======Principle=====
<code>
Adenosine ------> Inosine
            ADA
            
NH3 + Nitroprusside + HOCl 
</code>
{{:nchsls:c:biochemistry:document:ada_principle.jpg|}} 
======Procedure for Adenosine Deaminase examination======
|**references**|{{ADA Ref Literature 620.full.pdf|ADA reference literature}}|

|**environmental and safety controls**|{{Adenosine MSDS.pdf|Adenosine MSDS}}|
| |{{Ammonium Sulphate msds.pdf|Ammonium sulfate MSDS}}|

**Preparation of [[ADA reagent]]**

|The examination is done on Erbachem|  


**Preparation of ADA reagent for automated measurement**
|{{ :nchsls:c:biochemistry:document:ada_2021-06-28_with_pipes.ods |}}|
|{{ :nchsls:c:biochemistry:document:ada_2021-06-28_with_pipes...ods |}}|
|{{ :nchsls:c:biochemistry:document:ellis1973_good.pdf |}}|
|{{ :nchsls:c:biochemistry:document:0256-4947.1993.14.pdf |}}|
|{{ :nchsls:c:biochemistry:document:2182.pdf |}}|
|{{ :nchsls:c:biochemistry:document:ada016-en-ifu.pdf |}}|
|{{ :nchsls:c:biochemistry:document:ellis1973_good.pdf |}}|
|{{ :nchsls:c:biochemistry:document:enzyme_assays.pdf |}}|
|{{ :nchsls:c:biochemistry:document:bota2000-1.pdf |}}|
|{{ :nchsls:c:biochemistry:document:slaats1985.pdf |}}|
======Erbachem======
**Note:** Following instruction is useful only if operator is familiar with [[Erbachem]]

^Parameter^Value^
^Mode|Abs(absorbance)|
^Sample volume(ul)|10|
^Reagent volume(ul)|1000| 
^Filter|630 nm|
^Aspiration Volume(ul)|700|
^Factor|1|
^Decimal place|4|

^Calibration, Result calculation, Quality control and sample examination in Erbachem^

^Prepare batch of sample as below. This is batch calibration. Refer [[Calibrator and QC]]^^^^^^
|Standard 60|Standard 60|Low control|Patient Samples|High control|Standard 60|

|{{ADA record format.ods|Format for recording ADA results}}|
|{{ADA record format.pdf|Format for recording ADA results in pdf}}|

^Reagent/Sample^Calibrator Blank^Sample Blank^Calibrator^Sample^
|Adenosine Buffer|1000|1000|1000|1000|
|Sample| | | |10|
|Calibrator| | |10| |
|Incubate at 37'C for 1 hour|||||
|Phenol Reagent|50|50|50|50|
|HOCl Reagent|200|200|200|200|
|Incubate at 37'C for 20 minutes|||||
|Sample| |10| | |
|DI Water|10| | | |
|Read at 630 mm|||||
|All volumes are in ul|||||

  -{{:nchsls:c:biochemistry:document:ada_procedure.doc|ADA WDI}}
  -Switch on the incubator and set at 37'C.
  -Take out the required number of eppendroff cup of adenosine buffer from refrigerator And incubate at 37'C in the incubator for 10 minutes.
  -Mark ependroff cups test and blank for each sample id, standard (3 cups of T60(60 U/ml NH4+ calibrator) and one cup of water as blank) and control.
  -Add 10uL sample in respective test cups and 10uL standard in test standard.
  -Incubate at 37'C for 1 hour.
  -After 1 hour take out required amount of 1 ml phenol ependroff cup and 8 ml HOCL reagent bottle from refrigerator.
  -Now in both test cup and blank cup add 50 uL phenol and then 200 uL HOCl.
  -Incubate for 20 minutes.
  -And then add 10uL sample in respective blank cups only. In Blank standard add 10uL DI water.
  -Take reading at 630 nm filter.
  -Select "Run test" -> ADA in Erbachem menu.
  -When instrument asks "Aspirate water", aspirate DI water.
  -When instrument ask for "Aspirate standard", press 'No'. 
  -When instrument ask for "Aspirate sample",aspirate test and blank cups and note down the OD of all sample, standard and control.
  -Enter this OD in spreadsheet and calculate factor with OD of standard .
  -Calculate results=(Absorbance T-B)X(Average Factor) in spreadsheet.
  -Evaluate factor and control results. If factor/control results show major deviation from past, rerun batch.
  -If Sample OD is more than 2.7 OR result is more than 243 U/ml then dilute the sample with 1 part of sample and 2 part of DI water(1:3).
  -[[lis_calibration_entry|Enter Calibration in LIS]]
  -[[list_qc_entry_manual|Enter control results in repeat check manually in LIS]]
  -[[list_result_entry_manual|Enter sample results manually in LIS]]


======PROCEDURE FOR OPERATING THE INCUBATOR=====

To start the incubator, 
  -We have to confirm first that the incubator is properly set within the adequate range(36-38 *c). 
  -Then the water within the tray should be noticed if present in adequate amount so that the ependroff cups  get partially(75%) submerged in the tray of water. kindly look at the image for observation. {{:nchsls:c:biochemistry:document:c360_2018-09-03-14-41-22-249.jpg|}}
  -It also should be measured that the temperature of the water is within 36-38*c. 
  -To confirm the proper temperature first it should be calibrated and measured by separate external thermometer. 
  -Once the two requirements are fulfilled we can now keep our samples for further procedure.  
  -The door of the incubator should never be kept open for long and to be closed as soon as possible. And the switch of the incubator present in the clinical biochemistry lab should always be kept "switched ON". 

|{{29-12-2014-ADA linearity .ods|ADA linearity Check}}|  

here is the principle of ADA {{:nchsls:c:biochemistry:document:ada_principle.odt|}}