Discussion. Principle. Laboratory Procedure Manual EXERCISE 3. Blaney, Chapter 4 The ABO and H Blood Group Systems. Textbook: 20 points.

Transcription

1 Exercise 3 ABO and D Typing Textbook: Blaney, Chapter 4 The ABO and H Blood Grop Systems Skills: 20 points Objectives 1. Explain why the ABO blood grops are the most significant of all blood grop systems. 2. State the 4 major ABO blood grops. 3. List each blood grop and state the ABO antigens present on the red blood cells. 4. List each blood grop and state the ABO antibodies expected to be present in the serm/plasma. 5. State what is detected in performing the ABO forward type. 6. State what is detected in performing the ABO reverse type. 7. Given a set of forward and/or reverse typing reactions, interpret the reslts. 8. Define the term ABO discrepancy. 9. List 6 common cases of ABO discrepancies. 10. List 6 methods which may be tilized to resolve ABO discrepancies. 11. State the action to be taken when an ABO discrepancy occrs and patient rgently needs a transfsion 12. State the significance of the D antigen as it relates to transfsion. 13. Define immnogenic. 14. State the percentage of the poplation which is D positive and D negative. 15. State the principle of the weak D test. 16. State the significance of a weak D positive person as a patient and as a blood donor. 17. List 2 instances, when typing for the D antigen, in which a D control mst be rn. 18. Compare and contrast the prpose of the D control for an AB pos person verss a D negative person. Discssion The ABO typing is the most important test performed in transfsion practice today. The single most common case of transfsion-related fatalities is de to a patient being transfsed with ABO incompatible blood. These reactions occr becase individals form potent, natrally occrring antibodies to ABO red cell antigens which they do not possess. When transfsed with ABO incompatible blood, an immediate antigen/antibody reaction occrs which, if not detected in time, may be fatal. The terms D positive and D negative refer to the presence or absence of the D antigen on the red cell. Approximately 85% of the general poplation have the D antigen on their red cells. After A and B antigens, the D antigen is the most important antigen in transfsion practice. The D antigen is very immnogenic. Individals who lack the D antigen mst be given D negative blood to prevent antibody stimlation. Principle ABO antibodies are of the IgM class and react preferentially at 22 C (RT) or below. Incbation at warm temperatres may case a false negative reaction. Enhancement of weak reactions may be obtained by RT incbation or incbation at 4 C. MLAB 2431 ABO and D Typing 21

2 There are three (3) types of tests which mst be performed in order to determine an individal s ABO/D type: ABO forward, ABO reverse and D typing. 1. ABO Forward Typing This test is sed to detect the presence or absence of A and/or B antigens on an individal's red blood cells. An individal's ABO grop is determined by testing the red blood cells with reagent anti-a and anti-b sera. Aggltination of the individal's red cells by the appropriate antisera signifies the presence of the antigen on the red cell while no aggltination with the antisera signifies its absence. Additional testing of the red cells with Anti-A,B sera may be sed for the detection of weak sbgrops of A or B. Becase weak A or B sbgrops have fewer antigens present on the red blood cells, very weak or negative reactions may occr with anti-a or anti-b serm., bt will give stronger or positive reactions with anti-a,b serm. The se of potent moncolonal antibodies has dramatically decreased the se of anti-a,b. In the past Anti-A sera was obtained from grop B individals, anti-b serm was obtained from grop A individals, and anti-a,b serm was obtained from grop O individals. Most facilities crrently se monoclonal sorce antibodies for blood groping. 2. ABO Reverse Typing This test is sed to detect ABO antibodies in an individal's serm, and is sed to confirm the ABO Forward Typing. There are strctres present in natre on certain bacteria and pollens which are very similar to the A and B red cell antigens. Individals will prodce potent, natrally occrring antibodies directed against the ABO antigens they lack. The patient's serm is mixed with reagent grop A 1 cells. Aggltination indicates the presence of Anti-A in the patient's serm. Mixing the patient's serm with reagent grop B cells similarly allows for the detection of anti-b in the patient's serm. The otcome of the serm typing (Reverse Typing) is compared with the otcome of the cell typing (Forward Typing) to ensre the accrate ABO determination. Any discrepancy mst be resolved before final interpretation of the blood grop is made. Grop A individals lack the B antigen and their serm will aggltinate the reagent B cells de to their natrally occrring anti-b. Their serm will not aggltinate the reagent A cells since this antigen is present on their own cells. Grop B individals lack the A antigen and their serm will aggltinate A cells with their natrally occrring anti-a. Their serm will not aggltinate the reagent B cells. Grop O individals lack both A and B antigens and their serm will aggltinate both the A and B reagent red cells. Grop O individals have 3 natrally occrring antibodies in their serm: anti-a, anti-b and anti- A,B. Grop AB individals have both A and B antigens on their red cells, and their serm will not aggltinate the A or B reagent red cells. MLAB 2431 ABO and D Typing 22

3 These two (2) tests, forward and reverse typing, constitte the ABO typing of an individal and are one of the check systems in rotine Blood Banking. Remember that the forward typing is indicative of the antigens present on an individal's red cells, while the reverse typing is indicative of the antibodies present in the individal's serm or plasma. The forward type mst correlate with the reverse type and any discrepancy mst be resolved. An ABO discrepancy is a sitation in which reactions obtained in the forward type DO NOT match or correlate with reactions obtained in the revers type. For example, no aggltination when testing a patient with anti-a and anti-b indicates that a patient is grop O. The expectation then is that both the A and B reagent cells will be aggltinated by the patient s serm. If negative reactions occr with one or both reagent red cells then an ABO discrepancy has occrred. No blood may be issed for transfsion ntil the ABO discrepancy has been resolved. If the need is rgent the technician may need to conslt with the patient s physician and the pathologist. The type of discrepancy involved will determine how the sitation is to be handled. Reasons for an ABO discrepancies: 1! sbgrop of A (A 2 or A2B) with anti-a 1 - patient forward types as A bt aggltinates A rbcs.! strong cold atoaggltinins present - A, B or AB patients will aggltinate reagent rbc s! patient may be an infant, elderly or immnodeficient reslting in decreased antibody levels - lack of expected aggltination reactions of reverse cells.! roleax - nexpected positive reactions in reverse cells.! nexpected antibodies in the serm -nexpected positive reactions in reverse cells.! patient s red blood cells heavily coated with protein - nexpected positive reactions in forward type. Resoltion of ABO discrepancies: 1. Repeat the forward and reverse - this shold always be the first corse of action 2. If patient is grop A or AB, bt reverse types as an O (false positive), test the RBCs with anti-a 1. If the patient is A 2or A2B test their serm against A 2cells. Lack of aggltination of the A 2cells indicates that the patient has anti-a If the reverse type gives false negative reactions allow the serm and cells to incbate at RT for 5-10 mintes. Respin. False negative reactions are sally de to low levels of antibody being present. The incbation period allows time for additional antibody attachment. 4. False positive reactions in the reverse may be de to strong cold aggltinins additional testing mst be performed cold aggltinin titer, prewarmed techniqe to resolve this problem. 5. False positive reslts in the reverse may be de to roleax. Examine tbes microscopically to confirm. Use saline replacement techniqe (spin tbes down, remove serm, add saline, respin and read). Negative reactions will be obtained if roleax was present. 6. False positive reslts in the forward type may be de to the red blood cells being so heavily coated with immnogloblin that the cells spontaneosly aggltinate. Perform a DAT, a positive reslt confirms this as the case. An extensive work p wold be initiated. Transfse with O negative if this is an emergency or valid typing cannot be determined. 3. D Typing After ABO, the most important antigen in transfsion practice is D. The D antigen is a member of the Rh system. Unlike the ABO system, Rh antibodies are not natrally occrring, therefore, persons who lack the D antigen do not have anti-d antibody in their serm. Antibody formation reslts from exposre to immnizing red cells that possess the D antigen, either throgh transfsion or pregnancy. MLAB 2431 ABO and D Typing 23

4 A high proportion of D negative sbjects exposed to the D antigen will prodce antibodies. The immnogenicity of D (i.e., the likelihood of its provoking an antibody if introdced into a D-negative recipient) is greater than that of virtally all other red cell antigens stdied. Of D negative individals who receive a single nit of D positive blood, 50-75% can be expected to form anti-d. Exposre to amonts as small as 0.1 ml of red blood cells can case antibody formation. All D negative recipients mst receive D negative blood. Detecting the D antigen consists of testing the individal's red blood cells with anti-d. An Rh control is necessary if the anti-d reagent sed contains a high protein media to enhance the strength of the reaction. Some individal's red cells may spontaneosly aggltinate when placed in this high protein media, giving a dangeros false positive reaction. The Rh control consists of the high protein media only. If the control is positive, the test is invalid and alternate methods mst be sed to determine the individal's tre D type. If the D type cannot be determined, the individal mst receive D negative blood. A positive reaction with anti-d and a negative control indicates that the individal possesses the D antigen on their red cell and is D positive. If a negative reaction is obtained in both the anti-d and Rh control tbes, frther testing is reqired to establish D type. Most clinical blood banks are now sing saline anti-d or chemically modified anti-d which does not reqire a control to be rn nless the patient/donor is AB positive. If a person is AB positive there will not be a forward typing tbe having a negative reaction. One mst ensre the validity of reslts by rnning a negative control as specified by the manfactrers instrctions. A negative control tbe verifies that the individal is AB positive while a positive reaction in the control tbe indicates the need for additional testing to determine the individal's ABO and D type. Not every D positive cell sample reacts with the anti-d sera on immediate spin. The D antigen may actally be present, the cells may actally be D positive, bt additional testing is needed to demonstrate the presence of the weakly expressed antigen. This type of reactivity is de to variants of the D antigen collectively called weak D positive (previosly called D ). In the weak D (D ) test, the negative D tbe and control tbe are allowed to incbate at 37 C for 15 mintes. If the D antigen is present, the cells will be sensitized with the anti-d contained in the D tbe. To determine whether this has occrred, the cells are washed and anti-hman globlin (an anti-antibody) is added. If the cells have been sensitized, aggltination will occr, indicating that the individal is D positive. The correct term for a patient whose cells react at anti-hman globlin phase with anti-d is weak D positive. If negative reactions are obtained, the individal is D negative. The control tbe mst be negative in order for the test to be valid. If the weak D test and Rh control tbes are positive a DAT mst be performed. If the DAT is positive this indicates the patient cells are coated with immnogloblin. A false positive D test may be obtained on individals whose red blood cells are coated with immnogloblin. The weak D type may case confsion when transfsion is reqired. Weak D donors are considered D positive. The weak D recipient is considered D negative. The weak D test is not reqired for recipients by AABB Standards. Bt de to the confsion of when this test shold or shold not be performed, it is best to perform the test on all patients and donors. MLAB 2431 ABO and D Typing 24

5 4. Significance of the D control Many stdents have a difficlt time nderstanding the significance of the two sitations in which a D control mst be rn. When a patient types as AB positive there is no tbe in the forward type with a negative reaction. And even thogh the reverse type might match (no aggltination of A or B reagent rbc s) this cold be a false negative de to a decreased antibody prodction in the patient. The positives in the forward tbe cold be de to spontaneos aggltination of the red blood cells de to heavy protein coating of the red blood cells. If one to were erroneosly omit the D control and the patient was in reality an A positive, then transfsion of AB positive wold be potentially fatal de to the patients natrally occrring anti-b antibodies. A negative D control will verify that the patient s rbc s were NOT spontaneosly aggltinating. When a patient types as D negative an additional phase of the test, the weak D test, mst be performed to confirm the patient as trly D negative. The prpose of the D control for the weak D test is to control the antihman globlin (AHG) portion of the test. Some patient s rbcs may be coated with antibody de to disease (ato-immne hemolytic anemia) or in response to a recent transfsion. If the D control is positive at the AHG phase of testing this invalidates the test and will prompt additional testing of the patient s rbcs. If one to were erroneosly omit the D control then one wold incorrectly interpret the patient as a weak D positive. In an emergency sitation in which no D negative blood was available a weak D positive blood may receive D positive blood. The D antigen is very immnogenic. Transfsion with D positive blood may immnize the patient to the D antigen (the patient will prodce anti-d). This wold be devastating in a female patient who plans to have children. In smmary, the D control for an AB positive is to control the immediate spin phase of testing to ensre that the patient s rbcs are not spontaneosly aggltinating. The D control for D negative patients is to control the AHG phase of testing and prevent failre to detect a positive reaction de to patient s rbcs being coated with antibody. If after stdying yor text, lectre gide and lab procedre yo contine to have difficlty with this concept make an appointment with yor instrctor. Models of D and D complex antigens MLAB 2431 ABO and D Typing 25

