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388
particular, there is a weak subgroup of D (Rho) called weak D. Weak D red cells often fail to
give a positive reaction with some commercial Rh anti-D typing serums and so may be falsely
typed as D-negative. Therefore, many large blood banks screen D-negative red cells for weak
D as well.
Rh antibodies are usually univalent and react best in vitro at 37°C in high-protein medium.
Medical Laboratory Technology: Volume 1
other Blood group systems
Besides the ABO and the Rh blood group systems there are a number of other unrelated
blood group antigen systems that have some importance, either for medico-legal parenthood
studies or because sensitization to these antigens causes transfusion reactions or haemolytic
disease of the newborn.
The most important of these systems is Keli (К), a well-recognized blood bank problem.
The Kell antibodies are similar in characteristics and behaviour to the Rh system D (Rho)
antibody. Fortunately, only about a small percentage of population have the Kell antigen and
are thus Kell positive, so that opportunities for sensitization of the Kell-negative persons are
not great. Kell antibody is univalent and, like Rh, acts best in vitro in a high-protein medium
at 37°C. If reactions to Kell antibodies occur, results of the direct Coombs test are positive
(unless the aected red cells are destroyed). A similar situation exists for the Duy (Fy) and
Kidd (Jk) systems.
There are other systems that resemble the ABO system in their antibody characteristics, and
these include the MN, P, Lewis (Le) and Lutheran (Lu) systems. They are primarily bivalent
antibodies and react best in vitro in a saline medium at room temperature or below. Unlike
ABO antibodies, they are rare causes of transfusion reactions and when diculties arise, they
are clinically milder than problems associated with the univalent antibody systems.
There are a large number of minor blood group antigens, the discussion of which is beyond
the scope of this book. Only a few, more important ones will be mentioned here. One of
these is the I-I, or IH system. This system is very weak in the red cells of newborns and
becomes established at approximately after 2 years. Anti-I (or anti-IH) antibodies are IgM
cold agglutinins. Anti-I antibody is a frequent cause of viral or mycoplasma-assocmted cold
agglutinins or idiopathic cold autoimmune haemolytic anaemia.
In the vast majority of cases, other than high titre anti-I antibodies, the minor group antigens
or antibodies are not clinically signicant. However, they are a great source of frustration to
the blood bank when they occur, because any unexpected antibody must be identied and
because they interfere with the cross-match and cause donor red cells to appear incompatible
with the recipient.
Summary
The major blood group systems outside of ABO and Rh become important only after patients
develop unexpected antibodies. Then, a fundamental knowledge of antibody characteristics,
clinical signicance and antigen frequency is needed to help conrm antibody specicity and
to select appropriate units for transfusion.
Only a few antibody specicities are commonly seen: M, PI and I antibodies react at room
temperature and are considered clinically insignicant; K, S, s, Fya, Fyb, Jka and Jkb antibodies
react in the antiglobulin phase and are clinically signicant.
Not all antibody problems are easily resolved; panel reactions are sometimes inconclusive.
Resolution of antibody problems involving the unusual specicities described here might
require the assistance of an immunohaematology reference laboratory.
Introduction to Blood Transfusion Therapy
389
pretransfusion testing
Blood transfusion today is a major medical service that is rendered to patients needing
whole blood or its components. Major developments in the eld of transfusion therapy have
occurred since the beginning of the Second World War. It is accepted as a routine and relatively
safe procedure in the management of patients. However, as the number of transfusions
administered was increased, it was found that compatibility of major blood groups is not the
only criteria to avoid transfusion reactions. Many unexpected transfusion reactions occurred
due to the presence of unexpected antibodies in the serum of the recipient which recognize
and bind to the donor’s red cells. In many cases, due to incorrect blood typing, transfusion
brought disastrous results. To prevent this, the concept of a cross-match evolved. Today,
‘major cross-match’ is an essential pre-transfusion testing in which the red cells of the donor
are placed into the serum of the recipient under standard conditions. The older procedure
of ‘minor cross-match’ is no longer done where the red cells of the recipient were matched
with the serum of the donor. This is because the amount of serum coming from the donor is
relatively small and can be avoided by transfusing packed red Cells. The minor cross-match
has now been replaced by an antibody screening procedure on recipient serum.
antiBody screen
The antibody screen consists of the recipient serum and a commercially supplied panel of
dierent blood group О red cells that contain various other blood group antigens. If there is
free antibody in the patient serum (other than ABO antibody), it will either coat (sensitized)
or will agglutinate the test red cells. By this testing process the presence of the antibody
will be detected. The test is carried out by incubation at 37°C (mimicking the conditions in
vivo) and usually, with some enhancing agent like albumin or low ionic strength medium
which accelerate the reaction. This is followed by a direct Coombs test. If the test is positive,
presence of unexpected antibody is detected. This calls for the identication of an unexpected
antibody, which is done by looking into the paern of reaction of the red cells (that bears
known antigenic proles), with the serum containing unknown antibody. Most of the
peripheral laboratories do not have facilities to identify the antibody and hence, the serum
specimen is sent to a central laboratory as needed.
