Page 1 of 17 Red Cell Serology Introduction Red cell serological reactions form the basis of laboratory testing in a blood transfusion service. Antigen-antibody reactions in blood group serology are usually detected by •
Haemagglutination
•
Complement fixation
•
Neutralization
•
Absorption and Elution
•
Precipitation
An error in ABO or Rh grouping of a patient or a crossmatch incompatibility, can have the most serious effects on the patient and may even be fatal. It is therefore e ssential to perform all the technical procedures very carefully and precisely. In addition, it is very important to use recommended controls with each serological technique used. Reagents can deteriorate due to improper s torage or contamination and such errors can only be detected by correct application of controls for t hat reagent. The various basic serology techniques used in a blood transfusion laboratory are •
ABO grouping
•
Rh grouping
•
Antiglobulin testing
•
Antibody detection and identification
•
Pretransfusion testing
•
Specialized serology procedures o
Antibody titration
o
Saliva testing for A,B and H substance
o
Preparation and use of lectins
o
Preparation and use of enzymes
o
Preparation and use of LISS
o
Investigation of autoimmune haemolytic anaemia
ABO Grouping Aim of this subsection is to make the medical officers understanding the principle of ABO grouping so that they can perform accurate basic blood grouping test. ABO Grouping Techniques Principle The principle principle of ABO grouping is based on a s pecific agglutination reaction between antigens on red cells and IgM antibodies in the tyuping serum. Cell grouping is determined by testing an individual’s red cells with known blood grouping reagents and the corresponding serum grouping with known A cells, B cells and 0 cells. Practical Aspects of ABO Grouping 1. ABO grouping tests should be done at room temperature (2Oo24oC) or lower; testing in hot environment weakens the raction. 2. Routine ABO grouping must include both cell and serum testing as each serves as a check on the other. 3. Antisera used in the ABO grouping must be used as per the manufacturer’s instructions. 4. Adequate controls should be put with each batch of test s for quality control of the reagents,
Page 2 of 17 alternately once a day the reagents must be checked with appropriate cells. Before use all t he reagents must be checked grossly to rule out any t urbidity or contamination. 5. Tubes, slides, tiles, microplates or gel cards should be labelled properly. One should not rely on the colour of the dye to identify the antiserum. 6. The glassware used should be dry and clean. 7. Serum should be added before adding cells and each tube should be examined after serum has been added to ensure that none has been missed. 8. Results should be recorded immediately after observation. 9. Concave mirror (agglutination viewer) or microscope may be used to examine reactions that appear negative by the naked eye. ABO grouping sera It is essential to use reliable grouping reagents to obtain correct results. Commercially prepared polyclonal (human source) and monoclonal (tissue culture) antisera are available. Monoclonal antisera give more specific reactions and are very sensitive for weaker reactions. Adequate quality control must be done before purchasing commercial antisera. E ach batch of antisera must be checked against A1,A2, (if available, otherwise A group) B and 0 cells before use and should be put to routine use onlyw hen unequivocal results are obtained. The potency of any antisera deteriorates rapidly if kept for too long at ambinet tem perature, groupign sera should therefore, be kept at 4°C or as directed by the manufacturer when not in use. Frozen antisera must be completely thawed before use and no refreezing should be done.
Blood sample for ABO grouping 1. Properly labelled samples of clotted blood collecte d in screwcapped plain test-tube are most suitable for ABO grouping. Ths amples should be stored at 4°C and preferrably be tested within 48 hours. 2. Haemolysed samples are not suitable for testing. 3. The blood sample may be centrifuged at 1000-3000 rpm for 3 minutes for adequate serum separation. 4. Separated serum should be placed in a prelabelled tube for serum grouping and other serological tests. 5. Cells from the test sample should be washed in 0.9% nromal saline and a 2-5% cell suspension should be prepared.
Preparation of Red Cells for ABO Testing Preparation of reagent red cells in addition to patients’ and donors’ red cells is required daily while performing the serum grouping and cell grouping. Reagent red cells When performing an ABO serum grouping, it is important to ensure that the cells prepared are fresh and well-selected
Page 3 of 17 ‘A’ cells Pool known fresh A group cells from at least 3 donors ( to include Al subtype). Wash three times with normal (0.9%) saline, make a 2-5% suspension in saline for use. ‘B’ cells Pook known fresh B group cells from 1-2 donors. Wash three ti mes with normal saline, make a 25% suspension in saline for use.