6 Models of D and D cells MLAB 2431 ABO and D Typing 26

7 PROCEDURE: Spplies 1. Blood Bank Reagent Rack 2. 12x75 Test tbes 3. Plastic Blood Bank Pipets 4. Blood Bank Saline Sqeeze Bottle (make sre it is labeled 0.85% NaCl) ABO and D Typing - Tbe Test CAUTION: NEVER place any specimen or reagents in an nlabeled tbe. 1. Write patient's fll name and hospital nmber on TWO tbes, one for serm or plasma and one for cell sspension tbes. 2. With a pipette, remove serm or plasma from centrifged specimen (try not to contaminate with cells) and place in one tbe. (NOTE: If serm or plasma is already separated, check that the write the patient's fll name and hospital nmber on it, label an additional tbe for patient rbcs and proceed to #4.) 3. Place tip of the pipette at bottom of clotted specimen or top of cells in EDTA specimen and aspirate a small amont of cells (approximately 3-5 drops) and place in yor second labeled tbe. 4. Fill the tbe containing cells three-forths fll with saline, sing enogh force to thoroghly resspend the cells. Do NOT overfill. DO NOT place tip of saline bottle into tbe. 5. Place serm and cell sspension across from each other (if volmes are the same) in the serofge head. If the volmes ARE NOT the same yo will need to prepare balance tbes. Spin serm and cell sspension one (1) minte in the serofge. 6. While serm and cell sspension are spinning label 5 additional tbes with patient initials at the top of each tbe and the identity of the reagent to be added below the initials. Do this by placing the palm of yor hand over all tbes with the open ends at the top. Yo can then qickly label all tbes at one time. Place in rack as indicated below. Sample Pt RBCs anti-a anti-b anti-d space Pt Serm A 1 cells B cells 7. Decant the saline from the cell sspension completely by inverting the tbe over the waste bcket, allowing all saline to drain ot, shake the inverted tbe 3 times. DO NOT shake tbe while saline is draining ot, yo will case the rbcs to be rinsed ot of the tbe into the waste bcket. 8. Add enogh saline to the patient s cells to obtain a 4-6% cell sspension (compare color with that of the reagent red cells in the dropper). 9. Add one (1) drop of reagent antiserm in the forward typing tbes (anti-a in anti-a tbe, anti-b in anti-b tbe, etc.) 10. Add three (3) drops patient serm or plasma to the A cell and B cell reverse typing tbes. NOTE: Visally inspect all tbes at this time to make certain that all tbes have serm or anti-serm in them. 11. Add one (1) drop of patient cells to each forward typing tbe. 12. Add one (1) drop of well mixed reagent reverse cells to each appropriate tbe. 13. Mix tbes thoroghly by gently shaking. 14. Spin all tbes for 15 to 20 seconds. NOTE: Serofge MUST be balanced. MLAB 2431 ABO and D Typing 27

8 15. Read and immediately record graded reactions as they are read. Yo mst hold the tbe in yor hand, observe the reaction and record it. DO NOT place the tbe in the rack ntil yo have recorded the reslts! NOTE: The manner in which the RBCs are dislodged from the bottom of the tbe is critical. Shake the tbe gently ntil all cells are dislodged. This is most easily accomplished by holding the tbe between thmb and forefinger and gently agitating, then tilting the tbe. Avoid over shaking which breaks p fragile aggltinates (false negative reaction) while inadeqate mixing may reslt in a false positive interpretation. If yo get reactions of less than 3-4+ in the D or reverse typing tbes yo are shaking too hard which cold reslt in a false negative reslt. 16. If anti-d is positive and patient is not Grop AB positive, stop here. a. If tbe containing anti-d is negative, contine to step 17 (weak D test). b. If patient appears to be AB pos go to step Set p a tbe labeled with patient initials and D ctrl. Place one drop of Rh control in the tbe followed by 1 drop of the patient s washed rbc sspension. Spin, read and record the D ctrl tbe. Place both the negative D tbe and D ctrl tbe in the 37 heat block for 15 mintes. 18. After 15 mintes, remove the D and D ctrl tbes, wash three (3) times with saline as follows: a. Fill tbes three-forths fll with saline. b. Spin in serofge for one (1) minte. c. Completely decant saline by trning pside down and allowing all saline to drain ot of the tbe, then shake three times. NOTE: do NOT shake as the saline is draining ot or yo will LOSE yor rbcs and will need to repeat the test. d. Resspend cell btton by vigoros tapping with yor finger. e. Add saline with sfficient force to resspend cell btton. NOTE: after resspending cells with saline yo notice a cell btton at the bottom either parafilm each tbe and invert OR add an additional wash. f. Repeat a-e two (2) additional times, for a total of three washes. 19. After decanting saline after last wash contine to hold the tbes pside down and blot ends of tbes with biowipe to obtain a dry cell btton. 20. Add two (2) drops of anti-hman globlin serm to each tbe. Mix well and centrifge for 15 seconds. 21. Gently resspend cell btton, looking for aggltination as in step 15 above. 22. If negative macroscopically, observe microscopically also. Record graded reactions now. 23. To all tests showing a negative reaction, add one (1) drop of coombs control cells. 24. Centrifge for 15 seconds. Shold get 1 to 3+ reaction. If negative reaction is obtained, test is invalid and mst be repeated. Record graded reactions now. 25. If all tbes in the forward type are positive (patient appears to be AB positive) a negative control tbe mst be rn. a. Label a tbe with patient initials and "D ctrl" below it. b. Place 1 drop of the patient red blood cell sspension in the tbe. c. Add one drop of Rh control reagent to the D ctrl tbe, mix well. d. Centrifge for 15 seconds. e. Shake gently to resspend. f. If tbe is negative, it confirms patient is AB positive, if it is positive additional testing mst be done. MLAB 2431 ABO and D Typing 28

9 Grading Serological Reactions in Tbe Testing Grade Appearance 4+ Complete aggltination no naggltinated RBCs s 3+ Intermediate between 3+ and Strong reaction a few detached masses of aggltinated RBCs; no naggltinated RBCs w 3+ Intermediate between 2+ and 3+ s 2+ Intermediate between 2+ and Moderate reaction large aggltinates in a sea of smaller aggltinates; few naggltinated RBCs w 2+ Intermediate between 1+ and 2+ s 1+ Intermediate between 1+ and Weak reaction many aggltinates of p to 20 RBCs with some smaller aggltinates and naggltinated RBCs w 1+ Intermediate between ± and 1+ ± Granlar reaction scattered aggltinates of 6-8 RBCs with many naggltinated RBCs seen microscopically r 0 Rogh reaction RBC btton does not disperse smoothly; edge of btton appears rogh (characteristic of some HTLA antibodies) 0 No reaction s MF Mixed-field strong large complete aggltinates with a small nmber of naggltinated RBCs MF Mixed-field moderate-sized aggltinates together with naggltinated RBCs w MF Mixed-field weak few clmps of aggltinated RBCs, bt majority of RBC naggltinated s H Strong hemolysis few or no intact RBCs seen H Moderate hemolysis some intact RBCs present w H Trace hemolysis many intact RBCs present Marsh, W. L., Scoring of hemaggltination reactions. Transfsion 1972, 12: MLAB 2431 ABO and D Typing 29

10 Interpretation Forward Reverse Optional Blood Grop anti-a anti-b anti-a,b A cells B cells Interpretation O O O + + O + O + O + A O O B O O A,B D Typing Weak D Test anti-d D ctrl D D (D ) ctrl 3+ Pos Invalid O O 3+ O Pos w O O 3+ O Neg O O Invalid If the D control gives a positive reslt the D typing is invalid and additional testing mst be done.. Report reslts as ABO type followed by D type. Example: A pos: O pos; AB neg MLAB 2431 ABO and D Typing 30

