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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
ward typing and serum is used in reverse typing. Anticoagulated blood is used only as a
second choice over the cloed blood. Citrated or heparinized blood is used in case of infants.
Principle
Forward typing: A suspension of red cells of the specimen is reacted with known reagent
antisera—anti-A and anti-B. Positive agglutination indicates the presence of corresponding
antigen (agglutinogen) on red cells.
Reverse typing: Serum of the specimen submied is reacted with known Aı cells and В cells.
Agglutination indicates the presence of corresponding antisera in serum. The forward and
reverse reactions should support each other in determining the blood type of an individual
(see Chapter 15, Table 15.1). In case of any deviation from the normal paern, the technologist
should investigate for possible technical error, presence of subgroups or other unusual
circumstances.
Preparation of Red Cell Suspensions
Cloed blood or anticoagulated blood must be washed in saline in order to remove any
contaminating antibodies. Red cell suspensions are suspended in saline. A heavier suspension
is needed for the slide method (10%) than for the tube test method (3 to 5%). The procedure
for preparing 10% and 4% red cell suspensions (Figure 17.2) is described below:
Figure 17.2 Preparation of red cell suspension for blood grouping: (a) Take clotted blood specimen,
(b) Remove the serum, (c) T ransfer two drops of red cell concentrate in saline, (d) W ash
the cells three times with saline followed by centrifugation. Finally, add the requisite
quantity of saline to prepare 10% or 4% red cell suspension (v/v) to be used for (e) slide
method, or (f) tube method
Routine Laboratory Procedures in Blood Bank
Note Some laboratories prefer to use 45% cell suspension for the slide test (particularly
for the Rh typing). This is prepared by taking 10 drops (0.5 mL) of cloed blood
specimen and suspending it in 1 mL of saline. If whole blood (anticoagulated) is used, no
dilution is necessary. The whole blood of a normal individual has about 45% of red cell
suspension.
429
Using calibrated Pasteur pipette
If calibrated Pasteur pipee is available, then follow the procedure described below.
• For slide test: Take ve drops of red cell deposit (0.25 mL) in a test tube with calibrated
Pasteur pipee delivering 0.05 mL uid (Figure 17.2). Add approximately 2 mL of saline
solution and mix red cells in saline. Spin the tube at high speed 2500 rpm (250G) for
5 min. Discard the supernatant (saline) with a Pasteur pipee and save the red cell
sediment. Repeat the wash once more. Finally, discard the supernatant, add 2 mL of
saline and re-suspend the red cells. This gives a 10% red cell suspension required for the
slide method of ABO/D testing.
• For tube test: Take 5 drops of cloed blood or red cell deposit from anticoagulated
blood. Use the calibrated pipee (20 drops/mL) if available. Wash the cells three times,
with approximately 4 mL of saline, each time spin at 2500 rpm (250 G) for 5 min, discard
the supernatant saline and replace with fresh saline solution (4 mL) and gently resuspend the cells. Finally add 4 mL of fresh saline solution and re-suspend the cells by
gently shaking. This results in a 5% suspension of red cells needed for the tube method
of blood group testing.
Using uncalibrated Pasteur pipette
If a calibrated pipee is not available, take any two identical droppers. Use one dropper for
transferring blood and the other for transferring saline. (You can also use the same dropper if
you can wash the dropper with saline after transferring the blood.)
• For slide test: Take 5 drops of packed red cells from the clot or from the anticoagulated
blood. Wash once with about 2 mL of saline. Finally re-suspend the cells in 45 drops of
saline taken with the same uncalibrated dropper. This yields a 10% red cell suspension
in saline (5 drops in 50 drops).
• For tube test: Take 5 drops of cloed blood (or cell deposit from anticoagulated blood),
wash three times and add as much volume of saline as equivalent to 95 drops of uid
delivered by the uncalibrated dropper. This yields a 5% cell suspension (5 drops in 100
drops).
Slide Method
The slide method of blood group testing is quick and needs only limited equipment. However,
it is not as dependable as the tube method. It can be used in a blood donation camp and in
case of mass scale emergency (e.g., train accident, war front). Many laboratories perform slide
test routinely and questionable cases are checked by the tube method.
