Hawler Medical UniversityCollege of Health SciencesClinical Biochemistry Dept.Ass. Lec. Amer Ali Khaleel

(M.Sc. Clinical Immunology)

Lab.2 : Whole Blood, Serum & Plasma Collections

Practical Immunology and Serology

Copyright © 2016

Preparation and Separation of Plasma:

The blood is transferred from a person’s vein to a test tube and prevented from clotting (tube containing anticoagulant e.g. Heparin, EDTA, Sodium Citrate, Oxalate, Sodium Fluoride, Sodium Iodoacetate ) , this preparation should be mixed immediately and thoroughly to avoid clotting, and centrifuged at 2500-3000 rpm for 5-10 min.

Preparation and Separation of Plasma:

It separates into two layers. The upper liquid layer, called plasma, represents about 55% of the volume of whole blood. The lower layer consists of red blood cells (erythrocytes), white blood cells (leukocytes), and blood platelets (thrombocytes). Collectively, these are called the formed (cellular) elements and represent about 45% of the volume of whole blood.

Preparation and Separation of Serum:• If blood is transferred from a person’s vein to a test tube

without containing anticoagulant and then allowed the blood to clot at room temperature for 15 to 30 minutes, when the blood has clotted completely and then centrifuged at 2500-3000 rpm for 5-10 min. it separates into two layers, the upper liquid layer called serum and the lower layer consists of formed elements.

• Serum =Plasma- Fibrinogen(coagulant factor)

Laboratory Specimens:Most Common•Blood samples•Urine samples

Swabs•Vaginal swabs •Wound swabs•Nose swabs•Pernasal swabs•Ear swabs•Eye swabs•Throat swabs

Additional•Biopsy material•Cerebrospinal fluid •Stool samples•Fungal samples of hair, nail and skin•Naso-pharyngeal aspirate•Sputum•Semen •Vaginal secretion •Saliva•Tears•Cerumen (earwax)•Sweat

Body Fluid •Percardial fluid•Pleural fluid•Synovial fluid•Vitreous humour•Amniotc fluid

Important note:• The color of the serum and plasma must be clear yellow

and there is no any turbidity (cloudy) or if any white color it shows the high proportion of fat in it which affects the result of the analysis.

• Similarly, if the color is reddish, it indicates the breaking of red blood cells, which affects a significant impact on some of the results.

Which is more appropriate Plasma or Serum or whole blood for doing blood biochemical estimations?

• It depends on type of analyte to be determined.

• You better use serum rather than plasma. Anticoagulate in the plasma interferes with many biochemical parameters particularly trace elements as lead and mercury are bond preferably on erythrocytes, therefore for its determination, the whole blood is preferred.

Method for Serum Preservation (Storage):

• In normal condition and the right is that the samples are direct do test without delay but, if necessary keeping the sample must be following in mind:

• Must separated sample and stored at low temperature in the refrigerator or freezer.

• Never preserves whole blood samples such as complete blood count in the freezer.

• Some samples have special conditions, such as the conservation Bilirubin must be kept in the dark.

• There are samples should never delay ,such as analysis of prothrombin and erythrocyte sedimentation analysis .

Method for Serum Preservation (Storage):

1. Preservation for short time,freezing method is followed between (-4 to -28°C).

2. Preservation for Long time, freezing method is (-86°C).

Benchtop FreezerUpright Freezer

Notes: • How long can frozen human serum be stored?

Frozen blood can be stored for 10 years from the date of blood collection.

• It is important to avoid freeze-thaw cycles because this is detrimental to many serum components.

• The serum put in polypropylene microcentrifuge tube.

Common Serum or Plasma Preparation Errors:1-Failure to separate serum or plasma from red cells within 30 to 45

minutes of venipuncture.

2-Hemolysis (occurs when the membrane surrounding red blood cells

is disrupted and hemoglobin and other intracellular components

escape into the serum or plasma. Hemolyzed serum or plasma varies in

color from faint pink to bright red, rather than the normal straw color.

Grossly or moderately hemolyzed specimens may be rejected and even

slight hemolysis may alter certain test results).

