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October 2011

Editor:
Fredrick L. Kiechle, MD, PhD

Question Q. Can an erythrocyte sedimentation rate test be performed on a specimen with a strong cold agglutinin? What is the special handling?

A. Before answering the question, it will be useful to explore the methods, utility, and limitations of the erythrocyte sedimentation rate test.

The ESR is a very simple, and essentially nonspecific, test that detects inflammation by using erythrocyte sedimentation. The basic method is to put well-mixed venous blood in a calibrated vertical tube, allow the red blood cells to “fall” (sediment out) onto the bottom of the tube, and record the length at which the RBCs fall in a specified time frame. In the strictest sense, the ESR is not really a rate of fall but a measurement of RBC sedimentation at a given time point.1,2

The ESR can be used as a diagnostic criterion for giant cell (temporal) arteritis and polymyalgia rheumatica as well as in the surveillance of disease activity/progression in giant cell (temporal) arteritis, polymyalgia rheumatica, inflammatory arthropathies, and rheumatoid arthritis. It has also found utility as a marker of cardiovascular disease and metabolic syndrome.3,4

Many factors affect the ESR. In inflammatory processes, multiple factors often contribute to ESR elevation, including anemia (often anemia of chronic disease), elevated fibrinogen, and increased immunoglobulins, making the ESR a sensitive marker for inflammation. The premise is that these positively charged immunoglobulin or fibrinogen molecules can decrease the negative charge that would repel RBCs from one another. The reduction of this negative charge can thus lead to rouleaux formation, allowing more rapid RBC sedimentation than in blood without excessive numbers of such proteins. With anemia, the increase in plasma-to-erythrocyte ratio favors rouleaux formation and is seen regardless of the plasma protein concentration. Other factors that elevate the ESR include red cell macrocytosis and decreased albumin. Small RBCs (microcytes) fall/sediment out slower than macrocytes. Changes to erythrocyte shape also affect the ESR; spherocytes and sickle cells hamper rouleaux formation, thus lowering the ESR.1,2,5–7

Cold agglutinins are cold antibodies that are essentially immunoglobulins, typically IgM with anti-I or anti-i specificity.1 Thus, with the presence of cold agglutinins, the ESR is predictably elevated. Though the recommendation is to handle the pipette with the blood sample at room temperature (18°–25°C), we note that some labs store samples at 4°C to increase the stability period before performing the test.6,7 This handling would likely increase the ESR further. Also, when the titer of the cold antibody is very high (that is, strong cold agglutinin), the antibody activity can even perform up to 37°C.1 Thus, the ESR result must always be interpreted in the context of other clinical findings and laboratory results. If cold agglutinin disease is suspected, CBC with peripheral smear review and cold agglutinin titers may be indicated instead.

If the ESR interface gives a readable value, then this value can be reported out, and the physician must interpret the result in context. However, there are situations in which the cold agglutinins result in significant cell clumping and plasma trapping and cause the ESR interface to be uninterpretable. In such situations, the ESR result should not be reported.

In reviewing the CLSI document, “Procedures for the erythrocyte sedimentation rate test,” there are no specific recommendations for removing cold agglutinins,6 and an extensive search in the medical literature likewise does not turn up any guidance on how to approach blood samples in patients with cold agglutinins. In any case, the ESR is not the preferred test to assess for another generalized inflammatory condition in a patient with a known cold agglutinin because the ESR will be predictably elevated in such patients.

In summary, though the ESR is a nonspecific marker of underlying generalized inflammation, it has certain limitations—one of which is in detecting/monitoring other generalized inflammatory conditions in the setting of cold agglutinins. If the concern is surveillance/monitoring of generalized inflammatory disease activity, perhaps a different laboratory test should be performed, such as monitoring C-reactive protein.8

References

1. McPherson RA, Pincus MR, eds. Henry’s Clinical Diagnosis and Management by Laboratory Methods. 21st ed. Philadelphia, Pa.: WB Saunders. 2006:465–466.

2. Jessen CU, Bing J. Methods for differentiating the cause of increased sedimentation rate. Acta Medica Scandinavica. 1940;105:287–300.

3. Hunder GG, Bloch DA, Michel BA, et al. The American College of Rheumatology 1990 criteria for the classification of giant cell arteritis. Arthritis Rheum. 1990;33: 1122–1128.

4. Salvarani C, Cantini F, Boiardi L, et al. Polymyalgia rheumatica and giant cell arteritis. N Engl J Med. 2002;347:261–271.

5. Talstad I, Scheie P, Dalen H, et al. Influence of plasma proteins on erythrocyte morphology and sedimentation. Scand J Haematol. 1983;31:478–484.

6. Procedures for the erythrocyte sedimentation rate test; approved standard—4th edition. CLSI document H02–A4, Vol. 20, No. 27. Wayne, Pa.: CLSI. 2000.

7. Jou JM, Lewis SM, Briggs C, et al. ICSH review of the measurement of the erythrocyte sedimentation rate. Int J Lab Hematol. 2011;33:125–132.

8. Rifai N. High-sensitivity C-reactive protein: a useful marker for cardiovascular disease risk prediction and the metabolic syndrome. Clin Chem. 2005;51:504–505.

Maria Vergara-Lluri, MD
Surgical Pathology Fellow
AP Co-chief resident
Department of Pathology
University of California
San Francisco

Member, CAP Hematology/Clinical
Microscopy Resource Committee

Question Q. Which is more cost-effective, extracting DNA from buccal smears or from paraffin blocks?