Forward or cell typing (Figure 17.3)
1. Prepare a 10% suspension of red cells in saline or its own serum. Whole blood may be
used when cloed blood is not available.
2. Take two slides (commercially available ceramic tiles can also be used in place of slides)
and label them as A and B. Two halves of the same slide can also be used, marked in the
same way.
3. Place 1 drop of anti-Α on the slide marked A; add 1 drop of anti-B on the slide marked B.
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Medical Laboratory Technology: Volume 1
4. Add 2 drops of the cell suspension to each slide.
5. Mix the antiserum and cells with a stick or tooth pick or with the corner of a slide.
Spread to form a 2 cm circle.
Figure 17.3 Forward typing procedure by slide method: (a) Two drops of red cell suspension of the
specimen are taken (b and c) on two slides to which anti-A and anti-B are added and (d)
mixed; (e and f) Rock the slide and look for agglutination. If agglutination occurs on one
anti-B). If agglutination is seen on both the slides, it is AB and, if on neither, it is O. The
illustration suggests the blood group as B.
6. Tilt the slide back and forth to complete the mixing.
7. Examine for agglutination within 2 min. Protect the slide from evaporation (this should
particularly be observed in hot weather).
Routine Laboratory Procedures in Blood Bank
431
8. Reporting of the results:
Observation Report
Lile clumps of red cells are seen oating in a clear liquid +
No agglutination of red cells; cells are oating homogeneously –
9. Inference is shown in Table 17.2.
Table 17.2 Report of forward typing and probable ABO type
Report of reaction with Probable ABO type
anti-A anti-B
+ – Α
– + В
+ + AB
– – О
‘–’ = no haemagglutination ‘+’ = haemagglutination
Reverse or serum typing (Figure 17.4)
1. Take a microscope slide with three ceramic rings (or make three circles with a glass
marking pencil). Label them as A, В and O. Three separate slides can also be taken.
2. Add one drop of the reference cell suspensions (10%) of A, В and О on the respective
circles marked in Step 1.
3. Add two drops of test serum on the cell suspension on each circle.
4. Mix the serum with cell suspension, using a tooth pick or the corner of a slide or the end
of a test tube. Spread it over a 2 cm circle.
5. Record the results as described earlier with forward grouping.
(a) The agglutinates are usually smaller than those observed in forward grouping.
(b) Considerable variations exist between individuals regarding the degree of reaction,
which largely depends on the amount of anti-Α and anti-B antibodies in the serum.
Inference Many laboratories feel that reverse grouping should be performed in a test tube
only. The method of reverse grouping suers from certain deciencies: anti-Α and anti-B
antibodies may be so weak as to be virtually undetectable; certain atypical antibodies may be
present and capable of reacting with antigens other than A or B, hence confounding the blood
grouping; also, some A or weaker subgroups of A have anti-Α along with anti-B in the serum
and thus may react with both A and В cells (similar to О group).
Tube Test Method
Tube test method of blood typing takes longer and requires more equipment (e.g., centrifuge
with electric supply), but the results are more reliable. Many blood transfusion centres use
only the tube test technique.
Forward or cell typing (Figure 17.5)
1. Prepare a 4% suspension of red cells in saline.
Note The tube test is slightly modied for handling an anticoagulated whole blood
specimen. In this case, cells are not washed and one or two applicator sticks are used to
transfer an equivalent quantity of cells as present in two drops of 5% cell suspension in
saline. The volume of whole blood held between two sticks will be sucient (Figure 17.4).
2. Arrange three small test tubes (10 mm × 75 mm) and label them as A, В and AB.
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Medical Laboratory Technology: Volume 1
3. Add 1 drop of anti-Α in the test tube marked ‘A’, 1 drop of anti-B in test tube marked ‘B’
and 1 drop of anti-AB in the test tube marked AB (optional).
4. Add 2 drops of the cell suspension to each tube and mix gently.
5. Centrifuge immediately at 1500 rpm (150G) for one minute or incubate at room
temperature for 1 h.
Figure 17.4 Reverse typing by slide method: (a) Take a slide with three ceramic rings labelled A,
B, and O, (b and c) Add to these rings, the corresponding known cells and the serum
specimen, (d and e) Mix and rock, (f) Look for agglutination within 2 min. The reaction
Table 17.3).
