The large number of globin gene mutations
in the human genome has resulted in several hundred hemoglobin protein variants.
Although many of these are unimportant clinically,
some such as hemoglobin S and hemoglobin O-arab are associated with significant pathology.
There are two main detection and analytical methodologies currently in clinical use,
hemoglobin electrophoresis and high pressure liquid chromatography, H P L C.
Although isoelectric focusing and globe-in-chain electropheresis are also available.
This video concentrates on the analysis of hemoglobin variants
by electropheresis and uses the Helena Biosciences system.
You can see their SAS 1 plus automated electrophoresis unit,
the smaller unit on the right, coupled with the SAS 2 stain and D stain unit.
The larger unit in the middle,
you can see the stain and D stain reservoirs on the left.
Helena Biosciences produce a range of preformed gels
for various analyses supplied in foil packs with the acid
and alkaline hemoglobin gels being the appropriate ones
for the separation of hemoglobin variants.
Washed packed patient red cells need to be liced using the provided Triton X 100
based --- reagent to yield a released hemoglobin solution
that can then be diluted to about twenty milligrams per mill.
The white sample tray strips are produced for various formats of the Helena units
with different sample capacities.
The SAS 1 plus unit takes twelve samples
and therefore the strip needs to be shortened as it’s being shown here,
before being inserted into the holder.
The gel to be loaded in the next part of the presentation is an acid gel.
That is one where the incorporated buffer normally has a pH around six.
Under the acid conditions hemoglobin variants migrate variously
from the point of application towards the anode or the cathode,
and therefore the samples need to be applied in the middle point of the gel.
Whereas with the alkaline gel with a pH normally in the range 8.4 to 8.6,
the hemoglobin variants all migrate towards the anode,
so the samples are loaded on the gel more to the cathode end.
Thirty five microliter hemoglobin samples are loaded into the appropriate wells
of the disposable sample tray strip.
Here in close up you can see the loaded sample tray.
So for the alkaline gel, we insert the sample strip into the upper slot,
and with the acid gel we would use the lower of the two slots.
We've only loaded sample trays into both slots.
Here to illustrate the alternative positions.
The gel tray of the SAS 1 plus electrophoresis unit is then opened
and the sample holder placed in position.
Four hundred microliters of buffer is pipetted onto the heat sink
to ensure good contact between the gel and the heat sink.
The gel in this case, the acid hemoglobin gel is removed from its foil,
its protective foil and plastic pack, and carefully lowered into position.
A clean --- tip can be used to finally adjust the gel position.
The gel surface is then carefully blotted to remove any excess buffer
and to ensure that when the samples are applied,
they enter the gel rapidly exactly at the point of application.
The carbon electrode rods are placed in position on the gel buffer blocks at each end
of the separating part of the gel, again, ensuring that the electrodes
are in contact with the metal posts.
The gel cover is then put in place.
The samples now need to be applied to the gel using a sample comb.
The bar protecting the sample application teeth is removed.
The comb is put in position.
It only fits one way round for the acid hemoglobin gel the comb is located in the front pair of the two sets of slots.
With the alkaline hemoglobin gel, the comb would be located in the rear pair of slots.
The process from here is automated after programming the unit appropriately.
Once the program is started, the sample comb loads with the samples
and then applies moves to apply the samples to the correct point on the gel.
The comb then moves back to the sample tray reloads,
and then applies a second --- of sample to the same gel position as before.
The draw then closes and the electrophoretic step starts sixty volts for twenty two minutes
with the acid gel and two hundred volts for thirty minutes with the alkaline gel,
the sample comb is then discarded.
At the end of the electrophoretic run the display indicates that the run is finished,
the gel drawer is opened,
and the gel cover and carbon electrode rods are removed
together with the sample tray.
The gel is then carefully lifted
and using the gel slicer.
The gel buffer blocks are carefully cut away from the running gel.
The gel on its plastic backing is lifted carefully
and set in the metal holder of the stain destain unit.
This holder with the gel is then inserted into the slot in the top of the SAS two unit.
Again, this unit is programmable with the specific stain protocols.
Acid --- for the acid gel, acid blue for the alkaline gel,
and specific destain protocols for these two types of gel.
Again, the display indicates when the stain destain program is complete.
The gel is then ready for visual inspection
and may also be scanned to enable identification of weak bands
and to obtain an estimate of relative band concentrations.
The stained and dried gel film shown here is an acid gel with various hemoglobin variants.
The results shown here is an alkaline gel rather less than ideal
as the gel has retained too much background stain.
Hemoglobin A is in tracks three and five, hemoglobin A2 in track four.
Hemoglobin S is in track seven, and a mixture of A2 S, F and A are in track eight.
You can see that even if the gel background were much lighter,
visual inspection alone would almost certainly not allow you to distinguish all the components
of the mixture in track eight.
Next, you can see the scans of each of the sample tracks
with the protein concentration being displayed graphically.
In each scan, the anode is to the right and the cathode to the left.
In this first scan here of track three, you can see a strong fast running hemoglobin A band
with a much slower running, much weaker A2 band.
Here in track four, you can see a much stronger A2 band.
Track five shows a strong fast running A band this time with no A2 band.
Track seven shows a strong hemoglobin S band that runs between A2 and A.
And in track eight in the hemoglobin mixture as we move from the slow bands
to the faster bands, you can see that the scan has identified a weak A2 band, a stronger S band,
and strong F and A bands distinct but close to one another.
You can find a further account of hemoglobin electrophoresis
in the Daisy and Lewis Practical Hematology text.