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(Blood Bank Name and address) / Procedure number: 201 / Page 3 of 5Subject: General Guidelines for Serologic Testing
GENERAL GUIDELINES FOR SEROLOGIC TESTING
PURPOSE
To perform and record serologic tests in standard fashion by all technologists using appropriate materials.
PRINCIPLE
Routine tests in the blood bank are based on antigen-antibody (serological) reactions which are made visible by means of agglutination or hemolysis of red cells bearing the antigen(s). This procedure applies to both “tube" and "gel test" methods for detecting such agglutination reactions, but many of the principles outlined in this procedure are “good lab practices” applicable to other forms of agglutination testing. The interpretation of these tests is unavoidably subjective, but the gel test method removes much of the subjectivity as well as the requirement for manual dexterity from agglutination testing. Standardization of these tests is to be sought to the degree possible in order that results obtained by different technologists are comparable. For this reason it is necessary for all technologists to adhere to uniform serologic procedures, and to record their results in a uniform fashion.
PROCEDURE
General Rules
1. Only reagents and instruments which meet the criteria of the blood bank’s quality control program are to be used for testing. All reagents and instruments must be used according to the manufacturer's instructions, including use of proper controls and indicated expiration date. Reagents prepared ‘in-house” must be used according to procedures by which the reagents were validated. Rare antisera (e.g. antigen typing sera) may be used beyond the expiration date provided appropriate positive and negative controls are run and react as expected. If reagents are packaged in “kit” form, components should not be mixed between lots unless permitted by the manufacturer. Manufacturer's instructions are incorporated into the individual procedures using reagents and instruments listed in the "MATERIALS" sections. If special circumstances necessitate use of a different reagent or instrument from those listed in a procedure, the manufacturer's instructions must be followed and supervisory guidance may be necessary.
2. All test tubes and gel cards must be labeled so that there can be no confusion about the identity of the sample or the reagent in use. Patient identity can be indicated by initials, the first 3 letters of the last name, accession #, etc. Donor identity is indicated by the donor number. Reagents can be identified as the antiserum specificity or test cells such as "AC" for a group A reverse typing cell "SCI" for an antibody screening cell, etc. If code numbers or letters are used (e.g. "1,2,3...") to identify the specimens being tested, the key to the code must be written (e.g. on the patient specimen tube); the technologist must not depend on memory to decode the identifiers on the tubes.
When labeling gel cards draw a line on the blank side of the card after the last microtube you will be using to facilitate the use of the remaining tubes for other tests. Record the patient ID in the center box, the screen cell numbers either on the top or bottom row, and the date and time of testing on the gel card.
3. All results will be recorded IMMEDIATELY after observation without exception. If possible interpretations must be recorded immediately. (Exceptions: discrepant ABO and Rh typing tests, indirect and direct antiglobulin tests for which the Coombs’ control cells are nonreactive, other special tests with discrepant control results.)
4. Patient and donor cells should be washed at least once before testing, and a 3-5% suspension should be prepared after washing (see procedure #202). Specific procedures may require additional washes. For antiglobulin tests, make sure that the saline is completely decanted after each wash and that the last wash is completely decanted as well (the latter applies to automatic cell washers as well as manual washing). When manually washing, make sure that the tip of the saline bottle is outside and above the tube opening.
5. For gel cards the foil seal is removed only for the number of microtubes that will be used. (E.G. If you are performing an antibody screen for 1 patient, remove the foil from 2 microtubes, for 2 patients uncover 4 microtubes, for 3 patients remove the entire strip). Do not remove the foil until you are ready to perform the test. If more than 1 hour has elapsed since removing the foil, the gel card is unsuitable and must be discarded.
6. Contamination of the antisera and reagent test cells must be avoided. DO NOT TOUCH test tubes or test tube contents with a reagent dropper that is returned to the reagent bottle.
Note: Calibrated mechanical pipettes used in gel testing should NOT be touched to the sides of the microtubes in order to deliver the final drop.
7. In tube testing, the size of a "drop" of specimen or reagent can vary according to how the serologic pipet is held. In order to achieve standardization, all drops should be delivered with the pipet held at a uniform angle.
