AUSTRALASIAN SOCIETY OF BLOOD TRANSFUSION INC.

Topics in
Transfusion
Medicine
________________________________________________
February 2002
Vol 9, No 1

u Editorial Board
Editor:

Ken Davis

Board Members:

Margaret Buring, Wendy Erber, Jim Faed, John Gibson,

Robyn Minchinton, David Roxby

Editor's Note
This edition

I am indebted to Andrew Geczy for providing me with an update on HLA testing which obviously plays
a significant role in transplantation, both haematological and in solid organ.
To the often asked question is it OK to put drug in the infusion while blood is running I
have included a couple of articles from the e-Forum Network of the Californian Blood Bank Society.
A number of us have experience in the transfusion support of haematological transplantation but
fewer would need to consider this aspect with solid organ transplants. The article from Institute of
Transfusion Medicine, Pittsburgh hopefully is of value in this area.
Lastly in these days of tighter red cell inventories, the demand for emergency O blood is an ongoing
issue, especially in deciding who gets the Rh negative units as a priority. This discussion article will
hopefully stimulate some local opinions in this area.
Members are encouraged to let the Editor know what topics they would like addressed in future
issues.

Ken Davis
[ken.davis@imvs.sa.gov.au]
Extra copies of Topics in Transfusion Medicine are available from the ASBT office for $22 each (includes GST).
Please spread the word.
All Correspondence
The Australasian Society of Blood Transfusion Inc
145 Macquarie Street, Sydney NSW 2000

February 2002
Vol 9, No 1

CONTENTS

1.

HLA typing: an update

Dr Andrew Geczy; Senior Manager-Tissue Typing and Research, ARCBS, NSW

2.

Which solutions other than isotonic saline are approved for addition to
blood and components?
Reprinted with permission: e-Network Forum, Californian Blood Bank Society,
[Dec 26, 2000]

3.

Is it Safe for Anesthesiologists To Infuse Medications Along With

Blood?
Reprinted with permission: e-Network Forum, Californian Blood Bank Society
[March 9, 2001]
Comments from Dr Graeme Woodfield, Chair, ASBT Scientific Subcommittee:
"Until the relevant research investigations have been undertaken, we should take a
very conservative view on the addition of any drugs or other solutions to blood that is
being transfused. Such research is long overdue, and is a subject that would be a
good one for Australasian researchers. It was suggested as a topic several years ago
at ASBT ASM's."

4.

Transfusion Support in Solid-Organ Transplantation

Darrell J. Triulzi, M.D.
Reprinted with permission: Institute for Transfusion Medicine,
Pittsburgh, PA [April, 2001]

5.

Policies for Ensuring An Adequate Supply of O Rh Negative Blood in

Emergencies
Reprinted with permission: e-Network Forum, Californian Blood Bank Society,
March 24, 2001

February 2002
Vol 9, No 1

1. HLA typing: an update

Dr Andrew Geczy; Senior Manager-Tissue Typing and Research, ARCBS, NSW

Introduction
Histocompatibility matching or tissue typing refers to typing for Human Leucocyte
Antigens (HLA). The genetic region coding for the HLA proteins is located on the
short arm of Chromosome 6, and it forms part of the human Major Histocompatibility
Complex (MHC) which spans about 4,000Kb and includes more than 200 genes (1)
The fundamental role of MHC antigens is in the presentation of processed peptides
to T cells, and in transplantation these antigens are the principal barriers to the
acceptance of foreign grafts.
For the past fifteen years tissue typing techniques have been in transition from
serological to molecular methods which provide a better definition of HLA gene
polymorphisms. Nevertheless for both solid organ and bone marrow transplantation,
serological techniques will continue to be employed for donor-recipient crossmatch
and for the detection of anti-donor antibodies. For solid organ transplantation, low- to
medium-resolution typing is usually employed and this practice is dictated to some
extent by the chronic shortage of suitable organ donors and by the difficulty of finding
a closely HLA-matched graft. By contrast, for bone marrow transplantation, high
resolution matching for at least the Class II antigen is required, as this has been
shown to result in improved clinical outcome (2,3). In addition to the major HLA
antigens (Class I and II), graft survival and graft-versus-host disease (GVHD) can
also be influenced by minor histocompatibility antigens (mHag) which are recognised
in the context of certain Class I alleles (eg. HLA-A2) and detected by molecular
techniques (4). The testing for mHags is gaining popularity and will quite likely
become part of routine clinical practice in stem cell transplantation.

Serological typing techniques

Lymphocytotoxicity testing is a complement-dependent reaction in which viable T
and B cells are incubated with antibodies directed to Class I and II antigens; rabbit
serum is used as a source of complement (5,6). When antibody is bound to an HLA
antigen on the cell membrane, the complement pathway is activated and this process
leads to cell damage or lysis of the cell rendering the leucocyte membrane
permeable to vital dyes. Viable unstained cells indicates the absence of a particular
HLA antigen. A number of modifications, aimed at increasing sensitivity, have been
developed and many tissue typing laboratories, have adopted cytotoxicity testing
using immunomagnetic beads. These beads, which are coated with antibody to CD8
for Class I, or antibody to Class II ( chain monomorphic determinant), are used to
separate lymphocytes, and fluorochromes instead of a dye are used to distinguish
viable from damaged or dead cells (7). The main limitations of serological
techniques are: the necessity to isolate viable cells; the constant requirement to
acquire and characterise specific HLA antisera and lack of reliable HLA anti sera that
are sufficiently specific for the ever-increasing number of HLA specificities.

