Pre-analytic and analytic sources of variations in thiopurine methyltransferase
activity measurement in patients prescribed thiopurine-based drugs:
A systematic review☆
Evelin Loita, Andrea C. Triccob, Sophia Tsourosc, Margaret Searsd,
Mohammed T. Ansaric, Ronald A. Boothe,f,⁎
aDepartment of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada
bLi Ka Shing Knowledge Institute, St Michael's Hospital, Toronto, Ontario, Canada
cMethods Centre, Clinical Epidemiology Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada
dChildren's Hospital of Eastern Ontario Research Institute, Ottawa, Ontario, Canada
eDepartment of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, Ontario, Canada
fThe Ottawa Hospital, Division of Biochemistry, Ottawa, Ontario, Canada
a b s t r a c t a r t i c l ei n f o
Received 14 December 2010
Received in revised form 16 February 2011
Accepted 4 March 2011
Available online 12 March 2011
Objectives: Low thiopurine S-methyltransferase (TPMT) enzyme activity is associated with increased
thiopurine drug toxicity, particularly myelotoxicity. Pre-analytic and analytic variables for TPMT genotype
and phenotype (enzyme activity) testing were reviewed.
Design and methods: A systematic literature review was performed, and diagnostic laboratories were
Results: Thirty-five studies reported relevant data for pre-analytic variables (patient age, gender, race,
hematocrit, co-morbidity, co-administered drugs and specimen stability) and thirty-three for analytic
variables (accuracy, reproducibility). TPMT is stable in blood when stored for up to 7 days at room
temperature, and 3 months at −30 °C. Pre-analytic patient variables do not affect TPMT activity. Fifteen drugs
studied to date exerted no clinically significant effects in vivo. Enzymatic assay is the preferred technique.
Radiochemical and HPLC techniques had intra- and inter-assay coefficients of variation (CVs) below 10%.
Conclusion: TPMT is a stable enzyme, and its assay is not affected by age, gender, race or co-morbidity.
© 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Literature search and study selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Data extraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Quality assessment and synthesis of evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Survey of laboratories conducting TPMT analyses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pre-analytic variability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Specimen stability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Influence of gender on TPMT activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Clinical Biochemistry 44 (2011) 751–757
Abbreviations: (TPMT), thiopurine S-methyltransferase; (HPLC), high performance liquid chromatography; (CV), coefficient of variation; (6-MP), 6-mercaptopurine; (6-TG), 6-
thioguanine; (6-MMP), 6-methyl mercaptopurine; (SAM), S-adenosyl-L-methionine; (RBCs), red blood cells; (RFLP), restriction fragment length polymorphism; (PCR), polymerase
chain reaction; (EDTA), ethylene diamine tetra-acetic acid; (6-MTG), 6-methylthioguanine; (ALL), acute lymphoblastic leukemia; (ANOVA), analysis of covariance; (IC50),
concentration of inhibitor at which enzyme activity is 50% of uninhibited activity; (6-TG), 6-thioguanine.
☆ Disclaimer: This project was funded under Contract No. HHSA290-2007-10059-I (EPCIII) from the Agency for Healthcare Research and Quality, U.S. Department of Health and
Human Services. The authors of this report are responsible for its content. Statements in the report should not be construed as endorsement by the Agency for Healthcare Research
and Quality, the National Center for Complementary and Alternative Medicine, National Institutes of Health or the U.S. Department of Health and Human Services.
⁎ Corresponding author at: The Ottawa Hospital, Division of Biochemistry, 501 Smyth Rd, Ottawa, Canada, ON K2H 8L6. Fax: +1 613 737 8541.
E-mail address: firstname.lastname@example.org (R.A. Booth).
