Clinical and Laboratory Standards Institute (CLSI) simplifies the order of draw: a brief history

Medical Laboratory Observer May 2004 by Dennis J. Ernst & Roger Calam

Other articles by Dennis J. Ernst

The order in which tubes should be filled seems to mutate daily into increasingly creative incarnatios, In fact, it mutated right out of existence last year when one workshop presenter astonished her attendees by stating, "There is no order of draw." Adding to the confusion are Internet phlebotomy sites, training manuals, certification study guides, and textbooks that perpetuate many variations.The proper order in which blood-collection tubes should be filled is designed to prevent the carryover of additives from one tube to the next. Such carryover alters the composition of the next tube and can lead to erroneous results--with the potential to mislead physicians and invite catastrophic errors in patient management. For the record, the order of draw has been established by NCCLS--the National Committee for Clinical Laboratory Standards, which develops voluntary standards--through its consensus process with industry, government, and laboratory professinals and is published by NCCLS in document H3, Procedure for the collection of Blood Specimens for Diagnostic Testing by Venipuncture.The order was revised and simplified this past December to one that works for both glass and plastic tubes, regardless of whether the specimen is drawn by using a tube holder and needle assembly or syringe. To understand the necessity for the order of draw and to clarify the widespread confusionon the matter, let us examine the history on how the order came about and how it has evolved through the years.Historical perspectiveThe order has its origins in the literature as early as 1977, when evidence that additive carryover occurs was first published in the American Society of Clinical Pathologists; (ASCP's) Summary Report. (1) Authors Nilda Sun and Rita Knauf at the St. Barnabas Medical Center in Livingston, NJ, reported their observation of an asymptomatic patient who had a potassium level of 25.4 mEq/L (normal 3.5-5.3 mEq/L) obatained from a nonhemolyzed specimen. Upon Investigation, the authors discovered that the phlebotomist filled the lavender-stopper tube containing potassium EDTA immediately prior to filling the tube from which the elevated potassium was obtained.

The first recommendation on establishing a formal order of draw for blood collection was published in a letter to the editor of Clinical Chemistry by RogerCalam, PhD, and Marsha Cooper of St. John Hospital in Detroit, MI, in 1982. (2) Although tube manufacturer Becton Dickinson (BD) had been recommending as early as 1976 that all additive tubes be drawn after non additive tubes, with citrate tubes being the first additive tube filled, Calam and Cooper postulated that a more detailed order was necessary for additive tubes. They reported five cases in which they observed spuriously abnormal potassium and calcium levels, all of which resulted when a lavender-stopper tube containing potassium EDTA was filled immediately prior to the gel tube intended for chemistry tests. When specimens were re-collected, potassium and calcium fell within normal limits. Based on these observations and those of Sun and knoff, Calam and Cooper concluded that not only should additive tubes be drawn after nonadditive tubes, as BD had been suppesting, but that heparin, EDTA, and potassium-oxalate/sodium-fluoride tubes (gray stoppers) should have a specific "order of draw."The reasoning for their proosed order was based on the fact that if EDTA could carryover into nonadditive tubes, it could also carryover into tubes containing other additives. Such proof suggested that the additive of any tube could also carryover into a subsequent tube, threatening the accuracy of results. The authors proposed that heparin tubes be drawn before the EDTA tube, and the EDTA tube should precede the oxalate/fluoride tubes. Since oxalate/fluoride is disruptive to cell membranes, placing it subsequent to the EDTA tube prevents problems with cell morphology that might otherwise occur. Also, with the gray top drawn after the green top, neither potassium oxalate nor sodium flouride in the gray-top tubes would contaminate sodium or potassium testing.The order proposed by Calam and Cooper for filling additive tubes (following nonadditive tubes) was adopted by the NCCLS in 1984 in document H3-A2 as:

  1. sodium-citrate tube;
  2. heparin tube
  3. EDTA tube
  4. oxalate/fluoride tube
  5. Gel separator tubes and clot activators

The introduction of serum gel tubes (1976) and plasma gel tubes (1987) provided a convenience to specimen-processing personnel who had been required to remove the stoppers of nongel tubes after centrifugation and physically remove the serum or plasma for testing or storage. The gel tubes provided a physical barrier between the serum or plasma following centrifugation, preventing the changes that occur when serum or plasma is allowed to remain in contact with the cells for prolonged periods of time prior to testing. Serum separator tubes contain a clot activator (glass or sillica particles) to facilitate clotting. Although clot activators may shorten the time it takes for the specimen to clot, manufactureres stress that such tubes facilitate complete clotting, not rapid clotting, yielding serum that is less likely to contain fibrin strands that can interfere in testing.