February 2002
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Molecular typing techniques

Oligotyping
In this technique, nucleotide primers and probes are synthesised according to the
known nucleotide sequences of the alleles in question (2,8). The ability of these
oligonucleotides to hybridise to target sequences forms the basis of this technology.
Some of the advantages of oligotyping over serology are accuracy and
reproducibility.
Polymerase Chain Reaction (PCR)
This methodology, which was introduced in the mid-1980s, is used to amplify small
segments of DNA (9,10). The segment of double-stranded DNA is placed between
two oligonucleotide primers through many cycles of amplification. This amplification
takes place in a thermal cycler with one step taking place at a high temperature in
the presence of a thermostable DNA polymerase, an enzyme that can withstand the
high temperature. Within a few hours the original DNA fragment is transformed into
millions of copies. The development of PCR has seen the introduction of precise but
simplified techniques such as PCR-SSO (Sequence-Specific oligonucleotide) probing
and PCR-SSP (Sequence-Specific primers).
At present most Tissue Typing laboratories have adopted one or more of these PCRbased techniques that give different levels of resolution. Low resolution or generic
typing identifies "broad" HLA specificities while medium resolution will resolve all the
serologically defined specificities and also some specificity at the allelic level. High
resolution typing attempts to identify all the alleles of an HLA locus.
PCR-SSO
In this technique probes can be labelled with radioisotopes such as 32P, or nonradioactive markers such as biotin or digoxygenin. Hybridised probes can be
detected by radioactive methods or chemiluminescence detection systems (11).
PCR-SSO is relatively cheap and suitable for handling large sample numbers where
non-urgent typing is involved such as typing of donors joining a bone marrow donor
registry. However for organ transplantation more rapid methods such as PCR-SSP
are employed.
A variation of the SSO technique is the reverse dot blot or reverse PCR-SSO. In this
method sequence-specific oligonucleotide probes are bound to a solid support
membrane through an incorporated poly-(T) tail. This leaves the detection end of the
probe free to react with the target DNA. In the next step, labelled (eg biotinylated)
PCR amplified DNA, applied to the dot blot membrane, will hybridise with those
oligonucleotides that have a complementary sequence (2). Finally, the biotinylated
PCR DNA-probe hybrids are detected by a chemiluminescence or colour reaction
after binding of streptavidin-horseradish peroxidase or streptavidin-alkaline
phosphatase to the biotin molecule.

February 2002
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PCR-SSP
This method, based on the amplification refractory mutation system (ARMS),
involves the determination of HLA specificity by the use of sequence-specific primers
in which a 3' single base mismatch inhibits the priming of non-specific reactions (12).
The PCR-SSP technique involves the use of a large array of primer mixes and
multiple PCR-SSP reactions are set up where each reaction is specific for an allele
or a group of alleles. The presence of amplification is detected by agarose gel
electrophoresis. By comparison with SSO, the SSP method is costly but better
suited than SSO for urgent typing required for the matching of donor and recipient in
cadaveric solid organ transplantation (2, 13).
Sequence-based typing (SBT)
Although the SSO and SSP methods can usually detect single base differences in
DNA sequences, they may fail to detect new alleles. The value of SBT is that it
permits the precise assignment of HLA alleles by detecting directly the nucleotide
sequence of an allele. The basis of SBT involves the amplification of the
polymorphic regions of the HLA allele by flanking primers, followed by sequencing
the PCR product. Laborious manual sequencing techniques have been replaced by
dye-labelled primers and dye-labelled ddNTP terminators and fluorescent automated
cycle sequencing (14). Computer analysis is required to facilitate the interpretation
of the data. SBT was first applied to HLA-Class II typing where sequencing of exon 2
can detect most of the polymorphisms. On the other hand, sequencing of class I is
more complex and usually involves exons 2, 3 and 4.

Cellular assays
In this predominantly molecular era some may be surprised to learn that cellular or
functional assays still have a place in tissue typing.
The Mixed Leucocyte Culture (MLC) test, which measures T cell activation in
response to Class II disparity between donor and recipient cells, has largely been
superseded by molecular typing techniques which are better predictors of graft
survival and graft-versus-host disease. However, some cellular assays are still in
vogue and these include Cytotoxic T lymphocyte precursor (CTLp; 15) and Helper T
lymphocyte precursor (HTLp; 16,17) assays. T cell precursor analysis is performed
by limiting dilution assays in which sequential dilutions of donor responder cells are
co-cultured with a constant number of recipient stimulator cells and assayed for
cytotoxicity, proliferation or cytokine release. The effector function of cytotoxic
precursors is monitored by measuring the release of 51Cr from labelled stimulator
cells while proliferative cells are monitored by measuring tritiated thymidine
incorporation. The HTLp assay which involves the measurement of IL-2-secreting
donor helper T cell precursor responses, shows a positive correlation between high
HTLp frequency and the risk of developing GVHD (17). The HTLp assay in
conjunction with molecular typing can be used to select the best HLA-identical donor
- recipient pair with minimum risk of GVHD in the recipient.

The standard donor - recipient cytotoxic crossmatch

The T cell cytotoxic crossmatch which predicts hyperacute rejection has been the
accepted "industry" standard since the early days of solid organ transplantation (18).
However, even with improved definition of HLA, some well-matched grafts with
negative cytotoxic crossmatches continue to be lost.

February 2002
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Indeed, many of these grafts are lost within the first three months post-transplant,
which suggests the cause is undetected sensitisation. The significance of the
cytotoxic crossmatch involving B cells is far from clear. Although hyperacute
rejections have been reported due to HLA Class II - specific antibodies (19), grafts in
similar situations have proved successful (20). The detection of HLA-Class I specific antibodies is undoubtedly important, but the currently used B cell crossmatch
technique does not distinguish between Class I and Class II-specific antibodies.

