PROTOCOL: Dual Platform PanLeucogated (PLG) CD4+T cell enumeration.

 © AffordCD4.com, 2002.

 Debbie K. Glencross

Technical Assistance: Lesley Scott and Denise Lawrie

Please Note: This protocol is in a state of evolution. Constructive critical input and comment is always appreciated. Although this rough guideline has been written in a way to include all the relevant steps to ensure that testing can be adequately reproduced in other centres, we hope that those centres using PLG CD4 testing will help us to improve this method through follow-up and usage of the method.

Sample Collection:

1.   Use K₃EDTA appropriately filled Vacutainer™ tubes or equivalent collected blood.  Avoid use of heparin or ACD as anti-coagulants.

2.   Maintain temperature during transport @ ~ 25^oC or less.

3.   Storage of whole blood should preferably be at room Temperature 25^oC.

4.   Appropriately labelled as per GCP

Specimen integrity.

1.   Avoid use of obviously haemolysed, clotted or frozen samples.

2.   Samples that are up to 7-8 days old (received after the date of venesection can be used with the SP PLG CD4 method of testing (see below).

Dual Platform WCC requirement.

1. If the Dual Platform option is preferred, then heparin anti-coagulated samples should be avoided to ensure accurate White Cell Counting.
2. WCC should be performed within 18-24hours of venesection or as specified by the haematology analyser manufacturer.
3. The White blood cell differential or the Absolute Lymphocyte count is NOT used for the calculation of the PLG CD4!
4. Adequate internal quality control and external quality assessment is performed on the haematology analyser used to derive the WCC.

Single Platform Option.

1.      If an adequate WCC is unavailable or if the samples are received > than 36-48 hours after time of venesection, then a SP volumetric option for PLG CD4 enumeration is recommended (Ortho Cytoron Absolute).

 2.     If the use of beads is the only option, then BC Flow Count beads are preferred by the Glencross/ SA NHLS Flow unit for use with PLG as it allows for easier and automated gating of the CD45 population (FL expression of these beads is well beyond that of the CD45 expression). The PLG principle is possible on BDS instrumentation utilizing TruCount ™ beads provided that careful gating of the CD45 population is performed (preferably manually for each sample as the Trucount bead population sits very close to the CD45 expressing populations and gates need to be very carefully drawn so as not to include the beads in the CD45 gate).

Flow cytometry Beckman Coulter option: guidelines for optimal PLG CD4 testing

1.      The antibodies that are recommended for use in the PLG CD4 method include 2D1 or the BC CD45 FITC (KC56 clone) and the locally produced RFT4 PE clone.

2.      Titrate monoclonal antibodies appropriately for use in the testing prior to patient testing, and document dilution with Lot number and expiry date of reagents. Decide on the appropriate dilution to use. Make up dilutions as freshly as possible (especially commercial product) and avoid storing diluted product. File all titration experiment with Lot number and expiry date of product used and clearly indicate refrigerated storage site.  BC CD4 RD-1 is also recommended for use. In our experience this product can be optimally used after appropriate titration (often to 1:2-1:4 of product added to working solution) (final dilution proportional to actual working volume used). Separation of monocytes from is adequate for the purposes of defining the CD4 T cell subset, provided that SS is incorporated as a discriminating parameter (see below).

3.      Ensure that antibodies are prepared and ready for use prior to commencing preparation of samples. Double check!

4.      Set up the protocol/ panel for use on the BC XL or equivalent as per the described BC XL MCL analysis below. This includes setting the FL1 as the primary threshold, setting the sensitivity on the FL1 (CD45) PMT so that the granulocytes fall just inside of the third log decade of FL1, and well within the third log decade of the SS expression. A heterogeneous gating strategy improves the resolution of the respective white blood cell population and includes the use of SS (Side Scatter, recommended as a log scale although linear SS expression can be used). The use of log CD45 versus SS (log or linear) will define the CD45+++ lymphocytes that are used to calculate the CD4% of lymphocytes (see annexure 2).

5.      Sensitivities of the FL2 PMT should be adjusted so as to place the CD4+ T cells at least within the third log for FL2 for the adequate separation of the CD4 T cells. Use of this gating strategy ensures that the monocytes are easily discernable from the CD4+ T cells and fall towards the third log of the SS scale.

6.      Make sure that the ImmunoPrep/ Qprep instrument is in good working order, is properly calibrated (there should at least be a monthly calibration procedures in place) and that the instrument is dispensing the correct amounts of the Reagents ABC (laboratory staff will get used to the final volume of "QPrepped" samples and should notice straight away whether the Qprep is dispensing correctly or not).  Ensure that there is sufficient reagent for the number of samples that will be processed at that time.

