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Platelet freeze-storage for antibody testing
THROMBOSIS RESEARCH Printed in the'united
BRIEF
Vol. States
6, PP. 281-283, Pergamon Press,
1975 Inc.
COMMUNICATION
PLATELET FREEZE-STORAGE FOR ANTIBODY TESTING
Richard J. Hirschman and Harvey R. Gralnick Clinical Pathology Department, Clinical Center, National Institutes of Health, Bethesda,Maryland 20014 U.S.A.
(Received
26.12.1974; Accepted
in revised by Editor
A.L.
form
10.2.1975.
Copley)
Sensitive and simple methods for the detection of platelet isoantibodies
and the anti-plateletfactors in autoimmune thrombocytopenicpurpura (ATP) and systemic lupus erythematosis (SIR) have recently been reported (l-3). These methods have been useful in the diagnosis of ATP and in the selection of compatible platelet donors. (1,3,4) However, the rapid screening of a large panel of potential platelet donors has been hampered by the necessity of using recently drawn platelets for testing in the serotonin release (SR) and platelet factor 3(PF-3) release tests. Techniques for freezing human platelets for later use in transfusionhave improved in recent years with the use of dimethylsulfoxide(DMSO) as a cryopreservative.(5,6) We have modified a method for frozen platelet storage to develop a panel of platelets of various HL-A types which are readily available for use in the SR and PF-3 tests. Platelets were frozen in the following manner: Citrated platelet rich plasma (approximately800,000/nun3) was mixed in equal parts with a balanced buffered protein solution (7) composed of 0.5% human albumin, 140 n&fNaCl, 5 mM KCl, 5 mM glucose and 20% DMSO (v/v) in 0.1 M tris buffer pH 7.4. The mixture was then frozen to -120 'C in a mechanical refrigerationunit without attempting to control the rate of freezing and kept at this temperatureuntil used. Thawing was accomplished in a 37 OC water bath and the platelet suspensions were immediatelydiluted in an equivalentvolume of titrated platelet poor plasma. The PF-3 test was then performed as previously described. (3) 281
PI.,ATELETFREEZE-STORAGE
282
Vol.6,No.3
KDTA (0.15%)was added to the platelet suspensionsto be used in the SR test to facilitateresuspensionof platelets after washing. The SR test was then performed as previously described. (2) The platelet count in the PF-3 assay varied from 350,000-700,000/mm3while in the SR assay the platelet count was 4oo,ooo/mm3. Platelet complement fixation was performed in a microliter system (8) and RL-A typing of lymphocyteswas performed by Dr. Paul Terasaki with a lymphocytotoxicitytest. (9) Anti-plateletantibodies of known RL-A specificityas determinedby complement fixation, serotonin release and PF-3 release gave identical specificitieswhen tested with previously frozen platelets. Representative data are given in Table 1.
Table 1
Antibody G.D. with Known SpecificityAnti-M-A2 Reaction with Frozen Platelets Donor
Platelets
g
Test
10 Control Mean + S.D. (CPM)
Antibody Release (CPM)
(2)(13)
SR
6168 + 276
8731
(POS) @OS)
L.H.
(2,11)(5,60)
SR
2886 +
3717
WS)
E.E.
(2,10)(18,13) SR
4043 + 179
8307.
(POS)
B.C.
(2,3)(50,7)
SR
5128 + 267
7975
(POS)
M.MC.
(2102,141
SR
7052 + 361
14,727
J.R.
(2,29)(7,22)
SR
4867 2164
6142
@OS)
B.K.
(1,3)(7,8)
SR
4114 + 214
3798
@=I
B.C.
(2,3)(50,7)
PF-3
106.7 + 4.2 (set)
93
E.E.
(2,10)(18,13) PF-3
125.9 5 3.6 (set)
100
(POS) (POS)
L.H.
(2,11)(5,60)
PF-3
65.3 + 3.2 (set)
44
(POS)
B.K.
(1,3)(7,8)
PF-3
108.4 + 2.45 (set)
110
F,S.
Cl,21 (60)
SR
4739 + 131
7669
J.B.
71
WS)
ma
Similarly, sera from patients with ATP and SLE gave positive results with previously froeen platelets. Platelets frozen by the method described above and kept at -120 'C could be used up to one month after the freezing procedure, but after this time, the frozen platelets could not be used in the SR and PF-3 tests.
