mikro-grafOff ic ia l Publ icat ion of the Michigan S ociety of Histotechnolog i sts

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IHC Detection of Common Viral Infections in Placenta Amy S. Porter, HT (ASCP) QIHC

Patricia K. Senagore, M.D., Perinatal Pathologist

The placenta (plah-senʹtah) is an organ whose presence helps to define mammals; it develops in the uterus during pregnancy, connects the uterus to the fetus, and allows nutrient delivery, gas exchange, endo-

crine hormone production and waste removal to occur during gestation. It is normally expelled from the uterus following delivery of the infant and is thus readily available for pathologist examination.

Overview Of placental grOss examinatiOn

The placenta is comprised of three basic components: the disc, the extra-placental membranes that form the birth sac, and the umbilical cord. The placenta in its entirety may be weighed prior to removal of membrane sac and cord but by convention, for diagnostic evaluation, the cord and mem-branes are removed prior to weighing the disc. Measurements will record the umbilical cord length/diameter, number of cord blood vessels, and distance of cord insertion to the nearest margin. Also measured is the membrane length that represents the shortest distance between the membrane rupture site and the disc margin. Placental disc dimensions to be documented include its largest diameter, the perpendicular dimension through this diameter at the cord insertion site, and disc thickness. All examinations should describe and sample abnormalities of the cord and extra-placental membranes, as well as non-lesional tissue. The intact maternal (bloody, rough, and dark) and fetal (smooth and shiny) surfaces of the disc are also inspected for abnormalities prior to disc dissection.

Cross-sectional samples of the extra-placental membranes are taken from a “jelly roll” preparation that includes its entire length from site of rupture to disc margin, and of the umbilical cord taken at its proximal insertion into the disc and at a distal location.

Gross dissection of the disc involves making slices (“breadloaf”) 1–2cm apart, parallel to the smaller disc diameter and through its full thickness. Represen-tative tissue sections are taken that include both maternal and fetal surfaces. Such sections are most often taken to sample lesions but should also include central disc tissue that is grossly “normal” and without lesions. Research examinations may require additional sections of the disc from the margin and through the umbilical cord insertion site.

Whatever examination procedure is followed, it usually represents a routine protocol, such that information is gathered systematically and without omis-sions.

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this issue

President’s Message 2

Editor’s Note 2

Inbox 3

Test Your Knowledge 3

MSH News & Events 9

In the Spotlight 10

Spring Symposium 11

Signals: Dermatopathology 12

Around the Region 14

NSH Update 17

Officers And Chairpersons 19

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President’s Message

Make 2014 the year to get more involved in the MSH! Nominate yourself for

an MSH office; join us at the MSH board meetings; make a promise to attend

the Spring and Fall Seminars… in other words, invest in your professional

growth. Just do it!

Please feel free to contact me, or any member of the board, with suggestions or

concerns. We can only do our job of serving the membership if we know what

the membership wants!

Editor’s Note

Dee Wolfe, Editor

editor@mihisto.org

Snow, snow and more snow…the flakes keep falling…but Spring is around the corner and brings with it The MSH Annual Scientific Symposium. On April 11th and 12th we hope you will join us for education and fun at The Radisson Hotel of Lansing. This year is sure to be as successful as the last. The committed technologists involved with the MSH achieved many accolades at last year's National Society of Histotechnology Symposium Convention in Rhode Island. After that, our own society hosted a great Fall Seminar spotlighting the newly renovated histology facility at the Capitol Area Career Center in Lansing, whose hospitality program prepared our wonderful lunch and break foods.

The newly elected MSH officers will be announced at the Spring Symposium – keep your eyes open for election materials in this and coming issues of the Mikro-Graf, on the website, as well as email blasts. Make sure you participate – the society belongs to its members and your participation is paramount in its endeavors. Think about becoming involved, if not in an elected office, maybe with a small responsibility that you could share or be mentored in by an established board member. It is my sincere hope that will we have a new generation of techs that become active at the state level who continue to carry the mantle to the national level.

I have established an email specifically dedicated to all things MSH mihistoprez@mihisto.org; please consider this an open invitation to communicate with me at anytime.

Amy Porter, MSH President

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Inbox

we’d love to hear from you!Submit YOUR technical question for a response from one of our experts!

