WEEK 3: LAB DIAGNOSIS: FROM SPECIMEN COLLECTION TO DIAGNOSIS

Question 1

A patient’s specimen is a ‘reflection of the patient’s condition at the time of collection’. Maintaining quality/integrity of specimens is vital for accurate laboratory diagnosis results.

What does Successful collection depend on?

Answer 1

Successful collection depends on:
*Collection at appropriate time
*Use of correct technique
*Use of correct equipment
*Safe & timely delivery to lab

Question 2

What is the health care practitioner’s role in specimen collection?

Answer 2

Health care practitioner’s role:

-Know requirement(s) for a lab’s investigation
-Initiate the collection procedure
-Collect the required & appropriate clinical specimen into the correct sterile specimen container
-Arrange prompt delivery under optimal conditions to the laboratory

Question 3

Clinical specimens should be collected ‘before’ antibiotic or antimicrobial treatment.

Why?

When unusual specimens are to be collected double check ‘specific’ laboratory requirements.

Answer 3

There will be few, or no bacteria detected by the tests as there are killed by the antibiotic.

Question 4

How is the investigation for organ specific disease different from Generalized diseases.

Answer 4

For Generalized diseases, on top of the Radiology and microbiology done in Organ specific diseases, there is need for hematology and chemistry immunology.

Question 5

Outline specific guidelines & or standards i.e., Occupational health & safety (OSHA) or Centre for Disease & Control (CDC) for specimen collection.

Answer 5

Wear gloves for contact with blood or other potentially infectious materials

If specimens are accidentally touched wash hands immediately

Eating, drinking, smoking, applying make-up prohibited: pathogens can be transmitted hand-to-mouth

No licking of labels that are to be applied to specimen containers

Breaks on skin must be covered with plasters or bandages

Specimen spills on work surfaces must be cleaned with appropriate disinfectants.

Question 6

State general equipment required for specimen collection.

Answer 6

Disposable gloves
Labelled specimen container
Laboratory request form
Swab in transport medium
Spatula
Sterile water
Needle and syringe
Tourniquet

Question 7

Many of the
-Chemistry (e.g., blood chemistry),
-Hematology (e.g., full blood count),
-Serology (e.g., immunology: antibody-antigen detection)
lab tests are performed on:

After collection these should be promptly transported to the lab, at what temperature should they be maintained at, and the delivery should be done within how much time?

Answer 7

*Whole blood
*Serum
*Plasma

After collection these should be promptly transported to the lab, maintained at 2-8C & delivered within 2-4 hours.

Question 8

ATT: whole blood, serum & plasma are not interchangeable matrices for lab tests.

1. What is serum?
   II. How is it obtained?
2. What is plasma?
   II. How is it obtained?

Answer 8

1. Serum: fluid attained after the clotting of whole blood. External to the body, blood clots spontaneously, when it is in contact with a surface e.g., glass or plastic.

Serum is the liquid portion of blood that remains after the blood has clotted and the clot has been removed.

II.Serum is formed by allowing a blood sample to coagulate or clot.

During coagulation, the blood cells, primarily red blood cells, white blood cells, and platelets, become trapped in a fibrin mesh, forming a blood clot. The liquid portion that separates from the clot is serum.
In a lab, the clotted blood is usually centrifuged to sediment the clot (containing the RBC’s) to allow for collection of serum.

2. Plasma is the fluid component of blood & is obtained when an anti-coagulant is added to whole blood which is then centrifuged to separate the cellular material from the liquid portion (plasma).

Question 9

Differentiate between plasma and serum.

Answer 9

1. So certain components will not be present in serum in comparison to plasma e.g., WBC’s, RBC’s, fibrinogen, platelets (& other clotting factors) absent in serum but present in plasma.

So certain blood analytes tested for different lab tests will not have the same counts within blood vs. serum vs. plasma.

2. The main difference between serum and plasma is that plasma is obtained by anticoagulating the blood sample, preventing it from clotting.

Question 10

Outline components of serum.

Answer 10

*Water
*Electrolytes (sodium, potassium, calcium, etc.), *Proteins (such as albumin, globulins, and other serum proteins)
*Hormones
*Waste products, and various metabolites.

It is a vital component of the circulatory system and plays a key role in transporting nutrients, hormones, and waste products throughout the body.

Question 11

What is the color of serum?

How many % of blood is made up by serum?

Answer 11

It is a clear, pale-yellow fluid.

