Dr. James Manos (MD)
January 5, 2016
Review: Tips in Medical Biochemistry
Volume (12)
CONTENTS
EFFECTS OF HEMOLYSIS ON CLINICAL SPECIMENS
Hemolysis on clinical specimens
Effects of hemolysis on clinical samples
Prevention of hemolysis on clinical specimens
Advice to prevent hemolysis during venipuncture
THE LIPEMIC SPECIMEN
Lipemia
Lipemia on blood products
EFFECTS OF HEMOLYSIS ON CLINICAL SPECIMENS
· Hemolysis on clinical specimens
· Hemolysis due to the breakdown of red blood cells is essential to the laboratory because it can have an effect on laboratory results. The effects can be the result of products liberated from the red cells themselves, or due to interferences with laboratory analyzers. This may vary from one test to another depending on the formulation of the reagent.
· Hemolysis is the No1 cause of ED (emergency department) specimen rejection (52 – 85%).
· Hemolysis can occur in vivo (in the patient), due to a variety of medical conditions, including antigen-antibody reactions, hemolytic anemias (including autoimmune hemolytic anemia), transfusion reaction, toxins and poisons, mechanical RBC rupture due to artificial heart valves, as well as treatments such as hemodialysis and the use of the heart-lung bypass machine.
· Hemolysis can occur during suboptimal blood collection, or in vitro (e.g., in the collection tube) due to improper handling, transport, and storage.
· Hemolysis can be recognized in the laboratory by visual inspection of the plasma or serum sample, which appears rosy to bright red in color.
· Samples with slight hemolysis are analyzed and the results reported with a comment indicating the degree of hemolysis and the effect on the test result.
· Grossly hemolyzed samples can affect the results of many tests; therefore, a recollection will be requested for most grossly hemolyzed specimens.
· Effects of hemolysis on clinical specimens:
· Results from all laboratory disciplines can be affected by hemolysis, especially in chemistry. Some of the more routine tests involved are: potassium, sodium, calcium, magnesium, bilirubin, haptoglobin, total protein, aldolase, amylase, lactate dehydrogenase (LD; LDH), AST (SGOT), ALT (SGPT), phosphorus, alkaline phosphatase (ALP), acid phosphatase, gamma-glutamyltransferase (GGT), folate, and iron.
· The amount of hemolysis needed to affect a test is dependent on the test being performed. Generally, slight hemolysis has little effect on most tests; however, it will cause increased test results for specific tests like potassium (K+) and lactate dehydrogenase (LD; LDH).
· Effects of hemolysis on results:
· a) Slight change:
· i) Test result increased by hemolysis: phosphate, total protein, albumin, magnesium, calcium, alkaline phosphatase (ALP).
· ii) Test result decreased by hemolysis: haptoglobin, bilirubin.
· b) Noticeable change: Test result increased by hemolysis: ALT (SGPT), CK (creatine kinase), iron, coagulation tests.
· c) Significant change:
· i) Test result increased by hemolysis: potassium (K+), lactate dehydrogenase (LDH; LD), AST (SGOT).
· ii) Test result decreased by hemolysis: Troponin T.
· iii) Test results increased or decreased by hemolysis: (HGB) hemoglobin, RBCs (red blood cells count), MCHC, Platelet count.
· Overview of effects:
· a) Moderate effects: AST (SGOT), CK (creatine kinase), lactate dehydrogenase (LDH), potassium (K+), sodium (Na+), amylase, bilirubin, FVIIa (activated factor VII), PT (prothrombin time), CEPI, CADP.
· b) Slight effects: Cl (chlorine), lipase, magnesium (Mg++), alkaline phosphatase (ALP), creatinine, glucose, triglycerides.
Hemolysis may also affect D – Dimers, PT, and aPTT.
