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445 Cards in this Set

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Hemato / poiesis
blood production
Blood cell production normally occurs in the__________ and is carefully regulated
bone marrow
The ability of the cell to go through cell cycle and make more cells
cell proliferation
The ability to become a functional cell
Dedicated to a cell lineage
RBC, WBC, PLT
cell differentiation
Orderly process, identified by cell morphology
Carefully regulated
functional maturation
Normally occurs in fetal development or
During disease states in adult life when bone marrow cannot provide enough red cells
extramedullary hematopoesis
splenic
hepatic
both are what?
spleen
liver
extramedullary hematopoesis
medullary hematopoesis occurs where?
during what ages of child?
during what ages and where on a adult?
Bone marrow

Primary site at birth
100% BM involvement until 4 yrs when
fatty tissue begins to replace active BM

Limited to specific bones in adults
pelvis, vertebrae, ends of ribs, femur, humerus, sternum, skull
adult marrow is what percent adipose and what percent cellular?
what about older adult?
50:50
older adult 70adipose and 30 cellular
descibe living tissue
- soft, spongy
- very vascular
- rich blood supply
what is a precursor to platelets
megakaryocyte
what makes up 5% of all cells in the blood
hematopoietic stem cell
what part of the BM controls the growth
extracellular matrix
________ _____ is released from functional cells and causes further stem cell proliferation
c-kit ligand
chemical signals
May be lineage specific
Are synergistic
cytokines
binding sites for chemical signals
Are found on the cell surface
Bind cytokines and growth factors
Receptors
Hormones
c-kit ligand is released from functional cells and causes further stem cell proliferation
May be specific (EPO, G-CSF)
Growth factors
stem cells have what antigen
CD 34
Morphologically resemble a lymphocyte
high N:C
light blue cytoplasm
purple, open nucleus
stem cell
mitotic cycle
Overall cell size doubles
State of proliferation determines relative cell size
Resting state has condensed, non-active DNA

resting, g1, s, g2, m, resting
how much blood do we have
6-8L
plasma is composed of what and is what %
water, ions, nutrients
(55-60% of total blood volume)
plasma minus clotting factors
serum
Red cell mass measured as HCT
Spun HCT= what?
Spun HCT= (100 – [plasma + Buffy coat])
RBC
size and life span
6-8 microns in size

Life span = 120 days
what does hemoglobuin do?
binds to oxygen
carries H+ ions (buffers blood)
carries some carbon dioxide
leukocyte size
8-25 microns
thrombocyte
platelet
what are platelets- what do they do?
whats there size and lifespan
cytoplasmic fragments that are produced in BM

Function in hemostasis and survey the vascular system to keep it intact
Provide a reaction surface for attachment of circulating clotting factors
Form a stable fibrin/platelet plug
Size 2-3 microns
Life span = 7-10 days
what hematology test can be preformed from a EDTA or WB and with a cappilary specimen?
CBC
Hgb & Hct
Indices, RBCM
Differential
Reticulocyte count
Platelet count
what hematology tests can be preformed with citrated plasma and not a capp specimen?
Protime, aPTT
Factor Assays
What hematology test can be preformed with an EDTA, WB, or citrated plasma but can not be a capp specimen?
ESR
what kinda of stain is used for a PBS
wright
what kind of specimen can you use with PBS and where should they be collected?
use a EDTA, whole blood specimen
venous
capillary
what two tests should you not use with a PBS and why?
Heparin
distorts morphology
clumps platelets

Na citrate
causes a dilutional error
Blood smears should be made within __ hours
Smears made after ___ hours are unacceptable
Blood smears should be made within 2 hours
Smears made after 4 hours are unacceptable
Good for bone marrow comparisons and multiple stains
coverslip smears
what angle should you have with wedge smear
35-45
what is WRight stain composed of
Polychrome methylene blue
Eosin
Methanol (absolute)
Potassium and sodium phosphate buffers
Na2HPO4 buffer
KH2PO4 buffer
pH = 6.4-6.8
Segs & bands
(neutrophils)
Lymphocytes
Monocytes
Eosinophils
Basophils
Segs & bands ~60%
(neutrophils)
Lymphocytes ~35%
Monocytes ~10%
Eosinophils ~ 3%
Basophils ~0-1%
with a too acidic stain what do the following look like?
nucleus
cytoplasm
rbc
eosinophils
Nuclear characteristics are pale blue
Cytoplasm looks gray instead of blue
Red cells appear bright red/orange
Eosinophils granules are brilliant red-orange
Cells lack contrast
what would cause the stain to be to acidic
Stain time too short
Extended buffer time
Excess washing
Old stain
Buffer or wash water have acidic pH
what would an alkaline stain look like?
nucleus?
neutrophil?
rbc?
eosinophils?
lymphocyte?
Nuclear details appear deep, dark, purple-blue with little contrast in chromatin pattern
Neutrophil granules appear large and toxic
Red cells are blue-green
Eosinophil granules are blue-gray
Lymphocyte cytoplasm is gray-lavender
what would cause an alkaline stain?
Smear too thick
Extended staining time
Insufficient washing
Buffer or water too alkaline
Fresh stain, not aged
causes of stain precipitate?
unfiltered stain
PBS dried flat
excessive drying during staining process
insufficient washing
dirty slide
for a PBS at 10x what should be done?
Choose the correct area
50% of red cells should be touching
RBC morphology
Rouleaux
Agglutination
Cell size, shape, color
Check feathered edge for platelet clumping
Look for parasites-microfilaria at edges
COUNT WBC
1 WBC (LPF) = ? cells/uL
200
what should be done when using 100X
Immersion oil has the same refractive
index as glass so no light is lost by
refractive scatter
Higher power objectives require more light
Condenser up & fully open, field aperture fully open
what is done on HIGHPOWER
WBC DIFF
RBC morph
platelet estimate
1 platelet x 15 K = units?
1 platelet x 15 K = plt/uL
to get the absolute cell count what do you do?
Absolute Cell Count = (Relative %) x WBC count/uL

wbc you got from 10x
relative if from WBC diff
Coarse, dense chromatin
Fine pink granules
Life span = hours
pink to tan color
neutrophils
when are banded neutrophils seen
under stress
neutrophil ranges:
relative
absolute
Relative 47-80%
Absolute 1800-7700 cells/uL
cytoplasm scant
clear to medium blue cytoplasm
dark, dense, lumpy chromatin
nucleus usually round
Life span = years
lymphocyte

when active they are larger
what is specific about neutrophil chromatin?
coarse dense
Lymphocyte ranges:
relative
absolute

