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Medical Laboratory Technology: Volume 1
Red cell component
At no point should ice be allowed to come into direct contact with the blood as the
red cells nearest to the ice might freeze and haemolyse. Appropriate materials and
packing arrangements are therefore necessary. In boxes shipped long distances or at high
environmental temperatures, the volume of ice should at least be equal that of the blood. In
an insulated container, the temperature can be considered to be in the 1–10°C range as long
as unmelted ice is still present on arrival at destination.
Plasma
There should be at least as much wet ice in the cold box as there is plasma. It is important to
protect the frozen plasma units during transportation. If possible, they should have been
placed in cardboard boxes before freezing to protect bags from developing small cracks. A
simple method to determine if plasma units have thawed and refrozen is by placing a rubber
band around the unit at the time of preparation. Once the unit freezes it leaves an indentation
at the sides. If the unit has thawed, or thawed and refrozen, the indentation will not be there.
Platelets
Containers for transporting platelets should be equilibrated at a temperature of 20°C–24°C
before use. If outdoor temperatures are extremely high, special chemical, coolant pouches
are available that may be shipped with platelets and maintain temperatures of approximately
20–24°C for up to 12 h. Also available are containers with a power source that maintains
temperatures between 20-24°C. Platelets should reach their destination within 24 h, which is
the maximum time allowed without agitation.
delivery of Blood and Blood componentS to clinical areaS
When blood is issued from the blood bank, the time of issue must always be recorded. Blood
should be issued in a cold box or insulated carrier which keeps the temperature under 10°C. To
avoid wastage, only one unit of red cells should be removed from the blood bank refrigerator
at a time unless the rapid transfusion of large quantities of blood is required. Platelet
concentrates should be issued from the blood bank in a carrier that keeps the temperature
between 20°C–24°C. Platelets should be transfused as soon as possible. If unused, they should
never be placed in a refrigerator, but returned immediately to the blood bank. Fresh-frozen
plasma and cryoprecipitate are thawed at between 30–37°C in the blood bank before issue
and transported to the ward at ambient temperature. They must be used immediately and
should never be refrozen.
The blood bank personnel are responsible for the issue of blood to the respective hospital
ward on the understanding that the blood will be transfused within 30 min. If the transfusion
cannot be commenced within 30 min, the blood may be stored in an approved and monitored
blood storage refrigerator in the hospital ward until required for transfusion. In busy
facilities such as the operating theatre or the ICU, it is a commonplace to have a blood bank
refrigerator that stores blood components for immediate use. This may be cross-matched
blood or Group О Rh-negative blood. If an approved blood refrigerator is not available and
the blood component cannot be administered within 30 min, the blood should be returned to
the hospital transfusion laboratory or blood bank for storage until required.
Return and Reissue Policy of the Blood Bank
If a blood component is returned to the blood bank, the following checklist should be used to
decide whether it should be put back into stock or discarded.
Collection and Processing of Blood for Transfusion
• Check that the unit has been returned to the blood bank within 30 min of issue.
• Verify that the unit has not been opened, by squeezing it gently and looking for blood
at the entry port.
• Check the temperature by hand or by folding the unit around a thermometer.
• For red cell units, after mixing the unit gently, keep it in the upright position while
it ‘seles out’ in the refrigerator and look for signs of haemolysis or other signs of
deterioration in the unit.
Refuse to return the issued blood or its components, if:
• The unit has been out of the refrigerator for longer than 30 min, the seal of the unit is
broken;
• The bag shows signs of haemolysis;
• The temperature of the unit is over 10°C (for red cell units);
• The decision to discard or to re-use a unit of blood should only be taken after consulting
with the responsible physician at your hospital or blood bank.
419
review queStionS
1. On what grounds are donors rejected and why?
2. Why is a volunteer donor’s blood preferred over paid donors?
3. Why did the plastic bags replace the traditional glass boles?
4. What steps would you take to avoid wanning of the blood collected for transfusion in
the mid-summer of a tropical country?
5. How do you store red cell units? What is the shelf-life of the stored red cells? What
changes occur in the blood during storage?
6. Why is it necessary to screen a donor? Under what conditions the donor is permanently
barred from giving blood?
