Procedure number: 201
(Blood Bank Name and address)
Page 1 of 5
1
Subject: 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.
Procedure number: 201
(Blood Bank Name and address)
Page 2 of 5
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Subject: General Guidelines for Serologic Testing
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; undercentrifugation 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.
Procedure number: 201
(Blood Bank Name and address)
Page 3 of 5
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Subject: General Guidelines for Serologic Testing
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
4+
3+
2+
1+
w+
vw+
O
H
MF
R
Description of Agglutinates
One solid agglutinate, clear background
Several large agglutinates, clear background
Medium size agglutinates, clear to slightly turbid background
Small agglutinates, turbid background
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.
Agglutinates are only observed microscopically
No agglutination or hemolysis noted macroscopically or microscopically
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.
Mixed field agglutination. Definite agglutinates seen on a background of unagglutinated cells.
Rouleaux
Procedure number: 201
(Blood Bank Name and address)
Page 4 of 5
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Subject: General Guidelines for Serologic Testing
Gel Testing
Reaction Strength
4+
3+
2+
1+
W+
O
H
MF
Description of Gel Card Appearance
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.
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.
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.
RBC agglutinates are predominately observed in the lower half of the gel column. Unagglutinated
Cells form a pellet in the bottom of the microtube.
Few RBC agglutinates are present just above the RBC pellet. The pellet at the bottom appears
disrupted.
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.
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.
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.
2.
3.
4.
5.
6.
7.
8.
9.
10.
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).
Indelible marking pen (Sharpie) and ball point pen.
10 x 75 mm test tubes or 12 x 75 mm test tubes
13 x 100 mm test tubes with caps
Identification label "tape"
Test tube rack
Pasteur pipets (Unidrop)
Dropper bulb for Pasteur pipets
Squeeze washing bottle
0.85% NaCl
Procedure number: 201
(Blood Bank Name and address)
Page 5 of 5
Subject: General Guidelines for Serologic Testing
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
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
37o C incubation device, either dry heat block (includes gel card incubator) or water bath
Agglutination reading mirror
Serologic centrifuge and heads (includes gel test-specific centrifuge)
Automated cell washer
Microscope
Timer
Mechanical pipettes calibrated for gel test volumes as specified by the manufacturer
Pipette tips
Gel test cards containing anti-IgG
Diluent for RBCs in gel tests
Mechanical pipetting device for gel test RBC dilution
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