Blood Banking

LIFE'S BLOOD
Table of Contents CLASS NOTES

by M. Schroeder and M. Jensen
ABO DISCREPANCIES
DEFINITION: Any deviation from the expected pattern of antigen on the cell and the opposite antibody in the serum . ANTI-A ANTI-B A1 CELL B CELL 4+ 0 2+ 4+
ANTI-A 4+
ANTI-B 0
A1 CELL 0
B CELL 0
ANTI-A 4+
ANTI-B 4+
A1 CELL 4+
B CELL 4+
What are their ABO types ? ? ? ? ? ? ?
ABO Discrepancies MUST BE RESOLVED

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In RECIPIENTS the discrepancies must be resolved before any blood component is transfused . If not resolved before blood is needed, transfuse Group O (O NEGATIVE if there is a discrepancy in the Rh type also). In DONORS the discrepancies must be resolved before any blood is labeled with a blood t;ype..
GENERAL RULES TO RESOLVE:

1. Always re-test first. 2. Check for clerical/technical errors
3. Weakest reaction is usually the one in doubt. 4. Check results of the screening cells. 5. Check the patients age. 6. Check the diagnosis 7. Check the transfusion history.
KINDS OF DISCREPANCIES:
CLERICAL ERRORS (TRANSCRIPTION ERRORS)
Clerical errors are the most common.
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If you do not record the results as you read each tube, you run the risk of recording incorrect results. Always check which tube you are reading and record the results immediately. Make sure you are recording the results on the right worksheet. One way to prevent this error is to minimize the times you are working with more than one patient or donor at a time. Recording results in the wrong spot on worksheet could occur when you put some of the serum results in the cell typing area or vice versa. Be sure you have techniques that will prevent you from performing this error. (This is why
our labeling procedure uses capital A and B for the forward type and a 1C and bC for the reverse typing)
TECHNICAL ERRORS
There are a number of technical errors that m ay also occur:
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Sample mix-up such as wrong serum tube with wrong clot or 3% suspension. Failure to add serum or reagent can lead to technical errors where no reaction is occurring where one is expected. Remember for both ABO and Rh always add your reagen t antisera and serum before adding cells. Addition of wrong reagent such as screening cells, which are O, instead of A 1 and B cells can lead to significant technical errors. Contaminated reagents could result in either false negative or false positive results depending on whether the reagent added neutralized or added to the reactivity of the original reagent. Under centrifugation could lead to a negative reaction since the cells are not encouraged adequately to bind with the antibody. Over centrifugation can lead to you reading the reaction as positive while there is still a button on the bottom of the tube or your shaking to dislodge the button broke up the agglutination reaction. Warming the test could result in a false negative reaction since ABO
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antibodies are IgMs that react better in the cold. Too many cells in your cell suspension can lead to decreased or negative reactions since there are too many cells for the number of antibodies present in the reagents. Remember we want to be in the zone of equivalence for our reactions. Failure to detect weak results can occur if you are not watching the reactions while you are shaking them out or if you shake too hard. Failure to detect hemolysis can be a definite problem. Remember a positive reaction can be hemolysis as well as agglutination since the antigen -antibody reaction can bind complement. When complement is bound it can lead to hemolysis that is also an indication of a positive reaction. Dirty glassware can cause the cells to artificially clump.
PROBLEMS WITH SERUM TESTING

So where is the problem if it is actually a true discrepancy between the ABO cell type and the ABO serum test? Serum testing is more common than problems with cell typing. This is either manifest as an extra antibody present or an expected antibody missing Extra Antibody ANTI-A 4+
ANTI-B 0
A1 CELL 2+
B CELL 4+
Expected Antibody Missing ANTI-A ANTI-B 4+ 0
A1 CELL 0
B CELL 0
PROBLEMS WITH RED CELL TESTING

There are a number of problems that can occur with the red cell testing including:
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Mixed-field agglutination Weak or missing antigens Unexpected antigen Polyagglutinable cells
Mixed-field agglutination ANTI-A MF Weak or missing antigen ANTI-A 0 Unexpected antigen ANTI-A 4+
ANTI-B 0
A1 CELL 0
B CELL 4+
ANTI-B 0
A1 CELL 0
B CELL 4+
ANTI-B 2+
A1 CELL 0
B CELL 4+
Polyagglutinable cells ANTI-A 4+
ANTI-B 4+
A1 CELL 0
B CELL 4+
RESOLVING PROBLEMS WITH SERUM TESTING:

Weak or missing antibody(ies)
An extreme example would be no reaction for the forward and reverse typings.
ANTI-A 0
ANTI-B 0
A1 CELL 0
B CELL 0
The steps to follow to resolve this discrepancy is to: 1. Check birth date since newborns and the elderly are more likely to demonstrate this discrepancy. Newborn antibodies are not present until at least 6 months. DON'T ATTEMPT TO SERUM-CONFIRM NEWBORNS. As individuals ages they may also lose their ability to maintain their antibody levels. Therefore, the very elderly have decreased antibody levels. 2. Check diagnosis since patient conditions such as: Immune deficiencies, Chemotherapy, Radiation Therapy, and Bone marrow transplantation may explain the mi ssing antibodies.
Resolution of Missing Antibodies:
1. Add two more drops of serum just in case you forgot to add them the first time and centrifuge. If negative then incubate in cold (4 -18 oC) 15-30 MINUTES 2. Include autocontrol to rule out interference from natural anti-I when incubating at (4-18oC). 3. At 4oC Anti-A and Anti-B enhanced since they are saline, cold -acting antibodies as seen in this example for an O individual. ANTI-A 0 ANTI-B 0 A1 CELL 2+ B CELL 2+ AUTOCONTROL 0
4. Compare this with a 4oC Auto-Anti-I enhanced would have a positive autocontrol as seen in the example below ANTI-A 0 ANTI-B 0 A1 CELL 2+ B CELL 2+ AUTOCONTROL 2+
5. Group A or Group B can serve as its own negative control. 6. 4oC Anti-B enhanced is shown below: ANTI-A 4+ ANTI-B 0 A1 CELL 0 B CELL 2+ AUTOCONTROL 0
7. 4oC Anti-I enhanced on the other hand would have a positive autocontrol. ANTI-A 4+ ANTI-B 0 A1 CELL 2+ B CELL 2+ AUTOCONTROL 2+
8. If anti-I enhanced along with anti -A or anti-B, can re-set up and incubate at 18oC. As seen in this example of 18 oC: Anti-B enhanced, anti-I nonreactive ANTI-A 4+ ANTI-B 0 A1 CELL 0 B CELL 2+ AUTOCONTROL 0
Presence Of Unexpected Anti-A
The presence of Anti-A1 should be suspected when the antibody is reactive against the A cells but not the screening cells at immediate spin as seen in the example below. ANTI-A 4+ ANTI-B 0 Immediate Spin 0 0 0 37oC A1 CELL 2+ AHG B CELL 4+ CCC
Screening Cell I Screening Cell II Autocontrol
Naturally anti-A1 occurs in subgroups of A or are passively-transfused from Group O platelets and other blood products. How to Resolve the Issue of Unexpected Anti -A: 1. Check recent transfusion history for group O products, (especially platelets) that would explain the presence of this antibody. 2. Test patient cells with lectin -A1. Subgroups will be negative with this reagent but A1cells will be positive.
Lectin + A 1 CELL = 4+ Lectin + A subgroups CELLS = 0
3. Test patient serum with three A 1 cells and three A2 cells and if it is an antiA1 the following reactions will occur: Anti-A1: SERUM + A1 CELLS = + SERUM + A2 CELLS = 0 Anti-A1 will react only with the A 1 cells but not with the A 2 cells In the case of passive Anti-A from Group O Platelets the reactions would be the following: SERUM + A1 CELLS = + SERUM + A2 CELLS = + In this case if the antibody is strong enough you may need to transfuse group O blood .
UNEXPECTED A OR B ANTIBODY WHEN THE IMMEDIATE SPIN ANTIBODY SCREENING IS POSITIVE
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You may have a positive reaction with the reagent A 1 or B cell that is due to a room temperature antibody reacting with an antigen other than A or B on the cells ANTI-A 4+ ANTI-B 0 Immediate Spin 2+ 0 0 37oC A1 CELL 2+ AHG B CELL 4+ CCC
How to Resolve the Issue of Unexpected Anti -A that is probably another antibody due to the results of the Antibody Screening: 1. Identify the antibody by performing an identification panel at room temperature. 2. Pre-warm away (use caution) the effect of this antibody by doing the reverse typing with prewarmed serum and reagent cells. 3. Type reagent A 1 or B cell for the corresponding antigen once the antibody is
identified. For example, if the patient had an anti -N that was showing up at room temperature according to the antibody identification process, you would then type for N on the reagent cells used for the reverse typing. If anti-N is causing your problem, then the cells should have N antigen present.
ROULEAUX FORMATION GIVING UNEXPECTED AGGLUTINATION IN ALL SERUM TESTS
Rouleaux can give unexpected agglutination in all serum tests ANTI-A 4+ ANTI-B 0 Immediate Spin 2+ 2+ 2+ 37oC A1 CELL 2+ AHG B CELL 4+ CCC
Rouleaux may also give false positive cell typing if strong enough and cells are insufficiently washed. This phenomenon is due to alteration in serum protein concentration such as:
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Multiple myeloma Macroglobulinemia Liver disease (decreased albumin) Also seen with volume expanders
Characteristics of rouleaux is that it:

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Looks like agglutination macroscopically Microscopically it appears as "stacks of coins"
How would you resolve rouleaux problems?
Do saline replacement technique: 1. 2. 3. 4. 5. Re-centrifuge the test tube. Draw off serum without disturbing cell button Add two drops of saline Resuspend Rouleax disperses in saline; TRUE AGGLUTINATION REMAINS
RESOLVING PROBLEMS WITH CELL TYPING

Mixed-field agglutination
Mixed-field agglutination is seen as large or small agglutinates with many unagglutinated cells. Usually mixed -field agglutination means a MIXED -CELL POPULATION The causes of mixed-field agglutination can be:
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Mixed cell populations resulting from massive transfusion of another blood group such as an B individual receiving "O" red blood cell donor units since the transfusion center did not have enough B donor units. Bone marrow transplant patients may have both some of their original type of cells and the type of the bone marrow transplant. Weak subgroups of A 3 traditionally give a mixed field reaction. Rarely the condition called chimerism due to intrauterine exchange of erythrocyte precursors between twins or 2 fertilized eggs fuse into one individual.
You should try to find cause of mixed field agglutination before setting up blood to transfuse so be sure to check the patient's transfusion records and clinical history. If it appears to be a weak subgroup performed the tests discussed under Unexpected Anti-A
Weak or missing antigen
ANTI-A 0
ANTI-B 0
A1 CELL 0
B CELL 4+
Weak, or missing, antigen may be due to v ery weak subgroup of A or B, loss of transferase in acute leukemia, massive transfusion of GROUP O, or bone marrow transplant How would you resolve a weak, or missing, antigen?
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Obtain recent transfusion history a nd any clinical history of bone marrow transplant Read forward grouping microscopically Use anti-A,B and incubate at 4-22oC at least 15 minutes Use monoclonal antisera that is known to react with antigens like A x and B x Perform specialized tests if the above steps do not resolve the problem:
Specialized tests would include absorption/elution techniques and saliva studies.

Acquired B antigen
ANTI-A 4+
ANTI-B 2+
A1 CELL 0
B CELL 4+
Acquired B antigens are seen in problems with the colon or infections with Gram negative rods Bacterial enzymes modify the "A" antigen to a "B" antigen and the patient forward types as an AB but reverses as an A.
How would you resolve a possible acquire d B antigen?

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Set-up an autocontrol. The patient's own anti-B will not agglutinate their own AB cells. Check clinical history to evidence of colon problems or Gram -negative rods. Check monoclonal anti-B product inserts since some will not react with B acquired antisera Acidify some reagents anti-B to pH 6 and re-test. Modified (acquired) B antigens will not react in the acidified antiserum, normal B antigens will still react
Polyagglutinable cells
Most monoclonal anti-A and anti-B will show problems with polyagglutinable cells if it is a problem with the cell membrane that leads to the agglutination. The most likely causes of due to Wharton's Jelly, found in cord blood, and strong positive direct antiglobulin test due to a cold agglutinin. In the case of the strong positive DAT, it would appear to be an AB in the forward type and an O on reverse. ANTI-A 2+ 1. WHARTON'S JELLY
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ANTI-B 2+
A1 CELL 4+
B CELL 4+
Coats newborn cord cells and the child's type may appear AB. You do not do a reverse on newborn blood since they have not made any anti -A or anit-B yet. If the baby types as an AB recheck by washing cells several times and retesting since you need to make sure you have removed the Wharton's Jelly and the baby is truly an AB. Better yet ALWAYS WASH CORD BLOOD AT LEAST 4 TO 5 X'S BEFORE DETERMINING THE TYPE OF THE BABY.
2. Strong positive DAT

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May be seen in cold auto-immune hemolytic anemia If due to cold agglutinin, wash several times in warm saline and re -test Cells washed 3X at 37 oC would probably look like this: ANTI-A 0 ANTI-B 0 A1 CELL 4+ B CELL 4+
PROBLEMS WITH BOTH CELLS AND SERUM

Strong cold auto-agglutinins
ANTI-A 4+
ANTI-B 4+ Immediate 37oC
A1 CELL 4+ AHG
B CELL 4+ CCC
Spin 4+ 4+ 4+
3+ 3+ 3+
3+ 3+ 3+
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A strong cold auto-agglutinin is most often due to strong auto -anti-I. 1. To resolve cell typing difficulties:
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Wash cells 3-4X with warm (37 oC) saline Re-test warm-washed cells
2. To resolve serum typing difficulties:

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Perform serum testing at 37 oC (Use caution that weak isoagglutinins (anti -A and anti-B) are not missed using this technique) Autoabsorb cold agglutinins onto patient cells at 4 oC.
OBJECTIVES - ABO DISCREPANCIES

1. Describe how an ABO discrepancy would be recognized. 2. Explain what must be done if an ABO discrepancy cannot be resolved before the patient requires a transfusion. 3. Explain what must be done when a discrepancy arises in a blood donor. 4. List at least 6 technical or clerical errors that may cause ABO discrepancies. 5. Describe the reactions, list the clinical situations in which they may occur, and explain how to resolve each of the following causes of ABO discrepancies: *Decreased immunoglobulin levels *Weak subgroups of A with anti-A1 *Passively transfused anti-A1 *Unexpected alloantibody reacting at room temperature *Loss of A or B antigen *Acquired B antigen *Rouleaux *Cold agglutinins 6. List at least three causes for mixed-field agglutination.
Performance objectives:
1. Recognize when an ABO discrepancy exists. 2. Given any sample of blood showing an ABO discrepancy, correctly identify and perform the necessary procedures to resolve the discrepancy. Table of Contents
LIFE'S BLOOD
LABORATORY TECHNIQUES: REAGENTS, REACTION GRADING; CELL WASHING; SAMPLE COLLECTION BLOOD BANKING REAGENTS
The techniques used in Blood Bank involves mixing antigens, usually on red blood cells with antibodies. The environment where this reaction occurs can range in temperature from 4 oC to 37oC. With the most common being room temperature for ABO and the initial Rh(D) testing and 37 oC when screening and identifying other clinically significant antigen -antibody reactions. In a number of situations we are looking for particular antigens on the red cell such as looking for A or B antigens to determine a patient's ABO type. Other times we may be looking for particular antibodies that may cause transfusion reactions or hemolytic disease of the newborn. Depending on whether we are looking for a particular antigen or antibody will determine what reagents we are going to use. If we are looking for an A ant igen on a patient's red cells, we will use known anti -A reagent that will cause agglutination of the A antigens on the red cells. If the patient has on B antigens or no ABO antigens, as in the case of an O individual, their cells will not agglutinate with anti-A reagent.
Sources of Antigen Testing:
In almost all blood bank techniques we have red cells with antigens present. These red cells may either reagent red cells with known antigens, patient red cells, or donor red cells. The reagent red cells are commercially prepared and have all the red cell antigens identified. When we use red cells where the antigens have already been determined, we can identify the possible antibodies present. For example: Anti-A and Anti-B are expected antibodies in patie nt's who lack that particular antigen. Therefore if a patient or donor has only the A antigen on their red cells, then they should have anti B in their serum. When we test their serum with A 1 and B cells, agglutination will occur with the B cells and not with the A1cells since they have an anti-B. The reagent cells used for blood banking include the following:
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A 1 and B cells for confirmation of the ABO type in all patients and donors other than newborn babies Antibody screening cells are O cells that ha ve been studied to determine the
presence of a number of antigens for specific antibodies that are known to cause transfusion reactions and hemolytic disease of the newborn. The antibody screening technique is part of all compatibility tests done before b lood is transfused. Some of the more common antibodies detected are anti -D, anti-E, anti-K. Antibody identification cell panel are again O cells with the specific antigens known. Usually there are between 8 and 12 different cells in a cell panel. The pattern of positive and negative reactions help identify the antibody.
Sources of Antibody for Testing
Antibody is found in serum. If it is the patient's serum that is being tested, we do not know what antibody may be present so we are using one of the 3 types of reagent cells listed about. If the serum is commercial reagent, the specific antibody present is already known. The commercial serum reagent is referred to as antisera. Therefore, we use Anti-A antisera to determine if a patient or donor is Typ e A. If we are trying to determine if the patient is Rh + or Rh -, we will use anti-Rho (D) antisera. Table 1 is a summary of known and unknown sources of both antigens and antibodies.
Table 1
Known Source with known components
Unknown components - The source is either the patient or the donor Patient or Donor red blood cells Patient or Donor serum or plasma
Antigen Reagent Red Blood Cells Antibody Commercial Antisera
Testing Procedures Routinely Done in Blood Banking
In a transfusion service there are a number procedures routinely done. The ones noted in red are those done even in small hospitals whereas the rest are more likely done at larger hospitals and reference laboratories:
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ABO/Rh(D) typing Antigen typing from other blood group systems such as Rh antigens other than D, Kell, Kidd, and Duffy Antibody screening for antibodies form to blood group antigens other than A and B Antibody identification to determine the specific antibodies detected in the antibody screening Crossmatch, or compatibili ty testing, which determines whether donor blood can probably be safely transfused to the recipient
Table 2 summarizes the sources of both the antigen and antibody (Modified from Basic & Applied Concepts of Immunology, Blaney and Howard, lst ed., p. 39
Procedure ABO/Rh typing Antigen typing Purpose Detects A, B, and D Detects antigens of other blood group Source of Antigen Patient's RBC's Source of Antibody Commercial anti-A, anti-B, and anti -D Commercial antisera to the specific
Patient's RBC's or
systems (examples: K, Donor RBC's E, C, Fya, Jka)
antigens (examples: anti-K, anti-E, anti-C, anti-Fya, anti-Jka)
Detects antibodies Commercial Screening Antibody screening with specificity of RBC Patient's serum Cells antigens Identifies the Antibody identification specificity of RBC antibodies Commercial Panel Cells
Patient's serum
Crossmatch
Determines serologic compatibility between Donor RBC's donor and patient before transfusion
Patient's serum
GRADING REACTIONS
Grading agglutination reactions gives an indication of the relative amount of antigen or antibody present. All tubes tests should be graded. The technique used in the resuspension of the cells will affect the grading of the reaction. The correct procedure for resuspending and grading rea ctions follow:
1. Resuspension Procedure:
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use lighted agglutination viewer read only one tube at a time hold tube upright position cell button so it is facing you in the mirror very gently shake the tube and observe how the cells come off the cell button
2. Grading Reactions
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swirling off = negative coming off in chunks = positive continue shaking till all cells resuspended tilt tube, read and grade reaction
3. Grading system:
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4+ = solid clump 3+ = several large clumps 2+ = small to medium sized clumps; clear background 1+ = small clumps; cloudy background +w = tiny aggregates; cloudy background + micro = positive upon microscopic examination only MF = mixed field. Small clumps amidst many unagglutinated cells. Can be confused with 1+ hem = hemolyzed (a positive reaction) neg = negative, no agglutination (Never use - for negative; either write neg or 0)
WASHED CELL SUSPENSIONS