Flow cytometric crossmatch

The flow crossmatch is designed to measure alloantibodies by means of an indirect
immunofluorescence technique. In the first step, the target cell population and test
serum are incubated to allow test antibody to bind to a specific determinant on the
cell membrane of the target cell. The second step involves the incubation of a
fluorescein isothiocyante (FITC) - conjugated anti-human immunoglobulin reagent
directed to the bound antibody. Thus, the FITC will label cells that carry the specific
determinant against which the test antibody is directed. The FITC fluorescence is
then analysed on a Flow Cytometer, which detects the intensity of fluorescence of
each cell individually and produces an objective result in contrast to the sometimes
subjective observations encountered with the cytotoxic crossmatch technique. Flow
cytometry is increasingly becoming the method of choice for the detection of donorspecific sensitisation, as it is more accurate and precise than cytotoxicity (21) and it
allows one to analyse serum reactivity to specific cell types or cells with particular
characteristics.

Conclusion
A number of factors dictate the choice of typing method used in an HLA laboratory.
Some of these factors include the level of resolution required, sample numbers
involved, the clinical urgency of a typing, the preferences of the transplant physician
as well as the availability of equipment, money and technical expertise of the staff.
As mentioned earlier, many molecular typing methods, which offer accurate and
reproducible definition at the allelic level, are replacing serological and cellular
techniques. Nevertheless functional assays may still play a role in donor selection
and the prevention of GVHD.
Another challenge facing Tissue Typing laboratories is the accurate detection of
presensitisation in organ transplantation. While flow cytometric crossmatching has
contributed to the detection of previously unrecognised donor-specific pre-transplant
sensitisation, many of the mechanisms underlying pre-transplant sensitisation are
still poorly understood.

February 2002
Vol 9, No 1

References
1. Campbell, R. D. and Trowsdale, J. A map of the human major histocompatibility
complex. Immunology Today: January supplement, 1997.
2. Bunce, M., Young, N. T. and Welsh. K. I. Molecular HLA typing - the brave new
world. Transplantation 64: 1505, 1997.
3. Begovich, A. B. and Erlich, H. A. HLA typing for bone marrow transplantation:
New polymerase chain reaction - based methods. JAMA 273: 586, 1995.
4. Goulmy, E.G., Schipper, R., Pool, J. Blokland, E., Falkenburg, J. H. F., Vossen,
J., Gratwohl, A., Vogelsang, G. B., van Houwelingen, H. C. and van Rood, J. J.
Mismatches of minor histocompatibility antigens between HLA-identical donor
and recipient and the development of graft-versus-host disease after bone
marrow transplantation. New. Engl. J. Med. 334: 281, 1996.
5. Terasaki, P. I. and McClelland, J. D. Microdroplet assay of human serum
cytotoxins. Nature 204: 998, 1964.
6. Curtoni, E. S., Mattiuz, P. L. and Tosi, R. M. (Eds), Histocompatibility Testing,
Munksgaard, Copenhagen, 1967.
7. DYNAL Product Insert for DYNABEADS HLA Cell Prep I and II.
8. Lee, J. S., Trowsdale, J. and Bodmer, W. F, cDNA clones coding for the heavy
chain of human HLA-DR antigen. Proc. Nat. Acad. Sci. USA 89: 545, 1982.
9. Saiki, R., Scharf, S., Faloona, R., Mullis, K., Horn, G., Erlich, H. A. and Arnheim,
N. Enzymatic amplification of -globin genomic sequences and restriction site
analysis for diagnosis of sickle cell anaemia. Science 230: 1350,1985.
10. Mullis, K. B. and Faloona, F. Specific synthesis of DNA in vitro via a polymerase
catalysed chain reaction. Meth. Enzymol. 155: 335,1987.
11. Saiki, R., Chang, C. A., Levenson, C. H., Boehm, C. D., Kazazian, H. H. and Erlich,
H. A. Rapid genetic analysis of enzymatically amplified DNA with non-radioactive
allele-specific oligonucleotide probes. Am. J. Human Genetics 41: A237, 1987.
12. Newton, C. R., Graham, A., Heptinstall, L. E., et. al. Analysis of any point mutation in
DNA: the amplification refractory mutation system (ARMS). Nucleic Acid Res. 17:
2503, 1989.
13. Middleton, D. and Williams, F. A History of DNA typing for HLA. In: Terasaki, P. I.,
Gjertson, D. W. eds. Los Angeles, California: HLA, UCLA Tissue Typing Laboratory,
125, 1997.
14. McGinnis, M. D., Conrad, M. P., Bouwens, A. G. M., Tilanus, M. G. J and Kronick,
M. N. Automated solid-phase sequencing of DRB region genes using T7 sequencing
chemistry and dye-labelled primers. Tissue Antigens 46: 173, 1995.

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15. Kaminski, E., Hows, J., Man, S., Brookes, P., MacKinnon, S., Hughes, T., Avakian,
O., Goldman, J. M. and Batchelor, J. R. Prediction of graft-versus-host disease by
frequency analysis of cytotoxic T cells after unrelated donor bone marrow
transplantation. Transplantation 48: 608, 1989.
16. Schwarer, A., Jiang, Y., Brookes, P, et.al. Frequency of anti-recipient alloreactive
helper T-cell precursors in donor blood and graft-versus-host disease after HLAidentical sibling bone-marrow transplantation. Lancet 341: 836, 1997.
17. Weston, L. E., Geczy, A. F. and Farrell, C. Donor helper T-cell frequencies as
predictors of acute graft-versus-host disease in bone marrow transplantation
between HLA- identical siblings. Transplantation 64: 836, 1997.
18. Ting, A. The lymphocyte crossmatch test in clinical renal transplantation.
Transplantation 35: 403, 1983.
19. Ahern, A. T., Artruc, S. B., Dellapelle, P. et. al. Hyperacute rejection of HLA-ABidentical renal allografts associated with B lymphocyte and endothelial reactive
antibodies. Transplantation 33: 103, 1982.
20. Karuppan, S. S., Lindholm, A. and Moller, E. Characterisation and significance of
donor-reactive B cell antibodies in current sera of kidney transplant patients.
Transplantation 49: 510, 1990.
21. Kerman, R. H., van Buren, C. T., Lewis, R. M. et.al. Improved graft survival of flow
cytometry and anti-human globulin crossmatch-negative retransplant recipients.
Transplantation 49 52, 1990.