Comments, suggestions or technical tips are also welcome! Send to:

Mikro-Graf Editor8476 Pennfield RoadBattle Creek, MI 49017or email: editor@mihisto.org

Dear Mikro-Graf,

Thought your readers would enjoy hearing about the experiment we conducted today. We have been having problems with tissues received from a certain client. Troubleshooting led me to believe that we could have a fixation problem. But my questions regarding specimen collection were getting me nowhere! We don't normally see the tissues as the samples come down in cassettes ready to process. So, I asked for a representative grossed specimen– a jar filled with the amount of formalin normally used with a piece of tissue the size they would normally cut. We all gasped when the specimen was recieved! I couldn't resist sharing the photo (right).

I don't think this follows the rule 20-1 fixative to specimen ratio! Even worse was the fact that, since it is lung and wants to float, half of the tissue is not exposed to the formalin. This is the source of our cassetted tissue!

Our experiment consisted of taking multiple samples of lung, roughly the same size, and put ting them in various amounts of formalin–one of them being the recommended 20:1. We will gross them after 48 hours of fixation which is their normal process. I will be making this into a poster for them to use as a visual aid to reinforce our findings!

Trying all these different things has definitely increased our workload but it's been a fun learning process! Hopefully it will fix what ails this project and help others at the same time!

A Loyal Reader,

Dear Reader,

Thank you for an excellent reminder of the importance of PROPER fixation!

Q. Name the tissue.1.

Positive staining showing overamplification of this prog-2. nostic marker is required for Herceptin™ therapy.

Test Your Knowledge

[answers on page 3]

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[continued from page 1]

DevelOpment Of the placental Disc

The placental disc tissue is composed of highly vascular-ized fetal villous tissue that is bathed in maternal blood. A thin maternal decidua, developed from uterine endome-trium, forms the deep surface or “floor” of the disc (decidua basilis). Fetal membranes, carrying blood vessel branches of the umbilical cord that give rise to the villous placental tissue, form the “roof” surface of the disc (chorion fundo-sum).

Except for the amnion, the tissues of the placenta are derived from the outer cell layer of the blastocyst, a spheri-cal, hollow, cellular structure that forms several days after the ovum is fertilized, the result of a series of cell divisions. The embryo/fetus is derived from the inner cell mass of the blastocyst. Upon attachment of the blastocyst to the decidualized endometrium, the trophoblast shell further differentiates into an inner layer of proliferating cytotropho-blasts that move outward and fuse into a continuous outer mantle known as the syncytiotrophoblast. Within the syncy-tiotrophoblast mantle small blood lacunae develop that will interconnect and form the intervillous space of the maternal circulation, described below. The syncytiotrophoblast is involved in the production of many proteins, and exchange of nutrients and waste between mother and fetus.

The sustained proliferation of cytotrophoblasts into col-umns, continued formation of its covering mantle of syn-cytiotrophoblast and eventual ingrowth of mesenchymal cells that will become villous blood vessels, lead to the formation of the placenta’s tree-like villous architecture. Ultimately blood vessels in villi connect to blood vessels simultaneously forming in the umbilical cord and the fetus, thus establishing the fetal-placental circulation. The devel-opment and growth of the placenta continues throughout most of pregnancy as the fetus grows in size, guided by growth factors that allow branching and elongation of the villous blood vessels.

The maternal blood supply of the placenta is usually com-pleted by the end of the first trimester. Formation of the maternal- placental circulation is initiated when small uterine spiral arteries in the decidualized endometrium are invaded by specialized trophoblasts and then converted into coiled, large-diameter, and elongated vessels. Maternal arterial blood begins to flow through the remodeled spiral vessels into the intervillous space that surrounds the forming villi, eventually bathing them in the oxygen and nutrient-rich blood needed for growth of the fetus and its placenta. Deliv-ery of nutrients and removal of waste occurs as substances move across the vasculo-syncytial membrane of the villi, a thin tissue barrier that separates the maternal and fetal circulations. As nutrients are removed and exchanged for waste, oxygen-poor and nutrient-poor maternal blood is returned to the uterus through endometrial veins.