3% Red Blood Cell Concentration in Saline
Between 2-5% cell suspension provides optimum antigen concentration for the tube method for red blood cells typing. To make sure your suspension is within this range use reagent red cells for comparison.
Washing Red Blood Cells Before Making the 3% Suspension

The purpose of washing the red blood cells is to remove plasma, which contains substance that may interfere with antigen -antibody reaction. The following may be in the plasma and may interfere with testing:
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Soluble antigens such as A and B may be present and neutralize your reagent. Interfering proteins such as Wharton's jelly that is seen in newborn cord blood, cold acting autoimmune antibodies, and increased levels of immunoglobulins that may cause either agglutination or rouleaux.. Hemolyzed red blood cells due to a difficult draw will interfere in your grading interpretation of hemolysis Fibrinogen can result in fibrin strands forming that makes grading reactions difficult.
Good Technique when washing and making a 3% c ell suspension involves the following:
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Place 1 to 3 drops of blood in the tube Aim the tip of the saline bottle towards the center of the tube and forcibly squirt saline into the tube. Fill the tube 3/4 full of saline (there will be less splattering in t he centrifuge) Centrifuge long enough spin to pull most of cells into a button in the bottom of the tube. Decant the saline completely Shake the tube to resuspend cell button before washing the cells again. It will depend on the procedure being done as to how many washings are going to be done.
COLLECTING BLOOD BANK SAMPLES

Samples for Blood Bank Testing
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Most samples for blood banking are drawn into a red top tube - serum is preferred. (No clot activation tube should be used since the patient's red cells may also need to used and no other chemicals should be present) A few tests require an EDTA sample if complement is not to be activat ed. Serum must be tested while fresh to ensure good complement activity. Antigens on cells are stable longer (months) in a clot tube.
Patient Identification
The patient MUST be positively identified and preferably banded. Some institutions use specific Blood Bank arm bands.
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Ask patient to state his/her name. Responsible party should identify patient if he/she cannot. Verify information by comparing to ID band. Resolve any differences before proceeding with the blood draw.
Labeling of Sample
The information on sample MUST match information on ID band, which would also needs to be consistent with the order. Information on samples MUST include the following:
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Name (last, first, middle initial) and no nicknames. Unique identification number such as medical record number or possibly social security number. Date and time sample drawn along with the signature or unique identifier of phlebotomist (on sample or on orders) Gender and birthdate desirable but not mandatory . The date of birth provides another unique identifier along with the medical record number and full name of the patient.
Mislabeled Samples
Do NOT accept any sample not properly labeled. The following are what would warrant an improperly labeled specimen:
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Missing information Incorrect information Information on sample not matching information on orders
Improperly labeled samples must be discarded if the problem cannot be resolved. In the case of an emergency blood draw on a patient who is unidentified at that time, the blood specimen must also discarded when both name and medical record number have changed (ex. John Doe, #12240253 becomes Jack Adams # 37859012) unless ID tags with both sets of information remain in place
OBJECTIVES - GRADING REACTIONS; WASHED CELL SUSPENSIONS; BLOOD BANK SAMPLES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Identify sources of antigen and antibody used in testing. Explain why it is important to grade agglutination reactions. Describe the proper resuspension technique Explain how the resuspension technique affects the graded result Describe how each of the following looks: 4+, 3+, 2+, 1+, w+, micro +, hem., MF, neg State the optimum % cell concentration for blood bank testing Explain why the above concentration is best Describe the proper technique for adequate washing of cells Explain how you can tell if your cell suspensions are the proper concentration Name four plasma (or serum) constituents washing removes, and tell why it is desirable to remove them before doing blood ban k testing Describe the proper procedure for identifying a patient when obtaining a sample for blood bank testing Explain what should be done if there is a discrepancy in the patient's identification State the information required on all samples for blood banking testing Explain why gender and birthdate are helpful.
1. Correctly prepare washed 3% suspensions of patient or donor red cells for blood bank testing 2. Utilize the proper resuspension technique to read agglutination reactions 3. Properly obtain a sample for blood bank testing, including use of proper tubes, identification of patient, and labeling of samples
Table of Contents
LIFE'S BLOOD
CLASS NOTES (Under construction)
QUALITY ASSURANCE IN BLOOD BANKING

Quality Programs
When discussing Quality Programs it includes:
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Quality Control Quality Assurance Quality Improvement
For Blood Banking Quality Programs are essential requirements of 2 Federal Agencies: 1. Centers for Medicare and Medicaid Systems (CMS), formerly HCFA, under CLIA-88 which covers all Clinical Laboratory acti vities and related federal payments 2. Food and Drug Administration that has the following concerns: a. Responsibilities of the blood product requirements (anticoagulants and preservatives, shelf life etc.) b. Specific requirements related to independent qualit y control and quality assurance for overall quality of blood products and the processes related to dispersion of those products. A number of accrediting agents have quality requirements as well: 1. American Association of Blood Banks (Blood Banks and Transf usion Services) 2. Joint Commission on the Accreditation of Healthcare Organizations 3. College of American Pathologists
AABB Quality System Essentials & FDA Guidelines for Quality Assurance in Blood Establishments
Organization: active support of quality systems must be place for the following procedures;
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SOP's - Standard Operating Procedures Training plans and development of procedures Approval of lot release of reagents and quality control reagents Review and approval of practices relating to personn el, equipment, selection of suppliers, process control, final inspection and handling nonconforming components, methods in place for handling incidents, errors, and accidents.
Personnel Practices
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Methods for hiring of qualified personnel needs to be in pl ace Job descriptions for all positions need to exist and be available Training program and full documentation of that training for new and continuous employees. Whenever a new procedure or instrument is implemented a training program needs to be in plac e. Regular competency evaluations including direct observation and documentation of such must occur.
Equipment
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Validation of new equipment Calibration and preventative maintenance including standard equipment like refrigerators, complex equipment and computer systems Continual monitoring of blood bank refrigerators extremely important in both blood centers and transfusion services
Supplier issues
The Food and Drug Administration licenses blood bank reagents, antisera, reagent cells, other commercial additives. Specific criteria is set for both the specificity and the potency of the reagents. For example, anti-A will only react with A cells and will demonstrate a 3-4+ reaction with A 1 reagent cells. Once the reagents have met the FDA criteria for specificity and potency, a license number is assigned. Along with the license number and lot number an expiration date is also placed on the labeled. Other than very rare antisera, routine blood bank reagents CANNOT be used after the expiration date. Daily quality control testing needs to be done for ABO, Rh, and Antibody Screening. Typing antisera for other red cell antigens will be tested when performing the antigen testing on the patient and donors since this test is not done each day.
Each manufacturer is required to provide a product insert for each reagent. The product insert needs to include the following:
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Reagent's description Proper use procedures What to expect in regards to performance Limitations
When a new shipment of reagents is received, the product insert needs to be reviewed and any changes in the standard operating procedure needs to incorporated into the lab's procedure before the reagents are used by the laboratory. Total compliance with the manufacturer's directions must be followed. According to AABB, the following criteria and documentation must be in place in the individual laboratory.
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List of critical supplies and services Clearly defined requirements Evaluation of suppliers qualifications to meet requirements Included: requirements for manufacturer mechanisms to notify facility of changes Prior to use of incoming supplies they need to be tested. Determination needs to be made relating to whether they are satisfactory for intended use. Documentation of package, storage and transportation Documentation of testing needs to done by facility before being put into use and prior to each use for reagents related to ABO, Rh, antibody screening and infectious diseases before being used for patient or donor testing. (See attached worksheet for daily QC of blood bank reagents.)
Process control, final inspection, and handling elements

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Process control includes Development of SOP Control of changes in policies, processes or procedures Acceptance testing to new/revised software involved in blood bank procedures Validation of new policies, processes or procedures Monitoring and control of p roduction processes Participation in proficiency testing appropriate for each testing system in place Established QC procedures for supplies and equipment Supplier qualifications and product specification need to be in place Control processes for nonconforming blood and blood components and products.
Documents and records (4 levels)

Documents and records have 4 levels 1. 2. 3. 4. Policies (Level 1) relate to "What to do" in response to various situations Processes (Level 2) relate to "How it happens" Procedures (Level 3) "How to do it" Forms/Records, Supporting Documents etc. (Level 4) that need to be completed when you are performing the procedures and following the processes and policies.
Incidents, errors, and accidents

In order to continuous imp rove methods need to be in place to detect incidents, errors, and accidents. Therefore the follow should be in place:
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Process to capture incidents, errors etc. If incident occurs, the severity of the incident is determined by the facility If it is a one-time incident: "What is the likelihood it will happen again?" and what to do about it if it could happen again If there are multiple similar incidents "What might be the root cause?" Develop processes for continuous improvement to help eliminate both onetime incidents and multiple similar incidents.
Assessments: internal and external

A Quality Assessment Program includes both internal and external assessment:
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Internal assessment includes blood usage review committees within a hospital (transfusion audits) or institutional QA teams External assessments includes inspections, surveys, proficiency surveys performed by agencies like the FDA, AABB, and CAP
Process improvement
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Corrective actions that are educational not punitive Timely corrections Yearly reports relating to QA and CQI committees
Facilities and safety

Although both transfusion services and blood centers are primarily concern with safe transfusions and related issues, there also needs to be processes in place related to employee safety therefore the following needs should be met:
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Compliance with OSHA requirements: chemical and biologic Disaster preparedness Adequate space and ventilation Adequate sanitation and water systems etc. Evaluations of limitations of physical structure prior to implementation of new equipment or processes
OBJECTIVES - Quality Assurance in Blood Banking

1. Identify the agencies having requirements for quality assurance in Blood Centers, Transfusion Centers and Blood Banks. 2. Explain the value of the following a) SOP's b) Personnel Policies c) Standard Processes and their improvement d) Equipment and supplies contracting, validation etc.
LIFE'
lass t s
LOOD
Tabl f t t
ANTIG
IN TESTING
The anti l bulin test, hi h is also eferred to as the anti human lobulin test A or the oombs test, is the ornerstone of detecting clinicall significant unexpected antibodies that have coated cells either in vivo or in vitro. or a historical perspective, see "The Discovery of the Anti-Globulin Test" written by A. E. Mourant pages 180 to 18 Vox Sang. 45: 180-8 (198 )
P i
i l
Anti l
lin Test
Red cells coated ith complement or IgG antibodies do not agglutinate directl hen centrifuged. These cells are said to be sensiti ed ith IgG or complement.
IgG-coated red blood cells
Complement-coated red blood cells
In order for agglutination to occur an additional antibody, hich reacts ith the c portion of the IgG antibody, or ith the C3b or C3d component of complement, must be added to the system. This ill form a bridge" bet een the antibodies or complement coating the r ed cells, causing agglutination.
The light-colored antibody molecule represents the anti globulin reagent that binds ith the c portion of the IgG antibody attached to the red blood cells.
The light-colored antibody molecule represents the antiglobulin reagent that binds with the complement attached to the red blood cells.
Traditionally rabbits were immunized with human gamma globulin to make this antibody to IgG or C3d.
Types of Antiglobulin Tests

The original work done by Coombs and Mo urant was detecting those antibodies, especially in the Rh system, that would cause hemolytic disease of the newborn, which we now classify as the Indirect Antiglobulin Test. There are two types of antiglobulin tests:
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Direct Antiglobulin Test (DAT) - Detects antibodies or complement coating patient's cells in vivo. Indirect Antiglobulin Test (IAT) - Uses a 37oC incubation step so antibodies in serum can react with antigens on cells in vitro, After washing the cells antiglobulin reagent is used to detec t antibody coating of cells.
Reagents
Production Methods of Anti-Human globulin (AHG or Coombs) Reagent
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May be made by injecting rabbits with purified human IgG or C3, then harvesting the antibodies produced by the rabbit. Monoclonal technology may be used to make monoclonal antiglobulin reagent
Specificity types
Polyspecific Anti-human Globulin: blend of Anti -IgG & Anti-C3b, -C3d Monospecific reagents: Anti-IgG alone or Anti-C3b,-C3d alone Note: Reagent does not contain antibodies to IgM. Information about IgM coating of cells comes from the presence of C3 coating the cells since IgM is a strong complement activator.
Interpretation of Antiglobulin Tests

Whether the cells have been coated, or sensitized, in vivo or in vitro the final interpretation is based on the following
P siti e Anti l
lin Test
(Wash Bottle Image)

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ash cells three times to remove unbound antibody nly antibody attached to the cells remain
Add Anti-Human Globulin

(Anti-globulin Image)
Visible Agglutination in the test tube: Grade the reaction strength
Summary of the reaction:

Antigen-antibody reaction, which can take place either in vivo or in vitro Cells coated with IgG antibody and/or complement Cells washed 3-4X to remove unbound or free antibody or complement The only antibody or complement left is attached to red cells AHG (Coombs serum) added Antibodies in Coombs serum react with antibodies or complement on red cells, causing agglutination 7. If no agglutination add Coombs control reagent cells* (CCC).
1. 2. 3. 4. 5. 6.
*Coombs control reagent cells will be discussed under False Negative Reactions.
Negative Antiglobulin Test
Antibodies are not attached to the antigens during incubation.
Wash the cells 3 times to remove any unattached antibodies.

Add Anti-human globulin
No visible agglutination and therefore a negative test
Add Coombs Control Check Cells
Check cells agglutinated and original test cells remain unagglutinated.
Coombs Control Agglutinated by Anti-Human Globulin

Coombs Control Check Cells tell you if you did the test properly when you have a negative test.
1. NO antigen-antibody reaction occurred. 2. No attachment of antibody or complement to red cells 3. Cells washed three to four times = all plasma or serum antibodies was washed away. 4. Anti-human globulin, Coombs, serum added, which would react with antibody -coated cells if present. 5. But no agglutination, because no antibodies or complement on red cells for the anti human globulin, Coombs, serum to react with 6. Must add Coombs Control Check Cells to negative reactions
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CCC are cells coated with IgG antibody Will react with antibodies in Coombs serum still "floating around" in the tube. Agglutination will now result Agglutination following addition of CCC verifies negative result
False Positives and Negatives

False-Negative Reactions
False-negative reactions can occur whe n antigen-antibody reactions have occurred but WASHING IS INADEQUATE and free antibody remains when the anti -human globulin is added.
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Anti-human globulin (Coombs) antibody prefers to react first with free antibody and then with antibody -coated cells If the free antibody has already reacted with the anti -human globulin, no free Coombs serum to react with Coombs Control Check Cells (CCC)
False negatives that are detected by negative Coombs control cells includes
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inadequate cell washing delay in adding an tiglobulin reagent after the washing step presence of small fibrin clots among the cells inactive, or forgotten, antiglobulin reagent
Inadequate cell washing will lead to unbound antibody remaining in the red cell suspension that are available to neutralize the AHG (Coombs serum) so it will not react with red cells bound with antibody. Delay in adding Coombs serum after washing step will lead to antibody eluting off, detaching from, cell while cells are sitting in saline. Now free antibody present in the saline neutralizes the AHG, Coombs, serum so it will not be able to react with the cells bound with antibody.
Small fibrin clot among the cells that were not washed away will have immunoglobulins and complement present. The antibodies and compleme nt in the fibrin clot neutralizes AHG, Coombs, serum leading to a negative test. Inactive AHG (Coombs serum) or the failure to add AHG (Coombs serum) will also be detected by a negative reaction when adding Coombs Control Check Cells. There are also false negatives NOT detected by negative Coombs Control Cells that include:
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Too heavy cell suspension Delay during cell washing procedure, which can lead to antibody eluting off cells while they are sitting in saline and then the antibody is washed away during the remaining washes Improper centrifugation can either lead to lost of cells during the washing or the need to shake too hard during resuspension.
False positives
False positive reactions can also occurred when performing this test. These would not be detected by the use of Coombs Control Check Cells. Reasons for a false positive reaction could be the following:
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Using improper sample (clotted cells instead of EDTA for Direct Antiglobulin Test, DAT) Spontaneous agglutination (cells heavily coated with IgM) Non-specific agglutination ("sticky cells")
All of these reactions would be the result of cells appearing to agglutinate, or actually agglutinating. Using a clotted tube for the DAT may allow complement to become activated in the test tube since ca lcium ions are free to be part of the complement cascade.
Direct Antiglobulin Testing

Principle
The Direct Antiglobulin Test detects in vivo coating of patient cells - either IgG antibodies, complement, or both. Within the patient's blood stream antibodi es attach to their specific antigens on the red blood cells. This happens in Hemolytic Disease of the Newborn (HDN), in transfusion reactions, and in autoimmune hemolytic anemia. Certain drugs are also known to activate complement and it can also coat the cells in vivo. When the blood is drawn the antibodies and/or complement have already attached to the red cells. Those red cells from the EDTA tube will be washed 3 or more times and a 3% cell suspension is made. A drop of cell suspension and the anti -human globulin are mixed in a tube and then centrifuged. If agglutination occurs, it indicates
the patient has a positive Direct Antiglobulin Test due to antibody coating the cells in vivo. If IgM antibodies involved, DAT will be identified by complement binding since the polyspecific antisera has both anti-IgG and anti-C3. The meaning of a positive DAT is found under Clinical Causes of a Positive DAT.
Technique
1. Add 1 drop of patient cells from EDTA tube to tube 2. Wash these drops of blood 3 -4X to remove plasma antibodies and make a 3% cell suspension. 3. Add a drop of 3% cell suspension to a clean, labeled tube. 4. Add drop of Polyspecific AHG (C oombs serum) to the tube. 5. If test is positive with polyspecific reagent, set up again using monospecific reagents to see if it is antibody or complement or both coating the cells. 6. We want to make the test as sensitive as possible, so allow all negatives to incubate 5 minutes to enhance complement coating. 7. Read all negatives microscopically to detect weak coating. 8. False pos. possible if red top tube used to collect sample.
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In-vitro complement coating frequently happens when sample clots or cools down due to weak cold-acting auto-antibodies like anti -I Prevent by using lavender top tube to tie up Ca+ and Mg+ ions and prevent complement activation in vitro.
8. Whenever positive DAT is obtained, obtain the following information on the patient:
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Diagnosis (particularly autoimmune hemolytic anemia, hemolytic disease of the newborn and transfusion reactions) Medications Recent transfusion history of both red cell and plasma components Other lab values that may indicate red cell destruction (hematocrit, bil irubin, LDH)
Clinical Causes of Positive DAT

