from the time px was transfused(done periodically) ○ If transfuse reax observed, STOP IMMEDIATELY 4. Only normal Saline(infused w/ blood towards the vein of the px) ○ 5% dextrose cause hemolysis in vivo ○ Ringers lactate(contains calcium) - cause in vitro coagulation 5. Use of ﬁlters to remove gross blood clots and cellular debris ○ Necessary ○ 170-260 micrometers is the standard ﬁlter Remove cellular and plasma components ○ Use ﬁlter even if leukocyte reduction of product has been performed to ensure safety since there could be small clots present in blood bags Could be done on the bedside of the patient 6. Blood should be infused within 4 hours from the time it was released from the blood bank facility ○ If left at room temperature for more than 4 hours = bacterial contamination might happen. ○ If not infused w/in 4 hours - return blood bag to blood bank facility 7. Documentation and blood keeping are essential ○ Required with regards to the transfusion to verify if consent took place or if a transfusion reaction happened. ○ The following must be listed: Serial # of blood component Time transfused End time of transfusion Name of nurse who brought the blood from the laboratory Name of MT who released the blood STORAGE AND TRANSPORT Calibration must be performed on centrifuge & thermometers. Recording of equipments after every 4 hours For shipping; do the following: Whole blood & PRBC ○ 1-10oC ○ Containers must be validated periodically Frozen units ○ Dry ice ○ Must be wrapped carefully, very brittle bags Platelets ○ Close to 20-24oC(room temp) ○ During transport, the movement of the vehicle can serve as a ○ Discontinuation of platelet agitation should not exceed 24 hours Discontinuation of agitation is only allowable up to 1 hour. ISBB - LEC MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 13 BLOOD BAG PROCESSING 7. i. Directly store @1-6^C if no ﬁltration ii. Store @1-6^C after ﬁltration If we want to extract Cryoprecipitates, plasma must be THAWED at 1-6°C (average is 4°C) ○ Perform hard spin to express cryo-poor-plasma (CPP) thus cryoprecipitate will remain in the blood bag ○ Plasma will be expressed to another Satellite Bag ○ Pulling off of cryoprecipitate can be performed by freezing within 1 hour from the collection ○ CPP should be freezed as well but can be done within 24 hours from preparation ○ If plasma do not undergo preparation of cryoprecipitate, it must remain stored at -18°C NOTE: Plasma derivatives(coagulation factors, plasma proteins) ○ Derived from unfrozen plasma as long as it is separated w/in 8 hours of collection ○ Can also be derived from recovered plasma, pooled plasma, and fresh frozen plasma. [END] 1. 2. 3. 4. 5. 6. The presence of blood component preparation starts from the collection of whole blood form the donors Blood bag undergoes ﬁltration to reduce amount of WBC present in whole blood by attaching leukoreduction ﬁlters ○ From the initial blood bag, blood goes to reduction ﬁlter up to the blood bag proper NOTE: If we are going to retrieve RBCs and Plasma, we need to perform SOFT SPIN to avoid hemolysis ○ Commonly performed in the blood bank facilities in the Philippines Contrary, the AABB suggests to perform HARD SPIN in separating RBCs and Plasma However, in Platelet manufacturing, it must undergo with SOFT SPIN ﬁrst so that the platelets will not go with the RBC ○ Remains in the plasma after centrifugation Primary Bag contains the RBC while the Satellite Bag contains Platelet-Rich Plasma If in case that you are not going to manufacture platelets, perform HARD SPIN which is LONGER and much more FASTER ○ There is a separation of plasma BUT NO PLATELETS ○ Can be considered Platelet-Poor Plasma since plt has been included in the RBC If you have done the light spin, separation