0009-9120/$ – see front matter © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/clinbiochem
Influence of age on TPMT activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effect of race on TPMT activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Co-administered drugs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effect of hematocrit on measured TPMT activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effect of morbidity on TPMT activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Analytic variability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Laboratory survey. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pre-analytic considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Analytical considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thiopurine-based drugs (azathioprine (AZA), 6-mercaptopurine
(6-MP) and 6-thioguanine (6-TG)) are currently used for mainte-
nance therapy for leukemia, transplant rejection prevention and
chronic autoimmune inflammatory conditions. AZA and 6-MP are
effective to induce remission in 50%–60% of inflammatory bowel
disease patients, and permit steroid reduction or withdrawal in up to
65% of patients . Use of AZA or 6-MP in other chronic
inflammatory disorders including lupus and rheumatoid arthritis
has been variable, and thiopurines are often not the primary drugs of
choice, due partially to adverse effects. A particularly serious effect is
dose-dependent myelosuppression, thought to be caused by the
active metabolite, deoxy-6-thioguanosine 5′ triphosphate (6-tGN).
The most thoroughly characterized enzyme involved in the
metabolism of thiopurine drugs is thiopurine S-methyltransferase
(TPMT), which catalyzes the S-methylation of 6-TG and AZA. The
TPMT gene (HGNC:12014) is located on chromosome 6 at 6p22.3. It is
approximately 27 kb in size and contains 9 exons [42,58]. TPMT (EC
in lymphocytes and red blood cells (RBCs) is most clinically relevant, as
30 variant alleles of TPMT have been identified, the majority of which
have been associated with lower TPMT enzymatic activity or protein
The four most common variant alleles seen in Caucasians, Asians and
Africans(TPMT*2, TPMT*3A, TPMT*3B, and TPMT*3 C)accountfor 80%–
95% of individuals with lower TPMT activity [2,8,18,43,49,65]. Approx-
imately 0.3% of patients are homozygous for a variant allele and have
very low or absent enzyme activity; while 5%–15% of patients are
heterozygous and have intermediate enzymatic activity [2,8,19].
Currently, there is no evidence that the presence of one or more
variant TPMT alleles causes disease or places one at increased risk for
disease. However, lower TPMT enzyme activity or the presence of a
variant allele has been suggested to increase the risk of thiopurine-
related drug toxicity, particularly when using AZA or 6-MP [26,51].
Patients with intermediate or low TPMT activity may benefit from a
lower thiopurine starting dose to reduce the risk of drug-related
toxicity, while patients with absent TPMT activity may be candidates
for other therapies.
TPMT status is usually ascertained by analysis of either red blood
cell TPMT enzymatic activity or genotyping.
TPMT enzymatic activity is most commonly determined by
measurement of TPMT mediated formation of 6-methyl mercaptopu-
rine (6-MMP) from 6-MP, with S-adenosyl-L-methionine (SAM) as
the methyl donor. The original radiochemical method developed by
nonradioactive detectionby high performanceliquid chromatography
Genetic analysis in routine clinical laboratories targets specific
TPMT mutations, usually the three or four most common alleles.
Depending on the variant alleles targeted and the ethnic background
of the patient, genotyping can identify up to 95% of affected
individuals; however it will not identify those patients with rarer
mutations. Genotyping is most commonly performed by polymerase
chain reaction/restrictionfragment length polymorphism (PCR/RFLP),
genetic sequencing and site-directed PCR.
of the CDC's Office of Public Health Genomics) require assessment of
analytical validity in conjunction with clinical validity and utility;
therefore we examined pre-analytic, analytic and post-analytic require-
ments. Here we report a subsection of the review nominated by the
American Association for Clinical Chemistry (AACC) and commissioned
by the Agency for Healthcare Research and Quality (AHRQ), to
summarize the evidenceregarding TPMT testing inchronic autoimmune
gov/clinic/epcindex.htm) examined whether pretreatment determina-
tion of TPMT enzymatic activity (phenotyping) or TPMT genotype, to
guide thiopurine therapy in chronic autoimmune disease patients,
reduces treatment harms. Other objectives included assessing the
following: preanalytic, analytic, and postanalytic requirements for
status; and costs of testing, care, and treating drug-associated complica-
tions. The specific questions addressed in this review are as follows:
In terms of the analytical performance characteristics of enzymatic
measurement of TPMT activity and determination of TPMT allelic
1) What are the pre-analytical requirements for enzymatic measure-
ment of TPMT and determination of TPMT allelic polymorphisms
(e.g. specimen types and collection procedures, lab transportation,
interference of co-administered drugs, patient preparation and
2) What are the within and between laboratory precision and
reproducibility of the available methods of enzymatic measure-
ment of TPMT and determination of TPMT allelic polymorphisms
3) Are there any post-analytical requirements specific to measure-
ment of TPMT enzymatic activity or TPMT allelic polymorphism
Materials and methods
Literature search and study selection
The following databases were searched electronically: Ovid MED-
LINE(R) 1950 to May Week 3 2010; The Cochrane Library (CLIB 2009 3)
EMBASE 1980 to 2010 Week 21; Genetics Abstracts: May 7 2009; and
Ovid Healthstar 1966 to April 2010. No language or date restrictions
were imposed on any of the searches. Animal studies were excluded.
E. Loit et al. / Clinical Biochemistry 44 (2011) 751–757
All English language studies that measured TPMT status either by
enzymatic analysis or genotyping, and addressed pre-analytic
requirements, analytical precision and post-analytic requirements
were included. Non-English records, editorials, reviews, commentar-
ies, letters, news or case reports were excluded. One reviewer
screened record titles or abstracts to include studies; a second
reviewer independently verified exclusions. Two reviewers indepen-
dently screened full-text reports and resolved conflicts by consensus.
A third level of screening was undertaken by content experts (RB and
EL) to confirm records marked as relevant.
The followingdata was extracted from studies: first author'sname,
year of publication, country, sample size, type of genetic testing (e.g.
alleles tested, method of testing and source of DNA) and type of
enzymatic assay, reported sensitivity and specificity, coefficients of
variation and pre-analytic variables for TPMT activity.
When there were multiple reports of the same study the most
relevant record was referenced as the primary identifying study, and
additional data was extracted as available from companion report(s).
Evidence from the included studies was synthesized qualitatively.
Quality assessment and synthesis of evidence
Quality assessment of studies answering such questions remains
subjective, and no standard tools exist to appraise internal validity of
studies. Thus evidence was synthesized qualitatively and quality was
Survey of laboratories conducting TPMT analyses
To supplement the existing evidence, data regarding pre-analytic
and post-analytic requirements of TPMT laboratory analyses were
sought using a survey of laboratories that provided TPMT analytical
services. The survey questionnaire was approved by the Ottawa
Hospital's Research Ethics Board. Potential participating laboratories
were identified by the Technical Expert Panel, which also queried two
listservs: Clinical Chemistry General Topics (American Association for
Clinical Chemistry) and CSCC Listserv (Canadian Society of Clinical
Chemists). Based on these expert recommendations, two organiza-
tions and seven laboratories were contacted to determine their
willingness either to complete a questionnaire or to disseminate the
questionnaire to other relevant laboratories. The survey questionnaire
included 11 questions with multiple subquestions relating to TPMT
analytical methods (e.g., sample type and handling) and pre-
analytical requirements (e.g., specimen stability). We conducted a
descriptive analysis and summarized results using frequencies and
ranges as appropriate.
The full evidence report, including search strategies and PRISMA
flow chart, is available at http://www.ahrq.gov/clinic/epcindex.htm.
Thirty-five unique records for the first question, thirty-three for the
second and none for the third were included in the evidence base.
For the first question, all 35 studies pertained to the pre-analytical
performance characteristics of the TPMT activity assays and none
related to TPMT genotyping.
Thirteen studies assessed the stability of TPMT enzyme activity
(Table 1) [3,6,35,36,38,39,41,45–47,61,64,67]. Two studies were
conducted in the USA [47,64], and eleven in Europe [3,6,35,36,38,
39,41,45,46,61,67]. TPMT stability was assessed at room tempera-
ture, 4 °C, −20 °C, −21 °C, −23 °C, −25 °C, −30 °C, −70 °C, −80 °C
and −85 °C. Time periods from a few hours to 16 months were
studied. TPMT was found to be stable at room temperature for a
maximum of 7 days in control blood samples, while in the case of
acute lymphocytic leukemia, patient blood TPMT was stable for
3 days . At −20 °C, TPMT was stable for up to 3 months [36,39].
