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v i r a l   i m m u n i t y   &   p a t h o g e n e s i s   g r o u p

:: p r o t o c o l s  ::  F A C S  f o r  p l a t e l e t s  a n d  r e t i c u l a t e d  p l a t e l e t s  ::


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[  F A C S  f o r  p l a t e l e t s  ]

OUTLINE

FACS represents much more sensitive assay than ELISA is.

PROTOCOL
  1. Purify platelets.
  2. resuspend platelets in 200 µl of HAFA (ice-cold !)
  3. centrifuge at 3000 rpm, 7 min, 4ƒC
  4. resuspend platelets in 200 µl of HAFA (ice-cold !)
  5. centrifuge at 3000 rpm, 7 min, 4ƒC
  6. resuspend in 100 µl of ice-cold HAFA containing the primary Ab, incubate for 60 min at 4ƒC
  7. centrifuge at 3000 rpm, 7 min, 4ƒC
  8. resuspend platelets in 200 µl of HAFA (ice-cold !)
  9. centrifuge at 3000 rpm, 7 min, 4ƒC
  10. resuspend platelets in 200 µl of HAFA (ice-cold !)
  11. centrifuge at 3000 rpm, 7 min, 4ƒC
  12. resuspend in 100 µl of ice-cold HAFA containing the secondary (FACS-revealing) Ab, incubate for 60 min at 4ƒC
  13. centrifuge at 3000 rpm, 7 min, 4ƒC
  14. resuspend platelets in 200 µl of HCF (ice-cold !)
  15. centrifuge at 3000 rpm, 7 min, 4ƒC
  16. resuspend platelets in 200 µl of HCF (ice-cold !)
  17. centrifuge at 3000 rpm, 7 min, 4ƒC
  18. resuspend platelets in 200 µl of HCF (ice-cold !)
  19. add 200 µl of ice-cold HCF-PF 1.25%. mix gently.
  20. run FACS
SOLUTIONS
  1. HCF buffer = 137 mM NaCl + 5 mM KCl + 0.4 mM MgSO4 + 0.3 mM MgCl2 + 5 mM glucose + 4 mM NaHCO3 + 1 mM EDTA + 1 mM phosphate + 20 mM NaN3 + 100 U/ml penicillin + 100 µg/ml streptomycin, pH 7.4 (Coutelier J-P et al, 1994).
  2. HAFA buffer, 100 ml = HCF, 97 ml + decomplemented fetal calf serum, 3 ml, pH 7.4 (Coutelier J-P et al, 1994).
ADDITIONAL INFO
  1. respect the temperature and incubation time regimens.
  2. omit the incubation step with the primary Ab when performing the FACS for platelet-associated Ig.
SCHEME

[  enlarge  ]
 
REFERENCES
Flow cytometric analysis of platelet function in stored platelet concentrates. Transfus Sci. 1999 Apr;20(2):129-39 .Wang C, Mody M, Herst R, Sher G, Freedman J.
 
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[  F A C S  f o r  r e t i c u l a t e d  p l a t e l e t s  ]

OUTLINE

Reticulated Platelets are young platelets that contain residual mRNA and rRNA when they are released from the bone marrow into the peripheral circulation as a result of thrombopoiesis. These are often called "large, young" platelets or "stress" platelets and appear normally in the peripheral blood at low levels (up to 4.5% of total platelets). The correlation with size is generally proportional as large platelets often contain more RNA. However, platelets of all sizes can be found with increased RNA content which indicates that correlation with the Mean Platelet Volume (MPV) is subject to variability and that MPV alone cannot be used to enumerate all young platelets.

By analogy to reticulated erythrocytes (reticulocytes), an increased proportion of reticulated platelets in blood indicates increased thrombopoiesis and is therefore a reflection of megakaryocyte number and activity. Thus, it is frequently possible to distinguish decreased production or bone marrow failure from increased platelet consumption as the cause of thrombocytopenia using only a peripheral blood sample.

