Answering your questions

Answering your questions

Hemolysis in serologic specimens, infant stool collection
and capillary blood for blood gasses

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. 

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 multicenter 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.

Thus, in conclusion, there is evidence that moderate to severe hemolysis and lipemia will interfere with certain specific assays. Each assay system for a specific analyte must be evaluated individually.

Robert M. Nakamura, MD
Chairman Emeritus and Senior Consultant
Department of Pathology
Scripps Clinic

References

1. Bossuyt X, Blanckaert N. Evaluation of interferences in rate and fixed-time nephelometric assays of specific serum proteins. Clin Chem. January 1999;45(1):62-67. 
2. Hubl W, Wejbora R, Shafti-Keramat I, Haider A, Hajdusich P, Bayer PM. Enzymatic determination of sodium, potassium, and chloride in abnormal (hemolyzed, icteric, lipemic, paraproteinemic, or uremic) serum samples compared with indirect determination with ion-selective electrodes. Clin Chem. August 1994;40(8):1528-1531. 
3. Kazmierczak SC, Catrou PG, Best AE, Sullivan SW, Briley KP. Multiple regression analysis of interference effects from a hemoglobin-based oxygen carrier solution. Clin Chem Lab Med. April 1999;37(4):453-464. 
4. Ma Z, Monk TG, Goodnough LT, McClellan A, Gawryl M, Clark T, et al. Effect of hemoglobin- and Perflubron-based oxygen carriers on common clinical laboratory tests. Clin Chem. September 1997;43(9):1732-1737. 
5. Callas DD, Clark TL, Moreira PL, Lansden C, Gawryl MS, Kahn S, et al. In vitro effects of a novel hemoglobin-based oxygen carrier in routine chemistry, therapeutic drug, coagulation, hematology, and blood bank assays. Clin Chem. September 1997;43(9):1744-1748. 
6. F lood J, Liedtke R, Mattenheimer H, Rothouse L, Trundle D, Vroon D, et al. A multicenter evaluation of the Boehringer Mannheim/Hitachi 717 system. Clin Biochem December 1990;23(6):477-488. 
7. Hursting MJ, Butman BT, Steiner JP, Moore BM, Plank MC, Szewczyk KM, et al. Monoclonal antibodies specific for prothrombin fragment 1.2 and their use in a quantitative enzyme-linked immunosorbent assay. Clin Chem. April 1993;39(4):583-591. 

Infant stool collection

Q: What is the recommended
method of stool collection for children who are not toilet trained? Is it ever acceptable to use stool from a diaper? 

A: I consulted several people about
this question. Ten years ago, Peter Fuchs, MD, answered a similar question in this column. As a microbiologist, he restricted his reply to the suitability of diaper-collected samples for microbiology testing. His reply was, It is quite acceptable to test recognizable solid or semisolid stool that has been submitted in an infants diaper. On the other hand, liquid stool specimens that have been completely absorbed by the diaper are generally far less acceptable. An alternative that is preferable to the latter specimen, but still less desirable than an actual stool specimen, is the rectal swab. This method can provide acceptable specimens for fecal leukocyte smears and cultures for enteric bacterial pathogens. Nevertheless, they generally are not acceptable for C difficile toxin assays or for ova and parasite examinations.

Gillian Lockitch, MBChB, MD, at Childrens & Womens Health Centre of British Columbia, Vancouver, BC, Canada, told me that for microbiology specimens, a swab taken from the watery stool or a rectal swab was necessary. Stool absorbed into the diaper cannot be used.

Fecal testing in infants is performed for other analytes, too. For example, fecal fat, fecal alpha-1-antitrypsin and fecal elastase are quantitatively analyzed. Ching-Nan Ou, Ph.D., director of Clinical Chemistry at Texas Childrens Hospital, Houston, told me that their GI physicians who order quite a few fecal tests recommend a method of loose stool collection for children who are not toilet trained. It is to place a layer of kitchen plastic wrap on top of diaper and cut a hole for male to urinate outside of diaper. For female infants, frequent changes of diaper may be needed. For solid stool collection, it is acceptable to collect stool from a diaper. Dr. Lockitchs hospital uses the same method.

Daniel M. Baer, MD
Professor Emeritus
Department of Pathology
Oregon Health and Science University
Portland, OR 

References

1. Fuchs P, Tips from the Clinical Experts: Infant stool specimens. MLO, February 1993.
2. Campos JM, Lancz G, Fritsche TR, Specter S. Gastrointestinal Tract. In Howanitz JH, Howanitz PJ. Laboratory Medicine Test Selection and Interpretation. New York, NY: Churchill
   Livingstone; 1991:750-766.

Capillary blood for blood gasses

Q: We are using capillary blood to
collect arterial blood samples from newborns for blood gas exams. Is this an acceptable procedure or widely used? Is there an infection problem in a nursery environment? Is there a different reference range between an adult and newborn? In our lab, we are using the adult reference range for the newborn.

A: Arterialized capillary blood is
sometimes an acceptable alternative to arterial blood when blood losses must be minimized, when an arterial cannula is not available, or when repeated arterial puncture must be avoided. However, it is not acceptable when systolic blood pressure is less than 95 mm Hg, in cases of vasoconstriction, in patients on O2 therapy, in newborns during the first few hours after birth, or in newborns with respiratory distress syndrome. These situations pose a particular risk of admixture with blood from the venules, resulting in erroneously low PO2 values.¹

With the universal infection control implementation, there should be no infection problem in a nursery environment.

Yes, there is a different reference range between an adult and newborn, particularly in the first day of life.^1,2 For example, arterial blood PO2, low at birth, rises to an adult level of 83-108 mm Hg.

Ching-Nan Ou, Ph.D.
Director of Clinical Chemistry
Texas Childrens Hospital
Professor of Pathology
Baylor College of Medicine
Houston, TX

References

1. Burtis CA & Ashwood ER, eds. Tietz Textbook of Clinical Chemistry. 3rd ed. Philadelphia, PA: W.B. Saunders, 1999, 1082-1087.
2. Soldin SJ, Brugnara C, Gunter, KC, Hicks, JM, et al., eds. Pediatric Reference Ranges. 2nd ed., Washington DC, AACC Press, 1997.