(children)
12months
6years
Relative 13-40 %
Adult Absolute 1000-4800/uL
Pediatric
12 months 4000-10,500/uL
6 years 1500-7000 /uL
Abundant, gray-blue cytoplasm, fine ground glass granules
Nucleus is round or kidney bean shaped,brain-like convolutions
Chromatin is open “lacy”
has vacuoles in cytoplasm
monocyte
monocyte ref ranges:
relative
absolute
Relative 2-11%
Absolute
200-800 cells/uL
Color is pH dependent with Wright stain
Large red/orange granules contain antihistamine
Increases are found with parasitic infections and allergic reactions
Eosinophils
eosinophils are found when and what do their granules contain?
Large red/orange granules contain antihistamine
Increases are found with parasitic infections and allergic reactions
eosinophil ref ranges:
rel
abs
Relative 0-3%
Absolute
0-500 cells/uL
dense, segmented nucleus
large, coarse, blue-black granules
cytoplasm may be degranulated
granules contain heparin, histamine
basophil
basophil ref ranges
rel
abs
Relative 0-2%

Absolute
0-200 cells/uL
platelet ref range:
abs
150-450 K/uL
what is platelet satellitism
Platelets are functionally potent and react with a variety of protein receptors and negatively charged surfaces, such as glass,
collagen, basement membrane
Both rely on cells settling and measuring red cell mass
hematocrits and ESRs
microhematocrit
volume < 0.1ml
Spun crit
Capillary collection acceptable
macrohematocrit
volume 1.0 ml
ESR
Capillary collection unacceptable
hematocrit give you direct and indirect measurements of what?
Direct measurement of red cell mass
Indirect measurement of oxygen carrying potential of the blood
Volume of packed red cells/volume of WB
hematocrit
Expressed as L/L or % of WB
hematocrit
Hct is evaluated to assess _____
anemia
refers to a low volume of packed red cells/whole blood volume
anemia
Acute loss of red blood cells
Chronic loss of red blood cells
Inability to make red blood cells

all cause what?
anemia
when do you decide on a transfusion?

what is a critical call value for HCT
when HCT < 30%

<18%
HCT can tell you 4 things
transfusion need
renal function: Erythropoiesis
Function of EPO stimulating red cell production

determines blood loss

dehydration: HCT is elevated
Loss of tissue fluids (plasma) concentrates red cell mass
expressed as percent
hematocrit
if 15 g/dL is the hemoglobin value then what is the Hematocrit value
For Hct = HGBx3 +/- 3

so for 15g/dL Hgb
15x3 +/- 3 = 45% +/- 3

You would expect a 15g/dL Hb= 42%-48% Hct
what is the normal % for hematocrit
45%
how is HCT calculated?
Hct = MCV x RBC divided by 10

MCV is measured directly as the individual cell size and then a mean is calculated for the population of red cells
what are sources of error for manual HCT
Centrifugation
Maximum cell pack requires correct centrifuge speed
1-4% plasma is trapped in the packed red cells giving a falsely elevated manual HCT reading
Misreading of the red cell, clay or plasma interfaces
Insufficient mixing of WB specimen before sampling
what are sources of error for calculated HCT
Anything that alters the red cell size
Hyperglycemia – cells swell
Hypernatremia – cells swell
WBC counts > 35,000/uL affect the MCV
Coincidence counting
Cell size alters in storage
Insufficient mixing of whole blood alters total red cell count
what is hyperglycemia and what does it do to RBC
high glucose content in WB cause RBC to swell
hypernaremia
what is it and what does it do to RBC?
high sodium in blood and causes RBC to swell
what can cause error in bother calculated and manual HCT
Short draw has a dilutional effect with liquid anticoagulants

Incorrect anticoagulant – heparin crenates (spiky) RBCs upon standing

IV fluid from central lines may dilute sample

Tissue fluids alter according to time of day

Patient hydration/IV status

Hemolysis – destroys red cell mass
top 5 lab errors with HCT
Improper mixing #1 lab error
Age of specimen
Improper reading
Problems with high WBC count
-Buffy coat
Adult reference range for HCT AND HGB
Males and females
Adult reference range
Males 41 - 53%Hct
Hgb 13-17 g/dL
Females 36-46% Hct
Hgb 12-15.2 g/dL
REF RANGES FOR HCT AND HGB

BIRTH
8WEEKS
6MONTHS
12MONTHS
6 YEARS
Birth 63% Hgb ~20 g/dL
8 weeks 31% Hgb~ 10g/dL
6 months 36% Hgb ~12g/dL
12 months 35% Hgb ~12 g/dL
6 years 38% Hgb ~13 g/dL
HCT DECREASE AFTER 65 YEARS OLD WHY?
BM becomes less productive
70% adipose tissue
what does high altitudes do to HCT
increases
pregnancy _____ hct.... explain why?
decreases

Increase in red cell production
Greater increases in tissue fluids
Net decrease in red cell mass/total blood volume
smokers hct is ________ explain why
increased

Carbon monoxide binds to Hgb so that O2 cannot
Therefore, more red cells must be produced to carry the same amount of O2
newborns have an ______ HCT why?
increased
Intrauterine life is hypoxic
what is ESR? what is it measured in and how is it measured?
Rate at which cellular elements settle out of a sample of whole blood – no centrifugation

measured in mm

distance fallen in 60 min
What is the Principle of the ESR... how does it work?
RBCs have a negative charge
-Sialic acid residues
-RBCs repel each other
-Zeta potential

Asymmetric plasma proteins decrease negative charge, thus decrease zeta potential

As zeta potential is overcome, cells stack together

Cells stack in Rouleaux
Density overcomes surface area
Clumps of cells fall
what are the phases of ESR... in mins
10 min Rouleaux formation takes place
40 min sedimentation phase
10 min packing phase
agglutination happens due to what?
antigen antibody interaction
what do increases in ESR indicate?
is esr a good screening tool?
Increases indicate an active inflammatory process is occurring

yes
Hyperfibrinogenemia does what to ESR and is caused by what?
– increases ESR
Tissue necrosis
Infection
Pregnancy
Haptoglobin does what to ESR and is caused by what?
Haptoglobin increases ESR
increase Chronic infection
increase Malignancy
what is CRP
c reactive protein factor in ESR
High RBC counts/plasma volume result in less plasma and therefore a net ____ in proteins present to reduce zeta potential
decrease
Biconcave discs Rouleaux best
because ____ and _____ are slower at compacting...
Spherical cells are slower
Sickle cells are slower
what are some sources of error with ESR
Tube length and bore diameter