‘0’ cells Pool known fresh C group cells from 1-2 donors. Wash three time s with saline and make a 2% suspension in saline for use. Cells are washed in isotonic saline to remove all traces of plasma or serum. They m ust then be accurately diluted to give a standard suspension in saline by either using a graduated t ube (Wintrobe’s tube) or by counting drops to make a 2-5% cell suspension. When prepared, the cells should be checked against anti-A and anti- B. The cells should be kept at 4°C in the refrigerator when not in use. If low-ionic strength saline solution (LISS) is being used in the laboratory as an enhancing medium, then the last wash should be in LISS and the cells are thereafter suspended in LISS to make a 2-3% cell suspension. Patient and donor cells The test cells also should be washed at least once with saline and suspended to make a 2-5% suspension in saline. When dealing with large number of samples, adequate precautions must be taken to avoid mislabelling of tubes and the cells must be dispensed in already labelled tube for washing. Control cells for ABO grouping Positive
Negative
Anti-A
A2 (A2B)
B
Anti-A
Al
A2
Anti-B
B(Al,B)
A2
Anti-AB
A2B(A2)
0
Al
A2
B
AB
0
Anti-A
+
+
-
+
-
Anti-Al
+
-
-
+
-
Anti-B
-
-
+
+
-
Anti-AB
+
+
+
+
-
Blood grouping - Methods & Procedure Three manual methods can be used when performing blood grouping: Three - Glass slide or white porcelain tile - Glass test tube - Microwell plate or microplate Newer techniques - Column technique (sephadex gel) - Solid phase tests Slide or Tile Testing
Page 4 of 17 This technique may be used for emergency ABO grouping tests or for preliminary This grouping particularly in an outdoor camp, however it should always be supplemented with a cell and serum grouping using any one of the other above mentioned techniques. Slide or tile testing is not recommended for routine use because it is not reliable for - weakly reactive antigens on cells - serum grouping with low titre anti-A or anti-B Disadvantages - Less sensitive than the tube test - Drying up of the reaction mixture can cause aggregation of cells, giving false positive results. - Weaker reactions are difficult to interpret. Procedure 1. Place 1 drop of anti-A and 1 drop of anti-B reagent separately on a labelled slide or t ile. 2. Add 1 drop of 20% test red cell suspension to each drop of the typing antiserum (the suspension may be prepared by adding 20 parts of red cells to 80 part of normal saline). 3. Mix the cells and reagent using a clean stick. Spread each mixture evenly on the slide over an area of 10-15 mm diameter. 4. Tilt the slide and leave the t est for 2 minutes at room te mperature (22°-24°C). Then rock again and look for agglutination. 5. Record the results. Tube Testing Test tubes either of glass or plastic may be used, of lOx75mm size. T he tube technique is more sensitive than slide technique for ABO grouping. Advantages of tube testing - It allows for fairly long incubation without drying up of the tubes’ contents. - Centrigugation involved enhances the reaction allowing weaker antigens and antibodies to be detected. - Simplicity of reading and grading of results. - Clean and more hygienic. - Requires smaller volume of reagents. Procedure Saline agglutination test for cell and serum grouping by tube method 1. Cell grouping / forward grouping 2. Serum grouping / reverse grouping Reagents required 1. Known antisera (anti-A, anti-B, a nti-AB), monoclonal or polyclonal. 2. Red cell suspension - reagent cells (Ac, Bc and Cc); test red cells (patients or donor) Cell and serum grouping 1. Spin test sample to separate serum. 2. Set up 6 tubes correctly labelled with donor/patient no. Anti A, Anti-B, Anti-AB, Ac, Bc and Cc. 3. Prepare once washed 2-5% suspension of the test cells. 4. Add I drop of anti-A in tube labelled A, anti- B in tube labelled B, and anti-AB in tube labelled AB . 5. Add 1 drop of 2-5% test cell suspension in the three tubes A,B and AB. 6. Add 2 drops each of the test serum in tubes labellaed Ac,Bc and Cc. 7. Add I drop each of reagent A cells in labelled tube Ac, B cells in labelled tube Bc and 0 cells in tube labelled Oc. 8. Mix all the 6 tubes and centrifuge at 1000 rpm for 1 mi nute. 9. Resuspend cell button by gently shaking the tubes and read against well-lit background. 10. Record results according to grades of agglutination.