In four studies of storage at -80 °C, TPMT was stable up to at least
25 days [6,36,38,61]; however TPMT activity decreased by 15% after
16 months of storage . Repeated freeze–thaw cycles of the RBC
lysate were reported to result in a 16% decrease in activity ;
however, the decrease was not statistically significant after three
cycles (initial values: 9.2, 11.7, and 16.5 U/mL RBCs; final values: 8.1,
9.8, and 14.3 U/mL RBCs). TPMT activity was stable in blood samples
shipped via regular mail and received up to seven days post
sampling (CV, 5.6%) . The laboratories that conduct TPTM
testing on a regular basis store specimens (whole blood with EDTA
anticoagulant) at 4 °C or room temperature for between 1 and
Influence of gender on TPMT activity
Eighteen studies evaluated gender-related differences in TPMT
activity [1,4,6,22,24,33,36,38,41,46,52,54,55,61,64,68,71,72]. Seventeen
studies reported the TPMT activity in RBCs and one in renal tissue .
with gender [1,4,22,24,33,36,38,41,46,52,54,55,61,64,71,72]. One study
reported TPMT values to be higher for males in Caucasian and mixed-
race groups (30 U/g Hb in females versus 38 U/g Hb in males in
Caucasians, and 33 U/g Hb in females versus 39 U/g Hb in males in a
mixed race group) . TPMT values in renal tissue were 10% higher in
males than in females . Fifteen studies reported TPMT values,
whereas three studies stated only the outcomes of comparisons
Influence of age on TPMT activity
Ten studies investigated variation of TPMT activity with age
[4,9,24,27,33,38,54,61,68,71]. In eight studies TPMT activity was
analyzed in RBCs, while one study examined each of renal tissue
 and lymphocytes . Most studies reported no relationship
Stability of TPMT enzymatic activity.
24 h, heparin 
36 h, EDTA 
3 days, heparin ⁎
4 days, heparin 
5 days, heparin 
6 days, EDTA 
7 days, heparin ⁎
72 h, EDTA 
25% ± 6% decrease
in 24 hours
4 °C Whole
24 h, heparin 
4 days, heparin 
6 days, EDTA ⁎
8 days, unspecified
7 days 
21 days 
3 month 
Several month 
Few days [6,61]
25 days 
16 months 
−20 to −30 °CRBC lysate Stable
−70 to −80 °CRBC lysate
⁎ Control blood was stable 7 days. Leukemia patient blood was stable for 3 days, and
the median activity showed a small but statistically significant decrease after 6 days.
E. Loit et al. / Clinical Biochemistry 44 (2011) 751–757
between age and TPMT activity; however one study reported a
statistically significant difference in TPMT activities between adults
and children (12.0 U/mL RBCs in children and teenagers versus
12.9 U/mL RBCs in adults; p less than 0.001) . The authors
attributed this unexpected result to the higher frequency of
intermediate metabolizers in the pediatric population sampled.
Effect of race on TPMT activity
Two studies with small sample sizes examined TPMT activity
differences among different races [6,30]. One study reported a
difference between TPMT enzyme activity of Caucasians and a
mixed group of other races that was not statistically significant .
Overall, no significant differences were found among races studied
(Caucasians, blacks, Japanese and mixed races) [6,30].
Ten studies evaluating the influence of drugs on TPMT activity in
blood are summarized in Table 2 [4,15–17,27,36,52,59,61,69,70]. 3,4-
Dimethoxy-5-hydroxybenzoic acid decreased TPMT activity by 97% in
vitro . Concentration-dependent inhibition of TPMT, from 11% to
55% in the presence of 80 μM–640 μM sulfasalazine was also noted in
vitro . One study reported 147% stimulation of TPMT activity in
vitro by methotrexate and 148% by trimethoprim . Interestingly,
another study of methotrexate in vitro reported no effect . The
remaining studies reported no significant inhibition by any of the
Effect of hematocrit on measured TPMT activity
Three studies that examined the effect of hematocrit on TPMT
activity were identified [40,52]. One reported a 7% (range 1.2%–12.0%)
lower TPMT activity in samples with high compared with low
hematocrit levels from 12 participants . Between erythrocyte
fractions of 0.1 and 0.5, the slope of a plot of TPMT activity versus
hemoglobin was reported to be significantly different from zero (p
ranging from 0.02 to 0.0001) . Another study described no
hematocrit dependant difference in TPMT activity . A single study
reported young RBCs to have TPMT activity 8.8 U higher than old RBCs
(Wilcoxon median difference 8.8 U (95% CI 7.2–10.8, p=0.006)) .
