1. Vacuum Blood Collection
Terry Kotrla, MS, MT(ASCP)BB
Revised April 13, 2018
Revised with permission August 2018 by Lois Wagoner, MT(ASCP)

2. Introduction
The vacuum blood collection system consists of a double-pointed needle, a plastic holder or adapter, and a series of vacuum tubes with rubber stoppers of various colors.
The evacuated tube collection system will produce the best blood samples for analysis.
The blood goes directly from the patient vein into the appropriate test tube.

3. Multi-Sample Needle
The bevel is the slanted opening at the end of the needle.
Needle length (shaft) ranges from 1 to 1 ½ inches.
Threaded hub screws into needle holder
The rubber sheath makes it possible to draw several tubes of blood by preventing leakage of blood as tubes are changed.

4. Bevel Bevel is slanted opening at end of needle.
Needle must be oriented so that bevel faces up prior to insertion.

5. Needle Gauge
The gauge of a needle is a number that indicates the diameter of its lumen.
The lumen, also called the bore, is the circular hollow space inside the needle.
The higher the gauge, the smaller the lumen.
The most frequently used gauges for phlebotomy are 20, 21 and 22

6. Holder
The holder for vacuum blood collection is a plastic sleeve into which the phlebotomist screws the double pointed needle.
The most current guidelines require that all holders are for single use only.

7. Vacuum Collection Tubes
Vacuum collection tubes are sealed with a partial vacuum inside by rubber stoppers.
The air pressure inside the tube is negative, less than the normal environment, which creates the vacuum in the tube.
After inserting the longer needle into the vein, the phlebotomist pushes the tube into the holder so that the shorter needle pierces the stopper.
The difference in pressure between the inside of the tube and the vein pulls the blood into the tube, which allows the tube to fill with blood.
The tubes are available in various sizes for adult and pediatric phlebotomies
Plastic tubes have replaced most glass tubes for safety reasons

8. Additives Any substance added to the tube by the manufacturer
Found in almost all blood collection tubes used today
Are the precise amount needed for the amount of blood that will fill the tube
Important to fill the tube so the ratio of additive to blood is correct; failure to fill the tube can lead to inaccurate results or rejected specimens
Additive tubes must be mixed by gentle inversion
The color of the tube stopper indicates what additive, if any, the tube contains

9. Types of Additives
Anticoagulant - Most common additive, prevents clotting of the blood by binding or inactivating an element necessary for clotting to occur
Clot Activator – In most plastic tubes that produce serum; helps the clotting process start and go to completion
Glycolytic Inhibitor – Prevents the use of glucose by blood cells; Sodium Fluoride in gray stopper tubes most common
Polymer Gel – Will form a physical barrier between the cells and the liquid portion of the sample; found on tubes for both serum (red and gold) and plasma (green)

10. Blood Cultures
Is a special collection to detect bacteria growing in the blood.
Blood is usually placed in Blood Culture Bottles at the patient’s side, but may be collected into a Yellow stopper tube containing Sodium Polyanethol Sulfate (SPS)
Site preparation VERY important.
Will be covered later in a separate lab activity.

11. (Light) Blue Stopper Anticoagulant: Sodium Citrate
Ratio of blood to anticoagulant is 9:1 – Critical to accurate test results
MUST BE FILLED COMPLETELY!!! NO EXCEPTIONS
Tests include Coagulation studies such as PT, PTT and fibrinogen

12. Red Stopper Clot activator in plastic tube
Tests using serum for Chemistry and Serology
Must allow to clot for 30 minutes prior to centrifugation, do not exceed 60 minutes
No additive in glass tube (glass tubes no longer common)
Previously used for Blood Bank testing using serum, has been replaced with Pink tubes for Blood Bank testing using plasma

13. Serum Separator Tubes (SST)
SST = Serum Separator Tube
Can be red/black mottled, gold or red with black stopper.
Gel forms physical barrier during centrifugation that separates serum from cells permanently
All tests using serum except Blood Bank

14. Green Stopper One of the following formulations:
Sodium Heparin
Lithium Heparin
Ammonium Heparin (rarely seen in the Austin area)
Used for Chemistry test utilizing plasma
Often used for STAT Chemistry tests

15. Green Plasma Separator Tube
PST = Plasma Separator Tube
Contains a Heparin anticoagulant, so it can be centrifuged immediately
Gel forms physical barrier during centrifugation, permanently separates plasma from red blood cells