February 2002
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2. Which solutions other than isotonic saline are approved for

addition to blood and components?
e-Network Forum, Californian Blood Bank Society, Dec 26, 2000.

According to AABB Standards, except for normal saline, drugs or medications must
not be added to blood or blood components unless the added substance has been
approved by the FDA to be added, or records show it is safe to add the substance to
blood and components. An e-network member asked if anyone had ever
encountered a physician who ordered a solution other than normal saline to be
added to blood or a blood component, and if so, which ones? If such a practice
occurred, what documentation of safety was on file to justify the practice? Does
anyone know which solutions (other than normal saline) are FDA-approved for
addition to blood and components?
To which the following replies were received:
Reply #1: According to this member Normosol-R pH 7.4, [Abbott], is approved for
mixing with blood products. At this institution it is used in the rapid infuser during liver
transplants.
Reply #2: Morphine and Demerol are reported as having been added to blood for a
patient who was using a controlled analgesic device during blood transfusion.
[Transfusion, September 1993, Meeting Abstract Supplement S100]. This member
did not think that these drugs were FDA-approved for addition to RBCs.
Reply #3: A member reported that her facility, which is a level I Trauma center, uses
Plasmalyte as a diluent on occasion for trauma patients; stating that the FDA had
approved the addition of Plasmalyte to blood and blood products. In fact, her
institution provided proof that the FDA has approved addition of Plasmalyte to blood
to AABB inspectors during a past inspection.
Reply #4: An opinion that conflicts with replies #1 and #3 above was provided by a
member who said that there have been no solutions (other than normal saline)
approved by the FDA for mixing with blood. This member claims to have recently
looked into this issue extensively. According to the member, there have been some
abstracts published about certain medications that have been tested that the authors
consider safe when administered in the same tubing with blood; however, there has
been no FDA approval. There is an abstract from the last AABB meeting by a group
in Canada describing studies they performed with some drugs. The anesthesiologists
who work at the member's institution would like to allow concurrent administration of
blood and the following medications: morphine or meperidine, heparin [with NaCl
only], insulin, cefazolin. This member suspects that even though AABB accredited
facilities have procedures that state only 0.9% saline can be administered
concurrently with blood, on occasion in certain situations the use of other solutions
occurs. In fact one of the anesthesiologists at this member's institution stated
"it is frequent practice by anesthesiologists at other institutions to
concurrently administer medications with a transfusion in perioperative
patients." By concurrent administration he means that medication actually is in
contact with the red cells in the tubing.

February 2002
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10

WEB MASTER'S NOTE: It would seem prudent to examine the product package
insert for evidence of FDA approval to add a substance to a blood product. If
FDA approval is not evident based on the product package insert, it would seem
prudent to have documentation on file that the addition of a substance to a
blood product is a safe practice, before actually adding the substance to
blood products that will be used for transfusion. If members have such package
inserts and would FAX them to me at 503-212-9577, I will compile a list of approved
solutions, and share the list back with the membership.
And here is what the AABB has to say about this thorny issue in their
publication Standards Source, Sept, 1999 on J 8.300!
(This was the answer provided in that document. Please consider signing up to
receive additional insightful responses to life's challenges!)
J8.300 Addition of Drugs and Solutions
Question: With the exception of 0.9% sodium chloride, USP, drugs or medications
shall not be added to blood or blood components unless:
1. They have been approved for this use by the FDA.
2. There are records available to show that the addition is safe and does not
adversely affect the blood component.
Question: If a patient has a triple lumen catheter and blood is being administered
through one port, is it acceptable to run other fluids or medications through the other
ports?
Answer: Standard J8.300 limits the drugs or medications that can be administered
with blood. It is desirable that blood be the only fluid administered through a catheter
at one time. Hypotonicity of some solutions may result in hemolysis, and the
presence of excess calcium in others may result in clotting. Further, red cells stored
for some weeks in CPDA-1 acquire a storage lesion and lose some of the buffering
capacity of fresh human blood, and are theoretically less able to cope with the
injurious effects of some high- or low-level pH drug solutions.
In addition, when a solution is infused simultaneously with blood and there is
an adverse event, it becomes difficult to distinguish whether the blood or the
solution is the cause. In this case, the transfusion has to be interrupted and the unit
potentially discarded. The use of another unit increases the cost of the therapy and
exposure to multiple donors when another unit is selected.
There may be circumstances, however, when infusing separately is not practical. If
simultaneous infusion is practiced within an institution, procedures should
describe the circumstances under which infusing blood and solution
simultaneously is acceptable and which solutions can be used in these
circumstances. For example, Amphoteracin B and IL-2 are medications that
frequently yield adverse reactions. In addition, the package labelling of
medications and solutions should be examined for any contraindications for
administration with blood components.

February 2002
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11

Typically, multi-lumen catheters have the exit holes for each lumen physically
separated by a distance of approximately 2.5 cm and exit on different sides of
the catheter. This design allows for dilution in the blood vessel and minimises any
mixing of infusing solutions. Thus, at slow rates of infusion, it is unlikely that the
blood component exiting the catheter would be affected. Even under these
circumstances, however, an adverse effect on the blood component cannot be
excluded with all solutions. The risk of an adverse reaction should be weighed
against the benefit of giving both fluids simultaneously.
Solutions that are not approved for simultaneous infusion include lactated
Ringer's solution, 5% dextrose in water, and hypotonic sodium chloride solutions.
Acceptable solutions for this practice include calcium-free isotonic electrolyte
solutions that meet the requirements of the FDA and the Circular of Information for
the Use of Human Blood and Blood Components. These solutions include ABOcompatible plasma, 5% albumin, or plasma protein fraction. All solutions are to
be administered with the approval of the patient's physician.
For more information, see these issues of TRANSFUSION:
1975; 15: 250-255
1993; 33 (Suppl.): S100
1997; 37 (Suppl.): S333
ADDENDUM: Feb. 4, 2001
Web Master Comments: In response to my earlier request that members FAX
copies of package inserts pertaining to acceptable non-plasma diluents for red
cells for transfusion, a number were received and are shown below:
1. Package insert for Normosol-R: The dosage and administration section of the
package insert of Normosol-R states that the solution can be used for starting
blood transfusions.
2. Package insert for Plasmalyte-A injection pH 7.4: The indications and usage
section of the package insert of Plasmalyte-A injection pH 7.4 states that the
solution is equally compatible to normal saline with blood and components.
Plasmalyte-A injection pH 7.4 can be used as a priming solution for blood
components, and may be added to or used with blood components through the
same line, and may be used as a diluent for blood components.
Ira A. Shulman, MD
CBBS e-network Web Master

February 2002
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3. Is it Safe for Anesthesiologists To Infuse Medications Along With

Blood?
e-Network Forum, Californian Blood Bank Society [March 9, 2001]
A network member recently requested information about the practices of
anesthesiologists when it comes to adding or mixing medications to blood products.
To review a related discussion, go to http://www.cbbsweb.org/ and click on the enetwork table of contents link and then on the link entitled: Which solutions other
than isotonic saline are approved for addition to blood and components?
In response to the question about anesthesiologist practices regarding adding or
mixing pain medications and antibiotics to blood or the IV through which blood is
infusing, here are some interesting replies:
Reply #1: An anesthesiologist in Texas wrote that in general, anesthesiologists are
given very little formal education about the addition of medications to blood products.
Rather, the knowledge that anesthesiologists have about this practice is passed
down 'from generation to generation', as residents watch their seniors put
medications into the blood line. This anesthesiologist pointed out that if you look on
the blood warmer administration sets used in the OR, there is a place to add
medications in front of the warmer as well as after the warmer. In big cases,
anesthesiologists (at least the ones this physician knows about) put lots of three-way
stopcocks on the blood warmer to administer medications. It was also pointed out
that anesthesiologists give blood faster than almost anyone, since they often wait
until blood becomes absolutely necessary. As an example, the anesthesiologist
reported that he often gives a unit over 10-15 minutes, but in a trauma, that can be
shortened to 2-3 minutes. So, how are anesthesiologists really practicing? To quote
this contributor, "Nearly everything goes into the IV except calcium". Do all
institutions always start a second IV in these cases? "In practice, this would be a rare
and an unusual event." Finally, the anesthesiologist mentioned that a few years ago,
there was some discussion about using plasmalyte (instead of normal saline) for
blood administration. He says that most folks do this in order to prevent the
hyperchloremic acidosis that comes with NaCl resuscitation.
Reply #2: A second anesthesiologist reported that while she did not represent
anesthesiologists as a group, she was attuned in to their practice, being the chair of
the American Society of Anesthesiologists Committee on Transfusion Medicine. She
pointed out the ASA published a brochure entitled "Questions and Answers about
Transfusion Practices", 3rd edition in 1997. One of the questions is "Which
intravenous solutions are compatible with red blood cells?" This really deals more
with diluting RBCs, but it's the only written document of the ASA that addresses the
issue even peripherally. To quote this anesthesiologist: "Quite frankly, I don't think
the basic issue you are concerned about is something the ASA would comment
upon. They certainly would not publish anything that conflicted with Standards (at
least not as long as I am Chairman of the committee!) On a practical note, what is
really done? I understand the reluctance to start another IV. Sometimes there is no
other site. I suspect the most common situation is patient-controlled analgesia (PCA).
I really don't see a problem and am sure my colleagues "piggy back" the PCA line
into an existing IV which may also be used for transfusion. I agree that a problem is
especially unlikely to occur with a multi-lumen CVP.

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13

This is not an issue likely to be resolved. I'm sorry I can't be more helpful. I think it's
one of those situations where the clinician does what's practical, regardless of
policy. No one can put in writing that it's OK, but....."
Reply #3: The third reply came from a blood banker who said he would challenge
the anesthesiologist to provide the data that their practice of adding
medications to blood is safe, or to develop data. Blood bankers could help with
the design and completion of such a study if there are no relevant data. Another
option would be to ask some pharmacists as they mix iv solutions if they have
information on what drugs are compatible or incompatible with blood.
Reply #4: Another blood banker pointed out that the Circular of Information states
"No medications or solutions may be routinely added to or infused through the same
tubing with blood or components except 0.9% Sodium Chloride, Injection (USP).
Other solutions intended for intravenous use may be used in an administration set or
added to blood or components under either of the following conditions: a) They have
been approved for this use by the FDA or b) There is documentation available to
show that addition to the component involved is safe and efficacious. ABOcompatible plasma, 5% Albumin, or Plasma Protein Fraction, or other suitable
plasma expanders may be used with approval of the patient's physician." This blood
banker went on to say that from what he has observed at his facility, official policy is
not always followed. He would suggest involving the hospital quality assurance
committee with an investigation of practice patterns of anesthesiologists to determine
whether or not hospital policy is being followed.
Web Master Note: It appears from the above discussion that we have conflict
between practice and policy. It would certainly be reassuring to have data that it
is safe to run medications through the same line as blood products. Maybe
such data will be forthcoming, now that this is out in the open? If data were available,
perhaps policies could be more in line with practice, and practice could be more in
line with official policies.
Ira A. Shulman, MD
CBBS e-network Web Master