Defining the Stregth of Reaction (Grading of Agglutination)
Page 5 of 17 To record the difference in the strength of reaction, it is necessary to have a system of grading or scoring the reactions, as depicted i n figure. Grades 4+ 3+ 2+ 1+ w Zero or -
Description 1 big clump 2 or 3 clumps many small clumps with clear supernatent many small clumps with turbid supernatent granular suspension smooth suspension
H
partial or complete haemolysis (positive reaction)
Interpretation
Anti-A + +
Cell grouping Anti-B Anti-AB + + + + +
Ac + + -
-
-
+
-
Serum grouping Bc Oc + + +
+
Result A B O AB AB Oh or any other irregular antibody
Precautions 1. Always use throughly cleaned test tubes for the test. 2. While reading the result, shake very gently to dislbdge the button. 3. Use correct speed and time centrifugation t o avoid erroneous results. Microplate Technique Microwell plate consists of a small tray w ith 96 small wells each of which can hold about 200300u1 of reagent. Microplate technology is gaining widespread popularity due to increasing workload in blood transfusion laboratories and recent availability of packaged automated system. Three Thr ee types of microplates are available a. U-type well b. V-type well c. Flat-bottom The U-type well is generally used in red cell serological work as it is easier to r ead the results in U- bottom plates. Flat bottom plates are useful in EL ISA technology but are unsuitable for liquid-phase blood group serology. U-well are preferred to V-well plates for resuspension technique because of the case of suspension prior to reading and their superior optical quality when using automated plate reader. Advantages of Microplate ABO grouping - Small volumes and low concentration of sera and red cells are used, making it cost-effective. - Easy handling of a microplate, which can replace 96 test tubes. - Batching of samples can be achieved with considerable economy in space and time. - If larger laboratories acquire microplate hardware items e.g. reagent dispenser, sample handler and cell washer it may further reduce the operation time. - Large batches of plates can be predispensed with antisera and reagent red cells before testing. - The technique of microplate grouping may be automated by on-line data capture in larger laboratories, which may help in ---- reduction in reading and transcription errors ---- saving in staff time ---- use of bar codes for samples and microplate identification ---- integration into a comprehensive computer system for storage of data. Equipment required for microplate grouping Microplate - Untreated rigid polystyrene microplates -Pipettes -Manual / automated single or multi-channel dispenser -Variable volume dispenser (20 - 50 ul) -Centrifuges -Bench-top centrifuge with head for carrying microplates
Page 6 of 17 -Microplate shaker -Two/four place shakers -Reading aid -Automated plate readers -Automated plate reader based on ELISA reading system
Blood Grouping - Newer Techniques Column technique Recently interest has been generated in use of column te chnology for serological testing. The aims of this technology is to standardize red cell agglutination reactions and trap the agglutinates to permit simple and reliable reading. The column consists consists of special microtubes containing a sephadex gel m atrix. Red cells and serum or red blood cells alone are dispensed into the microtubes, incubated if necessary and then centrifuged under strictly controlled parameters. The gel within the microtubes acts as a sieve, unagglutinated cells form a button at the bottom of the microtube and agglutinated red blood cells are trapped in t he gel. The Sephadex Sephadex gel may be •
Neutral
•
Specific reagent o
AH G
o
Anti-A
o
Anti-B
o
Anti-D
o
Anti-Kell, etc.
Image : Reading and grading of haemagglutination in gel system
Advantages of gel system - Superior in sensitivity to conventional tube test without loss of specificity. - Easy to use. - Requires small amount of red cells and serum for t esting (1O-40m1), making it ideal for neonatal and paediatric use. - Reactions are easily visible and can be graded - Reactions using gel techniques are stable for at least 48 hours and the gel cards can be photocopied providing a permanent record for future reference.
Sold phase tests
Page 7 of 17 These The se depend on the immobilization of one of the reactant so that during testing the immobilized component captures additional reactant from the liquid phase and binds them to solid phase. These techniques have been successfully developed for a range of se rological testing including red cell grouping, antiglobulin testing and antibody detection. Grouping of Cord or Newborn Infant Blood - Wash cord red cells 3-4 times with saline to minimize errors due to Wharton’s Jelly. - Reactions in cell grouping may be weak as ABO antigens are not fully developed at birth. - Corresponding ABO antibodies are usually absent, therefore, only cell grouping is recommended till 6 months of age. Testing for Al and A2 Subgroups Anti-Al reagent is used to differentiate Al and A2 subgroups.
Sources of anti-Al reagent •
Lectin - Dolichus biflorus (most commonly used)
•
Naturally occurring anti-Al in A2 and A2B individuals
•
Human anti-Al(by adsorption of group B anti-A serum by A2 cells)
Dolichus biflorus lectin reacts specifically with Al antigen and causes agglutination. It i s stored at 4-8°C and may be frozen at -20°C for prolonged storage. The group A and AR blood groups can be subtyped into Al, A2 and A 2B and A2B. Approximately 8% of A2 individuals and 30% A2 B individuals develop naturally occurring anti-Al antibodies. Therefore, The refore, it may be necessary to determine the subgroups of A for both, donors and the patients. If a patient with anti- Al requires blood transfusion, it will be necessary to select blood from A2 donors. Procedure 1. Place 1 drop of anti-Al reagent into a clean, dry test tube. 2. Add 1 drop of 2-4% saline suspension of patient’s cells. 3. Mix and leave at RT for 30-60 minutes. 4. Gently agitate and examine for agglutination. Interpretation Reactions showing agglutination indicate Al blood group. Controls Perform the test using known Al cells and A2 cells instead of patients’ cell. These will act as positive and negative controls.