(AUTHOR’S NOTE: in our laboratory pipettes are held vertically, but many laboratories specify that pipettes be held at a 45o angle.)
8. The minimum time for saline indirect antiglobulin testing is 30 minutes. If enhancement media or gel test methods are used, follow the manufacturer's instructions.
9. The calibration information sticker on each serologic centrifuge must be referred to for the proper centrifugation time for each phase of testing or cell washing.
10. Gel cards can be re-centrifuged for the purpose of performing additional tests. However, reactions of previously interpreted tests that are centrifuged again can not be saved for later review.
11. An optical aid (reading mirror) should be used to observe serologic reactions in tubes. Anti-human globulin (AHG) phase reactions that are negative macroscopically should also be observed microscopically for “saline” tests, but not for tests using certain enhancement media as specified by the manufacturer or individual procedure.
12. For gel tests, reactions must be read within 1 hour of centrifugation.
13. Tube tests must NOT be re-centrifuged and re-read as false-negative tests may result.
Special considerations
The following conditions or errors may lead to discrepancies in serologic tests:
1. Technical errors:
A. An improper serum to cell ratio may cause false positive or false negative reactions.
B. Failure to observe hemolysis as a positive reaction.
C. Dirty or contaminated glassware may cause false positive results. Contamination may also cause false negative results due to inactivation of the reagent.
D. Over-centrifugation may cause false positives in tube tests or false negatives in the gel test; under-centrifugation may cause the opposite effect in the respective tests.
E. Improper incubation temperature may cause false positive or false negative results.
F. Careless shaking of tubes or failure to use an optical aid may lead to false negative results.
G. Misidentification of sample or reagents, and incorrect reading or interpretation of results may cause the appearance of false positive or false negative results.
2. Red Cell Problems:
A. Improper and inadequate washing of red cells can cause pseudoagglutination due to the presence of serum macromolecules in the cell suspension.
B. Spontaneous agglutination can be due to antibody coating the red cells.
C. The presence of abnormally high serum blood group substances may neutralize a typing reagent..
D. A mixed cell population may be present in a recently transfused patient.
E. Polyagglutination due to acquired or genetic surface abnormalities of the red cell may cause false-positive results with antisera of human origin.
F. Acquired B activity, usually in a group A1 patient may occur due to gram negative sepsis.
G. Weakened or missing expression of the A or B antigen may be due to the inheritance of an unusual genotype or due to disease (i.e., leukemia).
3. Serum Problems
A. Antibodies in the patient’s serum reacting with blood groups other than A and B on the reverse grouping cell(s) can cause an apparent ABO discrepancy.
B. Rouleaux formation may be seen when testing the serum of patients who have high serum concentrations of fibrinogen, abnormal proteins, altered globulins or who have received plasma expanders, such as dextran.
C. Antibodies against the preserving media or reagent solution may cause false positive reactions.
Grading of Serological Reactions
All reactions are graded and recorded according to the following schemes. A hyphen or ‘dash’ should never be used for negative reactions. The plus sign can be omitted for numerical grades.
Tube Testing
Additional designations ("r" for rough, "s" for strong, etc) may be used as superscript notations in antibody identification, but are not used for reporting of results outside the laboratory. Photographs depicting examples of the reaction grades described below are included as appendix 201.1.
Reaction Strength Description of Agglutinates
4+ One solid agglutinate, clear background
3+ Several large agglutinates, clear background
2+ Medium size agglutinates, clear to slightly turbid background
1+ Small agglutinates, turbid background
w+ Tiny agglutinates observable as granularity on a turbid background. Agglutinates obvious on microscopic examination. This is the designation for the weakest reaction that can be observed macroscopically.
vw+ Agglutinates are only observed microscopically
O No agglutination or hemolysis noted macroscopically or microscopically
H Hemolysis observed, cells may or may not remain. IF cells remain, for manual recording (e.g. in antibody identification) the reaction can be graded and the H placed above it.
MF Mixed field agglutination. Definite agglutinates seen on a background of unagglutinated cells.