16. Lavender Stopper
Contains the anticoagulant EDTA (ethylenediaminetetraacetic acid)
Used for Hematology studies: CBC, WBC count, Hemoglobin, Hematocrit, Platelet count, Reticulocyte count, Differential
May be used for Erythrocyte Sedimentation Rate (ESR)

17. Pink Stopper
Intended use is for blood bank testing using the gel system, which requires plasma
Anticoagulant is EDTA (ethylenediaminetetraacetic acid)

18. Gray Stopper Additive – always contains Sodium Fluoride
Sodium Fluoride is a preservative that prevents glycolysis
May also contain an anticoagulant
Sodium Fluoride and Potassium oxalate (plasma sample)
Sodium Fluoride and EDTA (plasma sample)
Sodium fluoride (serum sample)
Test include: Glucose, Blood Alcohol (ethanol) levels, Lactic Acid

19. Order of the Draw
It is critical that the order of the draw is followed to minimize the effect of carryover
Carryover of additive from one tube to the next can adversely affect test results
Each lab should document it’s order of the draw
The Clinical and Laboratory Standard’s Institute (CLSI) has developed an order of the draw that all phlebotomist should know
The order of the draw does not eliminate carry over, but is designed so that test results are not affected by it

20. CLSI recommended Order of the Draw
Sterile/Blood cultures or Yellow SPS tube
Coagulation tube (Sodium Citrate) Light Blue
Serum tube (with or without gel) Red, Gold, Mottled
Other additives
Heparin (with or without gel) Green
EDTA - Lavender/Pink
Glycolytic Inhibition tube - Gray
Sodium fluoride & potassium oxalate or
Sodium fluoride & sodium EDTA or
Sodium fluoride only

21. Specialty Tubes The following tubes are used less frequently
The phlebotomist needs to be aware of the additive and uses of any tube used by their facility
The order in which these tubes are drawn is usually based on the additive in the tube, but may vary from lab to lab
Some tubes may require a separate blood draw – follow facility policy
It is the phlebotomist responsibility to learn about the correct placement in the order of the draw of all tubes used in their facility

22. Black Stopper Buffered Sodium Citrate
Only used for Westergren sedimentation rate determination
MUST BE FILLED COMPLETELY!!! NO EXCEPTIONS!!!

23. Royal Blue Stopper
Stoppers in standard collection tubes contain several elements that can contaminate a specimen and interfere with testing
Color of tube label indicates additive, if any:
purple - EDTA
green - heparin
red – none
Order of the draw usually determined by additive present.
Used for
Trace elements – examples: Manganese, Chromium
Nutrients – examples: Zinc, Copper
Toxicology – examples: Lead, Arsenic, Cadmium

24. Tan Stopper Anticoagulant: K2EDTA Specifically for lead analysis
Has been replaced at most labs with Royal Blue with purple label (EDTA)

25. Yellow Stopper Sodium polyanethol sulfonate (SPS)
SPS used for blood culture specimen collections in microbiology.
Acid citrate dextrose additives (ACD)
ACD used for tissue testing, HLA phenotyping, genetic testing, DNA and paternity testing

26. Blood Composition
As blood flows through the body it is made up of plasma and cellular elements.
The cellular elements include red blood cells, white blood cells and platelets.
Plasma in the body is composed of water and dissolved substances, such as proteins, nutrients, carbohydrates, lipids, minerals, gases, vitamins, hormones, antibodies, fibrinogen and waste products.
The lab tests for these different substances and cellular elements require different types of blood specimens.
The 3 types of blood specimens collected by venipuncture are serum, plasma, and whole blood.

27. Types of Blood Specimens: Serum
Serum is the liquid portion of a blood specimen that has been allowed to clot
Serum samples must be allowed to sit upright for at least 30 minutes to allow the clotting process to occur and reach completion
The sample must then be spun down, which allows the serum to rise to the top of the sample as the clot moves to the bottom
Serum does not contain any coagulation factors
Many chemistry and serology test are preformed on serum
The most common tubes that produce serum include red, gold, red/black mottled and tiger-top tubes
Other tubes that produce serum but are not used as often include royal blue with a red label and grey with only sodium fluoride

28. Types of Blood Specimens: Plasma
Plasma is the clear liquid portion of an anticoagulated blood specimen
Anticoagulated specimens must be properly inverted immediately after drawing so the anticoagulant is evenly dispersed in the sample.
Anticoagulated samples may be spun down as soon as they arrive in the lab. This makes them an excellent choice for STAT lab test.
Plasma contains all of the coagulation factors except one. Different anticoagulants inhibit or interfere with different coagulation factors.
Plasma is used for a variety of test, including coagulation studies, stat chemistries, and blood banking.
The most common tubes that yield plasma are light blue, green, pink and grey stopped tubes which include an anticoagulant.
Other tubes that produce plasma include royal blue with a green or lavender label.

