Ch50 Assay

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Contents 1 Indications 2 Reference-interval 3 Principle 4 Clinical-significance 5 Increases 6 Decreases 7 Specimen 8 Procedure 9 Notes 10 More General Terms 11 Additional Terms 12 References

Indications

• evaluation for complement deficiency

Reference-interval

• 75-160 U/mL [7]

*  CH50 Values          % of Reference values     Interpretation 
*     <100                  0-50                    Absent or low  
*     100-300               51 to 150               Normal  
*     >300                  >150                    High

• * the CH50 unit is the reciprocal of the dilution of serum that will lyse 50% of the cells in a 1 mL suspension (see Procedure:)

Principle

• 
  

• A test for total complement that involves addition of complement in the presence of Mg+2 & Ca+2 to cause lysis of sheep red blood cells coated with antibody. The amount of hemolysis is evaluated quantitatively & compared with known controls.
  
• The complement system participates in the host immunological defense. Levels of total complement differ from normal levels in certain diseases, genetic deficiencies & other conditions. Thus, determination of total complement levels can be important in the diagnosis & management of these conditions.

Clinical-significance

• CH50 provides a quantitative value for the functional activity of total complement (classical pathway including complement factors C1, C2, C3, C4, C5, C6, C7, C8 & C9)
• may not detect C9 deficiency [6]
• test can determine abnormal whole complement levels, but does not identify component or components that are abnormal
• does not detect defects in complement alternative pathway
  

Increases

• acute inflammatory conditions
• leukemia
• Hodgkin's disease
• sarcoma

Decreases

• genetic deficiency
• decreased complement synthesis

• liver disease

• fixation of complement by:

• cellular or tissue bound immune complexes

• chronic glomerulonephritis
• rheumatoid arthritis
• hemolytic anemias
• graft rejection

• circulating immune complexes

• systemic lupus erythematosus
• acute glomerulonephritis
• subacute bacterial endocarditis
• cryoglobulinemia

Specimen

• 
  

• Aseptically collect a sample of blood (at least 2 mL) in an empty container, without anti- coagulant. Allow the blood to clot in ice. Separate the serum and store at -50 degrees C until tested. Do not use plasma.

Procedure

• Reagents & Equipment:

• Sensitized Cells. Standardized suspension of sheep erythrocytes, sensitized with antibodies to sheep erythrocytes, preserved with sodium azide, 3 mL per tube, 24 tubes. Diamedix Corporation, Catalog No. 789-001. Store tubes upright, at 2-8 degrees C. Do not freeze.
  
• Reference Standard. A reference serum with normal complement levels is required to standardize the assay.

• EZ Complement Standard, Diamedix Corporation, Catalog No. 789-006 (Store in the lyophilized state, at -20 degrees C), is a reference preparation suitable for standardizing the assay. This standard is prepared from human complement & is stabilized by lyophilization.
• Reconstitute with 0.3 mL cold (2 - 8 degrees C) distilled water. Swirl gently and refrigerate for 15 - 30 minutes before use. Remove from refrigerator and gently swirl again. Use immediately. Discard unused portion.
  

• Complement Reference Controls Low & High, Diamedix Corporation, Catalog Nos. 789-008 & 789-009. Packaged 8 x 0.3 mL lyophilized vials. Store at -20 degrees C. See Quality Control below for reconstitution instructions & handling.
  
• Pipet and disposable tips to dispense 5 uL.
  
• Centrifuge
  
• Diamedix EZ Reader Photometer, Diamedix Corporation Catalog #783-001.
  

• Calibration:
  
• Calibration of the EZ Reader is performed with the reference standard at the time of reading the test results.
• See procedure below.
  
• Quality Control:
• The reference controls (low positive and high positive) controls are prepared from human complement and are stabilized by lyophilization. Reconstitute with 0.3 mL. cold (2-8 degrees C) distilled water. Swirl gently and refrigerate for 15 - 30 minutes before use. Remove from refrigerator and gently swirl again. Use immediately. Discard unused portion.
  