Errors Encountered in ABO Grouping Errors during ABO blood grouping usually present as discrepancies in the cell and serum grouping. The important factors leading to such problems could be due to •
factors related to red cells
•
factor related to serum
Page 8 of 17 •
factors related to technical procedure
Reagents Blood sample The sample should be as fresh as possible. Venous blood may be drawn in a clean container and stored at 4°C (if not tested w ithin 12 hours). Use serum (instead of plasma) for reverse grouping as plasma may lead to non-detection of weak or complement binding antibodies. Blood grouping antisera All reagents which need to be restandardized for microplate use should be tested for potency and specificity. High concentration of proteins or polymers such as ficoll, polyvinyl pyrrolidine (PVP) and dextran cause red cells to stick to surface of the well. Antisera for use in microplate technique should be diluted to take care of the dye and to decrease the effect of additives. Colour of the antisera should be light or colourless otherwise it will interfere with the results on plate reader. In routine laboratory, ABO and Rh (D) grouping is usually performed in parallel. ABO monoclonal and Anti-D IgM reagents will give excellent results when diluted in phosphate buffer saline containing 1- 3% bovine serum albumin. Polyclonal anti-D sera available for sLide and rapid tube test are usually unsuitable for microplate use. Usually a dilution of 1:20 of ABO antisera and 1:10 of anti-D antisera give good results, however individual laboratories should standardize the technique depending on the antisera in use. Reagent and test red cells The cells can be suspended in phosphate buffer saline (PBS), low ionic strength saline solution (LISS) or isotonic saline. A 3% suspension of red cells may be used. Enzymes techniques are commonly used in mocroplate blood grouping as the forces involved in resuspending negative reactions in the resuspension technique may lead to fragmentation of weak agglutinates. One-stage (bromelain or papain-cystein) enzyme method is likely to give better results for the weaker phenotypes. Procedure Prepare a worksheet for proper layout of samples. •
I. o
Arrange test samples in serial number i.e 1-10
o
Separate cells and serum of the test s amples and arrange them as shown below
o
Using a fine tipped Pasture pipette, dispense 1 drop each of anti-A, anti-B, anti-AB, anti-Di and anti-D2 (anti-D from two different firms or batches) into each well of row A,B,C,D,E, respectively.
o
Add I drop of 2-3% cell suspension of known A cell, B cells and 0 cells t o the well of row F-i, G0i and li-i through 12.
Micropiate A4BO & Rh (D) grouping 1 Anti-A
A
Anti-B
B
Anti-AB
C
2
3
4
5
6
7
8
9
10
11
12
Page 9 of 17 Anti-D1
D
Anti-D2
E
A Cells
F
B Cells
G
O Cells
H
•
II . Starting with test samples 1, dispense 1 drop of se rum to well F,G and H vertically
o
i.e. row I o
Transfer T ransfer 1-2 drop of red blood cells to saline tube labelled- I and prepare 2-3% washed suspension of test cells.
•
o
Repeat with all test and control samples till-12.
o
Dispense 1 drop of 2-3% cells from test sample 1 to well A,B,C,D, and E inrowl
III. vertically i.e. row 1. o
Repeat with all test samples and control samples till 12.
o
Gently tap the microplate and incubate at room temperature (22-24°C) for 1 hour.
If the microplate centrifuge is available, spin after 15 minute incubation at bog for 40 seconds. •
IV . Resuspend the red cells using a microplate shaker. Overshaking will reduce the
o
strength of agglutination and this can be avoided by switching off the microplate shaker as soon as a known negative reaction is fully suspended and positive reactions dislodged. o
Record results
o
Results are preferrably read by two technician independently, one reads while the other records results in the register and then they reverse roles.
o
Typical T ypical reaction pattern may be interpreted visually or using an automated or semi-automated reader.
