Arch. Derm. Forsch. 252, 311--315 (1975) © by Springer-Verlag 1975

Fluorescence Microscopy Test in Porphyrias, Photodermatoses and Lead Exposed Persons Aleksej K a n s k y Department of Dermatology (Head: Prof. Dr. J. Fettich), Medical Faculty, Ljubljana (Yugoslavia) Received March 4, 1975

Summary. Fluorescence microscopy tests were carried out in different groups of patients Peripheral blood diluted with saline was used and 200 high power fields were inspected in every case. The results were presented as the number of fluorescing erythrocytes (FE) per 100000 red blood cells (or 200 fields). In the controls, porphyria cutanea tarda patients and patients with photodermatoses other than erythopoietie protoporphyria and pellagra almost no FE were detected. In erythropoietic protoporphyria the mean value was 10600, in lead poisoning 1032, in patients exposed to lead 48.2, in sideropenic anaemia 123 and in patients with pallagra 8.1 FE/100000 red blood cells. The conclusion is made that one has to take care, when using this test for detection of latent carriers in genetic studies of the relatives of patients with erythropoietic protoporphyria. The test is useful for the confirmation of the diagnosis of erythropoietic protoporphyria. Zusammen]assung. •luorescenzmikroskopische Untersuchungen des peripheren Blutes wurden bei einer Anzahl yon verschiedenen Krankheitszust~nden durchgeffihrt. Das venSse Blur war mit physiologischer Kochsalzl6sung 1 : 5 verdfinnt and bei einer VergrSBerung yon 400× im l~luorescenzmikroskop untersucht. In jedem Fall wurden 200 Felder durchblickt und die Zahl der fluoreszierenden Erythrocyten (FE) festgestellt. Die Werte der gefundenen FE warden auf 100000 Ery~hrocyten bzw. 200 Sichtfelder bezogen; in Vorversuchen war nimlich festgestellt worden, dab pro Selffeld etwa 500 Erythrocyten vorhanden waren. Folgende Mittelwerte wurden ermittelt: Bei den Kontrollpersonen, bei den Porphyria cutanea tarda und Patienten mit Photodermatosen wurden nur in einigen l~illen vereinzelte FE ge£unden. Bei erythropoetischer Protoporphyrie wurden 10600, bei Bleivergfftungen 1032, bei Blei-exponierten Personen 48,2 bei Eisenmangelanaemie 123 und bei pellagroiden Zust~nden 8,1 FE/100000 Erythrocyten ermittelt. Auf Grund solcher Beobachtungen wurde die Schlul3folgerung gezogen, dab man bei der Fahndung nach latenten Konduktoren unter den FamilienangehSrigen der Patienten mit erythropoietischer Protoporphyrie vorsichtig vorgehen solh In Fillen, bei denen FE gefunden werden, sollten zun~chst alle Zust£nde, bei denen FE gefunden werden kSnnten, ausgeschlossen werden. The fluorescence microscopy screening test for the detection of increased a m o u n t s of p o r p h y r i n in red blood cells (RBC) was first used b y K o s e n o w a n d Treibs (1953) a n d later b y L a n g h o f et al. (1961). A detailed description of the test was given b y R i m i n g t o n a n d Cripps (1965). W u e p p e r et al. (1968) used the m e t h o d t o detect l a t e n t carriers of the inborn error of p o r p h y r i n metabolism in their genetic studies of patients with erythropoietic p r o t o p o r p h y r i a (EPP). During the past four years we have investigated with the fluorescence microscopy screening test the peripheral blood of a n u m b e r of patients suffering from lead exposure, sideropenic anaemia, some photodermatoses a n d various other dermatoses.

312

A. Kansky Methods

Fluorescence Microscopy Screening Test A drop of venous blood was diluted with 7 drops of saline and mixed. One drop of the suspension was transferred to a microscope slide, covered and examined with the fluorescence microscope (Leitzl). The light source was an HBO 200 mercury lamp, the primary filter BG 12 3 mm with a 470 nm secondary filter. In every case we examined 200 high power fields (magnification 400 ×) and counted the fluorescing erythrocytes (fluoroeytes ~- FE). On the basis of preliminary tests we assumed that there were approximately 500 red blood cells (RBC) in a single high power field. The results were expressed as the number of F E in 100000 RBC or in 200 high power fields.

Quantitative Fluorescence Microscopy Test The already diluted blood specimen was further diluted to approximately 1:5. Using an oil immersion lens the number of FE in every field was noted and the total number of RBC in every field recorded. At least 20 fields were counted and the percentage of FE recorded.