One should bear in mind that the aforesaid antibody screening test does not detect errors
in ABO typing, since the reagent red cells are all group О and thus non-reactive with anti-Α
and anti-B antibody.
Major Cross-match
One can compare the major cross-match as transfusion process in vitro. Donor’s red cells
placed in the recipient’s serum and incubated at 37°C with the presence of albumin or
other enhancer. The goal is to have a condition where the antibodies in the recipient’s plasma
should not have any reaction towards the red cell antigens of the donor (Figure 15.7). The
reaction is detected by agglutination or haemolysis of donor red cells. Occasionally, however,
the red cells are sensitized (coated with the antibody) without agglutination or haemolysis.
In that case, the Direct Coombs Test recognizes the sensitization process by detecting the
antibody-coated red cells.
Major cross-match is the nal step before starting transfusion. It checks all the previous
preliminary steps taken in search of compatible blood—ABO blood grouping and antibody
screening. In spite of all the care taken by the laboratory in providing compatible blood,
clerical errors resulting in the transfusion of wrong red cells to wrong patient can occur.
Hence, the nurse should be very careful in comparing the name of the recipient with the red
cells supplied by the blood bank.
390
Medical Laboratory Technology: Volume 1
Figure 15.7 Blood transfusion therapy: (a) Blood selected for transfusion should be compatible with
recipient’s blood, (b and c) Recipient’s plasma should not have any reactive antibody
towards the red cell antigens of the donor or any complement binding antibody, (d)
Post-transfusion reactions could be haemagglutination, or (e) haemolysis. The latter
immunologic reaction is complement mediated.
The above method may recognize ABO incompatibility but will not detect errors in
Rh typing if no Rh antibodies are present in donor or recipient blood. This is because Rh
antibodies are not naturally-occurring.
The classical cross-match technique has gone through many changes since 1980. Initially
the testing was performed in three sequential parts:
• Immediate spin: React at room temperature in saline medium.
• Incubation at 37°C with 22% albumin added (or LISS, low-ionic strength saline, which
reduces the incubation time from 30 min to 15 min) for enhancing the immunologic
reaction process.
• Perform the Coombs’ test that detects weaker univalent antibodies.
In recent years, the rst two phases have been eliminated. This is keeping with the goal
of supplying blood within a short time under emergency situations and saving money spent
on treatment. The rst step of ‘immediate spin’, which recognizes ABO blood compatibility,
is no longer necessary as a pre-transfusion test. ABO blood typing of the donor and the
recipient are performed much in advance and hence the one step of the major cross-match
might provide enough information to start the transfusion process much more quickly. This
policy may have to be modied in case of patients who received repeated transfusions and
developed many immune antibodies. Search for unexpected antibodies in patient’s serum is
done only when incompatibility is detected in the cross-match.
A word of caution that should be highlighted here relates to the few patients whose blood
presents unexplained diculty in cross-matching. Although the laboratory tries its best to
resolve the problem by going back to the classical approach of cross-matching, in the absence
Introduction to Blood Transfusion Therapy
391
of complete cross-matching, blood is given as a calculated risk. One should bear in mind that
the life of the patient may be hanging on a greater risk.
compatiBle Blood groups
During blood transfusion, an identical ABO blood group of the donor is ideal. However,
other blood groups can also be used (Table 15.4). Persons belonging to blood group О with
Rh-negative blood type are considered to be ‘universal red cell donors’, i.e., their red cells
can be given to individuals of any other blood group. The red cells do not carry either A or
В antigen and hence they do not react with their corresponding antibodies. If whole blood
is transfused, any unexpected antibodies in donor serum against recipient red cells would
be diluted by the volume of the blood in the recipient. The current blood bank procedure,
however, is to use ABO group specic blood (including same Rh type), under emergency
situations, which is more meaningful than using О Rh-negative blood in a blind fashion.
Even so, there is a risk involved, since the recipient might possess antibody to some red cell
non-ABO blood group antigen of the donor (e.g., anti-Rh or anti-Kell). A cross-match would
detect this. Again, if whole blood is transfused, the anti-Α or anti-B in group О blood may be
in high titer and transfusion reactions might occur when this blood is used in recipients who
are group A, В or AB. It will be wise to maintain a certain amount of low-titer O-negative
blood for use in emergencies.
