1. Lab (1): Introduction to Clinical Laboratories
L. Nouf Alshareef
KAU-Faculty of Science- Biochemistry department
Clinical biochemistry lab (Bioc 416)
2012

2. Clinical Biochemistry Lab
Clinical labs is important in disease diagnosis, determine its severity and patient response to specific treatment
Sections of clinical laboratory:
1) Hematology 2) Clinical biochemistry 3) Clinical microbiology
4) Serology ) Blood bank ) Histology and cytology
Clinical Biochemistry Lab
Measure the concentration of one or more substances in biological specimen of patient and compare it with reference value obtained from healthy subjects.
Types of samples:
Body fluids: blood, serum, plasma, urine, cerebrospinal fluid (CSF), feces other body fluids or tissues

3. How clinical biochemistical tests are performed
Automated computerized machine
Manually
Kits

4. BIOCHEMSTRY TESTS
LFT
(AST, ALT, ALP, GGT, TP, Alb, globuline, bilirubin)
KFT
(urea, creatinie, creatnine clearance, uric acid, Na+, K+ )
Lipid profile
(cholesterol, TG, HDL, LDL)
Cardiac profile
(AST, LDH,CK,K+)
Bone profile
(ALP, minerals: Mg2+, Ca2+, phosphate)
Electrolytes
(Na+, K+, Cl-, Mg2+, phosphrous)

5. Lab Request
Lab request or (request form), is computerize or manually filled by the doctor and send to the lab.
There are different forms (multi-purpose or specific).

7. Laboratory Work Flow Cycle:
Three phases of laboratory testing:
- test ordering, specimen collection, transport and processing
- testing
- results transmission, interpretation, follow-up, re-testing.
Pre-analytical
Analytical
Post-analytical

8. Post-analytical
Pre-analytical
analytical

9. BLOOD COLLECTION (Phlebotomy):
Phlebotomy: blood withdraw from a vein, artery or bed capillaries for lab analysis.
The phlebotomy equipments:
Disposable syringes or
Tourniquet
Alcohol swap
Blood collection tubes
Gauze pads or adsorbent cotton
Waste container

12. Note:
minimum use of tourniquet is advised because blood constituents may be changed due to prolonged venous occlusion.

13. Vein Selection Veinpuncture procedure, using arm vein (adult).
Three veins in arm may be used:
median cubital vein or,
cephalic or
basilic veins
Median cubital vein is the best choice (why?)
because it has good blood flow than cephalic and
basilic which has more slowly flow
venous-blood.html

14. Hand veins can be also used.
Artery blood is rarely used in special cases as when blood gases, pH, CO2, O2 and bicarbonate is requested. It is usually performed by physicians.

15. Preparation of Blood Sample.
Preparation of Blood Sample
Blood contains: RBCs, WBCs and platelets
Serum and plasma are prepared from whole blood by centrifugation.
After centrifugation of blood, the blood separate into three layers
In biochemical tests, one of three type of blood sample can be used:
Whole blood ( HA1C)
Serum
Plasma

16. whole-blood specimens must be analyzed within limited time (why?)
Over time, cell will lyses in whole-blood which will change the conc. of some analytes as potassium, phosphate and lactate dehydrogenase
Some cellular metabolic processes will continuo which will alter analytes conc. like glucose and lactate.
When we use whole blood in analysis

17. Difference between Serum and plasma
Blood serum:
Serum is the same as plasma except it doesn't contain clotting factors (such as fibrin)
Mainly use in chemistry lab & serology.
Blood plasma:
Contains clotting factors
So, serum and plasma all has the same contents of electrolytes, enzymes proteins, hormones except clotting factors

18. BOOLD COLLECTION TUBES

19. They are specific tubes evacuated from air called vacutainer tubes.
There are two major types of blood collection tubes:
Serum separating tubes (SST): contain no anticoagulants.
Plasma separating tubes (PST): contain anticoagulant or preservative.

20. Blood anticoagulants:
They are substances which prevent blood coagulation.
They inhibit coagulation process by eliminating Ca2+ or by binding with thrombin.
EDTA, citrate and oxalate: binds with Ca2+
Heparin: binds with thrombin
Coagulation occurs in two major steps:
- Prothrombin Ca Thrombin
- Fibrinogen Thrombin Fibrin (Clot)

21. Choose the correct anticoagulant in blood collection is very important.
It depends on the test to be performed; anticoagulant shouldn't interfere with the substance to be analyzed (measured).
False positive or false negative results may be produced if blood collected in tube contains wrong anticoagulant.