Vo1.6,No.3
PLATELE'I FREEZE-STORAGE
283
The techniques described in this communicationallow for the short term (up to one month) storage of platelets for convenient testing.
REFERENCES
1.
KARPATRIN, S. and SISKIND, G.W. -In vitro detection of platelet antibody in patients with idiopathic thrombocytopenicpurpura and systemic lupus erythematous.Blood: 33, 795, 1969.
2. HIRSCHMAN, R.J., YANKEE, R.A., COLLER, B.S. and GRALNICK, H.R. Sensitivemethods for the detection and characterizationof platelet isoantibodies.Thromb. et Diath. Haem.: 2, 408, 1973. 3. HIRSCHMAN, R.J. and GRAJdICK, H.R. A simplifiedplatelet factor 3 (PF-3) assay for the rapid detection of platelet isoantibodiesand an anti-plateletfactor in ATP and SLE. J. Lab. Clin. Med.: 84, 292, 1974. 4.
GOCKERMAN, J.P., BOWMAN, R.P. and CONRAD, M.E. A simple method for platelet typing for use in platelet transfusionin alloimmunized patients. Blood: 40, 972, 1972.
5. DJERASSI, I., FARBER, S., ROY, A. and CAVINS, J. Preparationand 2 vivo circulationof human platelets preservedwith combined dimethylsulfoxide and dextrose. Transfusion:a, 572, 1966. 6. VALERI, C.R., FEURGOLD, H. and MARCHIONNI, L.D. A simple method for freezing human platelets using 6% dimethylsulfoxideand storage at -80 'C. Blood: 43, 131, 1974. 7. ROSSI, E.C. The effect of albumin upon the loss of enzymes from washed platelets. J. Lab. Clin. Med.: 2, 240, 1972. 8.
COLOMBANI,M., COLOMBANI, J., DEHAY, C. and DAUSSET, J. A microtechnique of platelet complement fixation. Results obtained with sera and eluates as the source of antibody. HistocompatibilityTesting: 553, 1970.
9. MITTAL, K.M., MICKEY, M.D., SENGAL, D.P. and TERASAKI, P.I. Serotyping for homotransplantation.Transplantation:2, 913, 1968.
BRIEF
Vol. States
6, PP. 281-283, Pergamon Press,
1975 Inc.
COMMUNICATION
PLATELET FREEZE-STORAGE FOR ANTIBODY TESTING
Richard J. Hirschman and Harvey R. Gralnick Clinical Pathology Department, Clinical Center, National Institutes of Health, Bethesda,Maryland 20014 U.S.A.
(Received
26.12.1974; Accepted
in revised by Editor
A.L.
form
10.2.1975.
Copley)
Sensitive and simple methods for the detection of platelet isoantibodies
and the anti-plateletfactors in autoimmune thrombocytopenicpurpura (ATP) and systemic lupus erythematosis (SIR) have recently been reported (l-3). These methods have been useful in the diagnosis of ATP and in the selection of compatible platelet donors. (1,3,4) However, the rapid screening of a large panel of potential platelet donors has been hampered by the necessity of using recently drawn platelets for testing in the serotonin release (SR) and platelet factor 3(PF-3) release tests. Techniques for freezing human platelets for later use in transfusionhave improved in recent years with the use of dimethylsulfoxide(DMSO) as a cryopreservative.(5,6) We have modified a method for frozen platelet storage to develop a panel of platelets of various HL-A types which are readily available for use in the SR and PF-3 tests. Platelets were frozen in the following manner: Citrated platelet rich plasma (approximately800,000/nun3) was mixed in equal parts with a balanced buffered protein solution (7) composed of 0.5% human albumin, 140 n&fNaCl, 5 mM KCl, 5 mM glucose and 20% DMSO (v/v) in 0.1 M tris buffer pH 7.4. The mixture was then frozen to -120 'C in a mechanical refrigerationunit without attempting to control the rate of freezing and kept at this temperatureuntil used. Thawing was accomplished in a 37 OC water bath and the platelet suspensions were immediatelydiluted in an equivalentvolume of titrated platelet poor plasma. The PF-3 test was then performed as previously described. (3) 281
PI.,ATELETFREEZE-STORAGE
282
Vol.6,No.3
KDTA (0.15%)was added to the platelet suspensionsto be used in the SR test to facilitateresuspensionof platelets after washing. The SR test was then performed as previously described. (2) The platelet count in the PF-3 assay varied from 350,000-700,000/mm3while in the SR assay the platelet count was 4oo,ooo/mm3. Platelet complement fixation was performed in a microliter system (8) and RL-A typing of lymphocyteswas performed by Dr. Paul Terasaki with a lymphocytotoxicitytest. (9) Anti-plateletantibodies of known RL-A specificityas determinedby complement fixation, serotonin release and PF-3 release gave identical specificitieswhen tested with previously frozen platelets. Representative data are given in Table 1.
