TABLE OF CONTENTS

• Vacutainer Tubes
• Venipuncture Procedure
• How to Obtain Serum, Plasma, Whole Blood
• Sources for Unacceptable Specimens
• Specimen Handling
  • Centrifugation of SST Tubes
  • Refrigeration
  • Frozen Specimens
• Microbiology Specimens
• Stool (Fecal) Specimens
• Urine Specimens (Culture, Urinalysis, Random, 24-Hour, Mid-stream)
• Test Result Inquiry
• Specimen Drop Box Use
• Minimizing Amount of Blood Collected
• Adult Critical Laboratory Values
• Reference Units of Measure

If you have any questions regarding specimen collection, contact RML Customer Service Department or ask to speak with the Technical Manager or Technical Director.

SPECIMEN COLLECTION

THE QUALITY OF RML TEST RESULTS DEPENDS, TO A LARGE EXTENT, ON THE QUALITY OF THE SPECIMEN WE RECEIVE. THEREFORE, PROPER PATIENT PREPARATION, SPECIMEN COLLECTION, PACKAGING AND TRANSPORTATION ARE OF VITAL IMPORTANCE.

Specific specimen requirements are listed in the Alphabetical Test Listing. The section for individual tests gives the type of tube with color of top and the specific volume of SERUM , PLASMA or WHOLE BLOOD needed. If you have any concerns about specimen requirements, please call our Customer Service Department for assistance.

VACUTAINER TUBES:

Guidelines for estimating the amount of serum or plasma needed — the amount of blood drawn should be approximately three (3) times the amount of sample specified under Specimen Requirements.

NOTE: To minimize the amount of blood drawn from the patient, several tests with the same specimen requirements may be run on one tube. If you have any questions concerning the sample volume required for multiple tests, please contact our Specimen Processing Department.

BLUE TOP (LIGHT BLUE) Sodium Citrate plasma: This tube contains Sodium Citrate as an anticoagulant. In order to prevent clotting, we recommend inverting the tube at least 6-12 times. Available tube sizes are 5 ml and 2 ml pediatric. Approximate specimen yield for a full 5 ml tube would be 1 1/2 ml plasma. For a full 2 ml tube, it would be ¼ ml plasma.

GREEN TOP Sodium Heparin or Lithium Heparin, heparinized whole blood or plasma: In order to prevent clotting, we recommend inverting the tube at least 6-12 times. Sizes available for either anticoagulants are 5 ml and 10 ml tubes. Approximate specimen yield for a full 5 ml tube would be 1 1/2 ml plasma. For a full 10 ml tube, it would be 3 ml plasma.

GRAY TOP (GLUCOSE TEST ONLY) Sodium Fluoride whole blood or plasma: This tube contains Sodium Fluoride as a preservative. In order to prevent clotting, we recommend inverting the tube at least 6-12 times. Size available is 5 ml tube. Approximate specimen yield for a full 5 ml tube would be 2 ml plasma.

LAVENDER TOP EDTA whole blood or plasma: This tube contains EDTA as an anticoagulant. Avoid exposure to extreme cold or hot temperatures. In order to prevent clotting, we recommend inverting the tube at least 6-12 times. Size available is 5 ml tube. Pediatric sizes available in 2 ml size and Capijects for finger stick collections. Approximate specimen yield for a full 5 ml tube would be 2 ml plasma. For a full 2 ml tube, it would be 3/4 ml plasma.

RED TOP (plain) serum or clotted blood: This tube contains no anticoagulant or preservative. Size available is 7 ml. Approximate specimen yield for a full 7 ml tube would be 2 1/2 ml serum. THIS TUBE SHOULD NOT BE MIXED BY INVERTING AFTER FILLING, as this may cause hemolysis.

RED TOP SST (serum separator tube): This tube contains no anticoagulant or preservative but contains an inert gel substance in the bottom and a clot activator. This tube is used for most tests requiring serum unless otherwise stated in the Alphabetical Test Listing. With proper centrifugation, the gel substance in the bottom of the tube will form an effective barrier between the clot and the serum. To enhance and accelerate the clotting process, invert the tube at least 6-12 times immediately upon filling. The specimen should then be allowed to clot for 15 minutes in an upright position and centrifuged for 20 minutes. Serum may be sent in tube with good intact barrier. For tests requiring a frozen sample, pour serum into plastic transfer tube and freeze. DO NOT attempt to freeze serum in SST tube. Size available is 10 ml tube. Approximate specimen yield for a full 10 ml tube would be 3 ml serum.