Effect of morbidity on TPMT activity
Two studies [27,72] evaluated the effect of morbidities on TPMT
activity (Table 3). Inflammatory bowel disease (ulcerative colitis,
Crohn disease, and indeterminate colitis), autoimmune hepatitis,
multiple sclerosis, myasthenia gravis, pemphigus and chronic renal
failure were studied. Minor yet statistically significant differences (p
less than 0.001) in TPMT activity were observed among some disease
groups, such as inflammatory bowel disease, autoimmune hepatitis,
multiple sclerosis, myasthenia gravis, and pemphigus . Most
interestingly, patients with chronic renal failure had almost doubled
the TPMT activity compared with the healthy control before
hemodialysis (34±12 U/mL RBCs versus 16±4 U/mL RBCs) .
Post-hemodialysis TPMT activity levels were comparable in patients
with and without renal failure.
Thirty-three studies reported information relevant to analytical
performance characteristics in enzymatic measurement [3,10–14,20–
22,24,25,29,31–39,41,44,45,47,48,50,53,55,59,61,71,72]. Two studies
were conducted in North-America [37,47], three studies in China
[44,71,72] and the remaining 28 in Europe. Nine studies used
radiolabelled SAM for TPMT assays [29,31–33,44,48]. The reported
Effects of drugs on TPMT enzymatic activity.
Study Drug ID Effect on TPMT activity
Tinel et al., 1991  SKF 525-A 3,4-dimethoxy-5-hydroxybenzoic acidNone
Decreased by 97%
No significant change after aminosalicylate withdrawal. TPMT activity
before withdrawal of aminosalicylate; whole group; 12.29 U/mL RBCs
(range 8.25±16.85); sulfasalazine subgroup; 12.14 U/mLRBCs;
mesalazine subgroup 12.43 U/mL RBCs. TMPT activity after aminosalicylate
withdrawal; whole group; 11.41 U/mL RBCs (7.3±14.5) (p=0.245, not
significant); sulfasalazine subgroup; 11.43 U/mL RBCs; mesalazine subgroup;
11.39 U/mL RBCs.
No significant differences between patients on azathioprine compared with
those on mesazaline, at baseline or at any further visit.
No differences between patients on azathioprine or 5-aminosalicylates versus
Azathioprine; no treatment 20.7 U/mL RBCs versus treatment 21.2 U/mL RBCs
5-Aminosalycilates; no treatment 20.9 U/mL RBCs versus treatment
21.2 U/mL RBCs
No differences between patients on mesalamine medications versus controls
(median 32.4 range 14.7–49.2) ELISA units versus 31.8 (15.3–49.1 ELISA units)
No significant difference between patients on azathioprine versus
no treatment 19.9±6.0 U/mL RBCs versus 19.7±5.8 U/mL RBCs.
No significant effects in vitro.
Sulfasalazine; 9.4 (±3.1)μM
5-aminosalicylate; 236 (±55)μM
Ac-5-aminosalicylate; 73 (±20)μM
No significant effect in vitro.
Residual TPMT activity was always more than 70% of control activity.
Dewit et al., 2002  Accetylated metabolite of 5-aminosalicylic acid,
with either sulfasalazine or mesalazine.
Dilger et al., 2007  Azathioprine versus mesalazine
Gisbert et al., 2007  5-Aminosalycilates
Dubinsky et al., 2002  Mesalamine
Menor et al., 2002  Azathioprine
Xin et al., 2005 [69,70] Sulfasalazine, 5-aminosalicylate,
Jacqz-Aigrain et al., 1994 Syringle acid, prednisone, prednisolone,
methotrexate, trimethoprim sulphamethoxazole
SulfasalazineShipkova et al., 2004  Significant concentration-dependent inhibition 11% for 80 μM to 45% for 640 μM
TPMT activity significantly increased in vitro.
