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3023MSC Clinical SciencesLaboratory

Laboratory Manual & Workbook 2013

School of Medical Science, Griffith University, Gold Coast campus,Queensland, Australia.

Updated July 2013 by Dr Andrew Bulmer and Ms Ana Gomez

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Index

Page

i Abbreviations

iii Important Information

vii Health and Safety Information

ix Laboratory Class Schedule

1 Prelaboratory Essentials

11 1.0 Introduction to Clinical Biochemistry

32 2.0 Introduction to Haematology

49 3.0 Introduction to Histology

63 4.0 Introduction to Clinical Immunology

85 Appendix A Light Microscopy

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i

Abbreviations

AA Amino acid

AAb Autoantibody

AAg AutoantigenAb Antibody

ABX AntibioticsADP Adenosine diphosphate

AFP Alphafetoprotein

Ag AntigenAHG Antihuman globulin

AIDS Acquired immune deficiency syndrome

APTT Activated partial thrombin timeATP Adenosine triphosphate

AU Absorbance units

BC Blood cellBGL Blood glucose levelBSA Bovine serum albumin

BUN Blood urea nitrogen

CHD Coronary heart diseaseCOPD Chronic obstructive pulmonary disease

CV Coefficient of variation

DH Dopamine beta-hydroxylase

DIC Disseminated intravascular coagulationDMPD NN-Dimethyl-p-phenylenediamine

DNA Deoxyribonucleic acid

EDTA Ethylenediaminetetraacetic acidELISA Enzyme-linked immunosorbent assay

FBC Full blood count

FGFR Fibroblast growth factor receptorGST Glutathione-s-transferase

GSTM1 Glutathione-s-transferase--1

Hb HaemoglobinHct Haematocrit

HDL High-density lipoprotein

H&E Haematoxylin & EosinHIAA 5-Hydroxyindole acetic acid

HIV Human immunodeficiency virusHPV Human papilloma virus

HRP Horseradish peroxidaseHRP-aHAb Horseradish peroxidase antihuman antibody

5-HT 5-Hydroxytryptamine

HWE Hardy-Weinberg equilibrium

HWP Hardy-Weinberg principleID Identification

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IDL Intermediate density lipoprotein

IgG Immunoglobulin-

INSR Insulin receptorITP Idiopathic thrombocytopenia purpura

Lac+ Lactose fermenting

Lac- Non-lactose fermentingLDL Low-density lipoprotein

LOD Logarithm of the odds

MAb Monoclonal antibody

MCA MacConkey agarMCH Mean corpuscular haemoglobin

MCHC Mean corpuscular haemoglobin concentration

MCV Mean cell volumeMW Molecular weight

NA Nutrient agar

NNT Number needed to treat

OGTR Office of Gene Technology RegulatorPAS Periodic acid-schiff

PBS Phosphate buffered saline

PCV Packed cell volumepH Pondus hydrogenii

PHA Phytohemagglutinin

PT Prothrombin timePr Probability

RA Rheumatoid arthritis

RBC Red blood cellRh Rhesus factor

RR Reference range

RT Room temperatureSG Specific gravity

sGOT Glutamic oxaloacetic transaminase

sGPT Serum glutamic pyruvic transaminase

SI System internationalSLE Systemic lupus erythematosus

TMB Tetramethylbenzidine

TMPD NNNN-Tetramethyl-p-phenylenediamineTPH Tryptophan hydroxylase

TT Thrombin time

UA UrinalysisUV Ultra violet

VLDL Very low-density lipoprotein

WBC White blood cell

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iii

Important Information

Pre-Requisites

1005MSC Cell Biology or equivalent

3005MSC Molecular Genetics or equivalentNot compatible with 3020MSC Molecular Diagnostics

At tendance

Attendance at all laboratory sessions is compulsory and will be recorded. If you are not able toattend a laboratory class, please see the Course Convenor as soon as possible to minimise any

penalty that may result.

Laboratory Safety

Staff and students at Griffith University have a legal obligation to comply with workplace health

and safety legislation and university policies. PC2 requirements will be strictly enforced.

Students are strongly urged to seek professional advice regarding appropriate immunisationsagainst infectious blood-borne diseases. We specifically recommend that students are vaccinated

against Hepatitis B.

Assessment Method

Assessment in this course is ongoing and cumulative. This course is based on the acquisition ofpractical skills and the assessment methods reflect the learning outcomes of the course and

associated theoretical components. Students will receive a grade in accordance with the Griffith

University grading system reflected by a numerical value from 2 (fail) 7 (high distinction).

There are five assessment items in this course, the details of which are included below.

Item one

Week one online quiz (20 questions)

Duration: 25 mins

Weighting: 15%

Number of marks: 20

Multiple choice/short answer

Due date: 11:15 am, Thursday June 27th

.

Notes: Students will have the preceding days to study and Thursday morning to participate in aQuestion and Answer tutorial regarding this assessment item. The topic of the quiz will beintroductory concepts, haematology and biochemistry (ie. Labs 1-3). Students will be provided

some additional practice questions in addition to studying the laboratory notebook, from which

the majority of questions will be drawn. Assessment will be held in class time (11:15-12pm), in acomputer laboratory, supervised by tutors/convenor. Students will be allowed to bring a non-

programmable calculator into the quiz. No resits of this quiz will be allowed.

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Item four

Practical exam

Weighting: 20%

Number of marks: 20

Duration: 17 minsDue date: 12:30-5pm July 6

th

Students will be randomly assessed on one of three core practical skills and will have Thursday

July 4th

to practice these skills in the lab. Skills to be assessed include 1) making

solutions/calculation of dilutions/pipetting skill; 2) simulated collection of blood, analysis ofblood (biochemistry) and assessment of urine composition; 3) simulated collection of blood,

analysis of haemoglobin and white/red blood cell count (haematology). Six students will be

assessed every 25 mins (individually by 6 tutors). Therefore, all testing will be completed in ~4.5hours (+30 mins for retesting). Practical examination will take place from two groups of students

(Group 1: 12:30-3pm; Group 2: 3:20pm-5pm).

Students will be marked by tutors using a criteria sheet for each skill. Students must pass this

assessment item to pass the course overall. Alternative assessments will be held at the end of theday for any failing students. Students will be allowed upto two resits if the student fails and will

be assessed on different skills in each resit.

Item five

Laboratory notebook

Weighting: 35%

Number of marks: 122

Due date: 5pm, Friday July 5th

Notes: Students will have their note books assessed for quality of answers, legibility, graphingquality, correctness of results/answers. Students will receive brief feedback on the quality of their

work after submitting it to tutors/convenor after each laboratory. As a minimum, student must

have completed all activities in the practical, before leaving class. Tutors/convenor, will assesswhether students have completed sufficient work, prior to leaving class, to obtain a passing

grade. If students cannot conduct sufficient work in class time, the tutor/convenor will indicate

room for improvement on the final page of the laboratory activity, to indicate that the studentneeds to work further on this item, to obtain a passing grade. If any practical task is not

completed in its entirety, the student will not receive a passing grade for this item. The students

are allowed to improve the quality of their work prior to submission and during class can

complete their books in 2B pencil. Final marking of laboratory books will be completed by theconvenor. Red pen will be used to indicate areas of deficiency. Students are required to pass this

component of assessment, to pass the course overall. If students submit a non-complete

laboratory notebook, it will be returned to the student for completion. Normal penalty for latesubmission applies, as per Griffith University assessment guidelines.

Students should be aware of Griffith University rules on plagiarism and academic integrity.Laboratory books will be assessed for deliberate copying of content (ie. paired students should

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submit lab books together). Responses will also be compared to previous years laboratory note

books.

General assessment notes: If students are away and cannot complete assessment the student will

be required to complete assessment at a time that is suitable with the convenor(presentation/quizzes) or catch up/copy (as approved by convenor) laboratory notes. Students

cannot not graduate from this course, prior to completion of all assessment items.

Course Communications

Learning@Griffith will be used to deliver announcements, data, lecture material, assessment

feedback and administrative material. Students are reminded to check the website regularly.Students may also request access to the ILOVELABSCIENCE facebook webpage, which will be

used as a secondary means of communication.

Please note that only Griffith University email addresses will be replied to, this is primarily toensure student privacy.

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HEALTH AND SAFETY INTRODUCTION

Health and Safety are essential aspects of laboratory work. The University is bound by the rules

set out in Queenslands Health and Safety Act and requires that whilst students are on campus,

especially during laboratories and field trips that students understand they have an obligation to:

1. Behave responsibly.2. Follow instructions.3. Not misuse equipment or cause damage or injury.

You have a Health and Safety obligation to yourself whilst in the laboratories but also to fellow

students, the demonstrators and technical staff supporting your lab. If youre not sure on how to

proceed with a task please ask your demonstrator. Furthermore, whilst in the laboratory

classrooms please adhere to the instructions the demonstrator may give you.

Laboratory Rules

You must abide by several basic rules which apply to how you conduct yourself within the lab.

Non conformity with any of the below requirements may result in your exclusion from thelaboratories. These rules are reiterated below.

1. Do not, at any time, endanger the health and well-being of your fellow students,tutors and supporting staff by acting irresponsibly within the laboratoryenvironment (ie. splashing reagents, playing with equipment, spreading blood

etc). Any such behavior will result in immediate expulsion from the class,

disciplinary action including a report to the Head of Department and/or Health

Faculty Dean.2. Laboratory coats and covered shoes MUST be worn, otherwise you will be

excluded from the laboratory.

3. Regardall substances as potentially hazardous or infectious.4. Smoking, eating or consuming beverages and the application of cosmetics is not

permitted.

5. In the event of an accident or injury notify your demonstrator immediately.6. All breakages must be reported to your demonstrator or the technical staff.7. Wear appropriate protective equipment when needed or instructed to. Gloves and

fume hoods are available (bring your own safety glasses) so think about the nature

of the substances or materials you are handling andprotect yourself.8. The recapping and removal of needles isbanned (most needle stick injuries occur

from this practice)

9. Dispose of all wastes to the correct bin,e.g.; Sharps Bin (syringes with needles, scalpel blades, Pasteur pipettes) Broken Glass (to appropriately marked bin)

Non infectious waste (paper, plastics, paper products)

Infectiouswaste (sample remains, disposable equipment, usedgloves,biological tissue and fluids into BiohazardBags)

10.Before leaving the laboratory wash your hands thoroughly.11.Never participate in any laboratory procedure unless you are well informed.

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12.Ensure that you are familiar with the location, and use of, the safety features of eachlaboratory.

13.Keep your work area free of any non-essential materials.14.The exits to laboratory should always be clear.15.In case of an accident, emergency or evacuation, obey staff instructions.16.Your workarea is to be left clean and tidy at the end of each session

Location of Safety Equipment in your Laboratory

Your laboratory contains an array of safety equipment for emergency situations; make yourself

familiar with their locations around the laboratory.

Emergency Shower Eye Wash/Hose Eye Wash Station Fire Extinguishers Fire Blanket First Aid Cabinet Emergency Gas Shut Off Valves Power Isolation emergency button

PERSONAL PROTECTIVE EQUIPMENT REMINDERS

Laboratory Coats

You will be excluded from laboratories if you forget your lab coat. Hire of the traditional cotton laboratory coats from technical staff has ceased. If you forget your lab coat, you may purchase a disposable lab coat from the Science

Store (see below)

Students may lose some class time while organising the purchase of disposable laboratorycoats.

Footwear

You will be excluded from the laboratory if you are wearing inappropriate footwear. There is currently no avenue to loan or hire appropriate footwear.

Safety Glasses / Timers / Marker Pens

Students are required to bring their own safety glasses, timers and marker pens tolaboratories. There are no facilities to loan these items. Limited stocks of these items areavailable for purchase on the Gold Coast campus (see below).

Laboratory Kit / Laboratory Coat It is compulsory for School of Medical Science students to purchase laboratory kits and

laboratory coats. These are available from the Science Store (on the GC campus; G26).

Student laboratory kit contents

Timer Marker Pen Safety Glasses

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HIGHLIGHT THE GROUP YOU ARE IN AND ENSURE THAT YOU ARE WELL PREPARED FOR CLASS/ASSESSMENT N.B. If no room number is given, meet at the lab

Week 1 MONDAY

24/06

TUESDAY

25/06

WEDNESDAY

26/06

THURSDAY

27/06; G16 1.11/1.12

FRIDAY

28/06; G16 1.11/1.12

GROUP 1

Pre-lab essentials &

pipetting

accuracy/reproducibility

Introduction to

Clinical BiochemistryHaematology

Morning (10-11): study and

question/answer session;

(11:15-12pm): Quiz

Morning (10-1 pm): Case

study preparation

Afternoon (2-5pm):

Presentations

BBQ (6pm)

GROUP 2 Pre-lab essentials &pipetting

accuracy/reproducibility

HaematologyIntroduction to

Clinical Biochemistry

Morning (10-11): study andquestion/answer session;

(11:15-12pm): Quiz

Morning (10-1 pm): Case

study preparationAfternoon (2-5pm):

Presentations

BBQ (6pm)

Week 2MONDAY

1/7

TUESDAY

2/7

WEDNESDAY

3/7

THURSDAY

4/7

FRIDAY

5/7; G16 1.11/1.12 (Q/A

only)

GROUP 1

Preparation for Clinical

Immunology/

Histo I (H&E)

Clinical Immunology IIHisto II

(PAS/Massons)

Practice core lab skills

(pipetting/solutions/blood/urine

analysis) and private study

Morning (10-11):

Question/answer session

Quiz (11:15-12pm)

Practical assessment

(12:30-3pm)

Practical assessment

(3:20-5pm + resits)

GROUP 2

Preparation for Clinical

Immunology/

Histo I (H&E)

Histo II

(PAS/Massons)Clinical Immunology II

Practice core lab skills

(pipetting/solutions/blood/urine

analysis) and private study

Morning (10-11): Question

and answer sessionQuiz (11:15-12pm)

Practical assessment

(12:30-3pm)

Practical examination

(3:20-5pm + resits)

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- 1 -

Pre-laboratory Essentials

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Introduction

Laboratory Essentials

The purpose of this lab session is to provide you with an opportunity to review fundamentalconcepts necessary for the successful completion of the practical components of this course.

This session will deal with the review of laboratory safety, dilutions, weights and molarity,

working with solutions, spectroscopy, the correct method for handling and care of pipettes,

making agarose gels, and the use of the light microscope.

Practical1.00 Laboratory Safety

Take a few minutes to familiarize yourself with the laboratory rules then answer then

following questions:

Do students owe a duty of care to others whilst attending lab sessions? Explain.

Name 2 types of gloves and explain when you would use them, respectively.

Name 3 pieces of personal protective equipment that must be worn at all times and what

they protect from.

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Name 6 items that constitute SHARPS waste? How do you dispose of SHARPS waste?

Name 6 items that constitute NON-SHARPS waste? How do you dispose of NON-SHARPS

waste?

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The Office of the Gene Technology Regulator (OGTR) has been established within the

Australian Government Department of Health and Ageing to provide administrative support

to the Gene Technology Regulator in the performance of her functions under the Gene

Technology Act 2000. This government department prescribes information, best practices,

and legislation regarding biological safety in research facilities. Inspect the PC2 basic

practices sign and answer the following questions:

What biological safety level is assigned to your laboratory? Explain what this means with

respect to working in the laboratory.

Whose responsibility is it to clean your workplace after experiments?

What should you do in the event of a chemical spill/any other emergent incident?

Technical Services Health provides high quality professional, scientific, and technical support

for academic activities at Griffith University. Technical officers are trained technicians that

spend hours of preparation behind the scenes ensuring that lab classes run smoothly. Always

obey the instructions of your technical staff and demonstrators whilst in the lab.

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1.01 Dilutions

Some methods require the dilution of reagents or samples (plasma, blood, urine etc) prior to

measurement. Accurate preparation of these dilutions is essential for reporting the actual

concentrations of the constituent being measured. Dilutions express the amount (volume or

weight) of a substance in a specified total final volume. The relationship betweenconcentration and volume is explored in this section.

A commonly used equation for preparing dilutions is

C1 X V1 = C2 X V2

Answer the following questions given the information supplied:

The reagent concentration (C1) is 20M. You transfer 3L of this reaction (V1) intoanother solution of water. The final volume (V2) of the solution is 80.0L calculate thefinal concentration of the reagent in the solution. Show all working.

The final concentration of a solution (C2) is 1.75mM in a final volume (V2) of 30.0L. Thevolume of the initial reaction transferred into the final solution was 2.15L (V1). Calculate

the initial concentration of the solution.

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1.02 Weights and Molarity

The basic expressions of concentration are

1. Weight per unit weight (w/w) Both solute and solvent are weighed, the totalequalling 100g. For example, 5% w/w solution contains 5g OF SOLUTE AND 95gOF DILUENT.

2. Volume per unit volume (v/v) The volume of solute/solvent per the total volume ofthe total solvent. For example, a 1% solution contains 1 mL OF SOLUTE PER 100

mL OF SOLUTION. (i.e. 1 mL + 99 mL)

3. Weight per unit volume (w/v) Concentrations of weight per unit volume arereported in grams percent or grams/100mL. For example a 5% solution contains 5 g

OF SOLUTE IN A FINAL VOLUME OF 100 mL.

If no expression is given, you can assume it is w/v.

The above units of concentration can also be expressed by the following equations:

1. x%_w /w =xg_solute

100g_total_solution

2. x%_v /v =xmL_liquid_solute

100mL_total_solution

3. x%_w /v =xg_solute

100mL_total_solution

4. The Molarity (M) of a solution expresses the concentration as the number of molesper litre of solution. One mole (mol) is the molecular weight of the substance in

grams.

5. Normality expresses concentration in terms of equivalent weights of substances.Equivalent weights are determined by the valence. A one normal (1N) solutioncontains 1 equivalent weight per litre. The equivalent weight of an element or

compound is equal to the molecular weight divided by the valence.

Explain in detail how would you prepare a 20% w/v solution of NaCl?

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Explain how would you prepare a 20% w/w solution of NaCl?

Calculate the following

a. What is the concentration (in M) of 50 M NaOH diluted 1 in 20?b. What is the concentration (in M) of 60M HCl diluted 1:4?c. What concentration is a 32 mmol/L glucose solution diluted 1 in 5 and then 1 in 2?

Describe in detail how you would make 500mL the following stock solution: 0.6 M Tris

(FW = 121.4), 60 mM SDS (sodium dodecyl sulfate is a denaturing detergent; FW = 257),

1% powdered milk (w/v), 1.3 M NaCl (FW = 45.5).

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Make a solution containing 50 mM NaCl (FW: 58.44) and 0.25% (w/v) skim milk in 100mL

of water. Ensure accurate weighing (

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1.03 Correct Pipetting

You will be working with air-displacement pipettes during this course. The correct handling

and care of these pipettes will be crucial to ensuring that you complete experiments

successfully. Read the following information and complete the subsequent questions:

Air-displacement pipettes are recommended for aqueous samples and for general lab work.

These pipettes have a cushion of air (termed dead volume) between the pipette piston and the

liquid sample. The piston is a permanent part of the pipette.

When the plunger is pressed the piston inside the instrument moves down displacing air. The

volume of air that is left in the pipette is inversely proportional to the volume of the sample

being aspirated.

Sororex Acura pipettes will be used for this course. There are 8 (Acura Micropipette) models

of these pipettes designed to pipette 0.1L through 1mL.

The pipettes are most accurate at their mid-range. The maximum volume for the pipettes is

shown on the top of the push button. To adjust the volume, hold the pipette in one hand and

use the other hand to rotate the thumb-wheel. To improve accuracy, always finish setting

clockwise. To avoid internal damage, never attempt to force the volume setting beyond

maximum limits. The volume ranges are shown in the following table:

Model Volume Range

P2 0.2-2L

P10 0.5-10L

P10Y 1-10L

P20 2-20L

P50 5-50L

P100 20-100L

P200 50-200L

P1000 100-1000L

There are two modes of pipetting that we will be primarily concerned with: forward pipetting,

and reverse pipetting. Reverse mode is useful for difficult/viscous samples, using this method

the liquid that remains in the pipette tip between aspiration and distribution compensates for

liquid film left inside the tip.

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Theforward mode entails 5 steps:

1. Preparation Hold instrument vertically, depress plunger to first stop

2. Aspiration Immerse pipette tip in liquid, allow plunger to return to rest position

3. Distribution Place tip at 10-45 inside receiving vessel, depress plunger to first stop

4. Purge Wait one second, depress plunger to second stop and hold, remove tip

5. Home Once tip is removed and all liquid is evacuated return plunger to rest position

The reverse mode entails 5 steps:

1. Preparation Hold instrument in vertical position, depress plunger to second stop

2. Aspiration Immerse pipette in liquid, allow plunger to return to the rest position

3. Distribution Place tip at 10-45 inside receiving vessel, depress plunger to first stop

4. Re-aspiration Repeat procedure aspirating/distributing not purging

5. Complete purge Depress to the second stop to completely purge once finished

Which pipette would you ideally use to pipette the following volumes of liquid:

1.2L, 12.4L, 50L, 85L, 186L and 200L, 320 L

Please have your demonstrator sign and date your book prior to leaving the laboratory class

Demonstrator: __________________________________Date: _______________________

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1.0 Introduction to ClinicalBiochemistry

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Introductory Case Study

The aim of 3023MSC is to introduce you to the laboratory techniques and diagnostic processes

that are commonly used in pathological laboratories. In this course you will study two patients

John Harris (Clinical Biochemistry, Haematology and Histology) and Steve Smith (Clinical

Immunology) and you will be performing a series of tests that will enable you to propose adiagnosis for them. While the diagnosis is important, how and why laboratory tests are

preformed and the quality control measures used to ensure accurate testing are equally important.

You will be assessed on your understanding of these procedures as well as your performance inthe laboratory class.

The case history is presented below.

J ohn Harris (46 years old) consulted his GP Dr. Andrews about joint pain, affecting hisknees and hips, increasing fatigue, right upper quadrant abdominal pain, shortness ofbreath and frequency of micturition.

Dr. Andrews noted that J ohn looked quite tanned, had evidence of osteoarthritis in hisknees and hips, and an enlarged firm liver.

J ohn revealed that his father had died of liver failure complicated by liver cancer, andthat two of his uncles had diabetes.

Thought Questions:

What is the definition of a symptom? List the symptoms described above. Dysfunction in which

organ system/s are associated the indicated symptoms?

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What tests might you run to determine the cause of the symptoms?

What are the implications for Johns family history?

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As part of your investigation, you collect blood and urine samples from John and order a full

blood panel and biochemical profile. The results are as follows.

Biochemical Profile

Test Result Reference range

Sodium (mmol/L) 140 135-145

Potassium (mmol/L) 4.1 3.5-5.0

Chloride (mmol/L) 97 96-109

Bicarbonate (mmol/L) 30 23-32

Anion gap (mmol/L) 10 5-15

Calcium (corrected; mmol/L) 2.42 2.10-2.60Phosphate (mmol/L) 1.2 0.8-1.5

Creatinine (mmol/L) 0.25 0.04-0.12

Total protein (g/L) 59 63-80

Total globulin (g/L) 28 20-40

Cholesterol (fasting; mmol/L) 7.6 3.9-6.19

Triglycerides (mmol/L) 3.1 0.6-1.7

Urea (mmol/L) 10.6 3 8

Uric acid (mmol/L) 0.40 0.24-0.42

List the test results that are abnormal. Indicate whether they are higher or lower than the

reference range.

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These results indicate dysfunction in which organ systems?

List three conditions that your patient might be suffering from.

Define the term differential diagnosis.

Name three additional biochemical test/s that could assist in diagnosing the patient? What

conditions could they EXCLUDE the presence of ?

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SD =( )

1

2

=

n

i xx

Co-efficient of variation (CV)The CV statistic is an estimate of the precision (reproducibility) of an assay and generally must

be below 5%. To calculate the CV, the standard deviation is divided by the mean.

100*(%)x

CV

=

If a population of results is normally distributed it can be completely described by the mean andthe standard deviation.

In this situation, approximately 68% of values are within one standard deviation away from themean, 95% are within two standard deviations and 99.7% are within 3 standard deviations of the

mean. These intervals are called confidence intervals and they are the basis of statistical quality

control rules for assay acceptance, and for the determination of population reference ranges formany analytes.

*** Note: 2 SDs should indicate 95.5%

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Quality Control Issues

A quality control may be assessed internally (within the lab performing the test) or externally

(by an external company or government organization). Generally speaking, laboratory assays are

monitored over time using quality control charts. Laboratory tests are performed on a dailybasis and the results for the control analyte are plotted daily as the mean standard deviation.

The control values should vary around the mean in a guassian (normal) fashion. A quality

control result should be within two standard deviations from the mean. A shift occurs when sixsuccessive values are on the same side of the mean. A shift is usually the result of a change of

reagent. A trend occurs when there are six successive values in one direction and is usually due

to degradation or denaturation of a substrate. An example of a quality control chart is shownbelow.

Levy Jennings Chart

-3

-2

-1

0

1

2

3

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Days

Quality control measures are included every time an assay is performed. Each time an assay is

run, one or more standard solutions (of varying concentration) will also be analysed in the same

way as the quality control and patient samples. This allows for the concentration of the analyte in

the quality control and patient sample to be determined. The variation of quality controls (overtime) will either be given to you (as a mean value plus or minus 2 SD) or you may be given a

quality control chart (like the example above) or a series of values which can be used to calculate

the variation of quality control values (usually a coefficient of variation

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the same way i.e. using standardized methods. A reference range is the range of values that

95.5% of the population would fall into (ie mean 2SDs).

Blood Biochemistry

Laboratory values of clinical importance may be presented in one of two ways, using the system

international (SI) or using what is termed conventional units (no abbreviation).

Conversions from one system to another can be made as follows:

1. mmol /L =mg /dL 10

molecular_weight

2. mg /dL =mmol /L molecular_weight

10

Reference Ranges:

Reference ranges (RRs) are influenced by a number of variables including the populationstudied, laboratory methods and instrumentation, and the type of containers used to collect

specimens. Also note that adult and paediatric values (particularly for analytes and special

function tests) differ significantly. Individual laboratory references must be used when

interpreting results.

Common measures of clinical importance include:

a) Haematological and coagulation measures (antithrombin III, bleeding time,

carboxyhemoglobin, differential blood count, erythrocyte sedimentation rate, ferritin,

fibrinogen degradation products, fibrinogen, folate, haptoglobin, Hct, iron, MCH, MCHC,MCV, PT, TT, methemoglobin, plasminogen, platelets, protein C, protein S, reticulocyte

counts, total eosinophil count, and vitamin B12 levels)

b) Clinical chemistry measures (albumin, aldolase, 1-antitrypsin, -fetoprotein,aminotransferase, aspartate, alanine, ammonia, amylase, angiotensin converting enzyme,

anion gap, arterial blood gases, -2 microglobulin, bilirubin, brain type natriuetic peptide,calcium, calcitonin, carbon dioxide tension, carbon monoxide tension, chloride, C-peptide,

creatine kinase, creatine, erythropoietin, gamma-glutamyltransferase, glucose, haptoglobin

A1C, homocysteine, lactase, lactase dehydrogenase, lipase, lipoprotein, magnesium,

myoglobin, osmolality, osteocalcin, oxygen saturation, pH, parathyroid hormone-relatedpeptide, phosphatase, phosphorous, potassium, prostate specific antigen, total protein,

albumin, globulin, sodium, transferrin, triglycerides, troponin I, troponin T, urea nitrogen,

and uric acid)

c) Metabolic and endocrine tests (adrenocorticotropin, aldosterone, cortisol, epinephrine,estradiol, follicle-stimulating hormone, gastrin, growth hormone, human chorionic

gonadatropin, 17-hydroxyprogesterone, 5-hydroindoleacetic acid, ketosteroids, lutenizing

hormone, metanephrine, norepinephrine, parathyroid hormone, progesterone, prolactin,renin, somatomedin-C, testosterone, thyroglobulin, thyroxine, thyroid stimulating hormone,

triiodothyronine, and vanillymandelic acid)

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d) Toxicology and therapeutic drug monitoring (acetominophen, amikacin, cocaine,

cyclosporine, digoxin, ethanol, gentamicin, ibuprofen, lithium, methadone, phenytoin,

procainamide, quinidine, salicylates, theophylline, tobramycin, and vancomycin)

e) Vitamins and selected trace minerals (aluminium, arsenic, folic acid, lead, mercury, vitaminsA, B1, B2, B6, B12, C, D, E, K, and zinc)

f) Cholesterol (total, VLDL, LDL, HDL)

g) Cerebrospinal fluids (pH, glucose, total protein, IgG, oligoclonal bands, CSF pressure,

erythrocytes, leukocytes, differential counts including lymphocytes, monocytes, and

neutrophils)

h) Urine analysis (amylase/creatinine clearance ratio, calcium, creatinine, eosinophils, glucose,

5-hydroxyindoleacetic acid, microalbumin, oxalate, pH, phosphate, potassium, protein,

sodium, specific gravity, and uric acid)

Lipid Profiles:

In this practical class you will be using the Cholestech LDX machine to perform a lipid profileusing the LIPID PROFILEGLU test cassette. This cassette is used to measure total cholesterol,HDL, triglycerides and glucose.

Cholesterol

Cholesterol (C27H45OH) is a monohydric alcohol (sterol) that is widely distributed in human

tissues, oils, fats, myelin, spinal cord, axons, liver, kidneys, and adrenal glands. Cholesterol is

synthesised in the liver and is a normal constituent of bile. It is the principal constituent of mostgallstones and of atherosclerotic plaques found in the arteries. It is important metabolite serving

as a precursor to various steroid hormones (e.g. sex hormones and adrenal corticoids). Total

cholesterol (TC) refers to the sum of both low and high-density lipoproteins.

Elevated blood levels of cholesterol increase a persons risk of developing coronary heart disease(CHD). Lowering elevated total cholesterol and in particular low-density lipoprotein (LDL)

reduces the risk of heart attacks.

Lipoproteins

Lipoproteins are conjugated chemicals in the bloodstream consisting of simple proteins bound to

fat. Cholesterol, phospholipids, and triglycerides are all fatty components of lipoproteins.

Analysing the concentrations and proportions of lipoproteins in the blood can provideinformation about a patients risk of atherosclerosis, coronary artery disease, and death.

Lipoproteins are classified as very low-density (VLDL), low-density (LDL), intermediate-

density (IDL), and high-density (HDL). Increased levels of LDL and total cholesterol directlyraise the possibility of CHD. By contrast, increased levels of HDL are linked with lowered riskfor CHD.

Elevated levels of lipoproteins may be the result of a diet high in fats, saturated fats, and

cholesterols or genetically determined. Symptoms unfortunately do not normally manifest until

patients develop artery blockages and at that time develop ischemic symptoms.

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Treatment of elevated lipoproteins include prescription of lifestyle modifying factors, exercise,

dietary changes, and drugs such as niacin, bile-acid binding resins, and statins.

Very Low-Density Lipoprotein (VLDL)

VLDLs are plasma lipids that are bound to albumin. They exist as chylomicrons and

prelipoproteins. This class of lipoprotein contains a greater ratio of lipid than the low-densitylipoproteins and is the least dense.

Low-Density Lipoprotein (LDL)

LDLs are plasma lipids that carry the majority of cholesterol in plasma. They are a proven cause

of atherosclerosis when bound to albumin. Treatment with a low-fat diet or drugs can lower LDL

and helps prevent and treat coronary artery disease.

Intermediate-Density Lipoprotein (IDL)

IDLs are plasma lipids that are bound to albumin and consist of lipoproteins containing less

protein than HDLs but more proteins than LDLs.

High-Density Lipoprotein (HDL)

HDLs are plasma lipids bound to albumin and contain the most protein of the lipoproteins. It iscolloquially known as the good-cholesterol.

Triglycerides

Triglycerides are any combinations of glycerol with 3 of 5 different fatty acids. They are also

known as triglycerols/triacylglycerols, or neutral fats. In the blood, triglycerides combine with

proteins to form the lipoproteins. The liver synthesises lipoproteins to transport fats to othertissues where they are used as a source of energy. Fat that is not used for energy is stored as

lipid.

Glucose Testing

Glucose and its metabolism

Glucose is a simple sugar/monosaccharide (C6H12O6). It is the end product of carbohydrate

digestion. The right-handed (dextrorotary) isomer (D-glucose) serves as the primary energy

source for humans. In healthy people, normal blood glucose levels are maintained at 3.8-6.1 mM.Low blood glucose (hypoglycaemia) presents as confusion, anxiety, and other neurological signs.

High blood sugar (hyperglycaemia) may result in the sugar coating (glycosylation) of body

tissues. Hyperglycaemia is characteristic of diabetes mellitus. Diabetes mellitus is diagnosed

when a fasting patient returns a blood glucose level exceeding 7 mM.

Glucose is the primary energy source for most cell types in the body. It is oxidised during cell

respiration to carbon dioxide and water to produce energy in the form of adenosine triphosphate

(ATP). Insulin facilitates glucose transport into cells. Excess glucose is converted to glycogenand stored in the liver or in muscles with the help of insulin and cortisol. Glycogen is a hormone

and stimulates the liver to turn glycogen back into glucose as needed.

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When glucose levels are low, fat stores are metabolised. Incomplete metabolism of fats leads to

the formation of ketone bodies, which are also a symptom of diabetes. Nervous tissue is

especially dependent on glucose as a source of its energy. The brain is able to oxidise glucosedirectly.

Diagnost ic Enzymes:Enzymes which are released from damaged cells into the plasma have been frequently used in

the last few years for diagnosis of organ-specific lesions. Some enzymes exist in different

molecular forms, called isoenzymes which can be used to assist in the diagnosis. For example,

following a myocardial infarction, there is marked elevation in serum levels of lactatedehydrogenase 1, and isoenzyme of lactate dehydrogenase which is found predominantly in

myocardium.

In this practical, we will be measuring levels of two enzymes which are markers of liver

function.

Alanine Aminotransferase (ALT)

ALT is an intracellular enzyme involved in amino acid and carbohydrate metabolism. It is

present in high concentrations in liver, muscle and brain. An increased level of ALT in the bloodindicates necrosis or disease in these tissues. The measure is primarily used in the differential

diagnosis of liver diseases and the tracking of the progression of disease. ALT was formerly

known as serum glutamic pyruvic transaminase (SGPT).

Aspartate Aminotransferase (AST)

AST is an intracellular enzyme involved in amino acid and carbohydrate metabolism. Like ALTit is also present in high concentrations in liver, muscle and brain. Elevated levels of AST in the

blood indicate necrosis or disease of these tissues. AST was formerly known as serum glutamic-oxaloacetic transaminase (SGOT).

Urinalysis

What is Urinalysis?

Routine urinalysis (UA) entails cellular, biochemical, and microbiological examination of urine.A clean-catch (mid-stream) or catheterised specimen is required and must be collected in a

sterile container.

Cell counts aim to determine if there are detectable levels of leukocytes, erythrocytes, or pus

(and sometimes also epithelium and sediment) in the urine. This session will not deal with cell

counts.

A convenient way to analyse biochemical measures such as SG, pH, glucose, ketones, hormones,

or drugs in the urine is to use a dipstick (such as SG10 Multistix). This session will entail the use

of SG10 Multistix for biochemical analysis.

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Methods of urine culture involve the preparation of cultures on selective or differential media.

Note that some media are both selective and differential. You will prepare cultures from supplied

urine samples in the Clinical Immunology practical.

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Practical

1.01Bradford Total Protein Assay

We will be using The Biorad Protein Microassay which is based on the Bradford method of total

protein estimation. It involves the addition of an acidic dye to a protein solution and subsequent

measurement at 595 nm. The dye primarily binds to basic and aromatic amino acids. Theabsorbance maximum of the dye (Coomassie Brilliant Blue G-250) changes from 465 nm to 595

nm when binding to protein occurs. We will be using Bovine Serum Albumin (BSA) of known

concentrations to construct our standard curve. The linear range for the microassay is 0 to 500g/mL.

1. Prepare your BSA protein standardsin Eppendorf tubes. For each concentration, calculate the

amount of stock BSA (10 mg/mL) and the amount of water you should add to each tube. The

final volume of your standard should be 1 mL.

Concentration

(g/mL)

Volume Stock

BSA (L)

Volume Water

(L)

0

100

250

400

500

Check your dilutions with your demonstrator before proceeding.

2. Pipet 10 L of each standard, quality assay control and patient samples into separatemicrotiter plate wells IN TRIPLICATE. Note that the patient samples have been diluted 1:500

for analysis.

3. Use the P200 pipette to add 200 L of dye reagent to each well. Mix the samples gently by

using the same pipette tip you add the reagent with (try not to make bubbles). Change the pipettetip for each well.

4. Incubate at room temperature for at least 5 minutes. Absorbance will increase over time;

samples should incubate at room temperature for no more than 1 hour.

5. Hand your plate to your demonstrator. Ensure that the plate is correctly labelled with your

name and the date. If you have bubbles in your wells, let your demonstrator know, before thetutor makes the reading (using a microplate reader). Your results will be returned to you.

Fill out the table on the next page by calculating the mean, standard deviation and co-efficient

of variation for your values.

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Concentration

(g/mL)

Abs 1 Abs 2 Abs 3 Mean SD CV

0

100

250

400

500

Using your data, draw a standard curve for your assay (include labelling and units on axes)

Calculate the concentration of each of your patient samples and the quality control.

Sample Abs 1 Abs 2 Abs 3 Mean SD CV Conc (g/mL)Patient 1

Patient 2

Quality/Assay

Control

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Compare your quality control value to the standard curve (range) given. What statement canyou make about your assay (is it working)? Note that the control is lyophilized serum and was

also diluted 1:500 prior to analysis.

Were any abnormalities observed in the patient samples? The reference range for total proteinis 60 80 g/L.

What conditions are indicated by high and low total protein levels? What are the two main

types of protein found within plasma/serum and the organ that synthesises them?

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1.02 Total Cholesterol, HDL, Triglycerides, and Glucose Panel

The Cholestech LDX LIPID PROFILEGLU test cassette is intended for quantitative

determination of total cholesterol, HDL, triglycerides, and glucose in whole blood. A TC/HDLratio and estimated values for LDL and non-HDL cholesterol are calculated.

1. Familiarise yourself with the Lipid Profile & GLU test

2. Perform the Lipid Profile & GLU test on your provided blood sample and interpret the results

Write out or paste your results below:

1.03 Alanine Aminotransferase (Glutamate Pyruvate Transaminase) and Aspartate

Aminotransferase (Glutamate Oxaloacetate Transaminase) Test

The ALTAST (GPTGOT) test cassette is an in vivo quantitative diagnostic test for

determination of ALT and AST in whole blood:

1. Familiarise yourself with the ALT & AST (GPT & GOT) test

2. Perform the ALT & AST (GPT & GOT) test on your own blood sample and interpret the

results

Write out or paste your results below:

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1.04 Multiple Reagent Strips for Urinalysis Method

SG10 Multistix are used to test for glucose, bilirubin, ketone (acetoacetic acid), specific gravity,

blood, pH, protein, urobilinogen, nitrite, and leukocytes in urine:

1. Familiarise yourself with the SG10 Multistix

2. Perform SG10 Multistix analysis on the supplied urine sample

What are the abnormal findings of the urinalysis?

What are the advantages and disadvantages of using the Multistix reagent strips?

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1.05 Glucose Tolerance Test

Your patient undergoes an oral glucose tolerance test. Using the data supplied, calculate your

patients blood glucose readings.

Results:

9 am 10 am 11 amQuality Control 0.62 0.56 0.55Standard Reading 1.05 1.03 1.04Patient Reading 1.30 2.33 2.91

Note: You assayed equal volumes of quality control plasma and standard solution. The

concentration of glucose in your standard is 5 mmol/L.

Time 9 am 10 am 11 am

Glucose (mM)

You must determine whether you can accept the patients results. To do this you are required to

establish the variation of quality controls for the assay (variation of quality control values over

time; ie. coefficient of variation). Calculate the glucose concentrations of the quality controls,knowing that the standard concentration is 5mmol/L. Use these data to answer the following

questions

Day Quality Control Standard Reading Glucose conc.

1 0.62 1.02

2 0.58 1.053 0.57 1.064 0.64 0.985 0.59 1.026 0.66 0.987 0.56 0.99

What is the concentration range and coefficient of variation of the quality controls for this assay?

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Can you report your patients results (ie. are they under the standard reading and is the quality

control coefficient of variation

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Based on the information you have so far, give a diagnosis for your patient.

Can you suggest a therapy for your patient?

Demonstrator: ____________________________________ Date: _______________________

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2.0 Introduction to Haematology

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Introduction

What is Haematology?

Haematology is the science concerned with blood and the blood forming tissues. Blood is a tissue

containing fluid that circulates through the heart, arteries, veins, and capillaries, carrying

nourishment, electrolytes, hormones, vitamins, antibodies, heat, and oxygen (O2) to the tissueswhilst taking away waste matter and carbon dioxide (CO2).

The Low-Down on Blood

Blood cells (BCs) are produced in the red bone marrow at a rate of approximately 2,400,000/s and

each red cell lives approximately 120 days. In healthy people the concentration of red blood cells

(RBCs) remains relatively constant with time. White blood cells (WBCs) and platelets are alsoproduced in the red marrow, however agranular WBCs are produced in the lymphatic tissues.

Human blood is approximately 52-62% plasma and 38-48% cells. The plasma component is

mainly water, ions, proteins, hormones, and lipids. The cellular components are the erythrocytes(red cells), leukocytes (white cells), and thrombocytes (platelets). The leukocytes compriseneutrophils, lymphocytes, and monocytes. Proteins in the blood include albumin, globulin, and

fibrinogen. The inorganic elements in blood include Na, Ca, K, Mg, PO, Fe, I. The organic

component of blood includes urea, uric acid, xanthine, creatinine, amino acids, neutral fats,phospholipids, cholesterol, and glucose, as well as enzymes such as amylase, protease, and lipase.

Blood constitutes approximately 7-8% of total bodyweight. The pH of blood varies from 7.35-

7.45. The specific gravity (SG) varies from 1.048-1.066, with this measure being higher in males

than females (the SG is also increased after exercise and at night).

In passing through the lungs, the blood gives up carbon dioxide and absorbs oxygen. It is then

carried to the tissues as arterial blood and subsequently returned to the heart via the venous

system. Blood cruises through the aorta at an average speed of 30cm/s and completes the entirevascular system circuit in about 60s. The main function of RBCs is carrying oxygen; the mainfunction of the WBCs is immune response to infection. Platelets are involved in clotting. The

plasma transports nutrients, waste products, hormones, carbon dioxide, and contributes to fluid-

electrolyte balance and thermal regulation.

Clinical Haematology

Clinical haematology is the science of determining whether the various components of blood are

present in the correct numbers and that the cells have normal morphology. This is called a full

blood count and includes a red blood cell count, white blood cell count and differential, plateletcount and measures of haemoglobin.

In the laboratory, haemoglobin can be measured in a certain volume of blood and functions as a

direct red blood cell measure. Other direct measures include the mean corpuscular volume

(MCV), which is the average volume of an individual cell, and a red cell count. A commonlyreported measure, the haematocrit, is the proportion, by volume, of the blood that consists of red

blood cells.

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Various RBC disorders, such as anaemia, can be identified by changes in RBC sixe or shape. The

qualitative analysis of RBCs is done microscopically by examining a peripheral blood smear.

Anaemia occurs when there is a decrease in red cell mass and is usually defined by haemoglobin

concentration less than 120 g/L in women, and less than 140 g/L in men. There are three generalmechanisms for the development of anaemia. First is decreased production of RBCs

(hypoproductive anaemia), which can result from nutritional deficiencies (iron, vitamin B12 orfolate), low levels of erythropoietin (such as chronic renal failure), bone marrow failure (aplasticanaemia), or replacement of normal marrow by malignant cells (leukaemia or metastatic bone

disease). Another cause of anaemia is increased destruction of circulating erythrocytes, as seen in

haemolytic anaemia.

Haemolysis is the destruction of RBCs due to disease (e.g. spherocytosis or sickle cell disease)or because of their exposure to drugs, toxins, artificial heart valves, antibodies, infections, or

venoms. The cell bodies are destroyed either directly or via antibody-mediated lysis.

Autoantibodies may develop as a result of disease (e.g. haemolytic cancers), in response to drugs

(e.g. -methyldopa) or in Rh-negative mothers carrying Rh-positive foetus. Viral and bacterialinfections are frequent causes of haemolysis in children, whose RBC membranes are fragile.

Organisms that can cause haemolysis include streptococci, staphylococci, and tetanus bacilli. It

may also occur in smallpox, diphtheria, and following severe burns.

When erythrocytes are destroyed, haemoglobin is released into the surrounding plasma and lostthrough the kidneys, turning the urine red. This condition is termed haemoglobinuria. When the

loss is gradual, patients compensate for the resulting anaemia reporting only fatigue and a slight

tachycardia, particularly with physical exertion. Laboratory tests show decreased RBC count,haemoglobin, haptoglobin, and haematocrit, and elevated levels of lactate dehydrogenase, and

conjugated bilirubin. Fragments of RBCs may sometimes be seen under the microscope.

Haematological diseases affect different components of the blood. It is important to consider the

possible source of disease (bacteria, virus, parasite, fungus) when considering diagnosis and

differential diagnoses. During this laboratory session we will revisit the concept of referenceranges (RRs) and reiterate the importance of building a logical symptom picture when

interpreting disease states.

Standard and Universal Precautions

Standard precautions are guidelines that are recommended by the Centres for Disease Control andPrevention to reduce the risk of the spread of infections (primarily) in hospitals. These

precautions apply to blood, all body fluids, secretions, excretions (except sweat), non-intact skin,

and mucous membranes of patients and are the primary strategy for successful nosocomialinfection control.

Universal precautions are guidelines designed to protect workers with occupational exposure to

bloodborne pathogens (such as HIV and hepatitis virus).

In the laboratory, treat all biological materials as potentially infectious.

Transmission of infection requires three things: a source of infection, a susceptible host, and a

means of transmission.

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Transmission may be direct or indirect, by droplet (e.g. coughing, sneezing, talking), by common

vehicle (e.g. food, water, medications, devices, equipment), or vector-borne (e.g. mosquitoes,

flies, rats, vermin).

Means of isolating transmission include hand washing and gloving, careful patient/infectiousagent placement, careful patient/infectious agent transport, use of personal protective equipment

(masks, respiratory protection, eye protection, face shields, gowns, other protective apparel), andeffective decontamination procedures.

Practical

The aim of this practical is to perform a series of basic haematological tests on your patientsample and to use this information to aid in diagnosis.

2.00 Haemoglobin (Hb) Determination Method

The main component of erythrocytes is a conjugated protein that serves as the vehicle for thetransportation of oxygen and CO2. A molecule of Hb consists of two pairs of polypeptide chainsand four haem groups each with one atom of ferrous iron. Haem is responsible for the

characteristic red colour of blood and is the site at which each globin monomer binds one

molecule of O2.

The concentration of Hb is expressed in grams per 100mL of blood, or alternately grams perdecilitre (g/dL). Haemoglobinometry can be grouped into four techniques:

a) Colorimetric - Hb is first converted to cyanmethemoglobin and its optical density measuredin a spectrophotometer or colorimeter then compared with a standard

b) Gasometric - indirect method which estimates Hb from the amount of O2 it bindsc) Densiometric - the weight of a volume of blood compared to the same volume of water.

Drops of blood are allowed to fall into a series of solutions of copper sulfate of known

density. These drops become temporarily coated with copper proteinate and settle if their

density is higher than the copper sulfate solution

d) Chemical based measures iron content by separating it from Hb, usually accomplished byusing acid1. Measure Hb in a blood sample using the Haemacue machine as demonstrated2. Add your results to the class results as directed by your demonstrator

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What is the haemoglobin measurement (including units)?

How does the measured Hb compare to the prescribed RR? Explain.

2.01 Haematocrit (Hct) Determination Method

The haematocrit represents the ratio of erythrocyte volume to whole blood and is also known as

the packed cell volume (PCV). It is expressed as a percentage or decimal fraction:

Hct=L1

L2

Where L1 is the height of the red cell column in mm and L2 is the height of the whole blood

specimen (cells and plasma).

A heparinised capillary haematocrit tube approximately 7cm with uniform bore of 1mm isgenerally used.

Perform the following procedure to determine Hct:

1. Use a heparinised Hct capillary tube (7cm in length, 1mm bore) to collect blood from the

blood sample

2. Seal the capillary tube with clay

3. Centrifuge the capillary tube at 5,000 RPM for 10 minutes

4. Determine the Hct (RBCs as a proportion of the total sample).

5. Add your results to the classes results as directed by your demonstrator

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What is the Hct measurement (including units)?

How does the measured Hct compare to the prescribed RR? Explain.

2.02 Blood Smear Method

The blood smear (also known as peripheral blood smear) consists of a drop of (anti-coagulated)

whole blood spread thinly on a glass microscope slide. It is used to examine, count, and

characterise blood cell types.

1. The slide must be grease-free; it is cleaned with alcohol, rinsed in warm water, andwiped clean with a lint-free towel or lens paper

2. A small drop of blood is placed on the slide with a pipette tip

3. The end of another slide (spreader slide) is placed against the first slide at a 45 angleand pulled back against the drop of blood so that the drop spreads between the points

of contact of the two slides

4. Then the spreader slide is pushed forward against the first slide creating a smear

5. The slide is air-dried and now ready for Wrights staining

Note: Changing the angle of the spreader slide, and increasing or decreasing the speed at which it

is pushed forward changes the thickness of the film. The ideal slide has a thick portion and a thin

portion with some overlap of red cells in the thick portion.

2.03 Wrights Staining The Blood Smear Method

Analine dyes are used extensively in blood work and are of two classes: basic dyes and acidic

dyes. Structures that take up basic dyes are basophilic while structures that take up acid dyes areacidophilic, oxyphilic, or eosinophilic. Structures that take up both are neutrophilic. Most stains

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are dissolved in methyl alcohol and thereby combine the fixing and staining processes. The best-

known stains are Giemsa and Wrights.

The Wrights stain is used to reveal malarial parasites and to differentiate leukocytes.

Wrights stain is a methyl alcoholic solution of eosin and a mixture of thiazines, including

methylene blue, azure B, and other derivatives.

Method

1. Cover the air-dried blood smear with Wrights solution

2. Equilibrate for 2 minutes

3. Add equal amount of distilled water

4. Leave undisturbed for 5 minutes

5. Gently wash slide with distilled water

6. Observe stained slide under microscope

What element of the blood smear did the Wrights stain highlight? Draw your results.

2.04 Blood cell counts using the haemocytometer method

Blood cell enumeration using a haemocytometer is a widely adopted method and continues to

have a place in most laboratories. The haemocytometer contains two chambers separated by an

H-shaped moat. Each surface contains a square area that is ruled into 9 big squares of 1mm3

each.

A cover slip is used and sits exactly 0.1mm above the ruled areas. When the chamber is filled thevolume of the cell over each square is exactly 0.1 mm

3.

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Counting leukocytes:

Use the four primary (corner) squares A, B, C, and D.

Counting erythrocytes:

Use five of the small squares at the centre square (the labelled ones).

Counting platelets:

Use all 25 of the small squares at the centre square.

The haemocytometer you will be using has two sets of grids, one at the top and one at the bottom ofthe slide. Use the top grid to carry out the RBC counts and use the bottom grid to carry out the

WBC counts.

RBC Count

1. Prepare a 1:200 dilution of blood in a final volume of 1mL in red cell diluent

2. Pipette 10L of the solution between the haemocytometer and a cover slip3. Count and record the number of cells in the 5 blacked out squares within the middlesquare. Include in your count all of the cells that are touching the left and bottom sides,

ignore the cells touching the top and right sides.

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Calculate RBC Count

Given the following formulae:

1. LCellsCountedSquares

CountedCells/1020025

_

_=

2.Cells_Counted

Squares_Counted2520010

4=Cells /mL

3.Cells_Counted

Squares_Counted 25 200107 = Cells /L

Calculate the red blood cell count.

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WBC Count

1. Prepare a 1:20 dilution of blood in a final volume of 1mL

2. Pipette 10L of the solution between the haemocytometer and a cover slip

3. Count and record the number of cells in cells A+B+C+D. Again, count the cells that are

touching the bottom and left sides of the squares but ignore the ones touching the top and

right sides.

Calculate WBC Count

Given the following formulae:

1. LCells

CountedSquares

CountedCells/1020

_

_=

2.Cells_Counted

Squares_Counted 20104 = Cells /mL

3.Cells_Counted

Squares_Counted 20107 =Cells /L

Calculate the white blood cell count.

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2.05 Mean Cell Volume (MCV) Method

The MCV is the average volume of a RBC expressed in m3

or in femtoliters (fL). Normal bloodcells have a MCV of 80-98fL. The MCV value helps differentiate macrocytic and microcytic

anaemia. Macrocytic anaemia can be caused by Vitamin B12 or Folate deficiency and is further

differentiated by testing the serum levels of those vitamins. Microcytic anaemia can be caused by

Iron deficiency or Thalassemia and is further differentiated by erythrocyte count (decreased inIron deficiency and elevated in Thalassemia).

Given the following formula:

MCV=Hct(%)10

RBC_Count(in_millions /L)

Complete the following questions:

Calculate the MCV. How does it compare to the prescribed RR? Explain.

2.06 Mean Corpuscular Haemoglobin (MCH) Method

The MCH is the weight of Hb in the average RBC of a specimen. The result is given inpicograms (pg). The normal MCH in adults is 27-31pg. The value is higher in newborns and

infants because their MCV is higher than adults. MCH differentiates hypochromic and

normochromic erythrocytes.

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Given the following formula:

MCH=Hb(g /dL)10

RBC_Count(in_millions/L)

Calculate the MCH. How does it compare to the reference range?

2.07 Mean Corpuscular Haemoglobin Concentration (MCHC) Method

The MCHC is the average concentration of haemoglobin in each individual red blood cell

expressed as a percentage. It is the percentage haemoglobin in the packed cell volume. Normal

values are rounded to tenths and range from 33-38 mg/dL.

Given the following formula:

( )%100)/(

Hct

dLgHbMCHC

=

Calculate the MCHC. How does it compare to the prescribe RR? Explain.

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Note that the MCHC is the ratio of the mass of the MCH and the mean MCV expressed as a

percentage. Therefore, any of these values can be calculated from the others if 2 of the 3 are

known. The following formulae express this relationship:

1. MCHC=MCH

MCV100

2. MCVMCHCMCH = 01.0

3.MCHC

MCHMCV

=

01.0

What are the common causes of anaemia?

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Name and describe three different types of anaemia. Describe what laboratory tests you would

use to distinguish between them.

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2.08 ABO Blood Typing

In this experiment you will perform an ABO/D blood grouping assay and a 3-well indirect AHG

Coombs test of your patients plasma using the Grifols perfect screen 3 cell blood typing system.

The patients direct AHG Coombs test result will be made available as a demonstration.

1. Familiarise yourself with the product procedure insert, in brief:

a) Prepare 1mL of 0.8% dilution of the providedblood sample in an eppendorf tube

b) Add 50L of prepared 0.8% dilution to each of wells A, B, and D

c) Add 50L of 0.8% screen cells 1, 2, and 3 to each of wells AHG1, AHG2, and AHG3

d) Add 25L of patient plasma to each of wells AHG1, AHG2, and AHG3

e) Incubate card at 37C for 15 minutes in a Grifols incubator

f) Centrifuge card in a Grifols centrifuge for 10 minutes

What is your patients blood type? Ensure that you show your demonstrator your blood typing

card.

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2.09 Collate your patients results into the following table and answer the questions.

Blood Panel

Test Result Reference Range

Haemoglobin 135-175g/L

Haematocrit 40-54%

Red cell count 4.2-5.3 x 1012

/L

Mean Corpuscular Volume

(MCV)

81-97fL

Mean Corpuscular HaemoglobinConcentration (MCHC)

300-360 g/L

Mean Cell Haemoglobin (MCH) 24-30 pg

Total white cell count 3.5-10.0 x 109/L

Neutrophils 4.62 x 109/L 1.5-6.5 x 10

9/L

Lymphocytes 1.79 x 109/L 1.2-4 x 10

9/L

Monocytes 0.70 x 109/L 0.0-0.9 x 10

9/L

Eosinophils 0.29 x 10

9

/L 0.0-0.6 x 10

9

/L

Basophils 0.07 x 109/L 0.0-0.15 x 10

9/L

Platelets 244 x 109/L

150-400 x 10

9/L

Erythrocyte Sedimentation Rate

(ESR)

7 1-15

Serum Iron 85 mol/L 9-29 mol/L

Ferritin 850 g/L 12-300 g/L (male)

Transferrin 150 g/L 204 360 g/L

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Which of these tests is abnormal?

What do these results indicate? Comment on the condition of your patient.

Identify 3 factors during testing (one for haemoglobin, one for WBC/RBC and one forhaematocrit testing) that might have affected the result obtained. How would these factors have

affected the results ?

Demonstrator: ____________________________________ Date: _______________________

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Introduction

What is Histology?

Histology is the study of the microscopic structure of tissue. The term normal histology is used to

denote the study of healthy tissue. The study of diseased tissue is known as histopathology

(histological pathology). Tissue samples are fixed in order to maintain the structural integrity ofthe sampled tissue, sectioned and mounted on glass slides. Tissue and cellular structure is

examined under a light microscope. Histological stains are used to highlight different features

and aid in diagnosis.

Histological examination of samples is used in the diagnosis of many diseases including cancer,infectious diseases as well as metabolic disorders.

Sampling and Processing Tissue

Tissue is sampled using a variety of techniques and is usually fixed in 10% neutral bufferedformalin. This is the foundation for subsequent stages in preparing sections. Fixatives are used

to prevent the tissue from degrading or changing shape and volume i.e. to stay as close aspossible to its living state. Fixed tissues are usually embedded into a solid matrix such asparaffin wax.

Paraffin is one of a series of saturated aliphatic hydrocarbons having the formula CnH2n+2.

Physically, it is a waxy, white, tasteless, odourless mixture of solid hydrocarbons obtained from

petroleum. Clinically it is sometimes used as a wound dressing or ointment base. Histologically,it is used to infiltrate and embed tissues for sectioning in the preparation of microscope slides.

The ideal section for most tissue is 3-4 m, this thickness gives good representation of cells andis convenient to transilluminate. Paraffin slides are often stained.

Haematoxylin & Eosin Stain

The haematoxylin and eosin (H&E) stain is a widely adopted method of staining tissues for

microscopic examination. Haematoxylin (C6H14O6) is an alkaline dye that stains acidic structures(such as cell nuclei) blue/purple. Eosin is any of several acidic dyes that stain alkaline structures

(such as cell cytoplasm) red/pink. Nuclei, ribosomes, and rough endoplasmic reticulum have a

strong affinity for this dye owing to their high content of DNA and RNA, respectively.

Periodic Acid-Schiff Stain

The periodic acid-Schiff (PAS) is a stain used in histology/pathology to demonstrate glycogen intissue by staining complex carbohydrates. Periodic acid selectively oxidises glucose residues and

creates aldehydes that subsequently react with Schiff reagent to create a purple/magenta colour.

This stain is often used to distinguish different types of glycogen storage disease. A sample issaid to be PAS-positive if it normally stains magenta.

Massons Trichrome Stain

The Massons trichrome is a three-colour staining protocol. The primary purpose of the stain is todistinguish cells from connective tissue. The stain may be formulated in different ways but

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generally produces red keratin and muscle tissue, blue/green collagen or bone, light red/pink

cytoplasm, and dark brown/black cell nuclei. Erythrocytes stain bright red with this technique.

The trichrome is applied by emersion in Weigerts haematoxylin, a plasma stain, and finally a

fibre stain.

Perls Prussian Blue Stain

In humans, iron is stored within a protein called ferritin in its ferric state (iron (III) oxidehydroxide). Several diseases result in deposition of iron(III) oxide-hydroxide in tissues in an

insoluble form. These deposits of iron are called hemosiderin. Although these deposits often

cause no symptoms, they can lead to organ damage. Perls Prussian blue is a commonly usedstain that is used to stain iron stores including hemosiderin.

The iron is released from hemosiderin with hydrochloric acid, forming ferric chloride. The iron

reacts with potassium ferrocyanide to form ferric ferrocyanide (Prussian blue).

What is Cancer?

In Australia, approximately 100,000 people are diagnosed with cancer each year. Despite thisthere is confusion and misunderstandings about the definition of cancer and the correct use ofterms such as neoplasm, tumour and cancer.

A neoplasm (literally new growth) refers to the uncontrolled growth of cells usually resulting

from changes in genetic material of the cells. These abnormalities can result from carcinogens

such as tobacco smoke, radiation, chemicals or infectious agents. Other cancer-promotinggenetic abnormalities may be randomly acquired through errors in DNA replication, or they may

be inherited.

By convention a neoplastic mass of cells is known as a tumour although the word tumour simply

means swelling.

Benign vs Malignant TumoursThere are two main patterns of neoplastic growth benign and malignant. A neoplasm is termed

benign if the margins of the tumour are well defined and the tumour grows locally. Malignant

neoplasms on the other hand have poorly defined margins and the neoplastic cells grow into and

destroy the surrounding tissues. Malignant neoplasms may also metastasize (spread to otherlocations in the body via lymph or blood).

A neoplastic tumour begins with transformation of a single cell which may present at any stage of

differentiation. This cell undergoes replication forming a distinguishable mass. In the early

stages, tumours are usually confined by an anatomic capsule or basement membrane and arereferred to as in situ. Encapsulated tumours are highly amenable to removal by surgical means.

However, if the membrane is breached and has invaded the surrounding tissue, the tumour canmetastasize and spread to other organs.

Rate of tumour growth depends on a number of factors including the rate of cell proliferation andthe rate of cell loss. Generally speaking, the faster the doubling time, the poorer the prognosis for

the patient if left untreated. Rapidly growing tumours are highly amenable to chemotherapy as

there are high numbers of rapidly proliferating cells. On the other hand, slower growing tumoursare more resistant to chemotherapy.

http://en.wikipedia.org/wiki/DNA_replicationhttp://en.wikipedia.org/wiki/Genetic_disorderhttp://en.wikipedia.org/wiki/Genetic_disorderhttp://en.wikipedia.org/wiki/DNA_replication

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Cancer Diagnosis

Cancer is diagnosed using a combination of techniques including clinical history of the patient,

physical examination, imaging (ultrasound, X-ray, CAT scan, MRI) and pathological

examination (blood tests, cytology, histology etc). You will cover all of these techniques in thelecture class but in the laboratory class we will focus on the histological evaluation of tumour

specimens.

Samples of suspected tumours can be collected in a number of ways, commonly, a biopsy is

performed to allow for diagnosis and the tissue is fixed, embedded and stained. Histology andcytology can also be used in the diagnosis of other diseases.

Practical

By now you should have an idea of your patients diagnosis (from Clinical Biochemistry). In

order to confirm your diagnosis and to check for other possibilities, you order a liver biopsy.

Once processed the liver samples are available for histological analysis. Prior to examining yourpatients tissues you will need to familiarize yourself with what normal and abnormal tissues look

like under the microscope.3.00 Identification of normal and abnormal tissues

The aim of this part of the practical is to identify normal and pathological tissues using a lightmicroscope. You will be provided with various prepared microscope slides (PMSs). Examine the

slides with naked eye first then under low, medium, and then high power.

Appendix B includes notes on the correct use of the light microscope.

Prepare scientific drawings of the following tissues paying particular attention to pathological

details:

Liver (normal; 4x objective magnification) Cirrhotic liver (4x objective magnification)

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Skin (normal; 10x objective magnification) Squamous cell carcinoma (10x objective

magnification)

Answer the following questions about microscopy:

What is the difference between objective and total magnification? Give an example, if the

objective magnification was set to 4x, what will be the total magnification observed ?

List 3 things that you should be cautious of when using slides and a microscope to prevent

damage to yourself and the microscope. What would these points help to prevent ?

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3.01 H&E Stain Method

Stain the following sections using the Mayers Haematoxylin and Eosin Staining Method (below)

then visualise the samples under the microscope:

1. 3-4m normal liver tissue2. 3-4m patient liver tissueMayers Haematoxylin and Eosin Staining Method

NOTE: Wear gloves at all times

Step Solution Time

1 Xylene (fume hood) 2 min Dewax

2 Xylene (fume hood) 2 min

3 100% Ethanol (fume hood) 1 min Rehydrate

4 90% Ethanol 1 min

5 70% Ethanol 1 min

6 50% Ethanol 1 min

7 Running water (sink) 1 min Stain

8 Haematoxylin 5 min

9 Running water (sink) 5 min

10 Eosin 2 min

11 Running water (sink) 1 min

12 70% Ethanol 1 min Dehydrate

13 90% Ethanol 1 min

14 100% Ethanol (fume hood) 1 min

15 Xylene 3 (fume hood) 2 min

16 Xylene 4 (fume hood) 2 min

17 Do not let the slide dry before cover-slipping with Slide Mounting Medium

(only perform with the help of a demonstrator). THEN LEAVE SLIDESTO DRY IN THE FUMEHOOD FOR 10 MINS.

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Prepare scientific drawings of your results paying particular attention to pathological details:

Normal liver sample (40x objective

magnification; be careful !)

Patient liver sample (40x objective

magnification; be careful !)

Answer the following questions about the H&E stain:

Identify the cell nuclei and the cytoplasm, what colour did each of these structures stain?

How does H & E stain your patients liver sample?

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3.02 Periodic Acid-Schiff Method

Stain the following sections using the Periodic Acid Schiffs Staining Method (PAS, below) then

visualise the samples under the microscope:

1. 3-4m normal liver sample

2. 3-4m patient liver sample

Periodic Acid Schiffs Staining Method

NOTE: Wear gloves at all times

NOTE: Ensure that xylene is used only in the fume hood

Step Solution Time

1 Xylene (fume hood) 2 min Dewax

2 Xylene (fume hood) 2 min

3 100% Ethanol (fume hood) 1 min Rehydrate4 90% Ethanol 1 min

5 70% Ethanol 1 min

6 50% Ethanol 1 min

7 Running water (sink) 1 min

8 1% Periodic Acid 10 min Stain

9 Running water (sink) 5 min

10 Rinse gently in distilled water

11 Schiffs Reagent 10 min

12 Running water (sink) 5 min13 Mayers Haematoxylin 1 min

14 Wash in running water (sink) 5 min

15 50% Ethanol 1 min Dehydrate

16 70% Ethanol 1 min

17 90% Ethanol 1 min

18 100% Ethanol (fume hood) 1 min

19 Xylene 3 (fume hood) 2 min

20 Xylene 4 (fume hood) 2 min

21 Do not let the slide dry before cover-slipping with Slide Mounting Medium(only perform with the help of a demonstrator). THEN LEAVE SLIDES

TO DRY IN THE FUMEHOOD FOR 10 MINS.

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Prepare scientific drawings of your results paying particular attention to pathological details:

Normal liver (4x objective magnification) Patient liver (4x objective magnification)

How does Periodic Schiff stain your patients liver sample?

Explain the mechanism of staining of the PAS-positive sample?

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3.03 Massons Trichrome Staining Method

Stain the following sections using the Massons Trichrome Staining Method then visualise the

samples under the microscope:

1. 3-4m normal liver sample

2. 3-4m patient liver sample

Massons Trichrome Staining Method

NOTE: Wear gloves at all times

Step Solution Time

1 Xylene (fume hood) 2 min Dewax

2 Xylene (fume hood) 2 min

3 100% Ethanol (fume hood) 1 min Rehydrate

4 90% Ethanol 1 min

5 70% Ethanol 1 min

6 50% Ethanol 1 min

7 Running water (sink) 1 min Stain

8 Distilled water rinse

9 Weigerts Iron Haematoxylin 5 min

10 Running water 1 min

11 Distilled water rinse

12 Massons red 2 min

13 Distilled water rinse

14 1% Phosphomolybdic-Phosphotungstic acid 10 min

15 2% Aniline blue 1 min

16 Distilled water rinse

17 1% Acetic Acid 1 min

18 90% Ethanol 1 min Dehydrate

18 100% Ethanol (fume hood) 1 min

19 Xylene 3 (fume hood) 2 min

20 Xylene 4 (fume hood) 2 min

21 Do not let the slide dry before cover-slipping with Slide Mounting Medium (only

perform with the help of a demonstrator). THEN LEAVE SLIDES TO DRY IN

THE FUMEHOOD FOR 10 MINS.

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Prepare scientific drawings of your results paying particular attention to pathological details:

Control Liver (10x objective magnification) Patient Liver (10x objective magnification)

What is the aim/purpose of the staining technique?

What colours were produced and what tissues do they represent?

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3.04. Perls Prussian Blue Stain

Your patients liver sample (and a control) has also been stained with Perls Prussian Blue. View

the results of this staining method and draw what you see below. Pay particular attention to

pathological details:

Control Liver (40x objective magnification) Patient Liver (40x objective magnification)

How does Perls Prussian Blue stain the liver samples?

What particular element does Perls Prussian Blue stain?

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Based on your findings, can you suggest a diagnosis for the patient?

What is the gene most commonly mutated in the genetic form of this condition?

What is the function of the protein product of the gene?

What is the risk of the John Harriss children being carriers of the mutation?

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3.05 Review Questions:

What is the difference between histology and cytology?

Why is tissue fixed in 10% formalin prior to histological analysis?

Name and describe two routine screening procedures for cancers?

Demonstrator: ____________________________________ Date: _______________________

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4.0 Introduction to ClinicalImmunology

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Introduction

What is Immunology?

Immunology is the study of the components of the immune system and their associated functions.

The immune system consists of lymphatic tissues, organs, and physiological processes that

identify antigens as abnormal/foreign and attempt to prevent these from harming the body. Theskin, mucosa, normal flora, of the gastrointestinal tract and skin, and chemicals contained in

tears, sebaceous glands, gastric acid, and pancreatic enzyme all protect the body from invasion

by pathogens. Leukocytes are produced in the bone marrow and are the primary internal defencemechanism. Lymphoid tissues, including the thymus gland, spleen, and lymph nodes, influencethe growth, maturation, and activation of leukocytes. Lymphoid tissue in the gastrointestinal tract

and mucous membranes contain leukocytes for site-specific protection. Physiologically active

protein mediators termed cytokines, help regulate the growth and function of immunologicallyactive cells.

Recapping the Immune System

Two sub-divisions of the immune system exist, innate and adaptive. The innate system is thenon-specific, non-memory, immediate response system. The adaptive is the specific, slower to

respond, memorising system.

The innate system involves inflammation, complement activation, and cellular barriers

(leukocytes). The adaptive system involves antigen presentation and antigen/antibody interaction.

An antigen (Ag) is a protein or oligosaccharide marker on the surface of cells that identifies the

cell as self or non-self, the type of cell that it is (e.g. skin, kidney, etc), stimulates the productionof antibodies by B-lymphocytes, and stimulates cytotoxic response by granulocytes, monocytes,

and lymphocytes. Matching antigens is important for tissue transplant and blood transfusion.

An antibody (Ab) is a protein produced by B-lymphocytes. Each antibody combines with a

specific antigen to destroy or control it. Antibodies are produced by B cells and are stimulated todo so by foreign antigens (protein, polysaccharide, or nucleic acid), except for neutral antibodies

(e.g. antibodies to different blood types).

The Role of Immunoglobulins

Immunoglobulins are a diverse group of plasma proteins consisting of polypeptide chains and are

a primary mechanism for protection against disease. Two forms of Ig exist. The first group exi