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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5543_Библиотеки_им_академика_М_И_Перельмана.pdf
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Medical Laboratory Technology: Volume 1
Figure 17.7 Rh typing by slide method: (a) Take three labelled slides and place them on a viewing
box which maintains a surface temperature of 40°C. (Note: If viewing box is not available,
warm up the slide on a spirit lamp with hand.), (b) Place the cell suspensions of specimen
(10%) and Rh-positive (+) and Rh-negative (–) controls; also add the anti-D separately,
(c) Mix the antisera and the cell suspensions and rock, (d) Look for agglutination within
3 min, (e) The illustration shows that the specimen is Rh-positive
2. Place a slide on the viewing box and allow it to warm up.
3. Add 2 drops of 40–5% cell suspension or whole blood on the slide.
4. Add one drop of anti-D serum (specially marked for slide test).
5. Mix cells with the antiserum by means of a toothpick or applicator stick or corner of a
slide.
6. Continue mixing by tilting the warm viewing box back and forth.
Routine Laboratory Procedures in Blood Bank
7. Look for agglutination which is recognized by the clumping of red cells. Do not observe
longer than 2 min.
8. A control must be run once a day in order to obtain reliable results. Take a slide and
mark the two ends as ‘+’ and ‘–‘ Place it on the pre-warmed slide viewing box. Wait
for a minute and then place a drop of anti-D serum on each end of the marked slide.
Then add one drop of control Rh-positive cell suspension to the side marked as ‘+’ and
one drop of Rh-negative cell suspension on the ‘–’ side. Follow the test procedure as
described above. If the Rh-positive cells show agglutination and the Rh-negative cells
do not show agglutination, the system is considered to be acceptable.
Tube Test Method (Figure 17.8)
1. Prepare a 4% suspension in saline of washed cells or use diluted whole blood with
saline. The applicator stick method is quick and does not require washed cells. Take two
applicator sticks and transfer sucient amount of cells, held between sticks, from the
cloed or whole blood specimen to a test tube with saline. Bring the concentration of the
cell suspension to about 4%.
Note With experience, the 4% red cell suspension can be judged from the density of the
suspended cells.
2. Place four small test tubes (10 mm × 75 mm) on a rack and label them as: ‘S’ for specimen,
‘+’ for Rh-positive control, ‘–’ for Rh-negative control and ‘SA’ for albumin control (22%
albumin) of specimen.
Note Only one tube is used in the illustration (Figure 17.8).
3. Add 1 drop of anti-Rh into the rst three tubes marked as ‘S’, ‘+’ and ‘–’ and albumin in
the tube marked ‘SA’.
4. Add 2 drops of the cell suspension of specimen (Step 1) in tube ‘S’ and ‘SA’ and 2 drops
of control reagent cells (Rh-positive and Rh-negative) in the tubes, respectively, marked
as ‘+’ and ‘–’. Mix all the tubes gently by shaking the rack.
5. Incubate all the tubes at 37°C for 30 min.
Note The duration of incubation is given by the supplier of the anti-D.
6. Centrifuge the tubes at 1500 rpm (150G) for one minute. If centrifuge is not available
incubate for one hour.
7. Examine the agglutination reaction in each tube by dislodging the buon gently. If
necessary, use a magnifying hand lens or a concave magnifying mirror aached to the
spotlight.
Interpretation Agglutination will be recognized by the formation of small clumps in
a clear liquid. As the boom of the test tube is tapped, the clumps whirl up and then
sele down. This will be marked as a ‘positive reaction’ and cells are identied as Rh-
positive. If red cells re-suspend homogeneously with no visible clumps, it should be
marked as a ‘negative reaction’ and cells are identied as Rh-negative.
Quality control The Rh-positive control cells (in the ‘+’ marked tube) should always
show agglutination and the Rh-negative control cells (in the ‘–’ marked tube) should
not show any agglutination. The albumin tube (marked ‘SA’) should also show lack of
agglutination of test cells without anti-D. Results will be invalid if agglutination is seen
in the test (‘S’) and the auto-control tube (‘SA’). This is due to autoagglutination.