7. What is the minimum haemoglobin required to be present in the donor? Why is this
important?
8. How does the technician check the haemoglobin concentration of the donor in the eld?
9. What is the principle of copper sulphate method of determining the haemoglobin
concentration of blood?
10. How are the following blood components prepared? Under what clinical conditions are
they transfused to the patient?
Packed red cells, Cryoprecipitate, and Platelet concentrate
11. Why are patients with bleeding disorders given fresh-frozen plasma and platelet
concentrate?
12. Under what conditions will the blood bank refuse to take back the blood component
which has been issued for transfusion?
13. How is the donor prepared before drawing blood? What is the gauge size of the needle
used in phlebotomy? Name the antiseptics used.
14. What is the response of the technologist or nurse if you nd the donor feeling
uncomfortable and dizzy?
15. Describe the method of transportation of red cells or plasma units in warm weather.
How are they stored?
16. How are platelet units collected and stored?
17. What precautions do the laboratory takes to protect blood components in case of power
failure?
420
Medical Laboratory Technology: Volume 1
Routine Laboratory Procedures in Blood Bank
Pampee Paul Young and Jay S Raval
Chapter Outline
• Signicance of Quality Control in Blood Bank
• Specimen Collection for Blood Bank
• General Laboratory Preparations in Blood Bank
▪ Equipment
▪ Reagents
• Preparation of Laboratory Reagents in Blood Bank
▪ Preparation of antisera for blood typing
▪ Preparation of reagent cells for blood typing
• Reporting of Haemagglutination Reaction
• ABO Blood Grouping
▪ Preparation of red cell suspensions
▪ Slide method
▪ Tube test method
▪ Final report
▪ Sources of error in ABO typing
• Rh Blood Typing
▪ Slide method
▪ Tube test method
▪ Sources of error in Rh typing
• Antihuman Globulin (AHG) or Coombs’ Test
▪ Direct antihuman globulin test or direct Coombs’
▪ Indirect antihuman globulin test or indirect Coombs’
▪ Sources of error in antiglobulin test
▪ Weak D testing
• Major Cross-Match
▪ Routine cross-match
▪ Emergency cross-match
▪ Sources of error in cross-matching
• Antibody Screening Test
• Identication of Unexpected Antibodies
▪ Elution of antibodies
• Titration of anti-D
• Review Questions
• Appendix
17
Routine Laboratory Procedures in Blood Bank
No other department in a pathological laboratory carries the same degree of responsibility
as the blood bank, for in no other department is an error so likely to result in the death of
a patient. Hence, the technician working in the blood bank must take appropriate quality
control measures in order to avoid crucial errors. A reliable specimen, standard technique,
proper functioning equipment, clean glassware, dependable reagents and their proper
storage and accurate recording are all essential parts of the quality control system. Whenever
possible, include a positive control to demonstrate that the reagent is potent.
421
Significance of Quality control in Blood Bank
Proper functioning of the blood bank can only be accomplished when the hospital sta
realize the signicance of accurate specimen collection, proper identication of the specimen,
appropriate handling of blood during transit and transfusion of compatible blood to the
correct recipient. Work must be organized in such a way that clerical, serological or other
errors will be detected before any harm is done.
All crucial steps related to transfusion therapy need double checking by two separate
individuals. In the ward, any blood specimen taken from a patient should be put into a
container that has been previously labelled with the patient’s full name, the name of the
hospital, hospital number, ward number, name of the phlebotomist, date and time. Similarly,
when a unit of a blood component is received from the blood bank for transfusion, two
people should check that the blood is administered to the right person. This is a ‘must’ for
any laboratory.
Within the laboratory, double checking of blood grouping can be performed by performing
the forward and reverse typing on two independent samples or by two independent
technicians and results compared. In case of compatibility testing, a single experienced
technician may perform all phases of compatibility testing—saline and albumin; thermophase
and antihuman globulin, and the results must be well documented. Some laboratories prefer
the compatibility testing to be performed by two independent technicians and results must
be tallied.
