Storage and Transportation of Samples for Analysis of
Terminal Transferase by Indirect Immunofluorescence
RONALD D. BARR, M.D. AND MARIJKE KOEKEBAKKER, ART
Expression of the terminal transferase (TdT) antigen, as revealed
by indirect immunofluorescence, was retained in samples of human acute lymphoblastic leukemia (ALL) cells, of various phenotypes, during storage at 4°C for almost six months. Similar
specimens of ALL cells, of common phenotype, were transported
successfully by air without notable loss of TdT. The unsatisfactory outcome of earlier studies may be explained on the basis
of methodologic differences alone. (Key words: Storage; Transportation; Terminal transferase; Indirect immunofluorescence)
Am J Clin Pathol 1984; 81: 660-661
DEMONSTRATION of terminal deoxynucleotidyl
transferase (TdT; EC.2.7.7.31) in single cells is a potenReceived July 22, 1983; received revised manuscript and accepted
for publication September 19, 1983.
. Supported by a grant from the Physicians' Services Incorporated
Foundation.
Address reprint requests to Dr. Barr: Room 3N27D, McMaster University Health Sciences Centre, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5.
Department of Pediatrics, McMaster University,
Hamilton, Ontario, Canada
tially powerful diagnostic test for the clinical pathologist.
Such assays of TdT, by indirect immunofluorescence,
contribute to the classification of lymphoproliferative disorders and the acute leukemias.24 For these purposes it
is convenient to process samples in batches and to conduct
the analyses in central laboratories. Accordingly, we have
examined conditions of storage and transportation that
will allow achievement of these objectives.
Materials and Methods
Bone marrow was obtained from children with acute
lymphoblastic leukemia (ALL) at original diagnosis or
relapse. Cytocentrifuged samples of single cell suspensions
Table 1. Influence of Storage on Expression of Terminal Transferase Activity
4°C
22 °C
-20°C
Duration of
Storage (wks.)
Sample
Identity
Fraction
Intensity
Fraction
Intensity
Fraction
Intensity
0
A
B
C
0.98
0.92
0.84
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
3
A
B
C
0.94
0.81
0.77
0.97
0.94
0.93
A
B
C
0.97
0.41
0.93
11
A
B
C
1.00
0.18
0.89
15
A
B
C
0.90
0
0
++
+++
+++
+
+++
+++
+
++
+++
+
+
+
18
A
B
C
++++
++++
++++
+++
+++
++++
++
++
+++
+++
+++
+++
+++
+++
+
0.98
0.93
0.91
7
++++
++++
++++
+++
+++
+++
+++
+++
+++
+
+
++
+
23
A
B
C
0.99
0.58
0.88
1.00
0.72
0.91
N/A
N/A
0.95
0.50
1.00
0
0
0
N/A
N/A
N/A
0.94
0.57
0.76
0
0
0
N/A
N/A
N/A
0.97
0
0.89
* Terminal transferase (TdT) activity, as demonstrated by indirect immunofluorescence, in
bone marrow cells from children with acute lymphoblastic leukemia (ALL) in relapse. The
fraction of cells expressing the antigen and the intensity of fluorescence are recorded in this study
660
++
N/A
++
0.99
0.34
0.94
1.00
0.52
0.94
0.68
0.38
0.93
0
0.48
1.00
N/A
0
0
0.26
N/A
N/A
+
+
+
of the impact of temperature and duration of storage on TdT activity. A • common ALL; B
= pre-B-ALL; C = T-ALL: N/A = not applicable.
BRIEF SCIENTIFIC REPORTS
Vol. 81 - N o . 5
were air dried on glass slides and either examined immediately or wrapped in plastic and stored for subsequent
analysis. Replicate slides from three samples were held
at 22°C, 4°C and - 2 0 ° C and processed at intervals of
three to five weeks. In addition, samples from six patients,
which had been stored at 4°C for one to two months,
were flown approximately 500 miies (Toronto to Washington, D.C.), duplicates being transported in the hold
of the aircraft (cargo) and in the cabin (hand baggage).
After storage at room temperature for three to five days
(during the course of the meeting of the American Society
of Hematology, December 1982), the samples returned
to our laboratory under the same circumstances. These
then were processed with replicates that had been retained
in the laboratory at 4°C. Indirect immunofluorescence
was performed without prior fixation as described previously in detail.3 A single batch of rabbit anticalf thymus
TdT (PL Biochemicals, Milwaukee, WI) was used
throughout these studies. The proportion of cells exhibiting nuclear fluorescence and the intensity of the reaction
were recorded in each instance.
Results
As described in Table 1, diagnostically useful fluorescence was obtained in all samples that had been stored
for up to three months. Beyond that interval there was
a clear advantage in storing samples at 4°C. These were
re-examined after 30 weeks, and no fluorescence was demonstrable. Transportation of samples was accomplished
successfully with good reactivity on indirect immunofluorescence (Table 2). There was a marginal advantage, as
reflected in the intensity of fluorescence, to carrying the
slides in hand baggage.
Discussion
The outcome of these studies indicates that samples
for TdT analysis by indirect immunofluorescence can be
stored and transported satisfactorily, so allowing centralized processing in batches. In earlier experience, other
investigators observed a decline in staining intensity after
661
Table 2. Influence of Transportation on Expression
of Terminal Transferase Activity*
Bone
Marrow
Fresh
Control
Cabin
Cargo
0.98
1.00
0.97
0.96
+++
++++
++++
+
0.91
1.00
0.96
0.98
+++
+++
+++
++
0.96
0.88
+++
0.96
+++
0.01
+
0.95
0.90
+++
++++
0.79
++++
++
0.95
++
1.00
0.95
0.96
0.96
++
+++
++++
+++
0.82
0.72
0.84
0.78
++
++
+++
+++
• Terminal transferase (TdT) activity, as demonstrated by indirect immunofluorescence, in
bone marrow cells from children with acute lymphoblastic leukemia (ALL) in relapse. The
fraction of cells expressing the antigen and the intensity of fluorescence are recorded in thisstudy
of the impact of transportation by air on TdT activity. All samples expressed the "common"
ALL phenotype.
storage for longer than two weeks at room temperature
and loss of reactivity in more than 70% of samples that
had been transported by air.' At least in part this difference
may reflect the comparative quality of reagents and
technic.3
Acknowledgment. The authors thank Dr. T. Zipf for performing the
phenotypic analyses on the samples of ALL cells used for this study.
References
1. Cibull ML, Coleman MS, Nelson O, et al: Evaluation of methods
of detecting terminal deoxynucleotidyl transferase in human hematologic malignancies. Am J Clin Pathol 1982; 77:420-423
2. Jani P, Verbi W, Greaves MF, et al: Terminal deoxynucleotidyl
transferase in acute myeloid leukemia. Leuk Res 1983; 7:17-29
3. Koekebakker M, Barr RD: Demonstration of terminal deoxynucleotidyl transferase in single cells by indirect immunofluorescence—a methodological reappraisal. Leuk Res 1983; 7:237241
4. McCaffrey R, Lillquist A, Sallan S, et al: Clinical utility of leukemia
cell terminal transferase measurements. Cancer Res 1981;
41:4814-4820