Laboratory

Bulletin June 2012

UPDATES AND INFORMATION FROM REX PATHOLOGY LABORATORY

Issue Number 186

There Will be Blood When performing pretransfusion testing for a patient who has
been pregnant or transfused within the previous three months,
or if the pregnancy history and transfusion history are uncertain,
Pretransfusion Testing
the pretransfusion sample used for testing must be no more than
Collection of a properly labeled pretransfusion blood sample
three days old at the time of intended transfusion because recent
from the intended recipient is critical to safe blood transfusion.
transfusion or pregnancy may stimulate production of unexpected
The majority of hemolytic transfusion reactions arise from
antibodies. For patients without a recent history of transfusion or
misidentification of patients or pretransfusion sample labeling
pregnancy, there is no specific requirement that a pretransfusion
errors. The patient identification armband is a critical control
sample be less than three days old. However, there is a limit on
point in the series of events required to assure a safe transfusion.
specimen age acceptability for successful performance of the
The body (of the patient) to which a transfusion is ultimately
testing. A reasonable compromise to cooperate with logistic realities
given must be the same body (with the armband) from which the
of presurgical evaluation is to allow a sample collected up to 14
sample for compatibility testing is obtained. If it is not, the result
days before transfusion for patients with no history of possible
may be a completely preventable and potentially fatal hemolytic
recent alloimmunization.
transfusion reaction. Therefore, correct patient and specimen
identification must be assured, and a unique patient armband
The goal of pretransfusion testing is to determine ABO Rh type and
that links the patient recipient to the sample used for compatibility
detect red cell alloantibodies in the transfusion recipient, i.e. the
testing must be in place. Two independent patient identifiers are
antibody screen. The goal is to detect as many clinically significant
required, including the patient’s first and last names and a unique
antibodies as possible, to detect as few clinically insignificant
identifier (the medical record number for hospital patients
antibodies as possible, and to complete the testing as soon as
and date of birth for physician office patients). Any discrepancy
possible. In general, an antibody is considered potentially clinically
in patient or specimen identification, no matter how seemingly
significant if antibodies of its specificity have been previously
small (including minor “misspellings”), cannot be allowed. The
associated with hemolytic disease of the fetus and newborn (HDFN),
phlebotomist must label each blood sample tube, in the presence
with a hemolytic transfusion reaction, or with notably decreased
of the patient, with the two patient identifiers. If the patient’s full
survival of transfused red cells. Antibodies reactive at either 37
name and medical record number do not match on the arm band,
C or in the antiglobulin test phase are more likely to be clinically
specimen label and blood bag; then compatibility testing and
significant than are cold-reactive antibodies. The antibody screen
transfusion may not proceed. Collection of a new sample that is
may be performed in advance of, or together with, an order for
correctly identified must occur before continuing any further. This
transfusion. Performing the antibody screen before intended
may lead to a delay in the availability of crossmatch compatible
transfusion permits early recognition and identification of clinically
blood products. This requirement obviously applies to both the
significant antibodies and thereby provides time for selection of
outpatient and inpatient setting. For inpatients, Rex Hospital uses a
units for transfusion. A history of previously identified alloantibodies
bar code scanning device that matches the patient arm band to the
is important since blood lacking relevant antigens should be
specimen label. If used properly, this adds a significant measure of
selected for transfusion when a patient has a clinically significant
safety to specimen collection. Of course if the “armband” bar code
antibody identified currently or historically, even if the antibody is
scan occurs away from the patient’s body, then the system adds
currently nonreactive.
no value at all. Now at Rex, patient arm bands will have a unique
code not present on other patient bar codes to prevent successful
It takes 45 − 60 minutes to actually perform an antibody screen.
specimen label scanning away from the patient’s body. If the
This time does not include the preanalytic phase of order, collection
original pretransfusion sample was collected in a physician office,
and transportation to the transfusion service. If the antibody screen
the Identifying information at registration for patients receiving
is negative, indicating the patient does not have any detectable red
transfusion at Rex facilities must exactly match the originally labeled
cell alloantibodies, the crossmatch may proceed. If the antibody
pretransfusion sample for name and DOB. Otherwise, another
screen is positive, then the time required to find compatible blood
sample with the new armband label identifiers will be required.
REX PATHOLOGY ASSOCIATES, P.A.