February 2002
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14

4 Transfusion Support in Solid-Organ Transplantation

Darrell J. Triulzi, M.D., Institute for Transfusion Medicine, Pittsburgh, PA
April, 2001

Introduction
Solid-organ transplantation continues to grow as a treatment modality. Currently
more than 74,000 patients are waiting for an organ transplant. However in 1999 only
21,990 transplants were performed, limited primarily by the availability of organs.
Transfusion support remains an integral part of solid-organ transplantation, imparting
demands on the transfusion service not only quantitatively in terms of blood product
support, but also due to the unique requirements for specialised blood components,
the complex serologic problems, and the immunologic effects of transfusion on both
the allograft and the recipient.

Blood component utilisation

The typical requirements for blood components for each type of organ transplant in
adults are shown in Table 1. Liver transplant procedures require the most blood
components despite the fact that blood use in liver transplantation has declined
dramatically over the last decade. The multiple factors contributing to the reduction in
blood usage include: improved surgical technique, organ preservation, and
anaesthetic management, as well as better intraoperative monitoring of coagulation
status and pharmacologic treatment of fibrinolysis. Heart transplantation is
associated with lower blood requirements that approximate blood usage observed in
complex cardiopulmonary bypass procedures. Blood usage in lung transplantation
varies by the type of lung transplant procedure. More than 2/3 of single-lung
transplant recipients do not require any transfusions. Double-lung transplant
procedures typically require more red cells than heart transplant procedures. The
majority of patients receiving kidney transplants do not require blood.

Strategies for selection of compatible components

For solid-organ transplant procedures other than liver, transfusion requirements are
generally not sufficient to require deviation from traditional selection criteria of ABO
identical/compatible red cells. However, in liver transplantation, transfusions
frequently exceed the available supply of ABO identical red cells, antigen-negative
red cells, or compatible plasma, and thus require blood bank blood group switching
protocols to optimally use available blood resources.
Patients with Clinically Significant Alloantibodies
Pre-existing potentially clinically significant red cell alloantibodies are found in approx
6% of liver transplant candidates. Usually at least 8-10 units of antigen compatible
blood can be found for surgery. In order to minimise the risk of haemolysis, these
patients are ideally managed by using antigen-negative units for the first 5-10 units,
switching to antigen-unscreened units in the middle of the case, and then switching
back to antigen-negative units for the last 5-10 U transfused. This strategy requires
close communication between the anesthesiologist and blood bank.

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Indications for specialised blood components (table 2)

CMV-Negative/Safe Blood Components
CMV infection is the most frequent infectious complication following solid organ
transplantation. In seropositive solid organ recipients, reactivation of latent virus
represents the major risk for CMV infection. Thus, there is no documented benefit to
providing blood CMV neg/safe components to patients who are CMV seropositive. In
seronegative patients, the major source for primary CMV infection is the seropositive
transplanted organ and, to a lesser extent, transfused blood components. Published
data indicate that although the overall rate of CMV transmission by transfusion is low
(5-15%), the morbidity associated with this complication would support the use of
methods to prevent CMV transmission from blood components. Historically, this was
accomplished by using blood components from donors who lack antibodies to CMV.
Published data suggest that leukocyte reduction by filtration is equally effective in
reducing the risk of CMV transmission from blood.1 It appears that this method would
also be effective in solid-organ transplant recipients.2

Irradiated Blood Components

Graft vs host disease (GVHD) is a rare complication of organ transplantation (59
cases) reported most frequently in liver recipients and is almost always due to the
donor organ. Transfusion associated GVHD is very rare in solid organ transplant
recipients with only 4 published cases. Only two of these had convincing supportive
evidence, and one of these had an under-lying hematologic abnormality. These few
cases do not support a policy of routine irradiation of cellular blood components for
organ transplant recipients.

Leukocyte-Reduced Blood Components

Alloimmunization to HLA antigens is of considerable importance in organ
transplantation. A sensitized patient presents problems in identifying a crossmatchcompatible donor and, for some organs, the outcome of transplantation is inferior.
However, white cells in blood components may be beneficial in inducing tolerance
and prolonging allograft survival. Thus, the decision to provide leukocyte-reduced
components must weigh the risks and benefits of these opposing effects. HLA
alloimmunization has clearly been shown to be associated with decreased graft
survival in renal transplantation and more recently in heart and/or lung
transplantation. However, liver allograft survival is not adversely impacted by HLA
alloimmunization. Thus, leukoreduction to prevent alloimmunization provides benefit
to kidney, heart, and lung recipients. The beneficial immunomodulatory effect of nonleukoreduced transfusions on allograft survival has only been demonstrated for renal
transplant recipients. This effect is only apparent when the blood donor shares an
HLA haplotype with the recipient. The magnitude of the effect has become less
apparent with currently available immuno-suppressive drugs, and thus transfusions
are not routinely used for this indication.

ABO blood group system in organ transplantation

The ABO system is clinically important in two aspects of solid-organ transplantation:
first, as a transplantation antigen that influences graft survival, and second as an
antigen-antibody system implicated in immune hemolytic anemias in ABO nonidentical organ transplant recipients.

ABO System as a Transplantation Antigen

Transplantation across ABO lines will typically cause hyperacute rejection of kidney
and heart transplants, although exceptions do exist. Successful transplantation of
kidneys and hearts across ABO lines has been accomplished by removing ABO
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antibodies in the recipient or by taking advantage of the variable expression of ABO

antigens such as in A2 individuals or neonates. In general, ABO-incompatible organs
are avoided in kidney and heart transplantation. ABO major mismatch liver allografts
are used in 6.9% of pediatric and 2.4% of adult liver recipients due to organ
shortages. Liver allografts are felt to be resistant to hyperacute rejection when
transplanted across ABO barriers although reports of hyperacute rejection do exist.
ABO-incompatible liver transplants are commonly associated with acute graft failure
with a 46% graft failure rate reported within 30 days of the transplant.
Plasmapheresis to remove recipient isohemagglutinins and/or splenectomy may be
of benefit. Currently, ABO-incompatible liver allografts are reserved for patients with
fulminant liver failure in whom death is imminent without transplantation and when an
ABO-compatible organ is not available. ABO compatible but non-identical (minor
mismatch) liver transplants are associated with modest reductions in 1- and 3-yr
survival.

Immunohematologic complications
Passenger lymphocytes transplanted with the donor organ are capable of producing
ABO antibodies and hemolysis in ABO mismatched organ recipients. A positive
Coombs test +/- hemolysis is typically observed 7-10 days after transplantation.
Blood Bank protocols for optimal component selection are required to minimize
hemolysis.

Table 1
Median Blood Use (Units) in Organ Transplantation
Organ

Red cells

Plasma

Platelets

Liver (n=118)
Heart (n=51)
Lung
Single (n=46)
Double (n=30)
Kidney

12
4

13
5

10
10

0-2
7
0-2

2
-

8
-

Table 2
Recommendations for use of specialized blood components in CMV
seronegative solid-organ transplant recipients
Type
CMV neg/safe
Filtered
Irradiated
Kidney
Yes*
Yes
No
Heart
Yes*
Yes
No
Lung
Yes*
Yes
No
Liver
Yes*
No
No
*CMV-negative pairs only. Components rendered CMV-safe by filtration are
a reasonable substitute.

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REFERENCES
1. Bowden RA, et al. Blood 1995; 86:3598-3603.
2. Lopez-plaza I, et al. Transfusion 1999;39:S385.
3. Triulzi DJ et al. Transfusion 2001;41:419-26.
Copyright 2001, The Institute For Transfusion Medicine

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5. Policies for Ensuring An Adequate Supply of O Rh Negative Blood

in Emergencies
e-Network Forum, Californian Blood Bank Society, March 24, 2001

On February 28, 2001, a member requested help in crafting a policy/procedure to address