of RBC and platelet-rich plasma must take place ○ For PRBC, additive may be added to preserve for longer shelf life and can be stored at 1-6°C to preserve i. Might undergo ﬁltration/leukoreduction to reduce WBCs ○ Platelet-rich plasma undergo HARD SPIN then collect plasma and separate it from platelet by the plasma expressor equipment ○ Platelets prior to storage must be allowed to rest for 1 hour before placing it to platelet agitator machine stored at 20-24°C ○ Plasma(w/ out platelets) can undergo freezing and stored at -18°C or colder If hard spin is performed, separation of RBC and plasma(Platelet-poor d/t plt going to the rbc) ○ Plasma(PRP) → freeze at -18^C ○ RBC → ﬁltration/leukoreduction ISBB - LEC MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 14 IMMUNOHEMATOLOGY / ISBB / IH LESSON#4: CLINICAL CONSIDERATIONS IN TRANSFUSION MEDICINE FINALS | A.Y. 2023 - 2024 | SIR JASON “AB” CHUA CLINICAL CONSIDERATIONS IN TRANSFUSION MEDICINE Donor selection is based on a criteria, accepts only those who are QUALIFIED to donate In donor screening and phlebotomy, it includes pre-transfusion testing ○ Testing of blood for any abnormality, discrepancy, or incompatibilities in donor-recipient No matter how much we are careful and keen to avoid any transfusion reactions, at some point, it can’t be helped CATEGORIES OF TRANSFUSION REACTIONS TRALI HEMOLYTIC TR-GVHD DELAYED BACTERIAL CONTAMINATION SEROLOGIC TACO FEBRILE TRANSFUSION HEMOSIDEROSIS NON-HEMOLYTIC CITRATE TOXICITY ALLERGIC POST TRANSFUSION PURPURA ANAPHYLACTIC TRANSFUSION REACTION HEMOVIGILANCE MODEL (NHSN, ABB, CDC) ○ Model used to track, analyze and ultimately to improve transfusion outcomes ○ Reports and collates incident reports in transfusion reactions or any transfusion-related occurrence ○ 3 Organizations that recognize transfusion reaction: 1. National Healthcare Safety Network 2. American Association of Blood Banks 3. Centers for Disease Control SIGNS OF TRANSFUSION REACTIONS MAY INCLUDE THE FF: Fever (greater or equal to 1oC Skin manifestations: urticaria increase in body temp) (beehives), rash, ﬂushing, edema Chills or Rigor Jaundice, Hemoglobinuria Respiratory distress (wheezing, Nausea, Vomiting coughing, dyspnea, cyanosis) Hyper/Hypotension Abnormal bleeding Pain - Abdominal, etc. Oliguria or Anuria HEMOLYTIC TRANSFUSION REACTION (HTR) Acute - < 24 hours ○ IgM ab Delayed - > 24 hours ○ IgG ab Immune-mediated - complement activation involved Non-immune mediated - caused by other factors (e.g technical errors) HTR: ACUTE (AHTR) Rapid destruction of red cells(hemolysis) during, immediately after, or within 24 hours. Severity ranges from fever to death. ○ When patients show signs and symptoms such as fever, immediately stop the transfusion. IMMUNE-MEDIATED TRANSFUSION REACTION ACUTE VS DELAYED TRANSFUSION REACTION IMMUNE MEDIATED VS NON-IMMUNE MEDIATED ACUTE (LESS THAN 24 HOURS) DELAYED (MORE THAN 24 HOURS) IMMUNE MEDIATED Hemolytic Febrile, nonhemolytic Allergic (Type 1 hypersensitivity) anaphylactic TRALI (Transfusion Related Acute Lung Injury) Hemolytic Serologic RBC HLA Alloimmunization TA-GVHD (HLA involvement) Post-transfusion Purpura NON-IMMUNE MEDIATED Sepsis (d/t improper disinfection) TACO (Transfusion Associated Circulatory Overload) Physical hemolysis Hemosiderosis Citrate Toxicity (occurs during massive transfusion) TRANSFUSION IS FURTHER DIVIDED INTO AHTR (Acute HTR) TAS (Transfusion-associated Sepsis) FEBRILE FNHTR (Febrile Non-HTR) ALLERGIC PULMONARY ISBB - LEC MILD (e.g pruritus) SEVERE TACO TRALI Immune complexes form due to Antigen-Antibody interaction When the immune complex is triggered: 1. Activation of Membrane Attack Complex (MAC) Starts at: C5b6789 If activated it can cause intravascular hemolysis 2. Release of Vasoactive Amines Vasodilation Blood vessels lumen become bigger Hypotension More crucial Low oxygen levels in the kidney or lungs can lead to Shock ○ Causing Bronchospasm Immune Complex deposition Thrombus formation Renal Failure ○ Immune complexes are deposited in the kidney ○ Diﬃculty to ﬁlter blood due to thrombin formation causing clots in the bloodstream. MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 1 3. 