Biochemical variables and trace element analyses

Workshop for Animal Health Professionals, 09-09-1999
Grange Research Centre, Dunsany, Co. Meath, Ireland

[Although Grange does not test samples for the public now, the data in this paper will be useful background material  for professionals]

| Anticoagulants | "Normality" in single sample v group mean | Tests available at Grange | Minerals in Bulk-Milk & Bulk-Blood | Milk Iodine (MI) | Major elements in blood | Trace elements in blood & Milk I | Blood Cu v liver Cu | Blood Cu | Blood glutathione peroxidase (GPx) | Plasma inorganic I (PII) v TOTI & T4 | Plasma Zn | Nutritional & metabolic variables | Plasma globulin | Plasma urea | Beta-hydroxybutyrate (bHB), glucose & non-esterified fatty acids (NEFA) | Glutamate Dehydrogenase (GLDH) | Antibody tests & haematology | Antibody tests | Antibody titres in respiratory disease | Bovine hematology reference values | WCC & differential WCC | Haptoglobin | Platelets | Red cell size & haemoglobin | Grange Breakpoints to assess blood, liver & milk tests in Cattle | Warning to Vets | Further reading |

Phil Rogers MVB, MRCVS

  philrogers@eircom.net

Bernadette Earley PhD

  bearley@grange.teagasc.ie

Joe Larkin CBiol, MIBiol

  jlarkin@grange.teagasc.ie

& Mary Munnelly DipAnChem

  mmunnelly@grange.teagasc.ie

Introduction

This paper will discuss the interpretation of Lab tests in relation to their use to support or refute a clinical diagnosis of herd- or flock- problems of illhealth or lowered productivity.

Anticoagulants used in vacutainers: Vets who submit incorrect sample types can not expect all tests to be done. Though many tests can be done on serum from clotted blood samples, Grange discourages the submission of clotted blood. This is to maintain a standard type of sample, and because certain tests can not be done on clotted samples, for example whole blood Cu or GPx. The required anticoagulants are:

Blood test needed

Sample needed

Stopper colour of vacutainer

Anticoagulant

Most tests

Whole blood or plasma

Green

lithium heparin

P and glucose only

Plasma

Grey

sodium fluoride

Haematology only (RCC, MCV, MCHC, WCC, differential WCC etc)

Whole blood

Purple

EDTA

"Normality" in individual animals versus group means: For any given test, we set a narrower "normal range" for groups of animals than for individual animals. For example, a bovine blood Cu value in the range 10.20-20.41 m moL/L is normal for a single sample, but we take a narrower range, 10.72-19.39 umol/L as the normal range for a group of cattle. Similar slight adjustments are made for the other assessment categories for each test. For example, breakpoints (umol/L) used at Grange to assess bovine blood Cu status are:

1=1 sample X=herd mean

VL

LO

ML

NL

HI

VH

X

<7.05

-8.78

-10.71

-19.39

-25.36

>25.36

1

<6.42

-8.78

-10.19

-20.41

-26.69

>26.69

Tests available from the Grange Lab include: Alb, Alp, Alt, Antibody titres to PI-3, IBR and RSV, Ast, B12 (not reliable), bHB, Ca, CK, Cu, gGT, GLDH, Glob, Glu, GPx, Haptoglobin, Hb, IgA, IgG1, IgG2, IgM, Liver Cu, MCH, MCHC, MCV, Mg, Milk I, NEFA, P, PCV, Peps, PII, Platelets, RCC, TP, urea, WCC & Differential WCC and Zn. Table 1 (near the end) shows the breakpoints used to assess their status.

Mineral Tests on Milk Samples and Bulk-Blood Samples: Milk tests can NOT adequately replace blood tests for routine and accurate assessment of mineral nutrition of cattle and sheep. Bulk milk is easy to obtain; it can be analysed for multiple components. Analysis of bulk-milk can help to assess protein and fat levels, urea, ketones, antibodies, mastitis risk, etc. Some commercial Labs offer analysis of milk samples as an aid to investigation of mineral deficiencies in cattle and sheep. However, with the exception of I (see below), milk tests for nutritional major elements (such as Ca, Mg, Na and P), or trace elements (such as Cu or Se etc) are unreliable to assess the animals' status of these nutritional elements.

