Test plan coverage: Domain 3, Routine Blood Collections — 28 of 100 scored items, the largest domain[1]. This chapter covers tasks 3A, 3B, 3D, 3J and 3N and knowledge k47, k54–k59 and k66: selecting and checking equipment, needles, evacuated tubes and their additives, the order of draw, fill levels and inversions. Chapter 6 covers the draw itself, capillary collection and complications.
A reminder from the edition notes at the front of this book: the order of draw below is the CLSI order as published in the dated, freely readable sources named here. CLSI's current venous standard is PRE02 (8th edition, February 13, 2025), which replaced GP41[2]. BD's own chart, dated June 2023, is headed "CLSI-recommended order of draw (GP41-A7)"[3]. Always follow your facility's order where it is written down; BD's chart itself says "Always follow your facility's protocol for order of draw."[3]
5.1 The three collection systems (task 3A; k54)
| System | Parts | When it is used |
|---|---|---|
| Evacuated tube system | double-ended needle, tube holder, vacuum tubes | Routine venipuncture; multiple tubes from one puncture |
| Winged (butterfly) set | short needle with plastic wings, flexible tubing, holder or syringe | Small, fragile or hand veins; children; older adults |
| Needle and syringe | hypodermic needle, syringe, then a transfer device into tubes | Very fragile veins where vacuum would collapse the vein |
WHO explains why closed systems are preferred: "Closed systems for blood sampling are preferable because they have proven to be safer than open systems."[4] Vacuum tube systems reduce "the risk of direct exposure to blood and has made it easier to take multiple samples from a single venepuncture"[4]. In the evacuated system the tube "is under vacuum. Once the needle is in the vein, the tube is pressed on to the needle and the blood is drawn automatically into the sample tube by vacuum until the required amount is collected"[4].
Do not push the tube on early. WHO: "Do not push the laboratory tube onto the needle inside the barrel until the needle is in the blood vessel, or the vacuum will be lost."[4]
Syringe transfers. When a syringe is used, fill tubes with a transfer device, not by pushing a needle through the stopper by hand. WHO warns not to "press the syringe plunger because additional pressure increases the risk of haemolysis"[4], and a hospital procedure says to transfer "as soon as possible using a Blood Transfer Device, as a delay could cause improper coagulation"[5].
5.2 Needles: gauge, length and safety features (k55, k56, k66)
Gauge runs backward. The larger the gauge number, the smaller the needle's bore. A 21-gauge needle is larger than a 23-gauge.
WHO's Table 3.1 gives recommended gauges by use[4]:
| Gauge | Use in WHO's table |
|---|---|
| 16–18 | Blood donation |
| 21 (1–1.5 inch) | Adults |
| 22 (1 inch) | Adults; children, older adults and small veins |
| 23 | Adults; winged set for children, older adults, small veins and neonates |
Why not smaller? WHO lists "use of a needle of too small a gauge (23 or under), or too large a gauge for the vessel" among causes of hemolysis[4]. "If the needle is too large for the vein for which it is intended, it will tear the vein and cause bleeding (haematoma); if the needle is too small, it will damage the blood cells during sampling"[4].
Choosing the device (k55). The test plan lists the factors: "current health status, stated history, vein size, requisition requirements"[1]. WHO: "Choose the gauge of hypodermic needle that fits comfortably into the most prominent vein with little discomfort"[4].
Safety features (k66). Needles must have engineered sharps-injury protection where available — OSHA defines these as devices "with a built-in safety feature or mechanism that effectively reduces the risk of an exposure incident"[6]. They come as hinged shields, sliding sheaths and retractable needles. WHO notes that some systems have "a mechanism that can be activated once the needle has been used; the mechanism retracts the used needle into the barrel and snaps it shut"[4]. Activate the feature immediately as the needle leaves the skin (Chapter 6).
5.3 Checking equipment before you use it (task 3B; k58)
Task 3B is to "Verify quality of equipment (e.g., sterility, expiration date, and manufacturer's defects)"[1]; k58 adds "inspection of needles, check for cracks in tubes, and check expiration dates"[1]. BD's package insert gives the manufacturer's rules:
- "Do not use tubes after their expiration date. Tubes expire on the last day of the month and year indicated."[7] An expired tube may have lost vacuum or have a degraded additive.
- "Do not use tubes or needles if foreign matter is present."[7]
- "Do not use needle if label has been torn before venipuncture."[7] The paper seal shows the needle is still sterile.
- "Tube interiors are sterile."[7]
- Cracked or chipped tubes can break in the centrifuge and release "sample, droplets, and an aerosol into the centrifuge bowl"[7].
Also tap tubes gently before use: BD instructs collectors to "Gently tap tubes containing additives to dislodge any material that may be adhering to the stopper"[7].