1. Normal patient with unexplainable reasons for a positive DAT 2. Transfusion reaction work -ups require that a DAT be performed on the post transfusion specimen since the patient's antibodies and/or complement ma y coat the transfused donor cells. These reactions are usually a weak positive or mixed field agglutination since you are testing a mixed population of patient and donor cells. 3. Warm-acting Autoimmune disease, can lead to patient antibodies coating their own cells. This results in a strong positive result. A cold-acting autoimmune hemolytic anemia would be due to IgM antibodies that in turn activate complement. The complement-coated cells would then be detected by the antiglobulin reagent. 4. Hemolytic disease of the newborn is due to the mother's IgG antibodies crossing the placenta and coating the antigens on the fetal red blood cells. Cord blood collected at the time of birth would be tested, but may need to followed up by a heel stick of EDTA blood. The reaction is usually a strong positive. 5. Complement on the red cells may be the result of antigen -antibody reactions which may t involve red cells. Complement can also be activated if immune complexes are
present in the plasma and the activated compl ement attaches to the red cells. Complement can also become activated by the C3 by -pass mechanism and the lectin activation process. Again once the activation of complement occurs in the blood stream, it can become attached to the red cells.
6. Passive transfer of antibody from donor units of plasma or platelets may attach to the patient's red cells since recipients are given ABO compatible blood but other unexpected red cell antibodies may not have been detected. These antibodies in donor plasma can co at antigens on patient cells when group AB, A, or B receive group O plasma products (and possibly platelets) 7. ABO mismatched transplants of particularly bone marrow can occur if an universal "O" donor bone marrow is given to an A, B, or AB recipient. "Passenger lymphocytes" from group O donor organ make antibody to group AB, A, or B recipient cells and these in turn can activate complement. It is also more common for "O" individuals to make an IgG anti -A,B, which would also contribute to a positive DAT. 8. Sensitization of red cells due to medications like penicillin and cephalosporins that usually involves non -specific coating of red cells. Other drugs like tetracyclines, antihistamines and sulphonamides cause the development of immune complexes that are capable of activating complement. Some drugs, like ibuproten, levodopa and methyldopa, are also known to cause autoimmunity. If a patient has a positive DAT, drug-induced probl ems should be considered.
Indirect Antiglobulin Testing

The indirect antiglobulin test is one of the most important and commonly used techniques in immunohematology. It is used to commonly for the detection of:
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Weak D's in donor bloods and pregnant females of individuals who type D ( -) at room temperature when doing ABO and Rh typing. The presence or absence of antigens on a person cells from particularly the Kell, Kidd, and Duffy Blood Group systems. Unexpected, clinically significant antibodies i n the patient's serum during the antibody screening procedure and the antibody identification procedure..
Principle
The purpose of the indirect antiglobulin test is to detect In vitro sensitization of red cells. This is done when sensitization does not l ead to direct agglutination. This occurs when there are too few antigens on the red cell, too few antibodies in the serum and those antibodies are in the IgG class.
Summary of the Indirect Antiglobulin Technique
1. Incubate cells with serum at 37 oC for the recommended time. (Usually 15 to 30 minutes.) 2. After incubation wash the cells three to four times. 3. Add AHG, Coombs reagent, centrifuge and read for agglutination. 4. If the test is negative, add Coombs Control Check Cells to check for false negatives.
Uses:
Screening Serum for Unexpected Antibodies Procedure
1. Involves patient serum plus reagent red cells (Screening Cells) Duet I and II (attach photo of screening cells)
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The patient's serum potentially has unknown antibody. Screening Cells have known antigens for the common clinically significant antibodies. (attach screening cell sheet)
2. If there is agglutination after Coombs step with either (or both ) Screening Cells, patient has an unexpected antibody. 3. If antibody screen positive, must do additional tests to specifically identify antibody
The uses for antibody screen are:

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Testing donor plasma to make sure no unexpected antibodies will be transfus ed to the recipient. Testing recipient serum before transfusion to make sure patient has no unexpected antibodies to react with donor cells. Testing maternal serum to make sure pregnant mother has no antibodies to react with fetal cells causing hemolytic disease of the newborn.
Red Cell Antigen Typing
Red cell antigen typing involves patient cells plus reagent antiserum. The patient's cells are the unknown antigen and the reagent antiserum is the known antibody. The antiglobulin technique is used for a ntigen typing for a weak D and a number of other clinically significant antibodies like the Kell, Kidd, and Duffy antibodies. If there is agglutination after the addition of anti -human globulin, or Coombs step, patient cells had that specific antigen. The specific procedure varies depending on what antigen is being tested for, and what brand of antiserum is being used. Remember you must always read and follow directions in product insert carefully
Uses for red cell antigen typing are:

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Typing donors for antigen if patient has antibody. You would want units that are negative for that antigen. Verifying that patient is negative for antigen if he/she has made the antibody. Typing patient to see what antigens he/she lacks so can predict what antibodies he/she is capable of making if they seem to be particularly likely to make additional antibodies.
Controls
When performing red cell antigen testing al ays run nown positive and negative controls. This will verify that antiserum is acting properly and hel ps you interpret your test results. The positive control should be heterozygous for the antigen to ensure antiserum is capable of detecting weaker antigens. or example, when performing antigen typing for K, you would want a cell that is K and k+.
P SITIVE CONTROL NEGATIVE CONTROL PATIENT CONTROL Heterozygous Positive Cells without Antigen cells Patient Cells
PATIENT TEST Patient cells
+
Reagent Antiserum
+
Reagent Antiserum
+
Rh control
+
Reagent. Antiserum May be Positive Negative or Mixed ield +)
Should be at least +
Should be Negative
Should be Negative
OBJE TIVES ANTIG OBULIN TESTING

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Explain the principle of the antiglobulin test. Explain the difference between the direct antiglobulin testand the indirect antiglobulin test. 3 Explain how Coombs reagents are produced. 4 Explain the role of Coombs control cells in antiglobulin testing. 5 Explain the mode of action of the Coombs control cells. 6 Name five reasons for a false negative antiglobulintest. 7 Name two reasons for a false positive antiglobulin test 8 Explain why a lavender top tube is best for AT testing. 9. escribe the differences in the procedure between testing for IgG on the cells and testing for complement on the cells. 10. Name two reasons for a false positive AT, and explain why each would produce a false positive result.
11. List six clinical situations in which the DAT would be positive, and explain why each would cause the positive DAT 12. List the patient information that should be o btained if a positive DAT result is obtained on his or her sample 13. Describe the basic procedure for indirect antiglobulin testing 14. List three applications of the indirect antiglobulin test to detect red cell antigens 15. List three applications of the indirec t antiglobulin test to detect serum antibodies 16. State the controls used in antigen typing and explain the purpose of each
1. 2. 3. 4. 5. 6.
Correctly perform and interpret the DAT, using good washing technique. Use monospecific reagents appropriate ly, and correctly interpret results. Use Coombs control cells appropriately. Correctly perform and interpret serum antibody screens. Correctly perform and interpret red cell antigen typings. Correctly select and use controls when performing red cell antigen typing.
Table of Contents
LIFE'S BLOOD
GENETICS IN BLOOD BANKING
Mendelian Inheritance and Significance Terms
Basic Principles: 1. Each parent contributes 1/2 of the genetic information. 2. The genetic information is contained on chromosomes composed of DNA 3. Humans have 23 pairs of chromosomes a. 22 matched (autosomal) chromosomes and b. 1pair of sex chromosomes (females have 2 X chromosomes and males a X and a Y chromosome). Examples of Chromosome locations for common Blood Groups are as follows: System ABO MNSsU P Rh Kell Lewis Duffy Kidd Xg Common Genes A, B, O M, N, S,s,U P1 D, C, E, c, e K, k, Kp a, Jsa,Kpb, Js b Le, le a, Fy Fyb, Fy3 Jka, Jkb Xg a Located on Chromosom 9 4 22 1 7 19 1 18 X
4. Genes are the units of inheritance within the chromosomes. 5. At each location, or loci, on the chromosomes there are possibilities of different forms of the genes, these different forms are called alleles . (For example the ABO Blood Group System, there are A 1, A2, B, and O as common alleles. or allelic genes) 6. When the inherited alleles are the same the person is homozygous such as OO, when the individual inherits 2 different alleles such as AO, they are heterozygous for both the A and O genes. 7. On occasion we will see examples of dosage where some antibodies will react more strongly with homozygous cells than with heterozygous cells. For example, an anti-E that reacts as a 3+ with EE cells and only 1+ with Ee cells. 8. A Punnett Square is used to determine the inheritance possibilities for a particular mating. For example if the mother's genotype (genes) are AO and the father's genotype (genes) are BO, you would have the following Punnet
square possibilities. In this example there three heterozygous possibilities AB, AO, and BO and one homozygous possibility OO Dad
Mom
A O
AB BO
AO OO
9. In the above Punnett Square, the AB genotype will have both A and B antigens, therefore the phenotype is AB since both are expressed. AO and BO genotypes will demonstrate only the A and the B antigens respectively and therefore the phenotypes are A and B respectively. The individual that is OO will have the O phenotype. 10. A and B genes are dominant, or c o-dominant, and the O gene is recessive. The dominant genes will be expressed if present. Recessive genes will only be expressed if they are homozygous. 11. Most Blood Group genes are co-dominant and therefore will be expressed if present.
Mitosis and Meiosis

Two kinds of cell division: Mitosis is cell division that leads to two identical cells that has the same number of paired chromosomes. (In humans there are 23 pairs or 46 chromosomes) Meiosis is the cell division that occurs when gametes (sperm and eggs) are formed and will not have pairs of chromosomes. (In humans there will be 23 chromosomes in the sperm that will match up with the 23 chromosomes in the egg when fertilization occurs to form the gametocyte.). The sex of the child is determined by the X and Y chromosomes. Males provide either X or Y chromosome and females provide only provide X chromosomes. Genes that are found only on the X chromosome are said to be sex-linked. Genes found on the other 22 pairs of chromosomes are autosomal.
Genotypes, Phenotypes, Amorphs, and Pedigree Charts

Here is a pedigree chart for three generations. The ABO phenotypes are listed for the known blood types.
y The mother in the first generation has the AB genes since her phenotype is AB. In the mating for the second generation, the genotype for the father could either be BB or BO since his father's phenotype is unknown. It would appear the mother is AA since both her parents are A, but..... Look at their children's blood types. What is the mother's genotype now since both children are B?
There are no O individuals in the above example but O is considered an amorph since it has no detectible traits. The lack of D antigen is considered another example of an amorph since no reaction with anti -D indicates the individual is D negative (Rh negative). These two examples are recessive genes that need to be homozygous for it to be demonstrated.
Other Concepts Relating to Blood Group Genetics

Contributions of Blood Genetics to the Field of Human Genetics
Certain characteristics that make Blood Genetics useful for the field of human genetics 1. 2. 3. 4. Simple and unquestionable pattern of inheritance Can test or determine the phenotypes readily More than 1 allele occurring fairly frequently Environment does not affect the expression of the genes.
Some discoveries that were found in blood genetics:

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Multiple alleles seen in ABO system Linkage between the secretor genes with the Lutheran genes on the same chromosome
Population Genetics Linkage

Linkage between the secretor genes with the Lutheran genes on the same chromosome was already noted. 1. We now know that the D gene is closely linked to the Cc and Ee genes. The most frequently inherited Rh positive set of genes is CDe and the most frequent Rh negative gene is cde or ce since d is an amorph. 2. The MNSs genes are also linked, MS, NS, Ms, Ns leading to a difference betwee n the expected frequency and the observed frequency.
Expected frequency MS = 0.53 (M) X 0.33 (S) = 0.17 Ms = 0.53 (M) X 0.67 (s) = 0.36 NS = 0.47 (N) X 0.33 (S) = 0.16 Ns = 0.47 (N) X 0.67 (S) = 0.31 Observed frequency 0.24 0.28 0.08 0.39
Silent Genes
As indicated already there are some amorph blood group genes that exist and lead to none expression of a blood antigen. The following are some examples of silent genes.
Blood Group Gene h = r Ko Lu Jk Fy Blood Group System ABO Homozygous Phenotype Oh or Bombay
Rh Kell Lutheran Kidd Duffy
Rhnull Knull Lu(a-b-) Jk(a-b-) Fy(a-b-)
Blood Group Nomenclature

Accepted terminology according to AABB Technical Manual - 50th Anniversary 1953 -2003, 14th edition, 2003, p221. 1. Genes encoding the expression of blood group antigens are written in italics (or underlined if italics are not available). If the antigen name includes a subscript (A 1), the encoding gene is expressed with a superscript ( A1) 2. Antigen names designated by a superscript or a number (eg, Fy a, Fy:1) are written in normal (Roman)script....Superscript letters are lowercase.... 3. When antigen phenotypes are expressed using single letter designation, results are usually written as + or -, set on the same line as the letter(s) of the antigen: K+ k-. 4. To express phenotypes of antigens designated with a superscript letter, that letter is placed in parentheses on the same line as the symbol defining the antigen: Fy(a+) and Fy( -). 5. For antigens designated by numbers, the symbol defining th e system is notated in capital letters followed by a colon, followed by the number representing the antigen tested. Plus signs do no appear when test results are positive (K:1), but a minus sign is placed before negative test results: K:1, K:-1. If tests for several antigens in one blood group have been done, the phenotypes is designated by the letter(s) of the locus or blood group system followed by a colon, followed by antigen numbers separated by commas: K: 1, 2, -3, 4. Only antigens tested are liste d;...
Table 10-4. Examples of Correct and Incorrect Terminology

(AABB Technical Manual, p.222
Term Description Phenotype Phenotype Antibody Antigen Antibody
Correct Terminology
Incorrect Terminology
Fy(a+) Fy(a+b-) Anti-Fya K anti-k K:1, K:-1 A Rh+, B RhM+NRh:-1, -2, -3, 4,5
Phenotype Phenotypes Phenotype Phenotype
Fya+, Fy(a+), Fya(+), Fya+, Fya(+), Duffya+ Fya+b-, Fy(a+b-), Fya(+)b(-), Fya(+)b(-) Anti Fya, Anti-Duffy Kell (name of system) Anti-Cellano k1+, K:1+, K(1), K:(1), K1-, K:1-, K1negative A+ (means positive for A antigen) B- (means negative for B antigen) M(+), MM (unproven genotypes) Rh: -1, -2, -3, +4, +5, Rh: 1-,2-,3-, 4+,5+
Public versus Private Genes

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Public Genes are found in most of the population. In the Kell Blood Group System, the Kp b is found in close to 100% of the population Genes that are very rare are referred to private genes . Kp a is very rarely found (2.3% in whites and almost never in African Americans) and therefore close to being a private gene.
Paternity Testing
Today most paternity testing is done using the following technology:
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Red Cell Testing for the following Blood Group System: ABO, MNSs, Rh, Duffy, Kidd, Kell, White Cell Testing using HLA antigens DNA testing
1. Can a mother who types A and the alleged father who types O have a child who types B? 2. Can a mother who types CC and the alleged father who types cc have a child who types CC?
OBJECTIVES - Genetics in Blood Banking

1. Describe the importance of blood group genetics as it relates to the overall field of genetics. 2. Demonstrate the basics of inheritance of blood group traits relating to chromosomes, dominant and recessive genes, alleles, genotypes, phenotypes, heterozygous and homozygous inheritance, autosomal and sex linked inheritance. 3. Relate DNA and RNA roles in inheritance. 4. Identify what are inheritance patterns and pedigree charts. 5. Distinguish between mitosis and meiosis. 6. Explain the inheritance of dominant versus recessive versus codominant traits. 7. Differentiate between pheno types and genotypes. 8. Identify the role of population genetics in calculating gene frequencies. 9. Explain crossing over and linkage. 10. Differentiate between public and private genes. 11. Explain the use of blood group genes as genetic markers.
LIFE'S BLOOD
by M. Schroeder and M. Jensen
ABO BLOOD GROUP SYSTEM

ANTIGENS AND ANTIBODIES
Definition:
Blood group system A series of antigens exhibiting similar serological and physiological characteristics, and inherited according to a specific pattern.
Importance of the ABO system:

Most important (clinically significant) Blood Group System for transfu sion practice Why?
This is the only blood group system in which antibodies are consistently, predictably, and naturally present in the serum of people who lack the antigen. Therefore ABO compatibility between donor and recipient is crucial since these s trong, naturally occurring A and B antibodies are IgM and can readily activate complement and cause agglutination. If ABO antibodies react with antigens in vivo, result is acute hemolysis and possibly death.
Indications for ABO grouping:

ABO grouping is required for all of the following individuals:
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Blood Donors-since it can be life threatening to give the wrong ABO group to the patient. Transfusion recipients -since we need to know the donor blood is ABO compatible. Transplant Candidates and Donors -ABO antigens are found in other tissues as well. Therefore the transplant candidates and donors must be compatible. Prenatal Patients -To determine whether the mothers may have babies who are
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suffering from ABO-HDN. It is also beneficial to know the ABO group should she start hemorrhaging. Newborns (sometimes) If the baby is demonstrating symptoms of Hemolytic Disease of the Newborn, the ABO group needs to be determined along with Rh and others. Paternity testing Since the inheritance of the ABO Blood Group System is very specific, this serves as one of the first methods to determine the likelihood that the accused father is the father or not.
Discovery of the ABO system:

In 1900 Karl Landsteiner reported a seri es of tests, which identified the ABO Blood Group System. In 1910 he won Nobel prize for medicine for this discovery. He mixed the serum and cells of all the researchers in his lab and found four different patterns of agglutination. From those studies he developed what we now know as Landsteiner's rules for the ABO Blood Group:
1. A person does not have antibody to his own antigens 2. Each person has antibody to the antigen he lacks (only in the ABO system) 3. Below are the four blood groups and the antigens a nd the expected, naturally occurring antibodies present.
BLOOD GROUP A B AB O ANTIGEN A B A and B neither anti -A or anti-B ANTIBODY anti-B anti-A neither anti-A,B
Incidence (%) of ABO Blood Groups in the US Population

