======Hemoglobin electrophoresis and Dithionite test-(use this)======
====== examination procedure======
  *Learn operation of [[Electrophoresis tank]] and [[Electrophoreis Power supply]]. 
  *Read following documents

|**References**|
|{{:nchsls:c:biochemistry:hemoglobin_book2.pdf|Hemoglobin reference literature}}|

|**environmental and safety controls**| 
| |{{:nchsls:c:biochemistry:document:msds.pdf|MSDS of agarose}}|
| |{{amido black msds.pdf|MSDS of Amido black}}|
| |{{:nchsls:c:biochemistry:document:msds_of_boric_acid.pdf|MSDS of boric acid}}|
| |{{:nchsls:c:biochemistry:document:msds_of_kcn.pdf|MSDS of KCN}}|
| |{{:nchsls:c:biochemistry:document:msds_of_triton_x.pdf|MSDS of triton X}}|
| |{{msds_Tris base.pdf|MSDS of Tris base}}|
| |{{msds_EDTA.pdf|MSDS of EDTA}}|
| |{{:nchsls:c:biochemistry:document:methanol_of_methanol.pdf|MSDS of methanol}}
| |{{AceticAcidGlacial.pdf|MSDS of acetic acid}}|
=====Introduction=====
Electrophoresis is a comprehensive term that refers to the migration of charged  solutes or particles of any size in a liquid medium under influence of an electrical field.

  *Document and revise [[common laboratory informations related to the examinations]]
=====Steps of Hemoglobin electrophoresis=====
====Sample preparation====
  -Whole blood sent in EDTA vacuttee is acceptable.
  -If required the procedure can be performed with clot of plain sample or flouride sample.
  -Perform the procedure within a week preferably on thursday or friday.
    -Complete analysis within the evening of run.
    -Do not wash samples till day of analysis, because RBC gets nutrients from plasma. They are better preserved in whole blood.   
  -If RBC have settled completely, remove supernatant plasma
  -If RBC have not settled completely, centrifuge 3000 rpm for 10 minutes before removing supernatant.
===New Procedure to wash RBC===
  -Prepare wash solution containing 10 mmol/L EDTA and 5% Glucose. [[Dextrose Wash Solution Preparation]]
{{:nchsls:c:biochemistry:document:1sample_preparation.jpg?direct&700|}}
  - Add Dextrose Wash Solution upto 4.0 ml in EDTA vacuttee.
  - Centrifuge the EDTA vacuttee at 3000rpm for 10 minutes.
  - Remove saline part from the vacuttee.
===Alternate Old Procedure to wash RBC===
  -__First wash__
    -Add Normal saline upto 4.0 ml in EDTA vacuttee.
    -Allow thoroughly mixing of Normal saline  & cells parts of sample By inverting a few times.
    - Centrifuge the EDTA vacuttee at 3000rpm for 10 minutes. 
    - Remove saline part from the vacuttee.
  -__Second wash__
    -as above
    -{{:nchsls:c:biochemistry:document:sample_preparation_final.jpg?700|}}
  -Prepare a set of eppendroff cups with sample_id label to match the sample batch.
  -Take 100 ul of [[Hb A Hb F Hb S control(s)]].Take 100 microliter of control & tests samples in eppendroff cups.Add 100ul [[Haemolysate solution]] in each cups for hemolysis.Mix it properly.Make sure that sample becomes completely clear.
  -[[Half diluted TEB buffer]]


====Gel preparation====
  -The surface of the slide on which gel has to be applied, is made rough using a glass paper.This is done for proper fixation of the gel on the slide.
  -Before applying the gel on the slide wash 2 slides(Non siliconized slide{{:nchsls:c:biochemistry:document:siliconize.pdf|Siliconization of Glassware}} on which gel will be applied and siliconized slide will be used as a slider) and dry it with tissue roll.
  -Place 2  slides on a  levelled surface as shown in above figure.Cut a rectangle of slide  size from a x-ray plate, cut a four boardered frame  from  a   x-ray plate,so  the thickness  of  gel is same as thickness of x-ray plate{{:nchsls:c:biochemistry:document:c360_2020-01-30-12-04-12-568.jpg|}}current slide
   {{:nchsls:c:biochemistry:document:ssssfriday_15_september_2017_-_03_26_32_utc.jpg?direct&700|}}
  -prepare [[1% Agarose with TEB buffer]]
  -Then pour hot agarose gel on non siliconized slide carefully.Make a wedge of siliconized slide on non siliconized slide containing agarose gel(as shown in figure).
  -{{:nchsls:c:biochemistry:document:gel_preparartion-3.jpg|}}
  -Carefully press the non siliconized slide over the siliconized slide,so that air do not get trapped in between two slides.
  -Gel will be ready to use after 10-15minutes. 
  -then carefully remove the non siliconised slide just before the sample application and should be away from the fan or heat.
    {{:nchsls:c:biochemistry:document:sslide.jpg?direct&800|}}
  -Use only freshly prepared slide as slides kept in refrigerator cause significant increase in endosmosis.
====Preparation of Applicator====
**OLD METHOD**
  -Bring used histopathology blade from histopathology department after permission of in-charge of that respective department.
  -Make 2mm slot at every 5mm distance to prepare comb.
  -Wrap 3 layers of cello tape at each end of applicator comb so that level of applicator comb will remain faction of a millimeter higher from the gel so can prevent through and through cutting of gel and spreading of samples beneath the gel.
  -{{:nchsls:c:biochemistry:document:applicator-11.jpg|}}
  -Make assembly as shown in photograph below for holding sample applicator over the gel.
  -{{:nchsls:c:biochemistry:document:elecrophoresis_apperatus.png|}}
  
**NEW METHOD**
  - use the blade(applicator) on which a magnet is attached at the back, fix it temporarily with the scale behind.
  - See proper alignment.
  - place an empty slide beneath the comb.
====Loading of samples====
   -Loading sample over applicator:
    -the applicator is already attached to the scale by the help of magnet
    -place 2 Ul hemolysed sample on the root of each comb, 
    -if there are less number samples, then preferably skip the first and last comb, 
    -repeat the sample at least twice.
    -{{:nchsls:c:biochemistry:document:c360_2020-01-30-12-03-53-482.jpg|}}
  -{{:nchsls:c:biochemistry:document:c360_2020-01-30-11-59-49-814.jpg|}} SLIDE'S COMB IS FEEDED WITH BLOOD SAMPLES
  -after applying the 2ul sample pull the applicator and wipe off extra blood from behind  the applicator with a little piece of tissue paper. {{:nchsls:c:biochemistry:document:c360_2020-01-30-12-05-07-873.jpg|}}
  -Loading sample over Gel: 
    -put prepared gel slide beneath the applicator.{{:nchsls:c:biochemistry:document:c360_2020-01-30-12-06-47-570.jpg|}}
    
    -The applicator  is  put  over one end of  gel loaded slide away from margin.
    -Wait for 2-3 minutes,allow the samples to get completely absorbed within gel otherwise samples will spread.{{:nchsls:c:biochemistry:document:c360_2020-01-30-12-07-07-284.jpg|}}
    -Then remove the applicator carefully.{{:nchsls:c:biochemistry:document:c360_2020-01-30-12-08-09-199.jpg|}}
    {{:nchsls:c:biochemistry:document:prepared_gel_slide.jpg?direct&500|}}
    -{{apperatus.jpg|}}

