The monoclonal antibody used in these assays is referred to as CD four. This is the basic reagent to see ERT lymphocytes. This is to measure the number of CD four positive lymphocytes in the blood, which are gradually being destroyed by HIV.
And so the number of CD four cells is related to the immuno deficiency of the patient. Another reagent CD 45 is used to count all of the white blood cells, which is also a useful routine assay. An additional test for the so-called activation markers decreases the cost of viral load assays, which can be very high.
These studies have been carried out at the Whit Waters Rand University Johannesburg in South Africa under the guidance of Professor Deborah Glen Cross and Professor Wendy Stevens. The aim of this film is twofold. In the first part, you'll see how a well-equipped laboratory performs a CD four, CD 45, and viral load related assay.
This is in six modules. These technical steps are then implemented in 70 laboratories throughout South Africa. In the second part, the relationship is discussed between a large central laboratory that carries out thousands of tests each day and the more peripheral laboratories which are close to the point of care of patients.
The discussion here is focused on how to facilitate support for the treatment of more HIV infected patients with antiretroviral drugs. My name is Neil Ney. I Work, I'm a sec.
I'm a flow cytometry technologist. I work in the General General Hospital in Johannesburg, South Africa. This is the flow cyle.
This is, we use the flow cytometry. Beckman put the exhaust. Basically what happens, the library works in rhythm waste.
We, the sum that we do daily, like running of the use cell counting and flow instrument checks. There's the things that we do weekly like the archiving of the result. The some work that we do monthly like the maintenance.
When the clean cycle is done, we initialize the machine. We are pressing the run button. We run two land strips, two blade tubes, or you can run two principle and two distilled water tubes.
After all the cleaning is done, the first thing we run is the floor check to check the laser alignment and to monitor the voltage so that you make sure that the voltage remain constant. If you run the floor check in the morning and it fails, you need to try it again. If you still face, then we need to call main FA emissions to come and look at the machine.
cause there might be something wrong with the laser alignment or the voltages. We run immuno trials, the normal and the high every morning. They can be run at least in the eight hour shift.
So now we are running the high immuno cho. I'm going to check if the, the our cell counts are within the ranges. My Name is Denise Dory.
I'm the laboratory manager in the city for laboratory in the Johannesburg Hospital. I serve a national role in that. I've been involved in setting up quality assurance procedures to ensure accuracy and refugee disability of CD four count, not only in our own laboratory but in a national basis in South Africa.
Preparation of ping CD four samples is very simple. We use two monoclonal antibodies of different colors, CD four and CD 45. These normally come pre-mixed in one brown bottle that contains both antibodies.
To begin, we would prepare 10 microliters of the CD 45 CD four antibody into the bottom of a clean tube. Next, all the next step would be to add the blood. For this method we're using a hundred microliters of blood.
It's got to be accurately prepared. The blood and antibody would then be mixed together next. So the last stage of this preparation would involve adding eising reagent.
The, and as you can see, once you add the LY in reagent, the blood changes from an opaque rate solution to a hemolyze solution where you can actually see through the tube. If this is a very simple method, which can be done manually, but if you are doing large volumes of samples as most laboratories are, you would use an automated system such as this, which would do the pipe preparing for you. But the preparing is not necessarily necessary to be done through an automated system if you use the bee count rate because the beet counts, the beets which we add will tell us if somebody has perpetuated the sample correctly.
The the last stage before we would put this sample onto an analyzer is to add a hundred microliters of the beads which come in a tube base like this. The sample would then be ready to load onto flow cytometer. My name is sit MoFA And I'm the quality assurance officer and this flow cytometry laboratory and I love low cytometry.
We've got very beautiful machines here, which are the epics excels and they are very stable.Actually. We add our bits, which also show us the stability of the instruments. So how do we monitor our flow count rate?
Low account rate is all about the team. More recently we've started the automated preparation of our instruments, of our our samples, but still the monitoring of the flow account rate is still the relevant QC control. Our flow count will have this straight line, which shows how perfect the flow count rate is, but they still stand.
There are still some outliers, so these outliers can either be caused by pipetting error, but if it's we, we exclude the possibility of pipetting error, we can then check our instrument if it's functioning okay, and then we can call the engineers to come fix our instrument so that it can go on to the holder. This is Zo ly from Beckman Ter. He's our technician.