Specimen collection for Blood Bank
A pretransfusion specimen from the potential recipient’s blood should accompany every
request for blood from the blood bank. Many hospitals aach a special blood bank band, at
the recipient bedside, at the time of specimen collection. The blood bank band must remain
aached to the patient’s wrist throughout the transfusion period. The same band may be used
throughout one hospital admission as long as the information printed on the band is legible
and the band is still securely aached to the patient’s wrist. The specimen for typing and
screening is valid for three days. One can use that sample for multiple component requests
over that time period. The phlebotomist must always sign on the label of the specimen.
The specimen should be collected in a dry sterile test tube without anticoagulant. A
minimum of 5 mL blood sample is required. Double checking of the sampled blood
specimen and the identication of the patient is required. This is usually done jointly by the
aending physician and nurse in the ward. The label should indicate the name of the patient,
hospital name, hospital number, ward, date and time of specimen collection. The specimen
must be accompanied by the prescribed form (Appendix 17.1) of the hospital, requesting
that the specimen be typed and, if necessary, serum samples be used to perform compatibility
tests on one or more units of blood. The design of forms for requesting blood components
varies according to the local needs, and Appendix 17.1 will serve as a guide. All writing on
labels must be done with a ballpoint pen validated to remain legible in the refrigerator.
422
If the patient is an infant and it is dicult to collect blood by venepuncture, then 10–20 drops
of blood should be taken by heel stab into a dry sterile tube containing 3 drops of trisodium
citrate anticoagulant solution (anhydrous salt, 3.8% in water, and stored in a refrigerator).
Heparinized blood can also be used.
Special Note Except in emergencies, requests for blood and cross-match grouping should
be submied at least 24 h before the transfusion is to be given to allow time for any special
investigations that may be necessary.
Medical Laboratory Technology: Volume 1
general laBoratory preparationS in Blood Bank
Suitable equipment and reliable reagents, other than the quality of the specimen, determine
the reliability of the results generated in a blood bank. Laboratory equipment of the blood
bank largely depends on kinds of services it renders, the available budget and the area in
which it is located. The following is a list of essential items any laboratory is likely to require.
The names of some of the notable suppliers are given in the Appendix of Volume III. All
equipment in the laboratory must be checked regularly regarding their proper functioning.
This includes refrigerator, freezer, centrifuge, and water bath.
Equipment
• Centrifuge:
• Small low-speed centrifuge, bench model (1000 rpm, 100 G) for separating serum. A
hand-driven centrifuge can be used in places where electric supply is not available
or is erratic.
• Large refrigerated centrifuge, oor model, for separating blood components.
• Special bench model blood bank centrifuge, which is of swing-out type. If electric
supply is not available (or erratic), hand-driven centrifuges can be used. These special
centrifuges can aain high speed (2500 rpm, 250 G) in a short time which helps to
centrifuge the red cells from suspension, forming packed-cell-sediment (called a
‘buon’), at the boom of the centrifuge tube. Standardized speed of the blood bank
centrifuge is important in order to obtain reproducible results of haemagglutination.
• Refrigerators and Freezers: Equipment needed to reliably maintain average temperatures
of 4°C and –25°C. The laer is used for storing antisera, cryoprecipitate, and frozen
plasma while red cell units are stored in the fonner. Both should be ed with an alarm
system.
• Platelet rocker with temperature control set at 22°C.
• Spotlight with concave minor: This helps in observing accurately haemagglutination
reaction of the tube test. A magnifying glass (8×) can be used in its place; for this, a welllighted white background is desirable.
• Glassware
• Volumetric asks
• Test tubes: Glass (neutral) test tubes, without lips, of various sizes (10 mm × 75 mm in
large numbers); these are also sold as Kahn tubes.
• Pasteur pipees: These are either made in the laboratory or purchased. Standardized
Pasteur pipees deliver 20 drops per millilitre.
• Graduated centrifuge tubes
• Graduated pipees: 1, 5 and 10-mL
Routine Laboratory Procedures in Blood Bank
423
Cleaning of laboratory glassware in blood bank
Clean glassware is important for laboratory work in a blood bank. Detergent is most deleterious
for the red cells; hence, the glassware must be thoroughly washed. Use of disposable test
tubes is not common in developing countries.
1. All test tubes and small glassware should be kept in a wire basket. Test tubes should be
bundled together with a rubber band, with the open end facing up and these (and other
glassware) should be rinsed with cold tap water.