VENIPUNCTURE PROCEDURE

1. PREPARATION.
   1. Correctly identify and reassure the patient. Specimens collected on the wrong patient are meaningless.
   2. Determine test(s) ordered and the specimen requirements (refer to Alphabetical Test Listing in RML Manual).
   3. Always draw blood for tests requiring serum before drawing blood in anticoagulants.
   4. Assemble within easy reach the proper tubes and blood drawing supplies required to obtain the blood specimen.
   5. Wash hands and put on a new pair of disposable gloves.
      
2. LOCATION OF OPTIMUM VENIPUNCTURE SITE.
   1. Apply tourniquet or blood pressure cuff. Check both arms.
   2. Antecubital fossa area is usual site of choice. This is the inside elbow area at the bend of the arm, approximately the size of a quarter.
   3. Palpate vein to determine — 1) size of vein, 2) depth of vein, 3) direction of vein.
   4. When best venipuncture site is determined, release tourniquet or blood pressure cuff.
      
3. VENIPUNCTURE SITE PREPARATION.
   1. Pump up blood pressure cuff between systolic and diastolic. (Tourniquet is routinely used.)
   2. Re-palpate vein.
   3. DO NOT HAVE PATIENT PUMP OR CLENCH FIST.
   4. Cleans venipuncture site by using alcohol prep or cotton soaked in alcohol. Cleanse in a circular motion, moving clockwise away from site. Allow to air dry.
      
4. VENIPUNCTURE (see diagrams next page).
   1. Syringe and needle technique.
      1. Check syringe and needle for sterility seal.
      2. Secure needle to syringe (bevel up).
      3. Remove needle cover. Visually inspect needle using 360 degree rotation, checking for burrs or imperfections. Discard all inferior needles.
      4. Holding syringe at approximately a 30 degree angle with steady, smooth, deliberate motion, perform venipuncture.
      5. Successful venipunctures are usually visible by blood flow back in the hub of the needle. Draw back syringe plunger gently and slowly (allowing blood to flow 1-2 mm. behind plunger).
         
         PLEASE NOTE HANDS MUST BE GLOVED.
         
      6. Release tourniquet or blood pressure cuff after specimens are obtained.
      7. Place clean gauze or cotton ball gently over puncture site and simultaneously remove needle from arm.
      8. Apply pressure over venipuncture site.
      9. Apply bandage over folded gauze.
      10. Make Hematology slides from end of needle if needed.
      11. Needle disposal - ATTENTION: Needles MUST NOT be manipulated by hand, re-capped, bent or CLIPPED prior to disposal but must be DISCARDED DIRECTLY into an approved puncture-resistant container (i.e., "sharps").
          
   2. Vacutainer system.
      1. Follow patient preparation as with syringe technique.
      2. With clockwise motion, thread needle into holder (should be snug but not over-tightened).
      3. Insert correct tube into the tube holder.
      4. Remove needle cover. With bevel up, perform venipuncture.
      5. Push tube to end of holder, puncturing diaphragm of stopper.
      6. At this point, blood should be visible in tube.
      7. When desired amount of blood is obtained, release tourniquet or blood pressure cuff, remove needle from arm and apply pressure.
      8. Apply bandage over folded gauze.
      9. Immediately following blood collection, the blood tubes must be properly labeled.
      10. Needle disposal (see A.11 section above).
          
          NOTE: Vacutainer tubes should be filled in the following order: fill non-additive tubes first, followed in order by blue top, SST, green top, lavender top, grey top and yellow top.
          
5. SPECIMEN LABELING AND HANDLING.
   1. Using gummed labels provided on Test Request slip, be sure all blood tubes are labeled with proper patient numbers. NOTE: Specimen submitted for prenatal screening, blood typing and antibody screening must be labeled with the patient's full name and date collected. A separate outpatient identification form must be completed and submitted for Blood Bank specimens when a transfusion is anticipated.
   2. The top hard copy of the Test Request slip and the labeled specimens are placed in a ziplock bag and refrigerated (unless otherwise specified in the manual) until picked up by RML courier.