Interpretation A positive reaction will show the button of cells having a strongly crenated edge, while a negative result will show a compact button with smooth edge that streams on tilting or gets suspended on shaking. A gentle shake of the plate will often assist in clarifying doubtful results. If a microplate centrifuge and shaker is not available, after the one hour incubation, tilt the microplate at an angle of 600 and leave for 5 minutes to allow negative reactions to stream . (same can be done in V-bottom plates also). Predispensed plates A multichannel pipette or automated microplate dispenser may be used, which can save considerable time. Predispensed plates should be kept covered at 4°C until required. In order to avoid evaporation of reagents, predispensed microplates should not be st ored for more than 24 hours. Rh Grouping Practical Aspects of Rh Grouping Rh grouping in routine use for donors and patients involves testing for Rh (D) antigen only, however tests for other important Rh antigens e. g. C,c,E and e may be done for Rh genotyping. The method of Rh grouping grouping mainly depends on the type of reagents available, for which the
Page 10 of 17 manufacturers’ instructions have to be strictly followed. Reagents for Rh (D) Grouping I Polyclonal human anti-D serum (IgG) Potentiating or enhancing substances such as albumin, enzymes and AHG reagent are used to bring about agglutination with human IgG anti-D. •
Anti-D serum (IgG) for saline or rapid tube test (high protein medium) This contains macromolecular additives and give reliable results.
•
Anti-D for saline tube test 2 types o
Anti-D IgM Anti-D IgG - Chemically modified
II Monoclonal antibodies •
IgM anti-D monoclonal reagent
•
IgM and IgG anti-D monoclonal reagent
•
Blend of IgM monoclonal + IgG polyclonal reagent
These The se antibodies are highly specific, react equally well at 20°C as well as 37°C and are reliable for slide and rapid test tube technique. IgM anti-D monoclonal reagent cannot be used for Du testing by indirect antiglobulin test (IAT) while IgM + IgG monoclonat reagent and blend of IgM monolconal and IgG polyclonal can be used for Du testing. It is preferrable to use potent anti- D reagents from two different batches of different firms following the manufacturers’ recommended technique (anti-Di, anti-D2) III Controls for Rh (D) grouping Known 0 Rh (D) positive and 0 Rh (D) negative cells may be used as controls with monoclonal anti-D reagent. Alternatively, AB serum or diluent control provided with the anti- D reagent or 22% bovine serum albumin may be used as negative control with the t est cells Rh (D) Grouping In most of the blood transfusion laboratories, Rh (D) grouping is performed along with the ABO grouping and same techniques as used for ABO grouping may also be employed for Rh typing ie.e 1. Slide technique 2. Tube technique
- Tube sedimentatio sedimentation’ n’
(saline, albumin,
- Tube spin
enzyme and Al-IG test) 3. Microplate technique
Slide Technique This Thi s technique may be used in emergency Rh (D) typing if a centrifuge is not available. The slide test is not recommended for routine test as it may not pick up weak reactions, thus giving negative results. Tube Technique
Page 11 of 17 a. Saline Agglutination test for Rh (13) Typing Procedure 1. Prepare 2-5% washed red cell suspensionof test sample. 2. Place 1 drop of anti-D (Dl) in cleaned t ube labelled Dl and place 1 drop .of anti-D (D2) from a different manufacturer in a clean tube labelled D2. 3. Place 1 drop of 22% bovine albumin/control reagent in another tube labelled C. 4. Add 1 drop of 2-5% test cell suspension to each tube. 5. Mix well and centrifuge at 1000 rpm for 1 minute (in case of using IgG anti-D, incubate at 37°C for 10mm. and centrifuge (spin tube method) or incubate at 37°C for 60 minutes (sedimentation method). 6. Resuspend the cell button and look for agglutination. All negative results must be confirmed under microscope. Interpreation Positive test : Agglutination in anti-D (bot h tubes) and smooth suspension in control tube. Negative test : Smooth suspension in all the tubes (test and control) Test is considerable invalid if - both test and control tubes show a positive reaction. - conflicting results in tubes Dl and D2. In such case, the test should be repeated using saline IgM anti-D. For all microscopically negative reactions in donor grouping, Du testing should be performed, hereas some workers suggest that if the two anti D reagents used are potent and specific, it is not necessary to perform Du testing. b. Albumin technique for Rh (D) typing Principle Albumin increases the dielectric constant of the me dium and thus reduces the zeta potential. Due to this effect, the electrical repulsion between the red blood cells is less a nd the cells agglutinate. Mostly 22% bovine albumin is used, as higher concentrations can cause rouleaux formation. Albumin can be used in = Albumin addition technique additive to serum & cell mixture. - Albumin layering technique layered on cell button Albumin addition technique Procedure 1. Add 1 drop of anti-D to a labelled t ube. 2. Add 1 drop of 2-5% test red cell suspension. 3 Add 1 drop of 22% bovine albumin. 4. Mix a nd incubate at 37°C for 15-20 minutes. 5. Centrifuge at 1000rpm for 1 minute. 6. Gently resuspend the cell button and observe for agglutination. 7. Confirm all negative reactions under microscope. Albumin layering technique Procedure
