Answering your questions.

Hemolysis control

QI have been trying to find a hemolysis control for our COBAS INTEGRA and have been unsuccessful. We perform donor testing, and some of the samples are hemolyzed to some degree. The instrument package inserts indicate testing cannot be done if hemolysis is above a certain level. Is making up a level the right approach?

A Hemolysis, or the degradation of red-blood cells (RBCs), has long been known to interfere with several analytical test results. (1-3) In recognition of this issue, many automated instruments currently on the market can assess the level of hemolysis and allow the user to define the concentration at which a flag is generated. This is typically accomplished by spectrophotometrically measuring the absorbance of light by hemoglobin between 400 nm to 440 nm where heme proteins exhibit maximal absorbance. Some instruments may not have this ca pability. For those, an alternate means to assess hemolysis is to compare the serum or plasma to a color chart.

For either semiquantitative or qualitative methods, hemolysis standards are needed. If a lab has automated capability, the laboratory can prepare standards at different concentrations of hemoglobin. For those without automated capability, it is possible to obtain commercially available hemolysis charts; however, with digital photography, a lab can prepare its own standards and create a color chart, although interpretation of any color chart is subjective. MLO published a hemolysis chart several years ago. (4)

Hemolysates can be made chemically or thermally. (5), (6) Two easy, straightforward methods of preparing hemolysates are the osmotic-shock procedure (diluting blood with water) or cold shock (freezing). There are also several red-blood-cell-lysing kits commercially available, but keeping the original solutions is not recommended, as the hemoglobin will denature. Once the hemolysates are made, an automated hematology instrument can determine the hemoglobin concentrations.

It is good practice to include hemolysis-, bilirubin--and lipid-interference levels during the initial validation of the instrument. This will verify that the levels set by the manufacturer correlate with the actual concentrations of hemoglobin and can be accomplished by using standard solutions outlined by CLSI.(6) The value of routinely running control solutions is debatable, unless recommended by the manufacturer. Regulatory agencies have not required QC for indices in the past, although confirming the validation results on an annual or biannual basis may be prudent.

--Valerie Bush, PhD

Director Clinical Laboratory and POCT Bassett Healthcare Cooperstown, NY

References

(1.) Laessig RH, Hassemer DJ, Paskey TA, Schwartz TH. The effects of 0.1 and 1.0 percent erythrocytes and hemolysis on serum chemistry values. Am J Clin Pathol. 1976;66(4):639-644.

(2.) Blank DW, Kroll MH, Ruddel ME, Elin RJ. Hemoglobin interference from in vivo hemolysis. Clin Chem. 1985;31(9): 1566-1569.

(3.) sonntag O. Haemolsis as an interference factor in clinical chemistry. J Clin Chem Clin Biochem. 1986;24(2): 127-139.

(4.) Baer DM, Ernst DJ, Willeford SI, Gambino R. Investigating elevated potassium values. Medical Laboratory Observer, November 2006;38:24-31.

(5.) Lippi G, salvagno GL, Montagnana M, Brocco G, Guidi GC, Influence of hemolysis on routine clinical chemistry testing. Clin Chem Lab Med. 2006;44(3):311-316.

(6.) Clinical and Laboratory Standards Institute. Interference Testing in Clinical Chemistry; Approved Guideline--Second Edition. Wayne, PA: Clinical and Laboratory Standards Institute; 1998. CLSI document EP7-A2.

Are verbal orders okay?

Q Our medical-records department is telling us that the written physician name with office-staff initials after it is not valid for billing purposes. We are required to call nursing homes, home-health departments, and regular physician offices to request the actual physician signature on the order. Staff has been allowed to write the orders on requisitions and record the physician name along with his initials for a very long time. If Medicare and/or other insurance request additional information and confirmation of order, the nursing home, home-health, and physician office would then be contacted for a copy of the patient's chart to send in for verification purposes. If this has changed, where can I get a copy of the regulations for this ruling?

