Decline in detectable galactomannan levels during storage of serum samples

G. L. Johnson,1 K. Hill2 and S. Agrawal3

Author address: 

Queen Mary University of London, London, UK, 2Division of Infection, Barts and the London NHS Trust, UK and 3Department of Haematological Oncology, St Bartholomew’s Hospital, UK

Abstract: 

Objectives: Invasive pulmonary Aspergillosis (IPA) remains a common life-threatening opportunistic invasive mycosis in immunocompromised patients. The Platelia Aspergillus enzyme immunoassay (PA-EIA, Bio Rad, France) provides non-invasive, quantitative Aspergillus- specific marker detection for serum galactomannan (GM). Data on the stability of the galactomannan molecule with sample storage is not readily available. We performed a retrospective study looking at the impact of serum storage on GM reactivity. Methods: Serum samples were collected from patients with AML and MDS undergoing intensive chemotherapy or allogeneic transplantation at St Bartholomew’s hospital between July 2005 and March 2010. GM serum monitoring of neutropenic patients occurred twice weekly using PA-EIA ELISA carried out as recommended by the manufacturer. A GM Index "¡ 0.5 was considered positive, as recommended by the US Food and Drug Administration. Long-term storage at )80 C; From July 2005 to September 2007 a total of 54 serum samples were collected and GM tested, with aliquots stored at )80 C. Retesting of stored samples was carried out in 2009. Short-term storage at +4 C; laboratory records from February 2009 to March 2010 were filtered to remove GM data: (i) for all nonserum samples, and (ii) for all samples repeat-tested after greater than 48 h. A total of 112 serum samples repeated within 48 h were identified for analysis. Results: Long-term storage at )80 C; 28/54 samples were GM Index positive (>0.5) prior to freezing (T0). Following long-term storage at )80 C, signal loss was seen in 27 of these 28 samples (96.4%) (T1). Mean reactivity reduction was 28.9% in samples with T0 GM Index >2 and 64.6% in samples with T0 GM Index 0.52. 18/28 (66.7%) positive samples at T0 became negative after long-term storage at )80 C. Of 26/54 negative samples at T0, all 26 samples remained GM Index negative at T1, with 19 samples (73.1%) showing signal loss. Although data is limited (n = 12), a decline in GM reactivity was also seen in broncho-alveolar lavage fluid; interestingly, the degree of signal loss was less marked. Short-term storage at +4 C; 72/112 samples were GM Index positive at T0. Upon repeat testing within 48 hours (T1), 60 of these 72 samples (83%) showed signal loss. The mean reactivity reduction was 54.1% in samples with a T0 GM Index between 0.52 and 45/72 (62.5%) samples positive at T0 had become GM negative. Of 40/112 negative samples at T0, all 40 samples remained GM Index negative at T1, with 19 samples (47.5%) showing signal loss. Conclusions: Serum GM testing is widely used in clinical practice and is included in EORTC/MSG mycological criteria for diagnosing probable IPA cases. Our data shows significant signal loss in serum samples stored long term at )80 C. More importantly, the clinical use of GM testing may be critically time-dependent as samples kept at +4 C and retested within 48 h (i.e. according to manufacturer’s recommendations) also showed signal loss, with greater than 60% of samples becoming negative. Further investigation of the factors impacting on GM testing are underway.
2011

abstract No: 

O1.5

Full conference title: 

Trends in Medical Mycology, 5th
    • TIMM 5th (2013)