Polycystic Ovary by Sonography
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Polycystic ovary syndrome (PCOS, also known clinically as Stein-Leventhal syndrome), is an endocrine disorder that affects 5–10% of women. It occurs amongst all races and nationalities, is the most common hormonal disorder among women of reproductive age, and is a leading cause of infertility. The symptoms and severity of the syndrome vary greatly between women. While the causes are unknown, insulin resistance (often secondary to obesity) is heavily correlated with PCOS. more...

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Other names for this disorder include:

  • Polycystic ovary disease (although this is not correct, as PCOS is characterised as a syndrome rather than a disease)
  • Functional ovarian hyperandrogenism
  • Hyperandrogenic chronic anovulation
  • Ovarian dysmetabolic syndrome


There are two definitions that are commonly used:

  1. In 1990 a consensus workshop sponsored by the NIH/NICHD suggested that a patient has PCOS if she has (1) signs of androgen excess (clinical or biochemical), (2) oligoovulation, and (3) other entities are excluded that would cause polycystic ovaries.
  2. In 2003 a consensus workshop sponsored by ESHRE/ASRM in Rotterdam indicated PCOS to be present if 2 out of 3 criteria are met: (1) oligoovulation and/or anovulation, (2) excess androgen activity, (3) polycystic ovaries (by gynecologic ultrasonography), and other causes of PCOS are excluded.

The Rotterdam definition is wider, including many more patients, notably patients without androgen excess, while in the NIH/NICHD definiton androgen excess is a prerequisite. Critics maintain that findings obtained from the study of patients with androgen excess cannot be necessarily extrapolated to patients without androgen excess.

Signs and symptoms

Common symptoms of PCOS include:

  • Oligomenorrhea, amenorrhea - irregular/few, or absent, menstrual periods; cycles that do occur may comprise heavy bleeding (check with a gynaecologist, since heavy bleeding is also an early warning sign of endometrial cancer, for which women with PCOS are at higher risk)
  • Infertility, generally resulting from chronic anovulation (lack of ovulation)
  • Elevated serum (blood) levels of androgens (male hormones), specifically testosterone, androstenedione, and dehydroepiandrosterone sulfate (DHEAS), causing hirsutism and occasionally masculinization
  • Central obesity - "apple-shaped" obesity centered around the lower half of the torso
  • Androgenic alopecia (male-pattern baldness)
  • Acne / oily skin / seborrhea
  • Acanthosis nigricans (dark patches of skin, tan to dark brown/black)
  • Acrochordons (skin tags) - tiny flaps of skin
  • Prolonged periods of PMS-like symptoms (bloating, mood swings, pelvic pain, backaches)
  • Sleep apnea

Signs are:

  • Multiple cysts on the ovaries. Sonographycally they may present as a "string of pearls".
  • Enlarged ovaries, generally 1.5 to 3 times larger than normal, resulting from multiple cysts
  • Thickened, smooth, pearl-white outer surface of ovary
  • Chronic pelvic pain, possibly due to pelvic crowding from enlarged ovaries; however, the actual cause is not yet known
  • The ratio of LH (Luteinizing hormone) to FSH (Follicle stimulating hormone) is 2:1 or more, particularly in the early phase of the menstrual cycle.
  • Increased levels of testosterone.
  • Decreased levels of sex hormone binding globulin.
  • Hyperinsulinemia.

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The laboratory's role in assisted reproduction
From Medical Laboratory Observer, 1/1/05 by Linda C. Rogers



To earn CEUs, see test on page 16.


1. Describe laboratory analyses for infertility diagnosis.

2. Define the steps involved in an assisted reproductive technology cycle.

3. Describe the use of laboratory assays in supporting ART.

4. Identify complications of ART.

The field of assisted reproduction is a relatively new medical specialty. Less than 50 years ago, in 1959, the first "test tube" animal (a rabbit) was born. Experimentation rapidly progressed from animals to humans. In vitro fertilization (IVF) was the first of the assisted reproductive techniques to be developed. The first successful IVF occurred in England with the birth of Louise Brown on July 28, 1978. IVF was successfully used for the first time in the United States in 1981. (1) Since then, procedures have been changed, improved, and optimized. More than 114,000 babies have been born in this country as a result of these techniques. (2)


According to the Centers for Disease Control and Prevention (CDC), assisted reproductive technologies (ART) are procedures that include all fertility treatments in which both egg and sperm are handled. In general, ART procedures involve:

* surgically removing oocytes;

* combining oocytes with sperm in the laboratory for fertilization;

* transferring embryos to the woman's uterus or donating them to another woman.