ABO Group O A B AB Whites 45 40 11 4 Blacks 49 27 20 4
ABO Typing
ABO typing involves both antigen typing and antibody detection. The antigen typing is referred to as the forward typing and the antibody detection is the reverse typing
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The forward typing determines antigens on patient's or donor's cells a. Cells are tested with the antisera reagents anti -A, anti-B, (and in the case of donor cells anti-A,B) b. Reagents are either made from hyperimmunized human sources, or monoclonal
antibodies. c. One advantages of the monoclonal antibodies are the antibody strength. d. Another advantage of monoclonals: human source reagents can transmit infectious disease (hepatitis). Reverse typing determines antibodies in patient's or donor's seru m or plasma a. Serum tested with reagent A 1 cells and B cells b. Reverse grouping is also known as backtyping or serum confirmation
Routine ABO Typing

Reaction of Cells Tested With
Anti-A 0 + 0 + Anti-B 0 0 + + Red Cell ABO Group
Reaction of Serum Tested Against

A 1 Cells B Cells + + 0 0
Reverse ABO Group
O A
B AB
+ 0 + 0
O A
B AB
Discrepancies in ABO typing

1. Results of forward and reverse typing must agree before reporting out blood type as seen in the about table. 2. If forward and reverse do not agree, must identify cause of discrepancy. 3. If cannot resolve discrepancy, must report out blood type as UNKNOWN and give group O blood
Characteristics of ABO antigens:

ABO antigens are glycolipid in nature, meaning they are oligosaccharides attached directly to lipids on red cell membrane. These antigens stick out from red cell membrane and there are many antigen sites per red blood cell (approximately 800,000) Besides their presence on red blood cells, soluble antigens can be present in plasma, saliva, and other secretions. These antigens are also expressed on tissues other than red cells. This last fact is important to consider in organ transplantation. ABO antigens are only moderately well developed at birth. Therefore ABO-HDN not as severe as other kinds of Hemolytic Disease of the Newborn. .
Characteristics of ABO antibodies:

1. These are expected naturally occurring antibodies that occur without exposure to red cells containing the antigen. (There is some eviden ce that similar antigens found in certain bacteria, like E.coli , stimulate antibody production in individuals who lack the
specific A and B antigens.) 2. Immunoglobulin M antibodies, predominantly 3. They react in saline and readily agglutinate. Due to the pos ition of the antigen and the IgM antibodies it is not necessary to overcome the zeta potential. 4. Their optimum temperature is less than 30 oC, but reactions do take place at body temperature 5. Not only are these antibodies expected and naturally occurring, t hey are also commonly present in high titer, 1/128 or 1/256. 6. They are absent at birth and start to appear around 3 -6 months as result of stimulus by bacterial polysaccharides. (For this reason, newborn blood is only forward typed.)
ABO INHERITANCE
Inheritance Terminology:
gene: determines specific inherited trait (ex. blood type) chromosome: unit of inheritance. Carries genes. 23 pairs of chromosomes per person, carrying many genes. One chromosome inherited from mother, one from father locus: site on chromosome where specific gene is located allele: alternate choice of genes at a locus (ex. A or B; C or c, Lewis a or Lewis b) homozygous: alleles are the same for any given trait on both chromosome (ex. A/A) heterozygous: alleles for a give n trait are different on each chromosome (ex. A/B or A/O) phenotype: observed inherited trait (ex. group A or Rh positive) genotype: actual genetic information for a trait carried on each chromosome (ex. O/O or A/O) dominant: the expressed characteri stic on one chromosome takes precedence over the characteristic determined on the other chromosome (ex. A/O types as A) co-dominant:
the characteristics determined by the genes on both chromosomes are both expressed - neither is dominant over the other ( ex. A/B types as AB) recessive: the characteristic determined by the allele will only be expressed if the same allele is on the other chromosome also (ex. can type as O only when genotype is O/O)
ABO Genes
The A and B genes found on chromosome #9. We inherit one gene (allele) from our father and one from our mother. The two co-dominant alleles are A or B. Anytime an individual inherits an A or B gene it will be expressed. The O gene signifies lack of A or B antigens. It is not expressed unless this gene is inherited from both parents (OO). Therefore the O gene is recessive. Below is the example of two individuals who are A. One inherited only one A gene along with an O gene and is therefore heterozygous. The other inherited 2 A genes and is homozygous for A.
1 = A/A
1 = Homozygous A Phenotype A Genotype A/A Can Contribute Only an A Gene to Offspring
2 = A/O
2 = Heterozygous A Phenotype A Genotype A/0 Can Contribute A or O Gene to Offspring
Inheritance Patterns
We can't determine genotypes of A or B people unless family studies are done. Some basic rules of ABO inheritance are as follows:
1. A/A parent can only pass along A gene 2. A/O parent can pass along either A or O gene
3. 4. 5. 6.
B/B parent can only pass along B gene B/O parent can pass along either B or O gene O/O parent can only pass along O gene AB parent can pass along either A or B gene
ABO phenotypes and genotypes

1. Group A phenotype = A/A or A/O genotype 2. Group B phenotype = B/B or B/O genotype 3. Group O phenotype = O/O genotype 4. Group AB phenotype = A/B genotype
Offspring possibilities
Possibilities of an A/O mating with a B/O: (Children's genotypes in purple) Father's Genes Mother's Genes B A O AB BO
O AO OO
Possibilities of AA mating with BB: (Children's genotypes in purple) Father's Genes Mother's Genes B A A AB AB
B AB AB
Possibilities of an A/A mating with a B/O: (Children's genotypes in purple)
Father's Genes Mother's Genes

A A Possibilities of an A/A mating with an O/O: Father's Genes Mother's Genes O O B AB AB O AO AO
A A Possibilities of an A/O mating with an O/O:
AO AO
AO AO

A O Possibilities of an A/B mating with a O/O: O AO OO O AO OO

A B O AO BO O AO BO
BIOCHEMISTRY OF THE ABO SYSTEM

The ABO antigens are terminal sugars found at the end of long sugar chains (oligosaccharides) that are attached to lipids on the red cell membrane. The A and B antigens are the last sugar added to the chain. The "O" antigen is the lack of A or B antigens but it does have the most amount of next to last terminal sugar that is called the H antigen.
Production of A, B, and H antigens

The production of A, B and H antigens are controlled by the action of transferases. These transferases are enzymes that catalyze (or control) addition of specific sugars to the oligosaccharide chain. The H, A, or B genes each produce a different transferase, which adds a different specific sugar to the oligosaccharide
chain. To understand the process let's look at the sequence of events:

1. Precursor chain of sugars is formed most frequently as either Type 1 or Type 2 depending on the linkage site between the N -acetylglucosamine (G1cNAc) and Galactose (Gal).
2. H gene causes L-fucose to be added to the terminal sugar of precursor chain, producing H antigen (shown in this diagram of a Type 2 H antigen saccharide chaine).
3. Either A gene causes N-acetyl-galactosamine to be added to H substance, producing A antigen, (shown in this diagram) or
4. B gene causes D-galactose to be added to H substanc e, producing B antigen.
5. If both A and B genes present, some H -chains converted to A antigen, some converted to B antigen. 6. If H gene absent (extremely rare), no H substance can be formed, and therefore no A or B antigen. Result is Bombay blood group.
Bombay blood group:

The Bombay blood group lacks H gene and therefore cannot make H antigen (H substance). Since the H substance is the precursor for the A and B antigens, these antigens also are not made. The cells type as O and the serum has anti -A, anti-B, and anti-H since the individual lacks all of these antigens. Anti-H agglutinates O cells. The only cells Bombay individuals do not agglutinate are from other Bombay blood people since they lack the H antigen,
Subgroups of A and B
The subgroups of A and B are caused by decreased amounts of antigen on the red blood cells. They are inherited conditions. The most common are subgroups of A. Approximately 80% of the A's and AB's have a normal expression of A 1. Most of the other 20% are either A 2 or A2B. This subgroup has fewer H chains converted to A antigen result is more H chains on
red cell, and fewer A antigens. the individuals
A small percentage of
There are other, weaker subgroups of A exist: A 3; Aint; Am , Ax; Ael. Each has a different pattern of reacting with anti -A, anti-A, and various antibody-like substances called lectins.
Lectins
Lectins are extracts of seeds of plants that react specifically with certain antigens. The two most common lectins used in Blood Bank are:
y y
Ulex europaeus , or lectin H, which agglutinates cells that have H substance. Dolichos biflouros , or lectin A 1, which agglutinates cells with A 1.
Lectin-H reacts strongest with O cells, which has a high concentration of H antigen, and weakest with A 1 cells, which have a low concentration of H.
Lectin O cells A2 cells A2B cells B cells A1 cells weak to negative positive A1B cells weak to negative positive Bombay cells negative negative
lectin-H
4+
3+
negative
2-3+ negative
2+ negative
Lectin-A1 negative
Differentiating Subgroups of A:
Use the following steps to help differentiate the subgroups of A:
1. 2. 3. 4.
Use lectin-A 1 to differentiate A 1 cells from all others - will agglutinate only A 1 cells Look for weaker or mixed field reactions Look for anti -A 1 in serum (serum reacts with A 1 cells but not A 2 cells) Look at strength of reactions with anti -A,B or with lectin-H A1 4+ 4+ 4+ 0-w no A2 4+ 4+ 0 1-2+ may A3 mf mf 0 2+ may Ax 0 2+ 0 2-3+ often in serum
GROUP Reaction with anti -A Reaction with anti -A,B Reaction with Lectin -A1 Reaction with Lectin -H Presence of anti -A 1
Problems with Ax:

Because A x cells initially type as O and serum usually has anti -A1, (along with antiB), patient forwards and reverses as O. Unfortunately when A x is transfused into an O individual, the naturally occurring anti -A,B will react with the donor cells causing a transfusion reaction. Therefore: To prevent A x from being erroneously typed as O,
confirm all group O donors with anti -A,B.
OBJECTIVES ABO SYSTEM

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Explain why the ABO system is the most important for blood transfusion practice. List the situations in which an ABO type would be required. Describe 6 significant characteristics of ABO antigens. Describe 6 characteristics of ABO antibodies. Explain how the ABO system was discovered. State Landsteiner's rules. List the blood groups in the ABO system, the antigen(s) present on the e cell in each blood group and the antibody(ies) in the serum for each, for adults. State the differences in ABO antigens and antibodies in newborns. State which ABO groups could safely receive a red cell transfusion from donors of each of the following blood types: A, B, AB, O State which ABO groups could safely receive a plasma transf usion from donors of each of the following blood types: A, B, AB, O Explain how ABO blood types are determined. Explain what is meant by forward and reverse grouping, backtyping, and serum confirmation. Explain what an ABO discrepancy is, and what must be done if the discrepancy cannot be resolved State the incidence of each ABO blood group in the Caucasian population, and how the percentages differ in the Black and Oriental populations. Define each of the following and give an example of each within t he ABO system: a.gene b.chromosome c.locus d.allele e.homozygous f.heterozygous g.phenotype h.genotype i.dominant j.co-dominant k.recessive State the alleles in the ABO system. State which alleles are co -dominant State which allele is recessive For each of the following phenotypes, give the possible genotypes: a. A b. B c. AB d. O Predict all the possible phenotypes and genotypes from all blood type matings Describe the sequence of events in the synthesis of the ABO antigens, beginning with the precursor substance. State the sugars that are associated with each different blood group system Describe the significant characteristics of the Bombay blood group. Explain what lectins are. Predict the reactions o f each different blood group, including subgroups of A, with lectin-H. Explain what reactions demonstrate a subgroup of A.
16. 17. 18. 19.
20. 21. 22. 23. 24. 25. 26.
Table of Contents
LIFE'S BLOOD
CLASS NOTES
Rh SYSTEM
History
In 1939, Hemolytic Disease of the Newborn was first described by Levine and Stetson. The cause of hemolytic disease of the cause was not specifically identified but maternal antibody suspected. A year later (1940) Karl Landsteiner and Alexander Wiener injected animals with Rhesus monkey cells to produce an antibody which reacted with 85% of human red cells, which they named anti -Rh. Within a year Levine made connection between maternal antibody causing HDN and anti Rh. Between 1943-45 the other common antigens of the Rh system were identified. For many years the exact inheritance of the Rh factors were debated Weiner promoting Rh and hr terminology and Fisher -Race utilizing DCcEe for the various Rh antigens. In 1993, Tippett discovered true mode of Rh inheritance using molecular diagnostics
Rh Antigens
D (Rh o) is the most important antigen after A and B antigens. Unlike the anti-A and anti-B antibodies, anti-D antibodies are only seen if a patient lacking D antigen is exposed to D + cells. The exposure of D+ cells usually occurs through pregnancy or transfusion.
There are 5 principle antigens that may be found in most individuals. They are:
y y y y y y
D found in 85% of the population C found in 70% of the population E found in 30% of the population c found in 80% of the population e found in 98% of the population (d) which has never been identified but refers to the 15% of the population who has no D antigen
There are at over 50 Rh antigens that have been identified including those that are
either combinations of these antigens or weak expressions of t he above antigens, but most Rh problems are due to D, C, E, c or e.
Alleles:
The common alleles are:
y y
C and c are alleles with C w occasionally seen as a weaker expression of C. E and e are alleles although E is seen only a third as often as e. The e antigen is referred to as a high incidence antigen since it is found in 98% of the population. D and the lack of D (or d) are alleles.
Characteristics of Rh antigens
The Rh antigens together are proteins of 417 amino acids. These proteins cross the red cell membrane 12 times. There are only small loops of the protein on the exterior of the cell membrane.
Therefore the Rh antigens are not as available to react with their specific antibodies and there are fewer antigen sites than ABO. Unlike the ABO system the Rh antigens are not soluble and are not expressed on the tissues. They are well developed at birth and therefore can easily cause hemolytic disease of the newborn if the baby has a Rh antigen that the mother lacks. Besides the antigens being well-developed at birth, they are very good immunogens. This is especially true to D, which if the most immunogenic after A and B antigens.
Rh Antibodies
Unlike the ABO antibodies that are mainly IgM, the Rh antibodies are commonly IgG. They are NOT naturally occurring and therefore are formed by immune stimulus due to transfusions or baby's red blood cells during pregnancy. The most common antibody to form is anti-D in Rh negative individuals. Since Rh antibodies are IgG they bind best at 37 oC and their reactions will be observed with the indirect antiglobulin technique. Agglutination reactions are enhanced by high protein (albumin), low -ionic strength saline (LISS), proteolytic enzymes (ficin) and polytheylene glycol (PEG). Rh antibodies will react more strongly with homozygous cells than with heterozygous cells. For example, an anti-E will react with strongly with E+E+ cells and more weakly with E+e+ cells. This is called dosage.
Both Hemolytic Disease of the Newborn and Hemolytic Transfusion Reactions can occur due the various Rh antibodies. Anti -D has been the biggest concern since it was recognized in the 1940's as being the most common cause of hemolytic disease of the newborn. Since the D antigen is so immunogenic we screen all donor units for the D antigen. Therefore if an individual is A+, it means both the A and the D antigens are present. On the other hand, if an individual is A -, the A antigen is present and the D antigen is absent. To prevent problems due to anti -D:
y y
we try to always give Rh-negative individuals Rh -negative blood and we give Rh oimmune globulin to Rh -negative mothers to prevent the formation of anti-D during pregnancy.
The incidence of Rh antibodies

y y y y y
Anti-D most common antibody seen in Rh(D) negative people Anti-E most common antibody seen in Rh pos people since only 30% of the population have the antigen Anti-C or Anti-c less common - most people have the antigen Anti-e often seen as autoantibody and will make it difficult to find compatible blood since 98% of the population have the e antigen Anti-C,e or Anti-c,E often seen in combination. If a patient lacks both a C and e and has made an anti-C, then enhancement techniques should be done to make sure that an anti-e is not also present.
Rh System Inheritance
From the 1940's to the 1990's the mechanism for inheritance of the Rh Blood Group System was in question. The terminology that is part of the Fisher -Race Theory is most commonly used even today.
Fisher-Race Theory
The Fisher-Race theory involved the presence of 3 separate genes D, C, and E and their alleles c and e and the absence of D since an anti -d has never been found. These three genes are closely linked on the same chromosome and are inherited as a group of 3. The most common group of 3 genes inherited is CDe and ce (D negative) is the second most common.
Weiner Theory
Weiner believed there was one gene complex with a number of alleles resulting in the presence of various Rh antigens. According to Weiner there were 8 alleles, R o, R1, R2, Rz, r, r', r", ry , which ended up with different antigens on t he red cells that he called Rh o, rh', rh", hr', hr". Weiner terminology is not use as often today, but you will often see Rh o(D) when a person considered to be Rh -positive. At times the gene terms are easier to use than Fisher -Race. If a person has the Fisher-Race genotype
of DCe/DCe, it is easier to refer to that type as R 1R1 2. Made up of combinations of genetic products
Tippett Theory
In 1986, Tippett predicted that there are two closely -linked genes - RHD and RHCE. The RHD gene determines whether th e D antigen that spans the membrane is present. Caucasians who are D negative have no gene at that gene loci. In the Japanese, Chinese, and Blacks of African descent have an inactive or partial gene at this site. The RHCE gene determines C, c, E, e antig ens produced from the alleles:
y y y y
RHCe RHCE RHcE RHce
Rh Gene Complexes, Antigens, Possible Combinations and Percentages Haplotypes Genes Present Antigens Present Phenotype Percentage 1 R RHD RHCe D,C,e R1 42% r RHce dce r 37% 2 R RHD RHcE DcE R2 14% RHD RHce (more common in Ro Dce Ro 4% Blacks) r' RHCe dCe r' 2% r" RHcE dcE r" 1% Rz RHD RHCE DCE Rz <1% ry RHCE dCE ry <1%
Translating From Wiener To Fisher-Race

There are times when you will need to convert Weiner to Fisher-Race or vice versa. It will be easier to do these conversions if you remember the following: 1. 2. 3. 4. 5. 6. R always refers to D whether it is R o, R1, R2, or the very rare R z. r always refers to the lack of D o refers to having no C or E 1 or ' always refers to C 2 or " always refers to E The very rare haplotypes that have both a C and E are given letters from the end of the alphabet z and y.
Determining Genotypes From Phenotypes

The following steps will be helpful in determining from the individual's phenotype. These rules are based on probability so the least likely genotypes will
involve Rz or ry. 1. Type patient for the five Rh antigens: D, C, c, E, e 2. Start with D: is it positive or negative? 1. If negative, the individual will be homozygous d. 2. If positive for D, you can't tell yet whether the individual is homozygous or heterozygous for D. Therefore, put D on just one chromosome. 3. Look at C: is it positive or negative? 1. If negative, put c on each chromosome. 2. If positive, look at c result to determine if the C is homozygous or heterozygous. If there is no c present, there would be two C and it would be homozygous. If a c is present as well as C, they are heterozygous. 3. If homozygous, then put C on each chromosome. 4. If heterozygous, put C on same chromosome as D; put c on other. 4. Look at E: is it positive or negative? 1. If negative, put e on each chromosome. 2. If positive, look at e result to determine if homozygous or heterozygous. 3. If homozygous, put E on each chromosome. 4. If heterozygous, put E on same chromosome as the D unless the D already has a C; put e on other chromosome. DCe is more common than DcE and DCE is extremely rare. 5. Put C and E together on same chromosome only if no other possible combinations
Most Common Genotypes

