Nucleosomes in serum as a marker for cell death.

Author(s): Holdenrieder S, Stieber P, Bodenmüller H, Fertig G, Fürst H, Schmeller N, Untch M, Seidel D

Publication: Clin Chem Lab Med, 2001, Vol. 39, Page 596-605

PubMed ID: 11522104 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to determine the effects of pre- and post-centrifugation storage, the addition of EDTA, freeze-thaw cycling, specimen mixing, and contamination with hemolysate or bacteria on nucleosome levels in plasma and serum.

Conclusion of Paper

The authors report that nucleosome levels increased with delayed centrifugation at all temperatures, but the effect was largest when blood was stored at 37 degrees C. Increasing post-centrifugation storage prior to the addition of EDTA led to decreased nucleosome levels, which, the authors report, was independent of storage temperature. Ucleosome levels were not affected by storage of serum after the addition of EDTA for up to 8 h at 4, 25 or 37 degrees C or for up to 6 months at -20 degrees C. Dilution of serum prior to storage led to 50% lower measured levels of nucleosomes than when serum was diluted after thawing. The authors report increased nucleosome levels with increasing numbers of freeze-thaw cycles but that post-thaw centrifugation or vortexing had no effects on nucleosome levels. Plasma nucleosome levels were lower than serum levels and appeared to be relatively stable during frozen storage, with or without EDTA. The authors report the measured levels of nucleosomes increased proportionally with the addition of hemolyzed serum or bacteria to specimens.

Studies

Study Purpose

The purpose of this study was to determine the effects of the addition of EDTA and the duration of frozen storage on nucleosome levels in serum and plasma.

Summary of Findings:

Levels of nucleosomes were higher in serum than in plasma under all storage conditions. Nucleosome levels in serum were most stable when 10 mM EDTA pH 8.0 was added prior to storage at -20 or -80 degrees C. In 3 serum pools with EDTA added, the coefficient of variance was between 4.2-9.2% during storage for 6 months at -20 degrees C. Nucleosome levels were elevated in serum stored at -20 or -80 degrees C for 1 day without EDTA and were decreased in serum frozen at either temperature for 3 days without EDTA compared to unstored serum. Dilution of serum prior to storage led to 50% lower measured levels of nucleosomes than when serum was diluted after thawing. The authors report increased nucleosome levels with increasing numbers of freeze-thaw cycles, but that post-thaw centrifugation or vortexing had no effects on nucleosome levels. Nucleosome levels in plasma appeared to be relatively stable during frozen storage, with or without EDTA.

Biospecimens

Preservative Types

Frozen

Diagnoses:

Not specified
Neoplastic
Other diagnoses

Platform:

Analyte	Technology Platform
Protein	ELISA
DNA	ELISA

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Storage	Storage temperature	-20 degrees C -80 degrees C
Storage	Storage duration	0 days 1 days 3 days 7 days 21 days 1 month 2 months 3 months 4 months 6 months
Storage	Storage conditions	Diluted Undiluted
Storage	Freeze/thaw cycling	1 cycle 2 cycles 3 cycles
Biospecimen Preservation	Fixative additive/buffer	EDTA None
Biospecimen Aliquots and Components	Blood and blood products	Plasma Serum
Biospecimen Aliquots and Components	Biospecimen mixing	None (centrifuged) Vortexed 1 s Vortexed 3 s Vortexed 5 s Vortexed 10 s Vortexed 20 s

Study Purpose

The purpose of this study was to determine the effects of delayed centrifugation and storage post-centrifugation on nucleosome levels in serum. All serum was stored at -20 degrees C after experimental storage.

Summary of Findings:

The authors report that nucleosome levels increased with delayed centrifugation of blood specimens for up to 6 h at 4, 25, or 37 degrees C, but the effect was largest when blood was stored at 37 degrees C. Increasing post-centrifugation storage of serum, prior to addition of EDTA, led to decreased nucleosome levels, which the authors report were independent of storage temperature. Storing serum for up to 8 h after the addition of EDTA had no effects on nucleosome levels, regardless of storage temperature.

Biospecimens

Preservative Types

Frozen

Diagnoses:

Normal
Neoplastic
Other diagnoses

Platform:

Analyte	Technology Platform
Protein	ELISA
DNA	ELISA

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Preaquisition	Diagnosis/ patient condition	Unspecified acute infection
Biospecimen Preservation	Fixative additive/buffer	EDTA None
Storage	Storage temperature	4 degrees C 25 degrees C 37 degrees C
Storage	Storage duration	0 h 2 h 4 h 6 h 8 h
Biospecimen Aliquots and Components	Blood and blood products	Serum Whole blood
Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated

Study Purpose

The purpose of this study was to determine the effects of hemolysis or bacterial contamination on nucleosome levels in serum. Blood was stored at 37 degrees C for 4 h with bacteria prior to centrifugation. Hemolysis was induced by shaking whole blood at 37 degrees C and hemolyzed serum was added to specimens.

Summary of Findings:

The authors report that measured levels of nucleosomes increased proportionally with addition of hemolyzed serum. While in the absence of blood, no nucleosomes were detected in bacteria, the addition of bacteria to blood led to elevated measured nucleosome levels in serum, regardless of subsequent treatment of blood with antibiotics. Average nucleosome levels were much lower in serum from healthy individuals than from patients with inflammation or solid tumors (particularly lung cancer). High nucleosome levels were associated with advanced stage cancer.

Biospecimens

Preservative Types

None (Fresh)

Diagnoses:

Normal
Other diagnoses
Neoplastic - Carcinoma

Platform:

Analyte	Technology Platform
DNA	ELISA
Protein	ELISA

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Biospecimen Aliquots and Components	Biospecimen components	No additive 1 ul hemolyzed serum 2 ul hemolyzed serum 3 ul hemolyzed serum 5 ul hemolyzed serum 10 ul hemolyzed serum E. coli added S. aureus added P. aeruginosa added Antibiotic added (0, 1, 10 or 100 mg/mL)
Preaquisition	Diagnosis/ patient condition	Normal Inflammatory disease Solid tumor
Biospecimen Aliquots and Components	Hemolysis	Hemolysate added No hemolysate added Shaking-induced
Biospecimen Aliquots and Components	Blood and blood products	Serum Whole blood

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