Page 12 of 17 1. Place 1 drop of anti-D in a labelled t ube. 2. Add 1 drop of 2-5% test red cell saline suspension. 3. Incubate at 37°C for 45-60 minutes. 4. Allow 1 drop of 22% albumin to run down the inside wall of the tube. Albumin will form a layer on top of the red cells. Do not mi x. 5. Incubate further at 37°C for 15-20 minutes. 6. Examine for agglutination after gentle shaking and confirm all negative res ults under microscope. c. Enzyme agglutination technique for Rh (D) typing Proteolytic enzymes such as papain, trypsin, bromelain and ficin digest the cell membrane partially and expose Rh antigens to react with IgG antibodies. When the membrane is partially removed, it brings about a loss of negative electric charge on the red cell which is responsible for keeping the cells a set distance apart, hence small IgG molecules are able to s pan the gap between cells and bring about agglutination. (for further details see Subsection 3.6 : Specialized serological techniques.) One stage enzyme technique (papain-cystein) Procedure 1. To 1 drop of anti-D (IgG), add 1 drop 1% papain-cystein enzyme. 2. Add 1 drop of 2-5% saline suspension of test red cell (put appropriate controls). 3. Mix and incubate at 37°C for 45 minutes. 4. Centrifuge at 1000 rpm for 1 minute and record the results. 5. Agglutination of test cells indicates positivity while no agglutination shows a negative test (Read macroscopically only). Two-stage enzyme technique (Pre-treatment of test red cells with exzyme) Procedure 1. Prepare a 1% papain solution and dilute it to 1:3 using normal saline. 2. To .1 drop of washed packed test cells, add 2 drop of diluted papain solution. 3. Incubate at 37°C for 15 minutes. 4. Wash the red cells 3-4 times with normal saline and prepare a 2-5% cell suspension. 5. To 1 drop of anti-D (IgG), add I drop of enzyme-treated red cell suspension. 6. Mix & incubate at 37°C for 45 minutes. 7. Centrifuge at 1000 rpm for 1 minute and record the result. 8. Agglutination of cells indicates a positive reaction and no agglutination indicates a negative reaction (Ready only macroscopically) d. Indirect antiglobulin test (Du Testing)
Page 13 of 17 If using IgG anti-D in routine saline agglutination Rh (D) grouping proceed from step 6 in all negative reactions otherwise perform the following procedure : 1. Take 1 drop of anti-D (IgG) in a cleaned labelled test tube (T). 2. Take 1 drop of appropriate diluent control in another tube ( C). 3. Add 2-5% washed test red cell suspension to both the tubes. 4. Mix and incubate at 37°C for 45-60 minutes. 5. Centrifuge at 1000 rpm for 1 minute. 6. Gently suspend the cell button and look for agglutination, if posit ive test, no need to proceed as the sample is Rh (D) positive. 7. If negative, wash the cells 3-4 ti mes with saline and decant the last was hing. 8. Add 1-2 drop of anti-human globulin reagent (AI-IG-Coombs’ reagent). Mix gently and centrifuge at 1000 rpm for 1 minute. 9. Resuspended the cell button gently, examine for agglutination and record the results. 10. All negative reactions should be confirmed by adding known IgG sensitized cells, recentrifuge and re-examine for agglutination. The presence of agglutination confirms the t est result. (For details, refer to Subsection 3.3 Antiglobulin test) Interpretation Agglutination in test sample and negative reaction in control sample shows a positive test and the sample is labelled Rh (d) positive. Microplate technique for Rh (D) grouping As described in ABO grouping Rh(D) Grouping In Haemolytlc Disease of the Newborn In haemolytic disease of the newborn, the baby’s r ed cells may be coated with immunoglobulin and a saline reactive Rh antiserum is usually necessary for tes ting. When the cells are heavily coated with antibody, no free antigenic sites remain for reaction, resulting in a negative test. This is suspected when the infant’s cells show a positive direct antiglobulin test (DAT) and a negative test with anti-D reagent. In such instances it is recommended that the a ntibody should be eluted by gentle elution (heating at 45°C for 30 minutes) to expose the antigenic sites before testing. Erroneous Results in Rh Grouping Inaccurate or incorrect results in Rh grouping may occur due to technical errors. These can result from defects in equipment, reagents, s pecimen and techniques or through wrong interpretation. Resolving Rh Problems 1. Perform clerical checks for validity of labels and requisition fo rms.
Page 14 of 17 2. Obtain a fresh blood sample of patient. 3. Check patient rçcords for history, diagnosis, pregnancy, medication and previous transfusion. 4. Check equipment and reagents for proper quality control. 5. Check the antisera and controls. 6. Perform alternative procedures such as washing of red cells with warm saline, enzyme treatment of red cells, absorption / elution studies. 7. Carry out family studies.
