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March 2008

Question Q. There is a lot of information about how to freeze serum and plasma specimens, but I cannot find anything on how to thaw them properly. Can you help?

A. This question addresses an issue that is rarely discussed in studies related to specimen preparation and subsequent frozen storage followed by thawing and testing serum or plasma, or both.1-3 Detail is usually provided on the method used for freezing (snap-freezing using liquid nitrogen, uniform freezing to avoid gradients,4 dry ice,5 or addition of sucrose6 or dimethylsulfoxide7 to stabilize low-density lipoproteins while frozen). Young8 has listed a variety of assays that are altered by freezing and thawing serum specimens. Frozen specimens may be subject to freeze-thaw cycles when stored at -20°C in frost-free freezers. Using insulated containers may help prevent thawing during these ­cycles.

In general, frozen serum or plasma specimens should be thawed at room temperature. Rapid thawing using heat may result in decomposition of components. To disrupt concentration gradients formed during thawing, the sample should be inverted 10 to 20 times9 (no foam formation). If undissolved material is present, controlled warming may bring the particles into solution.10 An automated workcell for thawing 760 specimens has been reported.9 Frozen specimens are placed in racks with an opening at the bottom that allows room-temperature air (2 L/min) to circulate, thawing specimens in 22 to 23 minutes. The specimens are inverted eight times with an arm equipped with a pneumatic pin cylinder to prevent the cap from loosening and causing leaks.

It has been found in proficiency testing that pools of cryoprotected11 or fresh frozen serum11-13 are commutable to patient samples compared with samples spiked with nonhuman enzymes or various simulated aqueous matrices used to dissolve the constituents. The guidelines for preparing these pools carefully detail the procedure for freezing, but not thawing, the specimen prior to proficiency testing analysis in the clinical laboratory.4,13 They also address the effect of the length of storage of frozen samples.14 The method for thawing serum units prior to preparing smaller-volume fresh frozen serum vials for shipping was used to prepare frozen serum pools to have an assigned creatinine value using the reference measurement procedure isotope dilution gas chromatography/mass spectrometry.13 A total of 670 frozen serum units were thawed for five hours at room temperature with forced air circulation and incubated for four hours at 4°C prior to pooling, centrifugation, repooling, and mixing under argon. These units were then filtered, dispensed into vials, and refrozen at -50°C.13 This meticulous procedure provides rare detail on the steps that are required for preparing commercial material used for quality control or proficiency testing.

Finally, the American Association of Blood Banks has defined methods for thawing units of fresh frozen plasma, cryoprecipitate, red blood cells, and other blood products.15 Units of fresh frozen plasma should be thawed between 30° and 37°C, usually in a water bath.15,16 Moreover, this method at 37°C required 17.6 minutes compared with 6.99 ± 1.3 minutes when a microwave device was used.17 The microwave provides a more rapid turnaround time for stat product requests. If a microwave device is used to thaw fresh frozen plasma, the device must be shown not to exceed temperature limits and not to damage the plasma proteins.15

In summary, frozen clinical serum and plasma specimens should be thawed at room temperature with mixing by inversion several times before they are used for clinical analysis. Exceptions to this simple rule should be defined and communicated to the clinical laboratory community.

