Fredrick L. Kiechle, MD, PhD
Q. Is using MUM1 and CD138 immunohistochemical and kappa and lambda light chain in situ hybridization stains on bone marrow clot and biopsy specimens the preferred method for evaluating plasma cell dyscrasias?
A. Plasma cell clonality can be established by flow cytometry or immunohistochemistry, or both. Immunohistochemistry is superior to flow cytometry in detecting cytoplasmic immunoglobulin; hence, immunohistochemistry is the preferred method for establishing clonality in plasma cells.
An initial panel to evaluate plasma cell dyscrasia by immunohistochemistry should include CD138, kappa, and lambda. CD138 is a highly sensitive and specific marker for identifying and quantifying normal and malignant plasma cells. Clonality of the plasma cells can be established by kappa and lambda stains performed by immunohistochemistry or in situ hybridization.
Once the clonality of the plasma cells is established, additional immunostains can be helpful. CD20 and PAX5 can be helpful if lymphoma with plasmacytic differentiation is in the differential diagnosis. CD56, which is expressed in more than 50 percent of the cases, can be helpful in detecting minimal residual disease after therapy. BCL1, which is expressed in 30 percent of the cases and correlates with underlying t(11;14), can be helpful in detecting minimal residual disease when present.
MUM1, though a sensitive stain for plasma cells, is not as specific as CD138. It can be seen in many lymphoid and myeloid malignancies and is not recommended for plasma cell dyscrasia workup.
1. Dunphy CH. Applications of flow cytometry and immunohistochemistry to diagnostic hematopathology. Arch Pathol Lab Med. 2004;128:1004–1022.
2. Gualco G, Weiss LM, Bacchi CE. MUM1/IRF4: a review. Appl Immunohistochem Mol Morphol. 2010;18:301–310.
3. Higgins RA, Blankenship JE, Kinney MC. Application of immunohistochemistry in the diagnosis of non-Hodgkin and Hodgkin lymphoma. Arch Pathol Lab Med. 2008;132:441–461.
Randa Alsabeh, MD
Cedars-Sinai Medical Center
Q. Why are changes in the APTT therapeutic range needed?
A. Unfractionated heparin has traditionally been monitored by altering the heparin dose using an activated partial thromboplastin time (APTT). Because the APTT therapeutic range varies drastically with different APTT reagents and instruments, the laboratory is charged with determining an appropriate therapeutic range based on the specific instrument/reagent performance. The APTT therapeutic range is determined using samples from patients on unfractionated heparin (about 30 samples).1 An APTT and a heparin activity using an anti-Xa assay are performed on each sample, and a plot is then constructed to show the relationship between APTT and heparin.1 The therapeutic range is the spectrum of APTT values that falls between 0.3 and 0.7 IU/mL of heparin (Fig. 1).2,3 Exclusion criteria for data points are APTT within the “normal” reference range or greater than the measurable limit, heparin activity <0.1 IU/mL or >1.0 IU/mL, INR>1.3, and obvious disparity between heparin activity and APTT. If samples are to be frozen before testing for the therapeutic range, the samples must be double spun to reduce the number of platelets. This is to prevent falsely low APTT and heparin activities that could be obtained after thawing owing to the neutralization of heparin by platelet factor 4 released from platelets.
The laboratory informs the clinical services of this therapeutic range and then revalidates the range when a new lot number of APTT reagent is implemented. A manufacturer is often able to provide the same lot number of the APTT reagent for one year. The range can be revalidated with ex vivo heparinized samples as necessary by tracking cumulative differences of the mean APTT values of the old and new reagent lots (Fig. 2).2 If the cumulative change of means is less than seven seconds, then the new lot of APTT reagent is acceptable and the previously established therapeutic interval remains unchanged. The therapeutic range should be maintained as long as possible, but change is sometimes unavoidable when new coagulation instruments or reagents, or both, with different performance characteristics, are acquired. When a change of the APTT therapeutic range is necessary, extensive communication about the new range is compulsory.
Recently our two laboratories independently introduced new instrument/reagent systems, and both laboratories found that the new APTT therapeutic range was significantly different than the one in use for eight to 10 years. Our laboratories approached this opportunity for change with two different solutions. One lab adopted the new therapeutic APTT reference range and added anti-Xa heparin levels to nursing protocols so that either could be used for patient management. The other lab moved to institute monitoring using heparin levels alone. After the latter institution evaluated several reagents on a new instrument, it became clear that many of the available APTT reagents yielded higher and broader therapeutic heparin intervals than the laboratory had established previously. Three examples of these reagents yielded ranges as follows: reagent No. 1, 120 to 200 seconds; reagent No. 2, 70 to 130 seconds; and reagent No. 3, 58 to 77 seconds. While reagent No. 3 was appealing at first, the APTT reagent was not used widely and would have excluded the institution from any peer groups for proficiency testing. Historically, clinicians are comfortable with APTT therapeutic range upper limits below 100 seconds, so reagents No. 1 and No. 2 were problematic. Convincing clinicians that patients were safely anticoagulated with an APTT at 130 seconds would be difficult. Moreover, concern arose that imprecision of the APTT assay at 130 seconds would lead to many unnecessary dose adjustments when the APTT was maintained in this range. Thus, the laboratory decided to use an alternative monitoring strategy,4 a functional heparin assay (anti-Xa method) with a widely accepted therapeutic interval of 0.3 to 0.7 U/mL.2
Both institutions launched multidisciplinary campaigns to cover the many interactions of health professionals involved in heparin monitoring. After discussing our separate experiences, we recognized there was a striking similarity in our implementation processes. A summary combining the efforts of the two institutions follows and could serve as a checklist for hospitals undertaking a change in heparin monitoring:
- Discuss policy change with appropriate committees
Pharmacy and Therapeutics Committee
Medical Executive Committee
Anticoagulation Safety Committee
- Update written policies with pharmacists and nurses
- Ensure that all Web sites and information systems are updated
- Educate clinicians, nurses, medical technologists, and pharmacists
- Develop new nomograms with pathologist
- Distribute institutional publications about new heparin policy
Laboratory and hospital information specialists
- Develop new order sets for heparin
- Apply flashing alerts/pop-up windows to hospital information systems that warn clinicians about the change in policy
- Add temporary comments to laboratory results that inform clinicians about changes
- Update standard operating procedures
- Field calls from nurses and clinicians
- Survey floors to ensure removal of old nomograms
- Distribute institutional publications about new heparin policy
The educational effort was particularly important and extensive. Educational activities included giving presentations to clinical services, distributing laminated cards with therapeutic ranges, and sending memorandums to clinical faculty. Articles in institutional publications that were written by and circulated among clinicians, pharmacists, and nurses aided the educational effort. The laboratory and hospital information systems were excellent tools for alerting clinicians that heparin monitoring had changed.
Although laboratories do not wish to change their systems for monitoring heparin, whether this means changing the APTT therapeutic interval or switching to a heparin assay, new instrument technology and APTT reagent characteristics sometimes force change. Such change is difficult and requires great effort and communication among pathologists, medical technologists, pharmacists, nurses, and clinicians.
1. Brill-Edwards P, Ginsberg JS, Johnston M, et al. Establishing a therapeutic range for heparin therapy. Ann Intern Med. 1993;119:104–109.
2. Olson JD, Arkin CF, Brandt JT, et al. College of American Pathologists conference XXXI on laboratory monitoring of anticoagulant therapy: laboratory monitoring of unfractionated heparin therapy. Arch Pathol Lab Med. 1998;122:782–798.
3. Brandt JT. Anticoagulant Agents. In: Marchant KK, ed. An Algorithmic Approach to Hemostasis Testing. Northfield, Ill.: College of American Pathologists. 2008:31–20.
4. Levine MN, Hirsh J, Gent M, et al. A randomized trial comparing activated thromboplastin time with heparin assay in patients with acute venous thromboembolism requiring large daily doses of heparin. Arch Intern Med. 1994;154:49–56.
Russell A. Higgins, MD
University of Texas Health
Science Center at San Antonio
Vice Chair, CAP Coagulation
Sandra C. Hollensead, MD
Department of Pathology and
University of Louisville Hospital
Member, CAP Coagulation
Dr. Kiechle is medical director of clinical pathology, Memorial Healthcare, Hollywood, Fla.