Pretransfusion Testing (Compatibility Testing) The purpose purpose of pretransfusion testing is to minimize the risk of blood transfusion to a patient by selecting blood and blood components that will have acceptable survival when transfused and will not cause destruction of the recipients red cells. The term compatibility testing or pretransfusion testing refers to a set of procedures required before blood is issued as being compatible. Collection, Identification and Storage of Patient Sample 1. Identification of recipient 2. Labelling of tubes before puffing in sample Name, Age, Date of Birth, l.D.No. Ward/Bed No. and Date 3. Nature of specimen * Clotted blood sample - serum sample * EDTA blood sample - for DAT * Sample should not be haemolysed * Sample should be less than 3 days old
4. Test on Neonate * Baby’s blood specimen for ABO & Rh(D) grouping * Mother’s serum for crossmatching
5. Identification of blood sample in the blood transfusion laboratory. - Request Form Transfusion Tran sfusion request form must contain sufficient information for definite identification o f the recipient. It should include: - Name - Age/Date of birth - Sex - Registration number (1.D.No.) - Ward/Bed No. - Address For proper selection of blood and blood components; and for record maintenance, the request form should also provide information on:
Page 15 of 17 •
Type Typ e of requirement (R outine/Emergency/Group & Screen)
•
Clinical diagnosis
•
Obstetric history
•
Indication for transfusion
•
Number of units required
•
Which component required
•
Date and time when required
•
History of previous transfusion and transfusion reaction
•
Previous tests and unexpected antibody
•
The blood sample alone with the request form should be signed by a doctor or a phelebotomist collecting the sample.
•
Laboratory personnel should check the information on the request form with the sample and record the time of receiving the request form.
6. Storage of sample - For 7 days at 2-6°C in sealed tubes 7. Checking previous transfusion records •
ABO & Rh(D) group
•
presence of unexpected antibodies
•
any problem in compatibility testing
•
previous transaction
•
any transfusion reaction
ABO and Rh (D) Grouping of Recipient Perform ABO and Rh(D) grouping using a recommended technique. Any discrepancy must be resolved before blood is issued. In emergency situations, if time does not permit detailed testing, issue C group red blood cells. If problem with Rh (D) typing, give Rh (D) negative blood. Screening and identification of Unexpected Antibodies * Unpooled C group cells should be used. * C cells with known antigen profile preferred. * Antibody identification if antibody screen positive. * Selection of blood lacking corresponding antigen for compatibility testing. Selection of Blood ABO & Rh (D) grouping and screening for transfusion transmitted diseases must be done on all donor blood or blood component before the compatibility test is performed. 1. Whenever possible select ABO group specific blood or blood component for the recipient. 2. When group specific bipod is not available, use alternate ABO compatible blood or blood component.
Component
ABO group of recipient
Alternate compatible gorup
1. Whole blood
O,A,B,AB
None (only ABO group specific blood)
2. Red cell concentrate
O
None use only 0 group
A
O
B
O
AB
A,B or O
O
A,B, or AB
A
AB
3. Plasma
Page 16 of 17 4. Platelet concentrate
B
AB
AB
Note use only AB
All ABO group
Compatible with red cells are preferred
3. Select blood of same Rh (D) type a s that of recipient particularly in women of child bearing age. 4. If Rh(D) negative blood is not available, give Rh (D) positive blood followed by Rh (D) immunoglobulins. 5. Change over to group specific blood after administering group nonspecific blood. 6. If recipient’s serum contains unexpected antibodies, identify t he antibody if possible before selecting the appropriate blood. When this is not possible, perform compatibility testi ng by indirect antiglobulin technique with multiple (>10) ABO and Rh group specific donor units to find compatible blood.
Special Transfusion Requirement 1. Exchange transfusion in the newborn select <5 days old blood. 2. Massive transfusion (transfusion of blood equal to or more than the recipients blood volume within a period of 24 hours) select 10-14 days old blood. 3. Compromised heart and brain circulation select <10-14 days old blood. Compatibility Testing (Major Crossmatch) The function function of a crossmatch is to detect in vitro incompatibilities between the donor’s cells and the patient’s serum. If antibody screening of recipient’s serum is negative, an ABO i mmediate spin crossmatch (ISXM) is sufficient to check for errors that may have occured in ABO grouping of the recipient and the donor. If antibody screening has not been done, perform crossmatch using antiglobulin technique. Routine procedure for compatibility testing •
ABO and Rh (D) group
•
Antibody screen
•
Crossmatch o
Saline RT 37”C
o
Albumin or
o
Enzyme
o
Antiglobulin test (using Broad Spectrum AHG)
Techniques of compatibility test Immediate spin technique (Saline technique)
Reagents required 1. 2-5% suspension of donor’s red cells. 2. Recipient’s serum.
