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Clinical and Vaccine Immunology, November 2006, p. 1294-1295, Vol. 13, No. 11
1071-412X/06/$08.00+0     doi:10.1128/CVI.00218-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Dark Immunofluorescence: Correlation with Serum Immunoglobulin Abnormalities

Department of Haematology (Immunology Section),¹ Department of Clinical Biochemistry, Colchester General Hospital, Turner Road, Colchester, Essex CO4 5JL,³ Department of Immunology, St. Bartholomew's Hospital, London EC1A 7BE, United Kingdom²

Received 10 June 2006/ Returned for modification 25 July 2006/ Accepted 29 August 2006

	ABSTRACT

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Occasional serum samples (<0.5%) tested by indirect immunofluorescence showed less fluorescence than did negative-control serum. A retrospective review of these patients' serum immunoglobulins revealed a high percentage of abnormalities (71%, versus 22% of controls). We suggest that this observation should be reported when seen and that the clinician should be alerted to an association with immunoglobulin abnormalities.

	TEXT

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Indirect immunofluorescence (IF) is used in the detection of autoantibodies. Positivity is recognized as bright green fluorescence at the site of antibody-antigen interaction (1). Normal human serum gives the appearance of dull, blue-green or yellow-green staining depending upon the use of a counterstain and the selected barrier filter on the fluorescence microscope. This normal background fluorescence results from the nonspecific binding of the secondary fluorochrome-conjugated antibody. A failure to add either secondary antibody or serum during testing results in a noticeable absence of nonspecific fluorescence.

During examination of rodent liver-kidney-stomach (LKS) sections, occasional samples (<0.5%) demonstrated this markedly reduced level of overall fluorescence. In some cases, the LKS sections appeared dull red, suggesting that a failure to add either the serum or the secondary antibody had occurred. However, the observation was reproducible and was also noted during testing for antiendomysial antibodies (EMA) with monkey esophagus (MO). This appearance is referred to as "dark IF" throughout this report. Dark IF was less apparent on HEp-2 cells, possibly due to the higher screening dilution of the test samples, reducing the level of overall nonspecific fluorescence; therefore, this report describes results with LKS and MO only.

Local Research Ethics Committee (North and Mid Essex, United Kingdom) approval was obtained to explore the hypothesis that abnormalities in serum immunoglobulins (Igs) may be responsible for dark IF, a theory prompted by our observation of this phenomenon in a patient with known hypogammaglobulinemia. Since this initial observation, it has been standard practice to note the presence of dark IF when present. Retrospective data could therefore be collected for all samples recorded independently by two experienced screeners to have dark IF from June 2001 to December 2002. IF screening was performed without knowledge of the serum Igs; these data were subsequently available for 49 samples (1 sample was later discarded due to incomplete data).

For the selection of a representative control group (five times the size of the dark IF group), all requests for autoantibody testing and serum Ig analysis received by the laboratory during this 18-month period were considered (9,500 autoantibody test requests; 4,100 serum Ig analyses). To be eligible for inclusion in the control group, subjects had to have autoantibody screens not demonstrating dark IF and serum Ig results (n = 357). The first 245 cases (ordered alphabetically) were selected. Data analyzed included age, sex, findings for indirect immunofluorescence, serum Igs, and serum protein electrophoresis (SPE). The male/female ratios were similar in the two groups, 0.6:1 (dark IF) and 0.54:1 (control), as were the mean ages (standard deviations [SD]), 46.1 years (26.9) in the dark IF group and 52.1 years (23.4) in the control group.

Serum tested on LKS substrate was diluted 1/20 in phosphate-buffered saline (PBS). PBS, LKS substrate, and fluorescein isothiocyanate (FITC)-conjugated anti-human IgG (heavy and light chains), containing Evan's Blue counterstain as the second-stage antibody, were from BioDiagnostics Ltd. (United Kingdom). Slides were viewed at a magnification of x100.

EMA screens were performed by using MO slides with FITC-conjugated anti-human IgA, containing Evan's Blue counterstain as the second-stage antibody. Serum was diluted 1/5 in PBS. All reagents were manufactured by Biosystems (Spain) and supplied by Launch Diagnostics (United Kingdom). Slides were viewed at a magnification of x100.

Serum Ig levels were determined by immunoturbidimetry with an Olympus AU640 (Olympus, Hamburg, Germany) instrument. SPE was performed with the Sebia Hydrasys agarose gel electrophoretic system (Sebia, Atlanta, Ga.).

Serum Igs were considered abnormal if any Ig isotype (IgG, IgA, or IgM) was reduced or if a paraprotein was present. For adult subjects (age, 16 years or greater), reduced Ig levels were defined as follows: IgG, <5.4 g/liter; IgA, <0.8 g/liter; IgM, <0.5 g/liter. For pediatric subjects, appropriate age-related reference ranges were applied (2).

The most notable finding was the more-than-threefold-higher incidence of serum Ig abnormalities in the dark IF group (70.8%) than in the control group (21.6%). Upon evaluation of the mean number of abnormal indices per subject, this was demonstrated to be statistically highly significant, with the control group showing 1.32 (SD, 1.15) abnormalities per subject compared with 0.29 (SD, 0.64) in the dark IF group (P < 0.0001).

Data from MO and LKS screenings were then analyzed separately (Table 1). For MO, 83.3% had abnormalities in serum Igs in the dark IF group, compared with 27.8% of controls. While the most common abnormality was an isolated decrease in IgA, all subjects with abnormal findings in the dark IF group had reduced IgA levels. This is in keeping with the IgA-specific second-stage antibody used on MO sections. Mean IgA levels for the dark IF group were significantly lower, at 0.43 g/liter (SD, 0.36), than those for the control group, at 1.84 g/liter (SD, 1.91) (P < 0.0001).

Among those subjects with a paraprotein, 11 of 11 (100%) of those in the dark IF group had coincidental immunosuppression, compared with 45.4% of controls.

In conclusion, these results support the hypothesis that dark IF, particularly on MO, is associated with abnormalities in serum Igs. Since background fluorescence is dependent on the presence of Ig to bind the secondary antibody, the presence of dark IF may be assumed to indicate the absence of or a significant reduction in the level of serum Igs; this has been confirmed in this study. We believe that the observation of dark IF is significant and should prompt laboratory comment, informing the user that this incidental finding has been associated with Ig abnormalities in a high proportion of cases.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Haematology (Immunology), Colchester General Hospital, Turner Road, Colchester, Essex CO4 5JL, United Kingdom. Phone: 01206 742406. Fax: 01206 742400. E-mail: jenny.list{at}essexrivers.nhs.uk.

Published ahead of print on 13 September 2006.

	REFERENCES

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1. Holborow, E. J., D. M. Weir, and G. D. Johnson. 1957. A serum factor in lupus erythematosus with affinity for tissue nuclei. Br. Med. J. 13:732-734.
2. Milford-Ward, A., P. G. Riches, and A. M. Smith (ed.). 1999. PRU handbook of clinical immunochemistry, 6th ed. PRU Publications, Sheffield, United Kingdom.

This Article Abstract Full Text (PDF) Other Versions of this Article: CVI.00218-06v1 13/11/1294    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by List, J. Articles by Heelan, B. Search for Related Content PubMed PubMed Citation Articles by List, J. Articles by Heelan, B. Pubmed/NCBI databases Substance via MeSH