8. The Rh-negative cells must then be tested for weak D.
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Medical Laboratory Technology: Volume 1
Figure 17.8 Rh typing by tube method: (a) Mix two drops of red cell suspension (4%) of the specimen
and two drops of anti-D in a test tube, (b) Mix and place in a water bath (37°C) for 30 min,
and (c) Centrifuge; if centrifuge is not available, incubate for 1 h, (d) Read the results with
Sources of Error in Rh Typing
The possible sources of error are the specimen, equipment operation, reagent, technique and
reporting. The following is only a partial listing and you must be alert at every step:
• Always ran Rh controls (positive and negative) at least once a day.
• Check the temperature of the water bath and slide-viewing box periodically.
• Follow the manufacturer’s instructions precisely for the use of anti-D.
• A fresh specimen is always desirable; specimens that are several days old may not yield
accurate results. Contaminated or haemolysed specimens might give unreliable results.
Improper identication of the specimen can be avoided by careful labelling of the blood
Routine Laboratory Procedures in Blood Bank
collection vial and test tubes (or slides) used during testing. Specimen mix up can lead
to serious consequences.
• Heavy cell suspension in the tube test, light cell suspension in the slide test, excessive
centrifugation, drying of the slide due to excessive heat or observing the reaction on the
slide beyond the 3 min period might lead to false results. Failure to recognize haemolysis
can yield false negative results.
• Coating of red cells with autoantibody might cause a false positive result by slide test
where antisera have a high concentration of protein. Such cells can only be tested with
saline tube test sera after thorough washing and suspension in saline. Hence, in routine
Rh testing, saline suspension is preferred and the test must include the auto-control.
• Rouleaux formation can yield false positive results in the slide test.
• Check the anti-D and reagent control cells every day for possible contamination.
Bacterial contamination of anti-D is recognized by cloudiness, and loss of activity
is seen from the lack of agglutination of Rh-positive control cells. Contamination of
antisera rarely gives false positive results and often gives a false negative reaction. On
the other hand, bacterial contamination of reagent cells can make them agglutinate in
any antisera (Thomsen phenomenon).
• Occasionally weak agglutination reactions are seen which creates doubt regarding the
presence of D-antigen on red cells. This is due to two principal causes: low potency of
anti-D or the cells have weak D antigen expression.
• False positive results have been noted in the presence of high titre cold agglutinins in
the test serum which results in autoagglutination. Antibodies of I/i blood group are the
commonest cause of autoagglutination; but cold-reacting antibodies directed against
other antigens (i.e., P, MNS) may also be involved. High titre cold antibodies may also be
found in Mycoplasma infection causing atypical pneumonia. Cold agglutinins generally
have no clinical signicance but the blood bank technologist must determine their
specicity before ignoring them. Pre-warming both the serum and cell suspension to
37°C before starting the test can avoid interference by the cold agglutinins. Immunologic
reactions due to cold agglutinins often do not occur at body temperature (37°C).
441
antiHuman gloBulin (aHg) or coomBS’ teSt
AHG technique is very useful in recognizing weak immunologic reactions or those that are
mediated by IgG antibodies. It is widely
used in the identication of weak D,
compatibility testing, antibody identication,
and identication of red cells that have been
sensitized in vivo.
AHG is made by injecting human globulin
into rabbits (see Figure 17.9) and purifying
the AHG produced by the rabbit immune
system. AHG is commercially available but is
expensive.
There are two types of AHG testing, also
known as Coombs’ test—direct and indirect.
Direct Antihuman Globulin Test or
Direct Coombs’
This procedure recognizes the sensitized red
cells when sensitization occurs within the
Figure 17.9 AHG production in rabbit
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Medical Laboratory Technology: Volume 1
body, e.g., in haemolytic disease of the newborn,
autoimmune haemolytic anaemias, and haemolytic
transfusion reactions. The sensitized red cells,
coated with unknown human globulin in vivo,
do not agglutinate inside the body but will do
so in the laboratory in the presence of AHG sera
(Figure 17.10). It is a one-stage procedure. In this
process, the Coombs’ serum reagent is simply
added to a preparation of red cells after red cells
are washed to remove non-specic serum proteins
and/or to prevent a false negative reaction via
AHG neutralization. If red cells are coated with
Figure 17.10 Agglutination of sensitized
red cells (coated with
human globulin) by AHG
(Coombs’reagent)
antibody, the Coombs’ reagent binds the Fc portion
of antibodies on the in vivo sensitized red cells and causes them to agglutinate to one another
(i.e., forming clumps).