A Verbal orders are addressed in Clinical Laboratory Improvement Amendments (CLI A), and the requirement is that a verbal order must be substantiated either in writing or electronically within 30 days. There is no requirement for a physician's signature on a test request. It is important to differentiate between an order and are-quest. An authorized individual must write the order in the medical record, whereas the request can be sent in by anyone carrying out the authorized individual's order.

The regulation citations are CLIA 493.1241 Standard: Test Request--"(b) The laboratory may accept oral requests for laboratory tests if it solicits a written of electronic authorization within 30 days of the oral request and maintains the authorization or documentation of its efforts to obtain the authorization."

Interpretive Guidelines 493.1241(b)--"Review the laboratory's policy for requesting written orders within 30 days of the oral requests. If no written order was received, verify the laboratory has documentation showing the attempt."

Since the billing office is suggesting otherwise, you may want to ask the billing staff to supply you with the source of that office's information. You can then verify if it is a regulation or simply an internal policy.

--C. Anne Pontius, MBA, CMPE, MT(ASCP)

Senior Director, Quality Systems Expression Analysis Inc. Durham, NC

Hemolyzed specimens from the ER

Q The emergency department (ED) collects all of its own patients. The ED blames the lab for the high number of samples that are hemolyzed, claiming that the samples sit too long, which causes this. Can you help us?

A This is a common problem. Specimens collected by non-laboratory personnel have a higher hemolysis rate than those collected by the lab. In one study, 12.4% of specimens drawn by the ED were hemolyzed versus 1.6% drawn by the laboratory. (1) Another study evaluated over 500 hemolyzed samples for the cause of the hemolysis. (2) It found that 78% of the cases were related to blood being drawn too vigorously into a syringe through a needle. IV catheter, or from an infusion line. In 5% of the cases, blood was forcibly squirted from a syringe into a tube. The authors of this study recommended replacing the syringe collection with an evacuated-tube system.

In another study comparing a syringe collection with evacuated tubes, 19% of syringe-collected specimens were hemolyzed versus 3% of the evacuated tube collections. (3) Another study, carried out by emergency-room nurses, compared hemolysis in specimens obtained through a catheter (hemolysis rate, 13.7%) with venipuncture specimens (hemolysis rate, 3.8%). (4) The hemolysis rate was inversely proportional to the diameter of the catheter, with the highest hemolysis rates in 24-gauge to 20-gauge catheters.

Drawing through a small-gauge needle causes cell-membrane shearing of the RBC., while using a large-diameter needle causes the blood to enter the evacuated tube with great force, rupturing some of the cells. The optimal needle size for evacuated collection tubes is 21-gauage. (3)

A problem involved in collecting blood through a catheter or IV infusion tubing is that the diameters of the catheter, tube-adaptor device, and cap-piercing needle may be mismatched, causing changes in pressure on the RBCs during collection, with rupture of some of the cells.

Practical measures to reduce specimen hemolysis are the following:

* Replace syringe collection with an evacuated tube system;

* Use 21-gauge needles for collection;

* Do not collect blood through an IV catheter smaller than 20-gauge;

* If a syringe is used for collection, do not use strong negative pressure to draw the blood into the syringe; and

* If a syringe is used for collection, let the blood flow into evacuated tube by the tube's vacuum without pushing on the plunger.

The ED staff needs to be educated about the problem and its solutions. The third reference contains a good summary of the causes of hemolysis, with a discussion of practical steps to prevent it. This could be the basis of an educational program for ED personnel.

--Daniel M. Baer, MD

Professor Emeritus Department of Pathology Oregon Health and Science University Portland, OR

References

(1.) Burns ER, Yoshikawa N. Hemolysis in serum samples drawn by emergency department personnel versus laboratory phlebotomists. Laboratory Medicine. 2002;33:378-380.

(2.) Carraro P, Servidio G, Plehani M. Hemolyzed specimens: a reason for rejection or a clinical challenge? Clin Chem. 2000;46:306-307.

(3.) Stankovic A, Smith S. Elevated serum potassium values: the role of preanalytic variables. Am J Clin Pathol. 2004;121:S112-S105.

