Cryptococcal Antigen Latex Agglutination System (CALAS) Procedure

Principle:

CALAS is for rapid detection of Cryptococcal meningitis. It is a qualitative and semiquantitative test system for the detection of capsular polysaccharide antigens of Cryptococcus neoformans in cerebrospinal (CSF).

Specimen Preparation:

CSF

• Centrifuge specimen at 1000 x rpm for 15 minutes. 
• Aspirate the spinal fluid into a sterile container with seal .
• Inactivate the CSF by placing it in a boiling water bath for 5 minutes. 
• Allow to cool for 3-4 minutes before testing.

SERUM

• Centrifuge clotted blood at 1000 rpm for 15 minutes. 
• Aspirate serum into a sterile container with seal .
• Add 200 ul of serum specimen to 200 ul of the pronase solution .
• Incubate serum/pronase solution in a boiling water bath for 15 minutes at 56ºC .
• Immediately place the solution in boiling water bath for 5 full minutes. If the serum coagulates the CALAS Pronase has lost significant activity and should not be used .
• Allows to cool for 3-4 minutes before testing.

Procedure:

• Label the test slide. 
• Add 1 drop of Positive Control and Negative Control to each of the designated rings .
• Add 25 ul of the Antibody Control and Negative Control to each of the designated rings .
• Add 25 ul of the patient specimens to each of the 2 designated rings .
• Add 1 drop of Detection Latex to each of the designated rings .
• Add 1 drop of Control Latex to each of the designated rings .
• Mix each well using a separate applicator stick to each ring .
• Rock the slide by hand or by a rotator at 125 rpm for 5 minutes .
• Read agglutination immediately and grades the strength of the reaction from negative to 4+ .
• Patient specimens that have a 2+ or greater reaction with either the Detection Latex or Control Latex should be titrated with both reagents ( see below procedure for titration).

Titration (If Result is Positive)

• Do titration ( when patient specimens have a 2+ or greater reaction with either the Detection Latex or Control Latex) .
• Prepare two-fold serial dilutions of the specimens as follows: 
• Place 0.25 ml of Sample Diluent in each of 5 test tubes, labeled 1-5 and mix well .
• Using a clean pipet, places 0.25ml of patient specimen in tube #1 and mix well .
• Transfer 0.25 ml from tube #1 to tube #2 and mix well. Continue dilution procedure through tube #5. Transfer 0.25 ml from the 5th tube into a “holding” tube since further dilutions may be necessary.
• Beginning with tube #5, transfers 25 ul of this solution to both the Detection Latex marked rings.
• Repeat step 4 for tube #4 through 1. This procedure allows the use of a single pipette tip to place the five dilutions on test slide.

Tube	1	2	3	4	5
Pronase Treated Specimen	1:14	1:8	1:16	1:32	1:64
Non-pronase Treated Specimen	1:2	1:4	1:8	1:16	1:32

Interpretation:

• The positive control should give a positive reaction with the Detection Latex. A positive reaction between the two may indicate contamination. 
• The Antibody Control detects the presence of rabbit globulin on the latex particles. Failure of the Antibody Control to give a positive reaction with Control Latex indicates that one of the reagents is unsatisfactory. 
• The Negative Control should give a negative reaction with both the Detection Latex and Control Latex. A positive reaction with either of the two may indicate possible contamination or freezing which could produce false positive result to patients’ specimens. A false positive reaction may also occur by neglecting to heat inactivate the Negative Control.