001 – Lab Diagnostics

[] Rules for collecting material for lab tests

-A field of medical science that provides information about the patient's health condition based on laboratory tests
performed on biological material collected from the patient.
Ex: Blood (Arterial, Venous, Capillary) Urine, CSF

[] 3 phases of laboratory testing and rates of errors:

-Pre-analytical (68%): Patient preparation, specimen collection, transport
Sources of preanalytical variability:
-controllable, such as: diet, exercise and drug intake;
-noncontrollable: inherent to the patient's age, sex, race;

Factors related to material collection:

Body position, stasis, specimen additives, order of draw, sample mixing, specimen volume, phlebotomy
technique, needle size;

Factors related to the transport and storage of material: time, temperature

Interference: hemolysis, heterophilic antibodies, lipemia..

-Analytical (13%): Quality assurance, analysis, operator competence

-Post-analytical (19): Results transmission, interpretation

[] Blood as biological material for laboratory tests

Plasma –
Contains Fibrinogen Serum – Fluid part of the blood
Clear and Yellowish and does not contain clotting agents.

Whole Blood collected tests:

-Blood gas analysis (heparin = Anticoagulant)
-ESR (Erythrocyte Sedimentation Rate= marker for inflammation rate, trisodium citrate)
-Morphology (EDTA) – from Venous Blood
-Glucometric tests – From Capillary Blood (Blood glucose for diabetes, strip test method)

Serum: Fluid part of the blood and does not contain clotting agent, contains products of consumption of these proteins.
-Biochemical tests (ammonia ↑40% and lactate ↑25% -serum not acceptable).

Plasma: Blood plasma + Anticoagulant

-Heparin (lithium heparin) -biochemical tests (protein electrophoresis is not performed)
-Citrate (trisodiumcitrate1:10): Coagulation tests
[] Patient preparation
-Blood for all tests should be drawn preferably in the morning from 07.00-09.00 a.m.,24 hours prior to collecting blood
for testing, avoid intense physical activity.
Stress and physical activity
•↑in cell number (WBC increase, Hemolysis also)
•↑in coagulation factors (VIII), tissue plasminogen activator (t-PA), fibrinolytic activity
•Changes in acid-base balance parameters
•↑serum enzyme activity (Creatine kinase, Pyruvate kinase, Albumin, Glu, Uric acid, urea)
-Documentation of the exact time of Sampling is very important!
-Fasting requirements: no food or liquid except water
(10-12h; drinking natural coffee & smoking nicotine on the day of collection is not recommended).
Caffeine Inhibits Phosphodiesterase ↑cAMPGlycogenolysis
↑ Adrenalin  GlucoNeogenesis  ↑Blood glucose conc.
-No medication -unless research is used to monitor treatment.

[] The effect of the diagnostic procedure on laboratory tests results

•Prostate biopsy: ↑ in PSA concentration (several times). – Is a tumor marker
Another determination after 6 w.is recommended.
•Transurethral prostate resection: ↑ in PSA concentration. Another test after 6weeks.
•Prostate examination: slight ↑in PSA. Another blood sample after 1 week.
•Transrectal ultrasound of the prostate: ↑in PSA conc. Another determination of PSA after 1w.
•Radical prostatectomy: PSA decrease.

[] Carcadian Rhythm

[] Materials used to take the blood sample

-Closed systems for blood sampling = They have proven to be safer than open systems.
Hemolysis with full system usage
Microclots -specimen integrity is preserved

-Open system
Hemolysis redraw (Major source of error – Most common cause of sample rejection!)
Microclots (delay in contact with additive and this is a variable factor)
[] Needles
20Gauge 0.90mm Yellow
21Gauge 0.80mm Green
22Gauge 0.70mm Black
-The diameter of the needle
Too large diameterContamination of the sample tissue with Tissue ThromboPlastin,
Tissue trauma
Too small diameterToo fast blood flow (Especially when using vacuum tubes)
-Hemolysis
-Platelet Activation
-Activation of coagulation factors
-MicroMethod for capillary blood collection for children:
Cant do coagulation test in capillary blood(b/c contamination with tissue fluid & thromboplastin)