the use of Rh negative RBCs during emergency and massive transfusion situations. In the
inquiring member's opinion, conservative transfusion practices and/or the reluctance to
transfuse Rh positive RBCs to Rh negative (or Rh type unknown) patients unless/until the Rh
negative inventory is depleted seems to be an overuse or misuse of a seemingly limited
resource. The member requests that the e-network share policy/procedures with respect
to the following:
1. Under what circumstances of urgent orders for uncrossmatched blood, do you:
......issue group O Rh negative RBCs only?
......issue group O Rh positive RBCs only?
......issue either group O Rh positive or group O Rh negative RBCs depending on age and
sex of the patient?
2. If/when you find you've transfused group O Rh positive RBCs to an Rh negative
patient, how do you proceed? Do you
......monitor the patient for development of anti-D?
......administer Rh immune globulin?
......some other approach?
3. Do you impose a limit on the number of uncrossmatched group O units that you will
'ordinarily' issue to a given patient (i.e. maximum of 2 units before issuing group specific
units)?
4. Is it feasible and permissible to stock both group O Rh positive and group O Rh
negative RBCs in the same location at the same time, for example in the ER, group O Rh
positive RBCs intended for males, group O Rh negative RBCs for females, in order to
maintain a ready supply for urgent transfusion situations?
5. In the case of anticipated 'heavy' RBC usage for known Rh negative, anti-D negative
patients, under what circumstances would you or do you switch from transfusing Rh
negative RBC units to transfusing Rh positive RBC units?
6. Do you limit in some way the number of Rh negative RBC units available for that
patient prior to switching?
7. How does your policy differ with respect to age and sex of the patient?
8. If the prevalence of anti-D in transfusion recipients is so low (<1%), is it still
appropriate to plan to utilize a scarce resource
(group O Rh negative RBCs) to meet the urgent transfusion needs of the entire population
when relatively few Rh negative patients might actually benefit?
9. Why not issue group O Rh positive RBCs to this population of patients, thereby
conserving scarce group O Rh negative RBC's for known group O Rh negative patients.
10. Can you suggest/supply a model policy (your own?) or recommend specific references
for such a policy?
To which the following suggestions were received:
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1. A member stated that while waiting for cross matched blood to be available,
emergency transfusion needs can be met in various ways, depending on the clinical
status of the patient and the equipment available. This member believes that most patients
can tolerate an acute decrease in hemoglobin and therefore oxygen carrying capacity,
provided resuscitation of their intravascular fluid volume occurs in an adequate amount and
in a timely fashion. Crystalloid or colloid solutions may be infused to increase intravascular
volume and stabilize the vital signs, although overzealous administration of crystalloid to
normalize blood pressure may be disadvantageous in certain patient subsets (e.g.,
penetrating torso injuries). If the patient has cardiac disease, pulmonary disease or
cerebrovascular disease, and acute anemia will pose increased risk, or if there is not time to
wait because the patient is in extremis, then either group specific or group 0 Rh-negative red
cells can be administered while awaiting a formal crossmatch to be performed.
Group 0 Rh-positive Red Blood Cells for males or postmenopausal females can be
transfused in this setting as well. Administration of uncrossmatched blood has been shown
relatively safe, in part related to the low incidence of unexpected antibodies observed during
cross match in patients with (1 percent) or without (0.1 percent) a previous history of
transfusion. Administration of Group 0 Rh-negative blood may lead to hemolysis if multiple
units of Group 0 Whole Blood (containing anti-A and anti-B antibodies) have been transfused
to patients with Group A or B blood; this concept has led to the recommendation that one
should not switch back to the patient's blood type after administration of two units of Group 0
Whole Blood. However, modern blood banking strategy does not allow for the use of Group
O Whole Blood unless the patient is tested and shown to be group O. Until the patients blood
group is known, blood banks should not release whole blood. The patient can be switched
back to his or her inherent type specific blood after subsequent testing by the blood bank
indicates it is safe to do so. The passively transfused anti-A and anti-B antibodies are seldom
a problem after Group 0 Red Blood Cells are transfused, especially if the Red Blood Cells are
preserved in an additive solution.
2. A second member commented that there may be a threshold for the number of group O
units transfused above which you would not switch away from group O RBCs back to
the patients genetic ABO group, because too much incompatible ABO antibody has
been transfused. The anesthesiologists at this member's institution seem to consider the
transfusion of 6 group O units as the threshold above which you should stay with group O
blood. Although this member has seen other institutional policies where they will not switch
away from group O blood once the patient has received 12 units. This member suspects that
with the use of Adsol (or similar additive solution systems) units, a threshold of 12
units would be realistic.
3. A third member suggested that the e-network look at an educational piece for
physicians that was written about this issue in 1998. Here is the web page:
http://www.arcnocal.org/onegshort.html
4. A fourth member submitted her hospital PROTOCOL:
"Uncrossmatched 0 positive packed red cell units are to be issued to trauma patients
who are male or to females older than childbearing age instead of uncrossmatched 0
negative packed red cell units:
..When the number of 0 negative units in inventory falls below ten,
..When the patient is an 0 neg male and the need is for more than ten 0 negative units.
..When six units of 0 negative have been issued to a Code Yellow male patient and no
specimen has been received for type specific blood.
..When the blood suppliers cannot supply more 0 negative and there is a continuing need for
0 negative blood.
..When both in-patients and trauma 0 negative patients need blood.
The 0 Negative blood supply shall be reserved for women of childbearing age or
younger, and for in-house patients known to be 0 negative. The Transfusion Service
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Director is to be consulted when there is a need to switch, or in time constraint

circumstances, to be notified when time permits."
5. And finally, the Web Master's facility has this to add:
The Los Angeles County + University of Southern California Medical Center
(LAC+USC) houses one of the busiest emergency departments in the country. The level one
trauma center cares for nearly 28% of all the trauma cases in the County of Los Angeles,
including nearly 4,000 trauma registry cases per year. Immediate transfusion support with
group O, Rh negative RBCs is available and used by about 4-5% of these trauma cases.
[During times of severe group O, Rh negative shortages, male patients in need of
emergency/immediate transfusion support may receive group O, Rh positive RBCs instead of
O negative.]
If a LAC+USC emergency or surgery physician determines that an emergency transfusion is
medically indicated, group O, Rh negative RBCs can be available within seconds. This
immediate access is possible by maintaining a blood bank refrigerator stocked with group O
RBCs (usually group O Rh negative) in the Emergency Department and within selected
surgical areas. Each refrigerator is monitored by the blood bank and stocked with 6-8 units of
group O Rh negative RBCs (Rh positive RBCs are stocked during Rh negative shortage
periods). The preferred preservative system for these RBCs is AS, since AS-RBCs flow more
freely than CPDA-1 RBCs. Nurses prefer AS-RBCs because this product does not need to be
diluted and therefore can be administered through a straight intravenous line and surgeons
prefer to transfuse blood products, which flow like whole blood.
The Mayo Clinic has shown that AS-RBCs have flow characteristics similar to whole blood.
Each RBC unit stocked in an emergency refrigerator is conspicuously labeled to indicate,
"Emergency Blood Release. Blood issued without complete compatibility testing," and given
an identifying letter or number (i.e. unit T, unit XX, etc.) When opening an emergency
refrigerator door, an alarm sounds in the blood bank which signals a technologist to pick up
the designated phone. This phone automatically calls the phone located adjacent to the
opened emergency refrigerator. The transfusionist who answers the phone is asked to
provide the following information: the identifying unit number and the name and hospital
identification number of the intended, recipient patient. The technologist then fills out
paperwork documenting the request, the ordering physician's name, the unit identifying
number, and the patient's name and hospital identification number.
The technologist requests that a blood specimen be drawn and sent as soon as possible to
the blood bank for the usual testing. The blood specimen is sent via a pneumatic tube (six
inch diameter tube) directly to the blood bank laboratory. A technologist performs stat
ABO/Rh determinations and an antibody screen. Additionally, a technologist begins
crossmatch testing on tubing segments retained in the blood bank from the O negative
emergency units which were transfused. If antibody screening detects an unexpected red cell
alloantibody, the patient's physician is immediately notified and the specificity of the antibody
is determined so that units lacking the corresponding antigen can be selected and
antiglobulin crossmatching of can be performed.
Ira A. Shulman, MD
CBBS e-network Web Master
Entered: Mar. 24, 2001

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