The antibodies aached to sensitized red cells and detected in the direct Coombs’ test are
most often alloantibodies to foreign red cell antigens or autoantibodies. Certain medications
tend to produce autoantibodies against certain red cell antigens. In addition, some reports
indicate an increased incidence of apparently non-specic positive direct Coombs’ reactions
in patients with elevated gamma globulin levels.
Indirect Antihuman Globulin Test or Indirect Coombs’
The indirect Coombs’ test is applied to detect and identify an antibody present in circulation
that could potentially cause sensitization and destruction of red cells, or to identify red cell
antigens.
The indirect Coombs’ test is a two-stage procedure. In the rst stage, the cells are
deliberately sensitized in vitro which may be done in either of two ways:
1. Using red cells with known antigenic composition. This is exposed to serum containing
unknown antibodies. If the antibody combines with the sensitized red cells, as detected
by the second stage (which is the same as described under Direct Coombs’). A positive
reaction (agglutination of the sensitized red cells) shows the presence of corresponding
antibodies, against one or more of the antigens present on red cells in circulation.
2. When antigens on red cells are sought, one uses serum containing antibodies of known
specicity and exposes it to those red cells of unknown antigenic composition. If the
antibody combines with red cells, as detected by the second stage, this identies the
cognate antigen on red cells.
In both methods, sensitization of red cells is performed in vitro by incubating red cells with
the corresponding antibody at 37°C for 30 min. Following incubation, the cells are thoroughly
washed before reacting with the AHG reagent. This initial phase of immunologic reaction
(sensitization) is recognized only after treating the washed sensitized cells with AHG.
One of the common uses of the second method is when the presence of weak D antigen on
the red cell is sought. In the rst phase one uses anti-D to sensitize with the weak D antigen
present on the testing red cells by incubation. This is followed by washing of the red cell
suspension in order to remove the free anti-D. In the second phase, the sensitized red cells are
treated with AHG. Agglutination indicates the presence of weak D on red cells.
In order to avoid false negative result (absence of agglutination), add Coombs’ control
cells or pre-sensitized reagent cells, which must show agglutination due to the presence of
functional AHG.
Routine Laboratory Procedures in Blood Bank
443
Specimen
Serum is the specimen of choice but plasma can also be used. Hence, either cloed blood or
anticoagulated whole blood is submied for direct AHG test.
Principle
The direct AHG test detects ‘sensitized red cells’ where red cells get coated with IgG antibody
but do not agglutinate. When the sensitized red cells come in contact with AHG reagent (or
Coombs’ reagent) in vitro, they agglutinate (Figure 17.10).
Reagents
• AHG reagent
• Pre-sensitized red cells (Coombs’ control cells)
• Saline
Procedure
1. Wash the red cells suspected of being sensitized 3 to 4 times in large volumes of saline.
Complete removal of free globulin is important.
2. Decant completely at the end of last washing.
3. Add 2 drops of AHG serum to the remaining sedimented cells (buon).
Note Follow the manufacturer’s instruction regarding the use of AHG.
4. Mix well and centrifuge at 1500 rpm (150G) for 1 min.
5. Examine for agglutination by holding against a lighted background and tapping the
boom of the tube. A small hand lens or magnifying mirror aached to a spotlight may
be used as optical aids.
Note The manner in which red cells are dislodged is critical; hold the tube at an angle,
shake gently until all cells are dislodged, then tilt the tube gently, back and forth, until
an even suspension of cells or agglutinates is observed.
6. If the agglutination is not seen, leave the tube at room temperature for 10 min, then recentrifuge and read. A weaker reacting antibody shows delayed reaction. Consider this
as positive.
7. If haemagglutination is not seen in Step 6, add one drop of pre-sensitized red blood cells
(5% suspension in saline). This should result in haemagglutination of pre-sensitized
cells indicating that the AHG is reactive and the result is valid. This is an important step
of quality control because it is the only way to monitor the adequacy with which the cells
were washed. If the initial washings with saline (Step 1) were incomplete, antiglobulin
will be neutralized by free globulin before reacting with the coated globulin.
Interpretation
Haemagglutination of red cells with the addition of AHG (positive result) indicates that cells
are sensitized inside the body. If antibodies are to be identied, they are eluted and then
tested in the same way as the serum. This will be further discussed.
Procedure for indirect Coombs’ test
The indirect AHG test detects cells which are sensitized in the laboratory. The general procedure
is described below. Application of this procedure in various other tests will be discussed in their
respective places (e.g., antibody screening, antibody identication, weak D testing).
1. Prepare a 4% saline suspension of test cells.
2. Add 2 drops of cell suspension to a small test tube (10 mm × 75 mm).
3. Add 2 drops of the antiserum to the cell suspension.
4. Incubate in a water bath at 37°C for 30 min.
5. Remove the tube from the water bath and wash 3 to 4 times with large volume of saline
(4 mL); decant completely after last washing.
Note All free globulin must be removed.
444
6. Add immediately 2 drops of AHG and mix well.
Note Follow the manufacturer’s instructions, if dierent.
7. Centrifuge at 1500 rpm (150G) for one minute.
8. Examine for haemagglutination.
9. In case of negative haemagglutination, add pre-sensitized reagent cells or Coombs’
control cells to test the reactivity of AHG. Agglutination must be seen with the addition
of Coombs’ control cells. If there is no agglutination of pre-sensitized cells, check the
AHG reagent and repeat the entire process.
Note If the washing is not complete in Step 5, presence of free globulin might neutralize
the AHG and render it inactive.
Medical Laboratory Technology: Volume 1
Sources of Error in Antiglobulin Test
False positive
• Improper preparation of antiglobulin serum.
• Colloidal silica in saline stored in glass boles that is leached from the container.
• Metallic ions in saline stored in metal containers or used in equipment with metal parts.
This may bring about non-specic protein sensitization of red cells.
• Improperly cleaned glassware or other forms of contamination of cells, serum or
reagents.
• Over centrifugation.
• Autoagglutination before washing that may persist through the washing phase.
False negative
• Incorrect technique for a particular antibody involved.
• Inadequate washing of red cells causing neutralization of the antiglobulin serum by
trace amounts of residual globulin.
• Improper storage of test cells, test serum or antiglobulin reagent or all of these, resulting
in loss of reactivity.
• Delays or interruptions in the test procedure, particularly during the washing phase,
which might result in the elution of the antibody from the red cells.
• Failure to add antiglobulin reagent.
• Under centrifugation or over centrifugation.
• Contaminated antiglobulin reagent.
• Incorrect technique followed for red cell suspension, incubation, washing of red cells
and all other steps.
Weak D Testing
The indirect Coombs’ test is applied in weak D testing. Weak D is a weakened form of the D
antigen present on red cells of some individuals. The weak D antigen reacts with anti-D but
does not result in haemagglutination. This is because the reaction is not strong enough to
be visualized. The cells, however, are sensitized and are coated with anti-D (IgG) following
incubation. The sensitized red cells, following repeated washings, are recognized by reacting
with AHG. Red cells carrying weak D can falsely be considered as Rh-negative if weak D
testing is not performed. Thus Rh-negative donors should be routinely subjected to weak D
testing. Weak D donors should be considered as Rh-positive and the blood of such donors
should not be transfused into Rh-negative individuals.
Principle
Cells with D antigen are sensitized with anti-D by incubating at 37°C for 30 min. This results
in sensitization via the adsorption of anti-D on the surface of the cell without producing
Routine Laboratory Procedures in Blood Bank
445
haemagglutination. The presence of bound antibody on the surface of weak D cells is recognized
by using AHG which reacts with the coated antibody and causes haemagglutination. If the
cells do not show agglutination, even after anti-D treatment and AHG reaction, the cells are
truly Rh-negative. It is important to note at this point that the cells following incubation with
anti-D must be thoroughly washed in order to remove any free-oating antibody which reacts
with AHG. A control with protein (22% albumin) must be run simultaneously in order to
eliminate the possibility of autoagglutination. The control should not show any agglutination.
