Q & A

January 2002

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 (5.11.6.2 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 refrigerator.

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 cancellations.²

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.

References
1.  Menitove 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 Science;1997:243-244.
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
Pittsburgh
Member, CAP Transfusion Medicine
Resource Committee

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

References
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
and Pathology
University of Minnesota
School of Medicine
Minneapolis