The following geno types are listed as the most common with 1 being the most common in Whites and 7 the least common. Rz and ry are so rare they are not included in the following table. Incidence of the most common genotypes Genotype Incidence(%) Weiner DCE Whites Blacks Haplotypes DCe/ce R1r 31.1 8.8 1 o DCe/Dce RR 3.4 15 Dce/Ce Ror 0.2 1.8 DCe/DCe R1R1 17.6 2.9 DCe/Ce R1r' 1.7 0.7 ce/ce rr 15 7 1 2 DCe/DcE R R 11.8 3.7 DCe/cE R1r" 0.8 <0.1 DcE/Ce R2r' 0.6 0.4 DcE/ce R2r 10.4 5.7 2 o DcE/Dce RR 1.1 9.7 Dce/ce R or 3.0 22.9 Dce/Dce RoRo 0.2 19.4 2 2 DcE/DcE R R 2.0 1.3
Antigens Present 1 D, C, c, e
2 3 4
D, C, e ce DCcEe
5 6 7
DcEe Dce DcE
DcE/cE
R2r"
0.3
<<0.1
Applications of Rh genotyping
Paternity testing of the blood group antigens is based on a process of exclusion. Since the RHD and RHCE are closely linked and Ce, ce, cE are produced by a single gene, there are limited combinations that the father can provide. HDN predictability by testing the father's Rh genotype. This helps predict likelihood of HDN due to D when mom has anti-D. The most common Rh genotype of the father will indicate whether the baby has O%, 50%, or 100% probability of being D positive.
y y y
If the father is also D negative (ce/ce), the baby will be D negative as well and there is a 0% probability of the baby suffering from Rh o HDN. If the father's Rh genotype appears to be either, R 1r, R2r or Ror, the baby has a 50% probability of being D positive and suffering from Rh o HDN. On the other hand if a father's Rh genotype appears to be any of the following, R 1R1, R2R2, R1R2, RoRo, R1Ro, or R2Ro, the baby has a 100% probably of getting a D gene from his father and therefore being D positive and suffering from Rh o HDN.
Variants
Weak D (Du)
Weak D is a weakly expressed D antigen that will only be demonstrated after incubation at 35-37oC followed with antiglobulin testing. (ie being demonstrated only by Coombs technique). An Rh control must always be run along with the weak D test. Always consult the product insert to determine if Rh Control needs to be run when performing the immediate spin D testing. The following results could be obtained when performing the D testing:
Immediate 37oC Anti-D Spin Anti-D Rh Co Anti-D Rh Co + 0 0 0 0 0 0 0 0 0 AGT Anti-D Rh Co
Interpretation
+ 0 +
0 0 +
D positive Weak D True D negative Or any time the Rh control is positive, you can not interpret the results and need to perform further testing
Testing for Weak D

AABB requires that all donor blood that originally fails to react with anti -D at
immediate spin must be tested for weak D. Units that test weak D positive would be labeled D positive and would be transfused only to D positive individuals. Hospitals may or may not test all Rh negative recipients for weak D. The cost of time and reagents is minimized if only the immediate spin. This may create some confusion with the recipient if their donor card indicates they are Rh positive but they type Rh negative when they are the recipient. Recipients that type D negative at immediate spin would be given D negative blood, which not create a problem for the patient. When performing testing prenatal and postnatal mothers, D -negative blood at immediate spin would be te sted for weak D as well to determine if they are eligible for Rh o Immune Globulin. Since Rh o Immune Globulin is actually anti -D it is safe for a true D negative, but not for a weak D positive mother.
Why do weak D's exist?
There are three explanations for weak D's.

y
Quantitative Weak D There are individuals that quantitatively produce fewer D antigen sites. This is more common in Blacks and is often seen with the Dce haplotype. On rare occasions among Whites an unusual DCe or DcE may also produce a quantitatively decrease weak D. Position Effect Weak D In this case the D is weakened by the position of a C on the opposite haplotype which is called the trans position. The two Rh genotype combinations where this type of weak D is seen are: Dce/Ce and DcE/Ce. Today this type of weak D would type as a regular D due to the improvement of reagents. Partial D antigen (Mosaic D) It has been found that some D-positive individuals make an alloanti -D that reacts with other D positive cells but not their own. Many of these will demonstrate a weak D type of reaction. In this type of weak D, the individuals are lack some of the components of the D antigen and therefore are able to make allantibodies to those specific components if they are transfused with D pos itive blood.
Other Rh System Variants

There are presently 46 Rh antigens identified and named. The following are the most common of those variants
y
y y y y
Cw is a low frequency antigen found in approximately 2% of Whites and 1% of Blacks. It is not an allele of C and c. Its allele is MAR, which is found in 99.9% of the population. V and VS are low frequency alleles found in 1% or less of the Whites, but are more common in Blacks. V is found in 30% of the Blacks and VS in 32%. G is present when D or C present due to the present of serine at the 103 position of the Rh polypeptide. Anti-G will react with both D+ and C+ cells. f is present when c and e together on same chromosome: Dce or ce. This is the most common of what are called cis product antigens. Rhnull has no Rh antigens on their red cells but these individual can transmit
normal Rh antigens to their offspring. In the most common type the core Rh polypeptide is missing. A less common type has the regulator gene that turns off the expression of Rh. There have been at least 43 individuals in 14 families that are Rh null. In these individuals the red blood cell membrane is abnormal and some of these have been identified when it was observed that they had hemolytic anemia and abnormal red cell morpholo gy. If these individuals develop an Rh antibody following a transfusion or pregnancy, it is considered a anti-total Rh antibody.
Rh Typing
False Positives
False positive D's occur: 1. When following through to AGT for weak D and will be identified as false positive by a positive Rh control. This is seen when a patient/donor has strong positive DAT. The cells are coated with antibody (not necessarily Rh antibody) in vivo. Albumin is necessary in reagent Anti-D to overcome the zeta potential allowing cells coated with IgG Anti -D to get close enough together to agglutinate, but cells coated in vivo with any IgG antibody will also agglutinate in albumin. These false positives are corrected by using form of Anti-D that does not require albumin. There are two types of alternative types of anti -D:
y
Monoclonal (IgM) anti-D will cause agglutination of D positive cells without the presence of albumin at room temperature. A number of facilities normally use this type of anti-D and therefore do not routinely use Rh control. Chemically modified anti-D has been modified by breaking the disulfide bonds closest to the hinge region so antibody can reach cells that are farther apart.
2. False positive can also be caused by rouleaux formation, which will look like agglutination macroscopically. Rouleaux would be identified microscopically due to the "coin-stacking" appearance of the red cells. This false positive would be corrected by washing cells 3 to 4 times and then retesting.
False Negatives
False negatives are not readily identifiable, but can occur in the following instances:
y y
The most common is the result of too heavy cell suspension due to too many cells for the amount of antibody in the antisera. They may also rarely be caused by extremely strong positive DAT. In this case a the patient's D antigen sites are coated in vivo and there are no sites left for commercial anti-D to attach to. This can be fixed by heating cells gently to elute off antibody without damaging cells, then re -test.
OBJECTIVES - Rh SYSTEM
1. Briefly describe how and when the Rh system was discovered 2. List the major Rh antigens and state the frequency each is seen in the Caucasian population. 3. Describe the characteristics common to the major Rh antigens and compare them to the ABO system. 4. Explain the Tippett theory of inheritance. 5. For any given Rh phenotype, predict the most likely genotype in both the Wiener and Fisher-Race nomenclatures. 6. For any given Wiener genotype, list the Rh antigens present. 7. Explain why Rh genotyping is important. 8. Give three explanations for the weak D phenotype. 9. Discuss how the weak D phenotype applies to donors, recipients, and obstetrical patients. 10. State the relative frequencies o f the Cw, V and VS antigens. 11. Explain the G, f, and Rh null phenotypes. 12. Describe characteristics common to antibodies in the Rh system. 13. List the more common antibodies seen in the Rh system. 14. Discuss the use of albumin and enzymes in identifying Rh antibodies. 15. Explain how false positives can occur when testing for the Rh antigens, and describe how the problem may be overcome. 16. Explain how false negatives can occur when testing for the Rh antigens, and describe how the problem may be overcome. 17. Differentiate between high -protein anti-D, chemically modified anti-D, and saline anti-D.
1. 2. 3. 4. Correctly perform, interpret, and record the Rh type of any given sample Recognize when chemically modified or saline Rh reagents must be used. Correctly perform, interpret, and record a weak D (D u)test. Correctly perform, interpret, and record the Rh phenotype of any given sample, and state its most likely genotype.
LIFE'S BLOOD
CLASS NOTES
LEWIS BLOOD GROUP SYSTEM

There are distinct differences in regards to the Lewis Blood Group System
y y y
Manufactured by the tissues Lewis antigens are secreted into body fluids Absorbed onto red cells from the plasma
The Lewis Blood Group System is also similar to the ABO system
y y
The antigens are part of the same oligiosaccharides that are part of the ABH antigens The Lewis antigen (fucose) is added onto the N -acetyl-glucosamine that is just before the galactose where the fucose is added for the H antigen in the secretions.
Lewis Antigens
Alleles
The development of the Lewis antigens is controlled by two alleles of the Lewis blood group system.
y y
Le is dominant and results in the presence of Lewis antigen. The recessive le (absence of Lewis gene) is recessive and therefore 2 le/le needs to be inherited
Genotypes
Both Le/Le or Le/le result Lewis positive antigen. Lewis antigen exists as either Lewis a (Le a) or Lewis b (Le b). Lewis negative results from le/le.
Phenotypes: Lewis System Phenotypes and Their Incidence

(Modified from AAB B Technical Manual, 2002, p. 287)
Reactions with AntiLea Leb + 0 0 + 0 0 + +
Phenotype Le(a+b-) Le(a-b+) Le(a-b-) Le(a+b+)
Adult Phenotype Incidence in % Whites Blacks 22 23 72 55 6 22 Rare Rare
Formation of Lewis Antigens

What Lewis antigens are formed depends on interaction between Lewis (Le/le), Secretor (Se) and H genes in the tissues to produce Lewis antigens in the secretions. Lewis antigens have a similar structure to ABO antigens: 3. Formed at terminal sugars of Type I precursor substanc e made by tissue cell in plasma a. If person has the Lewis gene, it adds fucose to second sugar from the end = Lea b. If person is also a secretor, H gene adds fucose to terminal sugar of precursor substance = Leb antigen c. If person NOT a secretor, no H added to precursor substance made by tissue cell, so it remains as Lea
4. NOT part of rbc membrane - synthesized by tissue cells, carried by plasma, adsorb onto rbc surface 5. Not present on newborn red cells 6. Can disappear during pregnancy
Lewis Antibodies
Types of antibodies 1. Anti-Lewisa 2. Anti-Lewisb 3. Anti-Lewisa and Lewis b B. Almost always produced by Lewis a negative; Lewis b negative people
C. Naturally occurring but not regularly occurring (unexpected antibodies) D. Frequently seen in pregn ancy (due to loss of Lewis antigen during pregnancy) E. IgM, therefore usually not clinically significant 1. Does not cross placenta 2. Reacts best at RT, but some MAY react at 37C 3. If anti-Lea present at 37C, may cause hemolytic transfusion reaction 4. Anti-Leb is usually clinically insignificant III. Secretor Status A. Secretor status controlled by Secretor (Se) gene 1. Secretor = Se/Se or Se/se (80%) 2. Non-secretor = se/se (20%) B. Secretors have soluble A, B and H antigens in body fluids - plasma, tears, saliva C. Can test saliva for presence of ABH antigens D. Practical application of saliva testing: 1. Determine ABO type in patients with ABO discrepancies 2. Determine ABO type in patients massively transfused with another blood type 3. Can also use Lewis types to determine secretor status: E. Lewis a positive, Lewis b negative = non -secretor F. Lewis a negative, Lewis b positive = secretor G. Lewis a negative, Lewis b negative = can't tell secretor status from Lewis types H. Can only use this method if patient has not been heavily transfused recently need to type patient red cells, not donor IV. Testing for Secretor Status A. Utilizes principle of AGGLUTINATION INHIBITION 1. Patient's saliva boiled and cleared
2. Cooled saliva mixed with reagent ant i-A, anti-B and anti-H 3. If soluble A, B, or H antigens present in saliva, these will react with antibodies in reagent antiserum, and neutralize it, so no antibody available to agglutinate test cells 4. if no soluble A, B or H antigens in saliva, antibodi es in reagent antiserum will not be neutralized, and will be free to react with test cells B. Group A Secretor: 1. A antigens in saliva 2. Addition of reagent anti -A: antibodies tied up by soluble A antigens 3. Addition of known A cells results in no agglu tination (no free A antibody to cause agglutination)
NO AGGLUTINATION = POSITIVE TEST FOR SECRETOR STATUS
C. Group A Non-Secretor: 1. NO ABO antigens in saliva
2. Addition of reagent Anti -A leaves free antibody in serum
3. Addition of known A ce lls causes agglutination. AGGLUTINATION = NEGATIVE TEST FOR SECRETOR STATUS OBJECTIVES - LEWIS BLOOD GROUP SYSTEM 1. State the alleles in the Lewis system 2. Explain how Lewis antigens are produced 3. Explain the difference between Lewis a and Lewis b
4. Explain the relationship between the secretor gene and Lewis antigen production 5. Describe the general characteristics of Lewis antigens, including presence at birth and effect of pregnancy 6. State the possible Lewis phenotypes, and their approximate per centages in the Caucasian population 7. State which Lewis phenotype is most likely to make antibodies in the Lewis system 8. Describe the typical characteristics of Lewis antibodies 9. State which of the Lewis antibodies is more clinically significant, and explain why. 10. Explain what is meant by the term "secretor" 11. Explain the principle of agglutination inhibition, and how it applies to saliva testing 12. Given the results of any saliva agglutination inhibition test, state whether or not the patient is a secretor, and determine his/her ABO type 13. Explain how secretor status can be determined from the Lewis phenotype 14. State limitations to the above system 15. Give two practical applications of saliva testing Performance objectives: 1. Correctly perform and interpret the saliva test for agglutination inhibition, to determine your own secretor status 2. Correctly perform and interpret the antigen typing for Lewis a and Lewis b on your own red cells 3. Correlate the results of your Lewis typings with t he results of you saliva testing
LIFE'S BLOOD
OTHER BLOOD GROUP SYSTEMS

MNS SYSTEM
Antigens and Their Inheritance
The antigens M and N are co -dominant alleles that are closely linked to the S and s antigens, which are also co -dominant. Chromosome 4 contains these linked genes. These antigens are inherited by a complex pattern similar to the Rh system. The Ms and Ns linkage is more common than the MS and NS linkages. All these antigens, however are fairly frequent in the population with the following overall frequencies:
y y y y y
M = 78% N = 72% S = 55% s = 89% U = Greater than 99%
U antigen is a high incid ent antigen NOT seen in individuals who lack both S and s antigens. Individuals who lack this antigen (<1%) have a high likelihood of forming anti-U as well as anti-S and anti-s.
AABB Technical Manual, 13th ed.: Modified Table 15-3 Phenotypes and Frequencies in the MNS System p.318
Phe
ype Whites 28 50 22 11 44
M+NM+N+ M-N+ S+s-U+ S+s+U+
Phe
ype Freq e cy % B cks 26 44 30 3 28
S-s+U+ S-s-U-
45 0
69 Less than 1
Biochemistry of the MNS antigens

The M and N antigens are glycoproteins containing sialic acid that cross the cell membrane. Carboxyl terminus extends into the red cells interior, a hydrophobic segment as part cell membrane and an amino terminal segment on the external environment of the red cell. The external components of the antigens are destroyed by the enzymes like fiacin, typsin and papain. The antigens of the MNSU blood group system are well developed in newborns. Therefore a mother who is negative for one of these antigens could be stimulated to make antibodies thatmay cause HDN. The antibody would have to be an IgG immunoglobulin that reacts at 37 oC)
MNS System Antibodies Anti-M is frequently seen as a saline agglutinin if testing is done at room temperature.
y
y y y
It is predominantly IgM and can be naturally occurring. It will show dosage and therefore M homozygous cells will react with antibody more strongly than heterozygous cells. Therefore the predominant form of the ant ibody is not clinically significant. There are instances where some or all of the antibody is IgG in nature. If you have an anti-M that strongly at 37 oC and/or AHG, it should be considered to be potentially clinically significant. Mild to severe cases of hemolytic disease of the new born have be reported. Since the M antigen can be removed by enzyme, the reactivity of anti -M can be destroyed by enzyme. When performing a crossmatch on a recipient's specimen that contains anti -M, a prewarmed crossmatch should be preformed.
Anti-N is very rare and has similar reactivity as anti-M. Most often seen in kidney dialysis patients as cross-Reacting antibody to formaldehyde. Formaldehyde is used to sterilize Dialysis equipment. Anti-S anti-s and anti-U usually form following red cell immunization due to transfusions and pregnancies.
y y y y
They are usually IgG and react best after 37 oC with AGT (Coombs) technique. All are capable of causing Hemolytic Transfusion Reactions (HTR) (delayed) and Hemolytic Disease of the Newborn (HDN) S is usually destroyed by enzyme but s is variable and U is not destroyed by enzyme treatment. Anti-U is rare but should be considered if a previously transfused or pregnant Black patient has an antibody to a high -incident antigen .
Summary of Antibody Characteristics
Reacti it
Antibody < RT* 37
% Compatible Bind In Vitro HTR** HDN*** Comments AHG Enzymes Comp ementHemolysis in US Pop lation
Some Some Most
Variable Some Effect Variable Few Effect
Few Few Most
*Room Temperature **Hemolytic Transfusion Reaction ***Hemolytic Disease of the Newborn
KELL SYSTEM
In Dr. Mourant's article on the discovery of antiglobulin test, he recounts that they tested the serum of a number of mothers, who were believed to have babies with hemolytic disease of the newborn. One of the reactions was not due to Anti -D. "The non-Rh antigen involved in this case was that subsequently known as Kell. Thus at the very outset the test had d etected a previously unknown blood group system which has since proved to be of some clinical importance." (" The Discovery of the Anti-Globulin Test") There are three pairs of alleles within the Kell system. Each pair has a high frequency and low frequency gene that are co -dominate if present. The three pairs are:
y y y
K (Kell), or K1, and k (Cellano), K2 Kpa (K3) and Kp b (K4)(Penney) Jsa (K6)and Js b (K7)(Sutter)
K, Kp a and Js a are low frequency antigens and k, Kp b and Js b are high frequency antigens. There is a Kell phenotype, K null (K o or K5), is very rare and K, k, Kp a, Kpb, Jsa, Jsb antigens are not expressed.
Rare Some Most
No (Rare Change
Most Few Few Destroy (Rare
No No
Rare Moderate28% Yes Mild MildSevere MildSevere 45% W 69% B 11% W 3% B <1%
No
Yes
No
Yes
Most Few Few Destroy (Rare
No
Few
MildSevere
22%
Us ally Clinically Insignificant
The Kell Systems antigens are fo und in only small amounts on the red cell carried on a single protein. K has approximately 3500 sites and k has between 2000 5000. The function of this protein is unknown.
AABB Technical Manual, 13th ed.: Modified Table 15-5 Phenotypes and Frequencies in the Kell System p.322 Phenotype Freq ency % Phenotype Whites K+kK+k+ K-k+ 0.2 8.8 91 Rare 2.3 97.7 0.0 Rare 100.0 Extremely rare
Kp(a+b+) Kp(a-b+) Js(a+b-) Js(a+b+) Js(a-b+) Ko [K-,k-,Kp(a-b-),Js(a-b-)]
Kell System Antibodies