2. All glassware should be left overnight in a suitable detergent (this can be shortened by
boiling in hot detergent for 10 min).
3. If the detergent is strong, it should be neutralized by rinsing with 3% hydrochloric
acid before washing thoroughly. If hand washing is done, the glassware should be
shaken vigorously to expel the detergent or acid before washing in fresh tap water. It is
recommended that the glassware be rinsed at least six times with repeated brisk shaking
between each rinse.
4. All glassware should be nally rinsed with distilled water (or deionized water).
5. Before removing rubber bands holding the test tubes, Pasteur pipees and other
glassware, shake o the water and pack them in wire baskets with the open end
downwards.
6. The glassware should be dried in an oven at 120°C for two hours.
7. Finally, the glassware needs to be inspected thoroughly under a good light to ensure that
all traces of blood, grease and detergent have been removed. Clean any dirty glassware
again or discard them.
Reagents
• Albumin (20% bovine albumin, w/v)
• Sodium chloride solution or saline (0.8% NaCl, w/v). Use fresh saline solution.
• Antisera and reagent cells: Anti-A, anti-B, anti-D, antihuman globulin, Al cells, В cells,
control Rh-positive and Rh-negative cells, O-positive cells (for antibody screening),
sensitized О cells (Coombs’ positive control).
Instructions of the manufacturer accompanying antisera or other reagents should always
be followed. Keep all antisera in the same place and with proper labels. The universal colour
code for anti-A (blue) and anti-B (yellow) should be followed if antisera are made in the
laboratory. Before using, the frozen antiserum must rst be brought to room temperature by
allowing it to thaw slowly, but care should be taken not to leave it at room temperature for
a prolonged period. The bole should be closed tightly when not in use. Every care must be
taken to avoid contamination of antisera with other reagents or with micro-organisms. The
expiry date given by the manufacturer should be checked and outdated material should be
discarded. If the antiserum is cloudy, the chance of bacterial contamination is high and it
must not be used. However, before discarding, it should be checked under the microscope
(400× magnications).
Storage of reagents
All the blood bank reagents should be stored in a refrigerator (4°C). Fresh saline solution can
be left at room temperature. Antisera can be frozen but not the reagent red cells.
Additional information
• A1 cells are agglutinated by both anti-Α1 and anti-A2 sera.
• Usually the anti-D supplied by the manufacturer is suitable for tube testing as well as
for slide testing; the laer is easier to perform. If the anti-D is specically for the tube
424
test, do not use it in the slide test. It is important that instructions for testing provided in
the insert by the manufacturer along with anti-D are strictly followed.
Medical Laboratory Technology: Volume 1
preparation of laBoratory reagentS in Blood Bank
Antisera and reagent cells are now commercially available in all developing countries. They
are reliable but expensive. If the laboratory can aord it, antisera and reagent cells should be
purchased from reliable suppliers. If the laboratory intends to prepare some of the reagents
while purchasing the ‘dicult to make’ ones from outside, the following section may be
referred.
Preparation of Antisera for Blood Typing
Known antisera are used in blood typing in order to identify red cell antigens. The antisera
to be used should be of high titre (at least 128) so that the results are not ambiguous. Anti-Α
is obtained from group В individuals, anti-B from group A individuals and anti-Α plus
anti-B from group О individuals. It is essential that every typing serum is carefully tested
for potency, specicity, avidity and freedom from Rouleaux-forming properties. Ideally, it
must contain a powerful antibody that reacts only with red cells carrying the corresponding
antigen and it must agglutinate such red cells quickly to form large clumps (4+ reactions).
Many laboratories identify persons for obtaining typing sera and bleed them intermiently.
Donor’s blood is obtained by venepuncture but without any anticoagulant. The blood
is allowed to clot and the serum is removed aseptically into another sterile container via a
Pasteur pipee. Test the serum against known reagent cells (A1 cells, В cells, A1B cells and О
cells). If the serum meets the following criteria, it is then transferred aseptically into smaller
labelled containers and stored at–20°C.
• The titre of antisera (anti-A, anti-B) should be at least 128.
• Procedure for determination of titre: Mix 0.1 mL of serum with 12.7 mL of saline to
yield 1:128 dilutions, which is the test point of titre determination. Mix the diluted
serum (1:128) with red cells bearing corresponding antigen (e.g., A1 cells with anti-Α).
The serum is acceptable for further processing if visual agglutination is present in the
diluted serum.
• The antisera should not haemolyse red cells carrying the corresponding antigens.
• Acceptable avidity of each antibody for the corresponding red cell antigen is required.
This is tested by the slide method by adding one drop of 10% saline suspension of
washed cells to an equal volume of serum on a microscope slide or opal glass tile and by
rocking the mixture gently. Agglutination of A1 cells and В cells becomes visible in 10 s.
Antibodies are best sterilized by ltration. This prevents contamination of the reagent
antibody. Put the sterilized serum in 4-mL quantities in sterilized containers of 5-mL capacity.
Preparation of anti-D
Routine blood bank laboratories do not have sucient facilities for preparing their own
anti-D (anti-Rh) sera. Nevertheless, the basic procedure is described here for unavoidable
circumstances.
Suitable donors for contributing anti-D are Rh-negative mothers who have recently
delivered infants suering from haemolytic disease. Previous laboratory records indicate the
presence of anti-D in the donor. The patient should be bled by the routine procedure and
blood is collected in a dry container. Undesirable anti-A and anti-B are removed by treating
with fresh D negative red cells (not more than ve days old) containing corresponding red
cell antigens A and B.
Routine Laboratory Procedures in Blood Bank
At the nal stage, the quantity of anti-D is determined by titration, reacting with Rh-positive
cells; a titre value of 32 is desirable. If agglutination is visible without albumin, it may be
used as a saline anti-D, and if albumin is needed, it is used as albumin anti-D. Anti-D serum
should be stored at –20°C in small vials containing only 0.5 mL of the antiserum.
425
Preparation of antihuman globulin antisera
Only reference laboratories with facilities for housing animals and possessing skilled sta
to carry out the complicated procedure of purication and standardization are capable of
preparing antihuman globulin. Routine laboratories purchase the antihuman globulin (AHG)
sera from reliable manufacturers.
Preparation of Reagent Cells for Blood Typing
Reagent cells are needed to identify corresponding antibodies. Three types of reagent cells
are needed to identify antibodies related to ABO—A1 cells, В cells and О cells. Blood group
A,B can also be included, but not essential.
Identify ve dierent donors with known blood groups of A1; В and O, ignoring their
Rh type. Collect the blood by routine procedure in a dry container without anticoagulant
(though anticoagulated blood may also be used). Cells are obtained from the clot.
Wash the cells three times with saline using medium speed centrifugation (1500 rpm, 150
G). Prepare a 2–5% cell suspension in saline for the tube method and a 10% cell suspension
in saline for the slide method. Some laboratories prefer higher concentration for the slide
method (45%, v/v). Test the suspensions of A1, В and О cells with commercially available
antisera—anti-A and anti-B. О cells will not be agglutinated by either of the antiserum.
Preparation of reagent O cells for antibody screening
Reagent О cells are used for the identication of irregular antibodies other than the ones
associated with basic blood groups (ABO). The following technique of obtaining ‘pooled Rhpositive О cells’, divided into two groups, provides a reasonable opportunity to screen the
presence of abnormal antibodies.
Select four dierent O-positive donors (blood group О and Rh-positive). Collect the blood
in four separate dry containers without anticoagulant. Then remove red cells from the clot
and suspend them individually in saline solution. Wash the cell suspension three to four
times with saline with repeated centrifugation.
Pool the washed cells of two donors together and prepare a 2–5% cell suspension in saline.
Repeat the same for the other two donors. Label these two groups of pooled O-positive cells
as I and II. These pooled O-positive cells will be used for the antibody screening but not for
antibody identication. Identication of specic antibody can only be performed by using
О cells with known antigenic characters (called panel cells). These antigens are identied by
reference laboratories after reacting them with antibodies of known specicity.
Preparation of coombs’ control cells or sensitized red cells
Coombs’ control cells are ‘sensitized’ O-positive cells. Sensitization of O-positive cells
is brought about by reacting them with weak anti-D in a way that the O-positive cells are
coated with anti-D (IgG) but the immunologic reaction is not strong enough to agglutination.