Subjects Investigated The following groups of subjects have been investigated: 10 patients with EPP, 22 persons exposed to lead, 4 showing symptoms of lead poisoning, 16 with porphyria cutanea tarda (PCT), 16 with cutaneous symptoms of pellagra, 14 with other photodermateses and 6 with sideropenie anaemia. Sixty three persons and dermatological patients with tinea, urticaria, psoriasis and other noncomplicated conditions, without any sign of photosensitivity were used as controls. Results

Controls I n one p a t i e n t w i t h e r y t h e m a e x u d a t i v u m m u l t f f o r m e 1 F E in 100000 R B C was d e t e c t e d , whereas in 62 no F E were observed. T h e a v e r a g e score was 0.016 F E / 100000.

Erythropoietie Protoporphyria I n 10 p a t i e n t s we f o u n d a m e a n v a l u e o f 10600 F E / 1 0 0 0 0 0 R B C (10.6°/0). T h e lowest v a l u e in a single p a t i e n t was 7.20/0 a n d t h e h i g h e s t 23.80/0 (Table 2). Some o f t h e p a t i e n t s h a v e b e e n followed u p for m o r e t h a n 5 y e a r s a n d t h e n u m b e r of FE remained virtually constant.

Lead Exposed Persons I n 22 persons e x p o s e d t o l e a d a t w o r k b u t showing no s y m p t o m s o f poisoning, t h e m e a n v a l u e was 48.2 F E / 1 0 0 0 0 0 RBC. I n i n d i v i d u a l p a t i e n t s t h e v a l u e s v a r i e d f r o m 0 t o 529 F E / 1 0 0 0 0 0 RBC.

Persons with Signs o/Lead Poisoning I n four m e m b e r s o f a f a m i l y all showing s y m p t o m s o f l e a d poisoning w i t h a n a e m i a , a b d o m i n a l colic a n d c o n s t i p a t i o n a m e a n v a l u e o f 1032.5 F E / 1 0 0 0 0 0 R B C was f o u n d (1.03°/0). The lowest v a l u e was 350 (0.350/0) a n d t h e h i g h e s t 3.80/0 .

1 The fluorescence microscope Leitz was a gift from the A. v. Humboldt foundation, Bad Godesberg, West Germany.

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Table 1. Fluorescence microscopy tests of peripheral blood. The number of fluorescing erythroeytes in 100000 (200 high power fields)--mean values in different patients groups N

FE/100 000 RBC rain

max

Controls

63

0.016

0

1

Erythropoietic protoporphyria

10

10600

7200

23800

Lead poisoning Lead exposure Sideropenic anaemia Porphyria eutanea tarda Photodermatoses

4 22 6 16 14

1032 48.2 123 0.44 0.14

350 0 0 0 0

3800 529 393 3 1

Pellagra

14

8.1

0

50

N ~ Number of patients investigated. I~E -~ Fluorescing erythrocytes. ~ Mean values. min -~ Minimum value observed in a single patient. max -~ Maximum value observed in a single patient. Table 2. Percentage of fluorescing erythrocytes and protoporphyrin concentration in erythrocytes in 10 patients with erythropoietie protoporphyria Patient

FE o/0

Protoporphyrin ~tg/lO0 ml packed RBC

1 2

D.M. A. Sv.

7.8 10.8

341 1352

3 4

A. S1. A. 8.

8.8 10.6

714 444

5 6

K.V. L.D.

9.3 23.8

772 434

7 8

K.I. K.D.

8.9 9.5

228 209

9 10

Z.J. O.G.

9.1 7.2

391 553

10.6

544

normal

0

0-- 35

FE = Fluorescing erythrocy~es. ~ Mean value.

Porphyria Cutanea Tarda I n 16 p a t i e n t s a m e a n v a l u e of 0.44 F E / 1 0 0 0 0 0 R B C was found. I n 1 p a t i e n t 1 F E a n d i n two 3 F E / 1 0 0 0 0 0 R B C were observed, i n 13 t h e results of the t e s t were completely negative.

314

A. Kansky

Other Photodermatoses In 14 patients investigated a mean value of 0.14 FE/100000 RBC was found. In 12 the test was completely negative and in two 1 FE/100000 RBC was detected. Pellagra In 14 patients a mean value of 8.1 FE/100000 RBC were found. In 6 the results were completely negative and in 8 values from 2--50 FE/100000 RBC were observed. Sideropenic Anaemia In 6 persons a mean value of 123 FE/100000 RBC (0.12°/0) was observed. The lowest value found was 0 (3 patients) and the highest was 393 FE/100000 RBC (1 patient). In 1 patient with acute intermitent porphyria and in 1 with xeroderma pigmentosum the tests were negative.