22. False positive and False negative
Here are some examples:
Tubes contain sodium and potassium anticoagulants are not used in measuring concentration of electrolytes; this will produce false positive result (Li, ammonia or heparin is used in electrolytes).
Tubes contain Na+-oxalate anticoagulant are not used for measuring Ca2+ level; because oxalate will react with Ca+2 and precipitated as Ca2+-oxalate.
EDTA and citrate are not suitable for enzymatic assays, because it binds with Ca2+ ion that is a cofactor for enzymes like alkaline phosphatase.

23. Plasma separating tubes: Lavender (EDTA)
Green (heparin)
Light blue (citrate)
Gray (floride oxalate)
Black
Serum separating tubes:
Red no additives
Yellow : gel
- Hematology
- HbA1C
- Enzymes, Hormones
- Electrolytes
- Coagulation (PT,PTT)
- Glucose
- ESR

24. Order of the Draw
To prevent contamination of tubes with additives from other tubes it is important to draw the tubes in a SPECIFIC order called "the order of the draw".
For example, if the additive in the purple stopper tube contaminates the green stopper tube this would cause a falsely decreased calcium and increased potassium.

25. The sequence of collection of evacuated tubes in a multi-draw should be in this order:
Sterile/Blood cultures (yellow top or bottles)
Royal Blue - red label for trace metal analysis
Light Blue coagulation tube .
Red - Non-Additive
Red Gel separator tube (speckled or “tiger” top)
Green (heparin)
Lavender/purple top and/or pink (EDTA)
Gray top (Oxalate/fluoride tube)

26. Plasma separating tubes (PST)
Top Colors
Additives
Principle
Uses
Lavender
EDTA
The strongest anti-coagulant
Ca+2chelating agent
preserve RBCs components
Hematology
Blood bank
ABO blood group
HbA1C
Light blue
Sodium Citrate
must be completely filled and inverted immediately after filling.
PT: Prothrombin Time
PTT: Thromboplastin Time
Green
Sodium or Lithium Heparin
Heparin binds acts as anti thrombin.
Enzymes
Hormones
Electrolytes
Black
Ca+2 chelating agent
ESR (Erythrocyte Sedimentation Rate)
Gray
Na- Fluoride and K- Oxalate
Fluoride inhibits Glycolysis
Oxalate is anti-Coagluant
Glucose tests

27. Procedure of Serum preparation:
Draw blood from patient. Select vacutainer with no anticoagulant.
Allow to stand for 20-30min for clot formation.
Centrifuge the sample to speed separation and affect a greater packing of cells. Clot and cells will separate from clean serum and settle to the bottom of the vessel.
The supernatant is the serum which can be now collected by
Dropper or pipette for testing purposes or stored (-20°C to -80°C) for subsequent analysis or use.

28. Serum separating tubes (SST)
Top Colors
Additives
Principle
Uses
Red
Sometimes it has gel or silicon at the bottom of tube to reduce hemolysis
Enhancing the formation of blood clot
Serology, Antibodies
Chemistry, Hormones, Drugs
Virology
Blood cross matching before blood transfusion
Yellow
It has gel at the bottom of the tube to separate serum from the blood
Serum separating from the blood through the gel in the tube
Serology
Chemistry
Procedure of plasma preparation: - Draw blood from patient. Select vacutainer with an appropriate anticoagulant. - Mix well with anticoagulant. - Allow to stand for 10min. - Centrifuge the sample to speed separation and affect a greater packing of cells. - The supernatant is the plasma which can be now collected for testing - Purposes or stored (-20°C to -80°C) for subsequent analysis or use.

29. http://qalphaada. en. ec21

30. Sample Haemolysis
Haemolysis: liberation of hemoglobin due to rupture of RBCs.
Plasma or serum appears pink to red color.
Haemolysis must be avoided because it interferes with the results of some investigations. Usually it is associated with elevation in K+, Ca2+, phosphate, AST, D.Bil, LDH and acid phosphatases (why?)
Classified as: H+, H++ and H+++.
H++ & H+++ not acceptable for any test

31. Causes of haemolysis: Sampling:
dispense of blood directly from syringe through needle, the applied pressure may cause rupture of RBCs.
Storage:
Refrigerate non-separated blood sample (too cold, too hot will also effects).
Transportation:
Mechanically due to blood sample shaking.

32. Types of samples Hemolyzed: rupture of RBCs so, HB released from RBCs
Icteric: serum appears yellow due to high bilirubin.
Lipemic: serum appears milky or turbid due to high lipid.

33. Specimen rejection Specimen improperly labeled or unlabeled
Specimen improperly collected or preserved
Specimen submitted without properly completed request form
hemolyzd sample.

34. Sample Storage Serum or plasma is stored in: 2-4oC for 3-5 days
-20oC for long time (months)