Procedure for preparation of whole blood samples

    1.      Label 1 x 2ml blue B Coulter flow cytometry tube with the patient’s given laboratory number.

    2.      Dilute monoclonal antibodies

                                                 i.      CD4 RFT4 (AffordCD4)

 1.      Dilute 1:100 from “Neat” (1µl mAB plus 99µl PBS),

 2.      Add 20µl to the blue tube

                                                ii.      CD45 FITC (Beckman Coulter, KC 56 Coulter Clone)

 1.      Dilute 1:4 (30µl mAB plus 90µl PBS)

 2.      Add 5µl of the diluted mAB to the same blue tub

3.      Add 100µl the whole blood sample (wipe the pipette tip with gauze) to the pooled mAB’s in the blue tubes and mix gently (NO vortex!).

4.      Incubate for 15 minutes in the dark at 20-25oC.

5.      Set up the Immunoprep/ Qprep.

6.      Store tubes with covers at 4°C until time of analysis on the XL MCL.

XL-MCL analysis

1.      Select protocol “PanLeucogating CD4”

   The Protocol should ideally be set up as follows:

i.           The PanLeucogated CD4 gating strategy includes use of FL as the primary identifier and SS (side Scatter) as the secondary identifier.

ii.         Set the threshold for data capture on FL1 such that all the CD45+events are clearly and easily seen, and that background FL1 noise is excluded.

iii.       Ideally, set the sensitivity on the FL1 (CD45) PMT so that the granulocytes fall just inside of the third log decade of FL1, and well within the third log decade of the SS expression (the “dilution”/ titrated amount of antibody can also affect the position of the CD45+ populations on the histogram).

iv.        Sensitivities of the FL2 PMT should be adjusted so as to place the CD4+ T cells at least within the third log for FL2 for the adequate separation of the CD4 T cells. Use of this set-up ensures that the monocytes are easily discernable from the CD4+ T cells and fall towards the third log of the SS scale (once again, the “dilution”/ titrated amount of antibody can also affect the position of the CD4+ cells on the histogram).

v.          Use of SS with FL2 as a heterogeneous gating strategy aids and improves the resolution of the CD4 T cells from the monocytes respectively (a log scale of Side Scatter is recommended although linear SS expression can be used if preferred). This is important as CD3 is not used to define the T cells in this instance but the intrinsic SS with CD4 expression is used instead (and is equally as good as using CD3, which only adds expense to the test if CD4 enumeration is the primary objective).

vi.        The use of log CD45 versus SS (log or linear) will also aid in the definition of the CD45+++ lymphocytes that are used to calculate the CD4% of lymphocytes (see calculation of CD4% of Lymphocytes.

vii.      Set-up three (3) gates.

a.      The first gate is set around all CD45+ events. Label this gate “A”.  (See Fig 1)

b.      Exclude very few, very low SS events from this latter gate that appear due to the fact that the threshold is set on FL1. In fresh samples, FS may also be used as a threshold but it is not recommended for use in aged samples! (see Figure 2).

c.      DO NOT USE Forward Scatter (FS) AS A THRESHOLD in samples that are older than 24-36 hours. The number of ”true” WCC/ CD45+ event capture rate will decrease and result in an underestimation of the flow calculated WCC and CD4 T cell count. (Dead and dying cells in ageing samples fall below the typically set FS thresholds and are excluded or “lost” in the acquisition of data. Use of a FL threshold and identification of specific CD45 expression ensures that all CD45+ events are captured in spite of significantly “morphological” disintegration of the blood cells.

d.      The second gate is set around the low SS, CD4++/ +++ cells (third log decade FL2) to capture CD4 T cells. Label this gate “B”. Monocytes should be easily discernable on this histogram and should fall within the second/ third log decade on SS and within the second log decade of FL2 (see Fig 1), and should not be included in “Gate B”.

e.      The third gate is set around the bright CD45+++ cells to measure the % of lymphocytes of the total white blood cells. Label this gate “C”.  NB: The value obtained from this gate is NOT used in the calculation of the absolute PLG CD4 T cell count result, but is only used as a means of calculating the percentage of CD4 T cells of Total Lymphocytes for clinical use. The calculation of this value is optional but may be if value especially for paediatric CD4 reporting.

viii.             Generally a rectangular gate is optimal to define Gate A/ the CD45+ population. Amorphous or rectangular gates can be used to define Gates B and C, although rectangular gates are more easily used as a “fixed gated”. The BC XL only allows the use of a single “auto” gate. We have not typically used this facility for PLG CD4 testing.

ix.                 Set up the gates and use them as described in Figure 1 below.

x.                   All samples should be analysed accumulating a total of at least 30-35000 events to ensure that at least 5000 relevant lymphoid events are counted and at least 1000 (preferably 2000 beads for better accuracy) are counted (absolute minimum number of lymphoid events acceptable for adequate statistical analysis is 2000).

xi.                 It is advised to save all data in Listmode format (especially if it is research related data). Remember the Shapiro law!