In 1997, Rogers did an exhaustive search of published veterinary literature (including Focus on Veterinary Medicine) and online databases (including Medline). He also e-mailed several thousand members of the American Association of Veterinary Laboratory Diagnosticians, Beef List, Dairy List, and colleagues in State Veterinary Diagnostic Labs abroad. He failed to find reliable scientific evidence that milk (whether from individual cows, or from the bulk-tank) can be used to assess accurately the mineral status of cattle and sheep. Grange, in line with the advice from State Veterinary Labs abroad, does not recommend mineral tests on bulk samples of blood or milk for that purpose. Analysis of 7-10 individual blood samples provides the best practical assessment of the mineral status of a herd or flock.

Internationally, blood is the standard tissue used for accurate routine assessment of the mineral status of ruminants. For milk to replace blood for this purpose, levels of the relevant minerals in blood and milk should correlate very highly. Unfortunately, in most cases, the correlation for a given mineral are very poor. One exception to this is the Milk Iodine (MI) test.

Milk Iodine (MI) Tests: On specific request, Grange Lab assays MI levels, but these tests must be interpreted with caution. An MI value <50 m g/L may indicate a low iodine intake. As many farmers use iodine-rich teat-dips and disinfectants, iodine-contamination of milk is common. Thus, an MI value >50 m g /L can arise in cows whose bloods are severely low in iodine.

Major elements in blood: The following table indicates the main reasons for low or high values of major elements in bovine plasma.

Test Comments Low values can arise in High values can arise in
Ca Plasma Ca usually very stable in cows except in the days around calving. Note that high Ca inputs in the weeks before calving exaggerates hypocalcaemia at calving. Cows near calving, when hypocalcaemia is usual; all types of cattle under stress (starvation, cold stress) and acidosis; growers on Ca deficient diets (rare and usually on concentrate diets where balanced mineral mix omitted in error) Rare
Mg Plasma Mg levels reflect current daily Mg intake. Low values indicate possible clinical or subclinical problems. Mg shortage relative to requirement for production; stress (starvation, cold stress). In milking cows, low levels can occur at any time, but especially near peak lactation. In dry cows low levels may be associated with a depression of calcium mobilisation from bone, and increased risk of milk fever. Rare, but can arise if excessive Mg is fed
Inorg P Plasma inorganic P levels reflect current dietary intake. Cows can tolerate low levels at grass for some weeks. P deficiency relative to requirement for production. Deficiency is rare on well-run dairy farms. Chronic deficiency may cause infertility, poor thrive and lameness. Rare, but can arise with excess dietary P.

Trace elements in whole blood or plasma, and milk I: The following table indicates the main reasons for low or high values of trace elements in bovine plasma (P) or whole blood (B).

Test Low values can arise in High values can arise in
B12 (P) Not reliable in ruminants not reliable in ruminants
Cu (B) Cu deficiency, when liver Cu is exhausted Haemolytic stage of Cu toxicity, when liver Cu is released into the circulation
GPx (B) Se deficiency Se toxicity
Liver Cu Chronic Cu deficiency Cu toxicity
Milk I I deficiency Sample contamination with I-dips or disinfectants, or when cattle have excess I in their system
PII (P) I deficiency Sample contamination with I-disinfectants from the handler's hands, or when cattle have excess I in their system
Zn (P) Zn deficiency (very rare) Stress, infection and sample contamination, e.g. from Zn-containing stoppers on older vacutainers

Blood v liver Cu tests: Liver is the main reserve of mobilisable Cu in ruminants. Cu levels in whole blood usually remain in the normal range until liver Cu reserves fall to <30 mg/kg liver DM, or so. Compared with blood Cu levels, liver levels are is a better index of herd Cu status. However, because practitioners seldom use liver biopsy, they use blood Cu as the main field test of bovine Cu status. Where it is feasible, practitioners should consider abattoir- or post-mortem samples of liver for a better assessment of Cu status in a herd in cases of suspect Cu deficiency or toxicity.

Low Cu status may reduce immunity, fertility and growth, and increase neonatal calf deaths, placental retention, scour, pneumonia, anaemia and lameness. High status may indicate subtoxic / toxic conditions.

Whole blood Cu: Blood Cu is used as the standard test of copper status. Liver Cu is a better test, but is not practical for wide scale use. See comments above.