Procedure (Figure 17.11)
Weak D testing should be performed in addition to testing described earlier for all samples
reported as Rh-negative.
1. Prepare a 4% suspension of red cells in saline.
2. Place 2 drops of the suspension in two small test tubes (10 mm × 75 mm) marked as ‘S’
(serum) and ‘A’ (albumin).
Figure 17.11
3. Add 1 drop of anti-D slide test serum to the ‘S’ marked tube and 1 drop of 22% albumin
in ‘A’ marked tube.
4. Place the tubes in the water bath at 37°C for 30 min (read the manufacturer’s instructions
for actual timing).
5. Remove the tube from the water bath and wash the cells 3 to 4 times with large volumes
of saline (4 mL) with repeated centrifugation.
6. Add 2 drops of AHG to the sedimented cells, dislodge the buon and mix the cells
gently with the antiserum.
7. Centrifuge at 1500 rpm (150G) for 1 min.
8. Examine for agglutination. Cells showing agglutination in ‘S’ marked tube have D
antigen. The ‘A’ marked tube should show no agglutination unless autoagglutination
occurs. Specimens showing autoagglutination should be referred to the physician.
9. Include a positive and a negative control each day.
446
Quality control
Adequacy of saline washing of cells and eectivity of AHG are assessed by adding a drop of
sensitized 4% red cell suspension in the ‘A’ marked tube. The sensitized cells must agglutinate.
Medical Laboratory Technology: Volume 1
major croSS-matcH
Before the recipient receives a red cell transfusion, a compatibility test must be run within
the laboratory with the donor’s red cells and the recipient’s serum. This is called major cross-
match or compatibility testing. The primary purpose of major cross-match is to identify
any incompatibility between donor’s cells with patient’s serum in order to avoid transfusion
reactions. The minor cross-match is rarely requested when the compatibility of the recipient’s
red cells is tested against donor’s serum (Figure 17.12).
The purpose of major cross-match is to detect unexpected antibodies in the serum of the
recipient, so that it acts as a check on a previous antibody screen. It also serves as a check
on ABO typing, since a mistake in ABO typing might result in red cells from the donor
being incompatible with naturally occurring ABO antibodies in the serum of the recipient.
Unfortunately, it will not detect the most common ABO incompatibility errors, which are
patient identication mistakes, either from cross-match specimens obtained from the
wrong patient or properly cross-matched blood transfused into the wrong patient. Also,
the procedure will not detect errors in D typing if no anti-D antibodies are present in the
recipient’s blood. Since Rh antibodies do not occur naturally, the recipient would have to be
previously sensitized before anti-D antibodies appear.
Compatibility testing or cross-matching is performed subsequent to ABO and D typing of
the recipient’s and donor’s blood. The recipient’s blood is obtained fresh while the donor’s
blood is obtained from the pilot tube aached to donor’s bag.
Routine Cross-Match (Figure 17.13)
Routine cross-matching procedure involves three phases of reacting donor’s red cells with
recipient’s serum:
1. Saline phase: In this phase the immunologic reaction between red cells suspended in
saline medium and the antibody occurs at room temperature. It is often referred to as
‘Immediate spin’.
2. Thermophase with protein phase: Here the red cells are suspended in the antibody
(serum) with 22% albumin (protein) and incubated for 30 min at 37°C. The high protein
environment enhances agglutination of univalent antibodies such as the Rh system. The
modern use of LISS (low ionic strength saline) has helped to reduce the incubation time
from 30 min to 15 min. Use of LISS eliminates the use of albumin.
Routine Laboratory Procedures in Blood Bank
447
Figure 17.12 Principle of cross-matching: (a) Immune antibodies do not react with its corresponding
antigen in saline medium at room temperature, Immunologic reactions with immune
antibodies are recognized under three laboratory conditions-protein phase at (b) room
temperature, (c) thermophase, and (d) AHG phase