-Blood sampling Procedure (Cubital fossa)

1. Median Cubital vein (Most preferable, b/c most prominant)
2. Cephalic vein
3. Basilic vein

[] Procedure for sampling blood – Always ask before drawing blood = Name & date of birth

1.Disinfect the selected puncture site with alcohol (70% isopropyl or ethyl) and thorough drying of the field
2.Description of the tube in the presence of the patient
3.The use of needles, an optimum diameter (vacuum systems)
4.Apply tourniquet
5.Puncture the vein
6.Release the tourniquet as blood starts to flow
7.Collect blood with due regard to the order of draw
8.Aspiration of blood volume
[] Influence of short-term venous stasis on clinical chemistry testing
-Tourniquets cause a temporary occlusion of veins and temporary venous stasis.
-If applied for a long period of time (longer than 1 min) a tourniquet induces a substantial variation of blood composition,
due to extravasation of water and small molecules such as ions from the intravascular to interstitial space.
-The tourniquet should be applied and quickly removed when the needle is safely in the vein.

Case 4:

[] Order of blood draw tubes and additives

Follow the CLSI and EFLM recommended order of draw.
The correct order of draw follows:
1.Blood culture tube or bottle
2.Sodiumcitratetube
3.Clot activator tube(serum)
4.Heparin anti-coagulant tube
5.EDTA tube (Blood count analysis)
6.Glycolysis inhibitor tube Sodium fluoride/potassium

[] Problems associated with incorrect order of draw

K2 EDTA Cause is Suspected potassium EDTA Contamination
•Hyperkalaemia
•Hypoalcaemia
•Zinc
•Iron 
•ALP
•Prolonged coagulation = Transfer of anticoagulants
•Dilution effects = Tipping of samples
[] Mixing
-Most tubes contain an additive. Regardless of the additive type, all tubes should be gently inverted to ensure thorough
mixing of the blood with the additive.
-Tubes with anticoagulants such as EDTA, heparin etc., must be mixed to ensure that the specimen does not clot.

[] Blood Gas Analysis & Acid-Base Balance!

-Blood gas analysis provides information on a critically ill patient’s respiratory & metabolic status via measurement of
pH, carbon dioxide (pCO2) & oxygen (pO2) together with other vital information such as electrolytes, lactate &
hemoglobin.
It gives the possibility of:
-The immediate application of appropriate treatment or modification of current treatment, especially oxygen therapy.
-‘In blood gas analysis, using unreliable data is worse that not having data!’
-Accurate result is the best of all
-For blood gas testing, a small error can have disproportionately large consequences
The special importance of the pre-analytical phase – TIME is the key to sample quality.

[] Sample transportation and storage

•Samples must be analyzed within 30 min from collection.
•When analysis is performed >30 min after sample collection, the specimen should be maintained in a water and ice
bath, for not more than 60 min.
-Analysis within 5min for samples with = High pO2, special studies(ex:Shunt), High leukocyte or platelet count.

-Specimens requiring special handling = Place in crushed ice or a mixture of ice and water (Chilling) when transporting
Arterial blood gases, ACTH, ammonia, lactic acid, parathyroid hormone (PTH), renin

-Continued cellular metabolism in sample 

pCO2=Since carbon dioxide will still be produced
Ca2+=Since the change in pH will influence the binding of Ca2+to protein
Lac =Due to glycolysis
Glu=Since glucose will be metabolized
pH=Primarily due to the change in pCO2and glycolysis
pO2=Since oxygen will still be consumed
The effect of this will be Lactic acid  pH (Metabolic acidosis)
[] Variability of measurement parameters in vivo
In vivo pH / PCO2 and PO2 are susceptible to rapid changesunder the influence of various factors.
Patient's concerns:
Impact of psychological factors
Avoid words or phrases with negative meaning, ex:"Artery puncture"
Some patients hyperventilate (increase in pH and PO2 and decrease in PCO2) for fear of collection.
Others hold their breath (decrease in pH and PO2 and increase in PCO2).

[] Patient stabilization
•Assess the “real” metabolic and respiratory conditions of the patient:
•Checking that the patient is in a stable condition of ventilation for at least 5 minutes
•Keeping breathing aids unchanged for at least 20 minutes
•Minimizing the anxiety from arterial collection

[] Sample types used for blood gas analysis

CLSI46-A2guidelinestate:
•“Blood gas measurement for the purpose of evaluating the gas exchange function of the lungs (pO2andpCO2) should be
performed on arterial blood only....
•The blood should be collected under anaerobic conditions, mixed immediately to dissolve heparin anticoagulant and
promptly analyzed”

[] Alternative?
CLSI 46-A2 guideline state:
•If arterial blood can not be collected directly, peripheral capillary blood may be collected using an arterialization
technique (warming the skin to 42°C with a warm towel or vasodilating cream).
•Blood gas results may differ, especially those for pO2, sO2.
There is really no substitute for arterial blood if accuracy of pO2 measurement is important (oxygen therapy).

-Arterial blood sampling

CLSI H11-A4: Procedures for the Collection of Arterial Disadvantages
Blood Specimens ✓discomfort and pain
Sampling: ✓possible complications
•from radial artery ✓bleeding (more dangerous, even thrombosis)
•indwelling arterial catethers

[] Arterialized Capillary Blood

-Capillary blood gas sampling is recommended for adult patients only in situations when arterial blood puncture cannot be
performed (patients with severe burns, tendencies to thrombosis, obese and geriatric patients).
-Capillary blood is often the specimen for infants, very young children.

[] Tubes for blood gas analysis

Collection systems for arterial, and capillary sample collection:
•electrolyte balanced heparin syringe
•capillaries with a coating of calcium-balanced lithium heparin

[] There is a significant risk of contact with air during capillary blood collection
PCO2 about 0mmHg
PO2 about 150 mmHg

Air contamination of the sample:

↑pH ↑pO2 ↑sO2 ↓pCO2
[] Capillary sampling Capillary blood collection-step by step
1. Disinfection of the puncture site with 70% aqueous solution of isopropanol;
2. Air drying of the puncture site;
3. Skin puncture and wiping away of the first drop and Anaerobic collection of the blood from the centre of the second
spontaneously formed blood drop.

[] The principle of arterialization of the peripheral capillary vascular bed

This technique consists of use of a warm, moist towel at a temperature not higher than 42 °C on a puncture site not longer
than three to five minutes or use of other vasodilator agent. 10-fold increase in capillary flow in the tissue where
metabolism is low, so that the pH / PCO2 / PO2 values on the venous side will be very similar to those on the arterial side.

Example:

[] Well arterialized & properly collected capillary blood gives information about the acid-base balance of the body
For PO2the results greater than
100 mmHg should be treated with caution.

They may provide on gas exchange with

the environment during blood collection.

Case 8
Lab receives arterial blood sample from emergency department. Blood gas testing is requested. Sample was transported
by pneumatic tube within 10 min from sampling. You notice an air bubble in the syringe. What do you do?
1. Sample is acceptable. I would expel the air bubble and perform the analysis
2. Sample is not perfect, I would expel the bubble and perform the analysis. I would report the result with a
comment
3. Sample is not acceptable. I would reject the same and request repeated sampling
4. I would call a physician and ask him to decide what todo.

[] Arterial Blood Gas analysis - Synthesis of BEST PRACTICES

1.Use appropriate procedures for patient and sample identification.
2.Test patient in basal and stable conditions, avoiding anxious states (e.g., hyperventilation).
3.Maintain strict anaerobiosis (no bubbles).
4.Appropriately mix the sample before analysis.
5.Prefer balanced, heparin formulations.
6.Analyzethe sample (within 30 min) or refrigerate at 2-4 °C for less than 1 hour.