PROTOCOL
  1. collect blood on ACD and BSGC.
  2. centrifuge at 600 rpm, 5 min, ƒ4C
  3. save supernatant (SN).
  4. add 1ml BSGC (ice-cold)
  5. centrifuge at 600 rpm, 5 min, ƒ4C
  6. save SN
  7. add 1ml BSGC (ice-cold)
  8. centrifuge at 1000 rpm, 5 min, 4ƒC
  9. save SN
  10. add 1ml BSGC (ice-cold)
  11. centrifuge at 1000 rpm, 5 min, 4ƒC
  12. save SN
  13. pool SNs
  14. centrifuge at 600 rpm, 5 min, ƒ4C
  15. save SNs and centrifuge at 3000 rpm, 5 min, ƒ4C
  16. resuspend in 400 µl of  HCF-PF 1.25%. Let to stay for 45 min, at 4ƒC
  17. centrifuge at 3000 rpm, 5 min, ƒ4C
  18. resuspend in 100 µl of BSGC
  19. add 900 µl 0.1% of NaN3-PBS to negative control  ::  to aget platelets add 900 µl 0.1% of NaN3-PBS and 2.5 µg of rat-anti mouse CD61 Ab conjugated to PE  ::  for TO-labeling add 900 µl of RETIC-Count (Becton Dickenson)  ::  for double staining add 900 µl of RETIC-Count and 2.5 µg of rat-anti mouse CD61 Ab conjugated to PE. Incubate for 60 min, at RT, in dark.
  20. centrifuge at 3000 rpm, 5 min, ƒ4C
  21. resuspend in 400 µl of HCF
  22. run FACS
SOLUTIONS
  1. BSGC
  2. HCF
  3. HAFA
ADDITIONAL INFO
  1. [  r e a d  m o r e  ] about the interactions between the fluorescent dye thiazole orange and DNA. 
SCHEME

[  enlarge  ]

 REFERENCES
  1. Quantitation of reticulated platelets: methodology and clinical application.Br J Haematol. 1995 Oct;91(2):445-51. Richards EM, Baglin TP.
  2. Platelet Size, Platelet Surface-Associated IgG, and Reticulated Platelets in Dogs with Immune-Mediated Thrombocytopenia. Vet Clin Pathol. 2001;30(3):141-149.  Wilkerson MJ, Shuman W, Swist S, Harkin K, Meinkoth J, Kocan AA.
  3. Reticulated platelets in the evaluation of thrombopoietic disorders. Arch Pathol Lab Med. 1993 Jun;117(6):606-10. Rinder HM, Munz UJ, Ault KA, Bonan JL, Smith BR.
  4. Flow cytometric analysis of thiazole orange uptake by platelets: a diagnostic aid in the evaluation of thrombocytopenic disorders. Blood. 1990 Jan 1;75(1):116-21. Kienast J, Schmitz G.
  5. The rise of reticulated platelets after intensive chemotherapy for AML reduces the need for platelet transfusions. Ann Hematol. 1999 Jun;78(6):271-3. Stohlawetz P, Stiegler G, Knobl P, Hocker P, Panzer S.
  6. The significance of platelets with increased RNA content (reticulated platelets). A measure of the rate of thrombopoiesis.Am J Clin Pathol. 1992 Dec;98(6):637-46. Ault KA, Rinder HM, Mitchell J, Carmody MB, Vary CP, Hillman RS.
  7. A new approach to detect reticulated platelets stained with thiazole orange in thrombocytopenic patients. Thromb Res. 2000 Mar 15;97(6):431-40. Fujii T, Shimomura T, Fujimoto TT, Kimura A, Fujimura K.
  8. Flow cytometric analysis of platelet function in stored platelet concentrates. Transfus Sci. 1999 Apr;20(2):129-39 .Wang C, Mody M, Herst R, Sher G, Freedman J.
  9. B lymphocyte and macrophage expression of carcinoembryonic antigen-related adhesion molecules that serve as receptors for murine coronavirus.  Eur J Immunol.  1994;24:1383-1390. Coutelier J-P, Godfraind C, Dveksler GS, et al.


created by Andrei Musaji          ::          updated : 2004