John D. Benson, M.D. (919) 784-3059 Preeti H. Parekh, M.D. (919) 784-3060
Timothy R. Carter, M.D. (919) 784-3058 John P. Sorge, M.D. (919) 784-3062
Keith V. Nance, M.D. (919) 784-3286 Keith E. Volmar, M.D. (919) 784-2506
F. Catrina Reading, M.D. (919) 784-3255 Suzanne White, Pathology Service Specialist (919) 612-7272
Vincent C. Smith, M.D. (919) 784-3056 Rhonda Humphrey, Practice Manager (919) 784-3063
 LB PAGE 2
is completely unpredictable. For single alloantibodies for which
antigen negative units are common in the donor population, this
could be very fast. When alloantibodies are multiple or directed
against common red cell antigens, the process could take many
hours to a few days. Therefore, the Transfusion Service must have
sufficient time prior to surgery or the intended transfusion to
complete all necessary pretransfusion testing to avoid delays in
blood availability. Our routine policy for patients with a positive
antibody screen is to find two units of compatible blood even if
transfusion has not yet been ordered; with the goal of reducing
the turn around time should blood become required. However,
coordination with the transfusion service regarding clinical needs
will always help us meet those needs more efficiently.
The Rex Hospital Transfusion Service performs an electronic
crossmatch for all patients with negative antibody screens. The
electronic crossmatch takes five minutes. An electronic crossmatch
(also known as computer crossmatch) requires the following:
• the computer system has been validated on-site to ensure that
only ABO-compatible whole blood or red cells are selected for
transfusion;
• two determinations of the recipient’s ABO group are made
with one on a current sample, and a second one by retesting
the same sample, by testing a second current sample, or by
comparing with previous records;
• the computer system contains the blood unit number,
component name, ABO group, and Rh type of the component;
• two unique recipient identifiers;
• the recipient’s ABO group, Rh type, and antibody screen
results; and interpretation of compatibility;
• a method exists to verify correct entry of data before the release
of blood or components;
• the system contains logic to alert the user to discrepancies
between the donor ABO group and Rh type on the unit label
and the interpretation of the blood group confirmatory test,
as well as to alert the user to ABO incompatibility between the
recipient and donor unit.
Traditional immediate spin and antiglobulin crossmatches are
performed for patients with a positive antibody screen, and the turn
around time is variable and dependent on the complexity of the
alloantibody(s).
Weak D, HDFN, and Rh Immune Globulin
Determination of ABO Rh type and antibody screen are the most
important laboratory tests required to identify blood components
compatible for transfusion. The ABO blood group system is
more complex than the simple letter designation system implies.
Variations of A, B, and O groups challenge the daily practice of
transfusion medicine, but go unnoticed outside the laboratory
transfusion service because the final ABO type is eventually
determined and designated simply as A, B, AB or O. The Rh
positive or negative label belies the underlying complexity of the
Rh blood group system. Despite best efforts to communicate the
essential clinically meaningful Rh type, occasional vagaries and
inconsistencies in Rh designation cannot be avoided. The label Rh
Weak D (formerly called Du) is one result of these Rh D complexities.
The implications of Weak D depend on the clinical situation, and
most commonly influence the use of Rh Immune Globulin (RhIG) to
prevent Hemolytic Disease of the Fetus or Newborn (HDFN).
HDFN results from the destruction of fetal and newborn red cells
by maternal alloantibodies specific for sensitizing alloantigen(s). Rh
D is the clearly the most common and important red cell antigen
involved. However, antibodies targeted against other antigens
can also cause HDFN. The most common are K, and c. Unlike
HDFN caused by anti-D, HDFN caused by anti-K uniquely produces
suppression of fetal erythropoiesis in addition to hemolysis. Other
antibodies that have been less commonly reported to cause
moderate or severe disease include k, Kpa, Kpb, Ku, Jsa, Jsb, Jka,
Fya, Fyb, S, s, and U. The maternal IgG antibody is transported
across the placenta into the fetal circulation where it binds to the
corresponding fetal red cell antigen, targeting the antibody-coated
red cells for destruction. Women can be alloimmunized to red cell
antigens by previous transfusions as well as previous or current
pregnancy.
Complex factors influence the ability of individuals to respond to red
cell antigens. Rh D is the most potent stimulant of an immunologic
response, and a 200-mL transfusion of red cells stimulates anti-D in
about 85% of Rh D negative non-immunosuppressed individuals.
