Specimen Collection and Processing

GIGO

Garbage In, Garbage Out — a sophisticated analyzer cannot compensate for a poor-quality specimen.

Specimen quality

The quality of every laboratory result depends primarily on the quality of the specimen submitted for analysis.

Total Testing Process

The ten-step chain in which errors can occur at every step: physician orders test → patient preparation → specimen collection → labeling → transport → specimen processing → laboratory analysis → result verification → reporting → clinical interpretation.

Underfilled tube

A short draw, so the blood-to-additive ratio is wrong. Affects RBC morphology and lipids in EDTA tubes, and the binding of electrolytes. A short draw is also a stated reason for specimen rejection.

Re-spinning a gel tube

Causes increased potassium.

Hemoconcentration

Caused by a prolonged tourniquet; listed both as a common error in specimen collection and as an immediate local complication of venipuncture.

Delayed separation

Glycolysis consumes glucose, unstable substances are lost, electrolytes shift between cells and plasma, and hemolysis develops.

Unseparated serum

Serum or plasma held at 4 °C to 6 °C when analysis is delayed beyond 4 hours — decreases LDH isoenzymes 4 and 5 and increases ALP.

Mislabeling

The result is correct for the wrong patient. If the discrepancy is caught the specimen is rejected; if it is not, the error reaches the chart.

Types of specimen

Blood · cerebrospinal fluid (CSF) · urine · synovial fluid · pleural fluid · pericardial fluid · peritoneal fluid.

Blood

Volume: an average human body contains approx. 5 quarts (4.72 L) of whole blood; adult males approx. 5–6 L; adult females approx. 4–5 L.

Blood

Composition: 60 % plasma — 3 quarts (2.84 L); 40 % cells — 2 quarts (1.89 L).

Plasma

• Is: the liquid portion of anticoagulated blood, obtained after centrifugation.
• Contains: clotting factors, proteins, electrolytes.
• Used for: coagulation studies, chemistry, molecular testing.

Serum

• Is: the liquid remaining after blood clots.
• Contains: electrolytes, proteins, hormones, enzymes.
• Does NOT contain: fibrinogen, and most clotting factors.
• Used for: clinical chemistry, serology, immunology.

Plasma vs serum

Anticoagulated → plasma, with fibrinogen; clotted → serum, without it.

Steps of collection

• Step 1. Verify test request
• Step 2. Patient identification
• Step 3. Patient preparation
• Step 4. Proper collection technique

Verify test request

Check: patient name · requested tests · physician · date and time.

Patient identification

Use TWO identifiers. Examples: full name · date of birth · hospital number. Never use the room number.

Patient identification

The first step in sample collection — the prime factor in order to attain accurate results in the clinical laboratory.

3-Way ID

• The patient’s verbal ID statement
• A check of the ID band
• A visual comparison of the labeled specimen with the patient’s ID band before leaving the bedside

Conscious inpatients

Verbally ask their full names. Verify the name using the identification bracelet, which includes first and last names, hospital or unit numbers, room and bed number, and the physician’s name.

Sleeping patients

Identified in the same manner as conscious inpatients. They must be awakened before blood collection.

Unconscious patients

Unconscious and mentally incompetent patients are identified by asking the attending nurse or relative, and by the identification bracelet.

Infants and children

A nurse or relative may identify the patient, or identification is by means of an identification bracelet.

Outpatient identification

Verbally ask their full names, address or birth date, and countercheck with driver’s license or an ID card with photo. If the patient has an identification card or bracelet, proceed in the same manner as with hospitalized patients.

Patient preparation

• Prior to blood collection, patients must be given correct instructions on how to prepare for each laboratory test.
• Utmost care must be observed to minimize factors that may influence laboratory results.
• Examples: fasting · medication restriction · exercise restriction · diet restriction · timing.

Collection technique

Follow: standard precautions · aseptic technique · correct order of draw · proper equipment · correct tube · adequate volume.

Venipuncture

A process by which blood is obtained from a patient’s vein. Venous blood — deoxygenated — dark red colour.

Venipuncture

Sites: the antecubital fossa region; veins of the wrist and dorsal aspect of the hands; veins on the ankle.

Median cubital vein

Best site for venipuncture.

Cephalic vein

Second choice within the antecubital fossa.

Basilic vein

Third choice — the median nerve and the brachial artery lie alongside it.

CLSI Standards

An attempt must have been made to locate the median cubital vein on both arms before considering an alternate vein.

Antecubital fossa

• The plate labels thirteen structures: brachial artery, cephalic vein, median nerve, basilic vein, median basilic vein, median cephalic vein, median cubital vein, median antebrachial vein, median antebrachial cephalic vein, median antebrachial basilic vein, antebrachial basilic vein, ulnar artery and radial artery.
• The median nerve and the brachial artery lie alongside the basilic vein — which is why the basilic is the third choice and not the first.

Forearm and hand veins

Cephalic vein, basilic vein, median cubital vein, median vein of the forearm, radial vein, superficial dorsal veins, dorsal venous arch.

Leg and thigh veins

Femoral vein, long saphenous vein and its tributaries, popliteal vein, short saphenous vein.

Petechiae

If petechiae appear after venipuncture, it indicates that minute amounts of blood have escaped into the skin epithelium.

Order of draw

Evacuated tube and syringe, in sequence:
• 1 Yellow — blood culture tubes (SPS)
• 2 Light blue stopper — trisodium citrate
• 3 Serum tube — with or without clot activator or gel separator
• 4 Green stopper — heparin (Li, Na, NH₄)
• 5 Lavender / purple stopper — EDTA
• 6 Gray stopper — NaF and K oxalate

Order of draw

Fourteen containers on the photograph, left to right: BacT/ALERT FA aerobic bottle (green cap) → BacT/ALERT FN anaerobic bottle (orange cap) → yellow SPS → royal blue → light blue citrate adult → light blue citrate paediatric → red serum → gold SST → green sodium heparin → lavender K₂EDTA → pink K₂EDTA → black ESR → gray NaF / K oxalate → pale yellow ACD.

Order of draw

Containers the slide 28 list omits: the royal blue tube, the pink tube, the black ESR tube, the ACD tube, and the adult-versus-paediatric citrate distinction.

Citrate tube volumes

On the photograph the light blue buffered sodium citrate tube comes in two sizes: 2.7 mL adult and 1.8 mL paediatric.

Tube label amounts

Read off the photograph: red serum 6.0 mL · green sodium heparin 68 USP units, 6.0 mL · lavender K₂EDTA 10.8 mg, 6.0 mL · pink K₂EDTA 10.8 mg · gray NaF / K oxalate 15 mg / 12 mg, 6.0 mL · pale yellow ACD Solution A 8.5 mL.

Needle gauge

Gauge is inversely related to size — larger gauge = smaller bore and length.

Needle length

1 inch or 1.5 inches for 21–23 gauge; ½ to ¾ inch for the butterfly needle.

21 gauge

Use: standard for venipuncture.

23 gauge

Use: children and small or difficult veins.

25 gauge

Use: collect blood from the scalp or tiny veins (neonates).

21 gauge

Colour code: green. Bore: 0.8 mm.

22 gauge

Colour code: black. Bore: 0.7 mm.

23 gauge

Colour code: blue. Bore: 0.6 mm.

24 gauge

Colour code: purple. Bore: 0.55 mm.

25 gauge

Colour code: orange. Bore: 0.5 mm.

Tubes containing gel

Not used in blood bank and for immunologic testing; not used for TDM samples.

Gray top tubes

Fluoride oxalate: lactate, glucose, lipid. Sodium fluoride: ethanol.

Gray top tubes

Can also be used for lactate and alcohol determinations.

Polymer barrier

Has a specific gravity between the blood clot and the serum. It moves upward to the serum–clot interface, creating a barrier separating serum from fibrin — serum may be aspirated or measured directly from the collection tube during analysis, without transferring to another container.

Microcollection tubes

Lithium and ammonium salts of heparin are the preferred anticoagulants.

Anticoagulants

The five, in order: 1 oxalate · 2 citrate · 3 ethylenediamine tetraacetic acid (EDTA) · 4 fluoride · 5 heparin.

Oxalate

Combines with calcium to form an insoluble salt. Interferes with Na, K, and most BUN (urease) measurements.

Oxalate

Concentration: 1–2 mg/mL of blood.

Citrate

Combines with calcium in a non-ionized form.

Citrate

Concentration: 3.2–3.8 g/dL, in a ratio of 1 part to 9 parts of blood.

Citrate

Tube label, figure-only: light blue BD Vacutainer, buffered sodium citrate, 0.109 M, 3.2 %, in adult and paediatric sizes. The molarity 0.109 M appears nowhere in the slide text.

EDTA

Combines with calcium in a process called chelation.

EDTA

Two forms: versene and sequestrene (Na₂, K₂, K₃).

EDTA

Concentration: 1–2 mg/mL of blood.

EDTA

Uses: carcinoembryonic antigen (CEA), TDM and lead testing.

EDTA

Tube label, figure-only: lavender BD Vacutainer, K₂ EDTA 7.2 mg, 4.0 mL, REF 367861. That is 1.8 mg/mL — the arithmetic behind the 1–2 mg/mL rule.

Fluoride

Forms weakly dissociated calcium components. Interferes with Na, K, and most BUN (urease) testing.

Fluoride

Concentration: 10 mg/mL of blood.

Fluoride

Tube labels, figure-only: gray BD Vacutainer, sodium fluoride / potassium oxalate 100 mg / 20 mg, 10 mL, REF 367001 (slide 37); and 5 mg / 4 mg, 2.0 mL, REF 367921 (slide 43).

Heparin

Mucoitin polysulfuric acid. Acts as antithrombin and antithromboplastin; anti-Factor X; the ideal universal anticoagulant.

Heparin

Salts: available as sodium, lithium, potassium and ammonium salts.

Heparin

Concentration: 0.2 mg/mL of blood.

Heparin

Uses: NH₃, carboxy- and methemoglobin, plasma hemoglobin, pH and blood gas, cytogenetic studies (sterile tubes).

Lithium heparin

Uses: glucose, BUN, ionized calcium, electrolyte studies and creatinine.

Heparin

Tube label, figure-only: green BD Vacutainer, lithium heparin 75 USP units, 4.0 mL, REF 367884.

Red top

Additive: none. Yields serum. Clinical use: general chem. test.

Yellow and red top

Additive: polymer barrier. Yields serum. Clinical use: general chem. test.

Gray and red top

Additive: polymer barrier. Yields serum. Clinical use: general chem. test.

Yellow and gray top

Additive: thrombin. Yields serum. Clinical use: general chem. test.

Gold top

Additive: polymer barrier. Yields serum. Clinical use: general chem. test.

Orange top

Additive: thrombin. Yields serum. Clinical use: general chem. test.

Royal blue top

Additive: none. Yields serum. Clinical use: trace elements, toxicology, nutritional studies, TDM.

Royal blue top

Additive: sodium heparin or EDTA (Na₂). Yields plasma / whole blood. Clinical use: toxicology and nutritional studies.

Brown top

Additive: none. Yields serum. Clinical use: lead testing.

Brown top

Additive: polymer gel. Yields serum.

Brown top

Additive: sodium heparin. Yields plasma / whole blood. Clinical use: lead testing.

Green and gray top

Green and gray, or light green. Additive: polymer barrier and lithium heparin. Yields plasma / whole blood.

Pink top

Additive: none. Yields serum. Clinical use: general chemistry, blood bank and serology.

Pink / purple top

Additive: EDTA. Yields plasma / whole blood. Clinical use: blood bank, ABO and Rh, antibody screening, TDM.

Tan top

Additive: EDTA (K). Yields plasma / whole blood. Clinical use: lead testing.

Black top

Additive: buffered sodium citrate. Yields whole blood. Clinical use: ESR (Westergren).

Yellow top

Additive: sodium polyanethol sulfonate (SPS) or acid citrate dextrose (ACD). Yields whole blood. Clinical use: blood culture (SPS) or HLA typing (ACD).

White top

Additive: EDTA and gel. Yields plasma. Clinical use: molecular diagnostic.

Light blue top

Additive: trisodium citrate. Yields plasma. Clinical use: coagulation tests.

CTAD

Additive: citrate, theophylline, adenosine and dipyridamole. Yields plasma. Clinical use: coagulation tests and heparin monitoring.

Intravenous lines

The drawn blood is contaminated by the infusing fluid. Avoid; draw from the opposite arm. IV lines in both arms are listed as a site to be avoided.

Previously active IV sites

Residual fluid and altered local blood composition. Avoid.

Burned or scarred areas

Burns, scars and tattoos. Scarred tissue has poor vein access and impaired circulation; burns are painful and prone to infection. Avoid; select another site.