Posted in ascpi review, Online Classes, The Phoenixx App©

#TheCheckPointOnline ~ Online Review for International Med Tech Exam

Plasma-chan

PLASMA CELL EDITION (PCE) –

A Self-Paced Online Review for ASCPi and other International Exams

  • There are no scheduled live classes (except when I invite special guest lecturers occasionally).
  • ALL VIDEO LECTURES AND QUIZZES ARE ALREADY UPLOADED ON THEPHOENIXXAPP AND MAY BE ACCESSED ANYTIME, ANYWHERE YOU WANT.

Our online review courses are powered by #ThePhoenixxApp© (TPA)

Create an account here and try TPA for free!

Description:

  • A comprehensive review of relevant topics in the international certification exams for Medical Technologists such as the ASCPi (may also be used for AIMS and CSMLS).

Validity of Subscription:

  • Your subscription will be VALID UNTIL COVID-19 PANDEMIC ENDS* OR UNTIL YOU TAKE YOUR INTENDED EXAM/S, WHICHEVER IS SOONER
  • *Once pandemic ends, 1-year validity will follow

Review Fee: P15,000

Inclusions:

  • Use of ThePhoenixxApp
    • Unlimited replays of video lectures
    • Download option for offline viewing of videos
    • Practice exams/quizzes with feedback, item analysis and progress tracker
    • More in-app features to help you be more productive
  • Printable and annotatable soft copy of personalized PCE Notes
  • Comprehensive exams and practice quizzes
  • Final Coaching Session with notes

IMPORTANT: APPLICATION ASSISTANCE NOT INCLUDED
(We may answer questions you may have but you will have to personally apply for the exam. For more information, please check these links.)


See ONLINE REVIEW testimonials here.

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STEPS FOR ENROLLMENT

  • REQUIREMENTS:
    • Proof of payment (screenshot of transaction or photo of deposit slip/receipt)
    • Decent photo to be used for digital ID (preferably graduation pictures)
  • Slots for enrollment are unlimited. We can always accomodate you and your friends.

1) When you join, YOU AGREE TO THE FOLLOWING TERMS & CONDITIONS.
a. No recording in any form (audio, video, screenshot) will be allowed. Programs are in place to monitor your in-app activity and once you’ve violated our agreement, we will be compelled to file charges against you.
b. Your personal information will be kept strictly confidential and will not be sold, reused, rented, loaned or otherwise disclosed.
c. Only fully paid students will be allowed to join. Please BE HONEST and DO NOT invite students to join without paying individually.

2) To proceed with enrollment, please pay first using the following modes of payment. Fees are NON-REFUNDABLE and NON-TRANSFERABLE.

BANK DEPOSIT
BANCO DE ORO (BDO)
ACCOUNT NAME: Krizza-Almond S. Aguilar
ACCOUNT NUMBER: 00 732 001 4669

BANK OF THE PHILIPPINE ISLANDS (BPI)
ACCOUNT NAME: Mark Anthony N. Salido (account of my husband)
ACCOUNT NUMBER: 2349 2711 52

SECURITY BANK (SB)
ACCOUNT NAME: Krizza-Almond S. Aguilar
ACCOUNT NUMBER: 00000 17563681

GCASH (See QR code below)
If GCASH to GCASH or GCASH to bank, no extra fee
If kiosk (i.e., 7-11, palawan) to gcash, please add the required service fee to the amount to be transferred
GCash via PALAWAN: add 2% service fee
GCash via 7-11: add 3% service fee


PAYMAYA (See QR code below)

PLEASE DO NOT SEND PAYMENT VIA PALAWAN PERA PADALA.
HUWAG MAGPADALA SA PALAWAN EXPRESS PERA PADALA.
MODES OF PAYMENT

CLICK THIS FOR THE PCE ENROLLMENT FORM

3) Register using this PCE form. Please make sure you have the proof of payment ready as well as a decent photo which we will be using for your digital ID.

4) Wait for confirmation via text or email along with the instructions. Sir Axel will assist you. Kindly wait for his message.

5) Review notes (watermarked with your names) are included in the fee and will be sent in digital/soft copy via email/messenger. Please DO NOT SHARE them to anyone WITHOUT PERMISSION.

If you still have any questions or clarifications, please contact me.
Thank you very much and looking forward to meeting you online. 😊

Thank you very much and looking forward to meeting you online. 😊

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ALSO AVAILABLE! Get 50% discount if you enroll in both MCE and PCE courses.
Posted in Checkpoint Tutorials

#CheckPoint Tutorials for Medical Technologists

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MUST KNOWS (or not? XD)

FIRST, if you are too lazy to read everything here, then my tutorial is not for you. I’m sorry.

SECOND, if you are a fresh graduate/first timer, I suggest that you try in a review center first. But if you insist to join us, you are more than welcome.

THIRD, I do not accept everyone. I only want to do this for those who are fully willing to help themselves too. If you are looking for spoon feeding type of learning then I’m not the one who can help you.

FOURTH, please be aware that this is NOT the usual tutorial set-up nor the usual review center set-up you know…As in everything is NOT the usual.

FIFTH, please bear in mind that I am not forcing you to join. If you find it in your heart that you really want to do your review with us, then we are waiting for you. But if you don’t like any of the above rules, then thank you for your time.

There are a lot to explain (and some are really not explainable by words) but here’s a runthrough of #CheckPoint tutorials.

  • CheckPoint Tutorials for March 2019 will start on November 2019. Actual dates will be announced when PRC releases the final board exam dates.
  • We are currently having the tutorials in 2 areas. 
    • CheckPoint Manila version 10.0
    • CheckPoint Davao version 3.0
  • We don’t meet everyday.

Our schedule is not fixed since we (the tutors) are also working and so we will be developing a schedule that fits everybody.

  • Venues
    • CheckPoint Manila: OUR HOME at SAN MIGUEL (or PALATIW), PASIG CITY
      • We have a room for tutorials just in front of our home. I am very sorry if I cannot grant your request to do the tutorial anywhere other than this venue.
    • CheckPoint Davao: No permanent venue as of yet, we usually do tutorials in different coffee shops around Davao. 🙂
  • I do the teaching but I also get help from my colleagues.
    • CheckPoint Manila: I do most of the teaching but there’ll be subjects to be handled by Sir Ross Axel Rufino (Unciano Colleges).
    • CheckPoint Davao: Most subjects will be handled by Sir Jayson Sanchez (Davao Doctors College) while I teach certain subject/s whenever I have a schedule in Davao.

We have a tradition not to move to another topic until everyone in the group understands. And yes, you can tell us everything you want to understand and we will try our best to explain but we don’t guarantee being all-knowing. If we don’t know the answer to your question, then we are willing to study again for you. 

  • If you want an easy life and would just want to rely on recall questions, then you’re in the wrong place. 

You will pass because you understood the topics, not because you remember the recalls. But if you believe otherwise, then I can’t help you. That’s not how I roll.

  • We will also have a lot of practice exams with explanations why this and not that is the answer moments.
  • There will be unpleasant moments (typhoon, super traffic, dysmenorrhea days) to teach personally so that we’ll just have an online review.
  • I will require at least 50% downpayment for our expenses.
    • FEE: Manila – P11,000 & Davao – P12,000 (already inclusive of P1,860 worth of personalized #CheckPoint Notes – all available subjects)

I am not rich, I cannot support our growing #CheckPoint family with just my usual budget so, please pay. Haha. Usually, I use the money for our refreshments (food and coffeeeeee) and enjoyment (like ice skating, karaoke, arcades, haha, no kidding).

  • I DO NOT ACCEPT QUITTERS.

If you are joining us, make sure that you are not a quitter or if you really are, be prepared to change. To tell you the truth, I’m not willing to do this for someone who will QUIT on me, so, let’s have a contract as early as now.

  • I do this because I live to teach.

This has been my passion ever since and has been a part of my promise to my God (long story, you’ll know on the way). I choose to teach MedTech students who are willing to sacrifice, change for the better, believe in themselves and fight for their dreams. Even if it is hard, we will not quit.

SO, are you ready to be like us? ^^,

If yes, then SEND ME A MESSAGE, like right now. 🙂

P.S.
For those who’d like to know the in-between-experiences of the previous #CheckPointees, you are free to contact them.

CheckPoint v1.0

Ross Axel Rufino, RMT

Erwin Pagkatipunan, RMT

Kayla Lara, RMT

Josel Losada, RMT

Fatima Yao, RMT

CheckPoint v2.0

Hillary May Cuizon, RMT

Ahrjay Atienza, RMT

RF dela Cruz, RMT

Davicca Hwang, RMT

Chrismari Sison, RMT

CheckPoint v3.0

Wilfred Lawas, RMT

Mark Salvador Mabesa, RMT

Princess Alyssa Suerte, RMT

CheckPoint v4.0

Mia Cortez, RMT

Shirley Mae Magadang, RMT

Maria Corazon de Villa-Obnamia, RMT

John Gabriel Pagsanghan, RMT

CheckPoint v5.0

Patricia Inocencio, RMT

Sharmane May Almazan, RMT

Dette Nuñez, RMT

CheckPoint v6.0

Dexter de Vera, RMT

Dafy Macalalad, RMT

Aianna Valenton, RMT

CheckPoint v7.0

Michelle Reyes, RMT

Julia Aira Marie Sanchez, RMT

CheckPoint Manila v8.0

Alicia Abril, RMT

Lara Mae Ganaden, RMT

Jam Shane Supan, RMT

Georgina Belo, RMT

CheckPoint Manila v9.0

Gene Tulawie, RMT

Theodor Carandang, RMT

Almira Felias, RMT

CheckPoint Davao v1.0

Francis Jade Labatos, RMT

Geric Sam Lopez, RMT

Rey Marcelo Caupayan, RMT

Elspeth Katherine Luig, RMT

Denden Espero, RMT

CheckPoint Davao v2.0

Jamaica Cadorna, RMT

Shedney Mae Kawaling, RMT

Fauziyah Pagayao, RMT

Joanna Marie Guiraldo, RMT

Caryl Anne Abeto, RMT

GJ Gaetos, RMT

TO GOD BE ALL THE GLORY!

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Tara, aral? 🙂 Or manga na lang. Hehe!

Posted in Checkpoint Tutorials, Online Classes

#TheCheckPointOnline ~ Med Tech Online Review

  1. Memory Cell Edition (MCE) — The Comprehensive Online Review Course
  2. End Cell Edition (ECE) — The Final Coaching Course

SEE REVIEW TESTIMONIALS HERE


Our online review courses are powered by #ThePhoenixxApp© (TPA)

Create an account here and try TPA for free!


MEMORY CELL EDITION (MCE)

Description:

Highlights and Inclusions:

  • REVIEW STYLE OPTIONS:
    • Scheduled review
      • If you like interacting with the lecturer, review assistants, and your online study buddies, join our scheduled review.
      • Live Sessions: 9am to 4pm
      • See our schedule below
    • Self-paced review
      • If you are working, or still an undergraduate, or simply prefer to study by your own, you can do self-paced review.
    • Hybrid review (combined scheduled and self-paced)
      • If you wish to do both, you can. Just maintain your communication with us so we can help you in any way we can.
  • VALIDITY OF ENROLLMENT:
    • Your enrolled subject/s will be VALID UNTIL COVID-19 PANDEMIC ENDS* OR UNTIL YOU TAKE THE BOARD EXAM, WHICHEVER IS SOONER
    • *Once pandemic ends, 1-year validity will follow
    • Example scenario: If you enroll today (March 2022) but you plan to take the board exam on March 2023, you can join all the batches of the review with no additional cost. (This offer is valid only during the COVID-19 pandemic.)
  • ENROLLMENT OPTIONS:
    • Per subject
      • Choose this option if you just want to enroll in selected subject/s only or if you do not have the budget yet for a full course review.
    • Full course
      • Choose this option if you wish to access all subjects right away.
      • Get a FREE END CELL EDITION (ECE) COURSE – THE ONLINE FINAL COACHING when you enroll in all subjects (whether in full course or per subject basis)
  • INCLUSIONS:
    • Use of ThePhoenixxApp
      • Unlimited replays of video lectures
      • Download option for offline viewing of videos
      • Practice exams/quizzes with feedback, item analysis and progress tracker
      • More in-app features to help you be more productive
    • Printable and annotatable soft copy of personalized MCE Notes and other review materials
    • Every subject (except MolBio*) will have the following academic activities (to be explained in detail during orientation)
      • MCE Lectures
      • Pre and Post-lecture Exams with rationalization
      • Evaluation Exams with rationalization
      • Mastery Sessions
      • Drills
      • Special lectures for selected subjects
      • *MolBio will only include lecture and pre/post-lecture exams
    • GENERAL ACTIVITIES
      • Co-MEMORY-ate
      • Mock Board Examination
      • Brainstorming sessions or virtual group study sessions (Study-With-Me via Discord)
      • Small group tutorial (by request)
    • EXTRA-CURRICULAR ACTIVITIES
      • Worship service/Bible studies
      • Mental health hygiene
      • Support group
      • Chillout sessions
      • ZOOMba sessions
      • Gaming sessions

Schedule and Review Fees for AUGUST 2022 MCE:

  • Lecture sessions: 9 am to 4pm
  • Pre/Post exams: Exam-taking is from 12:01 am to 5:59 pm, Ratio is at 6pm to 9pm
  • Evals: Exam-taking is from 9am to 11am, Ratio is at 2pm to 5pm
  • Dates include INDEPENDENT STUDY TIME (ISTs) which means FREE DAY
  • NO SUNDAY CLASSES
  • You may actually enroll now and use the currently available review materials and then join again when the new batch intended for August 2022 starts in May without paying another fee.
  • UNDERGRADUATES ARE WELCOME TO ENROLL.
ACTIVITYTARGET DATESREVIEW FEE
REFRESHMENT WEEKTO BE ANNOUNCED FREE FOR ALL MEMORY CELLS AND END CELLS
ORIENTATIONTO BE ANNOUNCED FREE FOR ALL MEMORY CELLS AND END CELLS
MOLECULAR BIOLOGY*TO BE ANNOUNCEDP1500
CCTO BE ANNOUNCEDP3000
BMVP (Bacte, MycoViroPara)TO BE ANNOUNCEDP3000
CMTO BE ANNOUNCEDP1500
HEMATO BE ANNOUNCEDP3000
ISBBTO BE ANNOUNCEDP3000
HTMLETO BE ANNOUNCEDP1500
DrillsTO BE ANNOUNCEDFREE FOR EVERY SUBJECT ENROLLED
MOCK BOARDSTO BE ANNOUNCEDFREE FOR ALL MEMORY CELLS AND END CELLS
Co-MEMORY-AteTO BE ANNOUNCED FREE FOR ALL MEMORY CELLS AND END CELLS
TOTAL FEEAll schedules inclusive of lecture days, ISTs, exams, ratio, and mastery sessions except MolBio

*Only includes lectures, notes and pre & post-lecture exams
P16,500
TARGET SCHEDULE OF ACTIVITIES FOR MARCH 2023 MTLE REVIEW

Watch this for a more detailed explanation of the MCE review course.

STEPS FOR ENROLLMENT

  • REQUIREMENTS:
    • Proof of payment (screenshot of transaction or photo of deposit slip/receipt)
    • Decent photo to be used for digital ID (preferably graduation pictures)
  • Slots for enrollment are unlimited. We can always accommodate you and your friends.
  • To get the materials ahead of schedule, please enroll at least 3 days before the subject starts (see schedule here)
    • If you were not able to enroll on time, you may still register any time and use ThePhoenixxApp to replay the video lectures

1) When you join, YOU AGREE TO THE FOLLOWING TERMS & CONDITIONS.
a. No recording in any form (audio, video, screenshot) will be allowed. Programs are in place to monitor your in-app activity and once you’ve violated our agreement, we will be compelled to file charges against you.
b. Your personal information will be kept strictly confidential and will not be sold, reused, rented, loaned or otherwise disclosed.
c. Only fully paid students will be allowed to join. Please BE HONEST and DO NOT invite students to join without paying individually.

2) To proceed with enrollment, please pay first using the following modes of payment. Fees are NON-REFUNDABLE and NON-TRANSFERABLE.

You can pay on a full or per subject basis. (SEE THIS FOR FAQs ABOUT PAYMENT)
To be able to get the materials on time, please enroll at least 3 days before the start of the subject/s.

If the subject/s you are enrolling in is/are already finished, you can still enroll any time as you wish and you will just be replaying the lectures in the app.

BANK DEPOSIT
BANCO DE ORO (BDO)
ACCOUNT NAME: Krizza-Almond S. Aguilar
ACCOUNT NUMBER: 00 732 001 4669

BANK OF THE PHILIPPINE ISLANDS (BPI)
ACCOUNT NAME: Mark Anthony N. Salido (account of my husband)
ACCOUNT NUMBER: 2349 2711 52

SECURITY BANK (SB)
ACCOUNT NAME: Krizza-Almond S. Aguilar
ACCOUNT NUMBER: 00000 17563681

GCASH (See QR code below)
If GCASH to GCASH or GCASH to bank, no extra fee
If kiosk (i.e., 7-11, palawan) to gcash, please add the required service fee to the amount to be transferred
GCash via PALAWAN: add 2% service fee
GCash via 7-11: add 3% service fee


PAYMAYA (See QR code below)

PLEASE DO NOT SEND PAYMENT VIA PALAWAN PERA PADALA.
HUWAG MAGPADALA SA PALAWAN EXPRESS PERA PADALA.
MODES OF PAYMENT

CLICK THIS FOR THE MCE ENROLLMENT FORM

3) Register using this MCE form. Please make sure you have the proof of payment ready as well as a decent photo which we will be using for your digital ID.

4) Wait for confirmation via text or email along with the instructions. Sir Axel will assist you. Kindly wait for his message.

Enroll in all subjects and automatically get a free online final coaching.

WHO CONDUCTS THE LECTURES?

Dra. Krizza-Almond S. Aguilar-Salido will handle most of the subjects but there will be invited special lecturers for a few subjects.

For a list of special lecturers, please click this.


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Posted in Lecture Notes

Med Tech Mycology Notes

FUNGI
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CHARACTERISTICS:

  • Eukaryotic
  • Thallophytes
    • have true nuclei
    • heterotrophic members of the plant family that lack stems and roots
  • Lack chlorophyll
  • Larger and with more complex morphology than the bacteria
  • Chitin in the cell wall
  • Ergosterol in the cell membrane
  • Saprophytic nature (derive nutrition from organic materials)
  • Lack of susceptibility to antibacterial antibiotics

TWO PHASES:

  • Multicellular MOLD – fluffy, cottony, woolly, or powdery mycelial mass, grows at 25°C
  • Unicellular YEAST – moist, creamy, opaque or pasty, resembling bacterial colony, grows from 35°C to 37°C
DIMORPHIC FUNGI – capable of two phases
Mold at 25°C to 30°C – INFECTIVE TO MAN
Yeast at 37°C – TISSUE/IN VIVO/INVASIVE

PARTS:

  • MYCELIUM – intertwining structure composed of tubular filaments known as HYPHAE
    • Vegetative portion or thallus – grows in or on a substrate and absorbs water and nutrients
    • Reproductive or aerial part – contains fruiting bodies that produce reproductive structures (conidia or spores); extends above the agar surface
  • HYPHAE – microscopic unit of fungi
    • Septate – contain cross-walls
      • All fungi except Zygomycetes
    • Aseptate/Coenocytic – continuous, without cross-walls
      • ZYGOMYCETES (Rhizopus, Mucor)
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FUNGAL REPRODUCTION:

  • SEXUAL – meiosis (reduction division of two fertile cells) followed by merging of the cells and nuclear fusion occurs
    • PERFECT FUNGI – fungi that exhibit sexual phase
    • ASCOSPORES – contained in a saclike ASCUS
      • CLEISTOTHECIUM – large, round, multicellular structure that surrounds the asci until it ruptures, releasing ascospores
    • BASIDIOSPORES – contained in a club-shaped BASIDIUM
    • OOSPORES – fusion of cells from two separate, nonidentical hyphae
    • ZYGOSPORES – fusion of two identical cells arising from the same hypha
  • ASEXUAL – involves only mitosis with nuclear and cytoplasmic division
    • IMPERFECT FUNGI – do not exhibit sexual phase
    • SPORANGIOSPORES – asexual spores contained in sporangia (sacs) and produced terminally on sporangiophores or aseptate hyphae
      • UNIQUE TO THE ZYGOMYCETES
    • CONIDIA – asexual spores produced either singly or multiply in long chains or clusters by specialized vegetative hyphae (conidiophores)
      • MACROCONIDIA – large, usually septate
        • Club, oval, or spindle shaped
        • Thick or thin walled
        • Spiny (echinulate) or smooth surface
      • MICROCONIDIA – small, unicellular
        • Round, elliptical, or pyriform (pear) shape
    • BLASTOCONIDIA or BLASTOSPORES
      • Develop as daughter cell buds off the mother cell and is pinched off
      • Blastoconidia of yeasts (including Candida) may elongate to form pseudohyphae
    • CHLAMYDOCONIDIA or CHLAMYDOSPORES
      • Thick-walled, resistant, resting spores produced by “rounding up” and enlargement of the terminal hyphal cells
      • Germinate into a new organism when favorable environmental conditions exist
        • Terminal – form at the hyphal tip
        • Sessile – form on the hyphal sides
        • Intercalary – form within the hyphal strand
    • ARTHROCONIDIA or ARTHROSPORES
      • Simple fragmentation of the mycelium at the septum into rectangular-, cylinder-, or cask-shaped spores
      • Thick walled spores which may be adjacent or alternate (empty spaces or disjunctor cells in between each arthrospores) in arrangement
      • Useful identification characteristic of Coccidioides immitis and Geotrichum candidum
CLASSIFICATION

Botanical Taxonomy

  • Zygomycota –Mucor, Absidia, Rhizopus
  • Ascomycota
  • Basidomycota
  • Deuteromycota – most medically important fungi
    • With septate hyphae
    • Asexual reproduction

Type of Mycoses

  • Superficial and cutaneous mycoses
  • Subcutaneous mycoses
  • Systemic mycoses
  • Opportunistic mycoses
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IDENTIFICATION METHODS

MICROSCOPIC

  • SALINE MOUNT – quick and simple method to observe fungal elements (budding yeast, hyphae, pseudohyphae)
    • Major disadvantage: Lack of contrast
  • 10% KOH PREPARATION – rapid and simple method to examine hyphae, budding yeast and spherules
    • KOH dissolves keratin in skin, hair, or nail
    • Chitin is resistant to effects of KOH
    • Hair can be examined to determine type of infection
      • ENDOTHRIX – fungal invasion within the hair shaft
      • ECTOTHRIX – infection outside the hair shaft
    • Disadvantage: Lack of contrast
  • CELLUFLUOR – chemofluorescent brightening agent
    • Can be added to the KOH solution
    • Binds to the chitin in fungal cell wall
    • Provides excellent contrast when examined with a fluorescent microscope
    • Fungi fluoresce intense apple green
  • INDIA INK/NIGROSIN – used to identify hyaline capsule of the yeast Cryptococcus neoformans
    • Capsules do not stain with India ink and appear as clear halos against a dark background
    • May be difficult to interpret; WBCs and artifacts can be mistaken for yeast or capsules
    • Cryptococcus may be capsule negative in immunodeficiency
    • Replaced by direct antigen testing for the cryptococcal capsular protein
  • LACTOPHENOL COTTON BLUE (LPCB) (AMAN) – imparts blue color to the fungal cell wall
    • Slides can be permanently sealed for later study with either Permount or clear nail polish
    • Can also be used in the tease preparation (wet mount) and slide cultures
  • HUCKER MODIFICATION OF GRAM STAIN – recommended for mycology
    • Fungi generally stain gram positive
    • Oval or budding yeast, hyphae, arthrospores generally stain well
    • C. neoformans may appear pale lavender with blue inclusions (capsule prevents adequate staining)
    • Fungi are 2 to 3x the size of gram-positive cocci
    • Hyphae are 2 to 3x wider than gram-positive bacilli
  • GIEMSA or WRIGHT’S STAIN – used for the detection of intracellular Histoplasma capsulatum in blood smears, lymph nodes, lung, liver, or bone marrow
    • H. capsulatum appears as small, oval yeast cell staining light to dark blue
    • C. neoformans also stain well
  • METHENAMINE SLIVER NITRATE STAIN – useful for screening of clinical specimens
    • Provides good contrast and staining for fungal elements
    • Fungi appear outlined in black, with an inner dark rose to black color against a pale green background
    • Viable and nonviable fungi are stained using this method
    • GOMORI METHENAMINE SILVER (GMS) NITRATE MODIFICATION – used in histology to detect fungi in specimens
  • PERIODIC ACID SCHIFF (PAS) – stains hyphae of molds and yeast
    • Periodic acid oxidizes the hydroxyl in the carbohydrates of the cell walls to form aldehydes which react with basic fuchsin dye to form a pink-purple complex
    • Counterstain of fast green can be used to provide contrast
    • Useful in staining tissue in histology

CULTURE

  • Must include a source of nitrogen (nitrite, nitrate, amino acids, or urea) and a carbon source (usually glucose)
  • Incubated at 30°C or RT (25-30°C)
  • Chloramphenicol – inhibits bacteria
  • Cycloheximide – inhibits saprophytic, contaminating fungi

PRIMARY ISOLATION MEDIA

SABOURAUD DEXTROSE AGAR (SDA)
Main general isolation medium Primary recovery of saprobic and pathogenic fungi Primary agar for initial culture Contains peptone and glucose Inhibitor for bacteria: ACIDIC pH (5.6)
SDA WITH CYCLOHEXIMIDE AND CHLORAMPHENICOL (SDA-CC) Recovery of pathogenic fungi Bacteria and saprophytic fungi inhibited Available commercially as Mycosel or Mycobiotic medium
MYCOSEL OR MYCOBIOTIC AGAR
Isolation of dermatophytes from hair, skin, and nail specimens Contains the inhibitory agents, cycloheximide and chloramphenicol Similar to DTM
DERMATOPHYTE TEST MEDIUM (DTM)
Can be substituted for SDA-CC for the recovery of dermatophytes from specimens contaminated with fungi or bacteria Isolation of dermatophytes from hair, skin, and nail specimens Dermatophytes produce alkaline metabolites, which raise the pH and change the color of the indicator from yellow to red Indicator: PHENOL RED Antibiotics inhibit saprophytic fungi and bacteria
BRAIN-HEART INFUSION (BHI) AGAR
Isolation of saprophytic and pathogenic fungi from sterile sites Bacteria also grown in BHI Can be supplemented with blood
BHI AGAR WITH ANTIBIOTICS, CYCLOHEXIMIDE AND CHLORAMPHENICOL
Isolation of pathogenic fungi exclusive of dermatophytes; useful for specimens that may be contaminated with bacteria or saprophytic fungi
BHI BIPHASIC BLOOD CULTURE BOTTLES
Recovery of fungi from blood or bone marrow
 

DIFFERENTIAL MEDIA

BIRDSEED (NIGER SEED) AGAR/ STAIB’S MEDIUM
Isolation of Cryptococcus neoformans: brown to black colonies in 4 to 7 days C. neoformans produces phenol oxidase which breaks down the medium resulting in the production of melanin Similar to caffeic acid agar
CORNMEAL AGAR WITH TWEEN 80
Stimulation of conidiation and chlamydospore production in Candida species; useful for species differentiation of Candida Cornmeal agar + 1% glucose: differentiates T. rubrum from T. mentagrophytes based on PIGMENTATION
COTTONSEED AGAR
Conversion of mold phase of Blastomyces dermatitidis to its yeast phase
NITRATE REDUCTION MEDIUM
Confirmation of nitrate reduction in C. neoformans
POTATO DEXTROSE AGAR
Stimulation of conidia production in fungi Useful in slide culture Also demonstrates pigment production of Trichophyton rubrum
RICE MEDIUM Identification of Microsporum audouinii
TRICHOPHYTON AGARS
Nutritional requirement tests for the differentiation of
Trichophyton #1: casein agar base (vitamin free)
#2: casein agar base and inositol
#3: casein agar base, inositol, and thiamine
#4: casein agar base and thiamine
#5: casein agar base and nicotinic acid
#6: ammonium nitrate agar base
#7: ammonium nitrate agar base and histidine
UREA AGAR
Detection of urease production by C. neoformans and differentiation of Trichophyton mentagrophytes from T. rubrum
YEAST ASSIMILATION MEDIA (CARBON OR NITROGEN)
Detection of carbohydrate assimilation through utilization of carbon (or nitrogen) by yeast in the presence of oxygen
YEAST FERMENTATION BROTH
Identification of yeasts by fermentation reactions with various carbohydrates

ORGANISMS AND THEIR DESCRIPTION TO FOLLOW 🙂

Posted in Checkpoint Notes, Lecture Notes

Clinical Microscopy – REAGENT STRIPS

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TEST PRINCIPLE TIMEREAGENTSFALSE (+)FALSE (-)
Glucose (correlated with ketones)  
Double sequential enzyme reaction  

ENZYMES: Glucose oxidase Peroxidase  

30 s
M – glucose oxidase, peroxidase, potassium iodide  
C – glucose oxidase, peroxidase, tetramethylbenzidine  

CHROMOGENS:
O-toluidine (pink to purple) Potassium iodide (blue to brown) Aminopropryl-carbazol (yellow to orange-brown) Tetramethylbenzidine (yellow to green)
Contamination by oxidizing agents and detergentsHigh levels of ascorbic acid, ketones, specific gravity
Low temperature   Improperly preserved specimens
Ketones (correlated with glucose)  

Sodium nitroprusside reaction  

40 s
M – sodium nitroprusside (acetoacetic acid)  

C – sodium nitroprusside + glycine (acetoacetic acid & acetone)
Phthalein dyes, highly pigmented red urine, levodopa   Medications containing free sulfhydryl groups (MESNA)Improperly preserved specimens
Specific Gravity  

pKa change of polyelectrolyte

pKa = dissociation constant

45 s
M – poly (methy lvinyl ether/maleic anhydride) bromthymol blue

C – ethyleneglycol-Bis (aminoethylether) bromthymol blue
High concentrations of proteins because of protein anionsHighly alkaline urines (>6.5) Add 0.005 to S.G. readings
pH
(correlated with Nitrite, LE, microscopic)  

Double-indicator system

60 s
Methyl red
Bromthymol blue
NoneRunover from the adjacent CHON pad may lower pH
Protein (correlated with blood, nitrite, LE, microscopic)

Protein error of indicators  

60 s
M – tetrabromphenol blue

C – tetrachloropenol tetrabromosulfonphthalein

ACID BUFFER: Citrate
Highly buffered alkaline urine High specific gravity   pigmented specimens, phenazopyridine quaternary ammonium compounds (detergents)   antiseptics, chlorhexidine   loss of buffer from prolonged exposure of reagent strip to the specimenProteins other than albumin
Blood (correlated with protein and microscopic)  

Pseudo-peroxidase activity of hemoglobin  

60 s
M – diisopropylbenzenedehydroperoxide tetramethylbenzidine

C – dimethyldihyroperoxide- tetramethylbenzidine    

spotted blue for intact RBCs uniform blue for Hb and myoglobin
Strong oxidizing agents, bacterial peroxidases   menstrual contaminationHigh specific gravity/ crenated cells   high concentration of nitrite   formalin, captopril, ascorbic acid > 25mg/dl   unmixed specimens
Bilirubin (correlated with urobilinogen)  

Diazo reaction

60 s
M – 2,4-dichloro-analine diazonium salt

C -2,6-dichorobenzene-diazonium-tetrafluoroborate  
Highly pigmented urines, phenazopyridine, indican metabolites of LodineSpecimen exposure to light, ascorbic acid >25 mg/dL, high concentration of nitrite
Urobilinogen (correlated with bilirubin)  

Ehrlich’s reaction

60 s
M – para-dimethylaminobenzaldehyde
C – 4-methoxybenzenediazonium-tetrafluroborate  
M – Porphobilinogen, indican, p-aminosalicyclic acid, sulfonamides, methyldopa, procaine, chlorpromazine, highly pigmented urine

C – highly pigmented urine
M – old specimens, preservation in formalin

C – old specimens, preservation in formalin, high concentration of nitrite
Nitrite (correlated with protein, LE and microscopic)  

Greiss reaction

60 s
M – p-arsanilic acid Tetrahydrobenzo(h)-quinolin-3-ol

C – Sulfanilamide, hydroxytetrahydro benzoquinoline      
Improperly preserved specimens  


Highly pigmented urine
Nonreductase-containing bacteria insufficient contact between bacteria and urinary nitrate (should be 4 hours) lack of urinary nitrate, large quantities of bacteria converting nitrite to nitrogen, presence of antibiotics, high concentration of ascorbic acid high specific gravity
Leukocytes (correlated with protein, nitrite and microscopic)  

Leukocyte esterase  

120 s
M – derivatized pyerole amino acid ester, diazonium salt  

C – indoxylcarbonic acid ester diazonium salt  
Strong oxidizing agents, Formalin, highly pigmented urine, nitrofurantoinHigh concentration of protein, glucose, oxalic acid, ascorbic acid, gentamicin, cephalosporins, tetracyclines
Posted in Lecture Notes

Clinical Microscopy – Automation

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WHAT’S IN HERE?

  • Principles Used in Automated Urinalysis and Microscopy
  • Automated Urinalysis Systems
  • Automated Body Fluid Analysis Systems
Principles Used in Automated Urinalysis and Microscopy
  • REFLECTANCE PHOTOMETRY
    • Used by automated reagent strip readers
    • Measure the light reflected from the reagent strip color pads and compare the amount of reflected light with a known standard
    • PRINCIPLE: light reflected from the colored reagent pads DECREASES in DIRECT PROPORTION to the INTENSITY OF THE COLOR produced by the reaction with the specific substance in the urine sample
      • the darker the color, the less light reflected
      • the lighter the color, the more light reflected
    • The concentration of a specific substance and concentration units are displayed on the reader’s display
  • LASER-BASED FLOW CYTOMETRY, IMPEDANCE & LIGHT SCATTER
    • Identify sediment elements in a urine sample
    • measures sediment conductivity and light scatter
      • Conductivity is based upon the impedance (the amount of resistance that occurs when a sediment passes through an electrical field) of sediments and counts the numbers of pulses (sediments).
        • The size of the pulse indicates the size of the sediment.
      • Light scattering characteristics of the sediments are determined by their movement through the laser light beam.
        • Identification depends on how the light is scattered by the sediment.
  • HARMONIC OSCILLATION
    • Assesses SPECIFIC GRAVITY
    • Method based upon densitometry in which a sound wave of a specific frequency changes in proportion to the density of the urine sample
    • Change in wave frequency is measured by a microprocessor and translates the reading to specific gravity
  • HYDRODYNAMIC FOCUSING
    • Identifies specific sediments
    • involves the movement of single urine sediments past the optics of a microscope to allow sediments to flow in several planes plane past the microscope objective
    • A flow cell also measures sediment conductivity, size, and light scattering traits
    • Questionable findings are viewed on a monitor for operator identification and confirmation
Automated Urinalysis Systems
  • INDIVIDUAL STRIP  READERS
  • SEMIAUTOMATED  ANALYZERS
    • dependent on an operator for specimen mixing, test strip, dipping,  and  inputting  of  physical  and  microscopic  results
  • FULLY  AUTOMATED CHEMISTRY ANALYZERS
    • add urine to the reagent strip
  • AUTOMATED URINE CELL ANALYZERS
    • mix, aspirate, dilute, and stain urine to classify urine sediment particles
  • COMPLETELY  AUTOMATED  SYSTEMS
    • perform a complete urinalysis that includes the physical, chemical, and microscopic parts of a routine urinalysis
WAIVED URINE CHEMISTRY INSTRUMENTS
Roche Diagnostics Criterion II Siemens Medical Solutions Diagnostics Clinitek®50 Siemens Medical Solutions Diagnostics Clinitek®101 Siemens Medical Solutions Diagnostics Clinitek®Status
SEMIAUTOMATED URINE CHEMISTRY INSTRUMENTS
Dirui Urine Analyzer H-50, 100, 200 Iris Diagnostics Division iChem®100 Roche Diagnostics URISYS®1800 Roche Diagnostics Chemstrip Criterion II Siemens Medical Solutions Diagnostics Clinitek®200, 200+, 500
FULLY AUTOMATED URINE CHEMISTRY INSTRUMENTS
Iris Diagnostics Division AUTION MAX® Iris Diagnostics Division iChem® VelocityTM Roche Diagnostics URISYS 2400® Siemens Medical Solutions Diagnostics Clinitek® Atlas
AUTOMATED SEDIMENT ANALYSIS
Iris Diagnostics Division iQ®200ELITETM Iris Diagnostics Division iQ®200SELECTTM Iris Diagnostics Division iQ®200SPRINTTM Sysmex UF-100® Urine Cell Analyzer
TOTALLY AUTOMATED URINALYSIS SYSTEMS
Iris Diagnostics Division iQ®200 Automated Urinalysis System iRICELL2000 (iChem® VelocityTM plus iQ®200ELITETM) iRICELL3000 (iChem® VelocityTM plus iQ®200SPRINTTM) Siemens Medical Solutions Diagnostics ADVIA Urinalysis WorkCell System (Clinitek® Atlas plus the Sysmex UF-100)
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DESCRIPTION OF SOME SELECTED INSTRUMENTS

  • CLINITEK 50 & CLINITEK STATUS
    • well  suited  for  small  volume  laboratories  and  physician’s offices
    • Memory storage for test results -100 for Clinitek 50 and 200 for Clinitek Status
    • automated  reading  of  microalbumin-to-creatinine  and  protein-to-creatinine  ratios and human  chorionic  gonadotropin  (hCG)
  • CLINITEK 200
    • For medium-volume to large-volume urinalysis laboratories and features a high specimen output of one strip every 10 seconds.
    • Multistix reagent test  strips  are  used,  and  the  instrument  has  the  ability  to report semiquantitative (mg/dL) results or plus (+) and SI units.
    • The reflectometer  is  calibrated  daily  and maintenance is required each day for all areas in contact with urine test strips
  • SYSMEX UF-SERIES
    • Fully automated sample analysis with automatic classification of all 10 formed element groups with SCATTERGRAMS and HISTOGRAMS for reference
    • Laser-based FLOW CYTOMETRY along with impedance detection, forward light scatter, and fluorescence
    • Sample is stained with 2 dyes
      • PHENATHRIDINE – orange dye, stains DNA
      • CARBOCYANINE – green dye, stains nuclear membranes, mitochondria, and negatively charged cell membranes
  • SYSMEX UF-SERIES
    • Stained sample is passed through the flow cell, where it is HYDRODYNAMICALLY FOCUSED and presented to a laser light beam that produces fluorescence and light scatter
    • Particles are identified by measuring the change in impedance of the sediment elements, as well as the height and width of the fluorescent and light scatter signals, which are presented in scattergrams and histograms
  • iQ 200 Automated Urine Microscopy Analyzer (IRIS)
    • Automatically analyses and classifies urine particles into 12 categories
    • Uses AUTO PARTICLE RECOGNITION (APR) software that classifies urine particles in the photographs based on size, shape, texture, and contrast
Automated Body Fluid Analysis Systems
  • cells are first mixed with reagent fixative and then counted
  • differentials counting enumerates numbers of neutrophils, lymphocytes, monocytes, and eosinophils
  • automated cell counters use larger numbers of cells, enhancing precision and accuracy
BODY FLUID ANALYZERSFDA APPROVED FOR USE WITH THESE FLUIDS:
Iris iQ Body Fluid ModuleCSF, Pleural & Peritoneal Peritoneal lavage, peritoneal dialysate, pericardial, general serous fluids, synovial
Siemens Medical Solutions Diagnostics ADVIA120 and 2120CSF
Sysmex XE-5000 Automated Hematology SystemCSF Serous body fluids Synovial fluid
Medical Electronic SystemsSemen

ADVIA120 Hematology System

  • First automated instrument with an FDA-approved automated CSF assay
    • Uses flow cytometry, light scatter, and absorbance to count the RBCs, WBCs, and performs a WBC differential that includes percentages and absolute numbers of mononuclear cells and PMNs on samples with >20WBCs/µL

AUTOMATION OF SEMEN ANALYSIS

  • SQA-V automates sperm counts and motility
    • has a two-channel measurement system that interacts with a specially designed testing capillary that contains the semen sample
    • one channel  “measures”  light  absorption  and  refraction  in sperm cells and translates this into concentration
    • one channel “counts”  light  interruptions  (signals)  caused  by sperm  cells  moving  across  the  field  of  light
    • In approximately 1 minute, thousands of signals are “read” resulting in exceptional accuracy and precision.
    • Automating the motility analysis eliminates reader subjectivity and variance among technologists.

AUTOMATION OF URINE PREGNANCY

  • Quantitative human chorionic gonadotropin (HCG) is one such test that is interpreted by the VEDALAB Easy Reader.
    • Immunochromatographic rapid test cards are read by the meter using a high-resolution CCD camera.
    • Integrated software analyzes the images and records the results.
Posted in Lecture Notes

Clinical Chemistry – Carbohydrates

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WHAT’S IN HERE?

  • Carbohydrates
  • Glucose and Its Metabolism
  • Hyperglycemia
  • Hypoglycemia
  • Genetic Defects in Carbohydrate Metabolism
  • Laboratory Analysis of Glucose
  • References

DEFINITION

  • Compounds containing C, H and O with general formula Cx(H2O)y
  • Contain C=O and –OH functional groups
  • Derivatives can be formed by addition of other chemical groups such as phosphates, sulfates and amines
  • Commonly called “SUGARS” and use the suffix –ose

CLASSIFICATION

  • Based on four different properties
  • SIZE OF THE BASE CARBON CHAIN
    • TRIOSES: with three (3) carbons
    • TETROSES: with four (4) carbons
    • PENTOSES: with five (5) carbons
    • HEXOSES: with six (6) carbons
  • LOCATION OF THE CO FUNCTION GROUP
    • ALDOSE: has a terminal carbonyl group (O=CH) called an aldehyde group
    • KETOSE: has carbonyl group (O=CH) in the middle linked to two other carbon atoms called a ketone group
  • STEREOCHEMISTRY OF THE COMPOUND
    • STEREOISOMERS: have the same order and types of bonds but different spatial arrangements and different properties
    • ENANTIOMERS: images that cannot be overlapped and are non-superimposable
      • L-isomer: if the configuration of the highest-numbered asymmetric carbon is on the LEFT or if hydroxyl group farthest from the carbonyl carbon is on the LEFT
      • D-isomer: if the configuration of the highest-numbered asymmetric carbon is on the RIGHT or if hydroxyl group farthest from the carbonyl carbon is on the RIGHT
  • NUMBER OF SUGAR UNITS
    • MONOSACCHARIDES
      • Simple sugars that cannot be hydrolyzed to simpler form
      • Examples: glucose, fructose, galactose
    • DISACCHARIDES
      • Formed by two monosaccharides joined by glycosidic linkage
      • Hydrolyzed by disaccharide enzymes (i.e., lactase) produced by the microvilli of the intestine
      • Examples:
        • Maltose = 2 β-D-glucose in 1→4 linkage
        • Lactose = glucose + galactose
        • Sucrose = glucose + fructose
      • OLIGOSACCHARIDES
        • Chaining of 2 to 10 sugar units
      • POLYSACCHARIDES
        • Linkage of many monosaccharide units
        • Yield more than 10 monosaccharides upon hydrolysis
        • Examples: starch, glycogen

 MODELS USED TO REPRESENT CARBOHYDRATES

  • FISCHER: linear formula where the aldehyde or ketone is at the top of the drawing and can be depicted in the D- or L- form
  • HAWORTH: cyclic form that is more representative of the actual structure and is formed when the carbonyl group reacts with an alcohol group on the same sugar to form a ring and can be depicted in the α or β form

CHEMICAL PROPERTIES

  • REDUCING SUBSTANCES
    • Contain a ketone or aldehyde group
    • WITH FREE ANOMERIC CARBON
    • Can reduce other compounds
    • Examples: glucose, maltose, fructose, lactose, galactose
  • NON-REDUCING SUBSTANCES
    • Do not have an active ketone or aldehyde group
    • NO FREE ANOMERIC CARBON
    • Will not reduce other compounds
    • Example: sucrose (table sugar)
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Glucose and Its Metabolism

  • End product of carbohydrate digestion in the intestine
    • Enzymes involved
      • AMYLASE (salivary & pancreatic) – digests nonabsorbable polymers to dextrins and disaccharides
      • MALTASE (from the intestine) – digests disaccharides to monosaccharides
      • SUCRASE & LACTASE – hydrolyze sucrose & lactose respectively
    • FUNCTIONS:
      • Provides energy for life processes
      • The only CHO that can be directly used for energy or stored as glycogen
    • FORMS: ~35% alpha & 65% beta
    • MAJOR METABOLIC PATHWAYS
      • EMBDEN-MEYERHOFF PATHWAY or GLYCOLYSIS
        • Substrate: D-glucose
        • End-products: 2 moles of PYRUVIC ACID, 2 moles NADH and 2 moles of ATP
        • Can occur aerobically or anaerobically
          • If aerobic, pyruvate is formed
          • If anaerobic, lactate is formed
        • Other substrates can enter this pathway at various points
          • Glycerol (from TAG) enters at 3-phosphoglycerate
          • Fatty acids, ketones and some amino acids are converted to acetyl-CoA
          • Other amino acids enter as pyruvates or as deaminated α-ketoacids and α-oxoacids
        • HEXOSE MONOPHOSPHATE SHUNT OR AEROBIC/OXIDATIVE PATHWAY
          • G6P is converted to 6-phosphogluconic acid which permits the formation of NADPH (important to red cells because they lack mitochondria thus incapable of TCA cycle)
          • End-products: pentose phosphate, CO2 and NADPH
        • GLYCOGENESIS
          • Stores glucose as glycogen
          • Converts G6P to G1P
          • G1P → uridine diphosphoglucose→ glycogen by glycogen synthase
          • GLYCOGENOLYSIS – conversion of glycogen to G6P
PATHWAYS IN GLUCOSE METABOLISM
GlycolysisMetabolism of glucose molecule to pyruvate or lactate for production of energy
GluconeogenesisFormation of G6P from noncarbohydrate sources
GlycogenolysisBreakdown of glycogen to glucose for use as energy
GlycogenesisConversion of glucose to glycogen for storage
LipolysisDecomposition of fats
LipogenesisConversion of carbohydrates to fatty acids
  • MAJOR HORMONES CONTROLLING BLOOD GLUCOSE
    • PANCREATIC HORMONES
      • INSULIN – primary hormone for DECREASING blood glucose levels
        • Responsible for the entry of glucose into the cells by enhancing membrane permeability to cells in the liver, muscle and adipose tissues
        • synthesized by β-cells of the pancreas
          • released when glucose levels are high/increased
          • not released when glucose levels are low/decreased
        • EFFECTS:
          • increases glycogenesis, lipogenesis, and glycolysis
          • inhibits glycogenolysis
        • INSULIN IS THE ONLY HORMONE THAT DECREASES GLUCOSE LEVELS and can be referred to as a hypoglycemic agent
      • GLUCAGON – primary hormone for INCREASING blood glucose levels
        • released in response to stress and fasting states
        • synthesized by α-cells of the pancreas
          • released when glucose levels are low/decreased
          • not released when glucose levels are high/increased
        • EFFECTS:
          • increase glycogenolysis and gluconeogenesis
        • can be referred to as a hyperglycemic agent
      • SOMATOSTATIN
        • produced by δ cells of the pancreas
        • EFFECTS: inhibition of insulin, glucagon, growth hormone, and other endocrine hormones.
      • ADRENAL HORMONES
        • CORTISOL
          • produced by the adrenal cortex on stimulation by ACTH
          • EFFECTS: decreases intestinal entry into the cell and increases gluconeogenesis, liver glycogen and lipolysis
        • EPINEPHRINE
          • produced by the adrenal medulla
          • EFFECTS: inhibits insulin secretion, increase glycogenolysis and lipolysis
          • Released during times of stress
        • ANTERIOR PITUITARY HORMONES
          • GROWTH HORMONE
            • EFFECTS: decreases the entry of glucose into the cells
          • ACTH
            • EFFECTS: stimulates the adrenal cortex to release cortisol, increases glycogenolysis and gluconeogenesis
          • THYROID HORMONES
            • T3 & T4
              • EFFECTS: increases glycogenolysis, gluconeogenesis and intestinal absorption of glucose
HORMONAL ACTIVITY AFFECTING SERUM GLUCOSE LEVELS
HORMONESOURCEEFFECTACTION
Insulinβ cells of pancreasstimulates glucose uptake by cells
Glucagonα cells of pancreasglycogenolysis
ACTHAnterior pituitaryinsulin antagonist, glycogenolysis & gluconeogenesis
Growth HormoneAnterior pituitaryinsulin antagonist & glycolysis
CortisolAdrenal cortexinsulin antagonist, gluconeogenesis & lipolysis
HPLPlacentainsulin antagonist
EpinephrineAdrenal medullainhibits insulin secretion, glycogenolysis & lipolysis
T3 & T4Thyroid glandglycogenolysis, gluconeogenesis & intestinal absorption of glucose
Somatostatinδ cells of pancreasinhibits insulin, glucagon, GH

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Hyperglycemia

  • Increase in plasma glucose levels caused by imbalance of hormones
  • DIABETES MELLITUS
    • Group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or both
    • Categories of Diabetes (According to the ADA/WHO guidelines)
      • Type 1 Diabetes
      • Type 2 Diabetes
      • Other specific types of diabetes
      • Gestational Diabetes Mellitus (GDM)
  • PRIMARY DIABETES MELLITUS
Points of DifferenceTYPE 1TYPE 2
Former namesInsulin Dependent Diabetes Mellitus (IDDM)

Juvenile Onset DM

Brittle DM

Ketosis-prone DM

Non-Insulin Dependent Diabetes (NIDDM)

Maturity Onset DM

Stable DM

Ketosis-resistant DM

Receptor Deficient DM

OnsetBefore 20 y/oOver 40 y/o
Measurable circulating insulinNONELow
Insulin receptorNormal↓ or ineffective
Beta cell massMarkedly ↓Moderately ↓
C-peptide levelsUndetectableDetectable
Incidence10-15%85% (common)
Ketoacidosis*CommonRare
Physique/Stature**Normal or thinOften overweight
Pathogenesis -β-cell destruction

-Absolute insulin deficiency

-Autoantibodies

-Insulin resistance with insulin secretory defect

-Relative insulin deficiency

TreatmentParenteral insulin administraionOral hypoglycemic agent
  • SECONDARY DIABETES MELLITUS – associated with secondary conditions
    • Genetic defects of β-cell function
    • Pancreatic disease
    • Endocrine disease
      • Cushing syndrome – excessive cortisol
      • Pheochromocytoma – epinephrine excess
      • Acromegaly – growth hormone excess
    • Drug or chemical induced
    • Insulin receptor abnormalities
    • Other genetic syndromes
      • Maturity onset diabetes of youth (MODY) – rare; autosomal dominant
  • GESTATIONAL DIABETES MELLITUS (GDM)
    • any degree of glucose intolerance with onset or first recognition during pregnancy
    • due to metabolic or hormonal changes
    • Infants born to mothers with this kind of diabetes are at increased risk to respiratory distress syndrome, hypocalcemia & hyperbilirubinemia

Laboratory Findings in Hyperglycemia

  • INCREASED glucose (plasma & urine), urine specific gravity, serum and urine osmolality
  • Ketonemia and ketonuria
  • DECREASED blood and urine pH (acidosis)
  • Electrolyte imbalance (↓Na+, Cl and ↑K+)
DIAGNOSTIC CRITERIA FOR DIABETES MELLITUS
RPG                    ≥200 mg/dl (11.1 mmol/L) + symptoms of diabetes
Fasting PG         ≥126 mg/dL (7.0 mmol/L)
2-h PG               ≥200 mg/dl (11.1 mmol/L) during OGTT
CATEGORIES OF FASTING PLASMA GLUCOSE
Normal fasting glucose         FPG <110 mg/dL
IMPAIRED fasting glucose     FPG ≥110 mg/dl but <126 mg/dl
Provisional diabetes dx         FPG ≥126 mg/dl
CATEGORIES OF ORAL GLUCOSE TOLERANCE
Normal glucose tolerance   2h PG <140 mg/dL
Impaired gluc. tolerance     2h PG ≥140 mg/dl but <200 mg/dl
Provisional diabetes dx      2h PG ≥200 mg/dl
  • Screening test for GDM
    • Only high-risk patients should be screened for GDM
      • Age older than 25 years
      • Overweight
      • Strong family history of diabetes
      • History of abnormal glucose metabolism
      • History of a poor obstetric outcome
      • Presence of glycosuria
      • Diagnosis of PCOS
      • Member of an ethnic/racial group with a high prevalence of diabetes (e.g. Hispanic American,  Native  American, Asian American, African American, Pacific Islander)
    • METHODS:
      • ONE-STEP APPROACH – immediate performance of a 3h OGTT without prior screening
      • TWO-STEP APPROACH – initial measurement of plasma glucose at 1-hour postload (50g)
        • IF value ≥140 mg/dL (7.8 mmol/L) then do 3-hour OGTT using 100g glucose
        • GDM is diagnosed when any two of the following values are met or exceeded:
          • Fasting: >95 mg/dl
          • 1 hour: ≥180 mg/dl
          • 2 hours: ≥155 mg/dl
          • 3 hours: ≥140 mg/dl

Hypoglycemia

  • Decrease in plasma glucose levels
    • 65-70 mg/dl (3.6-3.9 mmol/L) – plasma glucose concentration at which glucagon and other glycemic factors are released
    • 50-55 mg/dl (2.8-3.0 mmol/L) – symptoms of hypoglycemia appear
    • Warning S/S are all related to CNS
  • Types of Hypoglycemia (Old)
    • Post-absorptive (Fasting) – MORE SERIOUS
      • Islet cell insulinoma
      • Insulin-producing tumors
      • Ethanol induced
      • Propanolol & salicylate
  • Post-prandial (Reactive) – MILD FORM
    • there is spontaneous recovery of glucose level as a result of insulin level returning to normal
    • Excessive release of insulin
    • Gastro-intestinal surgery
CAUSES OF HYPOGLYCEMIA
Patient Appears Healthy 
No coexisting diseaseDrugs

Insulinoma

Islet hyperplasia or NESIDIOBLASTOSIS

Factitial hypoglycemia from insulin or sulfonylurea

Severe exercise

Ketotic hypoglycemia

Compensated coexistent diseaseDrugs
Patient Appears ILL 
Drugs

Predisposing illness

Hospitalized patient

 
  • Diagnostic criteria for INSULINOMA
    • Change in glucose level of ≥25 mg/dl coincident with an insulin level of ≥6 μU/ml
    • C-peptide levels of ≥0.2 nmol/L
    • Proinsulin levels of ≥5 pmol/L
    • β-hydroxybutyric acid of ≤2.7 mmol/L
  • Diagnostic tests for HYPOGLYCEMIA
    • 72 hour fast which requires the analysis of glucose, insulin, C-peptide and proinsulin at 6-hour intervals
    • POSITIVE RESULT: <45 mg/dl; hypoglycemic symptoms appear after 72 hours had elapsed
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Genetic Defects in Carbohydrate Metabolism

  • Glycogen Storage Diseases – deficiency of a specific enzyme that causes alteration of glycogen metabolism
TypesEnzyme DeficientClinical Features
von Gierke’s dse

Type I

Glucose-6-phosphataseSevere fasting hypoglycemia

Lactic acidosis

Pompe’s dse

Type II

α-1,4-glucosidaseAccumulation of ↑ amount of glycogen on all organs

Presence of abnormally LARGE LYSOSOMES

Forbe’s dse

Type III

Debrancher enzymeHypoglycemia, hepatomegaly, seizures and mental retardation
Andersen’s dse

Type IV

Brancher enzymeProgressive liver enlargement or cirrhosis and muscular weakness by age 2

Absence of storage glycogen

Unbranched AMYLOPECTIN

Other enzyme defects/deficiencies that cause hypoglycemia: glycogen synthase, fructose-1-6,biphosphatase, phosphoenolpyruvate carboxykinase and pyruvate carboxylase.

  • Galactosemia – a cause of failure to thrive syndrome in infants; congenital deficiency of one of three enzymes involved in galactose metabolism, resulting in increased plasma galactose levels
    • Galactose-1-phosphate uridyl transferase – MOST COMMON enzyme deficiency
    • Fructose-1-phosphate aldolase deficiency

Laboratory Analysis of Glucose

  • SPECIMEN COLLECTION AND HANDLING
    • Glucose concentration in whole blood is approximately 15% lower than in plasma or serum.
    • Glucose levels decrease approximately 10 mg/dL (7%) per hour in whole blood.
    • Serum or plasma must be separated within 1 hour (Bishop) to prevent substantial loss of glucose by the cellular fraction, particularly if WBC count is elevated. (within 30 minutes – Henry)
    • Glucose is metabolized at a rate of 7 mg/dl/h at room temperature; and 2 mg/dl/h at 4°C
    • Refrigerated serum or plasma is stable up to 48 hours.
    • Sodium fluoride (2 mg/mL) prevents glycolysis (gray top tube) for up to 48 hours.
    • Glycolysis decrease serum glucose by approximately 5-7% per hour (5-10 mg/dl) in normal, uncentrifuged coagulated blood at room temperature.
    • Fasting blood glucose should be obtained after an approximately 10-hour fast (not >16 hours)
    • Fasting plasma glucose values have a diurnal variation with the mean FBG higher in the morning than in the afternoon.
    • Fasting reference range for serum or plasma is 70-110 mg/dL
    • In the fasting state, arterial (capillary) values are 5 mg/dL higher than the venous concentration.
    • Urine glucose analysis (in 24h urine glucose) may be stabilized by addition of a preservative; should be stored at 4°C during collection because 40% of glucose is lost after 24 hours at room temperature.
    • CSF glucose analysis (if will be delayed) must be centrifuged and stored at 4°C-20°C
    • In normal CSF, values are two-thirds (approximately 60-70%) of plasma level.
    • RENAL THRESHOLD for glucose: 180 mg/dl
  • TYPES OF SPECIMEN FOR GLUCOSE ANALYSIS
    • Fasting Blood Sugar – blood collected after 8-10 hours of fasting (NV: 74-106 mg/dl)
    • Random Blood Sugar – test for INSULIN SHOCK (NV: <200 mg/dl)
    • 2 hour Postprandial Blood Sugar
      • Standard load of glucose: 75 grams
      • Glucose measurement taken 2 hours later
      • (NV : <120 mg/dl)
    • Glucose Tolerance Test – multiple blood and urine glucose test
      • Oral GTT
        • Janney-Isaacson (Single Dose)
        • Exton Rose (Divided Oral dose or Double Dose)
        • Not recommended for routine use
        • Fasting and 2h sample are measured except for pregnant patients
        • Adult load is 75g; children: 1.75 g/kg to 75g
        • Factors that affect tolerance
          • Medications (salicylates, diuretics, anticonvulsants, oral contraceptives and corticosteroids)
          • GI surgery
          • Vomiting
          • Endocrine dysfunction
        • Requirements:
          • Patient should be ambulatory
          • Patient must be in unrestricted diet of 150 grams CHO/day for 3 consecutive days prior to the test
          • Patient must be free from undue stress or severe illness
          • Alcohol intake and smoking are not allowed prior to the test
          • Patient should be fasting at least 10 hours and not more than 16 hours
          • Test should be performed in the morning because of hormonal diurnal effect on glucose
        • IVGTT – blood sample is collected every 10 minutes for 1 hour
          • 5g glucose/kg body weight (given within 3 minutes) administered intravenously
          • fasting is also required
          • NV: 1.4 – 2.0 %
          • Indications of IVGTT
            • Patients who are unable to tolerate large CHO load
            • Patients with altered gastric physiology or GI d/o
            • Patients with malabsorption syndrome
  • Self-Monitoring of Blood Glucose (SMBG)
    • Type 1 DM – should monitor blood glucose 3-4 times per day
    • Type 2 DM – optimal frequency is unknown
  • Glycosylated hemoglobin/Glycated hemoglobin/HbA1C
    • hemoglobin compound formed when glucose reacts with amino group of hemoglobin
    • test for long term diabetic control
    • reflects the average blood glucose level for the previous 2-3 months
    • for every 1% change in HbA1c value there is 35 mg/dl (2 mmol/L) change in the mean
    • in presence of hemoglobinopathies, there will be less time for glucose to
    • binding of glucose to HbA1 is irreversible
    • preferred anticoagulant is EDTA
    • NV: 4.5-8.5%
Methods of HBA1c Measurement
Methods based on STRUCTURAL DIFFERENCES
ImmunoassaysPolyclonal or monoclonal antibodies toward the glycated n-terminal group of the β chain of Hgb 
Affinity chromatographySeparates based on chemical structure using borate to bind glycosylated proteinsNot affected by temperature and other hemoglobins
Methods based on CHARGE DIFFERENCES
Ion-exhange chromatographyPositive-charge resin bedHighly affected by temperature and hemoglobinopathies

HbF – ↑

HbS and C – ↓

ElectrophoresisSeparation is based on differences in chargeHbF values >7% interferes
Isoelectric focusingType of electrophoresis using isoelectric point to separatePre-hb A1c interferes
HPLCForm of ion-exchange chromatographySeparates all forms of glycol Hb (a,b,c)
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  • METHODS FOR ANALYSIS
    • CHEMICAL
      • REDUCTION
        • Cupric Ion Reduction
          • FOLIN-WU – measure of ALL REDUCING SUBSTANCES in the blood
            • Reagent that binds with Cu+: phosphomolybdic acid
            • End product: phosphomolybdenum blue
            • End color: blue
          • NELSON SOMOGYI – MEASURE OF TRUE GLUCOSE
            • Reagent that binds with Cu+: arsenomolybdic acid
            • End product: arsenomolybdenum blue
            • End color: blue
          • NEOCUPROINE
            • Reagent that binds with Cu+: neocuproine
            • End product: cuprous-neocuproine complex
            • End color: yellow/yellow orange
          • Ferric Ion ReductionInverse Colorimetry – reduction of yellow ferricyanide to a colorless ferrocyanide by glucose
            • HAGEDORN JENSEN
    • CONDENSATION
      • Orthotoluidine (DUBOWSKI method)
        • can be also used for urine and CSF without protein precipitation
        • Absorbance: 630 nm
        • Reagent: aromatic amine, glacial acetic acid
        • End color: green
        • Interfering substances: galactose and mannose
    • Polarographic Glucose Oxidase
      • measures oxygen consumption with PO2 electrode (Clark)
      • used to avoid interference made by strong oxidizing agents in GOD
      • Molybdate – catalyzes the oxidation of iodide to iodine by H2O2
      • Catalase – catalyzes oxidation of ethanol by H2O2 forming acetaldehyde and H2O
    • Hexokinase
      • Generally accepted as the REFERENCE METHOD
      • MORE ACCURATE THAN HEXOKINASE
        • coupling reaction using G6PD is highly specific
      • Measured by quantitating reduced NADPH formation
      • NADPH is measured directly at 340 nm or coupled to chromogen and measured in visible range
      • Interfering substances: gross hemolysis & extremely elevated bilirubin (cause ↓ values)
      • May be performed using serum or plasma (heparin, EDTA, fluoride, oxalate & citrate)
      • Excellent for glucose determination in urine, CSF and serous fluids

OTHER IMPORTANT TESTS

  • KETONES
    • Produced by the liver through metabolism of fatty acids to provide ready energy source from stored lipids at times of low carbohydrate availability
    • THREE KETONE BODIES
      • Acetone (2%)
      • Acetoacetic acid (20%)
      • Β-hydroxybutyric acid (78%)
    • Causes of increased ketone levels
      • Diabetes Mellitus
      • Starvation/fasting
      • High-fat diets
      • Prolonged vomiting
      • Glycogen storage diseases
    • KETONEMIA – accumulation of ketones in the blood
      • KETONURIA – accumulation of ketones in the urine
      • MEASUREMENT OF KETONES
        • For patients with Type 1 Diabetes, it is recommended during acute illness, stress, pregnancy, or elevated blood glucose levels above 300 mg/dL or when patients have signs of ketoacidosis
        • SPECIMEN: FRESH SERUM or URINE tightly stoppered and analyzed immediately
        • METHODS FOR ANALYSIS:
          • GERHARDT’S TEST – historical test
            • Used FERRIC CHLORIDE reacted with ACETOACETIC ACID to produce a RED color
    • SODIUM NITROPRUSSIDE – more common method
      • Uses SODIUM NITROPRUSSIDE which reacts with ACETOACETIC ACID in an ALKALINE pH to form a PURPLE COLOR
      • If GLYCERIN is also added, ACETONE will be detected
      • Used in urine reagent strips and Acetest tablets
    • ENZYMATIC – newer method adapted in some automated intstruments
      • Uses β-HYDROXYBUTYRATE DEHYDROGENASE to detect either β-HYDROXYBUTYRIC ACID or ACETOACETIC ACID depending on the pH of the solution
        • pH of 7.0 causes the reaction to proceed to the right (decreasing absorbance)
        • pH of 8.5 to 9.5 causes the reaction to proceed to the left (increasing absorbance)

MICROALBUMINURIA

  • Defined as persistent albuminuria in the range of 30 to 299 mg/24 h or an albumin-creatinine ratio of 30 to 300 g/mg
  • Clinical proteinuria or macroalbuminura is established with an albumin-creatinine ratio of ≥300 mg/24h or ≥300 µg/mg
  • Powerful predictor for future development of diabetic nephropathy
  • Annual assessment of kidney function by the determination of urinary albumin is recommended for diabetic patients
  • METHODS FOR MICROALBUMINURIA SCREENING
    • RANDOM SPOT TEST – preferred method
    • 24-HOUR COLLECTION
    • TIMED 4-HOUR OVERNIGHT COLLECTION
  • A patient is determined to have microalbuminuria when two of three specimens collected within a 3- to 6-month period are abnormal.
  • Factors that may elevate the urinary excretion of albumin include exercise within 24 hours, infection, fever, congestive heart failure, marked hyperglycemia, and marked hypertension

ISLET AUTOANTIBODY AND INSULIN TESTING

  • Not currently recommended for routine screening for diabetes diagnosis but in the future it might identify at-risk, prediabetic patients
TESTS FOR CARBOHYDRATE DISORDERS
DIAGNOSTIC TESTSACTION
Fasting Blood SugarNormal – 70-110 mg/dl

Diabetes – >126 mg/dl

2hr Post Prandial Blood Sugar (PPBS)Normal – <126 mg/dl

Diabetes – >200 mg/dl

Post-Loading GlucoseSimilar to PPBS

*Glucose load is standardized

*Diabetics ≥200 mg/dl

Glucose Tolerance Test (GTT) Standard dose = 75g*Diagnostics of diabetes mellitus

>150 mg/dl after 2 hours

>200 mg/dl after 2 hours

*Perform if FBS and PPBS are normal

Intravenouse Glucose Tolerance Test (IVGTT)*Poor absorption (flat curve with OGTT)

*Patient who cannot tolerate large glucose load (vomiting)

O’Sullivan Test

(for gestational diabetes)

*Standard dose 50g

*Probable gestational diabetes

>150 mg/dl at 1 hour

*Follow up with OGTT

TESTS FOR MONITORINGNOTES
Glycosylated hemoglobin*Assessment of long term control

*Average glucose level over 60 days (1-2 months)

Microalbumin*Detects small amounts of protein in urine of diabetic patients to assess renal damage
C peptide of Insulin

(reflects pancreatic insulin secretion)

Normal 1:1 (insulin:C-peptide)

Diabetes > 1:1

C-Peptide ↓after insulin injection

REFERENCES:

  1. Bishop, Michael L., et.al., Clinical Chemistry Techniques, Principles, Correlations, Sixth Edition
  2. PER Handbook
  3. Theriot, Betty, Clinical Laboratory Science Review: Bottom Line Approach
  4. McPherson, Richard, et.al., Henry’s Clinical Diagnosis and Management by Laboratory Methods, 22e
Posted in Lecture Notes

Water Bacteriology

Source: Alba’s Medical Technology

Water-Borne Diseases

  • Salmonella – typhoid & parathypoid fevers
  • Shigella – bacterial dysentery
  • E. histolytica – amebic dysentery
  • Vibrio – cholera

The bacteriological examination of water usually consists of

  1. Estimating the number of bacteria present by TOTAL PLATE COUNT
  2. Detecting the presence or absence of coliforms & estimation of the number of coliforms present by the “MOST PROBABLE NUMBER” (MPN) method

DRINKING WATER STANDARDS

U.S. Public Health Service Drinking Water Standards publication

  • standard for public water supplies
  • states that NO MORE THAN 10% OF ALL THE STANDARD 10 ml PORTIONS OF WATER EXAMINED IN A MONTH SHALL SHOW THE PRESENCE OF COLIFORM BACTERIA

Sampling

  • collected in sterile glass containers with ground glass stoppers
  • should be packed in ice or stored at 6-10°C
  • examination of contents should be done within
    • 6 hours – IMPURE WATER
    • 12 hours – PURE WATER
  • 20-50 mg of sodium thiosulfate is added to sample bottle when testing water from swimming pools to NEUTRALIZE RESIDUAL CHLORINE and prevent chlorine from killing bacteria in the interval between collection and testing

Two (2) Procedures carried out routinely:

  1. TOTAL BACTERIAL PLATE COUNT – report as number of bacteria (each colony is counted as one bacterium) per mL of undiluted H2O. When bacterial total plate count exceeds 100 organisms/mL at 37°C, the water is usually CONSIDERED UNSAFE FOR HUMAN CONSUMPTION.
  2. TESTS FOR COLIFORM GROUP OF BACTERIA

Coliform group – all AEROBIC & FACULTATIVE ANAEROBIC GRAM NEGATIVE NON-SPOREFORMING BACILLI which FERMENT LACTOSE WITH GAS FORMTION WITHIN 48 HOURS at 35°C.

PRESUMPTIVE TEST

  • LACTOSE BROTH or LAURYL TRYPTOSE BROTH
  • POSITIVE: Formation within 48± 3hours of GAS in ANY AMOUNT IN THE FERMENTATION TUBE
  • NEGATIVE: Absence of gas formation at the end of 48± 4hours incubation

CONFIRMED TEST

  • BRILLIANT GREEN LACTOSE BILE BROTH FERMENTATION TUBES
  • POSITIVE: Formation and presence of gas in any amount within 48± 3hours
  • ENDO or LEVINE’S EMB AGAR PLATES
    • Results on Endo or Levine’s EMB
      • Typical nucleated with or without metallic green sheen
      • Atypical, opaque, unnucleated after 24hour incubation. PINK.
      • Negative (others)
  • POSITIVE: Growth of TYPICAL COLIFORM COLONIES with 24± 2 hours
  • NEGATIVE: Growth of NEGATIVE COLONIES

COMPLETED TEST

  • BRILLIANT GREEN LACTOSE BILE BROTH FERMENTATION TUBES showing gas used for confirmed test
  • POSITIVE: Formation of acid and gas in Lactose broth; demonstration of Gram Negative non sporeforming bacilli in the agar
  • NEGATIVE: Absence of gas formation or failure to demonstrate Gram Negative non sporeforming bacilli in a gas forming culture
  • THE detection of the coliform group in significant number is takes as evidence of FECAL CONTAMINATION.

Fecal origin: E. coli IMViC (++–)

Non Fecal origin: E. aerogenes IMViC (–++) 

Posted in Checkpoint Notes

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CheckPointNotes are review notes you can use as a guide in preparing for both the local board exam and international certification exams (such as ASCPi, AMT, HAAD, AIMS, CSMLS) in Clinical Laboratory Science/Medical Laboratory Science/Medical Technology. They are also used by many pathologists for their diplomate exams.

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CHECKPOINTNOTES BY
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