The early maternal spiral artery conversion process described above allows for a progressive increase in the

amount of maternal blood flowing to the placenta as the pregnancy advances. Incomplete conversion of these blood vessels is thought to underlie some abnormal condi-tions that present later in pregnancy (pre-eclampsia).

fetal circulatiOn

The three large blood vessels of the umbilical cord seem to divide into branches within the chorionic connective tissue membrane that cover the fetal surface of the placental disc and course deep into the most terminal portions of its vil-lous tissue. Paradoxically, because of their muscular struc-ture, two vessels are actually arteries that carry “venous” blood from the fetus to the placenta. The third is a vein that carries “arterial” blood from the placental villi to the fetus. To explain: capillaries in the most terminal portions of the villi are the site of nutrient and waste exchange. Villous capil-laries that carry oxygen and nutrient-rich blood TO the fetus (“arterial” blood) gradually join together to form increasingly larger branches, and finally come together to form one large thin-walled vein that extends the length of the umbili-cal cord to the umbilicus. The umbilical vein then delves deep into the abdomen to deliver its contents into the fetal liver for processing and eventual delivery TO all body tis-sues. Fetal capillaries that exist in all tissues and organs collect blood, from which all nutrients have been removed, (venous) and gradually join together to become increas-ingly larger vessels that empty into the fetal heart. Since the fetus does not “breathe” through its lungs in-utero, venous heart blood bypasses the lungs and is pumped out of the heart through muscular arteries, and eventually is carried through the two umbilical arteries and then the placental vessel branches to the terminal villi of the placenta for nutri-ent/waste exchange.

This arterio-capillary-venous system brings the fetal blood exceptionally close to maternal blood in the intervillous space that bathes the villi. No true intermingling of fetal and maternal blood occurs, as the systems are separated by the vasculaosyncytial membranes of the placental villi.

PLACENTAL CIRCULATION. REPRODUCTION FROM GRAY'S ANATOMY.

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placental functiOn

Nutrition, excretion, endocrine, and immune functions are provided by the placenta during pregnancy. Nutrients are delivered through active and passive transport to the fetus; active transport allows vastly different plasma concentra-tions of a variety of large molecules to be maintained on both sides of the placental barrier. Excretion carries waste products from the fetus into the maternal blood via diffu-sion across the placenta; wastes include urea, uric acid, and creatinine. Endocrine functions allow for the secretion of hormones that are necessary to maintain pregnancy, which can include hCG – (human Chorionic Gonadotro-phin,) hPL – (human Placental Lactogen,) estrogen and progesterone. As part of its immune function, maternal IgG molecules are allowed to pass across the placenta to the fetus, usually between week 20 and week 24 of gestation, providing antibody protection to the fetus while in-utero. This humoral immunity is identical to that present in the mother and can remain with the newborn infant for several months after birth. However, because the much larger IgM antibodies cannot cross the placenta, some mother-to-child infections can be transmitted during pregnancy, which may have detrimental effects on the fetus. Cytomegalovirus and Parvovirus B19 are two such pathogenic viruses that may be transmitted vertically from mother-to-child.

pathOgenic viruses

Cytomegalovirus or CMV is from the viral family Herpes-viridae or herpesviruses; with the name from the Greek being cyto–, “cell” and –megalo, “large”. The viral species that infects humans is commonly known as human CMV (HCMV) or human herpesvirus-5 (HHV-5). This species is the most commonly studied of all cytomegaloviruses and is the most common congenitally-acquired infection in infants. About 1% of pregnant women become infected with CMV and of these; about 40% transmit the infection to the fetus. Placental infection most often occurs during primary infection of the mother within the period of viremia, when the virus can pass from the maternal to fetal circulation across a destroyed trophoblast layer. The resulting placen-tal inflammatory response is a lympho-plasmacytic chronic villitis. CMV viral cytopathic effect (CPE) can be histologi-cally identified with Hematoxylin and Eosin staining, rep-resented by the classic morphology of large “owls–eye” nuclear inclusions plus cytoplasmic inclusions that add to the enlarged size of infected cells. However, inclusions are often so widely scattered that careful inspection of many sections is required for diagnosis.

Most likely to be infected and show inclusions are villous capillary endothelial cells, and stromal cells. Immunohis-tochemistry will confirm the infection in most instances. Because capillaries are destroyed by the virus, the finding of hemosiderin pigment plus plasma cells in villi is strongly suggestive of CMV infection and should prompt a search for the inclusions. The CMV virus can also be detected in maternal cervical secretions and breast milk.

Infected infants that are symptomatic at birth (0.1% of all infants) are more likely to be born to mothers without pre-existing immunity (specific antibodies) to CMV. Manifesta-tions include congenital defects, liver and spleen enlarge-ment, jaundice, anemia, low platelets, low birth weight, microcephaly, and inflammation of the retina and its vas-cular membrane. Most (90%) infants with in-utero infection are born without symptoms and are healthy, yet after birth long term follow-up has shown that 10-20% infants pres-ent with conditions that can vary from neurological dam-age, to learning difficulties (Sherris). Hearing loss due to sensory nerve damage is the most frequent and long term outcome. Excretion of CMV in urine and saliva is common and prolonged, up to 5 years, in children with the congenital infection and may represent a source of infection for other children and daycare workers.

Parvovirus B19 (from Latin word ”parvus” for small) is sometimes known as erythrovirus B19 (it replicates in the nucleus of erythroid cell precursors), erythema infec-tiousum, or “Fifth Disease” (it is the fifth disease known to commonly cause a viral skin rash in childhood). This was the first and only virus in the family of parvoviruses known to infect humans until 2005. The B19 virus most frequently affects children, however infection can also be contracted by adults; about 20% of infected individuals are asymp-tomatic (CDC). This virus is the cause of the classic rash in children known as “slapped cheek syndrome” accompa-nied by runny nose, fever, headache, and sometimes pain-ful swollen joints. It was discovered by chance in 1975 in Australia by virologist Yvonne Cossart, who named it for the infected sample’s position in “well B19” of a large number of micro-titer plates. Parvovirus is transmitted primarily by respiratory droplets from an infected individual.

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CYTOMEGALOVIRUS (CMV) INFECTION. THE DRAMATICALLY ENLARGED NUCLEI ARE CHARACTERISTIC OF CMV.

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The virus preferentially targets and infects the nuclei of erythroid (red blood cell) precursors, which leads to the destruction of these cells and severe anemia. By adult-hood, approximately 50% of the population is thought to be B19 immune due to a previous infection; re-infection is possible in a minority of cases.

Maternal parvovirus B19 infection that occurs prior to the 20th week of pregnancy may result in pregnancy loss in about 5% of cases; but losses become minimal in the second half of pregnancy (CDC). Prenatal antibody test-ing enables a pregnant mother to determine her immune status and risk of infection. In infected fetuses, intrauterine transfusion therapy to treat anemia has been successful, and cases have been reported in which recovery occurred without treatment.

Congenital Parvovirus B19 infection acquired by the fetus during pregnancy may lead to hydrops fetalis, a life-threatening form of body-wide tissue edema resulting from severe fetal anemia, which sometimes leads to miscarriage or stillbirth. The placenta also appears hydropic; it is large, heavy, pale, and edematous. Because the chronic hemoly-sis of infected erythroid precursors results in a compensa-tory increase in erythrocyte production (erythroblastosis), large numbers of nucleated red blood cell precursors are evident in fetal blood vessels of the placenta, especially villous capillaries

The hallmark histologic finding in cases of hydrops fetalis caused by Parvovirus B19 infection is viral inclusion bodies within erythroid precursor nuclei (“lantern cells”) that can be identified with routine Hematoxylin and Eosin staining and later confirmed by immunohistochemistry. The classic morphology is of a nucleus with a dark, thin, peripheral rim of chromatin surrounding a central, glassy (“ground-glass”), amphophilic cleared area, filled with viral particles. The cells are resistant to autolysis and may be detected in cases of stillbirth in which tissues are macerated; immuno-histochemistry can highlight infected cells even in autolysed tissue. There is no accompanying inflammatory response to

placenta parvovirus infection, specifically no chronic villitis.

Immunohistochemistry can be routinely performed to detect these two common viral inclusions utilizing the following protocols; please keep in mind that every laboratory is dif-ferent and all immunohistochemical protocols should be developed and validated at the laboratory performing the assay. Polymerase chain reaction (PCR) is a more sensi-tive detection method that can be performed on paraffin embedded tissue in cases of CMV and parvovirus infection (nested PCR).

Routinely fixed, processed and embedded samples are sectioned at 4 to 5 microns; placed onto adhesive or charged slides; once sectioned slides are dried in a slide drying oven with the temperature not to exceed 60°C to preserve antigenicity. Slides are handled in the following manner:

Deparaffinize slides in 2 changes of Xylene – 10 min-1. utes each

Absolute Ethanol – 2 changes of 2 minutes each2.

95% Ethanol – 2 changes of 2 minutes each3.

Distilled water – several changes4.

Tris Buffered Saline without surfactant – 5 minutes at 5. room temperature to adjust the pH for epitope retrieval with heat or enzymes if needed for immunohistochemi-cal staining.

Staining protocols are performed on an Autostainer. See Table 1 Staining Protocols, next page.

Slides are counterstained with Gill 2 Hematoxylin for 6. 1½ minutes, differentiated in 1% Glacial Acetic Water, then blued followed by dehydration through 95% etha-nol, absolute ethanol and cleared through xylene.

When using Fast Red Phosphatase chromogen, these 7. reactions must be coverslipped with Fisher Scientific™ Permount™ Mounting Medium–other mounting medias will remove the reaction and cause staining to break-down. This recommendation has been given by more than one vendor of chromagens in relation to the Fast Red compounds.

This protocol is reflective of what we perform in our labora-tory. The development of polymer technology has allowed many changes to occur with reduced incubation times and reagents. Working with archival samples and specimens that have been fixed in aldehyde fixatives for an extreme period of time sometimes requires staining with increased incubations. With this protocol, our laboratory has been able to detect Parvovirus B19 in archived wet tissue fixed for up to one year without the need for retrieval. Please refer any questions related to vendor information to me directly if you wish to have more information as we do not wish to promote any specific vendor in this article at por-tera@msu.edu.

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PARVOVIRUS INFECTION. PLACENTA. H&E. 60X

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Early Development of the Placenta. Pathology of the Human Placenta • 6th Ed, Benirschke K, Ed Burton G,J and Ed Baergen RN. Heidel-burg: Springer (2012) pages 41–45.

Parvovirus Anemia and Other Causes of Myocarditis. Pathology of • the Human Placenta 6th Ed, Benirschke K, Ed Burton G,J and Ed Baergen RN. Heidelburg: Springer (2012) pages 447–450.

Virus Infections and Villitides. Pathology of the Human Placenta 6th • Ed, Benirschke K, Ed Burton G,J and Ed Baergen RN. Heidelburg: Springer (2012) pages 599—603, 608–609.

Kindelberger DW, Sirois KF, and Boyd TK . Evaluation of the Pla-• centa. Diagnostic Gynecologic and Obstetric Pathology 2nd Ed. Christopher P. Crum, Marisa R Nucci, Kenneth R. Lee. Philadelphia: Saunders Elsevier (2011) p. 1046, 1069, 1072-1074, 1076.

Boyd TK, Parast MM, Tantbirojin P, and Saleemuddin A. Placental • Correlates of Unanticipated Fetal Death. Diagnostic Gynecologic and Obstetric Pathology 2nd Ed. Christopher P. Crum, Ed Marisa R Nucci, ED Kenneth R. Lee. Philadelphia: Saunders Elsevier (2011) pages 1097, 1099–1101, 1106–1107.

Parast MM and Boyd TK. Complications of Previable Pregnancy. • Diagnostic Gynecologic and Obstetric Pathology 2nd Edition Ed. Christopher P. Crum, Ed Marisa R Nucci, Ed Kenneth R. Lee. Phila-delphia: Saunders Elsevier (2011) pages 1011–1012.

Mumps Virus, Measles, Rubella, and Other Childhood Exanthems. • Sherris Medical Microbiology 5th Edition. Ed Ryan KJ and Ed. Ray CG, New York : McGraw Hill Medical, (2011) pages 189-190, 201-203

taBle 1. staining prOtOcOls

cytOmegalOvirus parvOvirus B19

0.04% Pepsin in 0.2N HCl – 20 minutes at 37°C No retrieval requiredRinse in running tap water – 5 minutesRinse in several changes of distilled waterPlace in Tris Buffered Saline + Tween 20 – 5 minutes Place in Tris Buffered Saline + Tween 20 – 5 minutesNormal Horse Serum – 30 minutes Normal Horse Serum – 30 minutesTris Buffered Saline + Tween 20 Tris Buffered Saline + Tween 20Avidin D – 15 minutes Avidin D – 15 minutesTris Buffered Saline + Tween 20 Tris Buffered Saline + Tween 20d-Biotin – 15 minutes d-Biotin – 15 minuteTris Buffered Saline + Tween 20 Tris Buffered Saline + Tween 20Monoclonal Mouse anti – Cytomegalovirus diluted 1:40 – 60 minute – Room temperature

Monoclonal Mouse anti – Parvovirus B19 diluted 1:40 – 60 minute – Room temperature

Tris Buffered Saline + Tween 20 Tris Buffered Saline + Tween 20Biotinylated Horse anti-Mouse IgG H+L dilutedto 11µg/ml – 40 minute – Room temperature

Biotinylated Horse anti-Mouse IgG H+L dilutedto 11µg/ml – 40 minute – Room temperature

Tris Buffered Saline + Tween 20 Tris Buffered Saline + Tween 20Alkaline Phosphatase Enzyme Reagent –60 minutes – Room temperature

Alkaline Phosphatase Enzyme Reagent – 60 minutes – Room Temperature

Tris Buffered Saline + Tween 20 Tris Buffered Saline + Tween 20Fast Red Phosphatase Chromogen – 8 to 15 minutes Fast Red Phosphatase Chromogen – 8 to 15 minutesTris Buffered Saline + Tween 20 Tris Buffered Saline + Tween 20Distilled Water – Rinse Distilled Water - Rinse

references

Earn 1.0 contact hours of continuing education by reading articles in the Michigan Society of Histotechnologists newsletter MIKRO-GRAF.

MSH contact hours can be used for CMP required by ASCP BOR to maintain certification.

For previous TechPoint articles/tests, go to the MSH website: http://www.mihisto.org Click on Education

It is the responsibility of the participant to retain their MSH CE certificates as proof of continuing education. DATE OF ARTICLE: Winter 2014 TITLE: IHC Detection of Common Viral Infections in Placenta AUTHOR:       Amy Porter, HT (ASCP) QIHC; Patricia K. Senagore, M.D., Perinatal Pathologist

DIRECTIONS: 1. Answer the following questions by circling the one (1) BEST answer for each question. 2. Complete the information required at the bottom of the page. 3. Submit questions & check made out to “MSH”(in US funds) to: Sarah Bajer 35669 Impala Dr, Sterling Heights, MI

48312 To earn Continuing Education credit from MSH, completed form must be submitted within

Three (3) years of original date of the article.

1. In which of the following organs does the placenta develop during pregnancy? A. Cervix B. Fallopian Tube C. Ovary D. Uterus

2. TRUE or FALSE (circle one): The vein located in the umbilical cord carries venous blood from the placental villi to the

fetus.

3. TRUE or FALSE (circle one): No true comingling of fetal and maternal blood occurs in the placenta.

4. All of the following are provided to the fetus by the placenta, EXCEPT: A. Genes B. Endocrine functions C. Excretion functions D. Nutrients

5. CMV viral cytopathic effect can be identified on a routine H&E as: A. Large clusters of infected cells B. Lantern Cells C. Owl Eye Inclusions D. Small Cells

6. Which of the following testing methods is most sensitive for detecting CMV and Parvovirus B19? A. Enzyme Histochemistry B. IHC C. PCR D. Routine H&E staining

PLEASE PRINT NEATLY DATE and YEAR Completed/Submitted Test: __________________________

NAME: _________________________________________________________________________________________

STREET: ________________________________________________________________ APT. _____________

CITY: _________________________________________ STATE: __________ ZIP: ____________________

PHONE: ____________________________________ Email: _____________________________________________

_____ Yes _____ No I am a Michigan Society of Histotechnologists (MSH) member _____ Yes _____ No I have included a check made out to “MSH” in US funds

**FEE: $5.00 for MSH members, $10.00 for non-MSH members _____ Yes _____ No I require a fee receipt for reimbursement from my employer

Certificate documenting 1.0 hours MSH continuing education (CE) will be mailed to the participant within 4 weeks.