Fertilization may occur either in the laboratory by in vitro fertilization, or in the uterus (intrauterine insemination). This article will review the process of IVF and provide guidelines for clinical laboratories that provide testing and support for IVF clinics.

Infertility diagnosis

Infertility is a medical problem and is simply defined as the inability of a couple to conceive a child after one year of regular, unprotected sexual intercourse or the inability to carry a pregnancy to live birth. Infertility affects about 6.1 million people in the United States--about 10% of the reproductive-age population. Infertility can be related to the female, the male, both, or may be unexplained.

The laboratory plays a key role in the infertility diagnostic work-up of both males and females. The work-up of the male partner includes a semen analysis, identification of antisperm antibodies, and measurement of blood levels of hormones. A semen analysis can differentiate abnormalities of the number, morphology, or motility of sperm. Sperm autoimmunity can be determined by detecting antisperm antibodies. The presence of antisperm antibodies has been reported in approximately 10% of infertile men compared with less than 1% of fertile men. But successful fertilization can occur using micromanipulation techniques such as intracytoplasmic sperm injection. (3) This technique ensures fertilization by injecting one sperm directly into the egg. Hormones analyzed include testosterone, prolactin, luteinising hormone (LH), and follicle stimulating hormone (FSH). The interrelationship of these hormones is important for normal sperm production. Abnormalities in one or more of these hormones might help determine the location of the problem (testes, pituitary, or hypothalamus).


Evaluation of the female is more extensive and may include ovulation monitoring, a radiographic study of the uterus and fallopian tubes (hysterosalpinogram), a laparoscopy to directly view the reproductive organs, and endometrial biopsy. Initial laboratory analyses include thyroid-releasing hormone (TSH) and free thyroxine (free T4) to assess thyroid function, FSH, LH, prolactin to evaluate hypothalamic and pituitary function, and dihydroepiandrosterone sulfate (DHEA-S) and testosterone levels (total and free or bioavailable) to rule out hyperandrogenism. Additional hormone analyses are performed at specific times during the menstrual cycle. Day 3 FSH and estradiol (E2) levels are used to estimate ovarian reserve. LH and prolactin levels are evaluated during the follicular phase, and progesterone is measured at the midluteal stage to evaluate ovulatory function.

The ART process

The steps in an ART cycle include:

1. determination of ovarian reserve;

2. controlled ovarian hyperstimulation with exogenous gonadotropins;

3. induction of ovulation by human chorionic gonadotropin (hCG) administration (triggers final egg maturation);

4. harvesting of the oocytes (egg retrieval);

5. fertilization of the oocytes; and

6. transfer of the embryos.

Determination of ovarian reserve

Ovarian reserve is a measure of ovarian function. It has also been referred to as "reproductive potential." Each woman has a fixed number of oocytes at birth. Both the number of follicles and the function of the ovaries decline with age, and the level of FSH increases with decreasing ovarian function.

FSH is the most commonly used marker of ovarian reserve. The level of FSH on cycle day 3 has been shown to reflect the ovarian reserve and the quality of the residual oocytes. (4) Day 1 of a menstrual cycle is the day the menstrual period begins. Ovarian failure can occur in premenopausal women with regular menstrual cycles and affect their fertility prior to menopause. Elevated day 3 FSH levels may be an early warning of poor stimulation outcomes and a very low pregnancy rate and, therefore, used as a prognostic indicator for women considering ART. The level is predictive of ovarian response to stimulation protocols and the probability of pregnancy. Patients with low basal FSH levels have been shown to yield adequate numbers of oocytes following stimulation. In contrast, patients with high FSH levels may not be candidates for ovarian stimulation and may consider using donor eggs for an ART cycle, rather than go through an unsuccessful stimulation protocol. The day 3 FSH is valuable in patient selection for IVF and for counseling and managing expectations for couples in fertility programs.

Estradiol levels, measured on cycle day 3, may also be used to estimate ovarian reserve. (5) Other markers that have been studied for this purpose are inhibin B (6) and antimullerian hormone. (7) These analytes may become more widely used as automated methods of analysis are developed.

Controlled ovarian stimulation

Although the first IVF baby was born using a natural cycle, the pregnancy rate in couples undergoing ART can be greatly improved if multiple follicles are produced by hyperstimulation of the ovaries. This process results in multiple eggs for retrieval, fertilization, and transfer into the uterus. Therefore, the aim of controlled ovarian stimulation is to obtain multiple follicles from which good quality eggs can be retrieved. There are different protocols using drugs or combinations of drugs for this purpose. Ovarian stimulation needs to be individualized as the response to these drugs may differ.

Most protocols begin with pituitary suppression with a gonadotropin-releasing hormone agonist. Adequate suppression is ensured by measuring E2 levels. FSH and LH may also be assayed at this time. Pituitary suppression is followed with ovarian-stimulation drugs. Stimulated ovarian cycles are monitored closely and always involve E2 levels and ultrasound examination of the ovaries. In addition, cervical mucus examination and progesterone and LH measurements may be performed. The main objectives of monitoring E2 levels in treated cycles are to control follicular maturation, to detect poor ovarian response, to adjust medications accordingly, to time the administration of hCG for triggering of final egg maturation, to predict the outcome of the cycle, and to screen for ovarian hyperstimulation syndrome (OHSS).

Following ovarian stimulation, an initial rise in E2 level is used to determine ovarian response. Dose adjustments of the stimulation medications can be made based on the E2 level. As the protocol continues, E2 levels are measured periodically, along with transvaginal ultrasound, to determine the size, maturity, and number of developing follicles. Ultrasound is performed, and E2 levels are measured from the initiation of stimulation drugs (day 1) until ovulation induction, usually on days 3, 5, 7, 9, and 11. Each mature follicle produces approximately 200 pg/mL to 250 pg/mL, although this value may differ according to the assay used. The E2 level and ultrasound data are used together to time ovulation induction and the retrieval of the oocytes. This occurs when the "lead" follicle is 17 mm to 18 mm in size and the estradiol levels are consistent with maturity.

Complications of ovarian stimulation

One of the most serious complications of ART is ovarian hyperstimulation syndrome. OHSS is a severe medical condition that can result in cancellation of the cycle and/or hospitalization. E2 levels are utilized to monitor the patient for OHSS. OHSS ranges from mild ovarian enlargement to severe multisystem failure, with death occurring in approximately 1 in 45,000 to 50,000 infertile women. (8) The pathogenesis of OHSS is not completely understood. Risk factors for severe OHSS include young age, lean body build, high E2 levels, and a very large number of follicles. The basic features of OHSS are enlarged multicystic ovaries and increased systemic vascular permeability, with intravascular fluid loss into the third space. Mild to moderate OHSS is treated on an outpatient basis, while women with severe OHSS should be hospitalized. The goal of treatment for severe OHSS is to restore intravascular volume, to reverse the hemoconcentration that has occurred, and to maintain urine output. Surgical treatment for OHSS is indicated in the presence of ovarian torsion, ectopic pregnancy, or cyst rupture.

Prevention of the syndrome is a primary objective, and controlling hyperstimulation by individualizing ovulation-stimulation protocols and early cancellation of the cycle based on elevated E2 levels helps reduce the occurrence.

Ovulation induction

Ovulation is induced by administering an injection of purified, urine-derived hCG. Due to its similarity to LH, urine-derived hCG has been used to trigger ovulation and luteinization and to support the corpus luteum. An injection of recombinant hCG is used as a surrogate LH surge.

Most clinics draw a blood sample for the measurement of progesterone just prior to hCG injection to evaluate luteal status. Progesterone levels are a reflection of luteinization, and an elevated progesterone level may suggest a suboptimal uterine environment for successful implantation and pregnancy. Premature luteinization (PL) prior to egg retrieval, in vitro fertilization, and embryo transfer may be detrimental to establishing a viable pregnancy. Patients with an elevated progesterone level can have a greater chance of success achieving a pregnancy by utilizing cryopreservation and transferring the embryos to the uterus during a more optimal cycle.

Egg retrieval

Between 34 and 36 hours after hCG injection, the oocytes are harvested transvaginally through a needle guided by ultrasound. The procedure is performed while the patient is under sedation. Each follicle is punctured and the follicular fluid removed. The eggs are placed in nutritive media and incubated under strict conditions.


About 50,000 to 100,000 motile sperm collected from a fresh semen sample are added to each egg and incubated overnight. Eggs are checked for fertilization the next morning. When sperm quality is severely compromised or if male ejaculation is not possible, intracytoplasmic sperm injection may be performed to ensure a fertilized egg. In this procedure, a single sperm is obtained and injected directly into the egg. Following fertilization, embryonic development is closely monitored. By 48 hours, the embryos are at the four-cell stage.

Preimplantation Genetic Diagnosis: In IVF, genetic abnormality is the major reason for failure of an embryo to implant. Preimplantation genetic diagnosis (PGD) is a means of genetically analyzing a single cell from an eight-cell embryo to determine if it is normal and a candidate for uterine transfer. Specifically, parents who are carriers of severe genetic diseases like cystic fibrosis, muscular dystrophy, sickle cell anemia, Tay-Sachs disease, and Marfan's syndrome may be able to avoid having children with these heartbreaking genetic diseases. The most common reason for PGD is for infertile couples to improve the selection of embryos most likely to lead to a viable pregnancy.

Embryo transfer

Within 72 hours following fertilization, the embryos are transferred through a catheter into the uterus. Bed-rest for two days is recommended. The "window" for successful implantation is approximately three and a half days. Progesterone supplementation, via vaginal suppositories, injections, or micronized oral tablets, may be given. Progesterone levels are monitored periodically for dose-adjustment purposes.


Monitoring Pregnancy: The first blood sample to test for pregnancy (quantitative hCG measurement) is usually drawn 10 to 12 days following embryo transfer. If the results are positive (i.e., indicate a progressive pregnancy), progesterone and hCG levels are measured to monitor the early stages of pregnancy.

Guidelines for laboratories supporting ART

Many laboratory analyses are performed during an ART cycle. Tests are run frequently during each stage of the ART cycle (see table 2) and minimally include:

1. Prior to ART:

* initial infertility work-up: estradiol (E2), progesterone, FSH, LH, testosterone DHEA-S, prolactin, TSH, free T4; and

* circulating levels of FSH on day 3 of a normal cycle.

2. During an ART cycle:

* estradiol levels during ovarian stimulation;

* progesterone level on the day of hCG administration; and

* semen analysis.

3. Following an ART cycle:

* progesterone level; and

* hCG measurement.

Fertility clinics typically use a small but important menu of laboratory tests. Accuracy, reliability, reproducibility, and timeliness of test results are critical for successful outcomes.


In order to effectively support ART clinics, the laboratory must be able to provide a small but specific array of hormone assays. These include a basic panel for the assessment of infertility, critical assays utilized during the process of ART, and the follow-up of a successful ART cycle (e.g., pregnancy). A minimal menu includes the following assays: TSH, free T4, LH, FSH, estradiol, progesterone, prolactin, testosterone, DHEA-S, and hCG.

The three most critical assays utilized during ART are FSH, estradiol, and progesterone.

These tests are run at specific times during assisted reproduction, and critical decisions are made with the aid of these results. Accuracy, precision, reliability, and rapid turnaround time are essential considerations.

Turnaround time

Turnaround time is very important to fertility clinics. Many patients have blood drawn for analysis during an office visit. Treatment adjustments or other critical decisions are made based on test results. Typically, these patients cannot leave the office until the test analysis and decision making are complete. Thus, the time to first result is an important parameter to consider for a laboratory supporting ART.


Analysis range

The measuring range of an assay can significantly contribute to total turnaround time. An out-of-range result requires a technologist to stop and make a sample dilution followed by re-analysis, resulting in a doubling of the total analysis time. An analysis range that spans the majority of potential results can reduce the need for time-consuming dilutions.

Why do results differ from analyzer to analyzer?

Immunoassays differ from each other in many ways. These differences may be attributed to the material used for calibration, the antibodies used in the reagent system, and/or the method of detection of the end product. These differences are challenging to the physician using the results to make critical decisions pertaining to ART. Such critical decisions include the interpretation of prognostic factors, determining appropriate drug dosages, and timing of egg retrieval and embryo transfer.

Consequences of poor laboratory results

Poor laboratory performance can have a large impact on the success of IVF. Many steps can be affected. Unreliable FSH levels can result in erroneous prognostic indicators and choice of drug protocols. Estradiol measurements are critical for dose adjustments and timing of egg retrieval. The ultimate consequence can be no pregnancy with the accompanying emotional distress as well as financial loss. One cycle of IVF costs thousands of dollars and may or may not be covered by insurance.

Communicate with physicians

Physicians must be notified of any changes that the laboratory is considering before the new test results are reported. Let the fertility specialists know what changes to expect and when to expect them. Keep correlation data available if the physician requests more detail.

The following steps can help your physicians anticipate, understand, and prepare for the changes they may see in their test results:

1. Notify physicians of assay changes and the laboratory's implementation plan;

2. Provide concurrent testing with old and new platforms for a predetermined period of time;

3. Make available correlation data for the old and new methods; and

4. Distribute support materials:

a. correlation data to the old method;

b. testing guidelines for critical assays (FSH, estradiol, and progesterone); and

c. proof sources for the new methodologies, such as journal article reprints.

Struggle vs. success

The chance of a normal pregnancy in any given month for a healthy couple is 20% to 25%. According to the latest CDC statistics, the success rate for IVF is 29.4% deliveries per egg retrieval. (1) Table 3 shows a graphic representation of these results. This success rate requires state-of-the-art medical procedures and excellent and dedicated physicians. Complications may occur, particularly multiple births. The incidence of ectopic pregnancies is only slightly higher than in unassisted pregnancies (Table 4).

Infertile couples undergo physical, emotional, and financial stress and sacrifice. The clinical laboratory plays a critical role in medical specialty with timely, reliable, precise, and accurate results.


MLO and Northern Illinois University (NIU), DeKalb, IL, are co-sponsors in offering continuing education units (CEUs) for this issue's article on THE LABORATORY'S ROLE IN ASSISTED REPRODUCTION. CEUs or contact hours are granted by the College of Health and Human Sciences at NIU, which has been approved as a provider of continuing education programs in the clinical laboratory sciences by the ASCLS P.A.C.E.[R] program (Provider No. 0001) and by the American Medical Technologists Institute for Education (Provider No. 121019; Registry No. 0061). Approval as a provider of continuing education programs has been granted by the state of Florida (Provider No. JP0000496), and for licensed clinical laboratory scientists and personnel in the state of California (Provider No. 351). Continuing education credits awarded for successful completion of this test are acceptable for the ASCP Board of Registry Continuing Competence Recognition Program. After reading the article on page 12 answer the following test questions and send your completed test form to NIU along with the nominal fee of $20. Readers who pass the test successfully (scoring 70% or higher) will receive a certificate for 1.0 contact hour of P.A.C.E.[R] credit. Participants should allow four to six weeks for receipt of certificates.

The fee for each continuing education test will be $20.

All feature articles published in MLO are peer-reviewed.

The learning objectives and the CE test were prepared by Jeanne M. Isabel, MSEd, CLSpH(NCA), MT(ASCP), associate professor, School of Allied Health Professions, Northern Illinois University, DeKalb, IL.

1. One of the first assisted reproductive techniques developed was in vitro fertilization.

a. True.

b. False.

2. In vitro fertilization was used successfully for the first time in the United States in

a. 2003.

b. 2000.

c. 1996.

d. 1981.

3. Surgical removal of oocytes is not a procedure of assisted reproductive technology.

a. True.

b. False.

4. Infertility may result from both male and female causes.

a. True.

b. False.

5. Semen analysis can detect all of the following abnormalities except

a. sperm motility.

b. hormone level of FSH.

c. sperm morphology.

d. number of spermatozoa.

6. Male hormone production occurs in the

a. testes.

b. pituitary.

c. hypothalamus.

d. All of the above.

7. The female hormone level analyzed to rule out hyperandrogenism is

a. LH.

b. FSH.

c. DHEA-S.

d. TSH.

8. Estradiol levels in the female are used to evaluate

a. ovarian reserve.

b. ovulatory function.

c. thyroid function.

d. pituitary function.

9. The level of FSH decreases with decreasing ovarian function.

a. True.

b. False.

10. The hormone used as a prognostic indicator for women considering ART is

a. prolactin.

b. LH.

c. FSH.

d. testosterone.