====Running of samples in electrophoresis Tank====
    -take clean, dry electrophoresis tank 
    -fill the both compartment of the tank with TEB buffer with the container till the arrow mark, below separately in each compartment (*same buffer should not be used twice and should be discarded) {{:nchsls:c:biochemistry:document:c360_2020-01-30-16-38-14-723.jpg|}}
    -prepare wick from filter paper. Cut filter paper 7cm (width) X 17 cm (Length) and fold it 1 CM inwards. Wet with TEB buffer. wick should not be used twice and should be discard after one use.
    -After successful sample application,put the gel slide in electrophoresis chamber. 
    -Slide should be kept in such a way that the gel is directly in contact with wick. 
    -Connect the electrode  with the electric box  red to red and black to black) of the power supply.{{:nchsls:c:biochemistry:document:c360_2020-01-30-16-39-45-748.jpg|}}
    -Set voltage at 350 to 370 V, ampere 7-15 A and run for 15 to 30 minutes until the bands reach the mid portion.
    -After run,switch off  the power supply & remove the slides.
====Staining of samples====
  -Put the sample slide in methanol for 10 minutes for removal of excessive water molecules around proteins.Everytime use fresh methanol.
  -Preheat the Microwave oven or incubator for 5 to 10 minutes.
{{:nchsls:c:biochemistry:document:pre_heat_microwave.jpg?direct&500|}}
  -Take away the slide from methanol solution, With a piece of tissue paper,wipe away methanol from the opposite surface of the gel slide .
  -Allow it to dry in pre heated oven after switch off it 
  -Do not touch the surface containing gel with samples.
  -Denature protein and fix the gel by heating in pre heated oven.
  -Do not heat the slide constantly as it may get broken down due to excessive heat.
  -Allow the slide to be cool.
  -Remove the X-Ray film carefully without disturbing the gel.
  -Take around 2 ml of a staining solution [[Amido black stain]] on the slide and spread it with glass rod on slide so as to stain the proteins over the gel surface.
{{:nchsls:c:biochemistry:document:staining.jpg?direct&500|}}
{{:nchsls:c:biochemistry:document:staining2.jpg?direct&500 |}}
  -Remove the excess stain by putting in the destaining solution [[5% glacial acetic acid]] for 15 minutes.
{{:nchsls:c:biochemistry:document:destain.jpg?direct&500|}}
  -Allow the slides to dry at room temperature or inside the micro wave.

====Reporting of result====
  -label the slide with appropriate sample id according to sample application.
  -take a picture of the the slide applying a grid, make it black.
 {{:nchsls:c:biochemistry:document:img_20200123_115144.jpg?600|}}
  -open gel analysis, segregate the individual sample {{:nchsls:c:biochemistry:document:screenshot_from_2020-01-23_15-33-02.png?800|}}
  -save each sample in the computer for entering in the data base(cutting of each slides used as attachments){{:nchsls:c:biochemistry:document:104098.jpg|}}
  
====Entering of result in database====
  -Go to Application menu----->Click on web browser----->Open 10.207.3.240
  -Do Login from your login ID.
  -Click on "Edit"----->Click on "Manage attachment" as shown in below figure.
  -{{:nchsls:c:biochemistry:document:result_screenshot-2.jpg|}}
  -2 attachments should be done - 1st is "electrophoresis" and 2nd is "Interpretation" file.
  -{{:nchsls:c:biochemistry:document:screenshot_of_1st_attachment.jpg|}}
  -Do 2nd attatchment as shown in below figure.
  -{{:nchsls:c:biochemistry:document:2nd_atttchment_of_electrophoresis-a.jpg|}}
  -After click on "Browse",follow the sequence from where you find the "interpretation file(txt file)".
  -Application menu---->File manager---->Back up1---->Biochemistry---->Department---->Parameter details---->HBe---->hb interpretation---->select appropriate attatchment.
  -Click on "Edit" and enter relavant sample id as shown in below screenshot.
  -{{:nchsls:c:biochemistry:document:final_mstep_of_result_enterina-a.jpg|}}

======Dithionite test======
|**References**|
|{{:nchsls:c:biochemistry:document:dithione_ref.pdf|Dithionite reference literature 1}}|
|{{:nchsls:c:biochemistry:document:hemoglobin_dithionate_reference_literature_2.pdf|Dithionite reference literature 2}}|
{{:nchsls:c:biochemistry:document:dithionate_test_latest.ods|}}
 
===Interpretation===

  -{{Hb Variants.ods|Various types of hemoglobins}}
  -Hb A is moved fastest,then Hb F And Hb S moved very slowly, so its band is near application sites. 
  -Correlate result of Hb electrophoresis  with  Dithionite test.