11 Name Exercise 3 ABO and D Typing Recording Reslts For each of the following record the patient name (last name first) and the identification nmber directly from the tbe of blood. 1. Record GRADED reactions (0-4+) in EACH colmn by observing the reaction of the tbe while it is held IN YOUR HAND. 2. Leave space blank if test was not done. 3. Report interpretation as blood grop followed by pos if D pos, or neg, for D neg. Example: O pos 1. Forward Type D Type Weak D Test Reverse Type Patient Name ID Nmber Anti-A Anti-B Anti-D *D Control D cells D Control cells A Cells B Cells Interpretation *This is performed only if a high protein anti-d reagent is sed or the patient appears to be AB positive. MLAB 2431 ABO and D Typing 31

12 Name Exercise 3 ABO and D Typing Stdy Qestions 1. What is the most common case of transfsion related fatalities? (1 point) 2. State the immnological reason why ABO typing is the most important test performed in pretransfsion testing. (1 point) 3. Define the terms D positive and D negative. (1 point) 4. What is being detected in the forward typing test? (O.5) 5. What is being detected in the reverse groping test? (0.5) 6. State the prpose for sing anti-a,b typing sera.(1 point) MLAB 2431 ABO and D Typing 32

13 7. List the three ABO typing seras AND state the blood grop which prodces each. (1.5 points) 8. For each of the blood grops listed below state what antibodies will be detected in the serm and fill in 1 the expected reactions (positive or negative) with the A and B cells. (4.5 points) Blood Grop Antibodies Present in the Serm Expected reaction of serm with A cells Expected reaction of serm with B cells a. 0 b. A c. B d. AB 9. State 4 conditions/reasons which may reslt in an ABO discrepancy. (2 points) 10. List 4 techniqes which may be tilized to resolve ABO discrepancies. (2 points) MLAB 2431 ABO and D Typing 33

14 11. Define immnogenicity as it relates to the D antigen? (1 point) 12. What percentage of the poplation is D positive? D negative? (1 point) 13. Correctly interpret the following D typing reactions according to the information provided in the laboratlry instrctions. (1.5 points) Anti-D D ctrl D D ctrl Interpretation A. 4+ NA NA NA B C D controls are nnecessary when sing saline or chemically modified anti-d EXCEPT when the patient is which AB pos or D neg. Explain in detail why a control mst be rn in these sitations. (2.0 points) a) AB post MLAB 2431 ABO and D Typing 34

15 b) D neg 15. State the prpose of performing the weak D test. (1 point) 16. Describe the transfsion stats (D interpretation) of the weak D individal as a donor and as a recipient. (1 point) 17. The weak D test is positive. What test mst be performed to ensre that this is not a false positive reslt? (0.5) 18. According to the reporting format demonstrated, interpret the following ABO/D types. Notice that yo are to write yor interpretation of the forward type in the Interp Forward colmn, the reverse type in the Interp Reverse colmn, then compare the 2 determine validity of the reslts. If the reaction given are NOT valid write invalid for that portion of yor interpretation. Once yo have compared the reslts write yes for valid (forward and reverse match) or no for invalid (forward and reverse did not match). (2 points each) MLAB 2431 ABO and D Typing 35

16 A anti- Forward Typing anti- D anti-d B control Interp Forward Reverse Typing A 1 cells B Cells Interp Reverse Valid? Yes/No a. O O O O b. O O 3+ O 4+ c. O O d O O e O O O O f O 1+ O If yo answered No in the Valid? colmn describe the discrepancy yo observed and se the information provided in the laboratory to provide an explanation.. Extra credit awarded for correct problem solving. 18. Next week yo mst trn in an index card with the ABO and D tbe test written and condensed on it. Only one index card is needed. This is to be written in a simplified manner to aid yo in performing this procedre. THIS IS NOT OPTIONAL. This card will be sed throgh ot yor blood bank corses this semester and dring yor clinical rotation at the hospital. MLAB 2431 ABO and D Typing 36