Presentation on theme: "Life without Albumin Encounters with Analbuminemia"— Presentation transcript:
1 Life without Albumin Encounters with Analbuminemia Andrew W. Lyon, PhD FCACB, DABCC.Department of Pathology and Laboratory Medicine, University of Calgary & Calgary Laboratory ServicesBoras, Sweden. May 2007
2 Objectives: Review the pathophysiology of serum albumin Review clinical laboratory methods of albumin determinationDescribe Analbuminemia and four cases found in SaskatoonDepict how common laboratory method of serum albumin measurement confound the diagnosis of analbuminemia.
3 The History of ‘albumin’ 400 Hippocrates: Foam on urine with renal failure.1500 Paracelus: precipitated protein from urine with acid1894 Kander: Crystallized horse albumin1896 Starling: Suggests role of albumen in maintaining circulation.1926 Svedberg: Measures mass by ultracentrifugation1937 Tiselius: Separated serum by electrophoresis1947 Klotz: Studies how dyes bind to albumin1950 Peters: Biosynthesis of albumin in liver slices1954 Benhold: First report of analbuminemia, 2 cases.1957 Knedel: Report of the genetic cause of bisalbuminemia
4 The History of ‘albumin’ 1970 King: Studied tryptic fragments of albumin1975 Brown / Meloun: Amino acid sequence BSA & HAS1979 Sargent: Isolated the HAS gene1981 Lawn: Genetic sequence of HAS cDNA1986 Dugaiczyk: Complete HAS gene sequenceMutation studiesCrystallization studiesParenteral albumin utilization studies
5 Physiological Roles of Albumin Circulatory Role……….80% oncontic pressureTransport of metabolitesBilirubin, calcium, fatty acids, bile acids, drugsLow affinity reservoir for thyroxine, vitamin DSequestration of toxins… delivery to the liverBilirubin, carcinogens.Metabolic Effects: enhances lipoprotein lipaseMiscellaneous Effects: limits fibrin fiber thickness
6 Clinical Utility of Serum Albumin Levels Assess protein malnutritionRenal disease… nephrotic syndromeG.I. pathologyLiver disease… impaired synthesisHypoalbuminemia, intestinal edema, diarrhea
7 Methods of albumin quantification Turbidometry: following acid or salt precipitationDye-binding methodsProtein-error of pH indicating dyesProtein electrophoresisImmunoassay
8 Dye-binding Methods High pH causes a colour change: High pH Dye-H Dye H+Add albumin at Constant pHAlbuminDye-H Dye--AlbuminApplications: Urine dipstick, routine clinical chemistry.
9 Dye-binding Methods Urine Dipsticks: Urine Protein detection Largely measures urine albuminFalse positive colour at high pHPoor detection of Bence Jones proteins/ light chains … as they don’t bind the dyes.Serum AlbuminBromcresol Green , Bromcresol PurpleShort incubation times to improve specificity
12 Calgary Laboratory Services Serum Protein Electrophoresis
13 BisalbuminemiaNot particularly rare in Canada! Often seen in aboriginal peoples> 50 known Albumin mutations.Bisalbuminemia results from two copies of different albumin genes, resulting in different charges.
14 Albumin ImmunoassayMethod usually reserved for urine or CSF albumin determination: “microalbumin”Various immunoassay methods: rate nephelometry, nephelometry, turbidometry, radial immunodiffusion.
15 Objectives: Review the pathophysiology of serum albumin Review clinical laboratory methods of albumin determinationDescribe Analbuminemia and four cases found in Saskatoon, SK, Canada.Depict how common laboratory method of serum albumin measurement confound the diagnosis of analbuminemia.
16 Our first case…Paul Meinert (pediatric resident) complains “why are serum albumin levels in your lab fluctuating so much?”“What do you mean by fluctuating?”
17 Variable levels of serum albumin over 6 months, (7 month old infant) <10
18 Impact of low albumin?? Bilirubin-Alb Adipose Tissue T4 - Alb Fatty Acids - AlbT4 - AlbCalcium - AlbAlb for Oncotic Pressure, to avoid edema
19 Patient #1Admitted to NICU as a newborn for hypoglycemia and perinatal asphyxia, mild tubular necrosis & brain edema.Low serum albumin noted: 17 g/LNormal 24 hr urine protein level.Three admissions during first 6 months related to respiratory distress and wheezing.Nutritional status: Good.Gaining weight, growing, apparently normal liver function.
20 Patient #1At 6 months of age, still no explanation for the low serum albumin (13 – 18 g/L, routine chem).Serum albumin was still lower by electrophoresis (3-5 g/L).Tc99-labelled albumin scan: negative for protein loosing enteropathy.Clinical Biochemistry consult to review the results.
21 Analbuminemia Albumin (Dye-binding) : 10 – 17 g/L Albumin (electrophoresis): 2 – 3 g/LAlbumin (Immunoassay) : < 0.01 g/L
22 Consistent with: Analbuminemia A genetic lack of albuminWhat is analbuminemia and what is the prognosis ?Why did our routine laboratory methods detect 17g/L of albumin is there was NONE?