Note A titer over 50 is usually considered to be too high.
Table 15.4 Blood group compatibility (ABO) in blood transfusion
Blood group of recipient Blood group of donor
Group A Group В Groap AB Group О
A Yes* No No Yes
В No Yes* No Yes
AB Yes Ye s Yes* Yes
О No No No Yes*
*Group specic: Yes-compatible; No-incompatible
Similarly, those with blood group AB are called ‘universal red cell recipients’. They can
receive red cells from any of the blood groups—A, B, AB or O. This is because they neither
have anti-Α nor anti-B.
Transfusion of specic component of blood, as needed by the patient, is preferred over
transfusion of whole blood. Multiple blood components (RBC, plasma, platelets) are needed
in case of large-scale blood loss (haemorrhage). Anaemic patients with adequate blood
volume but who are decient in red cells require packed red cells if increased oxygencarrying capacity is required. In preparing packed red cells for transfusion, the plasma is
removed and the red cell concentrate (up to 70 to 80% haematocrit) is diluted with saline and
then transfused. Transfusion of plasma, plasma components and platelet concentrates may be
needed for those patients with a clinically signicant coagulopathy and/or thrombocytopenia.
When repeated transfusion is needed, a new specimen should be drawn from the patient
(recipient) for cross-matching purposes if blood was last given more than 72 h earlier. Some
patients demonstrate marked anamnestic (immunologic memory) responses to red-cell
antigen that they lack and might produce clinically signicant quantities of antibody in a few
hours. Thus, this antibody would not have been present in the original specimen from the
patient.
392
Medical Laboratory Technology: Volume 1
review Questions
1. Dene the following terms.
Antibody, Antigen, Complement, Natural antibody, Immune antibody, Agglutinin,
Agglutinogen, Immunologic reaction
2. Which is the most important blood group in humans? How will you identify them?
3. If the father is AB and mother O, what are the possible genotypes of the children?
4. Which is the most common blood group in the Indian population?
5. In a laboratory test an A blood group person indicated the presence of anti-A in serum.
How could this be possible?
6. What is the Bombay blood group O? What are its characteristics? Why is it that a person
of Bombay blood group cannot receive blood group O?
7. What is an immune antibody? How is anti-Rh dierent from anti-A or anti-B?
8. What is the dierence between Wiener and Fisher–Race nomenclatures for Rh-associated
antigens?
9. What is weak D? Why is it important to determine weak D for an Rh-negative
individual? How do you recognize the presence of D antigen on red cells?
10. Name a few antibodies which are recognized only in the antihuman globulin phase.
11. Which blood group is known as ‘universal red cell donor’? Which one is known as
‘universal red cell recipient’? Give reasons.
12. What are the steps involved in classical cross-match. Which is the most essential step of
cross-match?
13. Under what conditions does the physician ask for blood transfusion without any crossmatch?
14. Imagine this situation: You are working in a blood bank when two units of red cells
were ordered by the Emergency Department without any clue of the patient’s blood
group. The patient is reported to be heavily bleeding and is likely to collapse. Which
will be your best choice of red cells? If the blood group of the patient is known, which
will then be your rst choice and why?
15. If a recipient is O-negative, what should be the donor’s blood group?
16. Which blood group should be reserved for use under emergency conditions?
17. Which blood component should be given to a patient with a history of haemophilia?
18. Under which clinical conditions should the patient receive packed red cells?
19. Under what clinical conditions are the following blood components given to the patient?
Packed red cells, Fresh-frozen plasma, Platelets, and Whole blood
Collection and Processing of Blood for Transfusion
Pampee Paul Young and Jay S Raval
Chapter Outline
• Selection of Blood Donors
▪ Criteria for rejecting donors
▪ Registration of donor
▪ Field test for the haemoglobin content of donor
• Method of Blood Collection
▪ Use of disposable plastic bags
▪ Preparation of blood drawing
▪ Adverse reactions of donor
▪ Basic laboratory tests
• Transportation of Blood after Collection
• Storage of Blood
• Common Equipment in a Blood Bank
▪ Refrigerators and freezers
▪ Power supply
▪ Platelet agitators
▪ Miscellaneous equipment and supplies
• Reagents
• Preparation of blood components
▪ Clinical signicance of using blood components
• Autotransfusion
• Plasmapheresis
• Transportation of Blood
• Delivery of Blood and Blood Components to Clinical Areas
▪ Return and Reissue Policy of the Blood Bank
• Review Questions
16
(The reviewers acknowledge the help extended by Mr. Kevin Cochrane, MS, MT (ASCP)
SBB of St. Luke’s Hospital in Bethlehem, PA, USA, for providing the current information on
‘Blood Collection Procedure’.)
