The charge to prepare the leukocytes in our laboratory is $130.00. It is occasionally necessary to assay cystine in cultured cells. If we receive cells already established in culture, the charge to prepare these cells for cytine assay is also $130.00. The charge for the cystine assay is $375.00 for all tissues.
If the Cystine Determination Laboratory establishes and grows the cell cultures, and then prepares the cells (fibroblasts, amniocytes or chorionic villi cells) for cystine assay, the charge is $450.00.
The charge for preparing placental cells for cystine assay is $350.00. This requires identifying the fetal portion of the placenta and carefully teasing away the cells that will be assayed.
For heterozygote detection, polymorphonuclear (PMN) leukocytes must be isolated and prepared for cystine assay. Because of the critical nature of this procedure, PMNs are always prepared for cystine assay from a normal control and a known heterozygote individual, as well as the at risk individual, at the same time. Since it is possible to prepare four PMN samples at one time, the preparation charge is the same whether we prepare the cells from one or two at risk individuals. The preparation charge is $400.00 for one test and $200.00 for each additional test if more than one sample is prepared at the same time. There is no charge to prepare the PMNs and assay the cystine in the samples from the normal control and the heterozygote individual.
For questions concerning billing and payment information, sample preparation or cystine assay, call
Bernadette Libatique at (619) 471-0426.
Cysteamine bitartrate (Cystagon
- Mylan) an orphan drug, has been approved by the US Food and
Drug Administration for oral treatment of nephropathic cystinosis, an autosomal
recessive metabolic disorder that affects only a few hundred people in
the USA (WA Gahl et al, in The Metabolic Basis of Inherited Diseases
7th ed., CR scriver et al, eds, New York: McGraw-Hill, 1995, p 3763).
THE DISORDER - In patients with cystinosis, cystine accumulates in lysosomes in the kidney, cornea, muscle, and other tissues, forming crystals that eventually destroy the cells. In the most common type of the disease, untreated patients have signs of renal dysfunction by the age of two and are usually in renal failure by the age of 10. Renal transplantation was the only effective treatment for the nephropathy before cysteamine became available. Patients with renal transplants who survived into adulthood developed other problems due to cystine accumulation, including retinal degeneration, recurrent corneal ulceration, pancreatic insufficiency, severy myopathy, thyroid dysfuncion, and mental deterioration (DS Theodoropoulos et al, JAMA, 270:2200, 1993).
MECHANISM OF ACTION - Cysteamine converts cystine to cysteine and cysteine-cysteamine mixed disulfide, both of which, unlike cystine, can pass through the lysosomal membrane of these patients. The disease can be diagnosed and the effectiveness of therapy monitored by measuring cystine levels in leukocytes.
CLINICAL STUDIES - Cysteamine cannot reverse renal damage that has already occurred, but 17 patients treated adequately with the drug starting before the age of two and continuing for an average of seven years all had nearly normal renal function and grew at a normal rate (TC Markello et al, N England J Med, 328, 328:1157, 1993). In addition to preserving renal function, long-term treatment with cysteamine also can markedly decrease the amount of cystine in muscle and presumably in other tissues as well (WA Gahl et al, Biochem Med Metabol Biol, 48:275, 1992). Ophthalmic treatment with cysteamine can decrease photophobia and prevent corneal ulcerations (JA Bradbury et al, Eye, 5:755, 1991).
ADVERSE EFFECTS - Anorexia, nausea, vomiting and diarrhea have been the most frequent adverse effects of cysteamine and may be difficult to distinguish from manifestations of the disease. Skin rash, encephalopathy, and seizures occur rarely and may require temporary discontinuation of the drug and resumption at a lower dosage. Reversible leucopenia and abnormal tests of liver function may also occur; blood counts and liver function can be monitored at the same time as leukocyte cystine concentrations.
DOSAGE - The usual maintenance dosage of cysteamine for patients less than 12 years old is 1.3 gm/m2/day divided into 4 doses. After age 12, the usual dosage is 2 gm/day. Leukocyte cystine concentration are monitored every three months, and the dosage is adjusted to maintain them as close to normal as possible. The drug is available in 50-mg and 150-mg capsules, which cost the pharmacist about $400 for a month's supply at 2 gm/day.
CONCLUSION - Cysteamine is effective for treatment
of cystinosis and may lead to a greatly increased duration and improved
quality of life for patients with this rare disease.
Cystinosis is a rare metabolic disease in which non-protein cystine accumulates intracellularly. Although the intracellular cystine content of cystinotic leukocytes is 80 times greater than normal, this value is still extremely low and cannot be determined accurately by routine amino acid analysis. The Cystine Determination Laboratory utilizes an isotope dilution technique with a specific cystine-binding protein to assay intracellular cystine (1, 2).
Unfortunately, leukocyte intracellular cystine content is not stable and the cells must be prepared within a few hours of blood drawing. The Cystine Determination Laboratory works closely with laboratories in other cities to help them prepare leukocytes correctly.
