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Transfusion 2007;47:1972-1983.
TMR Journal Club - March 5, 2008
Maggie Constantine, MD, FRCPC (Hematology)
Resident, Transfusion Medicine (UBC)
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What is CMV?
Cytomegalovirus structure
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http://www.biografix.de/biografix/english/images/2/p_2b2a.jpg
Why is CMV important?
Immunocompromised patients
Disease
type
Probable
Definite
CMV
syndrome
Fever, malaise, leukopenia, atypical
lymphocytes, transminitis, evidence of CMV
in blood
Clinical/lab data and no other
cause identified
Pneumonia
Evidence of CMV in blood (or BAL), no
other cause of pulmonary disease
Probable plus detection of CMV in
lung tissue
GI disease
Symptoms of GI disease without other
cause PLUS endoscopic evidence +/- CMV
in blood
Probable plus detection of CMV in
GI tissue
Hepatitis
Elevation of bilirubin +/- liver enzymes in
absence of other cause PLUS evidence of
CMV in blood
Probable plus detection of CMV in
liver tissue
CNS disease
CNS symptoms in absence of other cause
plus CMV in CSF
CNS symptoms plus detection of
CMV in CNS tissues
Retinitis
Not applicable
Lesions typical of CMV retinitis
confirmed by opthalmologist
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Ways of preventing TT-CMV
Major recommendations
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Boeckh. ASH-ED 2001.
Ways of preventing TT-CMV
Canadian Consensus

View One - one panelist


Recommend abandoning CMV serologic testing
entirely, with careful follow-up of high-risk patients
View Two - two panelists

Advocate abandoning the use of CMV-tested blood
components except in pregnant women during the
first two trimesters of pregnancy and for intra-uterine
fetal transfusion
• Diagnosis of CMV infection/disease cannot be made in
time for antiviral agents to be administered
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Laupacis et al. 2001. Transfusion.
Ways of preventing TT-CMV
Canadian Consensus

View Three - seven panelists


Recommend continued provision of both WBCreduced and CMV-seronegative blood components for
CMV-seronegative pregnant women, intrauterine fetal
transfusions, and CMV-seronegative allogeneic
marrow transplant recipients.
Continued use of CMV-seronegative blood
components was felt to be probably indicated for
patients undergoing SOT, patients with conditions
likely to require allogeneic SCT and CMVseronegative HIV patients.
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Laupacis et al. 2001. Transfusion.
Ways of preventing TT-CMV
Meta-analysis
Leukoreduction vs Serologic screening
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Vamvakas, E. 2005. Transfusion Med Rev, 19(3); 181-199.
Ways of preventing TT-CMV
Meta-analysis
Leukoreduction vs Serologic screening

Conclusions

CMV seronegative recipients = 829
• 11 studies
• CMV infection = 12%
• Amongst BMT /hematologic malignancy patients =
1.63%

WBC-reduced recipients = 878
• 12 studies
• CMV infection = 2.73%
• Amongst BMT/hematologic malignancy patients =
3.01%
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Vamvakas, E. 2005. Transfusion Med Rev, 19(3); 181-199.
How do we detect CMV?
Serologic and other assays


Screening versus
diagnostic testing
Seropositivity amongst
blood donors


Between 20-80%
Serologic assays for
screening


Latex, particle
False negatives
• Window period
• Waning antibodies
• Genotypic variation
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


Detection of viral
antigens or nucleic acids
Likelihood of detection
highest soon after
infection
Unclear clinical
implications if



Seronegative with +ve
nucleic acid test
Seropositive with +ve
nucleic acid test
? Explain
seroconversion of CMV
negative recipients of
“seronegative” blood
products
How do we detect CMV?
Interpretation of results

Viral exposure


Acute infection – high
viral loads in peripheral
blood WBC and plasma
Seroconversion –
elimination of plasma
viremia and actively
infected cells
• 6-8 weeks
seronegative “window
period”


CMV DNAemia (marker
for acute infection)



Positive in
seroconverting donors
(seronegative)
Not in remotely infected
seropositive donors
“window period”
• 8 weeks to several
years
Leukoreduction does not
address this nor CMV
transmission of latent
virus within WBCs
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Drew WL and Roback JD. Transfusion 2007;47:1954-1957.
Ziemann et al. Transfusion 2007;47:1972-1983.
Review of article

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What is the correlation between
interdonation interval and prevalence
of CMV DNA in plasma sample of
newly seroconverted donors or the
variations in prevalences of CMV DNA
between different donor collectives?
Ziemann et al. Transfusion 2007;47:1972-1983.
Review of article - Methods


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Prospective study
University of Lubeck, Germany
August 2000 and June 2004
Volunteer regular blood donors

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12,800 donors -> 34,000 WB donations/year
41% female
82 well-defined CMV seroconversion cases
• Total number of CMV seroconversions during this
time unknown
• Grouped according to interval since last donation
(less than 120, 120, to 729, and 730 days or
more)
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Ziemann et al. Transfusion 2007;47:1972-1983.
Review of article - Methods

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598 latently infected blood donors
(seropositive for at least 1 year)
150 CMV-seronegative donors were tested
for CMV DNA as controls
To determine minimum rate of CMV DNApositive donations due to primary CMV
infection of donors

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All available samples from previously
seronegative donors who were repeat
reactive (IgG ELISA) between Jan and Dec
2006 were tested by PCR
Ziemann et al. Transfusion 2007;47:1972-1983.
Review of article - Methods