HOW TO OBTAIN SERUM, PLASMA, WHOLE BLOOD

1. SERUM. Fluid portion that is expressed by the clotted blood.
   
   SST (red top): Draw the specimen, mix the clot activator and blood by inverting the tube at least 6-12 times and let clot for at least 15 minutes in a vertical position. Spin for 20 minutes in your centrifuge, place labeled sample in ziplock bag with the Test Request slip and refrigerate.
   
   Plain red top: Draw the specimen, DO NOT MIX, let clot, place labeled sample in ziplock bag with Test Request slip and refrigerate.

   NOTE: When a patient is drawn by syringe, it is recommended that the red top vacutainer be filled in the following manner to avoid HEMOLYSIS:

   1. Remove top of tube.
   2. Remove needle from syringe.
   3. SLOWLY dispense the blood down the side of the tube. Be careful not to over-fill the tube.
   4. Replace top of tube SECURELY and allow to clot.
   5. Vent tube with needle to equalize pressure.

   NOTE: NEVER FREEZE SERUM IN THE COLLECTION TUBE BUT ALWAYS TRANSFER TO A PLASTIC TRANSFER TUBE, THEN FREEZE THE SERUM.

2. WHOLE BLOOD. Blood collected in tubes containing anticoagulants. Collect the blood in the appropriate tube as specified in the Alphabetical Test Listing under Specimen Requirement. Mix IMMEDIATELY, inverting gently at least 6-12 times, label and refrigerate. NEVER FREEZE WHOLE BLOOD.
   
3. PLASMA. Fluid portion that is derived from the anticoagulated blood and contains fibrinogen and other clotting factors when separated from cells. Draw the appropriate colored top tube. Mix immediately by gently inverting at least 6-12 times, then centrifuge for twenty minutes. Use a glass pipette, transfer the upper fluid portion or plasma to a plastic transfer tube. PLASMA SHOULD NOT CONTAIN ANY RED CELLS NOR SHOW HEMOLYSIS.

SOURCES FOR UNACCEPTABLE SPECIMENS

1. Contaminated Sample: Contaminated SAMPLE or LEAKAGE due to improper packaging. Examples would include fluid samples with untightened caps and blood tubes whose rubber caps have been taken off and then not vented to equalize pressure when caps were replaced, etc.
   
2. Hemolysis: This occurs when the red cell membrane is broken and allows hemoglobin and other intracellular components to escape into the plasma or serum.
   
   Hemolysis usually is the result of inadequate drawing technique or improper handling after specimen is drawn. Forcing blood through a needle on a syringe into a vacutainer tube will cause hemolysis.
   
3. Turbidity or Lipemia: Lipemic sera may be produced by the presence of fatty substances such as lipids in the blood. It is recommended that patients fast 12 hours for most routine chemistries and 14 hours for Lipid Panel studies.

SPECIMEN HANDLING

CENTRIFUGATION OF SST TUBES:

1. Be sure SST tubes are allowed to clot for AT LEAST 15 MINUTES in an upright position.
2. Before placing in centrifuge, make sure specimens are balanced with like tube and volume (i.e., within 1 mm. of blood level) and placed opposite each other (balance tube filled with water).
3. Spin a minimum of 20 MINUTES (a full head of four full tubes may require longer). Spinning longer will not harm specimens.
4. Upon completion of spinning, make sure the insert gel material is packed over the cells in an even layer. If any material appears to remain in the bottom or along the side of tube, RE-SPIN until NO material is visible but is evenly packed over the cells.
5. Package the centrifuged tube in a ziplock bag with the requisition slip and refrigerate.

REFRIGERATION:

Unless otherwise stated under Specimen Requirement, specimens of clotted blood (plain red top), whole blood (lavender, blue, green tops), serum (SST tubes), plasma, urine, sputum and stools are refrigerated.

FROZEN SPECIMENS:

Freeze SERUM or PLASMA specimens only if it is indicated under the Alphabetical Test Listing.