These sensitized O-positive cells agglutinate when they come in contact with AHG serum or
Coombs’ reagent.
Coombs’ control cells are used to test the reactivity of AHG serum or Coombs’ reagent. This
is crucial in the nal stage of cross-matching (AHG phase). Lack of haemagglutination in the
AHG phase of crossmatching could indicate compatible donor red cells with antibodies
426
Medical Laboratory Technology: Volume 1
of the recipient present in the serum. This negative haemagglutination can also be a false
indication of compatibility due absent or inactive AHG. In order to eliminate the laer, add
sensitized O-positive cells into the tube with AHG and donor’s red cell suspension; sensitized
cells must agglutinate indicating that the AHG is present and active.
1. Wash 0.5 mL of packed red cells 3 to 5 times with saline. After the nal wash, suspend
the cells in 1 mL of saline. Add 2 to 3 drops of weak or diluted anti-D and mix. If the
anti-D causes more than 1+ agglutination reaction, anti-D should further be diluted. The
1+ reaction is recognized by the formation of a few clumps with many free red cells and
a turbid background.
2. Incubate the Rh-positive cells suspended in weak anti-D at 37°C for 15 min. The cells get
sensitized with anti-D coating.
3. Wash the sensitized cells 3 to 4 times with large volumes of saline with repeated
centrifugation.
4. Prepare a 4% suspension of the sensitized washed red cells by adding saline to the
packed cell in ratio of 2.4 and 0.1 mL.
5. Take a drop of sensitized cell suspension in a test tube and add AHG reagent; mix,
centrifuge and read the agglutination reaction. It should give a 4+ reaction (Table 17.1).
Interpretation Agglutination indicates that red cells are sensitized and the AHG serum
is satisfactorily eective. A negative reaction of AHG is conrmed by adding the sensitized
О cells. Agglutination indicates the presence of active AHG and the negative reaction in the
previous step is truly negative and not due to the absence or inactivation of the AHG reagent.
reporting of Haemagglutination reaction
It is desirable that haemagglutination reactions are reported semi-quantitatively. This is
shown in Table 17.1 and Figure 17.1. It helps to assess the degree of immunologic reaction
and is also useful in the determination of titre.
Table 17.1 Macroscopic agglutination reaction in a test tube
Observation Report
One solid aggregate, clear background 4+
2–3 large agglutinates with clear background 3+
Several small agglutinates with many free cells, and clear background 2+
Several small agglutinates, reddish background of free cells 1+
Tiny aggregates, turbid background giving a granular appearance (weak) w
No agglutination or haemolysis (negative) 0
Note Haemolysis (pink colour of supernatant uid) should be considered as positive immunologic reaction between
the red cell antigen and the corresponding antibody.
Routine Laboratory Procedures in Blood Bank
427
Figure 17.1 Haemagglutination reaction: (a) Haemagglutination of red cells occurs when the red cell
antigens (agglutinogens) react with the corresponding antibody, (b) Positive agglutination
is observed by the clumping of red cells as seen on slide or by the button formation in the
test tube. Lack of agglutination (b) results in uniform distribution of red cells and lack of
button formation
aBo Blood grouping
ABO and Rh typing are the rst steps of analysis of a blood bank specimen. The techniques
described here are in conformity with the Technical Manual of the AABB (formerly known
as the American Association of Blood Banks) and standards laid down by the World Health
Organization.
The two basic methods to observe the haemagglutination reactions in ABO and Rh typing
are slide method and test tube method. The former is easier to perform and the laer is more
sensitive. Many laboratories in developing countries perform the test tube test only in case
where the results of the slide test are doubtful.
ABO blood typing is done in two ways—forward typing and reverse typing. In forward
typing, the antigens of red cells are determined by reacting with corresponding antibody that
leads to haemagglutination (Figure 17.1). In reverse typing, the specicity of the antibody is
determined which should concur with the expected antibody in the ABO type established by
forward typing.
Specimen
Centrifuge the cloed blood at low speed (2500 rpm, 250 G) and separate the serum into
another previously labelled test tube. Cells are separated from the clot via a Pasteur pipee
and suspended in saline in another pre-labelled test tube. Red cell suspension is used in for