Historically, tubes with anticoagulants (e.g., EDTA) or preservatives (e.g., sodium fluoride) were referred to as "additive tubes." In 1998, it was proposed that separator tubes containing gels and either a clotting activator or an anticoagulant also be considered as additive tubes. (4) The NCCLS incorporated these tubes into the order of draw in its 1998 revision (H3-A4). (5) The order evolved with the gel tubes (serum and plasma separators) following the citrate tube (blue stopper):

  1. Blood-culture tubes
  2. nonadditive tube
  3. additive tubes (in the following order):
    1. sodium-citrate tube
    2. gel separator tube
    3. heparin tube
    4. EDTA tube, ande. oxalate/fluoride tube

Since possible carryover of the clot activator could conceivably alter results, a gel tube with a clot activator had to be drawn after the blue top. Although not spelled out, the serum gel tube should be drawn before the plasma gel tube, as the latter often contains lithium heparin, which could affect the accuracy of a patient's lithium test in the serum tube.If required, a plastic red-stopper tube (plastic tubes contain a clot activator) can precede a heparin (green top) or EDTA (lavender top) tube without concern for carryover. The logic is that any carryover of the clot activator into tubes other than blue tops is irrlevant, since carryover should be neutralized by the excess anticoagulant. In contrast, carryover of a clot activator into a sodium citrate tube (blue top) could interfere with clotting factors, leading to inaccurate results.Some facilities maintain the necessity for a different order for syringe transfer because not all syringe draws go quickly and, therefore, the potential exists for clotting to occur in the syringe prior to blood transfer to successive tubes. (6,7) To prevent clotting within the syringe, an order is implemented that fills all additive tubes first and then the nonadditive tubes. NCCLS consensus concluded, however, that the potential for carryover from the needle of the syringe had the potential to be a more significant problem than any clotting that might take place in the syringe during a properly performed venipuncture. Therefore, the 1998 NCCLS revision also recommended that the same order of draw be followed when transferring blood specimens from a syringe to multiple blood-collection tubes.

Plastic tubesMotivated by increasing concern over broken-glass exposures, high biohazardous waste-disposal costs, and Occupational Safety and health Agency (OSHA) guidelines mandating substitution, many laboratories began switching from glass collection tubes to plastic (see "Richard Fairfax of OSHA Talks About the Bloodborne Pathogens Standard" February 2003 MLO, p.32) This industry-wide transition from glass to plastic necessitated a modicication to the order of draw. Plastic serum tubes are now positioned the same as gel separator tubes, which contain a clot activator. The revised order, "published in December 2003, is now as follows:

  1. Blood-culture tube
  2. Sodium-citrate tube (e.g., blue-stopper)
  3. Serum tubes with or without clot activator, with or without gelseparator (e.g., red-, gold, speckled-stopper)
  4. Heparin tubes with or without gel (e.g., green-stopper)
  5. EDTA tubes (e.g., lavendeer-stopper)
  6. Glycolytic inhibitor (e.g., gray-stopper)

(8)In the revised standar, NCCLS recognizes that some facilities may still be using glass serum tubes without a clot activator to serve as a waste tube before collecting special coagulation assays. Although the revised order functions well regardless of the presence of a clot activator in the facility's serum tube, a provision withitn H3-A5 affords the option of keeping the nonadditive serum tube before the citrate tube in the following passage: "The order of draw has been revised to reflect the increased use of plastic blood-collection tubes. Plastic serum tubes containing a clot activator may cause interference in coagulation testing. Glass nonadditive serum tubes may be drawn before the coagulation tube."The NCCLS order of draw has gone throughseveral revisions dictated by technology, publication, and common sense. The latest revision should make it easier for educators and trainers to teach the order of draw and prevent additive carryover, which can affect patient results. References(1) Sun N, Knauf R. Cross contamination solved by technique. ASCP Summary Report 1977;14(3):3.(2) Calam R, Cooper M. Recommended "order of draw" for collecting blood specimens into additive- containing tubes. Clin Chem. 1982;28:1399.(3) National Committee for Clinical Laboratory Standards. Procedures for the Collection of Diagnostic Blood Specimns by Venipuncture. Approved Standard, H3-A2, Villanova, PA; 1991(4) National Committee for Clinical Laboratory Standards. Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture. Approved Standard, H3-A5, Wayne, PA 2003