· In a study, sixteen (16) healthy volunteers were enrolled. Four hemolysis levels were constituted according to hemoglobin concentrations and they were divided into five groups: Group I: 0 – 0.10 g/L, Group II: 0.10 – 0.50 g/L, Group III: 0.51 – 1.00 g/L, Group IV: 1.01 – 2.50 g/L, Group V: 2.51-4.50 g/L. Lysis was achieved by mechanical trauma. Hemolysis interference affected lactate dehydrogenase (LD; LDH) and aspartate aminotransferase (AST; SGOT) almost at undetectable hemolysis by visual inspection (plasma hemoglobin < 0.5 g/L). Clinically meaningful variations of potassium and total bilirubin were observed in moderately hemolyzed samples (hemoglobin > 1 g/L). Alanine aminotransferase (ALT; SGPT), cholesterol, gamma-glutamyltransferase (GGT), and inorganic phosphate (P) concentrations were not interfered up to severely hemolyzed levels (hemoglobin: 2.5 – 4.5 g/L). Albumin, alkaline phosphatase (ALP), amylase, chloride, HDL-cholesterol, creatine kinase (CK), glucose, magnesium, total protein, triglycerides, unsaturated iron binding capacity (UIBC) and uric acid differences were statistically significant but remained within the CLIA limits. The study concluded that to avoid preanalytical visual inspection for hemolysis detection, improper sample rejection, and/or rerun because of hemolysis, it is recommended in this study that, routine determination of plasma or serum-free hemoglobin concentrations is important. For the analytes interfered with hemolysis, new samples have to be requested.
· In another study, nine aliquots, prepared by serial dilutions of homologous hemolyzed samples collected from 12 different subjects and containing a final concentration of serum hemoglobin ranging from 0 to 20.6 g/L, were tested for the most common clinical chemistry analytes. Lysis was achieved by subjecting whole blood to an overnight freeze-thaw cycle. Hemolysis interference appeared to be approximately linearly dependent on the final concentration of blood-cell lysate in the specimen. This generated a consistent trend towards overestimation of alanine aminotransferase (ALT; SGPT), aspartate aminotransferase (AST; SGOT), creatinine, creatine kinase (CK), iron, lactate dehydrogenase (LD; LDH), lipase, magnesium, phosphorus, potassium and urea, whereas mean values of albumin, alkaline phosphatase (ALP), chloride, gamma-glutamyltransferase (GGT), glucose and sodium were substantially decreased. Clinically meaningful variations of AST (SGOT), chloride, lactate dehydrogenase (LD; LDH), potassium (K+) and sodium (Na+) were observed in specimens displaying mild or almost undetectable hemolysis by visual inspection (serum hemoglobin < 0.6 g/L). The rather varied and unpredictable response to hemolysis observed for several parameters prevented the adoption of reliable statistic corrective measures for results by the degree of hemolysis. The study concluded that if hemolysis and blood cell lysis results from an in vitro cause, the authors suggest that the most convenient corrective solution might be the quantification of free hemoglobin, alerting the clinicians and sample recollection.
· An older study (1992), examined the effects of hemolysis on the results of 25 common biochemical tests. The scientists collected 60 blood samples of 15mL from inpatients and outpatients and mechanically hemolyzed 10 mL of the samples in a two-step procedure. They classified serum from these samples as being non-hemolyzed, moderately hemolyzed, or severely hemolyzed and then performed 25 common biochemical tests. Statistical analysis of the results showed that hemolysis had the greatest effect on the lactate dehydrogenase (LD; LDH), acid phosphatase, and potassium (K+) tests.
· Prevention of hemolysis on clinical specimens:
· a) Vein size and trauma. Puncturing small, fragile veins and probing or “fishing” the vein with a needle can lead to hemolysis. The phlebotomist should choose an appropriately-sized vein and use phlebotomy equipment suitable for the vein size. If the vein is fragile, he/she should not use large volume tubes. If a vein is traumatized during puncture, the first tube collected may be hemolyzed, while subsequent tubes are fine. The phlebotomist should avoid puncturing areas that have a hematoma.
· b) Alcohol preparation. The phlebotomist should allow the alcohol to dry completely before venipuncture. The needle can transfer wet alcohol from the skin into the blood specimen and cause hemolysis.
· c) Needle size. Using a large needle (larger bore=lower gauge) can cause hemolysis by allowing a large amount of blood to suddenly enter the tube with great force. Similarly, the use of needles that are too fine (higher gauge) can also cause hemolysis by forcing the blood through a tiny opening under a great force. The red cell walls become sheared on the needle as they enter the tube.
· d) Loose connections. The phlebotomist should ensure that all connections of the collection components are tightened, i.e., the connections between a blood collection set and luer adapter, between syringe and needle, and between catheter and luer adapter. Loose connections introduce air into the system and cause frothing in the specimen, which can result in hemolysis.
· e) Underfilled Tubes. The phlebotomist should fill all tubes to full capacity to ensure the proper blood-to-additive ratio. Certain additives in high concentrations, such as sodium fluoride, can cause varying degrees of hemolysis.
· f) Syringe collections. Improper syringe draws are notorious for causing hemolyzed specimens. Syringe use should be avoided, if possible, in favor of the evacuated tube system. A study evaluated the effects of specimen quality when using syringe draws, compared to the evacuated tube system. Visual hemolysis was found in 19% of specimens drawn by syringe, compared to 3% when drawn by the evacuated tube system. Also, syringe-collected samples exhibited clotted EDTA specimens in 11% of the patients, as opposed to none in the evacuated tube system. If a syringe must be used, the following recommendations can reduce the incidence of hemolysis:
· 1) The phlebotomist should pump the plunger 2 – 3 times before collection to loosen the plunger. Then tighten the needle and syringe connection.
· 2) The phlebotomist should use a 3 –10mL syringe, avoiding larger volumes if possible.
· 3) The phlebotomist should ensure that the speed of aspiration does not exceed 1mL of air space during collection. Excessive aspiration forces frequently cause hemolysis.
· 4) The phlebotomist should perform blood transfer into the tube immediately.
· 5) The phlebotomist should fill tube by vacuum only, but never push down on the plunger, as this increases the force of the blood flow, creating a high degree of red blood cell (RBC) trauma. More importantly, positive pressure is produced in the tube, with a potential to cause either tube breakage or stoppers to pop out.
· 6 The phlebotomist should use a blood transfer device to transfer syringe-collected blood into a tube. It will enhance safety and improve specimen quality.
· 7) The phlebotomist should angle the syringe so that the blood runs down the side of the tube. By preventing the cells from hitting the bottom of the tube with such a great force, RBCs (red blood cells) trauma can be reduced.
· g) Peripheral Catheter Collections. The highest rates of hemolyzed specimens appear to come from the acute care setting, i.e., Emergency Dept (ED), Labor and Delivery (L&D), and Intensive Care Units (ICU). Studies have shown that the main source of hemolysis in the ER (emergency room) is the use of IV catheters for specimen collection. One study found that specimens drawn by nurses through an IV catheter were more than 3 times as likely to be hemolyzed than those drawn by venipuncture (13.7% vs. 3.8%). In another study, specimens were collected using IV catheters and venipuncture and then compared. The results revealed a 50% rate of hemolysis in the IV catheter collected specimens, compared to no hemolysis at all in the [peripheral] venipuncture specimens. Blood collected from the back of the IV catheter is pulled through several gauges; the catheter generally ranges from 18 to 22G, the Luer adapter “front” end is 15G, and the stopper-piercing needle is 20G. Slowing down this “pull rate” can reduce the hemolysis rate significantly. The use of partial draw collection tubes is an effective way to slow down the pressure exerted on the blood and, thus reduce hemolysis. The reduced vacuum in these tubes yields a slower, gentler draw. Partial drawtubes are designed to fill “part way” while maintaining the proper blood-to-additive ratio. The smaller volume of blood drawn into partial-draw tubes satisfies the CAP recommendation for minimizing large blood draw volumes and mitigates safety concerns as less blood is handled and discarded (partial-draw tubes should not be confused with small size pediatric tubes that are fully evacuated).
· h) Specimen Handling Techniques. Once the proper specimen collection techniques are applied, subsequent specimen handling factors must be considered to prevent hemolysis from occurring in the pre-analytical phase. The following factors should be considered to prevent hemolysis:
· 1) Mixing Tubes. The phlebotomist should mix the blood with the tube additive through gentle tube inversions. The phlebotomist should not shake the tube after collection.
· 2) Transport Methods. The phlebotomist should be cautious with pneumatic tube systems and other rough transport conditions that can create turbulence and RBC trauma within the tube. The phlebotomist should hand deliver specimens when feasible. Specimens should be stored in an upright position following centrifugation.
· 3) Rimming clots. The phlebotomist should not use wooden applicator sticks to rim clots, which can shred the red cells. With the current evacuated serum tubes available, rimming clots is unnecessary.
· 4) Temperature. The lab technicians should store and transport specimens in controlled temperature conditions, as temperatures that are too high or too low can rupture red cell membranes. They should make sure that centrifuge temperatures are acceptable. They should abide by the recommended transport and storage temperatures specified by the laboratory that is performing the assays.
· i) Centrifugation. Optimally serum should be separated from the clot by centrifugation within one-half hour after collection. This not only maintains the sample integrity but will also prevent the sample from hemolysing after venipuncture.
· Advice to prevent hemolysis during venipuncture:
· The venipuncture site should be allowed to dry after cleansing
· The phlebotomist should use the largest bore needle that’s appropriate
· The phlebotomist should never draw blood through a hematoma
· The phlebotomist should remove the tourniquet as early as possible to decrease flow velocity and turbulence
· The phlebotomist should not remove the collection tube until full
· If using a syringe, the phlebotomist should ensure the needle is fitted securely to avoid frothing
· If using a syringe, the phlebotomist should avoid drawing the plunger back too forcibly
· When mixing is required, gentle inversion is adequate.
THE LIPEMIC SPECIMEN
· Lipemia: After hemolysis, lipemia is the most frequent endogenous interference that can influence the results of various laboratory methods by several mechanisms.
· The most common preanalytical cause of lipemic samples is an inadequate time of blood sampling after the meal or parenteral administration of synthetic lipid emulsions. Although the best way of detecting the degree of lipemia measures lipemic index on analytical platforms, laboratory experts should be aware of its problems, like false positive results and lack of standardization between manufacturers. Unlike for other interferences, lipemia can be removed, and measurement can be done in a clear sample. However, a protocol for removing lipids from the sample has to be chosen carefully, since it is dependent on the analytes that have to be determined.
· Lipemia in a serum sample is most commonly the result of a non-fasting status. However, this may also occur in rare inherited metabolic disorders that result in the overproduction of fats in some people. An excessive amount of triglyceride in a serum sample is responsible for lipemia. Other laboratory results are known to be affected by lipemia. In non-fasting samples, it is possible to separate out, via ultracentrifugation, the excess lipids and obtain more accurate values that approximate those found in a fasting sample.
· 15-minute ultracentrifugation will not separate serum lipids present in genetic hyperlipidemia.
· A study examined the effect of lipid removal using ultracentrifugation of lipemic samples, on some routine biochemistry parameters. Among all the samples obtained daily in the laboratory, the ones who were visibly muddy were selected and underwent to a process of ultracentrifugation, being determined a variety of biochemical tests before and after ultracentrifugation. A total of 110 samples were studied. The results showed significant differences in all the parameters studied except for total bilirubin, glucose, gamma-glutamyl transferase (GGT) and aspartate aminotransferase (AST). The most significant differences in the parameters analyzed were found in the concentration of alanine aminotransferase (ALT; SGPT) (7.36%) and the smallest ones in the level of glucose (0.014%). Clinically significant interferences were found for phosphorus, creatinine, total protein, and calcium. The study concluded that lipemia causes clinically substantial interferences for phosphorus, creatinine, total protein and calcium measurement and those interferences could be effectively removed by ultracentrifugation.
· Another study concluded that hyperlipidemia caused errors in indirect ISE electrolyte measurements. All 3 electrolytes (Na, Cl, and K) determined by the indirect ISE system were affected, showing artifactual decreases as a result of hyperlipidemia. The sodium (Na+ ) and chlorine (Cl−) were decreased by about 1 mmol/L and potassium (K+) by about 0.04 mmol/L for each 10-mmol/L increase in total lipid concentration. When direct ISE methods and ultracentrifuges are unavailable to handle severely lipemic samples, corrective formulas can be used.
· The following tests are generally not affected: Glucose, Creatinine, Calcium, GGTP, Total Protein, although Cholesterol HDL may show a slight increase.
· The following tests are usually flagged with an instrument error and require re-analysis either after ultracentrifugation or dilution: BUN (blood urea nitrogen), ALT (SGOT), AST (SGPT), lactate dehydrogenase (LD; LDH), bicarbonate.
· The following tests are usually increased as a result of lipemia: uric acid, bilirubin, albumin, fructosamine, and triglyceride.
· If triglycerides are >400, the calculated tests (i.e., VLDL, LDL) are not reported. If triglycerides are >800, ultracentrifugation is performed. Results are indicated on the clarified sample.
· One way to avoid a grossly lipemic specimen is to ask the patient to fast before specimen collection.
· Lipemia on blood products: on blood products: lipemia is a naturally occurring phenomenon that may cause the appearance of blood products to be ‘milky’ but does not affect their safety or effectiveness in treating patients. Lipemia is the presence of a high concentration of lipids (or fats) in the blood. Lipemia is a condition in which increased amounts of lipids are present in the blood and frequently occurs after eating. The most common cause of a lipemic blood product is a subject who ate a high-fat meal just before donating blood.
· On blood products, lipemia will appear as:
· a) On whole blood/ red blood cells (RBCs): a grossly lipemic whole blood/RBC will look similar to a strawberry milkshake.
· b) On plasma or platelets: opaque (milky) appearance.
No comments:
Post a Comment