394
Collection of whole blood from a healthy donor is one of the most important functions of
the blood bank. Good health of the donor should be carefully assessed and veried before
collecting the blood, which is important not only for the donor but for the recipient as well.
The vast majority of hospitals acquire blood from a healthy individual. The hospital may
work independently or in collaboration with various community organizations to acquire
blood. Volunteer donors provide the majority of donated blood in developed countries. In
developing countries, blood may also be obtained from paid donors. The collected blood
is stored temporarily in appropriate environment, processed for obtaining its various
components, tested fortransmiable diseases, initially ABO/D type and then supplied to
the site of the recipient (patient). Blood collection agencies do not perform the compatibility
testing, which is required before the blood is transfused so that the recipient may not have
any adverse reactions. These laboratory testing processes will be discussed separately. In this
chapter, we shall focus on the initial phases of blood collection, separation of its components,
storage and transportation. Careful organization of the ow of this precious gift of life, at
every step from the donor to the recipient, is important (Figure 16.1). Contaminated blood
can cause adverse eects in the recipient and may even be fatal.
Medical Laboratory Technology: Volume 1
Figure 16.1 Flowchart from blood collection to blood transfusion
Blood bank laboratories of developing countries face an additional challenge of obtaining
a reliable supply of energy to run their equipment. Hence they have to stay prepared with
alternate sources of energy to protect the precious blood, the gift of life. This is discussed
separately in Manual on the Management, Maintenance and use of Blood Cold Chain Equipment,
WHO Publication, Geneva, 2005.
Selection of Blood donorS
Prospective blood donors must meet several requirements to be eligible to donate whole
blood:
• The interval between donations must be at least 8 weeks.
• Donor should be an adult between 17 and 65 years of age.
• An adult donating 450 mL (± 45 mL) of blood must have a minimum weight of 50 kg
(110 lb). In case the donor weighs less than 50 kg, less amount of blood may be drawn.
Collection and Processing of Blood for Transfusion
• Minimum level of haemoglobin should be 12.5 g/dL. (The procedure for the eld test for
haemoglobin is given later.)
• Oral temperature should be 99.5°F (37.5°C).
• Pulse rate between 50 and 100 beats/min without any pathologic irregularity.
• Blood pressure should be less than 180.
• Donor should be without any skin lesions.
• Without any drug inuence, including alcohol or excessively nervous.
• Without any history of jaundice, viral hepatitis, tuberculosis, asthma, AIDS (acquired
immunodeciency syndrome), Creufeldt–Jakob disease (CJD), protozoan diseases,
infectious diseases.
• Person with sexually transmied diseases are deferred for one year after receiving the
negative report.
• Abnormal bleeding tendency are also rejected from blood donation.
• A drug addict should be barred from donating blood. Both arms should be checked for
evidence of sclerotic veins.
• Cases with skin piercing, acupuncture performed under questionable conditions, taoos
and skin allograph may also be reasons for deferral.
• Inmates of penal or metal institutions should be barred from blood donation for
12 months.
• Prospective donors should be deferred for 12 months after receiving hepatitis В immune
globulin (HBIG).
• A pregnant woman should not be allowed to donate for 6 weeks after the end of
pregnancy.
395
Criteria for Rejecting Donors
The donor is rejected if the person has:
• Received dental surgery within 72 h.
• Received transfusion within preceding months.
• Received immunization against smallpox, mumps, rabies or others within 2 weeks and
against rubella within 2 months.
• A history of malaria (for red cell products only) for last 3 years.
• AIDS (acute immunodeciency syndrome), jaundice, hepatitis and sexually transmied
diseases.
• Drug-related problems.
Registration of Donor
Healthy donors are rst registered (Table 16.1) before the blood is drawn. The registration
number must tally with the identication number given on the blood collection bag. The
use of barcode has greatly facilitated the process. This may not be available in peripheral
laboratories. The record is saved for at least ve years. The phlebotomist must sign the donor
record after completing the procedure. The record must show:
• Date of donation
• Name of the donor
• Identication, telephone number, address of residence
• Age and sex
396
Table 16.1 Blood collection record sheet
Date Location Signature of technician
Bag No. SerialNo. Donor Name Address Blood group Rh type
4260 1 Amitabh Singh
4261 2
4262 3
4263 4
Medical Laboratory Technology: Volume 1
Before drawing blood, the phlebotomist should explain the process to the donor and
gain his/her condence. If necessary, the donor might receive literature pertaining to blood
donation and the signicance of blood donation. Signing the consent form by the donor is
mandatory, which is subsequently countersigned by a witness. In case the donor is deferred,
the reason must be recorded.
field teSt for the haemogloBin content of donor
To be eligible to donate, donors must meet minimum haemoglobin of 12.5 g/dL. (Note: This
value might vary under certain conditions, e.g., ethnic origin.) During blood collection in the
eld without any electrical resource, the following copper sulphate method may be used. It
may not be very accurate but serves the purpose of accepting the donor.