2.4 μM trimethoprim; 148%
0.01 μM methotrexate; 147%
Vinscristine, dexamethasone, L-asparaginase had nonsignificant inhibitory effects
on TPMT activity.
Brouwer et al., 2005 Methotrexate, trimethoprim, vinscristine,
E. Loit et al. / Clinical Biochemistry 44 (2011) 751–757
inter-assay coefficient of variation (CV) ranged from 0.51 to 8.4%. The
intra-assay CV ranged from 0.72%to6.8%.Thedaytodayvariability was
8.5±1.7%. Seventeen studies used an HPLC assay to measure 6-MMP
[3,13,14,20,24,25,34,35,37–39,45,50,53,59,71,72]. The inter-assay CV
ranged from 0.2% to 9% and the intra-assay CV ranged from 0% to 9.5%.
Five studies used an HPLC method to measure 6-methylthioguanine
(6-MTG) formed using 6-thioguanine (6-TG) as a substrate
[12,21,22,41,47] and reported CVs from 2% to 5% for intra-assay and
from 4% to 10% for inter-assay performance.
Two studies measured precision and reproducibility between
laboratories [24,40]. The intra-day and inter-days CV values ranged
from 1.7% to 4.3% and 0.8% to 6% respectively, for six specimens. Inter-
assay CV values were 5.8% for 20 samples, and 5.2% for 5 samples of
quality control material. Klemetsdal et al (Department of Pharmacol-
ogy, Institute of Medical Biology, University of Tromso, Norway)
compared RBC TPMT activity in five samples also measured at the
Mayo Clinic (Rochester, USA), finding CV values of 1.8% to 2.6% for 20
analyses . Overall, no obvious trend over time or study
characteristics was observed between studies reporting lower versus
those reporting higher accuracy and reproducibility.
No study was found that specifically investigated the reproduc-
ibility, accuracy and precision of TPMT allelic polymorphism deter-
mination, but three studies reported 100% concordance between
denaturing HPLC and restriction fragment length polymorphism
(RFLP) tests of variant TPMT allelic polymorphism genotyping
[7,16,57]. One group developed a novel multiplex assay using
matrix-assisted laser desorption/ionization, with a time-of-flight
mass spectrometer (MALDI-TOF mass spectrometry) based on
Sequenom iPLEX technology .
All six laboratories that were surveyed collect whole blood with
EDTA as the anticoagulant for TPMT testing. Specimens are stored at
4 °C or room temperature for between 1 and 8 days.
Six laboratories were contacted to collect quality control informa-
tion. Of the six, one conducts only genotyping and two laboratories
conduct only phenotyping; however they do refer samples to other
laboratories for genotyping. One laboratory refers out low enzymatic
activity (i.e. below 10 U/g Hb) specimens for confirmatory genotyp-
ing. The other three laboratories conduct both types of analyses.
Between one and four specific genotypes are determined as
follows: TPMT*2 (four laboratories); TPMT*3A (six laboratories);
TPMT*3B (four laboratories); and TPMT*3 C (five laboratories).
Genotype determination is performed by PCR/RFLP, genetic sequenc-
ing and site-directed PCR. The methods are equally popular, each
being used by two laboratories.
TPMT enzymatic activity is determined using RBC lysates by
enzymatic assay followed by HPLC (three laboratories), or mass
spectrometry (one laboratory). Three laboratories report results as
nmol/g Hb/h, and one laboratory reports results as pmol/h/mg Hb.
Both are numerically equivalent to 1 U/g Hb, the unit chosen for use in
All six participating laboratories report a procedure for internal
quality control, which involves the inclusion of positive and negative
controls within each run, in some cases in duplicate (two laboratories
report conducting multiple runs per day). Results of internal quality
control procedures are similar across participating laboratories, with
enzymatic analysis repeatability ranging from 3% to 10% within runs,
and from 5% to 10% between runs. Two of the laboratories conducting
both genotyping and phenotyping analyses reported from 95% to
100% concordance between genotyping and phenotyping overall. One
laboratory clarified that concordance was lower (60%) for interme-
diate carriers. Positive and negative internal QC are obtained from
known staff, patients or pooled known samples.