Tube position
Tilt from vertical
3% tilt causes a 30% change in result

Temperature
-Use ambient
-Refrigerated, stored samples have increased viscosity
-Samples must come to room temperature before setting up

Surface vibrations
Centrifuge on counter with sed rate rack
Refrigerator motor

Hemolysis

Bubbles don’t allow cells to settle out
the older the patient the _____ in the ESR
increase
with ESR what anticoags should be used
EDTA
Na citrate
Oxalate shrinks cells
Heparin alters zeta potential
ESR is _______ in females
higher
westergren, wintrobe and mini ves are all what?
ESR tests
westergren
EXTRA DILUTION STEP and we used in lab
Tube: 230mm x 2.65mm
180 gradations
Sample: 1.0 ml of diluted blood
Time 1 hour
Normal ranges
0-15 mm/hr male < 50yrs
0-20 mm/hr female < 50 yrs
0-10 mm/hr children
> 65 years increased normal
wintrobe
Tube: 115mm x 3.0 mm
100 gradations
Sample: 1.0 ml WB
Time 1 hour
Normal ranges
0-15 mm/hr males
0-25 mm/hr females
mini ves
Velocita Erthrocyte Sedimentation
Semi-automated method decreases time to settle
Results read in 20 minutes
Tube deliberately set at 18 degree tilt
what is good about westergren and what is is closely compared to?
Westergren uses a larger scale

More sensitive to slight changes

Ideal for following changes in rheumatological disease monitoring

Decreased, or normal ESR indicates medication is working
or indicates disease remission

Mini-VES compares closely to Westergren ESR
buffers blood by controling pH and is ____ the RBC volume and ____% the RBC weight, and is a protein
hemoglobin
1/3
90%
Both the _______ and ______keep Hgb functional
RBC membrane and metabolic pathways
three things that could effect Hgb structure
Hb polymerization
Hb crystallization
Hb denaturization
describe hemoglobin structure
Hemoglobin is a tetramere


Heme + globin

4 units /hgb molecule; two dimer protein pairs

A heme unit is attached to each globin chain

Oxygen attaches to the heme unit
Each globin chain is a ____structure of folded segments of amino acids.
tertiary
what are the adult Hb Dimers
alpha2beta2 HbA1 97%
alpha2 gamma2 Hb F 1%
alpha2 delta2 HbA2 2.5%
when would you see:
Epsilon chains
Zeta chains Hb Gower I
Gamma chains Hb Portland
first 20 days - yolk sac
when would you see:
Gamma chains
Alpha chains HbF
6 weeks gestation- fetal liver
when would you see:
Alpha chains
Beta chains HbA1
20 weeks gestation -fetal BM
when would you see:
Alpha with Beta-Gamma-Delta chains
birth- BM
what chain does HBF have and where is it produced?
fetal hemoglobin
predominant in fetus
made in liver and spleen
2alpha and 2 gamma chains
___ comprises 90 -95% of HGB in fetus
HBF
____ has higher affinity for oxygen then HB A1
HBF
what is HGB - A1 composed of and who has it>
adults and alpha 2 and beta 2 chains
Beta chain synthesis does not exceed gamma chain synthesis until _____
after birth
Following birth, _____ will increase for the next 12 months
HB A
a small amt of _____ is produced after birth
HBF
when is HGB A2 found and what chains is it made of?
alpha and delta....
Occurs late in fetal life
Delta chain production begins 7.5-8 months gestation
2.5% found after birth
what is HGB is found in babies at birth
HBF - 70%
HGB A1 30%
what hgb is found in adults
Hb F - 1%
Hb A1 - 97%
Hb A2 – 2.5%
Once the red cell nucleus (DNA) is removed, no additional _________is made
hemoglobin
____% of hemoglobinization is complete while the nucleus is intact

____% of Hgb is assembled after the nucleus is extruded
65% of hemoglobinization is complete while the nucleus is intact

35% of Hgb is assembled after the nucleus is extruded
how is HGB still made after nucleus extruded
Assembly of chains occur while still attached to the ribosome
Subunits combine in the ______, pyrrole ring insertion occurs in the ________
Subunits combine in the cytoplasm, pyrrole ring insertion occurs in the mitochrondria
a porphyrin ring + ferrous iron (Fe+2) atom.
heme
heme is made of what?
a porphyrin ring + ferrous iron (Fe+2) atom.
Heme's pyrrole ring sits in a _________pocket of the globin chain, near the surface.
hydrophobic
Iron can only bind oxygen in the ________ state.
ferrous
Once the heme unit is complete, it goes into the cytoplasm to combine with the__________
globin chains
heme synthesis occurs where?
mitch and cytoplasm
ferrous iron is the ____ form
Ferric iron is the _____ form...
which can bind to oxygen?
reduced
oxidized

Ferrous
what is the rate limiting step in HEME synthesis>
Glycine + Succinyl Coenzyme A condensate to for 5 delta aminolevulinic acid
where does the rate limiting step happen>
mitch
where does heme synthesis begin?
begins in mitch-cyto-mitch(iron insertion)-
then heme moves to cyto for globin chains
protoporphyrin IX with iron in center is what? where does this occur?
heme
Hemoglobin ref range:
male and female
13-17 g/dl male
12-15 g/dl female
Hgb] is regulated by the control of _________
Hgb] is regulated by the control of erythropoiesis
Patients have shortness of breath, headache, lethargy, dizziness, increased heart rate
sign of what and what is there HGB
anemia
Hb < 10 g/dL
anemia is associated with what HGB
Hb < 10 g/dL
mild loss of red cell 20% 12g/dL
mod loss of red cells 30-40% 10g/d
severe loss of red cells> 50% 7g/dL
what do each lead to?
SOB
SHOCK
DEATH
Renal patients have Hb = 7-10 g/dL range
Chronic GI bleeders have Hb = 10 g/dL
Slow blood loss patients are admitted to ER with Hb < 4 g/dL
HOW IS THIS POSSIBLE?
Adaptations to red cell loss permit life below a 50% decrease in hematocrit/hgb (red cell mass)
HGB conc depend on :
age gender diet
HGB RANGES:
g/dL g/L
Newborns:
Children:
Male :
Female:

Critical call:
Critical call:
g/dL g/L
Newborns: 16-23 160-230
Children: 10-14 100-140
Male : 13-17 130-170
Female: 12-15 120-150

Critical call: < 6.6 < 66
Critical call: >19.9 >199
Conditions of increased red cell need:
high altitude
emphysema
cigarette smokers
long distance runners
pregnancy
Measured Hb < 10 g/dL
physiological need for more O2
associated with what?
anemia
with anemia what are some immediate physiological responses?
Increased heart rate
Increased respirations
Increased circulation
with anema what are some Slower physiological response--long term adaptations
Varying environmental conditions or physiologic demand for O2 changes the function of Hb and directly affects the release of oxygen. Physiological changes increase O2 utilization by the body.
shifting the Hb-O2 curve to the right does what?
allows HGB to give up O2 more easily
what would give the HGB - O2 curve a right shift?
pH
pO2
temp
pCO2
2,3 DPG
dec pH lactic acid build up

dec pO2 lack of oxygen in the tissues

inc temp higher body temperature

inc pCO2 build up of carbon dioxide

inc 2,3 DPG places Hgb tetramere in “stressed” configuration
Hb-O2 ~ __% saturated at normal pH in the tissues

Hb-O2 ~ __% saturated at acid pH in the tissues
50%

25%
what would cause a left shift?
pH
pCO2
2,3 DPG
temp
p02
Inc pH (dec H+)
Dec pCO2
Dec 2,3DPG
Dec temp
Inc in pO2
_______is produced from the glycolytic pathway.
2,3 DPG
what does 2,3 DPG do?
When inserted into the heme unit, Hb bonds are stressed, placing the pyrrole ring at the surface.
O2 is more easily delivered to the tissues
the bohr effect:
As [H+] builds up in the tissues, O2 is more easily released from Hgb.
Excess [H+] is then bound to the ____________
deoxyhemoglobin
The _________accounts for 70% of CO2 transport to the lungs. This is an_____ transport.
The chloride shift accounts for 70% of CO2 transport to the lungs. This is an indirect transport.
___% of CO2 is directly bound to Hb and transported.
25%
_____% of CO2 is dissolved and carried by the plasma
5%
when measuring hemoglobin what are the steps....
Whole blood is added to modified Drabkin’s reagent [K3Fe(CN)6].

The red cell is lysed; Hgb is released.

All Hgb is converted to the ferric state (Fe+3) of methgb.

MetHgb is converted to cyanmethemoglobin by KCN.

Absorbance of Hb is measured at 540 nm.

Concentrations are read off the standard curve.
true or false standard curve is not prepared for each lot of reagent
false
controls of _____ values are run along with each patient
known
what is done special with patients of unknown hemoglobin values.....
run duplicate
what do you all put on the Hemoglobin standard curve?
Your name, date, lot # of std
Title graph: Hgb Std curve
X&Y axis; Abs and Hgb conc.
Place abs readings on back.
Place any comments on back.
dilute _____ g/dL standard to make a curve
20
how do you find the conc of the unknown without a curve
Conc unk = (conc of stand x abs of unk )/ abs of stand
Oxyhemoglobin –
Deoxyhemoglobin –
Oxyhemoglobin – O2 attached
Deoxyhemoglobin – O2 released
Methemoglobin –
Methemoglobin – Heme iron is oxidized (Fe+3
who is more like to have METHEMOGLOBIN and why?
Infants are more susceptible
-HbF is more readily converted to MetHb
-Infant red cells lack enzymes to reduce Fe+3
what are signs of methemoglobin and what percentage will cause cyanosis and what percentage will cause death.
Blue color to lips and nails
10% methgb = cyanosis
60% methgb = death (hypoxia)
hypoxia
without oxygen
Sulfhemoglobin
Sulfhemoglobin – sulfur permanently bound to heme
Carboxyhemoglobin
CO bound to heme
what are signs of carboxyhemoglobim?
what is the affinity difference from regular O2 affinity?
what would happen if acute?
what do smokers have?
Lips and nails have a cherry red color

CO:heme binding is 200% stronger than 02:heme

Acute carboxyhemoglobinemia = death

Smokers have increased CO levels
types of hemoglobin to be measured....
Oxyhemoglobin
Deoxyhemoglobin
Carboxyhemoglobin
Methemoglobin
Sulfhemoglobin
what hemoglobin cant be measured with the cyanmethemoglobin?
sulfhemoglobin
which hemoglobin requires a longer reaction time to oxidize the hemoglobin to methgb.
carboxyhemoglobin
hemoglobin is recycled.... it breaks down into _____ and _____. where does they iron go?
heme(iron) and globin
rbc life span is ____ and then senescent cells are what?
120 days
engulfed by macrophages
or they degrade extra or intervascullary.....
which is good
extravascular degradation or intravascular degradation of hemoglobin/rbc?
extravascular since heme is toxic when intravascular....
extravascular degradation
hemoglobin catabolized outside of the vascular space (RES cells);

normal mode of recycling
intravascular degradation
red cell is lysed intravascularly;

heme, free iron are released into blood stream
Dec haptoglobin levels
Inc urine Hb
Inc methemalbumin;
Positive Schumm’s Test

are all signs of what?
intravascular degradation
Inc serum bilirubin
Inc urine urobilinogen
Inc fecal urobilinogen
Dec H&H levels
are all signs of what?
extravascular degradation
what are signs of extravascular degradation
Extravascular
Inc serum bilirubin
Inc urine urobilinogen
Inc fecal urobilinogen
Dec H&H levels
what are signs of intravascular degradation?
Dec haptoglobin levels
Inc urine Hb
Inc methemalbumin;
Positive Schumm’s Test
MCV-
MCH-
MCHC -
RDW-
MCV- mean corpuscular volume
MCH- mean corpuscular hemoglobin
MCHC - mean corpuscular hgb concentration
RDW- (average) red cell distribution width
true or false MCV is not patient specific
false
changes in MCV may indicate what?
lab error since it is patient specific
_____ help categorize anemias and indicate iron storage levels
Indices
what is the MCV equation and what are the units?
(HCt/ RBC)x10

femtoliters
what does MCV tell you?
red cell size
MCV ref ranges:
normocytic
microcytic
macrocytic
normocytic 80-100 fL
microcytic < 80 fL
macrocytic > 100 fL
what causes macrocytosis?
Young blood cells (shift cells)
Newborns
Post surgical
Post partum
Pregnancy
Iron replacement therapy
Hemolytic anemia
HDFN
B12 or Folate deficiency
Alcoholism
Liver disease
Chemotherapy
what causes microcytosis?
Iron deficiency
Thalassemia
Decreased iron utilization
RBC fragmentation
MCH tells you what and what is the equation, and what is the units?
hemoglobin content

(HGB/RBC) x 10

picograms
not sex dependent
related to HGB within a red cell
MCH
ref range for MCH
26-34pg
what are the times three rules?
Hb X 3 = HCT

RBC X 3 = Hb
MCH does not consider overall ___________, so it not as useful as the MCHC for QA
MCH does not consider overall cell size, so it not as useful as the MCHC for QA
what does MCHC tell you... what is the equation and what are the units
color of red cell

(HGB/HCT) X 100

grams/dL %
Reference Ranges for MCHC
Normochromic
Hypochromic
Hyperchromic
Reference Ranges (color)
Normochromic 32%-36%
Hypochromic < 32%
Hyperchromic > 36%*
Tightest RBC parameter
Best index for QA
MCHC
Markedly increased with RBC agglutination or spherocytes
MCHC
MCHC > 36 typically indicates
analytical error and RARELY spherocytes
RDW what is the equation and units?
Units = (%)


(SDt/MCV) x 100
RWD ref range:
11.5-14.5%
RDW > 16 indicates ?
anisocytosis
individual size difference of RBC
RDW
The bigger the RDW, the _____ size variance you will see under the microscope.
more
indices are used when reporting what?
Descriptors of red cell size and color are reported when the patient has anemia
closeness of the analytical result to the true value
accuracy
the ability to reproduce the result
precision
preanalytical error
any error that occurs before the sample is analyzed
Specimen problems
systematic error
analytical error that occurs because of variances in the procedure
Instrument problems
Reagent problems
QC material with known values ID systematic error
radom error
sporadic systematic error that occurs without an identifiable source
Duplicate testing eliminates random error
post analytical error
errors that occur during the reporting phase of laboratory testing.
Data utilization errors
TAT too long
Reported result uses incorrect units
______ provide inside into the expected red cell morphology
red cell indices
mean cell volume measured in what?
fL
hematocrit units
%
mean cell hemoglobin units
pg
mchc units
g/dl %
rdw units
%
diffusely basophilic erythrocytes also known as
reticulocytes
reticulocytes describe color and properties
immature RBC
staing bluish and slightly bigger than RBC
carry less O2 since not fully hemoglobinized and hemoglobin assembly still takes place due to lots of ribosomes even though nucleus may be gone.

you dont want to see in a PBS
anisocytosis
variation in RBC size
Microcytosis causes:
iron def
thalassemia
sideroblastic anemia
macrocytosis causes:
megabloblastic anemia
hemolytic anemia
hemorrhage
liver disease
asplenia
endocrinopathies
poikilocytosis
alterations in red cell shape
burr cells formal name
echinocytes
what do echinocytes look like
evenly distributed pointy projections, crenated
why do echinocytes form?
increased pH (from slide)
septiciema
renal insufficiency
artifactual
why are echinocytes shaped the way they are?
outer layer has more lipids giving it a rough shape
mouth cells formal name
stomatocytes
uniconcave cells, increased cell membrane surface are. inner inner layer has more lipids than outer
stomatocytes
what causes stomatocyte formation
alchoholism
liver disease
these can be genetic of acquired .... no central pallor and have a MCHC over 36%
spherocytes
rbc fragments due to mechanical damage are called
schistocytes
true of false... schistocytes can still deliver oxygen
true
what are schistocytes caused by...
fibrin strands
severe burns
mechanical heart valves
glomerular nephritis
hemolysis makes cells ____ so there are no _____
explode
fragments
formal name of spur cells
acanthocytes
describe acanthocytes
thorn like spikes
and they are irregular bublbous projections
what causes acanthocytes?
changes in plasma lipids
alchoholic cirrhosis,
lipid metabolism disorders
formal name for target cells
codocytes also called bulls eye or mexican hat
what causes codocytes
increase cholestrol and lipids
the skin is to lose for the body... increased surface area to volume ratio
codocytes
rbc fragments due to mechanical damage are called
schistocytes
true of false... schistocytes can still deliver oxygen
true
what are schistocytes caused by...
fibrin strands
severe burns
mechanical heart valves
glomerular nephritis
hemolysis makes cells ____ so there are no _____
explode
fragments
formal name of spur cells
acanthocytes
describe acanthocytes
thorn like spikes
and they are irregular bublbous projections
what causes acanthocytes?
changes in plasma lipids
alchoholic cirrhosis,
lipid metabolism disorders
formal name for target cells
codocytes also called bulls eye or mexican hat
what causes codocytes
increase cholestrol and lipids
the skin is to lose for the body... increased surface area to volume ratio
codocytes
what environmental factor can cause target cells?
high humidity
what is the formal name for tear drop cells
dacryocytes
why are dacryocytes formed?
inclusion are removed by spleen
what caues dacryocyte formation
myelodysplastic syndromes
another name for sickle cells
drepanocytes
genetic abnormality that result in HbS formation
sickle cell disease
drepanocytes
HbS tactoids and polymerization of Hb
sickle cell disease
drepanocytes
what causes sickle cells to unsickle
reoxygenation but can only do it a few cycles
what would Na metabisulfite be used for?
to test for sickle cell disease
When a fresh solution of sodium metabisulfite (reducing soln) is used, positive cases should show 10 to 75 percent sickling within 15 minutes. There is a positive correlation between the degree of sickling and the severity of the disease.

(3) Normal blood treated in this way shows no sickling.
formal name for cigar/pencil cells
elliptocytes or ovalocytes
what are elliptocytes/ovalocytes caused by?
hereditary membrane abnormality or can be acquired artifactual
formal name for helmet cells
keratocytes
another name for blister cells
keratocytes of helmet cells
hornlike projections cells
keratocytes
what causes keratocytes
cause by removing LARGE inclusions by spleen
or cells care caught upon intravascular fibrins strands
HbC crystals
Hemoglobin C comprises 2 normal alpha chains and 2 variant beta chains in which lysine has replaced glutamic acid at position 6. This unstable hemoglobin precipitates in red blood cells to form crystals. These intracellular crystals lead to a decrease in red blood cell deformability and an increase in the viscosity of the blood. The spleen effectively removes these crystal-containing cells.

Much like the mechanism in sickle cell hemoglobin, the amino acid change in the hemoglobin C molecule impairs malaria growth and development. It reduces parasitemia and confers protection against mild malaria attack.1, 2 Therefore, persons who are heterozygous for hemoglobin C have a survival advantage in endemic areas. The risk of malaria is lower still in persons who are homozygous for hemoglobin C.
when would crystals and lots of target cells be seen?
when a patient has inherited Hb C

few target cells seen in Hb AC which is a carrier state
Hbc is acquired how? and what gets rid of them?
genetically
and are removed by spleen
Polychromasia
prescence of reticulocytes normal to a certain extent

A slight degree of polychromasia is normal. Increases in polychromasia suggest an increased bone marrow response. No polychromasia correlates with non-responsive, anemic patients, which is characterized by all cells exhibiting the same coloration. This finding presents a guarded prognosis, pending resolution of the cause of the non-regeneration.
which cell types follow these guidelines?
occ: 0-2
moderate: 2-5
many: >5
stippling
howell jollu bodies
pappenheimer bodies
spherocytes
tear drops
ovalomacrocytes
which cells follow these RBC quidelines?
occ: 2-5
moderate:5-20
many: >20
burr cells
elliptocytes
target cells
which RBC types follow these guidelines?
occ: 2-3
moderate:4-6
many: >6
polychromasia
which RBC types follow these quidelines:
occ:5-20%
moderate:20-40%
many: >40%
macro/microcytes
what is dyserythropoiesis?
when rbc production goes wrong and nucleus hasnt been fully removed
what is left in howell jolly body cells and why?
DNA fragment....
from dyserythropoiesis
what is a cause for howell jolly bodies?
Common causes of asplenia are splenectomy due to trauma, and autosplenectomy caused by sickle cell anemia.
cabot rings are what and are caused by what?
Cabot rings are thin, red-violet staining, threadlike strands in the shape of a loop or figure-8 that are found on rare occasions in erythrocytes. They are believed to be microtubules that are remnants from a mitotic spindle.

Cabot rings have been observed in a handful of cases in patients with megaloblastic anemia, lead poisoning and other disorders of erythropoiesis.

such as dyserythropoiesis
what causes Heinz Bodies?
denatured Hb
how can you see Heinz Bodies?
supervital stains
how many heinz bodies per cell?
one
how many howell jolly bodies inclusions are in a cell?
few
pappenheimer bodies have what inclusions?
_______ deposits
_______ granules
iron deposits
siderotic granules
what are siderotic granules
iron deposits
how many pappenheimer bodies inclusions are seen in cells?
many
what are the two intracellular parasites we talked about
malaria - africa
babesia - USA
how is malaria and babesia acquired?
malaria - misquito
babesia- tick
what would you see with intracellular parasites?
ring froms with dark spot inside red blood cell
what stains can nucleated RBC be seen in and what is the inclusion
wright and supervital

dna
what stains can reticulocyte be seen in amd what is the inclusion?
supervital only

rna ribosomes
what stains can basophilic stippling being seen in and what is the inclusion?
can be seen with both the supervital and wright stain

the are RNA
what is basophilic stippling and what is it caused by?
rna inclusions
black to dark purple stuff with in RBC

caused by lead poisoning
with what stain can howell jolly bodies be seen with and what is the inclusion made of?
supervital and wright

DNA
with what stains can Heinz bodies be seen and what is the inclusion made of?
supervital only

hgb
what two cell type inclusions can only be seen with supervital stain?
reticulocytes and heinz bodies
with what stains are pappenheimer bodies seen and what are the inclusions made of?
supervital and wright
iron siderotic granules
what results from inc plasma proteins.....(rbc)
rouleaux
what disease causes rouleaux
multiple myeloma
this results from IgM cold Ab
agglutination
manual counts are counted in ___ ______ volumes
one microliter
to convert one microliter to one liter what do you multiply by?
10^6

5.5 x 10^3 = 5.5 x 10^9 cells/L
Normal WBC count
4500 - 11500 cells/microliter
how should WBC counts be reported?
##.# x 10^3/microliter

##.# x 10^9/liter

sometimes ##.# thou/microliter
what kind of blood should be used for manual cell counts
edta whole blood
body fluid samples
Manual counting procedure requires three things....
1. diluting fluid
2. dilutiong pipette
3. hemacytometer
what diluting fluids would you want to use when looking for WBC?
turks
1% HCl
2% glacial acetic acid

AMMONIUM OXALATE -lyses RBC
ammonium oxalate is used when?
diluting fluid used to lyse rbcs and read WBC or PLATELET counts
what diluting fluids would you use to read RBC counts
saline -reduces plasma proteins and stabalizes rbc
with the unopette system what dilutions do you wanna make for
RBC
WBC
PLATLET

and with what?
for RBC you wanna make a 1:200 dilution with saline

for platelet and WBC you wanna make a 1:100 dilution with ammonium oxalate
how do you calculate # of cells counted?
# cells counted/microliter = [(avg # of cells)x dilution]/(area x depth)
for unopette WBC count what is the equation?
(avg cells x 100)/(9mm^2 x.1mm)
what to do in high # of cells during manual count
increase dilution
what to do in low # of cells during manual count?
use larger volume
smaller dilution
count more areas
Neubauer Chamber
H chamber
used for manual counts
groud glass
precision instrument
depth is constant
two counting sides
for WBC counting how many of the squares do you count?
you count all the 9 lrg squares which are 1x1mm each
for rbc counting what squares do you count?
5 small squares within large center square
for platelet counting what squares do you count?
all 25 small squares within lrg center square
other Hemocytometers
FUCHS ROSENTHAL
SPEIRDS-LEVY
FR: 4mmx4mm depth=.1mm volume=1.6mm^3

SL: 5mmx2mm depth=.2mm volume=1.6mm^3
Cell counting procedure:
8 steps
1. make dilution
2. clean chamber
3. set coverslip
4. charge both sides
5. let PLT counts sit in humidity chamber while they settle to avoid dehydration
6.count cells that fall at top and left line
7.exclude right bottom line cells
8. count both side and avg within 10%
with cell counting what must the avg of both sides must be within what percent?
10%
sources of error with Manual counting
1.poorly mixed sample
2.not whipping off uniopettes
3.overzealous mixing and spilling dilutant
4.overfilling chamber
5.evaporation
6.uneven cell distribution due to ling or uneven charge
7.not letting PLT settle
8.using too much light
9.incorrecting identify cells
10.not using formula correctly
what does overfilling a chamber do?
falsely lowers cell count
-excess fluid flows into moat pulling cell particles out of counting area
what does evaporation do to a manual count?
falsely increases cell count
-large cell # takes to long to count
-fluid evaporates w/ light intensity
when doing manual count what must you do with the light...
low light and lower diaphragm
Coulter principle with counting....
impedence couting uses interruptions in an electrical current to detect cell particles

-voltage spikes = # of cells counted
-larger spikes (cell volume) is proportional to size of resistance

-relative frequency of occurence is plotted in a histogram plot of MCV
COULTER AcT
-sample size
-WBC and RBC dilutions done together or seperate?
-cell counts repeated how many times?
-what is the HGB measured from?
100 microliters of whole EDTA blood

seperate dilutions

cell counts done in triplicate

hgb measured from WBC dilution
Laser technology how does it work with cell counting
the way the cells flow through and the light scatters lets you know different characteristics of a cell
RALS
FALS

neutrophil would have what?
RALS measures how complex the interior is

FALS measures size

high fals and high rals
what would be Coulter machine errors?
1. poor mixing
2. fibrin/platelet clots
3. instrument error
4. patient specimen interferences
-lipemia
-cold agglutinins
-difficult draw
-short draw
-IV contamination
what is lipemia?
cloudy fatty plasma
what kind of counts can be done on peritoneal(dialysis fluid) and ventricular(brain ventricular space) fluid types?
RBC, WBC, Bacteria
what kind of counts can be done on Thoracic(upper chest) and Pleural(lung) fluid types
RBC, WBC, tumor cells
what kind of counts can be done on cerebrospinal fluid?
RBC, WBC, bacteria, tumor cells
what kind of counts can be done on Synovial joint fluid types
rbc,wbc,bacteria,crystals
what kinda of counts can be done on pericardual(heart lining) fluid counts
WBC, tumor counts
how are the following labeled:
WBC
RBC
HGB
HCT
MCV
MCH
MCHC
PLT
WBC- #.# x 10^9/L or ####/microliter

RBC- #.## x 10^12/L

HGB- ##.#g/dL

HCT- ##%

MCV- ## fL

MCH- ## picograms pg

MCHC- ## %

PLT- ### x 10^9/L
Rel cell type
absolute cell type

what are the units?
relative cell ##%

absolute cell #,###/microliters
what does the Hemocue measure?
HGB
what is an advantage of the hemocue over the coulter
smaller is to travel with and only needs cappillary blood.
much faster as well
when make a cyanmethemoglobin curve what is labeld on the axis's
Hemoglobin g/dL on X
Absorbance at 540nm on Y
the bohr effect refers to the cells ability of....
accepting H+
a person with renal disease would have
decreased hematopoiesis

erythropoeiten is not made and hematopoiesis is decreased.
3 phases of ESR
10 min forming rouleaux
40 min falling
10 min stacking
why do plasma proteins increase ESR?
the decrease the neg charge thus decreases the zeta potential of RBC
what is an acceptable agreement btwn a PBS WBC estimate and a Automated WBC count?
25%
and increase in macrocytic, polychromatphillic erythroctytes seen in wright stain smear should make you wanna do what test?
reticulocyte count
what are you lookin for in wright stain lower power
color and no precipitates
with lipemic blood sample which paramters will be affected?
WBC
RBC
HGB
HCT
MCV
MCHC
MCH
MCHC,MCH,HGB
allowing slide to dry vertically doesnt allow _______ to form
precipitate
specimen for wedge smear
and stain used
whole blood with anticoagulant EDTA
wright stain
if not stainin immediately let air dry and fix with methanol.. otherwise stain fixes with alcohol
wright stain effect is produced by the combinded action of ________ _____ and its oxidation products ________ and ___________
methylene blue

eosin Y and azure B
how to do a wright stain
1. place slide smear up on rack
2. flood with methanol, fix for 5 min
3. drain meth and blot with filter paper
4.flood with wright stain for 3 min
5.add wright buffer until greenish metallic sheen comes up... mix by blowing
6.allow mix to stand 8-10 min more wbc more time needed
7.float off green mix with distilled h2o
8.keep smear horizontal
9.blot smear immediately blow dry
10.wipe back of smear with meth.
11. mount coverslip with permount
lymphocytes have what color granules if present?
red
neutrophil granules are what color
purple violet
monocyte granules stain what color
pale red, fine dust particles
what to do on 10X
check color
wbc evenly distributed
look for RBC agglutination and rouleaux
look for immature abnormal cells, platelet clumps or satellitism
do WBC estimate
how to perform WBC estimate
units recorded?
avg wbc counted in 5 fields and multiply by 200....

should agree with automated WBC within 25%
##.# x 10^9/L or ####/microliter
what to do on 100x
platelet estimate and eval.
wbc diff
nucleated rbc
red cell morph
platelet ref ranges
rare: <50K/uL
decreased: 50-149K/uL
normal: 150-450K/uL
increased: >450k/uL
how to correct for WBC count when there are nucleated RBC
(WBC x100)/ (nRBC+100 )
packed cell volume of whole blood
hematocrit
what kind of specimen is used for Hematocrit (microcappilary)
whole blood with EDTA or Heparin
when measuring hematocrits blood collected by finder prick should be collected in a _______ ________. Blood collected from a venipuncture should be test in an _________ ______________.
heparinized capp tube

untreated capp tube
when the Coulter gives a WBC reading exceeding __________ a microcappilary method should be preformed to get hematocrit... .whY?
35.0 x 10^9/L

because high WBC maye elevate RBC and hematocrit reading....
coulter calc. HCT from rbc and mcv values
specimen for HCT....
whole blood in EDTA.... must be half full so cell done shrink and false lowered HCT

or if capp blood avoid edema areas
when doin HCT always make _________ capillary tubes and stopper with _____
2, clay
how long do you spin HCT tubes?
5 mins
when using the reader for HCT what do you do
clay/RBC is set to zero
plasma/air is set to 100
HCT is taken at RBC/plasma interface
duplicate microcapp tubes for HCT should agree within what %
+/- 3%
when recording HCT what two things do you record?
your duplicates
average as whole number %
ref range for HCT
male
female
male 41-53%
female 36-46%
RBC surface is ________ charged
neg
increased conc of fibrinogen, gamma globulin, and proteins does what to ESR
neg charge btwn RBC is reduced, they rouleaux more easily and an increased ESR
ESR is increased in anemia due to what?
easier settling of a small number of cells in a larger volume
used for the detection and evaluation of the conditions where tissue destruction and inflammation are present
ESR
advantage of using MINI VES analyzer over westergren
reduced biohazard exposure
greater accuracy
improved precision
shorter test only 20 min
what specimen is used for ESR
whole blood with EDTA less than 3 hours old if at Room temp
orrr lest the 12 hours old if refrigerated at 4 degree celcius

blood tube must be more than half full, free of clots,and at room temp before testing!
how to do ESR
1. fill dilution vial to lower line with .9% sodium chloride (.3mL)
2. add well mixed blood to top line (1.2mL) and mix with parafilm cover
3.pipette into westergren pipette until cotton plug is saturated
4.wipe off and place in level Sedirack
5.time for 1 hour
6.red interface of RBC and plasma
how to report ESR
report in whole number in mm/hour
ref ranges for ESR:
Male
female
children
Male:
18-50yrs: 0-15mm/hr
50-85yrs: 0-20mm/hr
85 and over: 0-30mm/hr

female:
18-50yrs: 0-20mm/hr
50-85yrs: 0-30mm/hr
85yrs and over: 0-42mm/hr

children: 0-10mm/hr
ESR sources of error slowing it.
bubbles
cold temp increase plasma viscosity
dehydration inc plasma viscosity
extreme microcytosis
excess EDTA
high RBC
old blood- rbc become spherical and less likely to rouleaux
poikilocytes
test read too soon
ESR sources of error accelerating it
low RBC
high fibrinogen or globulin levels- could be from infalmmation or pregnancy
macrocytosis
delay in reading
surface vibrations
tilted tube
warm temp
macrocytosis and ESR
large cells settle faster than smaller ones
to measure hemoglobin what method is used
cyanomethemoglobin
what decimal place is Absorbance measured too
third
what is all in DRABKINS reagent


undiluted _____standard is equivalent to _____
potassium ferriccyanide
potassium cyanide
potassium dihyrdogen phosphate
Triton X
can be stored for 1month in dark bottle

80mg/dL...... 20g/dL
when making curve what should your dilutions be for
5, 10, 15, 20 and blank g/dL?


patient sample are normally diluted what to what

how is this prepared with WB?
5: 1.5mL standard and 4.5mL D.R.
10: 3 and 3
15: 4.5 and 1.5
20: 6 and 0
blank: 0 and 6

1:250

pipette 5mL of Drabkins then add 30uL of blood(wipe tip) and mix well
let tube stand for 5 mins
zero out specto. and then measure abs
after mixing standard and drabkins how long must you wait?

what must you first do to spectrophotmeter?
3 minutes

zero with reagent blank
when measuring hemoglobin abs do you start with lowest conc or highest conc?
lowest
a ___________ must accompany each new lot of Drabkins....
new standard curve
principle of drabkins reagent and whole blood interaction
WB added to drabkins

RBC lyse and hemoglobin oxidizes to methemoglobin by K ferricyanide

during oxidation iron is converted to ferric iron

methemoglobin forms with K cyanide to for cyanomethemoglobin which is a stable pigment
what kind of specimen is used during the cyanomethemoglobin method?
WB with EDTA or Heparin
or
direct capp blood
what are the ref range and units for HGB
male
female
male 13-17 g/dL
female 12-15 g/dL

##.# g/dL
____ is a measurement of the coefficient of variation based on the red cell distribution histogram, rather than a calculation from other red cell parameters
RDW red cell distribution width
RBC morph reporting precedure
10x and 100x
10x look for rouleaux, agglut., stain quality, platelet clumps at edge

100x look at 10 field min (150-200 cells) RBC should not be touching and avg abnormalites reported
(platelets also evaluated)
if seen under low power report as present with RBC

these should be reported if seen at all
rouleaux or agglut

cabot rings
parasites
Hgb C crystals
how to use unopette system
repeat procedure twice and charge each side with one sample
1. puncture reservoir with protective shield
2.fill capp pipette with blood... wipe blood
3.add blood to reservoir
4.cap with shield to prevent evap.
5. let sit 10 -15 min to lyse cells
(WBC count should be preformed within three hours)
6.the convert pipette to dropper and charge one side of hemocytometer

first view at 10x then at 40x
sides should agree with 10%
what do you clean a hemocytometer with?
isoterge
distilled h20 and
ethanol
what specimen should be use for manual white cell counts?
whole blood in EDTA
what is the dilution for the UNOPETTE
1:100
20uL in unopette and 1.98mL of diluting fluid....
when doing WBC counts and using a 1:20 dilution only count the______
4 corner squares

with 1:100 dilution count all 9 squares
cells/ mm^3 is the same as what?
cells/uL
ref range for WBC
4.0-11.0 x 10^9/L
micro
femto
pico
milli
micro 10^-6
femto 10^-15
pico 10^-12
milli 10^-3
ONPG
beta-galactosidase
ONPG results
neg colorless

pos yellow
ADH
arginine dihydrolase
ADH results
neg yellow

pos red/orange
LDC
lysine decarboxylase
LDC results
neg yellow

pos red/orange
ODC
ornithine decarboxylase
ODC results
neg yellow

pos red/orange
CIT
citrate utilization
CIT results
neg pale green/yellow

pos blue green/ blue
H2S
H2S production
H2S results
neg colorless or grey

pos black/thin line
URE
urease
URE results
neg yellow

pos red/orange
TDA
tryptophane deaminase
TDA results
after adding 1 drop ferric cholride

neg yellow

pos brown/red
IND
indole production
IND results
after adding one drop KOVACs and waitng two mins

neg yellow

pos red ring
VP
acetoin production
VP results
after 1 drop K hydroxide and 1 drop alpha naphthol and wait 10 min

neg colorless

pos pink/red
GEL
gelatinase
GEL results
neg no black pigment diffusion

pos diffusion of black pigment
ALL SUGARS
fermentation and oxidation
ALL SUGARS results
neg blue/ blue green

pos yellow