11. In controlled ovarian stimulation, the triggering of final egg maturation occurs with

a. administration of hCG.

b. pituitary suppression.

c. cervical mucous examination.

d. administration of E2.

12. Retrieval of the oocytes occurs when the follicle is which size?

a. 5 mm to 10 mm.

b. 17 mm to 18 mm.

c. 24 mm to 25 mm.

d. 30 mm to 31 mm.

13. Enlarged multicystic ovaries with increased systemic vascular permeability is a symptom of ovarian hyperstimulation syndrome.

a. True.

b. False.

14. Evaluation of luteal status is achieved by measuring blood levels of

a. estradiol.

b. progesterone.

c. LH.

d. hCG.

15. Intracytoplasmic sperm injection is the method of fertilization for normal sperm.

a. True.

b. False.

16. Successful implantation of embryos into the uterus usually occurs in

a. three hours.

b. three days.

c. three weeks.

d. three months.

17. Laboratory analyses most used in the stage of ART following transfer of embryo are

a. hCG and progesterone.

b. hCG and estradiol.

c. estradiol and progesterone.

d. progesterone and FSH.

18. The three most critical assays utilized during assisted reproductive technology are

a. FSH, hCG, and estradiol.

b. FSH, estradiol, and TSH.

c. estradiol, LH, and progesterone.

d. estradiol, FSH, and progesterone.

19. Choosing an assay method that has a measuring range that spans potential results decreases turnaround time.

a. True.

b. False.

20. One of the complications of assisted reproductive technology is multiple births.

a. True.

b. False.


In the December 2004 Clinical Issues article "Responsible reporting in microbiology" by Colleen Gannon, credit for the information under "Nasopharyngeal (NP) cultures--Why do them?" was inadvertently omitted. The source of the material is a presentation by Ann Robinson, PhD, DABMM, entitled "Assessing Microbiology Test Orders and Specimen Quality: It's What's Up Front That Counts." Dr. Robinson presented this lecture at the annual American Society for Microbiology general meeting in May 2004. An article detailing the information included in this presentation will be published in the Clinical Microbiology newsletter in 2005.


1. U.S. Department of Health and Human Services. 2001 Assisted Reproduction Success Rates: National Summary and Fertility Clinic Reports. Available at: http://www.cdc.gov/reproductivehealth/ART01/PDF/ART2001.pdf. Accessed December 2, 2004.

2. Wood C, Trounson AD. Historical perspectives of IVF. In: Trounson AD, Gardner DK, eds. Handbook of In Vitro Fertilization, 2nd ed. Boca Raton, FL; CRC Press; 2000;1-14.

3. Clarke GN, Bourne H, Baker HWG. Intracytoplasmic sperm injection for treating infertility associated with sperm autoimmunity. Fertil Steril. 1997;68:112-117.

4. Sharara FI, Scott RT Jr, Seifer DB. The detection of diminished ovarian reserve in infertile women. Am J Obstet Gynecol. 1998;179:804-812.

5. Licciardi FL, Liu HC, Rosenwaks Z. Day 3 estradiol serum concentrations as prognosticators of ovarian stimulation response and pregnancy outcome in patients undergoing in vitro fertilization. Fertil Steril. 1995;64:991-994.

6. Bancsi LF, Broekmans FJ, Eijkemans MJ, de Jong FH, Habbema JD, te Velde ER. Predictors of poor ovarian response in in vitro fertilization: a prospective study comparing basal markers of ovarian reserve. Fertil Steril. 2002;77:328-336.

7. Fanchin R, Schonauer LM, Righini C, Guibourdenche J, Frydman R, Taieb J. Serum anti-Mullerian hormone is more strongly related to ovarian follicular status than serum inhibin B, estradiol, FSH and LH on day 3. Hum Reprod. 2003;18:323-327.

8. Brinsden PR, Wada I, Tan SL, Balen A, Jacobs HS. Diagnosis, prevention, and management of ovarian hyperstimulation syndrome. Br J Obstet Gynaecol. 1995;102:767-772.

By Linda C. Rogers, PhD, DABCC, FACB

Linda C. Rogers, PhD, DABCC, FACB, is manager of market development at Beckman Coulter Inc. in Fullerton, CA.

COPYRIGHT 2005 Nelson Publishing
COPYRIGHT 2005 Gale Group

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