Whole blood glutathione peroxidase (GPx): Blood GPx and blood Se have a good linear correlation, but GPx is a much cheaper and faster test to screen Se status in cattle and sheep. Levels of Se or GPx in blood reflect the long-term status of Se. Both take weeks to rise or fall with rising or falling dietary Se status. Although the clinical / subclinical signs may be controlled within days after Se treatment of deficient herds, blood GPx may take 2-3 months to reach its plateau. Without a detailed history of Se supplementation, low GPx may disguise an already improving situation, and normal GPx may disguise a severe deficiency that has arisen, and caused problems, very recently.

Low GPx status may reduce immunity, fertility and growth, and increase calf mortality, placental retention, muscular dystrophy, scour and pneumonia. High status may indicate Se toxicity due to excessive Se supplementation and/or high soil/herbage selenium.

Plasma inorganic I (PII) v TOTI & T4: In earlier attempts to assess the I nutrition of cattle, we examined many blood tests - total blood I (TOTI), protein-bound I (PBI) and thyroid hormones (T3, T4). They were of no practical value to assess I status of cattle. PII is the most sensitive test currently available to assess I status. It is a measurement of free iodide in transit from the digesta to the thyroid. (The thyroid I-trap is very efficient; it traps most of the I absorbed each day). After a rise or fall of I intake, PII rises within hours, and falls within days, respectively. Thus, PII helps to confirm a suspicion of current deficiency or excess of I.

PII is measured in minute quantities (m g/L, or ng/ml). It is very easy to contaminate a whole batch of samples if the handler's hands are contaminated. Therefore, vets, or others, who handle vacutainers must not handle disinfectants or other compounds high in I in the preceding 24 hours. To assess iodine nutrition of cattle and sheep accurately, we advise assay of PII levels in 7-10 individual blood samples. The PII test is more reliable than the MI test, but is time-consuming, labour-intensive and more expensive to do.

Textbook roles of I- and thyroid- function are in maintenance of reproduction (fertility, pregnancy, perinatal viability), placental expulsion, lactation, growth and basal metabolism. Textbooks often mention the birth of small, bald calves with visible or palpable goitre. That is very rare in Ireland. Most calves dead from I deficiency have a full haircoat and few show a visible or palpable goitre. Thyroid enlargement or pathology in these calves can be detected only by weighing both glands (weights >25g or so are "enlarged") and by histopathology for hyperplasia and colloid. Sometimes the histopathology shows hyperplastic / colloid cytology even though the combined gland weights are <25g. A vet practitioner will not detect this without the help of the lab! (Note: Se deficiency can also cause thyroid enlargement, as a Se-dependent enzyme is involved in thyroid metabolism of I).

I deficiency can have serious effects in Irish herds. They include heavy neonatal loss (late abortion, stillbirth, early postnatal loss); thyroid pathology (as described above) in the dead calves; lowered immunity (more scour / pneumonia) in young calves, esp. from first calvers; retained placenta in cows (especially first-calvers); subfertility (anoestrus, repeats, and embryonic / foetal loss) esp. in heifers. We have no solid evidence of I deficiency causing reduced growth or milk yield, but this may occur also. Note that some dietary goitrogens may induce deficiency even with I supplementation.

Plasma Zn: Although Grange can assay Zn in plasma, we have not seen a herd with low Zn for several years, and Zn levels in Irish forages and feeds are usually adequate.

Nutritional and metabolic variables: The following table indicates the main reasons for low or high values of nutritional and metabolic variables.

Test Low values can arise in High values can arise in
Alb Haemodilution, chronic undernutrition (protein or energy), anaemia, parasitism and chronic disease, especially liver disease Haemoconcentration (water deprivation, excitement, stress, shock)
Alp Not applicable  
Alt Not applicable Non-specific liver damage, soft muscle injury
Ast Not applicable Non-specific liver damage, soft muscle injury
bHB Not applicable Energy deficit, ketosis.
CK Not applicable Liver disease, muscular dystrophy, muscular exertion
gGT Not applicable Acute liver damage, biliary tract lesions
GLDH Not applicable Any agents (fluke, fatty-liver syndrome, many veterinary products) that stress the liver; general stress. The highest levels recorded in Grange were in cattle with very high weight gains on high-concentrate diets!
Glob Haemodilution, undernutrition, anaemia, parasitism and chronic disease, especially liver disease Infections and haemoconcentration (water deprivation, excitement, stress, shock)
Glu Energy deficit, ketosis Rare in ruminants, but can arise in stress.
NEFA Not applicable Energy deficit, ketosis, stress
Peps Not applicable Plasma pepsinogen rises when abomasal integrity is breached, especially in ostertagiasis and other forms of nematode parasitism
TP See Alb & Glob See Alb & Glob
Urea Protein deficiency, liver insufficiency?? Excessive intake of protein (especially NPN) and relative deficiency of energy. It can also arise in kidney failure (usually not a herd problem)