When indicated, freeze SERUM or PLASMA in the following manner:

1. Freeze as soon as the serum or plasma is separated from the cells.
2. ALWAYS freeze in plastic transfer tubes unless otherwise specified.
3. Freeze the tube overnight before shipping to insure solid freeze. Partial freezing and thawing will deteriorate specimen and could cause specimen to be rejected.

MICROBIOLOGY SPECIMENS:

Refer to Microbiology Section of RML Manual for all specimen requirements.

STOOL (FECAL) SPECIMENS:

Requirements are stated under the individual test in the Alphabetical Test Listing.

URINE SPECIMENS:

NOTE: All urine samples need to be kept refrigerated.

Urine culture specimen: Specimen should be collected in sterile container. The specimen must be collected by clean catch mid-stream procedure (see below). Refer to Microbiology Section of manual.

Urinalysis specimen: Specimen should be collected in a clean container. It is recommended that specimen be collected by mid-stream procedure (see below).

Random urine: First morning specimen is requested as it usually has a specific gravity greater than 1.010 and usually is acid in pH, which helps preserve the formed elements. It is recommended that specimen be collected by mid-stream procedure (see below). Sample should be collected in clean container.

MID-STREAM COLLECTION PROCEDURE:

1. Instruct patient to void a small portion of urine into the toilet.
2. Instruct patient to void remaining urine into the urine container.
3. Cap securely.

24 HOUR URINE:

A 24 hour urine collection is required for quantitative analysis. Some 24 hour collections require a preservative, such as Boric Acid or 6N HCL. A 24 hour urine collection container with or without preservative can be obtain through the RML Supply Department.

24 HOUR URINE COLLECTION PROTOCOL:

1. Empty bladder completely on awakening in the morning and discard this urine specimen.
2. Record the time the discarded specimen was voided and begin the test.
   
   (Example: Month/Day/Year/Time started).
   
3. All urine passed during the rest of the day and night must be poured into the container.
4. Include the first urine specimen of the next morning and record the final time and date. (Example: Month/Day/Year/Time finished [24 hours after the time started]).

NOTE: If possible, store container in the refrigerator during collection. Be certain patient understands cautions regarding handling container and the preservative provided. Container containing any caustic preservative must be labeled with appropriate warning.

When contacting RML Customer Service Department for test results or regarding a concern for a specimen already submitted, please state the following:

1. Test Request ID number.
2. Patient's full name.
3. Date specimen submitted.
4. Test(s) ordered.
5. Requesting physician.

SPECIMEN DROP BOX USE

The RML drop box is provided for your convenience to leave laboratory specimens for pickup after your office closes.

Please keep the boxes locked at all times. Provided with each drop box is a key, a hot bottle and a cold pack.

WHEN OUTSIDE TEMPERATURE IS GREATER THAN 60ºF: Take the cold pack from freezer. Wrap cold pack in paper toweling and place in box. Wrapping will prevent the specimen from resting directly on the cold pack.

WHEN OUTSIDE TEMPERATURE IS LESS THAN 45ºF: Place HOT tap water in the bottle, wrap it in paper toweling and place in drop box.

PLACING SPECIMENS IN BOX:

Keep CULTURES farthest away from cold pack and close to hot bottle. Lock box.

REMEMBER: The next day, open lock box, look for reports and courier service record slip.

Minimizing Amount of Blood Collected

It is important to note that we recommend collecting adequate amounts of specimen to allow the tests ordered to be processed efficiently. Even though we list the minimum specimen amount, these are generally more than adequate to do the test and repeat if necessary. Multiple serum tests can be performed on one SST tube. If you have a difficult draw or patient who will have blood collected repeatedly, please contact our Technical Managers for advice on how much specimen can suffice to do the tests.

ADULT CRITICAL LABORATORY VALUES

Laboratory values that fall beyond the following limits are considered critical and the facility submitting the specimen will be notified immediately by phone of any critical values. The person receiving these critical values is responsible for notifying the physician according to their established procedures. Please note that this notification is designed to be a backup measure for either hard copy reports or on-line viewing. It is possible that hard copy or on-line results could be available prior to receiving a telephone notification.