Anti-Kell is the most clinically significant antibody within this system. The Kell antigen is considered the next most antigenic after the D antigen of the Rh system. Individuals lacking the K antigen can make anti -Kell after only two exposures to Kell-positive blood. Because over 90% of the population are Kell negative it is not difficult to find donor blood that is compatible with the recipient.
Antibodies to other antigens in Kell system are very rare. Antibody Characteristics of the antibodies to the Kell Sy stem are:
y y y y y
IgG Cause HDN and HTR (delayed) React best in Coombs after 37 oC incubation Does not show dosage (homozygous KK and heterozygous Kk cells react with the same strength) Enzyme has no effect
Kp(a+b-
Blacks Rare 2 98 0 Rare 100 1 19 80
Reacti it
Antibody < RT* 37
% Compatible Bind In Vitro HTR** HDN*** Comments in US ComplementHemolysis AHG Enzymes Pop lation No Some change No (Some) change No (Some) change No (Some) change No (Some) change No (Some) change No Yes MildSevere Mild 91
Some Some Most
Few Few Most
No
Yes
0.2
Kpa
Some Some Most
No
Yes
Mild
99.7
Kpb
Few Few Most
No
Yes
Mild
<0.1 100 W 80 B
Jsa
Few Few Most
No
Yes
Moderate
Jsb
(No) (No) Most
No
Yes
Mild1B Moderate
DUFFY SYSTEM
The Duffy antigens Fy a and Fy b are a pair of co-dominant alleles found on chromosome 1. The phenotypes Fy(a-b+), Fy(a+b+), Fy(a-b+) are very common among the US white population. Fy(a-b-) is very rare in the white population but makes up 68% of the black population of the United States. Biochemically the Duffy antigens are glycoproteins that has an external loop. This external loop can be destroyed by enzymes such as ficin, papain, and trypsin. The Fya and Fyb antigens are receptors for the malarial parasite, Plasmodium vivax. Therefore individuals that are phenotypically Fy(a -b-) have a resistance to malaria. This particular phenotype is found up to 100% of western Africa and of course 68% of the American Blacks.
AABB Technical Manual, 13th ed.: Modified Table 15-6 Phenotypes and Frequencies in the Duffy System p.325 Phenotype Freq ency % Phenotype Whites Fy(a+b-) Fy(a+b+) Fy(a-b+) 17 49 34 Very rare
Fy(a-b-)
Duffy System Antibodies

Duffy antibodies frequently seen in multiply -transfused Blacks. Anti-Fya much more common than anti-Fyb and is more likely to cause HTR and HDN. 3. Characteristics
y y y y
IgG Anti-Fya can cause HDN and HTR (delayed) and anti -Fyb is milder and no HDN cases have been reported but could possibly be a cause. React best in Coombs after 37 oC incubation Reactions destroyed by enzyme of the red cells
Reacti ity
Antibody < RT* 37 AHG
% Compatible Bind In Vitro HTR** HDN*** Comments Enzymes Complement Hemolysis in US Pop lation No Yes MildSever (Yes) 34 W 17 W 77 B
Fya
Rare Rare Most Destroy Some
Fyb
Rare Rare Most Destroy Some
No
Yes
Blacks 9 1 22 68
KIDD SYSTEM
Jka and Jkb antigens are inherited on chromosome 18 where urea transport mechanisms are located. Cells that are Jk(a-b-) are less likely to to lyse in the presence of high concentration of urea. These antigens are inherited by the co dominant alleles Jk a and Jkb that are high frequency antigens. The Kidd antigens are thought to be grouped very close together in clusters on the red cell membrane. Due to the close proximity of the antigens when the antibodies are attached complement can be activated. The activation of complement can cause intravascular transfusion reactions. Approximate frequencies among the white US population are the following:
y y y
Jka = 75% Jkb = 75% Approximately 50% of the white population is Jk a and Jkb positive
The more specific frequencies are listed in the table below for both whites and blacks.
AABB Technical Manual, 13th ed.: Modified Table 15-7 Phenotypes and Frequencies in the Kidd System p.326 Phenotype Freq ency % Phenotype Whites Jk(a+b-) Jk(a+b+) Jk(a-b+) Jk(a-b-) 28 49 23 Extremely rare
Kidd System Antibodies

Both anti-JKa and anti-Jkb are hard to detect and identify since they are very weak and are detected primarily at the antiglobulin phase of testing. These antibodies are usually low titer as well as being weak reactions. The antibodies disappear rapidly from circulation and also in stored serum since their recognition is enhanced if complement is present. These antibodies will often show dosage. Enzyme can enhance the reaction as well as PEG enhancement solution. Characteristics of anti-Jka and anti-Jkb are as follows:
y
gG
Blacks 57 34 9
y y y
React best after 37 oC with Coombs technique Can cause HDN Can cause Hemolytic Transfusion reactions that are acute intravascular reactions, or they may be delayed transfusion reactions that show up after the patient's immune system is reexposed and the memory cells quickly produce antibodies to the antigens. While most of the antibodies discussed in newsletter are more likely to cause extravascular hemolytic transfusion reactions, the Kidd antibodies can activate complement and therefore cause intravascular he molytic transfusion reactions.
Reacti ity
Antibody < RT* 37
% Bind In Vitro Compatible HTR** HDN*** Comments AHG Enzymes ComplementHemolysis in US Pop lation Most Enhance All Some Yes Mild 23
Jka
Few
Few
Jkb
Few
Few
Most Enhance All
Some
Yes
Mild
28 W 57 B
Bg Antibodies
The Bg antibodies were first described by Bennett and Goodspeed. They are not considered clinically significant, but may mask clinically significant antibodies. They are antibodies to white cell antigen remnants on red cells
y y y
Anti-Bga reacts with Human Leukocyte Antigen (HLA) -B7 Anti-Bgb reacts with Human Leukocyte Antigen (HLA) -B17 Anti-Bgc reacts with Human Leukocyte Antigen (HLA) -A28
Reactions relating to these antibodies are weak, react possibly at room temperature or more commonly in the antiglo bulin (Coombs) phase. They react with only a few cells in a cell panel. They do not cause hemolytic disease of the newborn or hemolytic transfusion reactions. They are seen frequently seen in multiply transfused patients or in multiparous women (multipl e pregnancies)
HTLA ANTIBODIES = High Titer Low Avidity

A summary of these antibodies are as follows
y y
Not clinically significant, but serological reactions make them look like they are High Titer if the antibodies are titered. The titers are usually at least 1:64 and often will be over 1:1000
Associated with Severe Delayed HTR
Reactions are very weak and will break apart very readily due to the weak attraction between the antigens and antibodies (low avidity).
These antibodies basically have a high titer but a very weak reaction. Some institutions actually score the strength of the antigen reaction in points which will allow them to differentiate some of the differences seen in various reactions. Score = strength of reaction given points or score
y y y y y
4+ = 12 points 3+ = 10 points 2+ = 8 points 1+ = 5 points neg = 0 points
Specific serologic characteristics of the HTLA antibodies are:

y y y y y
IgG React best in Coombs after 37oC incubation W+ to 1+ reactions React with most cells (antibodies to high -frequency antigens) Not clinically significant since they are not known to cause hemolytic disease of the newborn or hemolytic transfusion reactions. Since they are antibodies to high frequency antigens they may mask clinically significant antibodies that are also in the serum.
OBJECTIVES - BLOOD GROUPS OTHER THAN ABO/RH

For each of the blood groups listed below:
y y y y
MNS Kell Duffy Kidd
y y y
Ii Lewis P
1. Recognize and translate the symbols used to denote antigens and antibodies 2. List the major alleles and the relative frequency of each in the Caucasian population. (High-incidence or low-incidence) 3. Describe the serological characteristics of the antibodies:
y y y y
class temperature of reaction effect of enzyme complement binding
dosage effect
4. Discuss the clinical significance of each antibody regarding transfusion reactions and hemolytic disease of the newborn 5. Describe the relationship between the Duffy system and malaria. 6. Describe the typical serological characteristics common to all HTLA antibodies. 7. Explain the difference between an antibody's titer and an antibody's score. 8. Describe the typical serological characteristics of the Bg antibodies 9. Describe the clinical significance of HTLA and Bg antibodies. 10. Explain the relationship betwe en white cells and the Bg antibodies.
Table of Contents
Life's Blood
I AND P BLOOD GROUP SYSTEMS

I Blood Group System
I/i Antigens
The I antigen is found on almost all adults and is part of the precursor component of the oligosaccharide that forms the A, B, and H antigens. There are two types of oligosaccharides: Type 1 and Type 2. These chains that are part of the ABH antigens form the i antigen found in infants.
By the time the child reaches 2 years of age a number of b1 -->6 linkages are added between the residual galactose components. In adults these precursor components are become branched with a F1-->6 binding between 2 galactose molecules forming a branched oligosaccharides that up the I antigen seen in most make adults.
y Therefore I (almost all adults) are formed from branched sugar chains on precursor substance. y All cord bloods form i antigen from straight-line sugar chains on precursor substance of the A, B, and H antigens.
As indicated: in most children i is converted to I by the age of 2 years.
Anti-I, Anti-IH, and Anti-i Antibodies

Anti-I antibodies are the most common antibodies found if antibody screenings are done at immediate spin, room temperature . Their characteristics are:
y y y y y y
IgM immunoglobulins and therefore are saline agglutinins with their optimal temperature at 4 oC. Since they are IgM they also do not cross the placenta. Anti-I is naturally occurring often due to a Mycoplasma pneumoniae infection or some lymphomas and leukemias. These antibodies are USUALLY not clinically significant Anti-I reacts with all adult cells (including patient s own, all reagent cells, all donor cells) Anti-I does not react with cord cells Auto-anti-I is a common cold agglutinin
High titers of auto-anti-I can interfere with serological testing:
In ABO typing, the forward grouping may have patient cells coa ted with IgM autoantibody, so all cells agglutinate. When this happens all results are positive and the group looks like AB+. On the reverse grouping anti-I in serum reacts with all adult cells, so A 1 and B cells always agglutinate. All results are positive and the group looks like an O.
Cells + anti-A 4+ Cells + anti-B 4+ Cells + anti-A,B 4+ Serum + Serum + B A1 cells cells 4+ 4+ Cells + anti-D 4+ Cells + Rh Interpretation Cont 4+ Unknown ABO Group
Method to resolve these Problems

y y
Correct forward typing by washing cells in warm saline and re -testing. Correct reverse by warming the serum before testing, or adsorbing the cold autoantibody out of the serum.
Crossmatch results will result in all donors being incompatible. Correct by:
y y y
warming the donor cells and patients serum before crossmatching, omitting potentiators like LISS and using monospecific anti -IgG.
Anti-IH antibodies are directed against both I antigens and H antigens. Therefore they react most strongly with group O ad ult cells, least with cord cells or A 1 cells. Comparison of Anti-I, Anti-IH, and Anti-H Reactions
Antibody Anti-I Anti-IH Anti-H Anti-i Adult A Cells 2+ 2+ 0 0 Adult O Cells 2+ 4+ 2+ 0 Cord A Cells 0 0 0 2+ Cord O Cells 0 2+ 2+ 2+
Anti-IH is seen most often in A 1 adults and is not clinically significant but may cause problems with the ABO grouping and crossmatch results just as anti -I does. Follow the same steps for resolving the problem as indicated for anti -I.
Diseases
1. Anti-I and anti-IH are only clinically significant when they cause Cold Hemagglutinin Disease (CHD). In this disease a strong autoanti -I is present. The blood in patient toes, fingertips, earlobes is cooler than than 37 oC and the antibody coats cooled cells, binds complement, and causes hemolysis. This disease is treated by keeping the patient's extremities warm so the blood is not allowed to cool. 2. High-titer anti-I also commonly associated with Mycoplasma. pneumoniae infections. Treatment would be giving the pa tient appropriate antibiotics. 3. Anti-i, which is fairly uncommon and transient, is associated with infectious mononucleosis.
P System
Antigens of the P system are P k, P, P 1 .
Basic structure is the precursor substance, but is called globoside (P) or paragloboside (p) in the P system as the diagram below shows:
from the AABB Technical Manual, 13th, 1999, p.291 y P antigens poorly developed at birth and therefore do not usually cause Hemolytic Disease of the newborn. y They are present in variable amounts on different red cells and seems to diminish in storage. y Like ABO and Ii antigens, they are composed of sugar chains as seen in the accompanying figure. y Like A and B antigens, they are present on tissue cells as well as red cells. y Similar antigens fou nd in nature (bird droppings, pigeon eggs, hydatid cyst fluid) and these substances can be used to neutralize anti -P in a patient's serum. These similar antigen solutions can be useful when the antibody is interfering with ABO and crossmatch testing. The Pk antigen occurs when the P k antigen does not convert to P
P Phenotypes
80% of people with P antigen are P 1 phenotype (have P and P1 antigens) and 20% of people with P antigen are P 2 (P antigen only - no P1). The P2 phenotype merely signifies absence of P1 antigen in people with P antigen (globoside). Individuals who are P k consistantly have an alloanti-P that is an IgM antibody. The absence of the P antigen is very rare and is designated as p.
P Antibodies
Alloanti-P1 is the most common of the antibodies in the P system. This antibody is commonly seen in P2 people. IAlloanti-P1'ss characteristics are:
y y y
IgM class and therefore is saline reactive, enhanced by cold incubation, may bind complement. Not clinically signific ant Naturally occurring but not regularly occurring. It is formed with no known red blood cell stimulus - either pregnancy or transfusion. If individuals are P 1 negative, they do not automatically have anti -P1 i, but it is fairly common Antigen-antibody reactions in P system enhanced by adding albumin to system, or by treating red cells with enzymes
Auto anti-P is an IgG antibody seen in P 1 or P2 persons and causes clinically significant Paroxysmal Cold Hemoglobinuria (PCH). The Donath-Landsteiner Test helps diagnose PCH. It is designed to detect biphasic anti -P. A biphasic antibody reacts with the antigen on the red blood cell at colder temperature, binding complement . When antigen-antibody-complement complex warms up, the red
blood cells are he molyzed. Donath-Lansteiner test is performed by incubating patient's serum with his own cells first at 4 oC, then at 37 oC, and then looking for hemolysis.
y y
A positive Donath -Landsteiner = no hemolysis at 4 oC, no hemolysis at 37 oC, hemolysis only in the tube that was incubated at both temperatures A negative Donath -Landsteiner = neg at both temps, OR hemolysis at 4 oCas well as 37oC
Donath-Landsteiner Test
4oC incubation:
37oC incubation:
4oC + 37 oC incubation: hemolysis: = POSITIVE
no hemolysis = NEGATIVE no hemolysis = NEGATIVE
OBJECTIVES - I AND P BLOOD GROUP SYSTEMS

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Differentiate between I and i antigens regarding structure and age when developed. State the optimal temperature of reaction of anti -I. Explain how anti -I interferes with ABO typing and crossmatch results. Explain how the above problems may be overcome. Explain why anti -I is always an autoantibody. Explain why anti -I or anti IH is not generally clinically significant. Explain the difference in reactions between anti -I and anti -IH Describe the cause, symptoms and treatment of Cold Hemagglutinin Disease. State what other disease auto -anti-I may be associated with. State what disease anti -i may be associated with. State the major antigens of the P system, and how they are distribut ed in the population. State which antigens make up the following phenotypes in the P system: P1, P2, p Explain what paragloboside or globoside is. List three other sources of the P antigen in nature, and explain when this knowledge may become useful. State the most common allo-antibody in the P system. Explain how antigen -antibody reactions in the P system may be enhanced. State the cause of paroxysmal cold hemoglobinuria (PCH). Describe the Donath -Landsteiner test and explain what it detects.
Table of Contents
Antibody Screening
PRINCIPLE AND APPLICATIONS
The patient's serum is tested for the presence of clinically significant antibodies using an indirect antiglobulin method. The serum is tested against unpooled Group O cells selected to possess the relevant blood group antigens. The antibody screen is rou tinely performed as part of compatibility testing; it is also performed upon request on prenatal patients, on Rh immune globulin candidates, as part of an organ transplant workup, or on other patients for whom an "indirect Coombs" is requested.
SAMPLE
Since the patient's serum is tested, a 10 ml clotted (red top) tube is preferred. A 4 or 5 ml red top is acceptable; a 2 ml red top is acceptable from a newborn. The sample should be tested when fresh; old or improperly stored samples may lose complement activity and lead to false negatives.
REAGENTS, EQUIPMENT, AND SUPPLIES

y y y y y y y y y y y y
12 x 75 mm test tubes Lighted agglutination viewer Plastic test tube holder Reagent Screening Cells I & II, and III Indelible marking pen PEG Large bore dispo pipettes Anti-Human Globulin (Coombs serum) 37oC waterbath or heat block Coombs Control Cells Serofuge Wash bottle with physiologic saline
PROCEDURE
1. Verify that patient information on the sample matches information on the worksheet. 2. Centrifuge the sample and separate the serum to a labeled tube. 3. Prepare a washed 3% cell suspension from the patient's cells. See WASHED 3% CELL SUSPENSIONS. 4. Label 3 tubes Patient last name/or #, I Patient last name/or #, II Patient last name/or #, III 5. Using a large bore pipette, add 2 drops of patient serum to all tubes. Hold the dropper at a consistent 45 -degree angle. 6. Add one drop of Screening Cell I to tube I; add one drop Screening Cell II to the II tube; add one drop Screening Cell III to the III tube.
7. Shake all tubes to mix and centrifuge at high speed the time appropriate for the saline spin calibration in the serofuge. 8. Gently resuspend and examine macroscopically for agglutination using the lighted agglutination viewer. 9. Record all negative reactions as ; grade positive reactions on a scale of w+ to 4+. 10. Again holding the dropper at a consistent angle, add 2 drops of PEG to all tubes. 11. Shake to mix and incubate at 37 oC at least 15 but no more than 30 minutes. (NOTE: IF A WATERBATH IS USED FOR THE 37 oC INCUBATION, THE INCUBATION TIME MAY BE REDUCED TO 10 MINUTES). 12. Wash all tubes 4 times, decanting well after each wash, mixing the cell button well between washes, and blotting the last drop of saline after the final wash. 13. Immediately add one drop AHG to each tube, shake to mix, and centrifuge the time appropriate for the Coombs spin calibration in the serofuge (usually the same as the saline calibration). 14. Immediately resuspend gently and check macroscopically for agglutination using the lighted agglutination viewer. 15. Read and record results as described above, along with your initials and the date, if not already recorded on the worksheet. 16. Add one drop Coombs Control Cells to all negative reactions and centrifuge 15-30 seconds in the serofuge. 17. Resuspend and macroscopically examine for agglutination. There must be agglutination in this step or the test is invalid. Record results.
INTERPRETATION
y y
The lack of agglutination indicates the absence of antibodies to antigens on the reagent test cells, and the test is reported negative. Agglutination in either Screening Cell tube prior to the addition of Coombs Control Cells indicates the presence of unexp ected alloantibodies. The test is reported positive and further testing is necessary to identify the antibody(ies). See ANTIBODY IDENTIFICATION. Agglutination in the autocontrol (AUTO) indicates coating of cells circulating in the patient. This may be due to an antibody to medications, autoantibody, passively transfused alloantibody, or alloantibody coating transfused cells in the patient. Perform a DAT and obtain the patient's medication list, diagnosis and transfusion history.
NOTES AND PRECAUTIONS