Discussion

The purpose of the present work was to find an answer to the following two questions: 1. In which disease could the fluorescence microscopy test be helpful in establishing the diagnosis? 2. What is the reliability of the test for the detection of latent carriers of the inborn error of metabolism in relatives of E P P patients. The mean value of FE/100000 RBC in different groups is important because the absolute number of FE/100000 RBC m a y not be exact. This is because the number of RBC in each high power field was assumed to be 500, a figure based on preliminary observations as explained earlier. A highly elevated number of F E was found in E P P and lead poisoning; the values in E P P are close to those observed b y Wuepper et al. (1968). A definitely increased number of F E as compared with the controls was established in persons exposed to lead and in some patients with sideropenie anaemia. However the fluorescence in E P P and sideropenie anaemia was much brighter than in lead poisoning and exposure, where it was usually faint, less bright and of shorter duration. In patients with PCT and with other photodermatoses the incidence of F E was also very low and such findings were in good agreement with the results of quantitative chemical assays of protoporphyrin in RBC. The most interesting finding however was the somewhat elevated number of F E in patients with pellagra, the quantitative protoporphyrin values being well within normal limits. Further studies are necessary for clarification of this observation. The percentage of F E in our E P P patients (7.2--23.80/0) is somewhat lower than the observation of Langhoff et al. (1961) or Wuepper and Eppstein (1968). The difference may depend on patients, the exact values are however difficult to establish. Extensive porphyrin studies in RBC of family members of E P P patients have been carried out by Haeger-Aronson and Krook (1966), Donaldson et al. (1967), Sehmidt et al. (1974) as well as by others. Wuepper and Eppstein (1968) were the first to t r y to detect latent carriers with the fluorescence microscopy test. We have carried out the fluorescence microscopy test in family members of 9 patients with EPP. I n three instances we detected in one of the parents a

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315

few FE. As there were no signs of sideropenic anaemia or history of exposure to lead we assumed that we have detected latent carriers in the respective families. After having found F E in sideropenic anaemia and also in pellagra we are now inclined to be very careful before making such a statement. I n all these family members quantitative protoporphyrin values in the RBC were within normal limits. Conclusions 1. Fluorescence microscopy is a quick and reliable test for detecting patients with EPP. 2. The method can be helpful in detecting latent carriers in families of the E P P patients; however before making a decision one has to eliminate sideropenic anaemia, pellagra and exposure to lead. 3. I n persons exposed to lead we frequently find F E but the fluorescence is less bright, sometimes it has a brownish hue and is of even shorter duration than in EPP. There is often poor correlation between this test and the quantitative assay of protoporphyrin in RBC. 4. The most interesting finding was that a certain number of F E were often detected in the peripheral blood of patients with pellagra. 5. An elevated number of F E m a y often be observed in patients with sideropenic anaemia. I n some cases a definite increase in protoporphyrin content of I%BC was observed on biochemical assay without an increase in FE. 6. F E are usually not found in healthy persons and in patients with noncomplicated dermatoses, porphyria cutanea tarda and other photodermatoses except E P P and pellagra. References Donaldson, E. M., Donaldson, A. G., Rimington, C. : Erythropoietic protoporphyria a family study. Brit. med. J. 1967 I, 659--663 Haeger-Aronsen, B., Krook, G. : Erythropoietic protoporphyria. A study of known eases in Sweeden. Acta med stand. 179, suppl. 445, 48--55 (1966) Kosenow, W., Treibs, A. : Lichtfiberempfindliehkeit und Porphyrinaemie. Z. Kinderheilk. 78, 82--92 (1953) Langhoff, It., 1Kfiller,H., Rietsehel, L.: Untersuehungen zur famil~rer protoporphyrin~misehen Liehturticaria. Arch. klin. exp. Derm. 212, 506--518 (1961) Rimington, C. D., Cripps, J.: Biochemical and fluorescence microscopy screening test for erythropoietic protoporphyria. Lancet 1965 I, 624--626 Schmidt, H., Snitken, G., Thomsen, K., Lintrup, J. : Erythropoietie protoporphyria. A clinical study based on 29 cases in 14 families. Arch. Derm. 110, 58--64 (1974) Wuepper, K., Epstein, J.: Genetic analysis of Erythropoietie protoporphyria (EPP) with erythrocy~e fluorescence method. Proceed. 13 Internat. Congr. Derm., Mtinchen 1968, Vol. I, pp. 566--568 Prof. Dr. Aleksej Kansky Dermatolo~a klinika Ljubljana Zalo~ka 2 Jugoslavia

Fluorescence microscopy test in porphyrias, photodermatoses and lead exposed persons.

Fluorescence microscopy tests were carried out in different groups of patients Peripheral blood diluted with saline was used and 200 high power fields...
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