About half of the remaining 15% will never become immunized,
even after repeated challenges with Rh D positive red cells. The
incidence of antibody production in volunteers undergoing repeat
immunization is 80% to 90%, but the incidence in Rh D negative
hospitalized patients switched to Rh D positive blood components
is much lower, approximately 32%. Although as little as 0.1 to 1
mL of Rh D positive red cells can stimulate antibody production,
the volumes of fetomaternal hemorrhage (FMH) are generally
small, which contributes to relatively low alloimmunization rates in
pregnancy. Without RhIG prophylaxis, Rh D alloimmunization occurs
in about 16% of Rh D negative mothers with Rh D positive infants.
Approximately 1.5% to 2% become sensitized at the time of their
first delivery, an additional 7% become sensitized within 6 months,
and the final 7% become sensitized during the second affected
pregnancy. Immunization is reduced to about 1.5% to 2% when the
mother is ABO incompatible with the fetus, due to the shortened
life span of the incompatible fetal red cells. The risk of an Rh D
negative mother becoming immunized by a Rh D positive fetus can
be reduced to less than 0.1% by the appropriate administration of
Rh Immune Globulin (RhIG). The mechanism of action of RhIG is not
completely understood. Current evidence shows that Rh D positive
red cells are opsonized by RhIG and removed by macrophages,
which release cytokines that result in immunomodulation. The
number of IgG molecules known to prevent immunization is much
fewer than the D antigen sites on red cells.
Weak D (formerly called Du) red cells have historically been defined
as having a reduced amount of D antigen, and are not agglutinated
by D typing reagent at immediate spin (Direct Antiglobulin Test
- DAT). They require an indirect antiglobulin test for detection.
The number of samples classified as weak D, however, depends
on the characteristics of the typing reagents, which have become
more sensitive over the years. Current anti-D reagents are potent
monoclonal and polyclonal/monoclonal blends that are capable of
agglutinating most weak D samples on direct testing. Samples

typed as Weak D in years past, may be typed as ordinary Rh D
positive with current more sensitive methods.
Weak D expression results primarily from single nucleotide
mutations that encode amino acid changes predicted to be located
intracellularly, or in the transmembrane regions of Rh D, rather than
on the outer surface of the red cell. The mutations affect insertion
of the protein in the membrane, reflected in the reduced number
of D antigen sites on the red cells. Many different mutations cause
weak D expression, and they are designated Type 1 through Type
73. Approximately 1% of Rh-positive individuals are considered
weak D. In addition, weak D expression can reflect inheritance
of partial D, a recombinant RhD-RhCE protein that is missing
parts of the normal D protein. Partial D individuals can make an
alloantibody-Rh D to high-incidence Rh D antigens that are missing
on their own red cells. Partial D individuals also express novel low-
incidence antigens that are generated by recombination events.
When exposed to Rh-positive red blood cells by pregnancy or
transfusion, persons with a partial D antigen are capable of forming
allo-anti-D, i.e., an antibody to their missing or variant D epitope(s).
These antibodies are capable of causing HDFN. Partial D individuals
are often identified by an apparent Rh discrepancy, which means
an Rh-positive individual has made an allo-anti-D reactive with Rh-
positive red cells but not with autologous red cells. Approximately
5 − 10% of weak D phenotypes are partial D. It should be noted
that partial D phenotype can yield an Rh typing result of either Rh D
positive or Rh D negative, depending on the reagents used. There is
no direct evidence that RHIG will prevent formation of anti-D after
delivering Rh D positive neonates in patients with partial D.
In many laboratories, including the Rex Hospital Laboratory, Weak
D testing is no longer routinely performed on patient samples. It is
recognized that patients with weak D phenotype will be reported
as Rh negative with this policy. The disadvantage of reporting these
patients as Rh D negative is considered negligible since their use of
Rh D negative red cell inventory is quantitatively insignificant due
to low incidence of weak D, and is no problem for the recipient.
They will be potentially identified as candidates for RhIG following
potential sensitization to Rh D through pregnancy, but this is
viewed as acceptable practice as the only harm is administration
of RhIG which may not be required. However, it also reduces the
impact of the uncommon partial D phenotype of weak D, with
its attendant risk of Rh D alloimmunization. In contrast, Weak D
testing is always performed on blood donors and infants born to Rh
D negative mothers since it is necessary to eliminate the possibility
of alloimmunization from the Weak D antigen.