It makes up approximately 55% of total blood volume.

Question 12

It is important to collect blood in the correct ‘vacutainers’ appropriate for the subsequent tests.

Some analytes do partition freely between the rbc’s & the plasma.

How will counts in blood vs. plasma be?

Ethylene-diamine-tetra acetic acid (EDTA) is used as an anticoagulant added to blood that will be used for several tests. But it is a chelating agent & sequesters metal ion e.g., Ca2+ & Mg2+.

How will counts for these in whole blood vs plasma be?

Why would serum (or plasma attained with a different anticoagulant from EDTA) would be more appropriate for testing for ALP?

Answer 12

Some analytes do partition freely between the rbc’s & the plasma. So counts in blood vs. plasma would be similar e.g. Parathyroid Hormone

Ethylene-diamine-tetra acetic acid (EDTA) is used as an anticoagulant added to blood that will be used for several tests. But it is a chelating agent & sequesters metal ion e.g., Ca2+ & Mg2+. So, counts for these in whole blood vs plasma, will be different.

Also, EDTA inactivates some enzymes that need a metal ion for their activity e.g., the liver enzyme ‘alkaline phosphatase’.

This may interfere with the accuracy of counts, so alternatively serum (or plasma attained with a different anticoagulant) would be more appropriate for testing for ALP.

Different anti-coagulants are used dependent on the test to be done.

Question 13

What are blood cultures bottles used for?

What do the following tops code for?

Yellow top –

Green top –

Orange top –

Black top –

Silver top –

Answer 13

Blood culture bottles

*Used for collecting blood for microbiological cultures, in cases of suspected bacteremia & septicemia:

Yellow top – pediatric (aerobic bacteria)

Green top – adult (aerobic bacteria)

Orange top – anaerobic bacteria

Black top – mycobacteria

Silver top – mycoplasma

Question 14

Tubes for blood collection

State the anti-coagulant found in the following tube and the samples they take.

1. Gold (also tiger top)
2. Red-top tube, plastic or glass
3. Lavender/ Purple-top tube
4. Grey-top tube
5. Light green-top tube
6. Dark green-top tube

Answer 14

Gold (also tiger top) top serum separator tube (SST)
Contains silica particles for clot activation & serum gel separator.
For serum collection for various chemistry, serology & immunology tests

2. Red-top tube, plastic or glass
   No clot activator; no anticoagulants or separator material.
   For serum collection, at times preferred to SST tubes because gel separator may interfere with analysis
   (for various chemistry, serology & immunology tests)
3. Lavender/ Purple-top tube
   Contains EDTA as an anticoagulant.
   Used for most hematology assays.
4. Grey-top tube
   Contains potassium oxalate as an anticoagulant.
   Sodium fluoride (to inhibit degradation of glucose in blood)
   Used for glucose determination in whole blood (& also lactate
5. Light green-top tube (lithium heparin)
   Contains lithium heparin & gel separator.
   Used for plasma determinations in routine chemistry tests (rarely used)
6. Dark green-top tube (sodium heparin)Contains sodium heparin.
   Used for the collection of heparinized plasma or whole blood for special tests i.e., ammonia, insulin, renin.

Question 15

What are microtainers also called?

Answer 15

Microtainers (pediatric tubes)

Question 16

To avoid cross-contamination of additives between tubes & altering the chemical composition of the blood, a specific order is used:
*Blood culture bottle
*Coagulation tubes - red top

Describe the Last draw - additive tubes in order.

Answer 16

Last draw - additive tubes in this order:
1. SST /red-grey or gold top (clot activator & gel separator)
2. Dark green top (sodium heparin)
3. Light green top (lithium heparin & gel separator)
4. Lavender top (EDTA)
5. Pale yellow top/ ACD (acid citrate dextrose)
6. Light gray top (Oxalate/fluoride

Question 17

Why is good aseptic technique imperative?

Answer 17

Good aseptic technique imperative to avoid ‘contamination’, by resident commensal bacteria or bacteria in environment.
*Contaminants may outgrow causative pathogen & falsify test results.

Supplies for specimen collection e.g., swabs & transportation (transport vials) must be sterile.

Clearly labeled: patients’ information, date, source, initials of person collecting

Question 18

Causative pathogens in clinical specimens can perish in-transit or be masked by the ‘overgrowth’ of contaminants.

State 3 precautions to be taken into account when transporting specimen.