• A low and a high positive control is run with each batch of CH50 assays. They should be within the range that is printed on the label of each vial used. If not, do not accept the run. The whole assay will have to be repeated. If still out of control, notify the supervisor for further investigation.
  
• General instructions:
  

• Set up as many tubes as there are specimens for testing, plus one tube for the reference serum, and one tube for the 'spontaneous lysis' control. The 'Spontaneous Lysis' control is simply one of the EZ Complement tubes containing only the sheep erythrocytes in buffer (no sample). Place test tubes containing the Sensitized Cells in a rack and allow to warm to room temperature (1 hour). Vortex or shake vigorously for 10 seconds to resuspend cells.
• Remove stopper and transfer 0.005 mL (5ul) of test serum sample, or reference serum, into a tube. Replace stopper & mix immediately by vortexing the tubes. The 'spontaneous lysis' tube should not receive a serum sample, but its contents should also be vortexed.
• Allow the tubes to stand at room temperature for 60 + 5 minutes. Mix the contents of all of the tubes again by vortexing.
• Centrifuge the tubes at approximately 1800 rpm for at least 10 minutes.
• Read the absorbance of the supernates in the EZ Reader Photometer within 15 minutes.
  

• Calculations:
  
• No calculations are required when reading the samples on the EZ reader. When a spectrophotometer other than the EZ reader is used to read the supernate,and results are to be reported out as '% of Reference,' the following calculations are necessary.
  
• Abs of Sample x % value of St'd = % of Reference
• Abs of St'd
  
• When results are to be reported out as 'CH50 Units', the following calculations are used.
  
• Absorbance of sample
• Absorbance of standard
  
• Reporting Results:
  
• Results can be reported as '% of Reference' or as CH50 . When reading the sample on the Diamedix EZ Reader, values that are printed under the 'conc' column on the printer tape are the '% of Reference' values for each sample if the % value of the reference standard is entered when the EZ Reader asks the user to 'Key value of S #1.' If the 'CH50 value' of the reference is entered column will be CH50 units. This lab reports the results out in CH50 units.
  
• Sources of Error:
  

• Serum not being properly stored prior to testing.
  
• Tubes not being well-mixed prior to & after adding patient serum, & before spinning tubes in centrifuge.
  
• Allowing reaction tubes to stand longer or less than 60 minutes, + 5 minutes.
  
• Not allowing tubes to spin for at least 10 minutes at 1800 rpm.
  
• Waiting longer than 15 minutes to read test after spinning down tubes.
  
• Distilled water not being chilled before reconstitution of standard and controls.
  
• Not swirling reconstituted controls and standard to dissolve all the lyophilized material on the sides and cap of vials. .

Notes

• 
  

• It is better that not more than 12 tubes be set up per run due to critical timing of assay

More General Terms

• special chemistry test

Additional Terms

• complement
• complement cascade

References

1. Package Insert, EZ Complement CH50 Assay, Diamedix Corporation, Miami, Fl, 1989
2. Tietz, Norbert W. Textbook of Clinical Chemistry, The W.B. Saunders Co., p. 579, 1986.
3. Rose, Noel R. and Freedman, Herman, Manual of Clinical Immunology, 2nd Edition, American Society for Microbiology, pp 166-167, 1980.
4. Stites, Daniel P., Stobo, John D., Wells, Vivian J., Basic and Clinical Immunology, 6th Edition. Appleton and Lange, pp. 277-278, 1987
5. Clinical Guide to Laboratory Tests, 3rd ed. Teitz ed., W.B. Saunders, 1995.
6. Medical Knowledge Self Assessment Program (MKSAP) 15, American College of Physicians, Philadelphia 2009
7. Interpretation of Diagnostic tests, 8th edition, Wallach J, Lippincott, Williams & Wilkens, Philadelphia, 2007, pg 14

CH50 Assay (serum)