1. 2. 3. Activation of Coagulation Cascade Disseminated intravascular coagulation(DIC) deﬁcient clotting factors causing internal bleeding KNOWN CAUSES(human-made errors): ○ Collection of blood from the incorrect patient ○ Incorrect labeling of blood samples ○ Misidentiﬁcation of sample at blood bank ○ Issuance of wrong unit from blood bank ○ Transfusion of blood to incorrect patient ○ Aliquoting a patient sample to improperly labeled test tube HTR: DELAYED Symptoms appear after 24 hours/1 day Less severe and require no treatment IgG antibodies ○ Second antibodies to respond Rapid decrease of hemoglobin to pretransfusion levels Presence of spherocytes ○ RBC membrane turns smaller due to decreased surface area to volume ratio Antibody(IgG) + Antigen interaction = Activation of RES (reticuloendothelial system) RES release cytokines Cytokines tap T cells and B cells to be activated and cause reaction CLINICAL SIGNS AND SYMPTOMS ACUTE HTR Fever, chills, ﬂushing, pain at site of infusion, tachycardia, tachypnea, lower back pain, hemoglobinemia, hemoglobinuria, hypotension; dramatic and severe; rapid onset DELAYED HTR >24 hours posttransfusion; fevertemperature increase >1° C (or 2° F) with or without chills: unexplainable decrease in hemoglobin and hematocrit: occasional mild jaundice MAJOR DIC, renal failure, COMPLICATIO irreversible shock, death NS No major complications; less severe reaction Complement activation; ABO incompatibility Anamnestic response to red cell antigen; alloantibody not demonstrating or missed CAUSES Clerical check; visual inspection of posttransfusion sample; DAT-positive; DAT-positive or negative: posttransfusion antibody CLINICAL repeat ABO testing; tests screen-positive: LABORATORY TESTS for hemolysis; ↑ plasma ↓hemoglobin/hematocrit; free hemoglobin; ↑ serum tests for hemolysis bilirubin; ↓haptoglobin; hemoglobinuria Treat hypotension and Provide antigen-negative DIC; maintain renal blood donor units: no additional MANAGEMENT ﬂow treatment necessary ISBB - LEC PREVENTION Avoid errors of mislabeled samples and patient identiﬁcation; design systems to decrease chances of technical error Check patient records HTR: NON-IMMUNE MEDIATED CAUSE BY: OTHER FACTORS Extreme Temperature - Can be due extreme heat or cold temperature resulting to lysed red cell Improper de-glycerolization on RBC unit Mechanical destruction Incompatible solutions Bacterial contamination of unit Intrinsic red cell defect DELAYED SEROLOGICAL TRANSFUSION REACTION Antibody development after 24 hours ○ Positive DAT ○ Antibody panel identiﬁcation FEBRILE NONHEMOLYTIC TRANSFUSION REACTION Febrile = Fever Most commonly observed Increase of 1°C or 2°F in temperature Cytokines ○ Transfused ○ In response to transfusion FEBRILE HEMOLYTIC TRANSFUSION REACTION Fever-temperature increase ≥1° C (or 2° CLINICAL SIGNS AND F): chills; nausea; vomiting; headache; SYMPTOMS and back pain MAJOR Usually not life-threatening for recipient COMPLICATIONS Recipient HLA antibodies to HLA antigens on donor lymphocytes CAUSES Cytokines released by WBCs during blood product storage CLINICAL LABORATORY DAT-negative No visible hemolysis TESTS MANAGEMENT Antipyretics-acetaminophen PREVENTION Prestorage leukocyte reduction of blood products AHTR: NON-IMMUNE MEDIATED CAUSES: ○ Chemical damage ○ Mechanical damage ○ Improper shipping or storage temperature ○ Incomplete deglycerolization of frozen red cells ○ Needles used for transfusion are of small bore size ○ Rapid pressure infuser or roller pumps ○ Improper use of blood warmers ○ Concurrent infusion of unapproved ﬂuids ○ Bacterial contamination IF AHTR IS SUSPECTED: 1. Discontinue transfusion 2. Perform clerical veriﬁcation 3. Notify recipient's physician 4. Maintain Intravenous line 5. Document the incident make an incident report 6. Draw post transfusion reaction samples 7. Collect urine sample MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 2 If blood samples appear hemolyzed, request for another sample PERFORM DAT! ALLERGIC & ANAPHYLACTIC TR Ranges from minor urticarial effects to fulminant anaphylactic shock and death Common with plasma transfusions IgE → mast cell degranulation IgA → IgA-deﬁcient patients Mild effects → itching Severe → could lead to death ALLERGIC CLINICAL SIGNS AND SYMPTOMS MAJOR COMPLICATIONS CAUSES ANAPHYLACTIC Rapid onset and severe, after small volume is transfused; wheezing, Hives, erythema, itching coughing, dyspnea, within 15-20 minutes bronchospasm, of transfusion respiratory distress, vascular instability: no fever Shock, loss of NONE consciousness; death Recipient antibodies to Associated with foreign plasma proteins genetic IgA deﬁciency or other substances in recipient; possesses such as drugs or food IgG, consumed by blood complement-binding donor anti-IgA antibodies CLINICAL LABORATORY TESTS DAT-negative; No visible hemolysis MANAGEMENT Transfusion interrupted and antihistamine administered Transfusion terminated; epinephrine administered; Oxygen administered and open airways maintained Premedication with antihistamine if patient history reveals repetitive allergic reactions. May necessitate washed cellular products Plasma-containing products from IgA-deﬁcient donors Washed red cell and platelet products PREVENTION TRANSFUSION-RELATED ACUTE LUNG INJURY (TRALI) Respiratory distress and pulmonary edema (1-2 hours post-transfusion) ○ Fluid build up in the lungs → respiratory distress ○ TRALI is not connected to circulatory overload Infusion of antibodies to leukocyte antigens (Class I and II HLA) ○ Activate neutrophils → capillary leakage → pulmonary edema Activated neutrophils: ○ Epithelial damage ○ Capillary leakage ○ Pulmonary edema Marked respiratory distress; fever, CLINICAL SIGNS AND hypotension, chills, cyanosis; rapid SYMPTOMS onset-occurs during transfusion or within 6 hours of completion Severe and dramatic presentation, can be MAJOR COMPLICATIONS fatal Interaction of granulocytes and HLA-speciﬁc donor antibodies, complement activation, CAUSES aggregation of granulocytes that leads to blockage of pulmonary microvasculature; ISBB - LEC CLINICAL LABORATORY TESTS MANAGEMENT PREVENTION capillary damage, vascular leakage, pulmonary edema DAT-negative; no visible hemolysis; WBC antibody screen in donor and recipient Respiratory support Defer implicated donor for plasma containing components. Avoid use of plasma components from multiparous women TRANSFUSION-ASSOCIATED GVHD GVHD (Graft Vs Host Disease) Rare but highly lethal (90% mortality rate) ○ Rarest in all of the reactions ○ can lead to death Immunocompetent donor lymphocytes ○ attacks and damages the organs 3-6 days post-transfusion Onset 3-30 days posttransfusion; fever, CLINICAL SIGNS AND erythematous maculopapular rash, abnormal SYMPTOMS liver function; nausea, vomiting, jaundice, abdominal pain, diarrhea MAJOR Sepsis and hemorrhage; 90% mortality rate COMPLICATIONS Transfused immunocompetent T lymphocytes CAUSES mount immunologic response against recipient Conﬁrmation by HLA typing to demonstrate a CLINICAL disparity between donor lymphocytes and LABORATORY TESTS recipient tissues