Analyte                           Less than (<) Greater than(>)
Bilirubin (infant)                -----------   18 mg/dl
Calcium                           6 mg/dl       12 mg/dl
Carbon Monoxide                   -----------   20%
CO2                               10 mEq/L      45 mEq/L
Glucose                           40 mg/dl      400 mg/dl
Glucose, CSF                      35 mg/dl      400 mg/dl
Lead, Blood                       -----------   40 mcg/dl
Magnesium                         1.0 mg/dl     5.0 mg/dl
Phosphorus                        1.0 mg/dl     9.0 mg/dl
Potassium                         2.5 mEq/L     6.5 mEq/L
Sodium                            120 mg/dl     160 mg/dl

Troponin                                        0.49 ng/mL

CK-MB                                           3.9 ng/mL

Acetaminophen                     -----------   150 mcg/ml
Alcohol                           -----------   0.4%
Digoxin                           -----------   2.5ng/ml
Gentamicin Peak                   -----------   12.0 mcg/ml
Gentamicin Trough                 -----------   2.0 mcg/ml
Lithium                           -----------   1.7 mEq/L
Phenobarbital                     -----------   50.0 mcg/ml
Phenytoin (Dilantin)              -----------   30.0 mcg/ml
Salicylate                        -----------   30.0 mg/dl
Tegretol (Carbamazepine)          -----------   15.0 mcg/ml
Theophylline                      -----------   25.0 mcg/ml
Tobramycin Peak                   -----------   12.0 mcg/ml
Tobramycin Trough                 -----------   2.0 mcg/ml
Valproic Acid (Depakane)          -----------   150.0 mcg/ml
Vancomycin Peak                   -----------   40.0 mcg/ml
Vancomycin Trough                 -----------   20.0 mcg/ml

Hemoglobin                       6.0 gm/dl      -----------
Platelet                         30,000 cu.mm   -----------

Fibrinogen                       75 mg/dl       -----------
INR                               -----------   5.0
Prothrombin Time                  -----------   28.0 seconds
Partial Thromboplastin Time (PTT) -----------   100 seconds
PTT (Heparin Therapy)             -----------   150 seconds

Positive findings on the following: Blood Culture;Blood Parasite Films; C. difficile; CSF smear or culture; Influenza A/B; New MRSA (BCHS, HGB Patients); New VRE (BCHS,HGB Patients); Pseudomonas or Gonorrhoeae in eye; RSV; Salmonella, Shigella or Campylobacter; Shiga Toxin 1 or 2; Systemic fungus culture; Positive AFB(CF+)on any culture or smear; Vibrio; or Yersinia. 

Positive ketones or sugars in neonatal patients.

Note: In addition to the above, all results received from reference laboratories designated critical will be called to the ordering account.

REFERENCE UNITS OF MEASURE

UNITS OF MEASURE

L:      Liter (1,000 ml)
dl:     Deciliter (100 ml)
ml:     Milliliter (.001 ml)
ul:     Microliter (.000001 ml)
kg:     Kilogram (1,000 gm)
g:      Gram
mg:     Milligram (.001 gm)
mcg:    Microgram (.000001 gm)
ng:     Nanogram
pg:     Picogram
mm:     Millimeter
u:      Micron
mEq:    Milliequivalent
mOsm:   Milliosmole
fmole:  Femtomole
mM:     Millimole
U:      Unit
mU:     Millliunit
uU:     Microunit
IU:     International Unit
mIU:    Milli-international Unit

UNITS OF TIME

hr:     Hour
min:    Minutes
sec:    Seconds

QUANTITATIONS

<:      Less than
>:      Greater than

METHODOLOGY

AA:     Atomic Absorption
CAE:    Cellulose Acetate Electrophoresis
EIA:    Enzyme Immunoassay
GLC:    Gas-Liquid Chromatography
HPLC:   High Pressure Liquid Chromatography
IEP:    Immunoelectrophoresis
IFA:    Indirect Fluorescent Antibody
NIA:    Nephelometric Immunoassay

OIA:    Optical Immunoassay
PCR:    Polymerase Chain Reaction
RIA:    Radioimmunoassay
TLC:    Thin Layer Chromatography