23 Analbuminemia Serum albumin test results: very low. Apparently a benign, recessive inherited disorder: elevated lipids and globulins, 30% have lipodystrophy below waist, 30% mild ankle edema, low capillary blood pressure.VERY RARE (approx. 42 reported cases)
27 Patient #2 Admitted at 2 days of age with cellulitis Admitted 3 times during the first 6 months for respiratory distress / infection.Mild hyperbilirubinemia, mild ALP elevation and low serum albumin: 10 – 15 g/L.Nutritional status, weight gain & growth were normal.Albumin by electrophoresis: 2 g/L
28 Patient #3Admitted at 6 weeks of age with tetany and prolonged diarrhea.Infant had low serum calcium and magnesium levels. Tetany resolved on magnesium administration.Low albumin levels were detected < 10 g/Li.v. albumin was administered prior to conducting serum electrophoresis.
29 A genetic lack of albumin AnalbuminemiaA genetic lack of albuminWhy did our routine laboratory methods detect 17g/L of albumin is there was NONE?
30 Re-evaluation of Dye-binding Serum Albumin Methods Roche Diagnostics: BCG method (rapid)Ortho Diagnostics: BCG method (slow)BCG: bromcresol greenLinearity of albumin methods with diluted serum (constant albumin : globulin ratio)Linearity of albumin methods with 100% human IgG or 100% human albumin.
34 Why did our assays report the presence of albumin in patients with analbuminemia ? Ortho Diagnostics Assay:Reacts with globulinsGives a positive result in the absence of albumin.The assay did not report that albumin was below the reportable limit.Roche Diagnostics Assay:Assay reported < 10 g/L in analbuminemiaAssay had no globulin interference.Serum Protein ElectrophoresisBaseline disturbances during densitometry lead to reports of 2-3 g/L albumin.
35 Our three patients with analbuminemia all appeared to have albumin present, according to the dye-binding albumin methods.What was the serum albumin concentration reported in the reported cases of analbuminemia?It depends on the albumin methods that were used
36 28 Cases of Analbuminemia: Levels of Albumin ‘detected’ A: Dye-binding methodsB: Salt Precipitation methodC: Protein ElectrophoresisD: Albumin Immunoassay
37 Awkward conclusions:Clinicians should consider a diagnosis of analbuminemia (a genetic lack of albumin) even when the clinical laboratory detects serum albumin up to 17 g/L.Albumin immunoassays and serum protein electrophoreses are capable of detecting analbuminemia, but routine serum albumin assays are NOT.
38 Patient #3 Diagnosis following i.v. albumin infusion. Establish a partnership with Monica Galliano and Lorenzo Minchiotti, Univ. Pavia.Isolation of genomic DNA14 exons were amplified by PCRSingle-strand conformation polymorphism (SSCP) and heteroduplex analysis.Sequencing
39 Exon # SSCP 1 Control 2 Mother 3 Patient 4 Control 5 Analb Codogno Exon #3 Heteroduplex Analysis 6 Control 7 Mother 8 Patient 9 Control 10 Analb. Codogno
40 ‘ ‘‘ ‘A: ControlB: Patient AT deletion“Kayseri albumin”‘ ‘
41 ‘AT’ deletion frameshift results in a stop condon and analbuminemia.
42 Three cases of analbuminemia…. Was this a coincidence? Dr. David Meyer, Dept Anthropology, Univ. Saskatchewan, Doctoral Thesis:Red Earth Crees 1860 – 1960Ethnogeographic and historical workNear-complete pedigrees 1860 – 1970Study of the Deme
48 Red Earth and Shoal Lake Population DatePopulationIn group marriage0 smallpox epidemic18506 men1870 – 190018760%1900 – 193022085%1930 – 196039120071500?
49 1971 Third Generation 75 Couples (married, widows/widowers). 8 marriages between first cousins.23 marriages between second cousins.35 marriages between relatives, > 2nd cousin.9 insufficient to establish kinship.
50 There is likely a ‘founder effect’ responsible for the local incidence of analbuminemia at the Red Earth and Shoal Lake reserves.Future Investigations??
51 September 2003: Patient #429 year old native female from Red Earth, SKreferred because of lower limb edema.History of presenting illness:admitted to Nipawin hospital for pneumonia (July 2002) and right calf cellulitisupon discharge the patient noticed that her legs were swollenthe family doctors consult note from June 2002 implies she has had a low albumin, large legs and lower extremities and an abnormal body habitus
52 And so the story continues… Life without Albumin
53 AcknowledgmentsGarth Bruce, Paul Meinert, Robin Casey, Pat Blakely: Pediatrics, Jill Newstead, Sharon Card: Medicine. Univ. Saskatchewan.Mark Salkie, Victor Laxdal Pathology, Univ. Saskatchewan.David Meyer, Anthropology,Univ. Saskatchewan.Lorenzo Minchiotti, Monica Galliano & Lab,Univ. Pavia.
54 ReferencesLyon AW, Meinert P, Bruce GA, Laxdal VA, Salkie ML. Influence of methodology on the detection and diagnosis of congenital analbuminemia. Clin. Chem. 1998; 44:Galliano M, Campagnoli M, Rossi A, Wirsing von Konig CH, Lyon AW, Cefle K, Yildiz A, Palanduz S, Ozturk S, Minchiotti L. Molecular diagnosis of analbuminemia: a novel mutation identified in two Amerindian and two Turkish families. Clin Chem ;48:844-9.Newstead J, Card S, Lyon AW. Low serum albumin and abnormal body shape in a young Canadian First Nations woman. Lab Med Volume 35, Number 6 / June 2004.For more details on analbuminemia, visit the Albumin Website: