Laboratory Issues David Winsemius, MD, MPH Heritage Laboratories/HooperHolmes

Transcription

1 Laboratory Issues David Winsemius, MD, MPH Heritage Laboratories/HooperHolmes Age and Gender: Association with Analytes The following Age-Sex distribution slides all have a common format: Males on left, Females on right Plot the 2.5 th, 25 th, median, 75 th, and 97.5 th percentiles for each analyte in 5 year age groups from age years of age Data appear sufficiently numerous to provide reasonably smooth curves

2 ALT: Age-Sex Variation ALT: Males : 'Ranges of Normal' ALT: Females : 'Ranges of Normal' ALT (UI) ALT (UI) AST: Age-Sex Variation AST: Males: 'Ranges of Normal' AST: Females : 'Ranges of Normal' AST (UI) AST (UI)

3 GGT: Age-Sex Variation GGT: Males : 'Ranges of Normal' GGT: Females : 'Ranges of Normal' GGT GGT Hepatitis C : Age-Sex Variation Percent Po s itive Hepatitis C Antibody: Within 5-Year Age Groups :18-83 Black line: Female; Das hed red line: Male [ 18, 23) [ 23, 28) [ 28, 33) [ 33, 38) [ 38, 43) [ 43, 48) [ 48, 53) [ 53, 58) [ 58, 63) [ 63, 68) [ 68, 73) [ 73, 78) [ 78, 83) Percent Positivity HepatitisC

4 Cholesterol: Age-Sex Variation Choles terol: Males : 'Ranges of Normal' Choles terol: Females : 'Ranges of Normal' Cholesterol Cholesterol HDL-Cholesterol: Age-Sex Variation HDL: Males : 'Ranges of Normal' HDL: Females : 'Ranges of Normal' HDL HDL

5 Alkaline Phosphatase: Age-Sex Variation Alkaline Phosphatas e: Males : 'Ranges of Normal' Alkaline Phos phatas e : Females : 'Ranges of Normal' Alkaline Phosphatase (IU) Alkaline Phosphatase (IU) Serum Glucose: Age-Sex Variation Glucos e : Males : 'Ranges of Normal' Glucos e : Females : 'Ranges of Normal' GLUCOSE (mg/dl) GLUCOSE (mg/dl)

6 Fructosamine: Age-Sex Variation (Note: Heritage Labs assay has ULN of 1.5) Fructos amine : Males: 'Ranges of Normal' Fructosamine : Females : 'Ranges of Normal' Fructosamine (mg/dl) Fructosamine (mg/dl) Serum Creatinine: Age-Sex Variation Serum Creatinine : Males : 'Ranges of Normal' SerumCreatinine : Females : 'Ranges of Normal' Serum Creatinine Serum Creatinine

7 egfr (Mayo E qn): Age-Sex Variation egfr: Males : 'Ranges of Normal' egfr: Females : 'Ranges of Normal' egfr egfr Urine Protein Assay False negatives(dipstick): Alkaline urine, ascorbic acid, ASA High dose penicillin High sp.gr. (>1.020) False positives: Benzalkonium Phenazopyridine Sulfosalicylic acid method( false pos.) Radiocontrast Aminosalicylic acid Peroxidase ingestion Tolbutamide; Sulfonamides

8 Pre-Analytical Factors Blood Draw: hemolysis Transport: Delay to centrifugation, Mislabeling, (Swapping serum tubes),heat, Hemolysis, Breakage, Loss of seal Processing Caveat about Interference Knowledge It has been my experience that concerns raised about analytical interference coming from self-educated agents, brokers and applicants about knowledge they have acquired from the Internet is based on a specific reports in the medical literature about methods not in current use.

9 Analytic Variation Variation in reagents, variations in sampling, variations in reaction time Coefficient of Variation: = Std. deviation of results/ mean of results Generally labs target 2-3% but CAP proficiency standards are not as tight as that. Some measures are thought to be acceptable with 15-20% Biological Sources of Variability Between-Person Variability --- Population Standard Deviation Within-Person Variability --- Diurnal variation ---Exercise --- Alteration in enzyme induction --- New onset disease --- Alterations in diet or alcohol consumption

10 NHANES-III Variability Stats: Clinical Chemistry 51(2), 2005, pp Between Persons Within Person Analyte (units) 2 n Mean SD CV g,% n Mean SD CV% Index of individuality Method CV i,% Creatinine, urine 3 (mmol/l) 19, Triglycerides 3 (mmol/l) 20, Bilirubin, total 3 (μmol/l) 15, Alanine aminotransferase 3 (U/L) 15, Urea nitrogen 3 (mmol/l) 15, γ-glutamyl transferase 3 (U/L) 11, Aspartate aminotransferase 3 (U/L) 15, HDL-cholesterol 3 (mmol/l) 20, Apolipoprotein B (g/l) 10, Glucose, plasma 3 (mmol/l) 15, Cholesterol, total 3 (mmol/l) 20, Apolipoprotein A (g/l) 10, Creatinine 3 (μmol/l) 16, Selenium 3 (nmol/l) 15, Alkaline phosphatase 3 (U/L) 15, Protein, total (g/l) 16, Calcium, total 3 (mmol/l) 16, Albumin (g/l) 16, Glycohemoglobin, blood 3 (%) 19, Proteinuria Patterns Glomerular: (albumin, transferrin); diabetic nephropathy, GN, MCD Interstitial: (alpha 1, alpha 2, beta 2 globulin, cystatin C, retinolbinding protein); interstital/tubular nephritis, chronic pyelo, congenital tubular nephropathies, calciuric renal diseases. (40% of dxs. leading to ESRD) Mixed: chronic pyelo Dysglobulinemias: MM, MGUS, macroglobulinemia, cryoglobulinemia