Plasma globulin: Glob is calculated by subtracting Alb from TP. Per se, Glob has no relevance to nutritional status, but is a rough index of antibody formation in response to chronic infection or inflammation. The level does not relate either to the severity of the condition or to how recently it took place. (See antibodies and specific immunoglobulins, below).

Plasma urea levels reflect the intake of effective rumen degradable protein (RDP, including ammonia and NPN) and the function of the liver and kidneys. Low blood plasma urea due to protein deficiency (which can reduce performance in all types of cattle) is easy to correct. In some herds, high plasma urea levels may be associated with reduced cow fertility but most Irish herds have high plasma urea on intensively fertilised lush grass.

Beta-hydroxybutyrate (bHB), glucose and non-esterified fatty acids (NEFA): When milking cows have high bHB and/or high NEFA, with normal or low glucose, this indicates a dietary energy deficit. High glucose levels may be associated with severe stress at sampling. High bHB levels within the last two weeks of pregnancy is a sign of negative energy balance which can have important implications for production and fertility in the following lactation.

Glutamate Dehydrogenase (GLDH) is concentrated in the liver, where cell damage allows it to be released into the blood.

Antibody tests and haematology: Grange can do routine haematology and assay for many antibodies in cattle blood.

Antibody tests available at Grange include Immunoglobulins (Ig series): IgG1, IgG2, IgA and IgM are tested in serum and colostrum by a single radial immunodiffusion (sRID) procedure. For example values range from 41.4 2.26 to 48.6 3.52 mg/ml for suckled calves to 17.5 1.82 to 21.5 0.87 mg/ml for purchased dairy calves. IgG1 represents >90% of the total Ig.

Antibody titres in respiratory disease: Antibody titres in response to PI-3, IBR and RSV are assessed in both serum and nasal washings.

Haematology reference values for cattle: The values are assessed in an automatic Celltac haematology analyser with bovine software. The range of bovine haematological values seen in samples from the field, and the ranges accepted as "normal" are:

 

Field Range

"Normal" (Target) Range

Hb (g/dl)

12.7 14.0

13.4 ( 10.4-16.4)

MCH (pg)

14.9 14.9

14.9 ( 13.9-15.9)

MCHC (g/dl)

10.9 63.0

37.0 ( 35.0-39.0)

MCV (fl)

13.0 67.7

40.4 ( 35.4-45.4)

PCV (L/L)

0.106 0.614

0.360 ( 0.30-0.42)

Platelets

275 - 1300

812 ( 750-875)

RCC (x1012/l)

8.5 9.5

9.0 ( 8.0-10.0)

WCC (x109/l)

6.5 25.5

7.2 ( 6.2-8.2)

WCC and differential WCC: White blood cells are responsible for both antibody and cell-mediated immune responses. Any serious change in the number and/or functioning of WCC impairs the natural defence mechanisms. Normal cattle blood has 97% lymphocytes, 2.7% granulocytes and 0.3% monocytes. The values are reported as % and absolute numbers.

Haptoglobin, an acute phase protein, is an index of haemoglobin binding capacity. Units are expressed as g Hb-binding capacity/l. Plasma values are always </=0 in young/adult healthy animals. High values (0.701.10 u) arise as a response to stress (for example surgical / Burdizzo castration) and infectious disease (for example in chronic respiratory disease in calves).

Platelet numbers: Platelets are formed in foetal liver, spleen and bone marrow. In adult animals bone marrow is the principal source, where platelets originate from the megakaryocyte. Calves have higher platelet counts than adults. The principal role of platelets is to prevent haemorrhage when blood vessels are injured. Thrombocytopenia (platelets <150 x103/l) may result from decreased platelet production in the bone marrow or by increased consumption or destruction. The survival time of platelets is relatively short. They survive 8-11 days in the circulating blood with a half-life of 2-3 days. Normal values are 812 30 (x103 platelets/m L).

