June 2011

Editor:
Fredrick L. Kiechle, MD, PhD

Q. Every diagnostic assay requires dilution of samples or reagents, or both. Some package inserts say the user must first dilute a reagent 1:9 or some other amount before use. I find it confusing and sometimes do not know exactly what I should be doing. What is the correct way to dilute samples and reagents?

A. I recently addressed this question in an article in the Journal of Chemical Education.¹ In the example you cited, “1:9” may mean to either mix one volume of reagent with nine volumes of diluent, for a total of 10 volumes, or one volume of reagent with eight volumes of diluent, for a total of nine volumes. Both dilution conventions can be correct, but only one is correct in a particular circumstance.

If a sample or reagent is to be diluted 1:100, for example, and it is diluted one volume plus 99 volumes diluent, the effect on the assay results may be relatively insignificant compared with one volume plus 100 volumes diluent. The problem may become more significant at greater dilutions. For example, a 1:1 dilution is either one volume in one total (that is, no dilution), or one volume in two total (that is, 50 percent), for a net difference of 50 percent. Furthermore, the issue may become magnified considering there are usually many different reagents in an assay that require dilution, often to different degrees. Errors may be cumulative, and this in turn might affect the sensitivity of an assay, with attendant consequences on its diagnostic capabilities and results.

The ratio a:b is generally defined as the quotient (a/b) and is described as a in b.^2–4 However, it may also be expressed as a to b.^5,6 It is noteworthy that a:b is used by different laboratory personnel to mean either the quotient a/b or the sum a+b.

Dynex Technologies is one manufacturer that recognized the use of different dilution conventions and provided users with assistance in this matter during assay setup with its DSX and DS2 automated ELISA instruments. Fig. 1 (PDF, 1.3 MB) is the dilution setup screen in the Revelation DSX Software version 6.13.

Here, the user has two options to choose from: “1 in...” and “1 to....” Dynex also provides further explanations regarding how it uses these terms, in the program help file and the DS-Matrix Software for the DS2 system (Fig. 2 PDF, 1.3 MB).⁷

This system also allows the user to simply specify the exact volumes to be pipetted—for example, if no dilution (a:b) is stated in the instructions.

Of interest is a dilution example in the Dynex Technologies DSX System Operator’s Manual—a 1:59 dilution, prepared as follows: “5 µL sample combined with 295 µL of diluent.”⁸ This dilution convention a:b means “a” volumes sample plus “b” volumes diluent. However, in other cases where instrument or assay manufacturers provide much less information, users might still use the dilution convention opposite that intended by the assay manufacturer, if they are unaware or unsure of which dilution convention their assay instructions intend for them to follow.

Some manufacturers avoid the entire problem of how dilutions are to be performed by offering unequiv­ocal dilution instructions with their assays. Two examples are the Roche polymerase chain reaction Cobas Am­pliscreen HIV-1 test, version 1.5, and the Euro-Diagnostica Diastat antinuclear antibody (ANA) ELISA. In these assays, the exact amount of each reagent, sample, and diluent are specified, leaving nothing to the user to interpret.

As part of a long-term solution, perhaps organizations such as the International Union of Pure and Applied Chemistry or American Chemical Society could establish a single dilution rule or convention that would then be taught in universities and professional schools.¹ As a more immediate solution, perhaps the CAP could require, or the government could mandate through CLIA, that only one dilution convention be adopted universally. In all cases, it is essential that package inserts for clinical assays in particular contain explicit instructions for the user as to how each dilution is to be performed. The most practical approach may be to simply require that all assay instructions carry such a statement at the beginning.

References

Acknowledgment

I thank Martin E. Gross of Dynex Technologies for providing information about the Dynex DSX and DS2 instruments.

Laurence Fishel, PhD, MT(AAB)
Molecular Laboratory Supervisor
Fertility and Cryogenics Laboratory
Downers Grove, Ill.

Q. Do hematology QC materials for daily calibration verification have to be handled, prepared, applied, and returned to storage in a consistent and timely manner to retain their integrity over their designated lifetime? If so, what would you recommend as an objective solution since QC manufacturers with whom I have checked do not officially state how to, and not to, prepare, use, and return QC material to cool storage.

A. QC material is very subject to the manner in which it is handled during the lifetime of the control vial. Extended warming periods, excessive mixing, and mixing before adequate warming are detrimental to QC materials and will accelerate the swelling of the red blood cells, thus increasing the MCV and affecting the MCH and MCHC. Excessive mixing will contribute to the degradation of the red blood cells and may increase the platelet count. A vial containing material that is inadequately mixed, then sampled, becomes tainted from that point. QC material handling is the most critical in minimizing shifts or trends in the QC data. Most manufacturers include specific instructions, which should be followed closely, on how to mix the QC material. When QC vials are mishandled, the point at which they were misused can often quite easily be seen on the Levy-Jennings charts.

According to CLSI guideline H26-A2,Validation, Verification, and Quality Assurance of Automated Hematology Analyzers, “Manufacturers of stabilized blood products should provide exact directions for storage, mixing and remixing, because these procedures may differ from fresh blood handling.”¹ The proper handling of QC material is necessary to retain the integrity of the material. In one study of analytic quality in hematology performed in Latin America, laboratories in seven participating countries were sent hematology control materials for determination of hemoglobin, hematocrit, and red and white blood cell counts. Analysis of results showed higher coefficients of variation for several parameters when compared with those reported from the United States and Europe. The authors concluded that “training in the preparation of quality control materials and the subsequent use of these materials... could provide a starting point for establishing... quality assessment systems in hematology.”²

While there is scant published literature on the recommended use of QC material such as that used in hematology, I believe many of us can attest to what should not be done with this material.

References

Tracy I. George, MD
Assistant Professor of Pathology
Director, Clinical Hematology Laboratory
Associate Director,
RBC Special Studies Laboratory
Stanford University
Stanford, Calif.

Chair, CAP Hematology/Clinical Microscopy Resource Committee

Q. Is there a consensus on updating and documenting Medicare Secondary Payer, or MSP, information on nursing home patients in which hospital personnel provide the phlebotomy service? We have copies of the MSP information from the nursing homes, but it needs to be confirmed at each encounter. Many nursing home patients cannot answer these questions.

A. As I understand and have referenced with the manuals, MSP information must be updated every 90 days. This applies to any hospital outpatient. Medicare defines outpatient as a patient who has had a face-to-face encounter with a hospital employee. This rule disregards the location of the encounter. In the case of nonresponsive nursing home patients, MSP information can be obtained from a health care surrogate or appointed guardian.

References

1. Centers for Medicare and Medicaid Ser­vices. Medicare Benefits Policy Manual. Chapter 6–Hospital Services Covered Under Part B. Rev. 137, 12–30–10. https://www.cms.gov/manuals/downloads/bp102c06.pdf.
2. Centers for Medicare and Medicaid Services. Medicare Secondary Payer Manual (MSP). Chapter 3–MSP Provider, Physician, and Other Supplier Billing Requirements. Rev. 57, 10–27-06. https://www.cms.gov/downloads/MSP105c03.pdf.

Ron Gemme
Former Administrative Director of Laboratory Services
Memorial Healthcare System
Memorial Regional Hospital
Hollywood, Fla.