(4.) Kennedy C, Agenmuller S, King R, et al. A comparison of hemolysis rates using intravenous catheters versus venipuncture tubes for obtaining blood samples. J Emerg Nurs. 1996;22:566-569.

Blood work after FFP or platelets

Q If a patient has received fresh frozen plasma (FFP) or cryoprecipitate, how long should you wait to draw a partial thromboplastin time (PTT) or prothrombin time (PT) test? If the patient received platelets, how long should you wait to check the platelet count? If the patient received a unit of packed red-blood cells (PRBC), how long should you wait to draw a complete blood count (CBC)?

A Unfortunately, there are no guidelines in the literature for the timing of sample draw for CBC, PT/PTT, or platelet counts after transfusion of packed red-blood cells. FFP. or platelets; however, from clinical experience and the practice of component therapy, it is my belief that (be timing for the sample draw will depend on the purpose of transfusion and purpose of the testing. The major purpose of testing for the above parameters after blood-component therapy can be 1) to determine immediate response or lack of response from component therapy (e.g., platelet refractoriness) and 2) to determine survival of cellular product or loss of response as the reversal of abnormal PT/PTT after few hours of FFP transfusion to guide for the necessity of further transfusion.

For the first purpose, a sample could be drawn within lOminules to 30 minutes of transfusion. The lab results for hemoglobin, hematocrit, platelet count, and PTand PTT should be pretty accurate to reflect the immediate response o( transfusion therapy. Also remember that clinical response from transfusion therapy depends on the circulating or intravascular portion of the transfused product irrespective of its distribution in body.

For the purpose of survival of PRBC or platelets, a sample can be drawn within 20 hours to 24 hours or as clinically necessary to guide the further component therapy for conditions such as autoimmune hemolytic anemia, immune platelet refractoriness, and others. Since the FFP is usually transfused to correct coagulopathy of massive transfusion, cirrhosis, or as a prophylactic for abnormal PT/PTT 1.5 times or greater than normal, prior to an invasive procedure, a sample can also be drawn within 10 minutes to 30 minutes of transfusion for the correction of coagulopathy; however, remember that due to the short half-life of factor VII (four hours), PT and PTT will turn abnormal again within seven hours to eight hours post transfusion, and to repeat the FFP transfusion may not be needed if the patient is not bleeding. For the same reason, FFP should be transfused immediately prior to a procedure for correction of PT and PTT, rather than six hours to eight hours prior to invasive procedure.

--Krishna Oza, MD

Department of Pathology University of Arkansas for Medical Sciences Little Rock, AR

Refrigerated urine for QC

Q Our urinalysis supervisor obtains urine specimens from the refrigerator to recheck tests from the previous day. For nitrite, I think this is not a good idea since the nitrite can be reduced to nitrogen gas, but I do not know if it would occur at 10[degrees]C. Would the white-blood cells continue to disintegrate from say 10/hpf to 4/hpf (high power field) in the refrigerator during 24 hours?

A In general, urine should be tested within one hour to two hours of voiding. If this cannot be done, it can be held under refrigeration. It is generally stable for six hours to eight hours when refrigerated--although both bilirubin and urobilinogen are especially susceptible to exposure to light. Degradation of nitrite to nitrogen is not generally believed to be a problem; however, neutrophils are quite labile and might well decrease in numbers under refrigeration. Remember that the life span of a neutrophil in the peripheral blood is only eight hours to 10 hours--this is reflected in the difficulty in retaining them in a urine specimen. For purposes of quality control, commercial urine-control specimens are preferred to duplicate specimens, especially when held for 24 hours.

--Karen M. Ringsrud, MT(ASCP)

Assistant Professor (Ret.) Department of Laboratory Medicine and Pathology University of Minnesota Medical School Minneapolis, MN

It is my belief that the timing for the sample draw will depend on the purpose of transfusion and purpose of the testing.

Daniel M. Baer, MD, is professor emeritus of laboratory medicine at Oregon Health and Science University in Portland, OR, and a member of MLO's editorial advisory board.

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Edited by Daniel M. Baer, MD