Red cell size and haemoglobin content: MCV (mean corpuscular volume, in ?m3 or fl) is the mean volume of a red cell (calculated as 10 x PCV / RCC). Volumes are highest in calves and decrease gradually to the adult level at 18-36 months of age. Low and high values arise in microcytic and macrocytic anaemia, respectively. Deficiency of Fe (very rare in ruminants) may manifest as a microcytic anaemia. MCHC (mean corpuscular Hb concentration) is the Hb concentration/unit of packed red cells, calculated as 10 x Hb / PCV, target 37.0 g/dl range 10.9-63.0 g/dl. Low values arise in hypochromic anaemia. High values are rare. MCH (mean corpuscular Hb) is the Hb concentration/red cell, calculated as 10 x Hb / RCC and measured in pg. Low and high values arise in anaemia and hypersplenism, respectively. Errors in erythrocyte counts contribute to variations in MCV and MCH. PCV, Hb and RCC: Low and high values of PCV, Hb and RCC arise in anaemia and haemoconcentration, respectively. PCV (l packed red cells/l whole blood) and Hb level are relatively high at birth. Mean target values for PCV are 34-0.38 L/L. Due to their dilution in blood, PCV and Hb levels fall as soon as the calf ingests colostrum. During the suckling period, when Fe intake is low and the calf is increasing in size, PCV and Hb may fall steadily for some weeks, to reach levels as low as 0.280.30 L/L for PCV and 89 g/dl for Hb. PCV (L/L) is approximately 0.033 times the Hb concentration (g/dl). RCC (red cell counts): The normal range for cattle >1 year of age is circa 5-10 x1012 erythrocytes/l of blood (5-10 x106/mL).

Grange Breakpoints to assess blood, liver and milk tests in Cattle: More complete, and more easily searchable, data (including breakpoints for sheep) are on the Grange Blood Lab page on the Internet at 0breaks.htm and readers are referred to that page.

The following breakpoints are used at Grange to assess the status of blood-test levels, milk-iodine and liver copper levels in samples from cattle. The assessment categories used for most tests are: VL = Very Low; LO = Low; ML = Marginal; NL = Normal (physiologically desirable range); HI = High; VH = Very High.

Click on any test below to see the individual (1) or group (X) breakpoints used for cattle.

Alb

Alp

Alt

Ast

B12

bHB

Ca

CK

Cu

gGT

GLDH

Glob

Glu

GPx

Hb

Lap

Mg

P

PCV

Peps

PII

Se

TP

Urea

Zn

Liver Cu

Milk I

Table 1. Breakpoints used at Grange to assess the status of blood-test levels, milk-iodine and liver copper levels in samples from cattle

Test 1=1 sample X=herd mean B=Bulk Milk

VL

LO

ML

NL

HI

VH

Alb g/L

X

<18.99

-23.09

-29.39

-35.15

-40.85

>40.85

 

1

<18.09

-23.09

-27.99

-37

-43

>43

Alp U/L

X

     

0-1246

-1600

>1600

 

1

     

0-1312

-2500

>2500

Alt U/L

X

     

0-91.2

-150

>150

 

1

     

0-96

-200

>200

Ast U/L

X

     

0-98.8

-180

>180

 

1

     

0-104

-200

>200

B12 pmol/L*

X

<78.00

-130

-194

-703

-1055

>1055

 

1

<74.00

-130

-185

-740

-1110

>1110

bHB mmol/L

X

     

0-0.95

-1.9

>1.90

 

1

     

0-1

-2

>2.00

Ca mmol/L

X

<1.71

-1.84

-2.1

-2.85

-3.33

>3.33

 

1

<1.63

-1.84

-1.99

-3

-3.5

>3.50

CK U/L

X

     

0-114

-240

>240

 

1

     

0-120

-300

>300

Cu umol/L

X

<7.05

-8.78

-10.71

-19.39

-25.36

>25.36

 

1

<6.42

-8.78

-10.19

-20.41

-26.69

>26.69

GGT U/L

X

     

0-51.3

-90

>90

 

1

     