R Rouleaux
Gel Testing
Reaction Strength Description of Gel Card Appearance
4+ There is a solid band of RBC agglutinates on top of the gel. A few agglutinates may filter into the gel, but remain near the predominant band. Occasionally a few unsensitized cells may migrate to the bottom of the microtube but the middle of the gel should remain free from agglutinates.
3+ The majority of RBC agglutinates are trapped in the upper half of the gel column where the agglutinates form a thick group or band with some RBCs dispersed below it. A 3+ reaction may also be characterized by an even distribution of agglutinates in the upper portion of the gel.
2+ RBC agglutinates are dispersed throughout the column. A few agglutinates may be observed at the bottom of the microtube, and the size of this pellet may vary. The position of agglutinates from side-to-side or front-to-back isn't important, but the upper and lower position of agglutinated RBCs in the gel is key to this interpretation.
1+ RBC agglutinates are predominately observed in the lower half of the gel column. Unagglutinated Cells form a pellet in the bottom of the microtube.
W+ Few RBC agglutinates are present just above the RBC pellet. The pellet at the bottom appears disrupted.
O Unagglutinated RBCs form a well-defined pellet in the bottom of the microtube. Debris, fibrin or other artifacts associated with serum, cord blood, or frozen samples may cause a few unagglutinated cells to trap on top of the gel, but these tests should be interpreted as negative.
H Hemolysis is typically observed in the liquid portion above or just into the gel. If hemolysis is observed, cells may or may not remain. IF cells remain, for manual recording (e.g. in antibody identification) the reaction can be graded and the H placed above it. However, in SOFTBANK, report only the hemolysis without grading associated agglutination.
MF A band of agglutinates is present on top of the gel, accompanied by a pellet of unagglutinated cells in the bottom. IgM antibodies such as cold agglutinins may produce a positive reading resembling a mixed field, but with small agglutinates spread diffusely throughout the gel column; record this appearance as "mf" as well. In SOFTBANK, report only the mixed field without an agglutination grade.
Material and Equipment
The following materials and equipment are used for routine serologic testing, but not all are necessary. For example, tube tests require RBC washing. Automatic cell washers greatly speed this process, but tubes can be washed manually. A centrifuge specifically designed for serologic work greatly improves the serologist’s efficiency, but other types of laboratory centrifuges can validated for serologic testing. Materials and equipment for serologic testing are listed here that are common to many serologic procedures so that they need not be listed separately in each of the following procedures. A materials and equipment section will be included only when additional materials are needed. Note however that the available materials and equipment will vary in different laboratories.
1. Blood Sample; 5 ml EDTA sample (purple top). A completely clotted blood sample (red top tube) can also be used. For pediatric patients, a smaller amount may be acceptable (1 ml).
2. Indelible marking pen (Sharpie) and ball point pen.
3. 10 x 75 mm test tubes or 12 x 75 mm test tubes
4. 13 x 100 mm test tubes with caps
5. Identification label "tape"
6. Test tube rack
7. Pasteur pipets (Unidrop)
8. Dropper bulb for Pasteur pipets
9. Squeeze washing bottle
10. 0.85% NaCl
11. Reagent Rack – typically should include one vial each of the following:
A. Anti-A
B. Anti-B
C. Anti-A,B
D. Anti-D
E. Anti-Human Globulin, polyspecific (Coombs Serum)
F. Anti-Human globulin, anti-IgG (Coombs Serum)
G. Set of reverse typing cells (A1 cells and B cells)
H. 0.8% suspension of antibody detection cells (“screening cells”)
I. IgG coated control cells (“Coombs control cells {CCC} or “check cells” {CC})
J. 6% albumin
12. 37o C incubation device, either dry heat block (includes gel card incubator) or water bath
13. Agglutination reading mirror
14. Serologic centrifuge and heads (includes gel test-specific centrifuge)
15. Automated cell washer
16. Microscope
17. Timer
18. Mechanical pipettes calibrated for gel test volumes as specified by the manufacturer
19. Pipette tips
20. Gel test cards containing anti-IgG
21. Diluent for RBCs in gel tests
22. Mechanical pipetting device for gel test RBC dilution
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