References

  1. Blaakaer J, Micic S, Høgdall CK. Immunoreactive inhibin concentration in blood tested under variable sampling conditions. J Steroid Biochem Mol Biol. 1996; 58:325-328.
  2. Sanlidag T, Akcali S, Ozbakkaloglu B. Serum hepatitis B DNA: stability in relation to multiple freeze-thaw procedures. J Virol Methods. 2005;123:49-52.
  3. Procedures for the Handling and Proc­essing of Blood Specimens: Approved Guideline. NCCLS document H18-A3 (ISBN 1-56238-555-0). Wayne, Pa.: NCCLS; 2004.
  4. Preparation and Validation of Commutable Frozen Human Serum Pools as Secondary Reference Materials for Cholesterol Measurement Procedures: Approved Guideline, NCCLS document C37-A (ISBN 1-56238-392-2). Wayne, Pa.: NCCLS; 1999:6.
  5. Sebire K, McGavin K, Land S, et al. Stability of human immunodeficiency virus RNA in blood specimens as measured by a commercial PCR-based assay. J Clin Microbiol. 1998;36:493-498.
  6. Rumsey SC, Galeano NF, Arad Y, et al. Cryopreservation with sucrose maintains normal physical and biological properties of human plasma low density lipoproteins. J Lipid Res. 1992; 33: 1551- 1561.
  7. Sigalov AB. Cryopreservation and long-term storage of human low density lipoproteins. Eur J Clin Chem Clin Biochem. 1995;33:73-81.
  8. Young DS. Effects of Preanalytical Variables on Clinical Laboratory Tests. 2nd ed. Washington, DC: AACC Press; 1997: 4- 180, 181,533.
  9. Hawker CD, Roberts WL, DaSilva A, et al. Development and validation of an auto­mated thawing and mixing workcell. Clin Chem. 2007;53:2209-2211.
  10. Guder WG, Narayanan S, Wisser H, et al. Samples: from the patient to the laboratory. The impact of preanalytical variables on the quality of laboratory results. Darmstadt, Germany: GIT Verlag; 1996: 40, 41, 69.
  11. Baadenhuijsen H, Kruypers A, Weykamp C, et al. External quality assessment in the Netherlands: time to introduce commutable survey specimens. Lessons from the Dutch "Calibration 2000" project. Clin Chem Lab Med. 2005; 43: 304- 307.
  12. Klee GG, Killeen AA. College of American Pathologists 2003 fresh frozen serum proficiency testing studies. Arch Pathol Lab Med. 2005;129:292-293.
  13. Miller WG, Myers GL, Ashwood ER, et al. Creatinine measurement: state of the art in accuracy and interlaboratory harmonization. Arch Pathol Lab Med. 2005; 129: 297- 304.
  14. Levey AS, Coresh J, Greene T, et al. Expressing the Modification of Diet in Renal Disease Study equation for estimating glomerular filtration rate with standardized serum creatinine values. Clin Chem. 2007;53:766-772.
  15. Brecher ME, ed. Technical Manual. Bethesda, Md.: AABB; 2005:191, 192, 815.
  16. Isaacs MS, Scheuermaier KD, Levy BL, et al. In vitro effects of thawing fresh-frozen plasma at various temperatures. Clin Appl Thromb Hemost. 2004;10: 143- 148.
  17. Churchill WH, Schmidt B, Lindsey J, et al. Thawing fresh frozen plasma in a microwave oven. A comparison with thawing in a 37 degrees C waterbath. Am J Clin Pathol. 1992;97:227-232.
  18. Frederick L. Kiechle, MD, PhD
    Medical Director, Clinical Pathology
    Memorial Regional Hospital
    Hollywood, Fla.
     
    Member, CAP Chemistry
    Resource Committee

Question Q. Is a differential cell count needed in CSF samples with normal cell counts? A. Cerebrospinal fluid, or CSF, is submitted to the laboratory most often to evaluate for infection, hemorrhage, or malignancy. A cell count and differential count are routinely requested as part of this evaluation. It is logical to perform a differential cell count on a CSF sample with an increased number of cells. However, the issue has been raised of whether a differential count is needed in CSF samples with normal cell counts. Literature addressing this question is scant, but the following are guidelines on how members of the CAP Hematology/Clinical Microscopy Resource Commi­ttee handle the differential cell count on CSF samples.

A Wright-Giemsa-stained cytocentrifuged slide is prepared for all CSF samples, regardless of the total cell count. In cases with a normal CSF cell count, the slide is used differently depending on the patient population. Cytocentrifuged slides from adults are always scanned for organisms or abnormal cells, particularly in patients with a history of malignancy. When a malignant cell is identified, it is always reported whether or not a differential cell count is performed. CSF-cytocentrifuged slides with organisms or malignant cells are always reviewed by a pathologist. A differential cell count is typically performed on CSF samples from children even if the cell count is normal. In cases where very few cells are present on the smear (as would be expected if the cell count is normal), the number of cells counted is also reported.

Beverly Nelson, MD
Northwestern University
Feinberg School of Medicine
Chicago, IL
 
Member, CAP Hematology/
Clinical Microscopy Resource Committee