Q. A decision was made to no longer store our blood samples
for crossmatch upright in racks. Instead, all tubes and sectors
involved in crossmatches are banded together and dropped in buckets.
Since they are placed on their sides, the serum, clots, and whole
anticoagulated samples are in constant contact with the tube stoppers.
Do stoppers or stopper byproducts cause crossmatch interferences?
A. The American Association of Blood Banks standards
(20th edition) mandate the minimum storage retention for recipients’
samples. The recipient’s blood specimen and a sample of the donor’s
red cells must be sealed or stoppered and kept at refrigerator temperature
for at least seven days after each transfusion.1
The proposed 21st edition (126.96.36.199 Recipient samples shall be stored
at refrigerated temperatures for at least seven days after transfusion)
has similar wording. Maintaining the patient’s and donor’s samples
allows for repeat or additional testing if there is any adverse
effect to the transfusion.
Individual institutions need to specify other sample storage conditions
such as temperature range, storage position, or maximum storage
duration that maintains sample stability. How long samples can be
stored depends most often on the available refrigerator space. Although
some transfusion service blood banks store patients’ samples at
1°-6°C in the same continuously monitored refrigerator with donor
units, the samples can be maintained at 2°-8°C in an unmonitored
The use of stored specimens in pretransfusion testing should be
based on the specimen storage limitations described in the manufacturer’s
reagent insert; if stored longer or under different conditions,
the institution should validate the reliability of test results.
Published studies of the stability of pretransfusion samples stored
for extended periods, such as with preadmission samples for patients
who have not been pregnant or transfused in the preceding three
months, is limited. Jefferies et al extended the time interval that
a sample could be used for pretransfusion testing to 30 days if
a patient had no history of recent transfusion or pregnancy. The
authors found that this policy resulted in a decrease in emergent
requests for pretransfusion testing and in last-minute surgical
When serum was the preferred pretransfusion sample to demonstrate
certain complement-binding antibodies, the deterioration of complement
was a limiting factor for sample storage. After about two weeks
at 4°C, serum complement activity falls below 60 percent, which
is the minimum level to detect complement activation by weak complement-binding
antibodies.3 In their recent study to evaluate the stability
of pretransfusion samples collected in plastic Vacutainer tubes
compared with glass tubes, Anderson et al demonstrated consistent
agreement of ABO, Rh, and antibody screen testing for EDTA plasma
and serum collected in both tube types over the 28-day period. The
strength of reactions remained fairly constant over time. However,
only two samples demonstrated alloantibodies, and after 48 hours
of storage at 2°-8°C, the remaining plasma or serum was separated
from the cells.4
No reports to date have been published that implicate rubber stoppers
as causing a problem with ABO, Rh, and/or antibody screen in pretransfusion
testing. Rubber tube stoppers that came in contact with blood have
in the past been a source of contamination in toxicology/therapeutic
drug-monitoring testing. The contaminant, trisbutoxyethyl phosphate,
or TBEP, interfered by displacing basic drugs from their protein-binding
sites, resulting in a redistribution of the drug into the erythrocytes
with a decreased amount of drug remaining in the serum or plasma.
Tube manufacturers have eliminated TBEP and it is no longer a problem.5
In our centralized blood bank laboratory, and in a few other transfusion
service centers I know of, both EDTA and non-anticoagulated tubes
are submitted for all blood bank testing, to include compatibility
testing. The serum or plasma is not separated from the red cells.
The rubber Vacutainer stoppers, though, are not reused after testing
is completed to decrease the risk of glass breakage and infectious
disease contact, and plastic caps are used to reseal the tubes.
According to AABB standards (20th edition) either plasma or serum
can be used for pretransfusion testing.1 Plain red-top
tubes (no anticoagulant), yellow-top tubes (acid citrate dextrose),
or purple/pink-top tubes (EDTA) are all acceptable sample types.
EDTA plasma is the preferred sample type in laboratories that employ
gel or solid-phase technology. In the past, serum was the preferred
sample for antibody screening and pretransfusion testing because
of the anticomplement property of the anticoagulants. Both EDTA
and citrate bind calcium, and thereby prevent complement activation.
When polyspecific antiglobulin reagent was used routinely in pretransfusion
testing, there was a concern that antibodies demonstrable only through
complement activation would not be detected if plasma was used.
However, almost all clinically significant antibodies implicated
in transfusion reactions, except for those of ABO blood group, are
known to be IgG and do not depend solely on the presence of complement
for their detection. The use of plasma was also discouraged in the
past because of the potential for small fibrin strands to be formed,
which, for the inexperienced blood bank technologist, could be difficult
to distinguish from agglutination. Since many crossmatches now are
performed by immediate spin or as electronic crossmatch and the
number of laboratories moving to gel technology is steadily increasing,
fibrin is less likely to interfere with interpretation. Another
important reason for using plasma rather than serum in pretransfusion
testing is the time that can be saved. Not having to wait for the
blood sample to clot is important in emergencies and when the patient
has been anticoagulated.
JE, ed. Standards for Blood Banks and Transfusion Services.
20th ed. Bethesda, Md.: American Association of Blood Banks; 2000.
2. Jefferies LC, Smith ME, Strobl FJ, et al. Improved
efficiency in providing blood to surgical patients using a novel approach
to preadmission testing. Am J Med Qual. 2000;15(6):251-256.
3. Mollison PL, Engelfriet CP, Contreras M. Blood
Transfusion in Clinical Medicine. 10th ed. Oxford, England: Blackwell
4. Anderson DR, Wiseman
J, MacLeod J, et al. Evaluation of polyethyleneterephthalate for ABO
and Rh typing and alloantibody screening. Transfusion. 2000;40:669-672.
5. H18-A: Procedures for the Handling and Processing
of Blood Specimens; Approved Guideline. Villanova, Pa.: National
Committee for Clinical Laboratory Standards; 1990.
Kathleen Puca, MD
Transfusion medicine fellow
Institute for Transfusion Medicine
Member, CAP Transfusion Medicine
Q. For Dade Behring’s troponin I assay (CTNI cat. No.
RF421A) on the Dimension RxL heterogeneous assay module, a reference
interval of 0-0.05 µg/L is expected in apparently healthy persons.
Determinations performed on outpatient specimens in our laboratory
accordingly show 69 out of 72 (95.8 percent) to be in that range.
Most people we talked to about this assay agree that values >1.5 µg/L
are diagnostic for acute myocardial infarction, as suggested by the
company’s product insert.
What is less clear with this
test is the lower limit of the so-called gray zone where one should
suspect the presence of unstable angina. A look at the frequency
distribution of results obtained from patients seen in the emergency
department showed that values <0.1 µg/L accounted for 61 percent
of 347 results, while 25 percent were in the 0.1-1.5 µg/L range.
A look at the frequency histogram suggested that a lower limit of
0.25 µg/L (77.5 percent of results) would be more realistic in our
hospital. Laboratories that served as beta sites for the test suggested
a practical lower limit ranging from 0.2 µg/L to 0.3 µg/L.
A publication (Morrow DA et al. Clin Chem. 2000;46:453-460)
suggests that a threshold of 0.1 µg/L, regardless of the assay used
in their study (Immuno 1, ACS 180, or RxL), should be prognostically
efficient for predicting future adverse clinical outcomes.
Is a consensus evolving regarding the lower limit of the gray
zone of the Dade assay? Should there be different followup protocols
for patients in the 0.1-0.3 µg/L range and for those above 0.3 µg/L?
A. The recent European Society of Cardiology/American
College of Cardiology consensus document regarding the redefinition
of acute myocardial infarction is heavily predicated on the use
of cardiac troponin testing (cTnI or cTnT).1-3 The guidelines
note that a patient with an increased serum cardiac troponin in
the clinical setting of ischemia is classified as an AMI. The upper
reference limit for troponin testing has been designated at the
99th percentile of a reference (normal) population measured using
an assay giving ≤10 percent imprecision (coefficient of variation
percent) at this cutpoint.1,2
However, because no manufacturer of troponin assays can currently
meet this criterion, it has been suggested the cutpoint be revised
to the lowest concentration that performs at an imprecision of 10
percent.4 Therefore, using the Dade Behring Dimension
RxL cTnI assay as an example, the 99th percentile has been shown
by the company to be 0.07 µg/L. However, the imprecision of the
assay at this level is greater than 20 percent. Dade Behring has
released internal data that demonstrates the lowest concentration
to give a 10 percent CV is 0.14 µg/L. However, the lowest concentration
that has been substantiated in the literature at 10 percent CV is
0.4 µg/L. Therefore, it would be my recommendation to use a cutpoint
at 0.3 µg/L, with values ≥0.4 µg/L indicative of myocardial
damage until the evidence-based literature reveals a different concentration.
I personally do not believe in gray zones. As for the studies
that demonstrate that a single-admission troponin concentration
at =0.1 µg/L imparts greater short-term risk of adverse cardiac
events,5-7 as well as potentially altering therapeutic
interventions to lower risk,8 the evidence speaks for
itself. As for ruling in and ruling out AMI, serial samplings taken
at admission (zero hour) and three to six hours, six to nine hours,
and 12 to 24 hours after admission are still the serial orders recommended.1-3
1. Jaffe AS, Raukilde J, Roberts R, et al. It’s time for a
change to a troponin standard. Circulation. 2000;102:1216-1220.
2. Alpert JS, Thygesen K, Antman E, et al. Myocardial infarction
redefined—consensus document of the joint European Society of Cardiology/American
College of Cardiology Committee for the redefinition of myocardial infarction.
Eur Heart J. 2000;21:1502- 1513.
3. Wu AHB, Apple FS, Gibler WB, et al. National Academy of
Clinical Biochemistry standards of laboratory practice: recommendations for
the use of cardiac markers in coronary artery diseases. Clin Chem. 1999;45:1104-1121.
4. Apple FS, Wu AHB. Myocardial infarction redefined: role
of cardiac troponin testing. Clin Chem. 2001;47: 377-379.
5. Morrow DA, Rafai N, Tanasijevic MJ, et al. Clinical efficacy
of three assays for cardiac troponin I for risk stratification in acute coronary
syndromes: a thrombolysis in myocardial infarction (TIMI) IIB substudy. Clin
Chem. 2000; 46:453-460.
6. Oltani F, Galvani M, Nicolini FA, et al. Elevated cardiac
troponin levels predict the risk of adverse outcome in patients with acute coronary
syndromes. Am Heart J. 2000;40:917-927.
7. Braunwald E, Antman EM, Beasley W, et al. ACC/AHA guidelines
for the management of patients with unstable angina and non-ST segment elevation
myocardial infarction. Circulation. 2000;102:1193-1209.
8. Bertrand ME, Simoons ML, Fox AA, et al. Management of acute
coronary syndromes: acute coronary syndromes without persistent ST segment elevation.
Eur Heart J. 2000;21:1406-1432.
Fred Apple, PhD
Medical director of clinical laboratories
Hennepin County Medical Center
Professor, Laboratory Medicine
University of Minnesota
School of Medicine