Cystine determinations are needed in two clinical conditions: cystinuria and cystinosis. In cystinuria, the urinary cystine (extracellular ) concentration is very high and can be measured easily by standard analytical techniques. The Cystine Determination Laboratory does not measure urinary cystine. In cystinosis, intracellular cystine is measured in very small samples as described in this review. This is much more difficult. Intracellular cystine is measured in the following circumstances:
1. For the Diagnosis of Cystinosis
Cystinosis is always suspected in children who have the renal Fanconi Syndrome. The diagnosis is established by measuring intracellular cystine in leukocytes . This requires only 5 ml of blood. An elevated intracellular cystine content in leukocytes is diagnostic of cystinosis. It is important to be 100% certain of the diagnosis since a positive diagnosis leads to a lifetime of therapy. It is also important to establish a baseline leukocyte cystine level before starting treatment.
Special circumstances sometimes make it easier to obtain cultured skin fibroblasts than peripheral leukocytes. Also, in unusual types of cystinosis the leukocyte cystine content may be equivocal and the diagnosis can be substantiated by measuring the cystine in cultured skin fibroblasts .
The standard method of in utero diagnosis involves by obtaining amniotic fluid at about 15 weeks gestation and establishing an amniotic fluid cell (amniocyte) culture . Choronic villi sampling for prenatal diagnosis has the advantage of providing an earlier diagnosis. In both procedures the cells are cultured and assayed for cystine, which is very elevated in cystinotic cells in both situations.
2. Heterozygote Detection
Parents of children with cystinosis, an autosomal recessive disease, are obligate heterozygotes (carriers) for this disease. Their leukocyte cystine content averages four to five times higher than normal. Family members are frequently anxious to know if they are carriers, especially when they marry and plan to start a family. The optimal way to determine this is to measure cystine in isolated polymorphonuclear leukocytes. In a recent report on this method, the heterozygotes were detected accurately in 29 of 29 attempts (6). Since that time, the Cystine Determination Laboratory has studied a few obligate heterozygotes whose leukocyte cystine values fell into the high normal range. For this reason it is preferable to study both members of couples who are at risk of having a child with cystinosis.
3. Monitoring Drug Dosage
The drug cysteamine was recently approved by the FDA for patients with cystinosis (7). The form was cysteamine bitartrate (Cystagon
, Mylan Pharmaceuticals). This drug depletes these patient's cells of cystine. If the drug is started at an early enough age, it improves growth and prevents renal deterioration (8-10). Unfortunately, the drug is very foul-tasting and must be taken four times a day for the rest of the patient's life. In addition, the dosage required to adequately deplete their cells of cystine varies from patient to patient and must be monitored periodically by checking the leukocyte cystine content. This also serves as a method of monitoring patient compliance in actually taking the drug.
1. Oshima, R.G., Willis, R.C., Furlong, C.E. and Schneider, J.A. Binding assays for amino acids. The utlization of a cystine binding protein from Escherichia coli for the determination of acid-soluble cystine in small physiological samples. J.Biol.Chem. 249:6033-6039, 1974.
2. Smith, M.L., Furlong, C.E., Greene, A.A. and Scheider, J.A. Cystine: Binding protein assay. Methods in Enzymology 143:144-148, 1987.
3. da Silva, V.A., Zurbrugg, R.P., Lavanchy, P., Blumberg, A., Suter, H., Wyss, S.R., Luthy, C.M. and Oetliker, O.H. Long-term treatment of infantile nephropathic cystinosis with cysteamine. N.Engl.J.Med. 313:1460-1463, 1985.
4. Reznik, V.M., Adamson, M., Adelman, R.D., Murphy, J.L., Gahl, W.A., Clark, K.F. and Schneider, J.A. Treatment of cystinosis with cysteamine from early infancy. J.Pediatr. 119:491-493, 1991.
5. Smith, M.L., Clark, K.F., Davis, S.E., Greene, A.A., Marcusson, E.G., Chen, Y-J. and Schneider, J.A. Diagnosis of cystinosis with use of placenta. N.Engl.J.Med. 321:397-398, 1989.
6. Smolin, L.A., Clark, K.F. and Schneider, J.A. An improved method for heterozygote detection of cystinosis using polymorphonuclear leukocytes. Am.J.Hum.Genet. 41266-275, 1987.
7. Cysteamine for cystinosis. Med.Lett.Drugs.Ther. 36:118, 1994.
8. Thoene, J.G., Oshima, R.G., Crawhall, J.C., Olson, D.L. and Schneider, J.A. Cystinosis. Intracellular cystine depletion by aminothiols in vitro and in vivo. J.Clin.Invest. 58:180-189, 1976.
9. Gahl, W.A., Reed, G.F., Thoene, J.G., Schulman, J.D., Rizzo, W.B., Jonas, A.J. Denman, D.W., Schlessman, J.J., Corden, B.J. and Schneider, J.A. Cysteamine therapy for children with nephropathic cystinosis. N.Engl.J.Med. 316:971-977, 1987.
10. Markello, T.C., Bernardini, I.M. and Gahl, W.A. Improved renal function in children with cystinosis treated with cysteamine. N.Engl.J.Med. 328:1157-1162, 1993.