I just come anytime there's a problem and yeah, and also I come every six months to do maintainance As our samples are analyzed through the instruments. As a QA officer, my duty is to always check these screen, which monitors our flow count rate. So I immediately, there's an outlier.
I am able to see that there's an outlier. There are actually five instruments running at the moment because our workload is incredibly high and these two here just show it is not the sample which which are acquired. It's just the in that we do at the end of each carousel, but we still do do see some outliers.
For example, this one on this instrument, which is Epic Excel four. This one will definitely needs to be re repair immediately before the results as as sent out of that whole carousel and then after the resetting it must be run driven immediately Before ization. To Recap, we've established that our machines are running optimally.
We've checked the reproducibility of the counting of the beads to ensure that the CD four counts, which follow will be accurate. We've checked that the lasers are properly aligned on our instruments and we've checked the accuracy of counts by running two levels of controls. This sample, no content, the CD four and CD 45, and I need to describe to you why CD 45 is so important in our analysis.
CD 45 is a cell marker, which marks all white blood cells in a sample. This is important for us in this context in that it can allow us to compare the white cell count we obtain, which has been determined by using CD 45 with the white cell count from a full blood count analyzer. If you see on these examples here, this is a full blood count printout from an LH seven 50 Beckman Coter analyzer and we can see we have a white cell count of 7.1 on the floor cytometer using CD 45 where we stain the total white cells.
We have a very similar CD weight cell count of 7.175. We would accept a value, a difference in weight cell cones of less than 5%The other added advantage of the inclusion of CD 45 is the differential staining of CD 45 on white cells. On the flow cytometer, we can use three say scatter, forward scatter and CD 45.
If we look in this histogram here, the lymphocytes are clearly defined. They tend to be the brightest staining with CD 45 and they've got the lowest complexity, they're the simplest cells and the greatest with CD 45. The next group of cells is our monocytes.
Our monocytes have slightly less CD 45 expression, but they're more complex than the lymphocytes. Then the next major group of cells, which in a normal sample would be the largest group of cells, is our granular cells. These populations are the most common blood cells seen in the peripheral blood.
The advantage of using CD 45 staining on a flow cytometer is that one. We can do a CD four current, an absolute white cell current and an automated differential current on the floor cytometer. The CD four five standing allows us to do differential cones for longer periods of tame.
We can do them up to 48 hours. If we're using CD 45 expression. The differential counts done in this method are less objective than other methods.
For example, the gold standard of doing a manual differential count, we can pick up leukemic populations and it also forms the basis for the pan lubricating cd, which is shown on the screen here. The advantage of CD 45 expression on white blood cells has not really been fully utilized in hematology laboratory so far. We have a variety of different full blood count analyzers which use different techniques to obtain weight cell differential counts.
Using CD 45 on a floor. Cytometer would be a brilliant way of standardizing the differential cones between different hematology analyzers because it would be one method that all of the other hematology analyzers could Be compared to. Hello, my name is Dr.Linda Sier.
I am actively involved in HIV research involving both CD four and CD eight testing. T lymphocytes can be divided into CD four positive cells and CD eight positive cells and CD four positive cells are used to monitor HIV in patients on treatment. The problem, however, with CD four is that it is not exclusively expressed on lymphocytes but also express CD four.
In the PLG protocol, we look at three different aspects of how to effectively discriminate between your monocytes and your lymphocytes. Firstly, we use complexity or granularity where you can see that if you look at complexity, your lymphocytes and monocytes can be separated based for that purpose. We also use CD 45 C where you can distinguish your lymphocyte population from your monocyte population.
CD four expression is bright on your lymphocytes and intermediate on monocytes used together with complexity. It is quite easy to distinguish between your CD four bright lymphocytes and your CD four dim monocytes and I will show you an example of a normal patient sample where you can see the lymphocytes and the monocytes this patient had is CD four count of over 700. If we look at a patient with a CD four count of 53, you can see that even though it's got a low CD four count, the discrimination between your positive lymphocytes and your CD four positive monocytes, it's still very clear.
Using the PLG method, we are able to do high numbers of analysis per day. In our laboratory, we currently do about approximately a thousand samples per day, which amount to 5, 000 samples per week. Two weeks worth of our work is approximately equivalent to that done in a whole year.
In a London hospital In South Africa, we have 70 NNRS laboratories and over a period of a year these laboratories do approximately 1.98 million CD four tests per year. Hi, I am Kni. I'm currently on my tea break, but after I'm done I will tell you about CD 38 and vital load.
So basically we all know that flow cytometers can count cells, but what they also can do is count the surface molecule, have molecules on the surface of cells. This is done by measuring the fluorescence intensity. Under standard conditions which have been previously mentioned, we use CD four and 45 known as the PLG method.
We are going one step further by putting two tests into one tube. Name the CD 4 45 PLG test method. We are gonna combine this with a two color test for activation marker or other viral load linked parameter, namely CD 38.
The Americans use four color immunofluorescence. They use CD four, CD 45, CD eight, and CD three. We use CD four, CD 45, CD eight, and we replace the CD three by using CD 38.
Firstly, it's important to note that a baseline CD 38 needs to be taken for a patient as the baseline 38 acts as a control for that particular patient, And that's when the high viral load is high, isn't it? Yes.And that's when a patient comes in with a high viral load and a low CD four count. That's at the start before therapy.
We also need to pay attention to CD 38 blips where there's an increase in a CD 38. This is evident in this patient here where there is a sudden increase in the CD 38 MFI and if you look, there's also a corresponding increase in the viral load. Is it possible that the patient was not taking the drugs at that time?
At this point, yes, it is possible that the patient stopped taking therapy because the levels go back to normal. So why are we so happy with this test? Firstly, it's combining two test methods in one tube.
Secondly, it is cost effective by giving us a CD 45 white cell count, a CD four count, as well as an activation marker. It is used on the instruments that are currently in our labs, so there will be no need to bring in new instruments and staff can be easily chained on how to gate and how to prepare these samples. Viral load costs will also decrease because currently every time a patient comes in for a CD four count viral load tests are also being done.
But if we monitor the CD 38 and if we notice that there's an increase in the 38 MFI, then only can we send the patient samples for the viral load test. On the Performance of our South African national program, which is the NHLE CD four program that uses PLG, where we support our labs a lot. So we have a central facility, central facility coordinates all the standardized procedures, all the quality control that's decentralized to the remote labs.
They run their system and they report back through EQA through our Africas back to the, through the support of the central lab. Also with additional and ongoing training initiatives and support services. And our program as a group performs significantly better than other national programs significantly so, like more than 50%on precision, not only within labs, but between labs.
So that means like CD four from a, from a lab in Berley will be very similar to a CD four from a lab from Durban, for example, using our standardized surface. So you take that concept and you decentralize it into point of care. Two, it means that the point of care will be facilitated through one Coordinating unit.
It was obvious that these efforts, which were shown by these dedicated people are conceptual advances, not just back one quarter flow cytometer, which can produce these results. The same concepts can be used with other instruments like Ton Dickinson machines or Partec machines or made by Accur or any other firm. In fact, recent developments from other countries, for example, India, are showing that they should be starting their own flow cytometry industry, which would use the same technology.
The only important issue here was the exceptional coordination. Now, those people who think that CD four counts are the only aims are mistaken because here we were dealing with wide blood cell counts, absolute numbers, wide blood cell count differentials. We were dealing with CD four and we were also dealing with activation markers which make the assays cheaper than the comparable viral load assays would be.
Nevertheless, this is not a competition between these activation markers and the viral loads, but a complementary kind of arrangement which saves money on the viral load assays. These have been scientifically cleared investigations, and the clinicians should look for these essays when the patient's compliance with the therapy is in question, whether the drug efficiency is in question or whether they need to change therapy because the patient starting to develop resistance. The essential conclusion have been the good quality assurance now, which leads us to the final point, and this is that it is much better to have excellent central laboratories, which can then provide flow cytometry and quality assurance, assess for the peripheral labs, and then the peripheral labs will be viable to catch more people into the antiretroviral therapy regimes.
So therefore, here we are seeing new development from the African soil. Thank you very much for your attention.