Page 17 of 17 Procedure 1. Label 1 tube for each donor sample to be crossmatched. 2. Add 2 drops of recipient’s serum to each tube. 3. Add 1 drop of 2-5% saline suspension of donor red cells from each donor unit to appropriate tube. 4. Spin tubes at 1000 rpm for 1 minute. 5. Look for agglutination. 6. No agglutination should occur if the donor’s and recipient’s blood are compatible. Do not proceed further if agglutination occurs since it indicates ABO mismatch. Regroup the recipient and the donor. If no agglutination occurs in the immediate spin crossmatch and no antibody s creening has been performed, proceed to the antiglobulin crossmatch. Enzyme technique Reagents required 1. 1% papain-cystein solution 2. Recipient’s serum. Procedure 1. Mix 2 drops of patient’s serum with 1 drop of papain-cystein enzyme and 1 drop of 2-5% washed saline suspension of donor’s red cells. 2. Incubate at 37°C for 45 minutes. 3. Read results after centrifugation at 1000 rpm for 1 minute. Interpretation Agglutination indicates that the unit is incompatible and absence of a gglutination indicates a compatible unit. Antiglobulin technique Reagents required 1. Anti-human globulin reagent (Al-IC) 2. Recipient’s serum. Procedure 1. Mix and incubate the tubes from step 6 of imm ediate spin test for 45 minutes at 3 7°C. 2. Spin and look for agglutination. Record the results. 3. Proceed to IAT. Enhancing techniques e.g. albumin, enzyme and LISS can be used with AI-IG t est to increase the sensitivity. It is recommended to use two enhancing techniques for increasing the s ensitivity of compatibility test. Interpretation
Page 18 of 17 No agglutination should be seen at any stage in compatibility t esting. Blood is compatible if no agglutination occurs. Use of low ionic strength saline (LISS) solution Use of LISS allows reduction in incubation time with relatively little loss of sensitivity. Instead of suspending cells in saline, make to the correct s trength suspension using LISS. The serum to cell ratio must be kept to unity. Increasing t he serum to cell ratio reduces the sensitivity of LISS tests. LISS may be used with Albumin, Enzyme or IAT. When incubation time is reduced, the second stage of haemagglutination must be assisted by gentle centrifugation. Great care must be exercised in reading the tests to distinguish between true agglutination and aggregation. Minor Cross match Minor cross match using patient’s red cells and donor’s serum is not essential, if every donor serum is screened for irregular antibodies at least with pooled 0 cells by saline and enzyme techniques.
Precautions Use the attached tubing with the blood bag for obtaining donor red cells in major crossmatch. Always place the sample from the segment of tubing from the blood bag into a prelabelled test tube and match the donor unit no on the bag and the te st tubes.
Pretransfusion Testing in Special Situations 1. Emergency transfusion During an emergency it is best to do as much as the time allows. After the blood has been issued, full routine procedure should be adopted. Minimum testing . Rapid ABO grouping of patient and donor unit . Immediate spin crossmatch (ISXM) technique 2. Massive transfusion a. When time does not allow for compatibility testing, issue C Rh (D)-negative blood. b. Do ABO & Rh (D) grouping of the patient and change over to the ABO and Rh (D) groupspecific blood as soon as possible. Issue the blood af ter doing an immediate spin crossmatch. c. After the emergency has been dealt with, antibody screening on pretransfusion samples may be performed. If negative, additional units can be issued afte r immediate spin crossmatch. d. If antibody screening detects presence of antibody, additional compatibility testing may be done using AHG technique. 3. Infants a. Determine ABO and Rh (D) group of the infant. Only cell grouping is necessary in infants less than 6 months. b. Perform a direct antiglobulin test (DAT) on baby’s cells. c. Screen maternal serum for expected antibodies.
Page 19 of 17 d. If baby’s DAT and maternal antibody screening are negative: * For neonates, select group ‘C’ Rh type specific blood using mother’s (if ABO compatible) or baby’s serum for compatibility testing. Transfusing group ‘C’ blood will also e nsure compatible blood in ABO-HDN without any additional tests. * For infants, select ABO and Rh group specific blood for compatibility test using infant’s serum. * If baby’s DAT and maternal antibody screening are positive. * Identify the antibody in the maternal serum. * Alternatively perform compatibility testing with multiple ABO and Rh specific donor units by the AI-IG technique against maternal and infant’s serum.
