IMMUNOLOGY Archives - LaboratoryTests.org

Cross Matching : Types, Principle, Procedure and Interpretation

Dhurba Giri — Tue, 02 Jul 2019 18:43:12 +0000

Cross matching is a procedure performed prior to transfusion of blood or blood products to detect any serological incompatibilities in the blood of donor and recipient. Before a donor’s blood is transfused into a recipient, there should be no antigens or antibodies in both, that would react with each other resulting in transfusion reaction. Cross matching is designed to prevent such transfusion reactions which may occur after transfusion.

Cross matching plays important role to detect:

Most recipient antibodies directed against donor’s red blood cell antigens.
Most donor antibodies directed against recipient’s red blood cell antigens.
Major errors in ABO grouping, labeling and identification of donor and recipients.

Principle of Cross Matching

Cross matching is based on the principle of serological detection of any clinically significant irregular/unexpected antibodies in either donor or recipient’s blood. There are two types of cross matches:

Major Cross Match: It involves testing the donor’s red cells with recipient’s serum to determine the presence of any antibody which may cause hemolysis or agglutination of donor red cells.This is more important than minor cross match.
Minor Cross Match: It involves testing of donor’s plasma with recipient’s red cells to determine the presence of any antibody which may cause hemolysis or agglutination of recipient’s red cells.

Type	Donor’s	Recipient’s
Major Cross Match	Red Cells	Serum/Plasma
Minor Cross Match	Serum/Plasma	Red Cells

Procedure of Cross Matching

There are different methods for cross matching, as shown in table. Among them most commonly used technique is Anti-human globulin (AHG) cross match.

Method of Cross Match	Detects Antibody of Type :
Saline Cross Match	IgM
Albumin Cross Match	IgG
Anti-Human Globulin (AHG) Cross Match	IgG

Major Cross Match

Prepare donor and recipient’s blood sample: Donor’s red cells and recipient’s serum/plasma.
Prepare 3-5% saline cell suspension of red cells.
Label a test tube.
Add two drops of recipient’s serum and one drop of donor cell suspension.
Mix and incubate the tubes at 37 degree Celsius for about 60 minutes.
Decant the serum completely and wash the cells three times in saline.
Add two drops of Anti-human Globulin (AHG) and mix. Allow to stand at room temperature for 5 minutes.
Centrifuge at 1500 rpm for 1 minute.
Observe macroscopically and microscopically for agglutination.
If macroscopic agglutination is not observed, transfer a small amount onto a glass slide and examine for microscopic agglutination. Rouleaux is not an indication of incompatibility.

Minor Cross Match

Prepare donor and recipient’s blood sample: Recipient’s red cells and donor’s serum/plasma.
Label a test tube.
Add two drops of donor’s serum and one drop of recipient’s cell suspension.
Mix and incubate the tubes at 37 degree Celsius for about 60 minutes.
Decant the serum completely and wash the cells three times in saline.
Add two drops of Anti-human Globulin (AHG) and mix. Allow to stand at room temperature for 5 minutes.
Centrifuge at 1500 rpm for 1 minute.
Observe macroscopically and microscopically for agglutination.
If macroscopic agglutination is not observed, transfer a small amount onto a glass slide and examine for microscopic agglutination. Rouleaux is not an indication of incompatibility.

Results and Interpretation

Compatible donor and recipient blood should show no agglutination in both major and minor cross match. Blood which shows incompatibility in major cross match should never be transfused, because the large plasma volume of the recipient blood containing antibodies can destroy the donor’s red cells easily. The minor incompatibility is less important because the donor’s serum which contains the antibodies is diluted in the recipient’s own plasma, making the antibodies very dilute and ineffective.

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Urine Pregnancy Test (UPT): Principle, Procedure, Interpretation and Limitations

Dhurba Giri — Mon, 25 Dec 2017 17:40:47 +0000

There are two types of tests for pregnancy testing: one uses blood sample and other uses urine. Both tests detect the presence of beta subunit of a hormone called human chorionic gonadotropin (hCG). The Urine pregnancy test (UPT) is an easy and rapid chromatographic immunoassay for the qualitative detection of human chorionic gonadotropin (hCG) in urine for the early detection of pregnancy.

Human Chorionic Gonadotropin (hCG)

Human chorionic gonadotropin (hCG) is a hormone produced by the developing placenta shortly after fertilisation. In normal pregnancy, hCG can be detected in urine as early as 7 to 10 days after conception. Levels of hCG rise rapidly, frequently exceeding 100mIU/mL by the first missed menstrual period, and peaking at 100,000-200,000 mIU/mL about 10-12 weeks into pregnancy. The appearance of hcG soon after conception and it’s subsequent rise in concentration during early gestational growth make it excellent marker for the early detection of pregnancy.

Principle of Urine Pregnancy Test

Most of the urine pregnancy test kits are based on lateral-flow technology. Most of them qualitatively detect the presence of hCG in urine specimen at the sensitivity of 25 mIU/mL. The test uses two lines to indicate results. The test line utilizes a combination of antibodies including a monoclonal hCG antibody to selectively detect elevated levels of hCG. The control line is composed of goat polyclonal antibodies and colloidal gold particles.

The assay is conducted by adding a urine sample to the sample well of the test device and observing the formation of colored lines. The sample migrates via capillary action along the membrane to react with the colored conjugate.

Positive samples react with the specific antibody-hCG-colored conjugate to form a colored line at the test line region of the membrane. Absence of this colored line suggests a negative result. A colored line will always appear in the control line region if the tests has been performed properly.

Specimen Collection

Collect urine into a clean, dry container. The first morning urine specimen is preferred since it generally contains the highest concentration of HCG; however, urine specimens collected at any time of the day may be used. Refrigerate specimens at 2° to 8°C (36° to 46°F) for up to 72 hours, if the testing is not performed immediately. If samples are refrigerated, bring them to room temperature before testing.