5.4 Tubes and additives (k57; k47)
The stopper color tells you the additive. Colors are standardized by the manufacturer, not by law, so always read the label as well. The table combines BD's tube guide and WHO's order-of-draw table.
| Stopper | Additive | What it does | Specimen | Typical use |
|---|---|---|---|---|
| Yellow (SPS) or blood culture bottles | Sodium polyanethol sulfonate (SPS) / broth | Preserves microorganisms | Whole blood | "SPS for blood culture specimen collections in microbiology"[8] |
| Light blue | Buffered sodium citrate, 0.109 M (3.2%) in plastic[8] | "Forms calcium salts to remove calcium"[4] | Plasma | "For coagulation determinations"[8] |
| Red | None (glass) or clot activator (plastic)[8] | Blood clots | Serum | Chemistry, serology |
| Gold / red-gray (SST) | Clot activator and gel[8] | Gel forms a barrier between serum and cells | Serum | "For serum determinations in chemistry"[8] |
| Orange (RST) | Thrombin-based clot activator | Fast clotting — "Blood clotting time: 5 minutes"[8] | Serum | STAT chemistry |
| Green | Sodium or lithium heparin | "Inactivates thrombin and thromboplastin"[4] | Plasma | "For plasma determinations in chemistry"[8] |
| Light green (PST) | Lithium heparin and gel[8] | Anticoagulates; gel separates plasma | Plasma | Chemistry |
| Lavender | K2EDTA or K3EDTA[8] | Removes calcium | Whole blood | "for whole blood hematology determinations"[8] |
| Pink | K2EDTA | Removes calcium | Whole blood | Blood bank; crossmatch label "required by the AABB"[8] |
| Gray | Potassium oxalate/sodium fluoride[8] | "Sodium fluoride is the antiglycolytic agent"[8] | Plasma | "For glucose determinations"[8] |
| Royal blue | Clot activator or K2EDTA; low trace elements[8] | Stopper formulated for "low levels of trace elements" | Serum or whole blood | "For trace-element, toxicology, and nutritional-chemistry determinations"[8] |
| Tan | K2EDTA | Certified low lead | Whole blood | "For lead determinations"[8] |
| Yellow (ACD) | Acid citrate dextrose | Preserves cells | Whole blood | "ACD for use in blood bank studies, HLA phenotyping, and DNA and paternity testing"[8] |
| White (PPT) | K2EDTA and gel | Separates plasma | Plasma | "molecular diagnostic test methods"[8] |
Three additive facts that turn into exam questions
- Lithium heparin and lithium levels. BD: "Do not use BD Vacutainer® Tubes containing lithium heparin for lithium measurement."[7] WHO's table says "For lithium level use sodium heparin"[4].
- Why EDTA wrecks a potassium or calcium result. K2EDTA and K3EDTA are potassium salts of EDTA, and EDTA works by removing calcium[4]. Carry a little of it into a chemistry tube and the potassium reads falsely high while the calcium reads falsely low. That is the main reason EDTA comes near the end of the order of draw.
- Fluoride stops glycolysis. Red cells keep consuming glucose in the tube; sodium fluoride, "the antiglycolytic agent"[8], stops them, and WHO notes it "preserves glucose up to five days"[4].
Matching the test to the tube (k47)
The test plan's example for k47 is "blue stopper for coagulation"[1]. Exam items usually give a test and ask for the tube, or give a tube and ask what it cannot be used for. Work from the department the test belongs to:
| Department or test type | Tube | What the sources say |
|---|---|---|
| Coagulation (PT/INR, PTT) | Light blue citrate | "For coagulation determinations"[8]; full draw required[4] |
| Routine chemistry (metabolic panels, lipids, liver tests) | Gold SST, red, or light green PST | SST "For serum determinations in chemistry"[8]; PST for "plasma determinations in chemistry"[8] |
| STAT chemistry | Orange RST | "For stat serum determinations in chemistry"[8] |
| Serology and immunology | Red or gold | WHO lists "immunology and serology" among clot-tube uses[4] |
| Hematology (CBC, hemoglobin, hematocrit) | Lavender EDTA | "For whole blood hematology determinations"[8]; WHO: "Haematology"[4] |
| Blood bank (type and crossmatch) | Pink EDTA (or lavender per facility) | Crossmatch label "required by the AABB"[8]; K2EDTA "may be used for routine immunohematology testing"[8] |
| Glucose, lactate (per lab) | Gray fluoride/oxalate | "For glucose determinations"[8] |
| Lithium level | Not lithium heparin | [7] |
| Ammonia | Heparin per WHO's table ("for ammonia level use either" heparin salt) or EDTA per some reference labs | [4] |
| Lead | Tan K2EDTA | "For lead determinations"[8] |
| Trace elements, toxicology | Royal blue | [8] |
| HLA typing, paternity, DNA | Yellow ACD | WHO: "HLA tissue typing ... paternity testing ... DNA studies"[4] |
| Molecular (PCR) | White PPT | [8] |
| Blood cultures | Culture bottles or yellow SPS | [8] |
Your laboratory's test directory has the final word: the same test can be validated on different tubes in different laboratories — ammonia is the classic example, drawn on heparin in some laboratories and EDTA in others[4, 9].
5.5 The order of draw (task 3J; k59)
Why it exists
WHO: "Draw blood collection tubes in the correct order, to avoid cross-contamination of additives between tubes."[4] The additive on the outside of the needle that punctures one stopper can be carried into the next tube.
The order
The CLSI order, as BD's 2023 chart and the CLSI 7th-edition summary give it[3, 10]:
- Blood cultures — blood culture bottles or the yellow SPS tube (sterile specimens first)
- Sodium citrate — light blue (coagulation)
- Serum tubes — red, gold/SST, orange/RST (with or without clot activator or gel)
- Heparin — green or light green (with or without gel)
- EDTA — lavender, pink, white (with or without gel)
- Sodium fluoride/potassium oxalate — gray (glucose)
A university hospital procedure gives the same six and adds the yellow ACD tube after gray[5]. BD's package insert states the principle in four groups: "1. Tubes for sterile samples. 2. Tubes for coagulation studies (e.g., citrate). 3. BD SST™ and Serum Tubes. 4. Tubes with other additives (e.g., heparin, EDTA, fluoride)."[7]
One way to remember it: say what each tube is for, in order — germs, clotting, serum, then the three anticoagulant families (heparin, EDTA, fluoride). If a mnemonic helps you, make one from the stopper colors; the order is what matters.
Rules that go with the order
- Same order for every method. "The order of draw is the same whether the specimens are collected by evacuated tube method or by syringe and is also the same for plastic or glass tubes."[10]
- What may come before the light-blue tube. "Only blood cultures, glass non additive tubes or plastic tubes without clot activator may be collected before the coagulation tube (light blue)."[10]
- The discard tube with a winged set. "When a winged blood collection set is used for venipuncture and a citrate tube is the first tube needed, a discard tube must first be collected. The discard tube is used to remove air from the tubing of the collection set, to ensure the proper volume of blood is drawn into the tube." It "should be a nonadditive or coagulation tube."[3] It "does not need to be filled completely"[7]. BD warns that its plastic serum tubes contain clot activator and "are not to be used as discard tubes before drawing citrate tubes"[7].
- Coagulation tube alone with a straight needle. WHO: "If a routine coagulation assay is the only test ordered, then a single light blue top tube may be drawn."[4]
- Blood cultures with a winged set. "When a winged blood collection set is used, the aerobic blood culture bottle is filled first, followed by the anaerobic bottle."[3]
- Trace elements. "Syringes must not be used for trace element collections that include testing for cobalt and chromium because the plunger tip contributes such elements to the specimen."[10]
- Capillary collections use a different order (Chapter 6).
Sources cited in this excerpt
- NHA Certified Phlebotomy Technician (CPT) Test Plan for the CPT Exam (based on a job analysis completed in 2024). National Healthcareer Association, 2024-06-24. https://info.nhanow.com/hubfs/Test%20Plans/nha-2025-cpt-test-plan.pdf
- CLSI PRE02, Collection of Diagnostic Venous Blood Specimens, 8th ed. (product page). Clinical and Laboratory Standards Institute, 2025-02-13. https://clsi.org/shop/standards/pre02/
- The BD Vacutainer Blood Collection System: Order of draw for multiple tube collections (BD-47766, June 2023), copy hosted by a hospital laboratory test directory. Becton, Dickinson and Company, 2023-06. https://www.testmenu.com/lifespan/TestDirectory/SiteFile?fileName=sidebar\BD+Blood+Collection+Tubes+Order+of+Draw++2025.pdf
- WHO guidelines on drawing blood: best practices in phlebotomy. World Health Organization, 2010. https://iris.who.int/server/api/core/bitstreams/10682984-b7a0-4e4b-bd32-0cbc7d22136b/content
- Routine Venipuncture Guidelines (UF Health Pathology Laboratories procedure). University of Florida Health Pathology Laboratories, 2019-10. https://pathlabs.ufl.edu/wordpress/files/2019/10/uf-health-pathology-laboratories-venipuncture-procedure.pdf
- 29 CFR 1910.1030, Bloodborne pathogens (eCFR, current). Office of the Federal Register / eCFR, 2026-09-24. https://www.ecfr.gov/current/title-29/subtitle-B/chapter-XVII/part-1910/subpart-Z/section-1910.1030
- Vacutainer Evacuated Blood Collection System For In Vitro Diagnostic Use (BD package insert, document VDP40436, valid from 12-Aug-2021), as posted by FDA. Becton, Dickinson and Company / U.S. Food and Drug Administration, 2021-08-12. https://www.fda.gov/media/151687/download
- BD Vacutainer Venous Blood Collection Tube Guide (VS5229-13, 07/10), copy hosted by a health-system laboratory. Becton, Dickinson and Company, 2010-07. https://www.chihealth.com/content/dam/chihealthcom/documents/services/laboratory/bd-tube-guide.pdf
- NH3V - Overview: Ammonia, Plasma (Mayo Clinic Laboratories test catalog). 2026-09-24. https://www.mayocliniclabs.com/test-catalog/overview/35130
- New CLSI Venipuncture Guidelines (ACAreer newsletter, vol. 22 issue 2), summarizing CLSI GP41 7th ed. American Certification Agency for Healthcare Professionals, 2017. https://acacert.com/files/newsletter/ACAreerSummer_Fall2017.pdf