How To Prevent Hemolysis: 1. Do not leave tourniquet for longer than one minute.2. Allow alcohol to dry completely before puncturing the

skin.3. Use a properly sized needle for routine collections.4. Do not remove the needle from the vein with the vacuum

tube still engaged.5. Make sure sample is not exposed to extreme heat or cold.6. Allow blood to clot completely prior to centrifugation.7. Avoid vigorous mixing or shaking of tubes.8. Do not centrifuge specimens at higher speed or for longer

than necessary.

Differences between in vitro, in vivo, and in silico studiesThere are three broad categories of experiments:

In vitro (Latin for within the glass) refers to the technique of performing a given procedure in a controlled environment outside of a living organism.

In vivo (Latin for “within the living”) refers to experimentation using a whole, living organism as opposed to a partial or dead organism.

In silico is an expression used to mean “performed on computer or via computer simulation.

Serological Reactions:

• Antigen-antibody reaction (binding) in vitro are known as serological reactions, its useful for detection and measurement either antibody (Ab) or antigen (Ag) in the serum.

Uses:1- Diagnosis of infections. 2- Evaluation of the immunological status.

• Antigens (Ag) : Are molecules (substances) recognised by the immune system, which induce an immune response examples bacteria, viruses, parasites & fungi.

• Antibodies (Immunoglobulin): Are proteins produced by plasma cells in response to stimulation of B cell by foreign antigen, basic structure of immunoglobulin is Y shaped structure, there are five immunoglobulin classes of antibody molecules found in serum: IgG, IgM, IgA, IgE and IgD.

Some Concept:

Serological Techniques and Immune Assays:

•Numerous types of serologic test differ in their speed and

sensitivity, so the most effective test have high specificity and

sensitivity, some are strictly qualitative (positive or negative)

and others are quantitative (amount measured).

Serological Techniques and Immune Assays:There are several different methods used in serological tests:

1.Agglutination test. 2.Precipitation test.3.Labeled Immune assay (immunoassay). A.Radioimmunoassay (RIA).B.Fluorescent immunoassay (FIA).C.ElectroChemiluminescent immunoassays (ECL). D.Enzyme immunoassay (EIA) & Enzyme linked immunosorbent assay (ELISA).

Principle of Serological Tests:

• A reaction between antigen (Ag) and antibody (Ab) to produce a DETECTABLE reaction.

• Antigen-antibody reaction are visible by clumps, precipitates, color changes, emitting photons & release of radioactivity.

Agglutination Reaction (test):• The interaction (immune reaction) between antibody and a

particulate antigen resulting a visible clumping called agglutination. Antibodies that produce such reactions are called agglutinins while antigens are called agglutinogen.

• Agglutination: This reaction done between Ab & insoluble Ag is part of the surface of some particulate material (such as Erythrocytes, Bacterial cells, Inert carriers such as polystyrene latex particles).

• Purpose: Agglutination reactions used to detect either the presence of antigen or antibody in a sample.

Figure: Phases of agglutination. (Stevens, 2010) A) Sensitization, physical attachment of Ag & Ab. B) Network formation.

Clinical Applications of agglutination test:(Reagents)• ANA (Antinuclear antibody) test for diagnosis nuclear autoantibodies.• ASO (Antistreptolysin O) test for diagnosis of Streptococcus bacteria.• Brucella agglutination test for diagnosis of brucellosis.• CRP (C-Reactive Protein) to check for inflammation in the body.• IM (Infectious Mononucleosis) or Monospot screening test for infections

mononucleosis.• Latex test for rheumatoid factor.• PT (Pregnancy testing) for detection Beta-HCG.• RF (Rheumatoid factor test) detects autoantibodies present in Rheumatoid

arthritis.• TPHP (Treponema pallidum particle agglutination assay) screening test for syphilis.• VDRL (Venereal Disease Research Laboratory test) screening test for syphilis.• Widal test for diagnosis of salmonellosis.

1-Prepartion of serum.2-Prepartion of plasma.3-Preservation methods.

Practical Part