MANAGEMENT Unresponsive to medical intervention Irradiation of blood products before transfusion in at-risk recipients; gamma irradiation (25 Gy) PREVENTION to prevent blast transformation of donor lymphocytes BACTERIAL CONTAMINATION of BLOOD PRODUCTS Bacteremia in donor (asymptomatic) ○ Sepsis ○ Bacteria in the blood Improper cleansing during collection Bacterial endotoxins ○ trauma or shock Common in platelet transfusions ○ d/t storage @RT CLINICAL SIGNS AND Same as acute hemolytic transfusion SYMPTOMS MAJOR COMPLICATIONS Shock Donor bacteremia, improper cleansing of CAUSES venipuncture site CLINICAL LABORATORY Gram staining, Bacterial culture and TESTS identiﬁcation Broad-spectrum antibiotic therapy MANAGEMENT (treatment prior to conﬁrmation) Use of diversion pouches PREVENTION Care during phlebotomy Use of plastic collection bags TRANSFUSION ASSOCIATED CIRCULATORY OVERLOAD (TACO) Cardiopulmonary system exceeds volume capacity Dyspnea, severe headache, peripheral edema, and signs of Chronic Heart Failure during or shortly after transfusion AT RISK: ○ Elderly and infants ○ Compromised cardiac and pulmonary status RBC infusion MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 3 CITRATE TOXICITY Occurs when multiple blood bag units is infused into one patient(Massive transfusion) Transfusion of large amounts of citrate AT RISK: ○ Massive transfusions ○ Impaired liver functions ○ Infants with hepatic or renal insuﬃciency PREVENTION: ○ Remove-plasma ○ Calcium chloride or calcium gluconate TRANSFUSION HEMOSIDEROSIS Accumulation of excess iron in macrophages ○ Hemolytic reaction occurs. Aftterwhich the release of iron leads to its accumulation in the blood. Excess iron in the blood will then be taken up by macrophages which could also lead to iron overload. Iron-overload ○ Thalassemia (target cell/codocyte) ○ Sickle-cell disease (sickle cell) PREVENTION: ○ Use of chelators Deferiprone Deferoxamine POST-TRANSFUSION PURPURA Platelet counts plummets 5-12 days post-transfusion ○ The body acquires Anti-Human Platelet Antigen(Anti-HPA) which will recognize the platelets as foreign, leading to sensitization of platelets and the production of alloantibody or autoantibody Anti-HPA-1a ○ Sensitization to HPA-1a (98% frequency) ○ Alloantibody ○ Autoantibody PREVENTION: ○ Plasmapheresis ○ Exchange transfusion (to dilute platelets) ○ Intravenous immunoglobulin ADDITIONAL TESTS in a TRANSFUSION REACTION ADDITIONAL TESTS in a TRANSFUSION REACTION INVESTIGATION TEST REASON ABO/D PHENOTYPING Errors in patient or sample identiﬁcation ANTIBODY SCREEN CROSSMATCH HEMOGLOBIN & HEMATOCRIT Newly detected antibodies Serologic compatibility between ABO and Rh blood group only Therapeutic effectiveness Hemolytic process Nonimmune hemolysis or bacterial contamination Bacterial contamination HAPTOGLOBIN BILIRUBIN URINE HEMOGLOBIN INSPECTION OF DONOR UNIT GRAM STAIN AND CULTURE POSTREACTION SAMPLE CLERICAL CHECK: ID errors? VISUAL CHECK: Hemolyzed or icteric? DIRECT ANTI-HUMAN GLOBULIN TEST [END] “God Bless on your Finals & Compre exams, future RMTs! See u sa pinning ” EVALUATION AND REPORTING A TRANSFUSION REACTION WHAT TO DO? Stop the transfusion ○ If patient temperature increases by 1^C degrees celsius, stop the transfusion. As little as 10 mL of blood transfused to patient could trigger an acute transfusion reaction Keep IV open with saline ○ Saline is isotonic preventing lysis of red cells Perform clerical check for ID errors Contact treating physician Monitor/record vital signs Contact transfusion service Collect post sample and return with blood bag attached IV ﬂuids to the lab ISBB - LEC MATUGAS, TUGAY, EMBOSCADO, PALCO, LAMELA, APIL, DELFIN, VALDIVIA, INTING BSMLS 3I & 3J 4

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