Answer 18

1. Clinical specimens should be transported speedily & under optimal conditions to maintain the ‘viability’ of the pathogens.
2. Use a leak-proof primary receptacle

Specimen container caps/lids should be securely closed before placing them in the transport carrier. Leaking specimens will not be processed.

3. A completed requisition must accompany each specimen.

Question 19

When there are anticipated delays, several methods have been designed to facilitate the preservation of clinical specimens.

1. Transport media

What are transport media for the following microorganisms?

Bacteria
Anaerobic bacteria
Viruses
Chlamydia
Protozoa, helminths

Answer 19

Bacteria
Anaerobic bacteria
Viruses
Chlamydia
Protozoa, helminths

Question 20

Boric acid: in urine at a concentration of 1.8% (v/w) inhibits bacterial replication. NB. Lower concentrations are ineffective & higher ones may kill the pathogen.

Name the specimen.

Answer 20

Boric acid: in urine at a concentration of 1.8% (v/w) inhibits bacterial replication. NB. Lower concentrations are ineffective & higher ones may kill the pathogen.

Question 21

State 3 other mechanisms of specimen transport.

Answer 21

Specimens on ice: specimen should be first placed in the transport bag & requisition forms in the outside pocket of the bag. Then the bag placed in the ice.

Refrigeration: Storage at 2-8°C before processing will prevent multiplication of most bacteria.

Freezing: Temperatures of <-70°C are achieved in liquid nitrogen or special deep freezes. These temperatures will preserve many microbes.

Question 22

In the lab, clinical specimens ‘are’ the patient (at a time/ condition)

Interferences i.e., substances or processes that falsely alter tests may lead to unnecessary further tests; incorrect diagnosis; incorrect treatment & unfavorable outcome for the patient. These may include:

Answer 22

These may include:

• Patients’ co-morbidities i.e., other existing conditions may result in the presence of certain substances within the collected specimen e.g., in blood, urine.

*Errors during collection & transportation

Question 23

State examples of pre-clinical errors before Specimen transportation.

Answer 23

*Incorrect or incomplete patient identity &/ or details

*Incorrect phlebotomy & other collection procedures

*Contamination with IV fluid or get hemolyzed

*Contamination by commensal bacteria i.e., blood culture, urine, wounds

*Incorrect order of draw in which tubes are filled

*Inadequate: vol.; mixing, leading to incorrect ratio of ‘anti-coagulant: blood’ or unwanted clots in anti-coagulated blood

Question 24

Patient conditions resulting in:

Hemolysis; icterus; lipemia alter results of analytes in blood chemistry tests.

What is Hemolysis?

Answer 24

Rupture of red blood cells (RBC), release of hemoglobin & intracellular components into plasma (& serum).

Most common reason for sample rejection.
Serum or plasma appears pale red to cherry red.

Question 25

State 3 causes of hemolysis in specimen collection.

Answer 25

Hemolysis can occur due to:

*Mechanical trauma e.g., use of inappropriate syringe size with IV-line collections; use of very small gauge needles, improper transfer of blood from syringe to tube.

*Osmotic shock (exposure to non-isotonic fluids).

*Exposure to Temperature extremes (e.g., during transportation)

Question 26

How does hemolysis interfere with blood tests results?

Answer 26

Mechanism of interferences from hemolyzed blood samples

RBC constituents esp. K+ & lactate dehydrogenase are significantly higher in RBCs than in serum or plasma.

Hemolysis can lead to incorrect higher counts in plasma or serum chemistry results.

Question 27

Discuss pre analytical errors caused by long storage of blood.

Answer 27

For measurements of blood electrolytes (salts & minerals e.g., K+, Na+, Cl-) in serum, the collected whole blood needs be centrifuged ASAP.

Error: whole blood that is left too long, without centrifuging can affect the electrolyte & glucose levels:

RBCs metabolize glucose, this can continue during long duration of storage of collected blood, leading to erroneous lower blood glucose results.

Outside of the human body & during long storage of uncentrifuged blood: the Na+-K+-ATP pump on RBCs is arrested, causing K+ to seep out of the RBCs & Na+ to move into the RBCs causing higher K+ & lower Na+ levels.

Question 27

Which of the following are increased and decreased in blood tests due to hemolysis?

Bilirubin
Mg++, Fe, Ca++
Amylase
Bicarbonate
Lactate dehydrogenase
Creatine Kinase
K+
SGOT/ ASTS, GRT/ ALT

Answer 27

DECREASED
*Bilirubin
*Amylase
*Bicarbonate

INCREASED
*Lactate dehydrogenase
*Creatine Kinase
*K+
*SGOT/ ASTS, GRT/ ALT
*Mg++, Fe, Ca++

Question 28

What is plasma?
What is serum?

Answer 28

Plasma: from centrifugation of anti-coagulated blood. Hazy color due to presence of fibrinogen (& some fibrin). Also has albumin, globulins.

Serum: watery portion of blood remaining after coagulation. Free of fibrin, albumin.

Question 29

How does plasma vs. serum affect blood tests?

Answer 29

Fibrin can retain some water, so plasma has a higher vol. of watery portion. Plasma contains fibrinogen, so total plasma protein conc. is approx. 3% higher than serum

Platelets & coagulation factors activated when blood vessels punctured. This continues in sample tubes, platelets release small amount of K+ into serum during clotting process

Lysis of platelets results in release of components & some causes increase in these i.e., potassium, acid phosphatase.

Question 30

Coagulants & effects on analyte counts.

Name 3 coagulants not appropriate for determinations of lithium, ammonium & sodium.

Why is EDTA unsuitable for iron & calcium analyses?

Answer 30

Examples:
Lithium heparin, ammonium heparin or sodium heparin anticoagulants: not appropriate for determinations of lithium, ammonium & sodium

EDTA chelates iron & calcium, so unsuitable for iron & calcium analyses.

Also, can have inhibitory effects on alkaline phosphatase & creatine kinase activities (due to chelation of metallic co-factors)

WHAT DOES CHELATE MEAN?
*Chelation is a type of bonding of ions and molecules to metal ions.

Question 31

Give example of how EDTA affects blood tests.

Answer 31

Examples:
Transferring whole blood that was collected in a purple top (with EDTA-K2 anticoagulant into other tubes e.g. red tops, will affect electrolytes counts i.e. Mg2+, Zn2+ and Ca2+

EDTA is a chelator of electrolytes, so it will falsely decrease electrolyte levels in serum.

Also, will affect levels of enzymes tests as enzymatic activity is dependent of Mg2+& Zn2+ (which are enzyme co-factors)

Question 32

What is icterus/ hyperbilirubinemia?

List at least 3 causes of high bilirubin.

Answer 32

Icterus/ hyperbilirubinemia: high levels of bilirubin in blood.

‘Icteric serum or plasma’: color ranges from dark to bright yellow.

Due to liver diseases, biliary tract obstruction or inappropriate excretion, resulting in increased bilirubin.

Question 33

Describe mechanism of interferences by high bilirubin.

Answer 33

Mechanism of interferences
*Decreased & increased analyte values
*Spectral interference during color measurements

Question 34

Which of the following will be increased and decreased due to high bilirubin levels in blood tests?
*Cholesterol
*Triglyceride
*Creatinine
*Bile Acids
*Mg++
*Lipase
*Total Protein
*Uric acid

Answer 34

INCREASED
*Mg++

DECREASED
*Cholesterol
*Triglyceride
*Creatinine
*Bile Acids
*Lipase
*Total Protein
*Uric acid

Question 35

What is lipemia?

Answer 35

Lipemia: presence of excess triglyceride-rich lipoproteins (lipids, fats) in bloodstream resulting ‘turbid or milky’ plasma or serum.

Question 36

List 3 mechanism of interferences by lipemia in blood tests.

Answer 36

Mechanism of interferences

*Vol. displacement greatly reduces no’s of some analytes esp. Na+, K+ electrolytes

*Lipemia increases chances of hemolysis

*Non homogeneity of sample

Question 37

Which of the following are decreased or increased due to lipemia in blood tests?

*Na+
*K+
*Cl-
*Bicarbonate
*Lactate dehydrogenase
*Bile acids
*Direct bilirubin
*Magnesium

Answer 37

INCREASED
*Bile acids
*Direct bilirubin
*Magnesium

REDUCED
*Na+
*K+
*Cl-
*Bicarbonate
*Lactate dehydrogenase

Question 38

Describe 4 activities which are done at the laboratory reception.

Answer 38

1. Integrity of samples checked
   Unsuitable specimens: REJECTED
2. Request forms matched with the specimens & specimen details entered into laboratory management systems.
   Errors can be disastrous e.g., mix-up of patient results.
3. Unique laboratory numbers i.e., barcodes allocated specimen: of specimens & request forms labeled.
4. After processing samples e.g., centrifugation of blood e.g.:

Plasma+ buffy coat + red blood cells

Laboratory numbers become identification for the specimen.

Question 39

Where are many of the tests for chemistry & hematology are automated?

Answer 39

Many of the tests for chemistry & haematology are automated in routine clinical laboratories

Question 40

Outline laboratory tests done at microbiology labs.

Answer 40

Microscopy

Culture

Identification i.e. biochemical assays, mass spectrometry

Non-culture methods e.g. Immunological assays

Serology assays

Antimicrobial sensitivity testing

Question 41

*Wet mounts: unstained fluid material used for examining cells, eggs, crystals in specimens e.g., urine, Cerebrospinal fluid, feces, vaginal secretions.

*Staining e.g., Gram & Acid-fast staining

*Fungi or parasites special stains or
concentration techniques required.

What microbiology test is used for the above?

Answer 41

Light microscopy

Question 42

What do we look for in microscopy?

Answer 42

1. Visual analysis:
   Color, consistency & inclusions e.g., blood, mucous, worms.
2. Wet preparation & microscopy:
   *Emulsify stool in saline & iodine. Examine at x10 & x40 magnification:

*White blood cells may indicate invasive infection NB. transport media may compromise nos.

*Red blood cells i.e., characteristic of E. coli serotype 0157

*Mucous

*Ova

*Parasites

Question 43

Describe the principle of culture in microbiology.

Answer 43

Culture media (solid, liquid) & incubation conditions mimic natural environmental & nutritional requirements of bacteria e.g.

*Source of carbon & energy

*Source of nitrogen

*Trace elements

*Environment. e.g., gaseous composition; temperature, pH

Question 44

Define the following terms:

Mesophiles
Psychrophiles
Thermophiles
Extremophiles

Answer 44

Pathogens that replicate on or in human body grow at ~36.5-40°C. referred to as’Mesophiles.’

Psychrophiles(cold loving): capable of growth under normal refrigeration temperatures (0-8°C)

Thermophiles(heat loving, e.g., 41-122 °C) i.e., hot springs, Sulphur springs.

Extremophiles (Most Archaea bacteria): bizarre physical conditions e.g., under enormous pressure (e.g., ocean floor)

Question 45

Define the following terms:
*Aerobic Bacteria:
*Microaerophilic Bacteria:
*Facultative Anaerobic Bacteria:
*Anaerobic Bacteria:
*Aerotolerant Anaerobes:
*

Answer 45

Aerobic Bacteria:

Oxygen Requirement: Aerobic bacteria require oxygen for growth. They can use molecular oxygen (O2) as the final electron acceptor in their metabolic pathways.
Metabolic Pathways: They primarily use aerobic respiration to generate energy.

Microaerophilic Bacteria:

Oxygen Requirement: Microaerophiles require low levels of oxygen (typically less than atmospheric levels) for growth. High oxygen concentrations are inhibitory to them.
Metabolic Pathways: They use aerobic respiration, but they are sensitive to high oxygen concentrations.

Facultative Anaerobic Bacteria:

Oxygen Requirement: Facultative anaerobes can grow in the presence or absence of oxygen. They are versatile and can switch between aerobic and anaerobic metabolism, depending on the availability of oxygen.
Metabolic Pathways: They can use both aerobic respiration and fermentation for energy production.

Anaerobic Bacteria:

Oxygen Requirement: Anaerobic bacteria do not require oxygen for growth and are often inhibited or killed by its presence. They can only grow in environments devoid of oxygen.
Metabolic Pathways: They primarily use anaerobic respiration or fermentation for energy production.

Aerotolerant Anaerobes:

Oxygen Requirement: Aerotolerant anaerobes can tolerate the presence of oxygen, but they do not use it for growth. They can grow in the presence of oxygen but do not perform aerobic respiration.
Metabolic Pathways: They primarily use anaerobic respiration or fermentation for energy production.
It’s important to note that these classifications are not always absolute, and some bacteria may exhibit flexibility in their oxygen requirements under different conditions. Additionally, oxygen tolerance can vary among different species and strains of bacteria.

Question 46

Describe the 2 types of Culture media.

Answer 46

Defined/ Simple media e.g., nutrient agar.

*Exact quantities of inorganic & organic components are known.

Complex media

(highly nutritious, due to addition of plant or animal extracts)
Enriched (i.e., for fastidious organisms)
e.g., blood (heated blood used for Chocolate media), beef extracts, yeast extracts, serum.