Procedure

Allow the Pregnancy Test Strip and urine sample to reach room temperature (15-30°C) before opening the foil pouch.
Remove the Pregnancy Test Strip from the pouch and use it as soon as possible.
Place the test device on clean and level surface. Hold the dropper vertically and transfer 3 full drops to the specimen well and start the timer. Avoid air bubble formation.
Wait for the colored line(s) to appear. Read the result after 5 minutes. Do not read the result after 15 minutes.

Results and Interpretation

POSITIVE: Two coloured lines appear. One line should be in the Control region (C) and another line should be in the Test region (T). This means there is a strong possibility that patient is pregnant.

NEGATIVE: One coloured line in the Control region (C). No apparent colored line appears in the Test region (T). This means patient is either not pregnant or has tested too early.

INVALID: The result is invalid if the Control Line (C) fails to appear. Insufficient volume of urine or incorrect procedure are the most likely reasons for an invalid result.

Limitations

In cases where very high levels of HCG are present (>500,000 mlU/ml) a false negative result can occur due to a “Prozone” effect. If pregnancy is still suspected, simply dilute specimen 1:1 with deionized water and retest.
If a urine sample is too dilute (ie: low specific gravity) it may not contain a representative level of HCG. If pregnancy is still suspected, a first morning urine sample should be obtained and retested 48 hours later.
A number of conditions other than pregnancy, including trophoblastic disease and certain non-trpohoblastic neoplasms including testicular tumors, prostate cancer, breast cancer and lung cancer cause elevated levels of hCG. Therefore, the presence of hCG in urine should not be used to diagnose pregnancy unless these conditions have been ruled out.
Drugs containing hCG may interfere with the test, and produce misleading results.

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Reverse Blood Grouping: Principle, Procedure, Interpretation and Limitations

Dhurba Giri — Thu, 21 Dec 2017 16:39:07 +0000

Several blood grouping systems have been described in humans. Among these, ABO blood grouping system is the most important system for screening, transfusion and transplantation practice today. ABO blood grouping system divides blood into 4 groups (A,B,AB,O) based on the presence or absence of antigens A and B on the RBC surfaces and naturally occurring antibodies anti-A and anti-B on serum. In normal human blood, a reciprocal relationship exists between antigens and antibodies as described below:

ABO grouping can be performed in two ways
Forward grouping/Cell grouping: Red blood cells are tested for A and B antigens using known anti-A and anti-B sera.
Reverse grouping/Serum grouping: Serum is tested for anti-A and anti-B anibodies using known A and B red cells.

Reverse Blood Grouping /Serum Grouping

Reverse blood grouping is a procedure to confirm ABO blood group based on the presence or absence of anti-A and anti-B in serum using known A and B red cells. It is cross check for forward typing. Performing both forward and reverse grouping provides a check for accuracy. Because of the lack of synthesized immunoglobulins, anti-A and Anti-B in newborns and very young infants, this procedure is not performed on infants below 4 months of age.

Principle

The reverse blood grouping procedure is based on the principle of direct hemagglutination. The erythrocytes of a person contain blood group antigens on the surface of the membrane. When these antigens are allowed to treat with corresponding antibodies, antigen-antibody reaction occurs and form agglutination.

Requirements

Specimen:
Serum is specimen for reverse blood grouping. No special preparation of the patient is required prior to collection. The specimen should be tested as soon as possible after collection, but specimens may be stored at 2 to 8?C if there is a delay in testing. Storage may result in weaker-than-normal reactions.

Cell Suspension:
Although red cell reagents for serum grouping are available commercially, most laboratories prepare their own A and B test red cells from persons known to be group A and group B. Make pooled cell suspension as follows:

Label tubes as A and B.
Tube A: Place 1 drop of red cells each from 3 of A group samples.
Tube B: Place 1 drop of red cells each from 3 of B group samples.
Add Normal saline and to suspend the cells. Centrifuge the tubes for at least 1 minute at 1000 rpm. To make 5% red cell suspension, add 1 drop of RBC to 19 drops of saline. Make 20% suspension for slide method.
Test the pooled cells prepared by adding the antisera (Anti-A, B) in use.

Procedure

The reverse blood grouping can be performed in two methods: Tube and Slide method. The Tube method is preferred to slide method.

Tube Method

Label two test tubes as A and B.
Add two drops of serum to be tested in each tube.
Add one drop each of A and B cells suspension to the corresponding test tubes.
Mix well and centrifuge both tubes at 1000 rpm for 1 minute.
Gently remove the tubes and completely resuspend cells and examine macroscopically for agglutination and if negative, microscopically.
Record the reactions and interpret the results.

Slide Method

Mark a clean slide into two halves, labeling the left and right side side as A and B.
Add a drop of serum to be tested on both sides.
Add one drop each of A and B cells suspension (20%) to the corresponding sides.
Using a clean applicator stick, mix the serum and cell suspension on both sides separately and spread into a smooth round circle.
Rock the slide gently for 2 minutes and look for agglutination.
Record the reactions and interpret the results.

Results and Interpretation

If agglutination is observed with A cells only, then the patient’s blood group is B
If agglutination is observed with B cells only, then the patient’s blood group is A
If agglutination is observed with both A and B cells, then the patient’s blood group is O
If agglutination is not observed with both A and B cells, then the patient’s blood group is AB

Limitations

Serum from persons with agammaglobulinemia may not contain detectable ABO antibodies.
As naturally occuring anti-A and anti-B are only formed 3-4 months after birth, it is not suitable for newborns and infants. Antibodies at this age are commonly of maternal origin.

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