29. Types of Blood Specimens: Whole Blood
Whole Blood is an anticoagulated specimen in which the cellular elements remain suspended in the plasma for testing purposes.
Whole Blood samples are never centrifuged; they are often placed on a mechanical rocker to keep the cellular elements suspended until testing can be preformed.
Whole blood samples are used for Hematology test, Blood cultures, Genetic and Tissue testing.
The most commonly drawn tube for whole blood testing is the lavender topped tube used for Hematology test.
Other whole blood samples are collected in yellow (both SPS and ADC), black, tan, and royal blue with a purple label.

30. Patient Identification
It is vitally important that the phlebotomist correctly identifies the patient.
Do not offer the patient a name to respond to. Always ask the patient to state their name.
All hospitalized patients have an identification arm band with their name, hospital identification number and other pertinent information.
Always compare the laboratory test request slip name, date of birth and ID number with the name, date of birth and ID number on the patient's hospital arm band AND with the name and date of birth the patient states for you.
If there is any discrepancy, do not draw the patient's blood.
For an out-patient – site specific protocols must be followed which may include:
Verify the patient's identity by having the patient give you additional identifying information such as a unique ID number, date of birth or address.
Patient may be asked to review and initial label.

31. Preparation Wash or disinfect your hands and put on gloves
Introduce yourself, state your mission
Properly identify the patient
Ask the patient if they have had any problems during previous blood draws
If they have had previous problems, discuss these further to help you prepare an appropriate plan for the patient
If the patient has not had a previous blood draw, explain the procedure to them
Choose the appropriate tubes for the tests requested

32. Tourniquet Application
Apply approximately 3-5 inches above antecubital fossa.
If the skin appears blanched above and below the tourniquet it is too tight.
If your finger can be inserted between the tourniquet and the patient's skin it is too loose.

33. Palpate After tourniquet application have patient clench fist.
Feel for a vein that rebounds (bounces) when pushed or tapped on.
PALPATE potential veins to help determine the following attributes:
size
direction
depth
A slight rotation of the arm or wrist may help to better expose a vein that may otherwise be hidden.
Use your index finger and middle finger to palpate.
Never use your thumb to palpate a vein.

34. Vein Selection Choose the vein that is large and accessible.
Choose the vein that feels the best, which may or may not be the most visible.
Avoid bruised and scarred areas.
Avoid veins which are sclerosed or occluded.
Palpate the vein above and the below the chosen venipuncture site to note the depth, diameter and direction of the vein

35. Can’t Feel the Vein? Tricks to Help Distend Veins:
Make sure the tourniquet is not loose
Rotate the patient’s wrist
Ask the patient to make a fist.
Do not allow the patient to pump their fist, this can lead to erroneous test results.
Hand the patient a couple of plastic tubes to grasp, as this is sometimes easier for the patient.
Have the patient dangle arm below the heart level for 1-3 minutes.
Warm the area with a hot pack or warm, moist cloth heated to approximately 42°C.
Remember to not leave the tourniquet on for more than 1 minute.
If you are unable to locate a usable vein consult an experienced phlebotomist for assistance and guidance.

36. Veins used for drawing blood
Median cubital vein - first choice, well supported, least apt to roll
Cephalic vein - second choice, on the thumb side of arm
Basilic vein - third choice, on the picky finger side of arm; lies close to the Basilic artery and Brachial nerve; often the most prominent vein, but it tends to roll easily and makes venipuncture difficult

37. Median Cubital – First Choice
This vein is located in the antecubital fossa (the area of the arm in front of the elbow)
Well anchored vein, usually large and prominent.
Very few problems. Offering the best chance for a close to painless puncture, as there are few nerve endings close to this vein.

38. Vein Selection

39. Cephalic Vein-Second Choice
Cephalic vein which is located on the upper or shoulder side of the arm.
This vein is usually well anchored.
The cephalic vein may lie close to the surface. A low angle of needle insertion must be used to avoid possible spurting or blood forming a drop at the puncture site. (15°)

40. Veins for Venipuncture
These are NOT listed in the order of preference but illustrates the usual position of the veins.

41. Basilic Vein-Third Choice
Located on the under side of the arm (pinky side).
In many patients this vein may not be well anchored and will roll, making it difficult to access with the needle.
Syringe draw should be considered as it gives the phlebotomist more control over a rolling vein.
Pooling of blood and hematoma formation possible.
Exercise caution when drawing from this area.
The basilic vein is close to the brachial artery so there is more risk of hitting an artery.
The basilic vein lies close to the brachial nerve which may result in injury to the nerve.
This area is often more sensitive, thus may be slightly more painful for the patient

42. Cleansing the Site After selecting a vein, remove the tourniquet.
Clean the selected site with a prepackage 70% isopropyl alcohol swab.
CLSI standards state that vigorous back and forth friction with an alcohol pad is superior to circular concentric cleansing.
Use enough pressure to remove all perspiration and dirt from the puncture site.
Discreetly look at the swab when finished; if it appears excessively dirty repeat the cleansing process with a fresh alcohol swab.
After cleansing do not touch the site, if the vein must be repalpated the area must be cleansed again.

43. Assemble Equipment
After cleaning the site, assemble the equipment. This will allow the site time to dry.
Twist needle into holder.
Select appropriate tubes; have them within easy reach of the proposed venipuncture site.
Reapply the tourniquet
DO NOT remove cap until right before you are ready to draw.

44. Re-Apply Tourniquet and Prepare to Draw

45. Anchoring the Vein
Using your non-dominant hand, position your thumb below the intended puncture site and pull the skin down and away from the intended site until the skin is taut
You may have to pull slightly off-center so that your thumb is not in the way of the holder
Use the rest of your fingers of your non-dominant hand to hold the patient’s arm steady
By pulling the skin taut, the puncture is more likely to be less painful to the patient
Large veins that are not well anchored in tissue frequently roll. Use extra care to anchor these veins for a successful venipuncture.

46. Performing the Draw
Make sure the tourniquet has been properly reapplied.
Uncap the needle using your non-dominant hand
Briefly inspect the needle for defects
Hold the prepared holder with the bevel of the needle facing up.
Use the thumb of the non-dominant hand below the puncture site to anchor the vein and pull the skin taut.

47. Performing the Draw (continued)
The needle entering the site should not touch the thumb of the phlebotomist.
Position the needle in the same direction as the vein, enter the skin and penetrate the vein at a degree angle in one swift, smooth motion to decrease the patient's discomfort.
If you enter too slowly
Blood may leak out at the puncture site creating a biological hazard
Your view of the puncture site may be obstructed by the pool of blood
The patient usually feels more pain and discomfort from the needle entry

48. Performing the Draw (continued)
The bevel of the needle should enter and remain in the center of the vein. You may feel a slight “give” or decrease of resistance when you are in the vein.
Using your non-dominant hand, carefully push the first tube onto the back of the needle in the holder using counter pressure; allow the first tube to fill.
Stabilize the needle and use counter pressure when putting tubes on or off the needle to insure that the needle does not move.
Change tubes as needed until all tubes have been filled.
Remove the tourniquet when your last tube is filling or within one minute of application, which ever comes first

49. Performing the Draw

50. Collecting the Sample

51. Ending Draw - Release Tourniquet
Tourniquet cannot be in place more than 1 minute.
Release the tourniquet as the last tube is filling.
Use one handed method of release.

52. Ending Draw -TTN Release Tourniquet Release last Tube from needle
Hold gauze over puncture site without applying pressure to the needle
Swiftly withdraw Needle
As soon as needle is withdrawn
Apply pressure to puncture site with your non-dominant hand
Immediately activate the needle safety device according to the manufacture’s directions and discard into sharps container
Ask patient to continue to apply pressure.

53. Ending the Draw TTN

54. Activating Safety Device
NEVER take your eyes off the needle until the safety device is activated.
CLSI standards requires the safety device to be activated with just ONE hand. Follow manufacturers directions on activation.
DO NOT USE YOUR OTHER HAND TO SNAP DEVICE INTO PLACE…EVER!
Carefully place activated device into Sharps.

55. Portex Needle Pro
The Portex Needle Pro requires a hard surface activation.
Do NOT activate with your thumb or other hand.

56. BD Eclipse
The BD Vacutainer® Eclipse™ Blood Collection Needle is a safety-engineered multi-sample blood collection needle.
It features a patented safety shield that allows for one-handed activation to cover the needle immediately upon withdrawal from the vein and confirms proper activation with an audible click
Look CLOSELY at the position of the thumb. DO NOT go higher with your finger as this may lead to a needle stick injury.

57. Needle Disposal
As soon as needle safety device is activated immediately dispose of entire assembly in a biohazard sharps container.
CAUTION: Never attempt to shove device into a full sharps container. This may lead to a needle stick injury if your finger slips inside the holder and your finger may be pierced by back end of needle.

58. Mixing and Labeling Tubes
While patient holds pressure on puncture site, gently invert all tubes multiple times to insure proper mixing of the blood and additive
Label all tubes appropriately at the patient’s side.
NEVER take unlabeled tubes from the patient’s presence.
Minimum information:
Patient’s full name, last name first
ID number and/or Date of Birth
Date & time of collection and your initials
Label the tubes from the stopper downward

59. Checking Site – The two-step check
Step One
Have patient apply pressure to site for 3-5 minutes while you invert and label tubes
Sometimes it is helpful to have patient elevate arm while applying pressure, this slows blood flow to the area.
Step Two
CLSI recommends the phlebotomist directly observe the puncture site for 5 – 10 seconds before bandaging
Check for both hematoma formation and superficial bleeding
If the site is still bleeding, phlebotomist must continue to apply pressure, rechecking site at one minute intervals

60. Post-venipuncture Care
Once bleeding has stopped place bandage or Co-flex over site.
Some patients are allergic to bandages
Co-flex is wrapped over gauze and around the arm
It sticks to itself and provides slight compression
Tell patient to remove in minutes
Caution patient about exerting arm or putting pressure on the site for several hours
Thank patient – allow out patient to leave
Discard used materials in appropriate trash receptacle
Disinfect work area
Perform proper hand hygiene

61. Leaving Discard all used materials Thank patient.
hint- place all wrappers, alcohol swab, needle cap in palm of gloved hand, remove glove.
Thank patient.
Wash or sanitize hands.
Leave

62. Problems with Needle Insertion
Swelling at site, hematoma, immediately withdraw needle.
Bevel against vein wall.
Collapsed vein.
Needle not in vein, move forward.

63. Problems with Needle Insertion
Needle inserted too far, back up.
Needle inserted into artery, IMMEDIATELY withdraw needle.

64. Sources of Error
Failure to insert the needle completely into the vein.
Puncturing the stopper before entering the vein.
Not anchoring the vein before inserting the needle. The vein must be held in place for successful needle penetration.
Not keeping the holder stationary during tube change – use counterpressure when changing tubes.
Retouching the site prior to inserting the needle.
"Bouncing" the needle on the skin before guiding it into the vein.

65. Rejection of Samples - Hemolysis
Hemolysis – caused by rupture of red cells, usually due to drawing technique, affects many lab test, must be recollected

66. Rejection of Samples - Clotted
Tubes with anticoagulant clot due to inadequate tube inversions.
Tube on right was not inverted properly, sample clotted.
Rube on left was inverted properly.

67. Rejection of Samples
Insufficient sample – Quantity Not Sufficient (QNS)
May be due to
Underfilling tubes which must be completely filled (light blue or black stopper tubes)
More sample is needed for testing than was submitted
Wrong tube collected for test ordered.
Improper storage - certain tests must be collected and placed in ice, protected from light or be kept warm after collection.
Improperly labeled - Failure to correctly label a sample will result in the sample being rejected.

68. Contaminated or Diluted Specimens
These samples were collected from the same patient.
Sample on right was drawn above an IV
Note the pale color of the serum and the ratio of red cells to serum.
Sample on left was collected properly

69. First Aid Following Needle Stick Injury
Be careful not to stick yourself with a used needle.
If an accidental stick does occur - immediately
Go to the sink, turn on the water, and bleed the site well by alternating squeezing and releasing the area around the site.
Do this for approximately 3 to 5 minutes.
Afterwards scrub the site with an alcohol swab.
Follow with a thorough hand washing.
Report it to your instructor immediately.

70. The End
Revised August 29, 2013
Revised with permission by Lois Wagoner, MT (ASCP) August 2018