So, if we don’t even test for Weak D in patients, why does it ever
get reported? Patients may have a blood type performed at another
laboratory, as part of blood donation, or during a previous HDFN
work up that yielded a discrepancy with the currently reported
blood type. When we investigate such a reported discrepancy, Weak
D testing becomes necessary for initially Rh D negative patients, to
resolve the discrepancy. If a pregnant patient is determined to be
Weak D positive, the decision regarding RhIG administration is not
universally agreed upon and varies by regional practice. However,
American College of Obstetricians and Gynecologists (ACOG) and
American Association of Blood Banks (AABB) recommendations are
LB PAGE 3
that RhIG is not required for Weak D positive mothers with a Rh D
positive infant, since most of these mothers will not form an anti-D
antibody as a result of exposure to the Rh D antigen. Those who
argue in favor of giving RhIG for Weak D positive mothers cite the
known, although small, incidence of Partial D phenotype among the
Weak D population, who can form anti-D antibody.
Women who are candidates for RhIG are:
• D negative with a negative antibody screen for anti-D after
delivery of a D positive infant or if the Rh D type of the
newborn is unknown or undetermined (e.g., stillborn).
Women who are not candidates for RhIG include:
• D-negative women (including without Weak D testing) whose
infant is known to be D negative (by definition - including
testing for Weak D),
• D negative women previously immunized to Rh D, and
• D-positive women (either with or without Weak D testing
according to AABB and ACOG guidelines).
One 300 mcg dose of RhIG protects against a Fetal Maternal
Hemorrhage (FMH) of 15 mL fetal RBCs (30 mL fetal whole blood).
To determine if additional RhIG dosing is required for adequate
immunoprophylaxis, quantitation of FMH is performed. If a fetal/
neonatal sample is Rh D positive and the maternal sample is Rh
D negative, screening for Fetal Maternal Hemorrhage (FMH) is
performed by the rosette test. The rosette test is a sensitive method
to detect FMH of approximately 10 mL or more. The maternal
sample is incubated with anti-D, and then indicator Rh D positive
red cells are added. The indicator red cells will form agglutinates
(rosettes) with the fetal Rh D positive red cells. A positive rosette
test indicates a large FMH. Although estimates suggest that only
0.3% of pregnancies are complicated by FMH greater than 15 mL
fetal RBCs (30 mL fetal whole blood), a large FMH is an important
and preventable cause of failed immunoprophylaxis. Following the
positive screening rosette test, the sample is sent for quantitation by
flow cytometry (performed at Mayo Medical Laboratory for Rex Lab
samples). The result is reported as ml of fetal RBCs detected and is
accompanied by a recommended dose of RhIG. Calculation of the
RhIG dose (vials) uses the formula:
X ml fetal RBC/15 ml = Y vials (doses)
To provide a margin of safety, if the calculated dose to the right of
the decimal point is >0.5 vial, it should be rounded up to the next
whole number plus one vial; if <0.5, rounded down plus one vial.
For example, if the calculated dose is 1.5 vials RhIG, then three vials
should be administered.
Postpartum RhIG should be given within 72 hours of delivery.
If prophylaxis is delayed, the prevention of alloimmunization is
somewhat decreased. Nonetheless, the ACOG recommends that
treatment still be administered, even if delayed past 72 hours,
because some studies have found partial protection has occurred as
late as 13 days after exposure and possibly as late as 28 days.
Administration of RhIG during pregnancy may produce a positive
antibody screen in the mother, but the titer is rarely greater than

four and thus poses no risk to the fetus. If the lab is able to obtain
the history of RhIG administration, a comment is added that the
anti-D detected in a prenatal sample is most likely due to the RhIG.
Occasionally, the DAT may be positive in the newborn without
evidence of hemolysis. About 10% (20 - 30 ug) of the 28-week
gestation dose will be present in the mother at delivery (half-life of
IgG is 25 days) and can be detected and identified as anti-D. This
anti-D should not be interpreted as active immunization, and the
postpartum RhIG dose should be given if the newborn is D positive.
Antibody titers in the mother do not correlate with the effectiveness
of the RhIG or the amount of FMH. Anti-D can be detected in the
maternal circulation for as long as six months.
Risks of Transfusion, a brief update on incidence
Blood component transfusion continues to carry risk of mortality
and morbidity for patients. However, advising patients of these
risks and factoring them into the decision to transfuse requires up
to date awareness of specific risk trends. As the risk of infectious
disease transmission has markedly decreased over the years,
greater awareness of other complications has emerged. Note
these incidence figures are difficult to establish and depend on the
reporting mechanism used. There is variability (sometimes quite
significant) in the figures quoted in the literature, professional
society annual meeting presentations, and various surveillance
program reports. However, these numbers are a reasonable
approximation based on review of a variety of sources by this
author. I believe it is generally recognized that the incidence of all
transfusion complications continues to be under-recognized and
under-reported, especially sepsis, TRALI, and TACO.
Mycobacterium tuberculosis Infection Determination by QuantiFERON-TB Gold®
The Tuberculin Skin Test (TST) remains the primary screening
method for latent tuberculosis infection (LBTI). We occasionally
receive requests for the QuantiFERON -TB Gold® test, an alternative
to TST. The QuantiFERON-TB test is an Interferon Gamma Release
Assay (IGRA) that measures the cell mediated immune response to
antigens simulating mycobacterial proteins. The main advantage
of the QuantiFERON test is a decrease in false positives due to BCG
vaccination, and avoiding the risk of a patient not returning for the
TST reading. Sensitivity, specificity and positive predictive value are
not significantly different for TST and QuantiFERON.
However, accuracy is significantly affected by the collection, in-lab
processing and transportation of the specimen for QuantiFERON-
TB. A critical step in the test is a 16-24 hour incubation that must
be carried out at precisely 37°C immediately after collection, prior
Rex Healthcare Laboratory (919) 784-3040. Telephone extensions are: Pathologists’ Direct Line (3063), Judy Allen (Clinical Laboratory Manager 2192), Stephen Finch (Laboratory Director 2400),
Janet Johnson, (Anatomic Pathology Manager 3888), Kellie Newman (Suburban Diagnostic Services Manager 7018), Michelle Tyson (Business Development Representative 6421), Robin Wagner
(Laboratory Outreach Manager 3136), Margaret Windett-Sims (Rex Blood Services Manager 4763).
Client Response Center (919) 784-6000 (phone) (919) 784-6299 (fax)
Rex Hospital Rex Hospital
Outpatient Laboratory Monday – Friday • 7 a.m. – 7 p.m.
Hours of Operation Saturday 7:30 a.m. – 4 p.m.
Sunday 7:30 a.m. – 3 p.m.
LB PAGE 4
Risk of Transfusion
(incidence per transfusion, increasing order)
HTLV I/II 1 in 3 million
HIV 1 in 2 million
Hepatitis C 1 in 1.6
million
Hepatitis B 1 in 350,000
Sepsis Red Cells 1 in 100,000
Sepsis Platelets 1 in 75,000
Anaphylaxis 1 in 50,000
Hemolytic Transfusion reaction: 1 in 10,000
Transfusion Related Acute Lung Injury (TRALI) 1 in 10,000
Febrile non-hemolytic reaction: 1 in 200
Transfusion Associated Circulatory Overload (TACO): 1 in 100
Questions or concerns regarding blood transfusion are welcome
and should be directed to the author or Judy Allen, (Clinical
Laboratory Manager).
Timothy Carter, M.D. • Medical Director Laboratory
1. AABB Technical Manual. 17th edition. John D. Roback, MD, PhD, Bethesda,
AABB: 2011.
2. ACOG Practice Bulletin. Clinical Management Guidelines for Obstetrician-
Gynecologist, Number 4, May 1999, reaffirmed 2010.
to sending the sample for completion of the assay at a reference
laboratory. Standard incubation temperatures in the microbiology
laboratory are 30°C and 35-36°C, thus hospital laboratories,
including Rex Laboratory, are not typically equipped to accurately
perform this critical incubation phase of the test. The temperature
requirement is strict and failure to incubate the specimen correctly
results in false negative results. So, even though our reference lab
(Mayo Medical Laboratory) offers the assay, we are currently unable
to reliably process samples for this test. Our understanding is that
LabCorp offers the appropriate specimen handling for this particular
test, and outpatients may be referred to a LabCorp testing location
for the few instances when this test may be indicated.
Timothy Carter, M.D. and Keith Volmar, M.D.
Copyright 2012 Rex Healthcare/Rex Pathology Associates (919) 784-3040. All rights reserved.
Rex Medical Plaza Cary & Wakefield Knightdale & Holly Springs
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