Routine Laboratory Procedures in Blood Bank
433
Figure 17.5
to them the known antisera, and (c) 4% red cell suspension of the specimen, (d) Mix
and centrifuge and if centrifuge is not available, wait for one hour at room temperature,
(e) Examine agglutination over magnifying mirror or use hand lens, (f) In case of weak
reaction, use a microscope
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Medical Laboratory Technology: Volume 1
6. Examine for agglutination: If the tube is centrifugea, red cell sediment will be seen at
the boom of the tube, which is called a buon. Gently tap the boom of the tube with
your right index nger and dislodge the cell buon. Watch the behaviour of the red cell
buon against a well-lit white background. If desired, readings may be conrmed with
the optical aid of a small hand lens or a magnifying concave mirror.
7. Red cells form one or more clumps with clear supernatant uid (reported as +, positive).
8. Red cells re-suspend easily, without any visible clumping (reported as –, negative).
9. In case of any doubt, take a drop of the suspension on a slide and observe under
l0× objective (l00× magnication). Clear agglutination (large clumps with clear
background), weak agglutination (smaller clumps with some free cells in the eld) and
no agglutination (all red cells are free) will be recognized without any diculty. Many
laboratories insist on grading the reactions.
10. Inference is shown in Table 17.2.
Reverse or serum typing (Figure 17.6)
1. Label three small-size test tubes as A, В and O. (Many laboratories skip the use of
О cells).
2. Add 2 drops of cell suspensions (reagent known cells) of A, В and О types into the
respective tubes.
3. Add 2 drops of serum under test to each of the two tubes.
4. Read the haemagglutination reaction immediately after low centrifugation for 1 min or
leave for one hour at room temperature.
5. If haemagglutination is not visible, or there is a weak reaction, re-suspend the cells
gently and incubate at room temperature (22–24°C) for 15 min and then centrifuge for 1
min at low speed (1500 rpm or 150G). Weakly reacting antibodies might require longer
times to react.
6. Read the haemagglutination reaction by holding the tube on a magnifying concave mirror
or against a lighted background at eye level. Dislodge the buon, as described earlier
and examine the agglutination. The presence of small clumps indicates a positive (+)
reaction; and if red cells are re-suspended easily without visible clumping, the reaction
is reported as negative (–). Haemolysis should also be considered a positive test. Hence,
while examining the agglutination reaction look for any possible haemolysis.
7. Inference is presented in Table 17.3.
Table 17.3 Report of reverse typing and probable ABO type
Agglutination reaction Serum group Probable ABO type
A cells B cell
+ anti-B A
+ – anti-A В
– – none AB
+ + anti-A anti-B 0
‘–’ = no haemagglutination ‘+’ = haemagglutination
Final Report
Matched results of forward and reverse typing (Table 17.4) are able to conrm the ABO type
of the specimen submied. In case of a discrepancy, an investigation must be conducted.
Routine Laboratory Procedures in Blood Bank
435
Figure 17.6 Reverse typing by tube method: (a) Label three test tubes, and (b) Add two drops of cell
suspension of known type
Mix and centrifuge or wait for 1 h, (f) Examine agglutination over a magnifying mirror and
for weak reaction, usea microscope
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Medical Laboratory Technology: Volume 1
Sources of Error in ABO Typing
False positive results
Drying on the slide, use of contaminated sera or specimen, presence of unexpected antibody
and Rouleaux formation (pseudoagglutinin) are some of the causes of false positive results.
Rouleaux formation is due to high immunoglobulin or brinogen concentration in the serum
following certain pathological conditions (e.g., multiple myeloma). In this, red cells, as seen
under microscope, collect in lines, like stacks of coins. In case of Rouleaux formation, cells will
disperse when a drop of saline is added on the microscope slide. In case of true agglutination,
cells will not disperse. Contaminated cell samples will sometimes agglutinate spontaneously.
Table 17.4 Matched results of forward and reverse typing
Cell grouping Serum grouping Interpretation Conrmed
anti-A anti-B A cells В cells Cell group Serum
group
+ – – + A A A
+ – + + A 0 A
– + + – В В В
+ + – – AB AB AB
+ + + AB В A
– – + + О О 0
* A person of A
or A2B blood type may occasionally have anti-A1 in serum
2
blood
group
*
2
B*
2
False negative results
There are various reasons for showing a negative reaction in haemagglutination. The serum
may be inactive, it may have been omied from the tube, red cells may not be in good condition
or the technique may have been incorrectly performed. Reliability of antisera is extremely
important for obtaining correct ABO typing. Avoid exposing antisera to room temperature
when not in use, and always store it overnight at 4°C; in case of prolonged storage, –25°C
is recommended. Check antisera periodically with known red cells. Discard outdated,
contaminated or weak reacting antisera which might yield false results.
Variable reactions
Most often the variable reactions are due to incorrect technique of ABO typing. For examining
all haemagglutination reactions, the suggested centrifugation is approximately 15 s at 900–
1000 RCF or 1 min at 100–125 RCF. The centrifugal force applied to cell/reagent mixtures
should be the minimum required to produce a ‘buon’ of red cells and a clear supernatant.
Over-centrifugation causes cells to adhere to the boom of the test tube so that vigorous
agitation is necessary before cells can be re-suspended. During such agitation, weak
agglutination may be dispersed causing a positive reaction to be missed. No one speed and
time of centrifugation can be recommended which will cover the wide variety of centrifuges
available; each laboratory must calibrate its own equipment and determine the time required
at a given speed to achieve the desired result.
While performing the slide test, do not interpret peripheral drying as agglutination. Blood
typing of the ABO system should be performed only at room temperature; do not heat slides
or incubate the tubes.
Routine Laboratory Procedures in Blood Bank
437
Rh Blood Typing
Rh typing (also called Rh blood grouping) is next important to ABO typing. Presence of
Rh antigen (i.e., D antigen) on red cells is determined by the haemagglutination reaction of
red cells after the reaction of laer with anti-D antiserum at body temperature or at room
temperature in the presence of protein. The technique is similar to ABO typing and hence Rh
typing is performed along with ABO typing.
Clinical significance
An Rh-negative individual should not receive Rh-positive blood. If this does occur, a
transfusion reaction will not occur with the rst transfusion. However, anti-D may be
produced in a few days to few weeks, and this will strongly react with the D antigen of Rhpositive cells in subsequent transfusions. Conversely, Rh-negative blood can be safely given
to an Rh-positive individual. The former does not have the D antigen to cause sensitization.
The Rh blood group is independent of the ABO blood group. Thus, there are eight types of
basic blood groups (ignoring subgroups) Rh-positive—A, B, AB and O; Rh-negative—A, B,
AB and O. More than 95% of the Indian population is Rh-positive.
Some individuals have weak D expression and can be wrongly typed as Rh-negative without
additional testing for weak D. A weak D donor should be considered as an Rh-positive donor.
Principle
The Rh typing refers to the determination of the presence (positive) or absence (negative) of
D antigen on red cells when reacted with anti-D serum. Rh-positive cells show agglutination
with anti-D in saline medium with incubation (37°C) or in the presence of protein (albumin)
at room temperature. Anti-D supplied by the manufacturer contains information about
the specic test conditions to be followed—slide method or tube method and temperature
of reaction. Rh-negative cells do not agglutinate with anti-D under any of the aforesaid
conditions.
Specimen
This is described under ABO typing. Either cloed or anticoagulated blood can be used.
Equipment and reagents
These are same as described under ABO typing with the addition of the following:
• Slide viewing box for slide test and water bath (37°C) for tube test.
• Anti-D: Follow manufacturer’s directions in the use of anti-D. Usually the anti-D
supplied by the manufacturer is good for tube testing as well as for slide testing; the
laer has high protein. If the anti-D is specically designated for the tube test, do not
use it in the slide test.
• Rh-positive and Rh-negative control cells.
• Cell suspension: Prepare a 4% red cell suspension (see Figure 17.2) for the tube method
and 45% cell suspension for the slide method according to the method described earlier
in this section. Suspension can be made in saline or in serum of the specimen.
The procedure for performing Rh typing is given below.
Slide Method (Figure 17.7)
1. Switch on the slide viewing box, the thermometer should read 40–45°C before the slide
is placed on it. If the viewing box does not have a thermometer, leave the box on for a
few minutes until it is suciently warm.