Table 1
Antibody G.D. with Known SpecificityAnti-M-A2 Reaction with Frozen Platelets Donor
Platelets
g
Test
10 Control Mean + S.D. (CPM)
Antibody Release (CPM)
(2)(13)
SR
6168 + 276
8731
(POS) @OS)
L.H.
(2,11)(5,60)
SR
2886 +
3717
WS)
E.E.
(2,10)(18,13) SR
4043 + 179
8307.
(POS)
B.C.
(2,3)(50,7)
SR
5128 + 267
7975
(POS)
M.MC.
(2102,141
SR
7052 + 361
14,727
J.R.
(2,29)(7,22)
SR
4867 2164
6142
@OS)
B.K.
(1,3)(7,8)
SR
4114 + 214
3798
@=I
B.C.
(2,3)(50,7)
PF-3
106.7 + 4.2 (set)
93
E.E.
(2,10)(18,13) PF-3
125.9 5 3.6 (set)
100
(POS) (POS)
L.H.
(2,11)(5,60)
PF-3
65.3 + 3.2 (set)
44
(POS)
B.K.
(1,3)(7,8)
PF-3
108.4 + 2.45 (set)
110
F,S.
Cl,21 (60)
SR
4739 + 131
7669
J.B.
71
WS)
ma
Similarly, sera from patients with ATP and SLE gave positive results with previously froeen platelets. Platelets frozen by the method described above and kept at -120 'C could be used up to one month after the freezing procedure, but after this time, the frozen platelets could not be used in the SR and PF-3 tests.
Vo1.6,No.3
PLATELE'I FREEZE-STORAGE
283
The techniques described in this communicationallow for the short term (up to one month) storage of platelets for convenient testing.
REFERENCES
1.
KARPATRIN, S. and SISKIND, G.W. -In vitro detection of platelet antibody in patients with idiopathic thrombocytopenicpurpura and systemic lupus erythematous.Blood: 33, 795, 1969.
2. HIRSCHMAN, R.J., YANKEE, R.A., COLLER, B.S. and GRALNICK, H.R. Sensitivemethods for the detection and characterizationof platelet isoantibodies.Thromb. et Diath. Haem.: 2, 408, 1973. 3. HIRSCHMAN, R.J. and GRAJdICK, H.R. A simplifiedplatelet factor 3 (PF-3) assay for the rapid detection of platelet isoantibodiesand an anti-plateletfactor in ATP and SLE. J. Lab. Clin. Med.: 84, 292, 1974. 4.
GOCKERMAN, J.P., BOWMAN, R.P. and CONRAD, M.E. A simple method for platelet typing for use in platelet transfusionin alloimmunized patients. Blood: 40, 972, 1972.
5. DJERASSI, I., FARBER, S., ROY, A. and CAVINS, J. Preparationand 2 vivo circulationof human platelets preservedwith combined dimethylsulfoxide and dextrose. Transfusion:a, 572, 1966. 6. VALERI, C.R., FEURGOLD, H. and MARCHIONNI, L.D. A simple method for freezing human platelets using 6% dimethylsulfoxideand storage at -80 'C. Blood: 43, 131, 1974. 7. ROSSI, E.C. The effect of albumin upon the loss of enzymes from washed platelets. J. Lab. Clin. Med.: 2, 240, 1972. 8.
COLOMBANI,M., COLOMBANI, J., DEHAY, C. and DAUSSET, J. A microtechnique of platelet complement fixation. Results obtained with sera and eluates as the source of antibody. HistocompatibilityTesting: 553, 1970.
9. MITTAL, K.M., MICKEY, M.D., SENGAL, D.P. and TERASAKI, P.I. Serotyping for homotransplantation.Transplantation:2, 913, 1968.