A. DNA extraction from buccal smears tends to cost less than DNA extraction from formalin-fixed, paraffin-embedded (FFPE) tissue. This is because extracting DNA from FFPE tissue usually requires more manual steps, including deparaffinization. Deparaffinization requires multiple washes in xylene, ethanol, and distilled water followed by rehydration in Tris. Also, if starting from scratch, obtaining DNA from a buccal smear is generally faster (averaging less than two minutes per sample) and simpler than obtaining DNA from FFPE tissue. However, an advantage of extraction of DNA from FFPE tissue is that enrichment of tumor cell DNA is possible with microdissection of tumor cells from thin sections of FFPE tissue under the microscope or by punching an area of tumor tissue in the paraffin block with the highest percent of tumor cells.

Also, there are DNA extraction and amplification methods from paraffin blocks that do not require deparaffinization, and it is possible that extracting DNA from FFPE tissue could be more cost-effective than from buccal smears under certain circumstances. Very simple extraction methods can be employed (boiling, for example), though the result is a crude extract and may be prone to PCR inhibition. Such simple extractions may work fine for some assays, but many, if not most, assays require more purified DNA. Therefore, the most cost-effective method may depend on the downstream application of the DNA.

Saab YB, et al.,1 reported a DNA extraction cost of only $0.06 per buccal smear specimen when testing 150 samples with a sodium hydroxide (NaOH) method. This cost included materials, reagents, and equipment, but it did not include the cost of the labor or sterile cotton swabs used to obtain the buccal cells. It took 16 minutes to extract DNA from 12 samples, with an average extraction time of 1.33 minutes per sample. The yield and quality of buccal cell DNA from this NaOH method were not significantly different from those of a kit-based method of buccal cell DNA extraction. The average DNA yield per specimen with the NaOH-based DNA extraction was 14.2 µg/mL. The quality of the DNA was assessed by amplification and gel electrophoresis detection of the angiotensin-converting enzyme gene insertion/deletion polymorphism. This polymorphism was detected successfully in 91.3 percent of the specimens (137/150).

It generally requires more time and money to extract DNA from paraffin-embedded blocks than to extract DNA from buccal smears. Muñoz-Cadavid C, et al., compared five DNA extraction methods from paraffin-embedded tissue and reported extraction times ranging between two and a half and six hours for 81 specimens using the different methods.2 By comparison, with an average extraction time of 1.33 minutes per specimen for buccal cell DNA extraction, it would have taken under two hours to extract DNA from 81 specimens. The prices of the kits for the five paraffin-embedded block extraction methods ranged from $42 to $180 ($0.52–$2.22 per specimen), which is higher than the cost per specimen of buccal DNA extraction ($0.06) without even including the cost of other materials and equipment.

Regardless of whether DNA is extracted from buccal cells or paraffin-embedded blocks, the extraction method the laboratory chooses will affect the cost in terms of the expense of materials and time. The materials for homebrew methods are usually less expensive than the purchase price of kit-based methods, but the time, labor, and sample success rates must also be considered in a cost comparison of methods. Bonin S, et al.,3 compared DNA extraction methods from paraffin-embedded tissue at multiple centers in Europe. Their study demonstrated that the success rates of DNA mutation testing from extraction protocols that did not include additional steps for purification or precipitation were not significantly different from the success rates of methods that did include these extra steps (such as NaAc/ETOH or isopropanol precipitation or silica-based absorption columns). However, the results for more complex genetic testing such as comparative genomic hybridization were significantly better with the silica-based absorption columns. This study highlights the importance of considering how the DNA will be used when deciding on the DNA extraction method.

References

1. Saab YB, Kabbara W, Chbib C, et al. Buccal cell DNA extraction: yield, purity, and cost: a comparison of two methods. Genet Test. 2007;11(4):413–416.

2. Muñoz-Cadavid C, Rudd S, Zaki SR, et al. Improving detection of fungal DNA in formalin-fixed paraffin-embedded tissues: comparison of five tissue DNA extraction methods using panfungal PCR. J Clin Microbiol. 2010;48(6):2147–2153.

3. Bonin S, Hlubek F, Benhattar J, et al. Multicentre validation study of nucleic acids extraction from FFPE tissues. Virchows Arch. 2010;457(3):309–317.

Theresa A. Boyle, MD, PhD
Stanford University Hospital
Palo Alto, Calif.

Member, CAP Molecular
Oncology Committee

Question Q. Is the hCG-like pituitary hormone two-stranded, that is, alpha and beta? Will the Siemens Immulite 2000 method detect it?

A. Although it is not widely appreciated, we have known for many years that the pituitary gland makes human chorionic gonadotropin (hCG) in addition to luteinizing hormone (LH). HCG is the trophoblast’s own version of LH, made to promote the continuation of progesterone production by the corpus luteum so the endometrium does not degenerate. The two beta-chain genes are part of an LH/hCG cluster on chromosome 19, and the two hormones share the same alpha chain. Pituitary cells stimulated by gonadotropin-releasing hormone (GnRH) make both hormones during the menstrual cycle; although pituitary hCG is present at a much lower concentration at the time of the mid-cycle LH surge, it is apparently much more biologically active.

While pituitary hCG has the same heterodimeric structure as trophoblast hCG, pituitary hCG is differently glycosylated. Most commercially available hCG immunoassays probably recognize pituitary hCG, however, which can produce puzzling pregnancy test results, especially during menopause.

Reference

Cole LA. Biological functions of hCG and hCG-related molecules. Reprod Biol Endocrinol. 2010;8:102.

James D. Faix, MD
Stanford Clinical Labs at Hillview
Stanford University School of Medicine
Palo Alto, Calif.

Member, CAP Chemistry Resource
Committee, Standards Committee,
Council on Scientific Affairs


Dr. Kiechle is medical director of clinical pathology, Memorial Healthcare, Hollywood, Fla.