2.   Important (as mentioned previously) to always ensure that the monoclonal antibody concentration (dilution) is adequate – cells will effectively be excluded or “lost” if careful attention is not paid to this.

     1.      If one is using the MCL carousel for batching samples, then run carousel in auto mode. Establish that fixed and relevant auto gates are appropriately positioned before commencing carousel acquisition. Move gates accordingly if required as mentioned above.

     2.      Enter data results in laboratory data base system/ Excel data sheet with WCC or download through interface to lab computer system.

FACS/ ~Calibur analysis

1.      The PLG principle can easily be applied for use on a BDS instrument (but not FACSCount at this stage). The protocol should be set-up using the exact same principles as described above for the BC XL machine.

2.      Actual examples are available on request.

3.      Lesley’s tip: Threshold first on FL1 (CD45 FITC) and set secondary threshold on SSC (this works the best actually – it is a pity that one cannot set a secondary threshold on the BC XL as well but this is effectively achieved by gating on CD45 anyway).

4.      Lesley S’s tip: Ensure event counts in the statistic box follows the correct gating logic and is appropriate to the specific histogram i.o.w set up your histograms properly and make sure that you understand Boolean logic or else your results will not be accurate.

5.      Remember that if one uses Flow Count beads on FACS machines then the absolute should be effectively doubled. Why? Well the Flow Count concentration is for the beads alone and the concentration is effectively halved if an equal quantity of blood is added. With Trucount, the concentration is that of the final working volume. Always read the package insert carefully because it can be very confusing if bead types are used interchangeably.

Absolute CD4+  T cell calculation

CD4 +, low SS cells (Gate B events) / CD45 positive cells (Gate A events) x WCC = absolute CD4 (cells/µl)

CD4 positive cells/(Gate B events) x 100/ CD45 +++ (bright) lymphocytes / (Gate C events)  =  CD4 %

Figure 1.  CD4 enumeration by CD45 assisted PanLeucogating of a fresh sample (Day 0/1).   Total leucocytes are initially identified on a [CD45 vs. side scatter (SS)] plot (Region A, Histogram 1.1). All gated events in Region A (PanLeucocyte) are then displayed in a second Histogram 1.2 using a [CD4 vs. SS] display where CD4++ lymphoid cells are identified (Region B, Histogram 1.2).  In this method, the total leucocytes serve as the 'common denominator' for DP absolute CD4 counting instead of the lymphoid population. This gating strategy also provides for the calculation of a CD4% of lymphocytes, i.e. the number of events in Region B (Histogram 1.2) divided by numbers of events in Region C (Histogram 1.1).  This value is however not used in the generation of the PanLeucogated CD4 counts, and is supplied as extra information when CD4% of lymphocytes may be clinically relevant, e.g. in paediatric cases.

Figure 2:

Day 3 sample preparation using the whole blood system Immunoprep and analysed on BC XL MCL. In histogram 2 some events are seen outside of the CD45 gate but are shown not to be relevant cellular events.

Also note that although the light scatter is not used for the analysis (first histogram above) it is interesting to see just how much the sample has disintegrated but how using the CD45 helps to “salvage” the sample and allow preparation way past its’ “morphological sell-by date”. This disintegration would have affected the results very badly if a typical primary gate based on light scatter alone had been used to define lymphocytes.

 More stability data available on request to D.K. Glencross.

Finally,

1.      The PLG principle can be applied to the enumeration of any lymphoid or White blood cell subset e.g. CD19/ 20 for B cells, CD16/ 56 for NK cells, etc.

2.      The PLG principle can be applied as a sophisticated 4- colour option to incorporate the improved dual platform accuracy in settings where costs are not so confining                 e.g. Tube 1 - CD45/ 4/ 8 /3 and Tube 2 - CD16 and CD56/ 19/ CD45/ CD3 for a complete lymphoid analysis using the CD45 PanLeucogating principle.

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