Principle
The copper sulphate oatation method is based on the principle of the relative specic gravity
of haemoglobin. When a drop of blood is dropped into a copper sulphate solution, the drop
encases the haemoglobin in a sack of copper proteinate, which prevents any change in specic
gravity for about 15 s. If the drop of blood has a satisfactory haemoglobin concentration, and
therefore a satisfactory specic gravity, it will sink in the solution within 15 s. If not, the drop
will hesitate, will remain suspended or will rise to the top.
The copper sulphate solution used should have a specic gravity of 1.053 (equal to 12.5 g/dL
haemoglobin). The solution should be stored at room temperature and should be tightly
capped to prevent evaporation.
Supplies
• Arrangement for nger stick
• Rubber bulb to discharge blood from capillary
• Tall glass jars to hold the copper sulphate solution
Reagent
Copper sulphate solution: Specic gravity—1.055 for men and 1.053 for women
Preparation of copper sulphate reagent solution
A. Stock solution of copper sulphate (Specic gravity 1.1000)
CuSO45H2O 170.0 g
Distilled water 1006.3 mL
To measure the volume of water, dispense 1 L of water from a volumetric ask into a
2 L bole and then add 6.3 mL water from a 10-mL graduated pipee. Measure the specic
gravity which will be close to 1.1000. If necessary, add a few drops of water to adjust.
Note Purity of copper sulphate is often variable. Hence you may have to adjust the initial
weight of the sulphate.
Collection and Processing of Blood for Transfusion
B. Working solution
(a) For men (1.055 specic gravity)
Stock solution 54.30 mL
Distilled water (q.s.) 100 mL
(b) For women (1.053 specic gravity)
Stock solution 52.25 mL
Distilled water (q.s.) 100 mL
Check the nal specic gravity with a hydrometer
(Figure 16.2) or hand refractometer. If necessary,
add a few drops of stock solution (to increase the
specic gravity) or distilled water (to decrease the
specic gravity). Prepared working solutions are
now commercially available. Exact specic gravity
is important for reliable results.
Procedure
1. Dispense 30 mL copper sulphate solution
(specic gravity 1.055 for men and 1.053 for
women) into a tall glass jar. (Note: The solution
must be changed daily or after each 25 tests).
2. Clean the site of skin puncture thoroughly
Figure 16.2 Hydrometer to measure
the specic gravity of
copper sulphate solution
with alcohol swab and wipe dry with sterile
gauze.
3. Perform nger stick by the method described earlier. The punctare must be a sucient
force so as to allow a free ow of blood using a sterile disposable lancet. Do not squeeze
the site of puncture, as this might dilute the drop with excess of plasma tissue and give
false results.
4. Collect the blood in a capillary tube avoiding air bubble.
5. Allow a drop of blood held in the capillary to fall gently into the tube from a height of
about 1 cm above the surface of the copper sulphate solution. You may use a capillary
bulb to force the blood out. This may not be necessary if there is sucient amount of
blood entered into the capillary tube.
6. Observe for 15 s.
7. If the drop of blood sinks to the boom without any hesitation, the donor has
haemoglobin concentration in the acceptable range (>12.5 g/dL). If the drop takes a
long time to sink or hesitate in the middle or oat to the surface, the donor has lower
haemoglobin concentration in blood (<12.5 g/dL).
8. Record result as: Acceptable or Non-acceptable
Caution No more than 15 drops of blood should be added to a cylinder that contains
20 mL copper sulphate to assure that the solution maintains its sensitivity and freshness.
The solution may have to be changed more often if the solution becomes cloudy and
at least once each day. Note that cylinders should be kept covered between tests as
evaporation changes the specic gravity of the solution.
397
Special Comment
It should be noted that the copper sulphate method is not a quantitative test and shows
only that the haemoglobin is equal to, above or below acceptable limits. Furthermore, a
donor with abnormally high-serum proteins (e.g., in multiple myeloma) passes the copper
sulphate test regardless of how low the haemoglobin is. The test may therefore be considered
inappropriate in some instances. Temperature of the copper sulphate solution and its
continued contamination with blood may also aect the result.