We provide an overview of the current state of research on the
pre-analytic and analytic requirements for determining TPMT status.
Although only one study was specifically designed to evaluate the
effect of storage on TPMT activity , a variety of relationships were
reported among 13 studies. The available literature suggests that
TPMT activity is stable in EDTA and heparin anticoagulated whole
blood for up to a week at room temperature or 4 °C, which is
consistent with laboratory practices to store specimens (whole blood
with EDTA anticoagulant) at 4 °C or room temperature for between 1
and8 days. RBClysateisstable for3 monthsat −20 °C.Longerstorage
should be at -80 °C, although in the range of 15% of TPMT enzymatic
activity may be lost after 16 months or with repeated freeze–thaw
Pre-analytic requirements for TPMT allelic polymorphism deter-
mination were not addressed specifically; however pre-analytic
requirements are common for genetic testing. The Clinical and
Laboratory Standards Institute (CLSI) has published excellent guide-
lines covering all pre-analytic requirements for collection, transpor-
tation, preparation and storage of specimens for genetic testing .
Age and gender did not affect TPMT activity, and in two studies
there was no significant difference in TPMT activity across races,
including blacks, whites, mixed races, and Japanese [6,30]. However,
more races with appropriate sample sizes should be studied to
confirm the lack of interracial differences. One large study published
in July 2004 in the journal Pharmacogenetics was excluded from the
review based on predetermined inclusion criteria (study population).
Among 1200 healthy German individuals, a statistically significant
difference in TPMT activity was observed between males and females.
They also showed a statistically significant difference between
smokers and non-smokers, for both male and female smokers.
While the differences were statistically significant, clinically they
are likely unimportant.
In vitro studies have demonstrated that various drugs can affect
the enzymatic activity of TPMT; usually decreasing the activity of
TPMT (Table 2). However in a clinical diagnostic laboratory in many
cases the red cells are rinsed prior to assay, which should remove
extracellular inhibitory compounds. Six in vivo studies demonstrated
no clinically relevant interactions [15,17,27,52,61]. While the co-
administration of interfering drugs will likely not affect the in vitro
measurement of TPMT, these drugs may alter the in vivo activity of
TPMT and hence the true in vivo TPMT may not be reflected by the
measured TPMT activity. Care should be taken when prescribing AZA
and 6-MP in combination with other drugs that may inhibit TPMT
TPMT activity in various disease groups.
Study Population TPMT activity
Gisbert et al.,
All study participants20.1±6
Inflammatory bowel disease (n=7046)
Autoimmune hepatitis (n=359)
Multiple sclerosis (n=814)
Myasthenia gravis (n=344)
Healthy control (n=241)
Chonic renal failure (n=30); before hemodialysis
Chonic renal failure (n=30); after hemodialysis
Zhang et al.,
Unless otherwise noted, TPMT is measured as the formation of 6-MMP from 6-MP, in
RBCs. The radiochemical method uses radio-labelled SAM for the methyl donor, while
the HPLC method measures 6-MMP formed from 6-MP and non-radioactive SAM.
E. Loit et al. / Clinical Biochemistry 44 (2011) 751–757
activity, although we did not find any evidence demonstrating
clinically significant sequealae.
The activity of TPMT is measured in RBCs or RBC lysate, and there
has been a suggestion that extremes of hematocrit may affect the test
results. Although two out of three studies did demonstrate a
correlation between hematocrit and TPMT activity, the effect was
small (less than 7% in the normal hematocrit range) and likely not
clinically relevant . Standardizing TPMT activity to grams of
hemoglobin or milliliters of packed RBCs should correct for any
significant effect of hematocrit on TPMT measurement.
