Answering your questions: hemolysis in serologic specimens, infant stool collection and capillary blood for blood gasses - Tips from the Clinical Experts

Medical Laboratory Observer, March, 2003 by Daniel M. Baer

Hemolysis in serologic specimens

Q: Our laboratory performs the following serological procedures: ANA (by indirect immunofluorescence) RA, Mono and Rubella immunity screen (by rapid latex agglutination). The package inserts all warn against using sera that are hemolyzed (all four tests) or lipemic (ANA and RA only). By what mechanisms do hemolysis and lipemia interfere with these tests? What degrees of hemolysis or lipemia are necessary for sample rejection? Do slightly hemolyzed or lipemic samples need to be redrawn? Calls to the manufacturers for clarification have been singularly unenlightening.

A: This is a very good inquiry concerning the interference of serologic antibody assays in hemolytic and lipemic specimens. It is correct that manufacturers state a disclaimer on the assays with specimens that have moderate to severe hemolysis, lipemia and high bilirubins. Most clinical laboratories will put cautionary disclaimers or reject specimens that have moderate hemolysis or severe lipemia. There are very few studies of the interference of hemolysis and lipemia on serologic assays.

The possible mechanism of interference by hemolysis or lipemia in serologic assays may be as follows:

1. Interference with antigen-antibody reaction.

2. Interference with the enzyme reactions in enzyme labeled antibody detection assays.

3. Very severe lipemia may make it difficult for accurate volume measurement in assays requiring precise sample measurement.

4. Interference with color measurement in assays.

Numbers 2 and 4 may be seen more in homogenous enzyme antibody assays than heterogeneous assays.

Qualitative antibody assays that are heterogeneous with a washing step do not show much interference with hemolysis or lipemia. Hemolytic and lipemic specimens may show variable results in antigen-antibody reactions, especially in nephelometric antibody assays. In general, lipemic specimens will interfere with enzymatic reaction assays. Below is a discussion of some of the reported studies.

In general, lipemic specimens will interfere with enzymatic reaction assays.

Bossuyt and Blanckaert have studied nephlometric assays for IgG, IgA, IgM, haptoglobin and alpha-1 antitrypsin in serum and compared the BN 100 from Behring and Array 360 assay of Beckman. (1) They found:

1. In lipemic samples, the BN100 gave higher values than the Array 360 for all analytes except IgG.

2. The interference of lipemia on the BN100 was also seen when the Beckman antibody was used.

3. BN100 gave higher values than the Array 360 for haptoglobin, but not for other analytes. The free haptoglobin will bind with hemoglobin released in serum of hemolyzed specimens.

Hubl W., et al. studied enzymatic determinations in abnormal hemolyzed, icteric, lipemic, paraproteinemic serum samples. (2) The observer found that none of the enzymatic methods could analyze grossly lipemic samples.

Several investigators have studied interferences from hemoglobin-based oxygen carriers used as "artificial blood" for transfusion.

Kazmierczak, et al. have studied the interference effects from a hemoglobin-based oxygen carrier on 29 different analytes using the Hitachi 747 and 911 systems, a Beckman CX3, and Abbott AxSym, Bayer Immuno I and a Dade ACA IV; a total of 60 analyte/instrument combinations. (3) They noted significant interference of cross-linked hemoglobin with 13 of 29 analytes tested.

With the Hitachi 747 and 911, interference of the cross-linked hemoglobin was noted in methods for albumin, alkaline phosphatase, bilirubin, cholesterol, total carbon dioxide, lactate dehydrogenase, magnesium, total protein and triglycerides. There was also interference of L-lactate assay with ACA IV and minor interference of glucose measurement with the Beckman CX3.

Ma, et al. also demonstrated that hemoglobin-based oxygen carriers demonstrated positive and negative interference on gentamycin and vancomycin assays, as well as some other analytes on different assay instruments and analytes. (4)

In contrast, Calles, et al. studied the effects of a glutaraldehyde polymerized bovine hemoglobin on routine chemistry, therapeutic drugs, coagulation, hematology and blood bank assays. (5) The polymerized bovine hemoglobin is an oxygen carrier; and the blood components containing this substance appear hemolyzed. The results of the study of Calles, et al. demonstrated that no significant analytical interference was noted with the polymerized bovine hemoglobin. (5)

Flood, et al. (6) reported on a multi-center evaluation of Boehringer Mannheim/Hitachi 717 system and reported no significant interference on many assays from hemolysis, icterus and lipemia. The 55 methods included end-point chemistries, enzymes, therapeutic drugs and specific protein assays.

Hursting, et al. (7) studied an enzyme-linked immunosorbent assay with use of monoclonal capture antibody for prothrombin fragment 1.2 and used a polyclonal peroxidase labeled detector antibody. They did not observe interference from lipemia, hemolysis, icterus or thrombolytic agents.