Activity No.

SPECIMEN COLLECTION AND PROCESSING

WRITE-UP SHEET

1. Why is complete clotting of blood (within 30 mins) in the preparation of serum necessary in blood

banking tests?

Serum provides the liquid portion of the blood without cells and clotting factors and, therefore, should
contain proteins and other molecules that represent the whole body system. The cells and clotting
factors must be removed from the blood sample by allowing adequate time for a clot to form. Most
manufacturers of collections systems for serum samples recommend 30–60 min at room temperature
for a clot to form and longer if the subject was taking any kind of anticoagulant at sample collection. The
selection of a collection tube was left to an individual’s discretion as long as it is without additives and
designated for serum isolation by the manufacturer. This matches standard practice in clinical
diagnostics. Serum samples that are allowed to sit less than 30 min are likely to retain cellular elements
and other contaminants impacting future analysis. Samples that sit longer than 60 min are likely to
experience lysis of cells in the clot, releasing cellular components not usually found in serum samples

Reference: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2655764/

Contributor:

ACTIVITY NO. 3

9. How is rouleaux formation differentiated from true agglutination? What are the possible causes?

Rouleaux are aggregates of red cells that, characteristically, adhere to one another on their flat surface,
giving a “stack of coins” appearance when viewed microscopically. This occurs when red blood cells are
sticky because of protein abnormalities or sometimes as a result of infusion of certain intravenous fluids.
This false agglutination, or rouleaux, may result in an ABO discrepancy. The most common cause of this
type of discrepancy is due to elevated protein levels, as might be seen in multiple myeloma,
Waldenstrom macroglobulinemia, and other plasma cell dyscrasias. Increased levels of protein may
interfere in cell grouping, serum grouping, or both. Interference in cell grouping tests may be overcome
by multiple washing of the cells to be tested so that all nonattached protein is removed.

Agglutination is formed by grapelike RBC aggregates and clumping reactions observed in vitro
result from the bonding of antigen and antibody with all the attendant variables influencing
the character and amount of observable reactivity. Agglutination of red cells occurs because
antibody molecules bind to antigenic determinants on multiple adjacent red cells, linking them together
to form a visible aggregate. In some tests, antibody directly bridges the gap between adjacent cells; in
others, antibody molecules attach to, but do not agglutinate, the red cells, and an additional step is
needed to induce visible agglutination or to otherwise measure the reaction.

Reference:

Quinley, E. (2011). Immunohematology: Principles and Practice. (3 rd edition) Lippincott Williams &
Wilkins, a Wolters Kluwer business.

AABB (2005) Technical Manual 15th edition

Amy C. Valenciano DVM, MS, DACVP, Ronald D. Tyler DVM, PhD, DACVP (Clinical and Anatomic
Pathology), DABT, in Atlas of Canine and Feline Peripheral Blood Smears. (2021). Red Blood Cells.
Retrieved from

https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/rouleaux#:~:text=Rouleaux
%20are%20orderly%20linear%20stacks%20of%20RBCs,%20whereas,and%20agglutination,%20a
%20saline%20dilution%20test%20is%20useful.

Contributor:

10. What is meant by the following:

a. Immune hemolysis

Immune hemolysis is destruction of RBCs as a result of antibody production and is an acquired

characteristic of the RBC membrane associated with demonstrable antibodies, as opposed to
intracorpuscular defects such as enzyme deficiencies and hemoglobinopathies, which represent
intrinsic abnormalities of the patient’s RBCs.

The three broad categories of immune hemolytic anemias are:

1. Alloimmune
2. Autoimmune
3. Drug-induced

In an alloimmune response, the patient produces antibodies to foreign or non-self RBC antigens
introduced into the circulation through transfusion, transplant, or pregnancy. In cases in which
an alloantibody clears the antigen-positive RBCs from circulation, intravascularly or
extravascularly, a transient hemolytic process may result (e.g., a hemolytic transfusion
reaction), sometimes causing anemia in the transfusion recipient or the affected fetus. For a
discussion of alloantibody production, refer to Chapter 9, “Detection and Identification of
Antibodies”;
An autoimmune response occurs when a patient produces antibodies against his or her own
RBC antigens.

A drug-induced hemolytic anemia is the result of a patient’s production of antibody to a

particular drug or drug complex, with ensuing damage to the patient’s RBC

b. Autoagglutination

Autoagglutination is the process by which erythrocytes adhere to each

other and form cohesive aggregates that do not dissipate when mixed
with equal parts of saline. Autoagglutination occurs when erythrocytes
are coated with surface antibody that interacts with adjacent
erythrocytes; therefore, autoagglutination indicates immune-mediated
hemolytic anemia. (Autoagglutination may indicate the presence of a cold reacting
antibody in the patient sample, which reacts with erythrocyte antigens during slide
preparation. Cold agglutinins are autoantibodies produced by a person's immune
system that mistakenly target red blood cells (RBCs). They cause RBCs to clump
together when a person is exposed to cold temperatures and increase the likelihood
that the affected RBCs will be destroyed by the body. This test detects and measures
the amount of cold agglutinins in the blood. When the presence of cold agglutinins in a
person's blood leads to significant RBC destruction, it can cause hemolytic anemia and
lead to a low RBC count and hemoglobin.)

Autoagglutination is usually associated with immunoglobulin M (IgM)

antibody deposition on the erythrocyte surface because IgM is more
efficient at cross-linking than any other class of antibody. High
concentrations of IgG complex deposition on the erythrocyte surface
also can result in autoagglutination.

c. Polyagglutination

Polyagglutination is the spontaneous agglutination of RBCs by all or almost all normal human sera.

The first type to be discovered was due to T activation. Bird showed that a plant lectin made from the
peanut, Arachis hypogaea, was able to detect an antigen on the surface of the RBCs of rare people who
seemed to develop an antigen labeled T on the surface of their RBCs.

Another type of polyagglutination is a result of unknown causes and may occur much less frequently.
This polyagglutination is permanent and results in Tn activation. It has not been simulated in vitro.
However, Tn is destroyed by enzyme, and thus enzymetreated cells may be used for further testing. Tn
activation results in cells that react in a mixed-field pattern and fail to react with the lectin from A.
hypogaea. Bird and Wingham showed that when properly diluted, a lectin from the plant Salvia sclarea
had specificity for Tn.

Reference:

Harmening, D. (2012). Modern Blood Banking & Transfusion Practices. (6 th edition) F. A. Davis Company.
Philadelphia

Labtestsonline. (2021). Cold Agglutinins. Retrieved from

https://labtestsonline.org/tests/cold-agglutinins
Shannon Jones Hostetter, Claire B. Andreasen, in Veterinary Clinical Pathology

(2021). Autoagglutination. Retrieved from

https://www.sciencedirect.com/topics/veterinary-science-and-veterinary-medicine/autoagglutination

Labce. (2021) Autoagglutination. Retrieved from

https://www.labce.com/spg28838_autoagglutination.aspx

Contributor: