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Answering your questions.

Du: Is it D positive or negative?

Q Our lab is in the practice of reporting the Rh type of weak D patients as Rh neg, Du positive. I think this can be misleading. The presence of the D antigen, whether it is weak or not, still means the person is Rh positive for D. I have talked to my co-workers about reporting such a reaction as Rh pos, Du pos. They say that anyone who is Du positive should be transfused with Rhnegative blood, especially female patients in childbearing years. But they believe that by reporting it as Rh neg, Du pos, blood-bank personnel will be prompted to cross match Rh-neg units, and other medical staff would expect Rh-negative units to be given. How should a weak D officially be reported?

A The Standards for Blood Banks and Transfusion Services (Standard 5.8.2) says if the initial test with anti-D is negative, the blood shall be tested using a method designed to detect weak D when either test is positive the label shall read "Rh positive." Thus, donor blood weak D is Rh positive. For the purpose of patient or recipients, however, according to Standard 5.13.2, Rh type shall be determined with anti-D reagent. The test for weak D is unnecessary when testing the patient. For the purpose of Rh immune globulin prophylaxis, the Standards start with cautioning about the Rh mistyping, and according to Standard 5.20.1, "Interpretation criteria shall be established to prevent the mistyping of an Rh-negative patient as Rh positive due to exposure to Rhpositive red cells." And to classify a pregnant woman Rh negative, in the standard (criteria for Rh immunoglobulin prophylaxis), it is clearly suggested. "A test for weak D is not required."

Weak D testing, however, is required when testing the fetus (Standard Thus, according to the standard, women of childbearing age can be classified as Rh negative without further testing for the weak D or Du. Standards avoided this issue and simplified it by avoiding testing for weak D. According to the standard, donors will be tested for weak D and classified D positive if tested positive, but recipients are not required to be tested and are classified D negative. Standards gives an easy answer for the above issue by avoiding testing for weak D and cautioning not to misinterpret a recipient as Rh negative; therefore, leaving it up to the transfusion services to interpret the results of the weak D testing.

Historically, weak D red blood ceils (RBCs) are defined as having decreased D antigen levels which requires the indirect antiglobulin test, or IAT, for detection. Weak D expression primarily results from single-point mutation in the RHD gene that encodes amino-acid change in the intracellular or cytoplasmic region of Rh D protein with the decreased number of D antigen sites. Up to 54 (1 to 54) different weak D types have been reported. The weak D types 4.2 and 15 have been reported to make anti-D and recommended to be classified as partial D. Since the difference in the D antigen is quantitative, not qualitative, the majority of weak D patients can be transfused with D positive RBCs. Thus, with few exceptions, from a historical perspective, one can safely classify the weak D as D positive.

The following factors are responsible for the difficulty in the D antigen typing:

* D antigen is defined by multiple conformational epitopes and their variation (about 175 RHD alleles are described genetically);

* multiple different methods of testing and monoclonal reagents (different clones and blends); and

* reactivity of monoclonal IgM to "D-like" epitope on Rhce protein.

Once we take in account above factors, it is not surprising why discrepancies in D typing are observed in routine testing. According to a 1999 CAP survey, 58% (may be lower now) of hospital services were performing weak D testing with striking differences in transfusion practices. The various methods used for D testing include slide, tube test, solid phase, gel columns, and automated analyzer with enzyme-treated RBCs. Of those surveyed, 44% were transfusing D negative units, while 42% were transfusing D positive units, and only 10% were transfusing D negative only to women of childbearing age. These survey results illustrate that various methods of testing cause variation in reactivity of weak D, as well as variation in D status assignment by the transfusion services. As seen at serological-level D typing, discrepancies can arise due to many D variants (see Table 1), and not only due to weak D (formerly Du). Therefore, DNA-based testing, as well as thorough serological testing, may be helpful prior to assigning the D antigen status--with the key consideration being whether it is for a blood donor or transfusion recipient, and what the risk of sensitization and formation of anti-D is.
  D variants     Reactivity with    Detection          Genetic basis

Weak D (Du)      Weak             Reacts in AHG      Point mutation of
                                  phase              RHD localized in
                                                     the intracellular
                                                     or cytoplasmic
                                                     region of RHD

Partial D        Strong           Forms anti-D       Point mutation of
                                  following          RHD localized in
                                  transfusion        the extracellular
                                                     region of RHD
                                                     (hybrid gene)

Del              Negative         Formation and      RHD mutation and
                                  elution of         altered epitopes
                                  anti-D             (r or D-C+ RBC)

D-like epitopes  Strong;          Negative with      Presence of
on Rhce          [D.sup.HAR],     some and Positive  D-like amino-acid
                 [ce.sup.CF]      with some          sequence in Rhce
                 antigen Weakly   monoclonal         protein reacting
                 reactive;        antibodies         to some
                 [ce.sup.RT]'                        monoclonal
                 [ce.sup.SL]                         anti-D

  D variants        Formation of anti-D       Rh consideration for
                                              transfusion purposes

Weak D (Du)      No, except D type 4.2 and  Can be considered D
                 15                         positive in most cases

Partial D        Yes                        D negative

Del              Yes                        Will type as D negative

D-like epitopes  Yes                        D negative
on Rhce

Table 1. D variants and Rh considerations for transfusions.

Most experts agree that a serologically or molecularly confirmed D-variant blood donor should be classified as D positive and a recipient as D negative. This is good for safer transfusion practices but could be confusing to patients and physicians. Also it creates a dilemma how to report and record the results. But the answer will not always be a simple Rh positive or Rh negative and will require an additional statement of explanation to both the patient and the physician.

One way to report could be the patient is a D variant (classify serologically and molecularly, if possible) and will be considered Rh positive as a blood donor and Rh negative for transfusion and Rh immune globulin administration. This may not apply to each ease; and, if possible, full serological and molecular testing should be used to assign the D antigen status in an individual at risk of formation of anti-D and risk of hemolytic disease of newborn.

--Krishna Oza, MD Hematopathology, US LABS Brentwood, TN

Further reading

(1.) AABB. Standards for Blood Banks and Transfusion Services, 24th ed. 2006. AABB: Bethesda, MD.

(2.) Westhoff CM. Rh complexities: serology and DNA genotyping. Transfusion. 2007;47(1 Suppl):17S-22S.

(3.) Westhoff CM. The Rh blood group D antigen ... dominant, diverse, and difficult. Immunohematol. 2005;21(4):155-163.

(4.) Denomme GA, Dake LR, Vilensky D, Ramyar L, et al. Rh discrepancies caused by variable reactivity of partial and weak D types with different serologic techniques. Transfusion. 2008;48(3):473-478.

(5.) Noizat-Pirenne F, Verdier M, Lejealle A, Mercadier A, Bonin P, et al. Weak D phenotypes and transfusion safety: where do we stand in daily practice? Transfusion. 2007;47(9):1616-1620.

(6.) Christiansen M, Samuelsen B, Christiansen L, Morbjerg T, et al. Correlation between serology and genetics of weak D types in Denmark. Transfusion. 2008;48(1):187-193.

RELATED ARTICLE: K2, Summit and Spice: Fake 'weed' is not so nice

By Laura James, MD; Jeffrey H. Moran, PhD; Cindy Moran, BS; and Keith R. McCain, PharmD, CSPI

The latestdrugs of abuse--marketed as herbal incense or aromatherapy blends under brand names like K2, Summit, and Spice--deliver a high reportedly similar to that induced by [DELTA]9-tetrahydrocannabinol, the psycho active cannabinoid found in marijuana. Early clinical reports, however, associate these synthetic cannabinoids with more extreme symptoms, such as agitation, paranoia, hallucinations, and seizures. There also have been several deaths associated with K2 product use.

Recreational drug users, including first-time drug users, seem to be attracted to these agents because they skirt existing regulations and avoid detection in common drug-screening programs. Yet, the potential for toxicity, addiction, and secondary effects, such as impaired driving, make these compounds a serious medical and public-health concern. Moreover, forensic data show that these herbal products are sometimes laced with numerous synthetic cannabinoids, all of which have varying degrees of toxicity, so users never know what they are getting.

Despite package labeling indicating "not for human consumption," these products are being ingested or smoked at increasing rates. In 2010, U.S. poison-control centers recorded 2,882 calls related to the use of K2-type products. As recognition increases, exposure cases are expected to rise; more than 700 calls to the poison-control center have been re corded in the first two months of 2011. (1)

To address this public-health threat, a translational-science research group formed by the Arkansas Department of Health-Public Health Laboratory (ADH-PHL) has developed an assay that detects and measures JWH-018, JWH-073--two of the more popular synthetic cannabinoids identified in products seized in Arkansas (and thought to be more potent than [DELTA]9-tetrahydrocannabinol)--and various oxidized metabolites of each. The validated assay has been in use for research purposes since the spring of 2010. It employs liquid chromatography tandem mass spectrometry and uses authentic standards. The assay provides high sample throughput and can be run in less than eight minutes with minimal sample preparation. The new assay has applicability for clinical, forensic, and public-health labs investigating a host of outstanding medical and epidemio logical questions. What are the clinical symptoms of JWH-018 and JWH-073 use and overdose? Is one more toxic than the other? Are certain patient groups more susceptible to toxicity? Are synthentic cannbinoids metabolized differently by different people? How does metabolism influence clinical symptoms and analytical detection?

The ADH-PHL workgroup--which includes the Arkansas State Crime Laboratory; University of Arkansas for Medical Sciences, Colleges of Medicine, Pharmacy and Public Health; Arkansas Children's Hospital; Centers for Disease Control and Prevention; U.S. Drug En forcement Agency, and several private entities--has collected several hundred human-urine specimens as part of an ongoing biomonitoring program aimed at determining use and prevalence in Arkansas. The workgroup also has begun to correlate clinical symptoms with analytical results and to use in vitro and in vivo mice models to study the pharmacology, toxicology, and metabolism of K2-type products. Knowledge gained through this translational workgroup is being used to develop educational and community-outreach programs aimed at reducing injuries and deaths related to these emerging drugs of abuse.

Laura James, MD, is section chief of Clinical Pharmacology and Toxicology at Arkansas Children's Hospital and professor of Pediatrics in the College of Medicine at the University of Arkansas for Medical Sciences. Jeffrey H. Moran, PhD, is branch chief of Environmental Chemistry at the Arkansas Department of Health, Public Health Laboratory, and a research instructor in the College of Medicine at the University of Arkansas for Medical Sciences, Department of Pharmacology and Toxicology. Cindy Moran, BS, is the quality assurance manager at the Arkansas State Crime Laboratory. Keith R. McCain, PharmD, CSPI, is an assistant professor in the College of Pharmacy at the University of Arkansas for Medical Sciences, Department of Pharmacy Practice, all located in Little Rock, AK.

This work was supported, in part, by the Association of Public Health Laboratories [Grant Innovations in Quality Public Health Laboratory Practice] (JHM), Centers for Disease Control and Prevention [Grant U90/CCU616974-07 and contract 200-2007-217291 (JHM). In addition, this work was supported by a pilot grant awarded to LPJ through the Arkansas Center for Clinical and Translational Research which is funded by the National Center for Research Resources [1 UL 1RR029884, Curtis Lowery, PI].


(1.) American Association of Poison Control Centers. E-mail correspondence. February 24, 2011.

MLO's "Tips from the Clinical Experts" provides practical, up-to-date solutions to readers' technical and clinical issues from a panel of experts in various fields. Readers may send questions to Brad S. Karon, MD, PhD, by e-mail at

Brad S. Karon, MD, PhD, is associate professor of laboratory medicine and pathology, and director of the Hospital Clinical Laboratories, point-of-care testing, and phlebotomy services at Mayo Clinic in Rochester, MN.

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Title Annotation:Tips from the clinical experts
Author:Karon, Brad S.
Publication:Medical Laboratory Observer
Article Type:Interview
Geographic Code:1USA
Date:Apr 1, 2011
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