0-54

-120

>120

GLDH U/L

X

     

0-19

-50

>50

 

1

     

0-20

-50

>50

Glob g/L

X

<17.84

-22.99

-30.44

-43.7

-51.3

>51.3

 

1

<16.99

-22.99

-28.99

-46

-54

>54

Glu mmol/L

X

<1.15

-1.6

-2.2

-2.95

-3.9

>3.9

 

1

<1.10

-1.6

-2.1

-3.1

-4.1

>4.1

GPx iu/g Hb**

X

<25.80

-32

-42

-161

-200

>200

 

1

<24.50

-32

-40

-169

-211

>211

Hb g/dl***

X

<8.50

-9.4

-11.13

-14.16

-15.87

>15.87

 

1

<8.10

-9.4

-10.6

-14.9

-16.7

>16.7

Liver Cu mg/kg DM

X

<15.75

-23.1

-31.5

-760

-855

>855

 

1

<15.00

-23.1

-30

-799.99

-900

>900

Mg mmol/L

X

<.52

-0.62

-0.73

-1.29

-1.37

>1.37

 

1

<.49

-0.62

-0.7

-1.36

-1.44

>1.44

Milk I ug/L

B

<25.00

-38

-50

-300

-400

>400

P mmol/L

X

<1.03

-1.16

-1.35

-2.76

-3.38

>3.38

 

1

<.97

-1.16

-1.29

-2.91

-3.55

>3.55

PCV L/L***

X

<.25

-0.27

-0.31

-0.40

-0.46

>.46

 

1

<.24

-0.27

-0.3

-0.42

-0.48

>.48

Peps U/L

X

     

0-0.95

-1.9

>1.90

 

1

     

0-1

-2

>2.00

PII ug/L**** 

X

<25.00

-51

-105

-285

-380

>380

 

1

<20.00

-51

-100

-300

-400

>400

Se umol/L

X

<.54

-0.69

-0.92

-2.4

-4.2

>4.20

 

1

<.51

-0.69

-0.88

-2.53

-4.43

>4.43

TP g/L

X

<43.04

-48.99

-59.84

-78.85

-87.4

>87.4

 

1

<40.99

-48.99

-56.99

-83

-92

>92

Urea mmol/L

X

<1.74

-2.49

-3.49

-7.1

-11.04

>11.04

 

1

<1.66

-2.49

-3.32

-7.47

-11.62

>11.62

Zn umol/L

X

<7.22

-8.8

-11.23

-29.07

-34.88

>34.88

 

1

<6.88

-8.8

-10.7

-30.6

-36.72

>36.72

* B12 is not a good test in ruminants. There is an artefact in the (human) test-kit that gives unreliable values that usually are too low in ruminants.

** GPx breakpoints were lowered on 2/1/1996

*** Normal Hb and PCV are higher in young cattle than in older cattle, and higher in beef cows than in dairy cows. Values usually fall steadily from November to April and rise steadily from May to October.

**** PII breakpoints may be lowered on Grange test soon. Work by Drs. John Mee & Maurice McCoy suggests that new lower limit of the normal range for individual samples should be +/- 70 m g/L, but more data are needed to define really critical normal levels

For example, in the table above, the normal range for albumin (Alb) in individual cattle is taken as 28-37 g/L, but is taken as 29.40-35.15 g/L for a group of cattle.

Some tests, such as the liver enzymes and bHB, have no VL, LO or ML categories, and the "normal range" is taken as from 0 to the value shown under the NL column.

Warning to Vets: Note that grossly abnormal values (VL or VH) for any of these tests arise in apparently healthy, thriving animals. Non-clinical deficiency or excess is very common in Ireland.

Vets are advised NOT to diagnose animal-health problems from the lab results alone. A specific condition may be suspected if the clinical examination and / or history suggest it, and the lab tests agree with that possibility.

Definitive diagnosis, however, should be based on a clear response to specific action to correct the suspected abnormality.

Further reading on the investigation and control of ruminant health problems: See the end of the paper "The Role of the Lab in the Investigation of Herd Health Problems: Strengths and Weaknesses of Lab Diagnosis" (Rogers & Earley 1999). Articles on many other problems of herd and flock-health investigation, and their control are available from the Internet at: Online Technical Notes for Vets & Nutritional Consultants.

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