11 Orthostatic Proteinuria Young persons Not present in first morning void AST Positive Interference: lipemia, hemolysis Enzyme induction: oxacillin, ampicillin, opiates, erythromycin Negative interference: DKA, beriberi, severe liver dz. Chronic hemodialysis Uremia (follows BUN, reason unknown) Pyridoxal deficiency, pregnancy

12 ALT Incr: Hepatobiliary dzs, Obesity (1-3x) Decr: Pyridoxal deficiency, pregnancy Positive Interference: Seasonal variation previously reported in literature and seen in our analyses Seasonal Variation: ALT: Percentiles versus Month of Draw Percentiles

13 Bilirubin Only if associated with AlkPhos is increase associated with increased risk: Low levels (< 0.5) actually increases mortality risk, perhaps due to a decreased antioxidant effect (and Heritage research shows this to be especially true with increasing GGT which further supports the oxidation hypothesis.) Alkaline Phosphatase Interference: EDTA (so cannot use plasma from whole blood tubes if serum tube damaged.) Stable at 25C, 80-90% 56C x 30 min Drugs: (incr.) acyclovir, amiodarone, amitriptyline, carbamazepine, erythromycin, INH, niacin, phenytoin, sildenafil, valproic acid Would expect many of these to raise GGT as well.

14 Glucose Metronidazole: (decr.) affects hexokinase method (which is the assay Heritage uses) Major artifactual decrease caused in the insurance setting by red cell and white cell metabolism prior to centrifugation. There is consistent predictable impact on serum creatinine for measured values below 50 mg/dl. Creatinine Transport issues: Consistent increases in the median, 25 th and 75 th percentiles of up to 0.3 mg/dl when associated with serum glucose values below 50. Appears to be a linear effect over that range. Red cells have higher levels of both creatine and creatinine than plasma. After consuming available glucose for energy, RBCs turn to creatine as energy source with resultant production of even further creatinine.

15 3) Can you discuss urine proteinuria, protein/creatinine ratio, light chains, albuminuria? Insurance labs use more accurate testing methods than are common in clinical practice. There is no accepted standard for urine protein. Urine has a mixture of proteins, some of the nonalbumin proteins are benign but others strongly associated with life-shortening diseases. Proteinuria vs. Albuminuria The focus on glomerular diseases blinds one by removing interstitial diseases. Filtration is not the only function of the kidneys. Proper recovery of useful filtered solutes and homeostatis are functions of the interstitium. 40% of diseases leading to ESRD are interstial. Specific urine proteins are increased in interstial diseases: cystatin-c, beta-2-microglobulin, alpha-1- microglobulin, retinol-binding protein

16 Proteinuria vs. Albuminuria So it is not just the multiple myeloma and MGUS cases with their (possible) light chain proteinuria that represent a challenge to the dominant view that micro-albumin is the sole arbiter of significant or unhealthy proteinuria There are a much greater number of unrecognized renal disease cases than there are MM cases. The level of proteinuria at which risk begins to increase is well below the conventional ULN of 0.2 gm/gm creatinine. IA reasonable level at which risk could be considered increase is 0.1, and by the time is is 0.15 the risk is at least doubled from a comparison group with levels below the median. Proteinuria vs. Albuminuria In the Heritage experience, among cases where both urine protein and microalbumin have been measured, it is the mixed proteinuria cases (those with an albumin to non-albumin ratios = ) that have the highest risk. The protein-creatinine ratio is an excellent mortality predictor across the full range of both protein and creatinine in our applicant cohort. My hypothesis is that both diabetes and the intrinsic renal diseases have an important inflammatory component. Further hypothesis: This inflammatory process interferes with proper tubular function and the measureable consequence is a greater than normal spillage of the small molecular weight proteins.

17 Interaction: egfr and Ur Prot/Creat ratio Age Last Birthday: Mayo egfr ---> Ur Pr/Cr [ 0, 15) [ 15, 30)[ 30, 45)[ 45, 60)[ 60, 75)[ 75, 90) [ 90,105)[105,120)[120,135)[135,150)[150,165) [ ) [ ) [ ) [ ) [ ) [ ) [ ) [4.00+] Interaction: egfr and Ur Prot/Creat ratio Age Last Birthday: Mayo egfr ---> Ur Pr/Cr [ 0, 15) [ 15, 30) [ 30, 45) [ 45, 60) [ 60, 75) [ 75, 90) [ 90,105) [105,120) [120,135) [ ) [ ) [ ) [ ) [ ) [ ) [ ) [4.00+]

18 Interaction: egfr and Ur Prot/Creat ratio Age Last Birthday: Mayo egfr ---> Ur Pr/Cr [ 0, 15) [ 15, 30) [ 30, 45) [ 45, 60) [ 60, 75) [ 75, 90) [ 90,105) [105,120) [ ) [ ) [ ) [ ) [ ) [ ) [ ) [4.00+] )If a urine specimen is in transit for 3 days from collection to arrival at the lab, and it has large hemoglobin but no RBCs: is there any estimate of the initial specimen s RBC count? Would it expected to usually have RBCs at the time of collection? This is a subject on which honest people apparently disagree. Inanalyses controlling for other known renal markers of mortality, we have found no increase in mortality risk for persons with positive urine hemoglobin who have no red-cells on microscopic analysis. Whether to believe there shouldhave been red cells, might well be moot. We believe that our analysis, which first stratifies by urine protein/creatinine ratio, is properly ascribing the blame (or credit?) for mortality in such cases to the more potent risk indicator: proteinuria. Or perhapsit is simply proteinuria that is adjudicating which cases really have renal disease. The sources of analytical interference that affect dipstick methods also affect the assay used for urine hemoglobin. The same reaction is measured.