1. Use of PEG requires a strict serum/PEG ratio. Also, add no more than 2 drops of serum when using PEG. 2. AHG must be added to the cells immediately following washing. Antibodies may elute from the cells if they are allowed to sit in saline without the additi on of AHG. 3. The Coombs Control Cells must be positive at the end of the procedure. Reasons for negative results here include:
y
Inadequate washing of the cells in the Coombs phase. Residual serum
y y
neutralizes AHG. A small fibrin clot has formed in the tube, neutralizing AHG. Be sure sample has thoroughly clotted. Coombs reagent was omitted or is inactive.
4. Weak or questionable reactions may be enhanced by:

y y y y
Increasing the amount of serum used to 3 -4 drops. PEG must be omitted, and the incubation time extended to 30 minutes. Omitting PEG and adding albumin to the test mixture. Extend the incubation time to at least 20 minutes. Using enzyme treated cells. See manufacturer's directions. Repeat testing using PEG to t he test mixture. See manufacturer's directions.
5. Occasionally you may get a moderately strong reaction at room temperature that persists weakly in Coombs. This may not be clinically significant if it is due to complement binding at room temperature from a cold-reactive antibody. To determine if the reaction is due to complement binding, do a pre warmed screen (SEE PREWARMED CROSSMATCH TECHNIQUE).
REFERENCES
AABB Technical Manual, current edition matc\abscrn.lab Author: Peggy Schroeder, Rose Dickinson
LIFE'S BLOOD
CLASS NOTES
THE CROSSMATCH
General Information
The Crossmatch also known as compatibility testing, pretransfusion testing or type and crossmatch (Type and Cross; T & C). The definition of a compatibility test (crossmatch) is a series of procedures use to give an indication of blood group compatibility between the donor and the recipient and to detect irregular antibodies in the recipient's serum. The main purpose for performing a crossmatch is to promote (not ensure) the safe transfusion of blood. We are performing testing to the best of our ability that will demonstrate that the donor blood is compatible with the recipient's blood. Crossmatch procedures should be de signed for speed and accuracy - get the safest blood reasonably possible available to the patient as soon as possible. In summary, the AABB Technical Manual states the goals of a compatibility test is to:
y y y
Detect as many clinically significant antibodies as possible Detect as few clinically insignificant antibodies as possible Complete the procedure in a timely manner. (p. 379)
Once donor blood is crossmatched with a potential recipient, the results of the crossmatch is good only 3 days. If the physicia n wants the donor blood available longer, we must get a new recipient sample and repeat tests. This protocol helps detect new antibodies that may be forming, especially when patient has been transfused within past three months.
Parts of the Crossmatch

The AABB Standards for Blood Banks and Transfusion Services requires that certain procedures are performed before blood is transfused to a recipient:
y
Identification of the recipient and recipient blood sample is crucial since the major of the hemolytic transfusion reactions are due to errors in patient or sample identification. ABO and Rh typing of the recipient's blood and resolving any ABO discrepancies. If the discrepancy can not be resolved before the patient needs the transfusion type O blood should b e given. If problems arise with the D testing, Rh negative blood
y y
should be given. Performing an antibody screen on the recipient's serum for clinically significant antibodies. These antibodies are most likely to occur in the 37 oC and AGT phases of testing. Each negative AGT test must be followed by "Coombs Control Check Cells." An autocontrol may or may not be used. Some labs prefer to perform this routinely during the antibody screen while others will only include it if an antibody needs to be identifi ed. The autocontrol has to be part of the antibody identification procedure. The SOP of each institution must be followed by all individuals performing these tests. Comparing present findings with previous records for the recipient. If previous testing has been performed on the recipient and should match current testing. These comparisons can give assurance that no identification errors have occurred, but it is not proof. Records would also show if clinically significant antibodies have been detected i n the past. These antibodies may be presently at undetectable levels. Any history of clinically significant antibodies, even if undetectable now in the patient, dictates an antiglobulin phase crossmatch needs to be done between the recipient's serum and the donor's cells. Confirmation of the ABO and Rh type of the red cell components being given when the shipment of blood is received in the laboratory. Selection of appropriate ABO and Rh component units for the recipient first would be the same ABO and Rh type. Transfused donor red cells must be ABO compatible with the patient's plasma and whatever antibodies may be present. Transfused plasma must be ABO compatible with the recipient's red cells.
AABB Technical Manual's Table 18-2 Selection of Components When ABO-Identical Donors Are Not Available, p 385 ABO Requirements Whole Blood Red Blood Cells (most plasma removed) Granulocytes, Pheresis Fresh Frozen Plasma Platelets, Pheresis Cryoprecipitated AHF Must be identical to that of the recipient Must be compatible with the recipient's plasma. Must be compatible with the recipient's plasma. Must be compatible with the patient's red cells. All blood groups acceptable; components compatible with the recipient's red cells preferred All ABO groups acceptable
Rh-positive components should be given to Rh-positive individuals and Rh negative units should be reserved for D -negative individuals. The physician needs to be involved in any decisions relating to giving Rh -positive blood to an Rh-negative individuals since those individuals have an 80% chance of making an anti-D following transfusion.
Perform a crossmatch either serologically or via a computer. If no clinically significant antibodies are found in the recipient the institution has t he option of choosing an immediate -spin crossmatch (serologic technique) or a computer crossmatch. If clinically significant antibodies are found, an antiglobulin crossmatch
must be performed. Label the components with the recipient's identifying informa tion
Type and Screen

The type and screen consists of ABO/Rh, antibody screen, and a records check. This order is used when likelihood of needing blood is low. Therefore, no donor blood crossmatched to patient . If need for blood suddenly arises, you can take sample that is already typed and screened, and perform a crossmatch with donor units from the specimen. Type and screen protocol cannot be used if patient has an antibody. Then an antiglobulin crossmatch must be performed.
Benefits of a Crossmatch
Performing a crossmatch before transfusing blood has the following benefits:
y y
Detects major ABO errors (ie. crossmatching an A donor with an O or B recipient ) Detects most recipient antibodies to antigens on donor red cells (if the antibody is in high enough titer to react) One of the most common clinically significant antibodies that are missed are the Kidd antibodies.
Limitations of a Crossmatch:
A crossmatch also has limitations:
y y y y y
Will not detect errors in patient identification (unless a prev ious record exists) Will not detect ABO mix -ups if blood types are compatible (can crossmatch group A donor blood for an AB recipient) Will not detect Rh errors (can crossmatch Rh+ donor blood with Rh negative recipient with no reaction if the patient ha s no anti-D) Will not detect all recipient antibodies to donor antigens (antibody may be too weak to detect, but still cause transfusion reaction such as the Kidd antibodies) Will not prevent alloimmunization of recipient (only ABO and Rh antigens matche d patient can potentially make antibody to all the other antigens) This is why many of the discovered antibodies are found in multi -transfused patients.
Immediate Spin versus Antiglobulin, Coombs, Crossmatch

The purpose of Immediate spin step of cross match is to detect major ABO incompatibility between donor and recipient. ABO incompatibility is the most common life-threatening type of transfusion reaction and is often due to clerical errors. It is permissible to stop at immediate spin step of crossma tch if:
1. Immediate spin is negative and 2. Antibody screen is negative in all phases and 3. There is no record of previous antibodies
It is NOT permissible to stop at the immediate spin step and you must incubate and
carry crossmatch through antiglobulin, Coombs, phase if:

y y y
Immediate Spin test agglutinated or Patient has an antibody (screening cells are positive) or Patient has a record of a previous antibody
Benefits of an Immediate Spin only crossmatch:

y y y
Makes blood available to patient faster More cost-effective 90% of patients are eligible for immediate -spin crossmatches
Electronic or Computer Crossmatch As An Alternative to the immediate-spin crossmatch

An institution may choose to perform computer crossmatches instead of an immediate-spin crossmatch. They must meet specific criteria in order to do electronic crossmatches. Electronic crossmatches have no mixing of patient serum and donor cells in test tube - computer verifies ABO/Rh compatibility of donor and recipient
1. 2. 3. 4. 5. 6.
Computer system must be FDA -approved and validated to do this Patient ABO/Rh must have been typed at least twice - by two different technologists Patient has no antibodies and no record of previous antibodies Donor information must be bar-coded into computer inventory for accuracy Computer does not allow use of donor unit until its ABO/Rh is verified Computer does not allow issue of ABO/Rh incompatible blood
Crossmatch Problems: OBJECTIVES - COMPATIBILITY TESTING

1. 2. 3. 4. 5. 6. 7. 8. 9.
y y y y y y y
Discuss the steps in compatibility testing and explain their purpose. Discuss the reasons for compatibility testing. Discuss the limitations of compatibility testing. Explain what a Type and Screen consists of, and when it would be used. Explain when a LISS -Coombs crossmatch would be done versus an immediate -spin crossmatch. Explain what an electronic crossmatch is. Describe what additional testing must be done when crossmatching a patient with an antibody. Describe how to determine the number of units to screen when crossmatching blood for a patient with an antibody Discuss how to resolve the following problems encountered in compatibility testing: Test results not matching previous records Screening cells positive at room temperature, negative in Coombs Screening cells positive in Coombs only - new antibody Screening cells positive in Coombs only - previously identified antibody Screening cells positive both at RT and in Coombs Negative screening cells, but records show a previously -identified antibody Negative screening cells but crossmatch positive at immediat e spin
Positve autocontrol
10. State how long you may keep blood crossmatched, before having to get a new sample and repeat the test
1. Correctly perform, interpret and report a compatibility test on any given sample 2. Correctly identify, document and resolve any problems associated with compatibility
testing.
LIFE'S BLOOD
CLASS NOTES
IMMUNE HEMOLYSIS & COMPLEMENT

Complement
Complement helps fight off bacterial and viral infections, eliminates protein complexes, and helps in the immune response complex. The three major roles of complement are:
y y y
promoting acute inflammation process that allow white cells and macrophages to migrate to the sourc e of the problem altering the cell surfaces to encourage phagocytosis (opsonization) modifying the cell surface that will eventually lead to cell lysis
Characteristics of Complement
Complement is a series of proteins found in fresh, normal serum. It is in the beta region on protein electrophoresis and is categorized as a beta globulin. The complement component that is found in the highest concentration is C3. Terminology commonly used for the various complement components:
y y y
Complement components are identified by C and their number: C 1, C2, C3 etc Complement products resulting from the splitting of these proteins during the activation process are followed by a lower case letter: C3 a, C3b, C1q If complement complexes develop that have enzymatic acti vity are written with a bar above the top: C5b678
Complement controls various biological process:

1. Cell destruction through lysis 2. Cell destruction through opsonization (enhanced phagocytosis) especially with C3b 3. Chemotaxis via certain split products ac ting as chemical signals to the phagocytic cells 4. Anaphylaxis again through the split products (C5a and C3a), which promote inflammation. C5a and C3a can bind with mast cells and basophils leading to the release of histamine. This is turn:
y y y
Increases vascular permeability Smooth muscle contraction to preserve blood for vital organs Increases cellular membrane adhersion
Complement is normally inactive in serum or if activate the activation pathways are inhibited by control mechanisms of oth er complement proteins. It becomes activated by:
y y
Antigen-antibody reactions - most often IgM (classical pathway) Bacterial polysaccharides, virus particles, enzymes, endotoxins (alternate and lectin pathway)
Complement Cascade - (Classical Pathway)

Two adjacent antigen sites bound by antibody capable of binding complement:
y y
IgM molecule since it has 5 immunoglobulin subunits Certain IgG molecues that are capable of binding with complement. (only IgG 1, 2, or 3) Complement needs 2 IgG molecules bound t o antigen sites that are within 30 40 nm of each other.
The steps of the classical pathway

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Antigen-Antibody reaction occurs Complement senses adjacent Fc receptors Complement is activated and forms C1qrs complex C1qrs activates C4 Activated C4 breaks down to C4a and C4b C4b activates C2 to form C4b2a complex C4b2a complex also known as C3 convertase In presence of Ca+, C4b2a activates C3 Activated C3 breaks down to C3a and C3b C3b attaches to rbc membrane - is sensed by RE system and removed from circulation (extravascular hemolysis) Activated C3a activates many C5 molecules that leads to the development of the Membrane Attack Complex. Each activated C5 activates many more C6 and C7 molecules C6 and C7 conversion proceed rapidly to C8 and C9 activation Activated C8 activates many more C9 molecules Activated C9 attacks red cell membrane, boring holes in it
11.
12. 13. 14. 15.
Contents of red cell leak out into plasma leading to intravascular h emolysis. As the complement cascade results in biologic side -effects such as chemotaxis, opsonization, and anaphylaxis.
What Determines Whether or Not Complement Will Be Activated:
Nature of antibody coating the cell
IgM antibodies are the best because they have more antigen -binding sites. They can achieve binding of two adjacent antigens by single IgM molecule. Only certain IgG subclasses are capable of activating complement: IgG subclasses 1, 2, and 3. Of these IgG subsets, IgG 3 is the best. IgG subclass 4 does not activate complement.
Number of antigen sites on red cell
The more antigen sites found on the red blood cell, the more likely two adjacent ones are bound by antibody.
What does this mean in relationship to the ABO System?
The A and B antigens are in very high concentration on the red blood cells. The natural-occurring, expected antibodies (anti -A, anti-B, and anti-A,B) are IgM. ABO incompatibilities are the most likely to result in intravascular transfusion reactions from the activation of the classical pathway.
Possible Outcomes of Complement Activation in Blood Banking

y
Normal survival of the red blood cells can occur but realize the other outcomes are more likely. Intravascular hemolysis (complement cascade goes to completion) an d the cells are lysed. Extravascular hemolysis where the complement cascade stops at C3b step. Cells coated with C3b are removed from circulation via macrophages and neutrophils "Damaged cells" (spherocytes and stroma fragments) lead to decreased ce ll survival and possible activation of Hageman factor that in turn leads to coagulation activation Disseminated Intravascular Coagulation (DIC) may also occur due to small clumps of agglutinated cells in the blood stream, fibrinogen consumption, activat ion of fibrinogen system and fibrin destruction. When leukocytes are exposed to various antigen -antibody complexes they will also respond by secreting various cytokines that will lead to: fever, a drop in blood pressure and additional release of white bloo d cells from the bone marrow as well as a number of other activities. Renal failure, which is the most common complication of an untreated hemolytic transfusion reaction. This occurs due to a combination of
1. Hypotension 2. Contraction of the blood vessel s in the kidney since it is one of the smooth muscles that responds in anaphylaxis. 3. Intravascular clots 4. Toxic effects of free hemoglobin
Non-Immune Hemolysis
Physical
y y y
Temperature extremes (outside body) Hypotonic solutions (IV solutions) Mechanical (pumps, intravascular clots)
Chemical- toxins
Immune-Mediated Cell Destruction (Hemolysis)

Causes of Immune Cell Destruction:
1. Antibodies that bind complement 2. Antibodies that do not bind complement 3. Complement activation only
Types of Immune-Mediated Hemolysis

Intravascular Hemolysis
Intravascular hemolysis is complement mediated since the hemolysis is due to activation of the complement pathway by the classical pathway. Intravascular indicates the cell destruction takes place within the blood vessels. This type of hemolysis can be life-threatening due to both the possibility of anaphylaxis and renal failure.
Extravascular Hemolysis
Extravascular Hemolysis may be antibody mediated or complement mediated. The cell destruction takes place within RE system (spleen, primarily) and is generally not life-threatening. The survival rate of the transfused cells will be decreased.
Immune Response Following a Blood Transfusion Leading to Alloimmunization
1. Donor red cells carrying foreign antigens die normally and are phagocytized by RE system. 2. Foreign antigens processed and stimulate immune response. 3. IgM antibody produced after several weeks, probably no cell destruction. 4. Gradual decline of red cells res ults in continuous re -exposure of the antigen to the immune system. 5. Primary and secondary responses can overlap each other. 6. IgG antibodies can be produced while IgM antibody productions is going on, from the same blood transfusion (same "stimulating even t"). 7. Low-titer IgM antibodies may not cause cell destruction, but higher -titer IgG antibodies will eventually lead to extravascular hemolysis.
Intravascular Hemolysis (complement mediated) Summary

Mechanism of intravascular hemolysis
Complement is activated when two adjacent antigen sites are bound by antibody. The activated complement rapidly proceeds through several chemical changes to membrane attack complex. The membrane attack complex bores a hole through red cell membrane, causing hemolysis. The rate of complement activation and amount of complement activated determines whether complement cascade goes to completion.
Signs of intravascular hemolysis
y y y y
y y
Sudden drop in blood pressure due to the anaphylactoxins: C5a and C3a Hemoglobinuria due to the lysis of of the red blood cells. If more hemoglobulin is released that can be carried by albumin. Hemoglobinemia (plasma hemoglobulin levels) occurs again due to the lysis of red blood cells in the blood vessels. Decreased haptoglobin since haptoglobi n can also carry hemoglobin. There are limited amounts of haptoglobulin produced so once it is used, it will be removed by the liver and the haptoglobulin levels decreased. No rise in hematocrit following blood transfusion since the donor blood is destroyed. Anaphylactic shock, death may result if large amounts of complement activated
Antibodies that bind complement (classical pathway)

y y
IgM antibodies = anti -A, anti-B, anti-I, anti-Lewis a IgG antibodies = anti -A,B, auto anti -P, anti-D, anti-Kidd, anti -Kell, etc.
Extravascular Hemolysis - Antibody Mediated

Mechanism of antibody -mediated hemolysis:
Antibodies attach to antigens on red cell membrane and are sensed by phagocytes in RE system. The the antibody-coated red cells are ingested by the phagocytic cells and destroyed at a rate faster than normal cell destruction. RE system plucks antibody off cells, leaving damaged membrane. Cell becomes a spherocyte with shorter lifespan.
Antibodies that do not bind compl ement but promote extravascular cell destruction
Extravascular hemolysis processes are caused by IgG antibodies:

y y y y y
Rh antibodies (anti -D, anti-C, anti-c, anti-E, and anti -e) Anti-Kell (anti-K, anti-k, anti-Kpa, anti-Jsa etc.) Anti-Kidd (anit-Jka and anti-Jkb) Anti-Duffy (anit-Fya and anti-Fyb) Anti-S
Signs of extravascular hemolysis:
Signs of extravascular hemolysis includes a falling hematocrit, increased bilirubin (unconjugated), increased LD, and an abnormal peripheral smear (polychromasia, spherocytes, fragments)
Extravascular Hemolysis - Complement Mediated
In extravascular hemolysis that is complement mediated the breakdown products of activated complement attach to red cell membrane (primarily C4b and C3b and C3d). Presence of C3b coated cells are sensed by phagocytes in RE system. Complement-coated cells are ingested and destroyed at a rate faster than normal cell destruction.
Causes of Complement -Mediated Extravascular Hemolysis:
Any of the IgM or IgG antibodies that are capable of activating complement can cause this type of hemolysis. Besides the classical pathway the alternate pathway of complement activation can also occur due to the presence of various components like cell walls of bacteria and yeast, dialysis membranes, dextran, and some tumor cells. Certain drugs can also activate complement and can lead to a complement-mediated extravascular hemolysis.
OBJECTIVES - IMMUNE HEMOLYSIS & COMPLEMENT

1. Briefly explain what compl ement is 2. List the effects complement activation can have on the body, in addition to cell destruction 3. Explain what is required for complement to be activated in the classical pathway 4. Explain how the alternate pathway differs from the classical pathway i n the complement cascade 5. Outline the steps of the complement cascade in intravascular cell destruction 6. Explain the role of complement in extravascular cell destruction, including the roles of C3b and C3d 7. Explain what C3 convertase is 8. Explain what the m embrane attack complex is 9. List two factors that help determine if complement will be activated during an antigen-antibody reaction 10. Explain why IgM is a better complement activator than IgG 11. List four possible results of complement activation regarding re d cell survival 12. List at least four causes of red cell hemolysis, other than immune -mediated 13. List the three main causes of immune -mediated hemolysis 14. Describe the mechanism of intravascular hemolysis 15. Describe the signs and physical symptoms of intravascu lar hemolysis 16. List the IgM antibodies that typically cause intravascular hemolysis 17. List the IgG antibodies that typically cause intravascular hemolysis 18. Describe the mechanism for antibody -mediated extravascular hemolysis 19. List the symptoms of extravascular hemolysis 20. List pertinent laboratory findings in extravascular hemolysis 21. Describe the mechanism for complement -mediated extravascular hemolysis
22. List three causes of complement -mediated extravascular hemolysis 23. Outline the steps in an immune response and subsequent cell destruction following
a blood transfusion, including types of white cells involved in each step, and the approximate length of time involved.
Life's Blood
CLASS NOTES
INTRODUCTION TO IMMUNOHEMATOLOGY
DEFINITIONS
y y y y y
Immunity: Protection against infection or disease caused by foreign particles Immunology: Study of the immune system and the immune response; study of antigen-antibody reactions in vivo Serology: Study of antigen-antibody reactions in vitro Hematology: Study of the cellular components of the blood Immunohematology (Blood Banking): The detection, identification, and/or quantitation of antibodies involved primarily with red cells, although white cells and platelets may also be involved
TYPES OF IMMUNOHEMATOLOGY FACILITIES

Transfusion Service:
1. Examples: UW Hospital, Meriter Park, St. Mary's, VA 2. Work primarily with patient's blood 3. Primary areas of responsibility:
y y y y y y y y
blood typing antibody detection and identification compatibility testing (crossmatching) blood component therapy (hemotherapy) transfusion reaction workups autoimmune hemolytic anemia workups hemolytic disease of the newborn (HDN) workups determining Rh immune globulin eligibility
Donor Center (Blood Center)

1. Examples: Red Cross, Blood Center of SE Wis. 2. Works primarily with donor blood 3. Major areas of responsibility:
y y y y y y y y
donor recruitment donor screening blood collection testing (typing, infectious disease screening) blood component preparation component preservation may provide reference lab services may store rare donor blood
Blood Bank
1. Appleton, Manitowoc 2. Combination of donor center and transfusion service
y y y
bleeds most of its own donors tests its own donor blood uses the donor blood for its own patients
The term "Blood Bank" used synonymously with transfusion service or donor center as well as the true Blood Banks, which collects donor blood and serve as a transfusion center.
NATURE OF BLOOD BANKING:

Blood banking involves less automation than Hematology and Clinical Chemistry. The results are not numerical other than grading reactions as 1+, 2+. 3+ and 4+. There is a heavy emphasis on the thought process es of logic, interpretation, decision making and problem solving. You need to think about what is happening in the test tube and whether the patient's red cell antigens or antibodies are involved. Blood banking can be very rewarding since there are visible life-saving aspects of providing the patient with the needed blood components. Unfortunately it is also prone to human error and the consequences of error can be fatal. There are not many places in the clinical laboratory where a patient may die from an erroneous result, but giving A or B blood to an O individual can do that.
OBJECTIVES - INTRODUCTION TO IMMUNOHEMATOLOGY

1. 2. 3. 4. 5. Differentiate between immunology and serology. Describe the study of immunohematology List the functions of a transfusion service. List the functions of a donor center. Explain the difference between a blood bank and a donor center and transfusion service 6. Explain how blood banking may differ from the other laboratory sciences in terms of:
y y y y
type of methodology kinds of results obtained type of thinking required consequences of error
LIFE'S BLOOD
Table of Contents
Class Notes
BLOOD BANK ANTIGENS AND ANTIBODIES

Antigen
Definition:
Antigens are defined as substances recognized by the body as foreign, causing the body to produce an antibody to react specifically with it
Characteristics of antigens:
In order to be an antigen to you it must be foreign (not found in the host): THE MORE FOREIGN THE BETTER ANTIGEN!
y y
Autologous antigens are your own antigens (not foreign to you) Homologous, or allogenic, antigens are antigens from someone else (within the same species) that are forei gn to you
Antigens must be chemically complex.

y y y
Proteins and polysaccharides are antigenic due to their complexity. On the other hand, lipids are antigenic only if coupled to protein or sugar. Besides being chemically complex, antigens must also be large enough to stimulate antibody production. Their molecular weight needs to be at least 10,000. Due to the complexity of these molecules there are specific antigenic determinant sites, or epitopes, which are those portions of the antigen that reacts specifically with the antibody.
Factors determining whether an antigen will stimulate an antibody response:

y y
Degree of foreignness . Only human blood is transfused to humans. Size and complexity. Although red cells are smaller than white blood cells, they tend to be more antigenic due to the complexity of the antigens on the cell surface. Some are proteins and others are oligosaccharides.
y y
Dose of antigen administered . How much antigen is the indiv idual exposed to and what is the frequency of that exposure. Genetic makeup of host may also dictate whether an antibody is produced. Some individuals have a greater ability to make antibody and others have the antigen so they would not make the antibody .
Blood group antigens:

y y y
There are over 300 known blood group antigens Over 1,000,000 different antigen sites on each red blood cell. These antigens are attached to proteins or lipids on the red cell membrane and are usually complex sugar groups. Some stick out far on the red cell membrane and some are buried within crypts on the membrane surface.
Antibodies:
Definition:
Proteins produced by lymphocytes as a result of stimulation by an antigen which can then interact specifically with that particul ar antigen.
Serum components
y y
Human serum can be separated into albumin and globulin components Globulins can be separated into several different parts: a. Alpha 1 and alpha 2 globulins b. Beta globulins (serum complement) c. Gamma globulins (immunoglobu lins or antibodies)
Parts of an antibody:
1. 2. 3. 4. 5. 6.
Heavy chains - made of alpha, gamma, delta, mu, or epsilon chains Light chains - made of kappa or lambda chains Disulfide bonds - hold chains together Hinge region - allows antibody to flex to reach more antigen sites Fab fragments - contains variable portion of antibody: antigen -binding sites Fc fragment - contains constant portion of antibody; also site of complement activation
Classes of antibodies:
1. IgG - provides longterm immunity or protection 2. IgM - first antibody produced in response to an antigenic stimulus 3. IgA - found in secretions. Protects against infections in urinary, GI, and respiratory tracts 4. IgE - involved in allergic reactions 5. IgD - not much known about it. Surface receptor of B lymphocytes 6. Most important classes of antibodies in blood banking are IgM and IgG , and to a certain extent IgA
Characteristics of IgG and IgM antibodies

1. Clinical significance
y
y y
Clinical of red cell antibodies in blood bank depend on whether they can cause in vivo hemolysis, which in turn will cause transfusion reactions or hemolytic disease of the newborn. IgG will frequently cause in vivo hemolysis due to antibody coating the red blood cells. IgM, with a few important exceptions, usually does NOT cause in vivo hemolysis. The most important of these exceptions are ABO antibodies.
2. Size of the antibodies

y y
IgG is relatively small since it is comprised of only one immunoglobulin subunit. (monomer) IgM is relatively large since it is comprised of 5 immunoglobulin subunits. (pentamer)
3. Serum concentration
y y y
IgG is found in the largest concentration of all immunoglobulins in the plasma. IgM is found in relatively small amounts IgG > IgA > IgM
4. Complement activation
y y
IgG = will do it if conditions are optimal IgM = very good complement activator
5. Placental transfer
y y
IgG is small enough to easily cross placenta and is the only immunoglobulin capable of doing so. IgM and the other classes do not cross placenta
6. Optimum temperature of reactivity

y y
a. IgG = 37 oC b. IgM = 4 oC (may react at any temperature below 30C)
7. Number of antigen -binding sites

y y
IgG has 2 binding sites IgM has 10 binding sites
Terms used to describe antibodies

Immunoglobulin : antibody formed as a result of immune stimulus (exposure to foreign antigen)
Naturally occurring antibody formed without prior exposure to foreign antigen
Autoantibody: antibody formed to one's own antigens (abnormal condition)
Alloantibody (unexpected, irregular, atypical): antibody formed to foreign antigens, but within the same species
Agglutinin: antibody capable of causing agglutination when reacting with corresponding antigen
Isoagglutinin : name commonly given to blood group antibodies anti-A and anti-B
Saline agglutinin: antibody capable of causing direct agglutination of antigens suspended in a saline medium without requiring any enhancement techniques
Hemolysin: antibody capable of causing hemolysis when reacting with corresponding antigen
Cold antibody (cold agglutinin) : antibody whose optimal temperature of reactivity is less than 30 C
o
Warm antibody: antibody whose optimal temperature of reactivity is greater than 35oC
Monoclonal antibodies
Monoclonal antibodies react with very specific antigenic determinants and therefore shows no cross-reactivity. They are not produced in humans or animals, but harvested from cells in cells grown in tissue culture. The tissue culture cells made from fusion of a plasma cell, which is the antibody producer and the myeloma cell, which provides longevity and ability to make large amounts of antibody
Monoclonal antibodies used in most reagent antisera today because contain high concentrations of highly specific antibod ies and lack infectious disease hazards associated with human-source antiserum.
ANTIGEN-ANTIBODY REACTIONS IN GENERAL

Rules of Thumb For in vivo Antigen-Antibody Reactions
1. If a person's cell have the antigen, the antibody should NOT be present in that person's serum 2. If an antibody to a blood group antigen is present in the serum of a person, his or her cells should lack that antigen 3. The antigens are on the cells and the antibodies are in the serum
Stages of Antigen-Antibody Interaction

The first stage is sensitization. Sensitization occurs when antibodies react with antigens on the cells and coat the cells.
The second stage of the reaction is agglutination. Agglutination occurs when antibodies on coated cells form cross -linkages between cells resulting in visible clumping.
FACTORS AFFECTING SENSITIZATION (In vivo or in vitro)

1. Specificity depends on the spatial and chemical "fit" between antigen and antibody
2. Since the immunoglobulins and the red cell membranes both have an electrical charge, there is an optimum pH. pH differences cause differences in chemical structures of antigens/antibodies, affecting the "fit".
3. The optimum temperature depends on the t ype of antibody involved. IgG antibodies react best at 37 oC; IgM react best at 4 oC.
4. Optimum incubation time: you need to incubate long enough to reach equilibrium, but not too long
5. The antigen's accessibility is also important since the antibodies must be able to reach antigens. Those antigens, like the ABO antigens, are on the surface of the red cell while others may be hidden in the crypts of the cell membrane.
FACTORS AFFECTING AGGLUTINATION IN VITRO

Number of Antigen Sites
The number of antige n sites on the red cell is important since the more antigen sites result in more antibodies being attached and forming cross -linkages. These cross-linkages result in agglutination
Size and Structure of the Antibody

The larger antibodies (IgM) can reach b etween more antigen sites on different red cells and therefore causing stronger agglutination reactions. IgM antibodies also have more binding sites to react with antigens and potentially causing cross linkages between 5 different cells.
Distance between Cells

Centrifugation of the cells attempts to bring the red blood cells closer together, but even then the smaller IgG antibodies usually can not reach between two cells. The larger antibodies, IgM, can reach between cells that are further apart and cause agglutination.
y
y y y y y
The concept Zeta potential is important to understand why the cells will maintain a certain distance from each other. Zeta potential refers to the repulsion between the red blood cells.It is due to an electric charge surrounding cells suspended in saline. It is cause by sialic acid groups on the red blood cell membrane which gives the cells a negative charge. The positive ions in saline attracted to the negatively charged red blood cells. The net positive charge surrounding ce lls in saline keeps them far apart due to repulsion from electric charges Smaller antibodies (IgG) cannot cause agglutination when zeta potential exists To overcome zeta potential techniques need to neutralize these charges One of the common techniques is: 1) Add albumin to test mixture 2) OH- groups of albumin neutralize positive charge
Antigen-Antibody Ratio
The optimum ratio is 80 parts antibody to 1 part antigen. There are specific terms for variations in this ratio.
Prozone - antibody excess: Antibodies saturating all antigen sites; no antibodies forming cross -linkages between cells; no agglutination Zone of equivalence: antibodies and antigens present in optimum ratio, agglutination formed Zone of antigen excess (Post -zone): too many antige ns - any agglutination is hidden by masses of unagglutinated antigens
In order to get optimum antigen -antiboy concentration in Blood Banking we make washed 3% saline suspension of red cells to mix with our reagents.
OBJECTIVES - ANTIGENS AND ANTIBODIES IN BLOOD BANKING

1. Define the following terms: Antigens Antigenic determinant Autologous antigen Homologous (allogeneic) antigen Antibody Immunoglobulin 2. Describe the characteristics common to all antigens 3. List the factors that determine whether an antibody will be formed in response to an antigen stimulus 4. Describe the characteristics of blood group antigens 5. Identify the parts of an antibody, and state the purpose of each part 6. Differentiate between: an autoantibody and an alloantibody an immune and naturally-occurring antibody an agglutinin and a hemolysin a warm and a cold antibody 7. Explain what is meant by "clinically significant" 8. List the classes of immunoglobulins 9. State the differences between IgG and IgM antibodies regarding: Size Number of binding sites Serum concentration Ability to cross placenta Ability to fix complement
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Ability to cause direct agglutination Optimum temperature of reaction Briefly describe how monoclonal antibodies are made State the main characteristics of monoclonal antibodies Differentiate between sensitization and agglutination List five factors that affect sensitization List five factors that affect agglutination Define specificity in antigen -antibody reactions, and explain how pH may affect it Explain how length of incubation affects antigen -antibody reactions Explain how accessibility affects antigen -antibody reactions Explain how the number of antigen sites on the red cells affect agglutination Explain how the size and structure of antibodies a ffects agglutination of red cells Explain what the zeta potential is, and how this may be overcome to promote agglutination Differentiate between prozone, zone of equivalence, and zone of antigen excess State the optimum ratio of antigen to antibody to promote agglutination
Table of Contents

Blood Banking

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Blood Banking

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LIFE'S BLOOD

Table of Contents CLASS NOTES

What are their ABO types ? ? ? ? ? ? ?

ABO Discrepancies MUST BE RESOLVED

GENERAL RULES TO RESOLVE:

PROBLEMS WITH SERUM TESTING

Expected Antibody Missing ANTI-A ANTI-B 4+ 0

PROBLEMS WITH RED CELL TESTING

Mixed-field agglutination Weak or missing antigens Unexpected antigen Polyagglutinable cells

Polyagglutinable cells ANTI-A 4+

RESOLVING PROBLEMS WITH SERUM TESTING:

Presence Of Unexpected Anti-A

Screening Cell I Screening Cell II Autocontrol

Lectin + A 1 CELL = 4+ Lectin + A subgroups CELLS = 0

Screening Cell I Screening Cell II Autocontrol

Screening Cell I Screening Cell II Autocontrol

Characteristics of rouleaux is that it:

Looks like agglutination macroscopically Microscopically it appears as "stacks of coins"

How would you resolve rouleaux problems?

RESOLVING PROBLEMS WITH CELL TYPING

Specialized tests would include absorption/elution techniques and saliva studies.

How would you resolve a possible acquire d B antigen?

2. Strong positive DAT

PROBLEMS WITH BOTH CELLS AND SERUM

ANTI-B 4+ Immediate 37oC

Screening Cell I Screening Cell II Autocontrol

2. To resolve serum typing difficulties:

OBJECTIVES - ABO DISCREPANCIES

Sources of Antibody for Testing

Known Source with known components

Antigen Reagent Red Blood Cells Antibody Commercial Antisera

Testing Procedures Routinely Done in Blood Banking

systems (examples: K, Donor RBC's E, C, Fya, Jka)

antigens (examples: anti-K, anti-E, anti-C, anti-Fya, anti-Jka)

WASHED CELL SUSPENSIONS

Washing Red Blood Cells Before Making the 3% Suspension

COLLECTING BLOOD BANK SAMPLES

OBJECTIVES - GRADING REACTIONS; WASHED CELL SUSPENSIONS; BLOOD BANK SAMPLES

QUALITY ASSURANCE IN BLOOD BANKING

Quality Control Quality Assurance Quality Improvement

Process control, final inspection, and handling elements

Documents and records (4 levels)

Incidents, errors, and accidents

Assessments: internal and external

Facilities and safety

OBJECTIVES - Quality Assurance in Blood Banking

Complement-coated red blood cells

Types of Antiglobulin Tests

Interpretation of Antiglobulin Tests

(Wash Bottle Image)

Add Anti-Human Globulin

Visible Agglutination in the test tube: Grade the reaction strength

Summary of the reaction:

Antibodies are not attached to the antigens during incubation.

Wash the cells 3 times to remove any unattached antibodies.

No visible agglutination and therefore a negative test

Add Coombs Control Check Cells

Check cells agglutinated and original test cells remain unagglutinated.

Coombs Control Agglutinated by Anti-Human Globulin

False Positives and Negatives

Direct Antiglobulin Testing

Clinical Causes of Positive DAT

Indirect Antiglobulin Testing