Definitions

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
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“date of seroconversion” = date of first
seropositive sample from a previously
seronegative donor
CMV serology “repeat reactive samples” =
samples retested in duplicate and at least
one of the two repetitions also gave a
positive result
“CMV DNAemia” = diagnosed by
reproducibly positive results
“surrogate markers for viral infections” = ALT,
neopterin
Ziemann et al. Transfusion 2007;47:1972-1983.
Review of article - Methods
CMV serology – automated enzyme
immunoassay to detect IgG Abs
against fusion proteins CG1 and CG2
(Biotest AG, Dreieich, Germany)
 Nucleic acid isolation – EDTA plasma
using Extractor (NucliSens, Nurtingen,
Germany)
 TaqMan PCR – standards, controls
and detailed methods included

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Ziemann et al. Transfusion 2007;47:1972-1983.
Review of article - Methods

Statistical analysis


Pre-determined p value of 0.05
Probability of CMV DNA in plasma of latently
infected blood donors
• Upper limits of 1-α confidence intervals of the
binomical distribution for an α level of 0.05
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Sensitivity of surrogate markers for detection
of CMV DNA-positive donation calculations
described
Ziemann et al. Transfusion 2007;47:1972-1983.
Review of article - Results

TaqMan PCR


1055 samples
1.2% (13) ambiguous
results
• Insufficient sample
volume
• Equivocal results even
with retesting

95% detection limit =
4.88 geq/PCR
procedure (3.66-8.22
geq/PCR)
• 13.5 geq/mL
• Mean in positive
samples = 166 geq/mL
• Max in positive
samples = 3200
geq/mL
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Ziemann et al. Transfusion 2007;47:1972-1983.
Review of article - Results
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Ziemann et al. Transfusion 2007;47:1972-1983.
Review of article - Results
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Ziemann et al. Transfusion 2007;47:1972-1983.
Review of article - Results
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Ziemann et al. Transfusion 2007;47:1972-1983.
Review of article - Results
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Ziemann et al. Transfusion 2007;47:1972-1983.
Review of article - Discussion
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Discrepant CMV DHAN detection in first time
seropositive donors
 44% in this study
 1% in Drew et al. 2003 study
 “window period” data is confirmatory (3% vs 0.5%)
Does CMV DNA detection correspond to viable CMV
detection?
 Yes
 Viral cultures and shell vial assays sensitivities too
low to use as screening assays
Seasonal reactivation not confirmed
Ziemann et al. Transfusion 2007;47:1972-1983.
Review of article - Discussion

Limitations
No analysis of whether residual WBCs
in WBC-depleted blood components
of newly seroconverted or latently
infected donors contained CMV DNA
 Cannot rule out if residual risk of TTCMV associated with LRD blood
products is related to viremia during
seroconversion

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Ziemann et al. Transfusion 2007;47:1972-1983.
Review of article - Discussion

Can “transfusion of LRD blood
components from seronegative
donors imply a greater risk of TT-CMV
than transfusion of LRD blood from
donors who have been seropositive
for at least 1 year”?

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because window-phase donations
were detected but not reactivation
donations
Ziemann et al. Transfusion 2007;47:1972-1983.
Review of article – Conclusion



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“…detection of CMV DNA was closely related to the
first detection of CMV IgG antibodies in up to 62% of
our newly seroconverted donors…”
“…probability of detection of CMV DNA in plasma of
blood donors at least 1 year after seroconversion was
lower than 0.5%.”
“Window-phase donations occurred in only 3% of
seroconversion cases.”
“…the main source of blood products containing free
CMV DNA were newly seroconverted donors.”
Ziemann et al. Transfusion 2007;47:1972-1983.
Critical appraisal
Are the results of the CMV DNA
testing valid?
 Is the following suggestion valid?
“transfusion of LRD blood
components from seronegative
donors imply a greater risk of TTCMV than transfusion of LRD
blood from donors who have been
seropositive for at least 1 year”

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Ziemann et al. Transfusion 2007;47:1972-1983.
Critical appraisal

Was there an independent, blind
comparison with a reference standard?

No reference standard to CMV DNA used
• Test for CMV infectious particles
• Unclear what the reference standard is for CMV
DNA
• Viral cultures or shell vial assays?

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Not explicitly stated in methods that
investigators conducting CMV DNA assays
were “blinded” to CMV serology status of
donor
Ziemann et al. Transfusion 2007;47:1972-1983.
Critical appraisal

Did donor sample include an appropriate
specturm of donors to whom the test will be
applied to at CBS?


Were the methods for performing the test
described in sufficient detail to permit
replication?

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Yes, a wide spectrum of “interval since last
seronegative donation” donors were
included
Yes
Ziemann et al. Transfusion 2007;47:1972-1983.
Critical appraisal

Will the results help me in managing donor
testing?



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Are the results applicable to my donor
population?
Unclear, but likely (is it necessary to
replicate the results for Canadian blood
donors?)
How large would a RCT have to be to
demonstrate a benefit if switched to
Ziemann suggested CMV transfusion
strategy?
Transfusion 2007;47:1972-1983.
TMR Journal Club - March 5, 2008
Maggie Constantine, MD, FRCPC (Hematology)
Resident, Transfusion Medicine (UBC)
Questions? Comments?
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