TPMT activity was significantly different in groups of patients with
inflammatory bowel disease, autoimmune hepatitis, multiple sclero-
sis, myasthenia gravis, pemphigus and chronic renal failure in
comparison with healthy control populations; however the differ-
ences were minor and not clinically relevant, with the exception of
patients requiring dialysis. Patients with renal failure showed
elevated TPMT enzymatic levels prior to hemodialysis, which dropped
by approximately 50% following hemodialysis to levels comparable to
normal individuals. The mechanism responsible for the elevated
TPMT activity pre-hemodialysis is unclear but may involve uniden-
tified TPMT activating uremic compounds . Although there are no
comparative studies of harms in a dialysis population directly
evaluating TPMT testing pre- and post-dialysis, the available evidence
suggests that dialysis patients should be measured post-dialysis, as
the levels most closely match those that would otherwise be seen in
them as healthier individuals. Measurement of TPMT activity prior to
dialysis may result in falsely identifying a low/absent or intermediate
metabolizer as a normal metabolizer, potentially placing them at
increased risk of drug toxicity. The remaining disease states studied to
date are organ transplant and acute lymphoblastic leukemia, which
were not included in this review.
The TPMT substrate 6-MP appears to be more widely used, but
enzymatic assays using either 6-MP or 6-TG, with either radiochem-
ical or HPLC detection method, were reasonably precise. Inter-assay
CVs were below 10% in all cases. With an analytical coefficient of
variance less than 50% of the biological variability, the amount of
variation added to the true test variability is 11.8% . In comparison
with other enzymatic assays, the currently achievable intra-laborato-
ry CV for TPMT enzymatic analysis is better than the minimum
acceptable performance for routine enzymatic analysis specified by
the U.S. Department of Health and Human Services: Clinical
Laboratory Improvement Amendments of 1988 (e.g. total creatinine
kinase (CK) below 30%, or aspartate aminotransferase (AST) below
TPMT allelic polymorphism detection appears to be reproducible
and accurate, with three reports of 100% concordance between
denaturing HPLC and restriction fragment length polymorphism
(RFLP) genotyping tests. The dichotomous nature of genetic results,
reported as either present or absent, does not permit precision and
accuracy calculation as is done for enzymatic determination. Howev-
er, a number of guidelines are available that address the complex
methodological issues. Recently, the Centers for Disease Control
released a Morbidity and Mortality Weekly Report detailing good
laboratory practices for molecular genetic testing. It reviewed the
need for adequate quality control of genetic testing and put forward
recommendations for laboratories . The Clinical and Laboratory
Standards Institute (CLSI) has also published guidelines for Molecular
Diagnostic Methods for Genetic Disease  and Validation and
Verification of Multiplex Nucleic Acid Assays . It is recommended
that any laboratories performing allelic polymorphism detection of
TPMT review the above guidelines to ensure the accuracy of their
Our systematic review revealed that sufficient pre-analytical data
were available to recommend preferred specimen collection, stability
and storage conditions for determination of TPMT status. There was
no clinically significant effect on TPMT activity of age, gender, various
co-administered drugs, or most morbidities (with the exception of
renal failure requiring dialysis). However, studies specifically
designed to investigate pre-analytic and analytic variables in TPMT
testing are needed.
Currently TPMT activity analyses are reported on one of two bases:
per milliliter of packed red blood cells or per gram of hemoglobin.
These are not readily or exactly comparable; so it is important to
establish a common standard. As well, cutoffs for low/absent,
intermediate, normal and high TPMT enzymatic activities should be
established, based on the common units. The available methods for
determination of TPMT enzymatic activity showed good precision,
with coefficients of variation generally below 10%. Based upon limited
evidence, the reproducibility of TPMT allelic polymorphism determi-
nation is acceptable; however, the sensitivity of genetic testing to
identify patients with low or intermediate TPMT enzymatic activity is
imprecisely known. Thus, if knowledge of TPMT status is desired and
there has been no recent transfusion of RBCs, with the current
evidence enzymatic assay (phenotyping) rather than allelic polymor-
phismdeterminationis preferred. Enzymaticassaywillcaptureeffects
of other polymorphisms that are not detected by genotyping the
common alleles; laboratories tend to use genotyping as a confirma-
tory test for low TPMT activity.
Supplementary data to this article can be found online at
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